Patent Publication Number: US-2017372030-A9

Title: Method and apparatus for computer modeling of the interaction between and among cortical and subcortical areas in the human brain for the purpose of predicting the effect of drugs in psychiatric and cognitive diseases

Description:
FIELD OF INVENTION 
     This invention generally relates to methods and apparatus for computer modeling of interactions between and among cortico and subcortical areas of the human brain, for example in a normal and a pathological state resembling schizophrenia, which pathological state has inputs representing the effects of a drug(s), for the purpose of using the outputs to predict the effect of drugs in psychiatric and cognitive diseases. 
     BACKGROUND 
     In a recent White Paper on Drug Development, the FDA has identified computer modeling as one of the new enabling technologies for improving drug development processes. Computational neuropharmacology, as described herein, is different from database management, data mining and pattern recognition, in that it attempts to predict outcomes based on mathematical models starting from well-defined physico-chemical principles, rather than applying statistical inference techniques. 
     Database mining is very useful, especially when applied to the integration of pharmacological with clinical information, but by itself it is usually not sufficient to identify drug targets with a sufficient probability of affecting a disease. 
     As an example, correlating binding affinity data on various human receptor subtypes for a number of neuroleptics with their clinical side-effects has enabled the identification of a number of key receptors involved in weight gain [56]. However, this particular approach uses only binding affinity data and does not take into account the actual dosage used in the clinical setting. Also, the interaction of neuroleptics at the receptor subtypes is also modulated by the affinity of the endogeneous neurotransmitter for the same receptor subtype, the presynaptic firing pattern, possible negative feedback via presynapic autoreceptors and the presence of pharmacologically active metabolites. Thus a mathematical approach encompassing all these interactions in a quantitative way is clearly necessary and has not been presented. 
     Another major issue is estimating the functional concentration of antipsychotic drugs in the brain. Fortunately, PET imaging using specific radioactive tracers can be used to determine the competition with added antipsychotic drugs at specified receptor subtupes, such as the dopamine D2R. A number of studies have made possible the prediction of brain D2R occupancy levels, measured by radio-active tracer displacement, in function of the plasma level [97], results that have been actually confirmed by experimentally in the human brain [94]. However, this approach as presented does not enable one to determine the actual concentration of the neuroleptic and its metabolite in nM in the human brain, which can be used at other synapses beyond dopaminergic synapses. In addition, it is not possible to determine the level of postynaptic activity, especially in the case of partial agonists. 
     Simulation has been applied to the problem of slow antagonist dissociation and long-lasting in vivo receptor protection; see Vauquelin et al. [99] which addresses the time-dependent evolution of receptor occupancy, dependent upon both affinity and half-life of the drug. Such approaches while interesting, do not address the level of receptor activation or determination of actual brain drug concentration in clinically relevant conditions. 
     At least one attempt to simulate the outcome of clinical trials with the antipsychotic quetiapine has been published; see Kimko et al. [51]. In this study, the relation between plasma drug concentration and BPRS scale as a measure of clinical efficacy was modeled as a linear, U-shaped, inhibitory Emax and sigmoidal Emax function, using standard statistical analyses. There was no attempt to include actual physiological interactions or pathology parameters. In view of the large discrepancy between drug plasma levels and actually measured functional brain receptor occupancies [94], it was of no surprise that the model deviated quite considerably from the actual data. 
     Another commonly used rule for determining the clinical efficacy or side-effect liability of neuroleptics is the degree of D2R occupancy, measured with tracer displacement. This is given by 
       D2R−occ=D2R−occmax*Dose/(Dose+ K   i )
 
     where Dose is the dose of the neuroleptic and K i  is a parameter determined from PET imaging studies with radio-active tracers, usually  11 C-racopride. This method at best yields modest correlations and is clearly not adapted to predict the clinical outcome of novel therapeutic agents with partial agonist effects at the D2R, such as aripiprazole. 
     The computational neurosciences approach is based upon mathematical models describing actual biophysical processes. In most cases, the readout of these models is neuronal action potentials, which obviously is related to behavior. This area of research has yielded an enormous array of novel insights in the way neuronal circuits code information and perform certain cognitive operations. However, prior to this invention these models have not been integrated together in practical way which would represent a disease state and predict the clinical effect of drugs thereon. 
     With regard to the problem of schizophrenia, the major brain regions involved are the cortex, the striatum and the hippocampus. There is evidence of a dysfunctional signal-to-noise ratio observed with EEG techniques in patients and siblings [105]. According to the hypothesis developed by Grace [39], the ventral striatum integrates inputs from cortical, amygdale and hippocampal regions. In order to develop an adequate mathematical model of the deficient information processing in the pathology of schizophrenia, there is a need for a detailed model of each of these brain regions, followed by an integration of the different inputs into the ventral striatal computation unit. 
     There are computational models in the neural areas of interest that have a basic science focus that are linked to schizophrenia. When human outcomes are of interest, the results are more generally linked to behavior rather than disease or particularly schizophrenia (e.g., Montague et al. [70] and Smith et al. [86]). For example, there have been computational models of the striatum and medium spiny projection neurons (e.g. Wolf et al., 2005), of dopamine signaling (e.g., Schultz et al. [83]), of cortical circuitry (e.g., Chen [21]), and of information processing in the frontal cortex and basal ganglia (e.g., Amos, 2000). None of those computational approaches, however, integrate a biologically-based simulation to human clinical data and derive therapeutic drug targets. The previous modeling did not correlate and compare the output of the simulations with clinical trial results to predict clinical outcomes for untested drugs or determine targets for novel therapeutic drugs. 
     A range of models have been developed directed at cortical and hippocampal processes relevant to schizophrenia (for review, 32). Recent modeling efforts have largely focused on understanding three fundamental cognitive processes: working memory, the decision process, and attention. 
     Based on the pioneering work of Pat Goldman-Rakic [37] and others, much research has focused on deficits in working memory function in prefrontal cortex (PFC) and its contribution to loss of executive control and disorganized thinking in schizophrenia. The major modeling efforts in this direction attempt to account for the ability of cortical networks to maintain a stable pattern of activity—e.g., a stable firing pattern—across a population of neurons, in the face of distracting stimuli. Durstewitz and colleagues [30] showed that a simple cortical architecture, based on interconnected pyramidal cells and interneurons, could maintain stable firing (so-called attractor behavior) with firing rates corresponding to experimental data from PFC, and that dopamine, acting through D1 receptors worked to stabilize the activity when distracting activity patterns were applied to “knock” the “goal” out of working memory. A succession of models has taken this attractor model further, notably X J Wang and Miller et al. [100, 67] have incorporated more accurate neuronal models with additional biophysical properties, and have shown more sophisticated attractor behavior. Additional interneuronal types have also been incorporated into the cortical network, based upon a different functional role for dendrite-targeting calbindin positive interneurons versus soma targeting palvalbumin positive interneurons, versus the calretinin positive interneurons that target other interneurons (100). 
     Thus, a substantial body of computational modeling has been developed to account for recent experimental findings on working memory, decision making, attention and other cognitive processes. The biophysical models of neurons used in these models vary from extremely simplified (integrate-and-fire models), to somewhat sophisticated multicompartmental models incorporating 6 or 7 intrinisic currents and several synaptic receptor currents (usually AMPA, NMDA, and GABA A ). 
     A final set of relevant work in the literature deals with the action of neuromodulators on brain circuit function. In a number of models 14, 27, 92, 31) however, the effect of dopamine is not explicitly calculated on model parameters—it is just assumed, loosely based on experimental literature, that dopamine increases NMDA conductance by a fixed percentage. Similarly, in Hasselmo&#39;s models of the effect of acetylcholine on functional connectivity in various hippocampal pathways [44], the effect of the neuromodulator is introduced only at a single effective concentration, and only through its effect on a model parameter, such as synaptic weight. 
     The dynamics of the neuronal circuits in the prefrontal cortex are important for addressing the issue of cognitive deficits in schizophrenia, an area which has recently been recognized by the NIMH and the FDA as a major unmet medical need. In fact, a very low fraction of ‘stable’ schizophrenia patients are able to return to their level of professional activity. 
     It is clear that for accurately describing the effects of antipsychotics and therapies used in psychiatric diseases on the neuronal dynamics in the prefrontal cortex, a new model is required integrating the pharmacological effects of other neurotransmitter systems, such as acetylcholine, serotonin and norepinephrine are needed. 
     With the advent of pharmacogenomics and functional data on patient genotypes, it is mandatory to have a model which can incorporate these functional genotypes in a rational way. 
     In the striatum, the most important cell type is a medium spiny neuron (MSN) GABA cell, a computational model of which has been published (40). This model has a dopaminergic D1 mediated input and a glutamate afferent input and was intended to demonstrate a dopamine-induced bifurcation and the link to expected reward. However, as all neuroleptics effective in schizophrenia antagonize the dopamine D2R, this model—which lacks D2 receptor—is clearly not sufficient to describe the effect of dopamine receptor modulation on the relation between incoming glutamatergic signals and outgoing GABAergic signals. In addition, both 5-HT2C [28] and D(3) receptor ligands (110) have been documented to modulate the dynamics of dopamine release in the striatum. At least these two pharmacological influences need to be incorporated in the model, as many neuroleptics also affect these receptor subtypes. In addition, novel insights in the pathophysiology of schizophrenia point to the idea of signal and noise (105). Also the gating of hippocampus and amygdala which is seen as major inputs into the N. accumbens [39] is not implemented in this model. Recent experimental data indeed have provided information on the electrophysiological interaction between prefrontal cortex, hippocampus and N. accumbens [38]. 
     There is a need for the integration of information on the interaction and pathways of the different neurotransmitter circuits with their different receptor subtypes in the human brain. Information is available from preclinical microdialysis and voltammetry studies on neurotransmitter levels and electrophysiological studies in well-defined brain regions in the monkey and rat brain. Human information relates imaging data (PET, MRI), functional genomic and postmortem data in appropriate patient populations. The specific affinity of each drug for different receptor subtypes defines its interaction with various neurotransmitter systems. 
     In particular, interactions between serotonin and norepinephrine are very important in the setting of schizophrenia. Indeed, many antipsychotics currently in use have pharmacological effects at serotonerge and noradrenerge receptor subtypes. In addition, a number of drugs used to treat depression have serotonergic and/or noradrenergic modulation as their primary mode of action. However, good models which can accurately describe the interaction between these two types of neurotransmitters are lacking, despite a wealth of information on the reciprocal interaction between Locus Coeuruleus, the source of noradrenergic neurons and the Dorsal Raphe Nucleus, the source of serotonergic neurons. Therefore a good mathematical model which can take all these interactions into account is necessary. 
     Computational neuroscience models have described the calculation of biomarkers such as fMRI [60] and EEG [64]. However, other parameters of interest in the field of drug development have not yet been published. There is a need for ways to adapt computational neuropharmacology approaches to (1) identify the ‘ideal profile’ of drugs, (2) estimate the effect of comedications, (3) perform power calculations based not only on pharmacokinetic variability, but on pharmacodynamic variability as well, (4) estimate the clinical effects of specific functional genotypes, and (5) estimate the influence of chronopharmacodynamics (i.e. the time of the day when drugs are given). 
     SUMMARY OF INVENTION 
     This invention fulfills a need for an integrated approach for predicting the effect of potential drugs in schizophrenia. However, it should be understood that the method and systems described herein are applicable to other psychiatric and cognitive diseases as well. In one embodiment, integration of a model of the striatum, model of the pre-fontal cortex, model of the hippocampus, and a receptor competition model is disclosed, in combination with an output from these models, and correlation of this output for many different drugs with a large database of prior clinical trials as a validation means. 
     Embodiments of the present invention relate to computer modeling of interactions among and between cortical and subcortical brain areas. For example, one embodiment of the present invention relates to a normal and a pathological state resembling schizophrenia, which pathological state has inputs representing the effects of a drug(s), for the purpose of using the outputs to predict the effect of drugs in psychiatric and cognitive diseases. 
     Another embodiment of the invention is a computer model of a human generic receptor competition model comprising a computer-readable memory storing codes and a processor coupled to the computer-readable memory, the processor configured to execute the codes. The memory comprises code to define biological processes related to the biological state of the receptor model and code to define mathematical relationships related to interactions among biological variables associated with the biological processes. 
     Another embodiment of the invention is a computer model of the biological state of a generic receptor competition model, comprising code to define the biological processes related to the biological state of the particular network, and code to define the mathematical relationships related to interactions among biological variables associated with the biological processes. At least two of the biological processes are associated with the mathematical relationships. A combination of the code to define the biological processes and the code to define the mathematical relationships define a simulation of the biological state of the cortico-striatal pathway. 
     In yet another embodiment of the invention one uses computer executable software code comprised of code to define biological processes related to a biological state of generic receptor competition model including code to define mathematical relations associated with the biological processes. 
     In one embodiment, the invention is a method for developing a computer model of interactions between and among cortical and sub-cortical brain regions. The method comprises the steps of identifying data relating to a biological state of the striatum and cortex; identifying biological processes related to the data, these identified biological processes defining at least one portion of the biological state of the striatum, hippocampus and cortex; and combining the biological processes to form a simulation of the biological state of the interactions between and among cortical and sub-cortical brain regions. The biological state of the interactions between and among cortical and sub-cortical brain regions can be, for example, the state of a normal or a diseased interaction. The diseases that can be modeled include psychiatric disorders, such as schizophrenia, bipolar disorder, major depression, ADHD, autism, obsessive-compulsive disorder, substance abuse and cognitive deficits therein and neurological disorders such as Alzheimer&#39;s disease, Mild Cognitive impairment, Parkinson&#39;s disease, stroke, vascular dementia, Huntington&#39;s disease, epilepsy and Down syndrome. 
     Another embodiment of the invention is a computer model of the biological state of interactions between and among cortical and sub-cortical brain regions, comprising code to define the biological processes related to the biological state of the striatum and cortex, and code to define the mathematical relationships related to interactions among biological variables associated with the biological processes. At least two of the biological processes are associated with the mathematical relationships. A combination of the code to define the biological processes and the code to define the mathematical relationships define a simulation of the biological state of the cortico-striatal pathway. 
     In yet another embodiment of the invention one uses computer executable software code comprised of code to define biological processes related to a biological state of interactions between and among cortical and sub-cortical brain regions including code to define mathematical relations associated with the biological processes. 
     Another embodiment of the invention is a computer model of a interactions between and among cortical and sub-cortical human brain regions, comprising a computer-readable memory storing codes and a processor coupled to the computer-readable memory, the processor configured to execute the codes. The memory comprises code to define biological processes related to the biological state of the interactions between and among cortical and sub-cortical brain regions, and code to define mathematical relationships related to interactions among biological variables associated with the biological processes. 
     Another embodiment of the invention is a computer model of the biological state of a neuronal network, which can be cortical or hippocampal, comprising code to define the biological processes related to the biological state of the particular network, and code to define the mathematical relationships related to interactions among biological variables associated with the biological processes. At least two of the biological processes are associated with the mathematical relationships. A combination of the code to define the biological processes and the code to define the mathematical relationships define a simulation of the biological state of the interactions between and among cortical and sub-cortical brain regions. 
     In yet another embodiment of the invention one uses computer executable software code comprised of code to define biological processes related to a biological state of the cortical network including code to define mathematical relations associated with the biological processes. 
     Another embodiment of the invention is a computer model of a human Dorsal Raphe-Locxus Coeruleus pathway, comprising a computer-readable memory storing codes and a processor coupled to the computer-readable memory, the processor configured to execute the codes. The memory comprises code to define biological processes related to the biological state of the cortical network and code to define mathematical relationships related to interactions among biological variables associated with the biological processes. 
     Another embodiment of the invention is a computer model of the biological state of a Dorsal Raphe-Locus Coeruleus, comprising code to define the biological processes related to the biological state of the particular network, and code to define the mathematical relationships related to interactions among biological variables associated with the biological processes. At least two of the biological processes are associated with the mathematical relationships. A combination of the code to define the biological processes and the code to define the mathematical relationships define a simulation of the biological state of the interactions between and among cortical and sub-cortical brain regions 
     In yet another embodiment of the invention one uses computer executable software code comprised of code to define biological processes related to a biological state of the Dorsal Raphe-Locus Coeruleus including code to define mathematical relations associated with the biological processes. 
     The computer model allows the estimation of the effect of an untested pharmacological agent on a well-defined clinical scale used in assessing psychiatric diseases together with a confidence interval, based upon the correlation between the computer model outcome of existing neuroleptics at their appropriate doses and the reported clinical effects on scales used in psychiatric disorders. 
     The computer model also allows for the effect of particular genotypes to be predicted, and if functional effects of these genotypes at the physiological levels are known such predictions can be used in improving the set up of clinical trials by supporting the decision of genotyping. 
     The computer model also allows for predicting the effect of specific dosage timing, when information over the circadian rhythms of receptor subtypes involved in the pharmacology of the drug are known; such prediction can improve the clinical outcome in trials by better synchronizing the pharmacokinetics of the compound with the endogenous rhythms of the brain. 
     The computer model allows also the identification of important receptor subtype targets for which the outcome is very sensitive; these receptor subtypes can form the pharmacological basis of a drug profile, and can lead to the identification and development of a novel drug with better anticipated clinical effects on various clinical scales, used in psychiatric diseases. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1 . Shows a schematic illustrating the inability of conventional methods to identify good drug targets. 
         FIG. 2 . Shows a schematic of the simulation platform which represents one embodiment of the invention. 
         FIG. 3 . Shows a schematic of the simulation modules which consists of a number of interconnected brain regions, known to play a role in psychiatric or cognitive diseases. 
         FIG. 4 . Shows flow chart of the generic receptor competition model, here represented as a dopamine synapse. 
         FIG. 5 . Shows a diagram illustrating the various processes used in the simulation of the Extra-Pyramidal Symptoms (EPS) Model. 
         FIG. 6 . Shows a flow chart of how the EPS liability index is calculated. 
         FIG. 7 . Shows a graph of the need for anticholinergic medication and D2R occupancy. 
         FIG. 8 . Shows a flow chart of the computer model of the interactions between and among cortical and sub-cortical brain regions yielding a signal to noise type output as a model for clinical efficacy. 
         FIG. 9 . Shows graphs of output from the Signal to Noise Model and observed changes on the clinical PANSS positive scale, and the D2R occupancy and observed changes on the clinical PANSS positive scale. 
         FIG. 10 . Shows a flow chart of the interactions between Dorsal Raphe Nucleus; Locus Coeuruleus, and efferent projections to the PFC or hippocampus. 
         FIG. 11 . Shows a schematic of how the mathematical model is validated through clinical correlation. 
         FIG. 12 . Shows a schematic of the PFC neuronal network of pyramidal cells and interneurons. 
         FIG. 13 . Shows an illustration of the concept of working memory span. 
         FIG. 14 . Shows a graph between clinical outcome on a CPT test and their respective effects on working memory in the Working Memory circuit. 
         FIG. 15 . Shows a flow chart of a model for identifying a pharmacological profile resulting in a substantially better clinical outcome. 
     
    
    
     DETAILED DESCRIPTION 
     The term “biological process” is used herein to mean an interaction or series of interactions between biological variables. Biological processes can include, for example, presynaptic autoreceptor regulation of neurotransmitter release; regulation of neurotransmitters bound to receptors; breakdown of the neurotransmitter; specific presynaptic firing patterns; appropriate facilitation and depression of presynaptic vesicle release. The term “biological process” can also include a process comprising of one or more therapeutic agents, for example the process of binding a therapeutic agent to a cellular receptor. Each biological variable of the biological process can be influenced, for example, by at least one other biological variable in the biological process by some biological mechanism, which need not be specified or even understood. 
     The term “biological variables” refers to the extra-cellular or intra-cellular constituents that make up a biological process. For example, the biological variables can include neurotransmitters, receptors, binding affinities, transportation rates, metabolites, DNA, RNA, proteins, enzymes, hormones, cells, organs, tissues, portions of cells, tissues, or organs, subcellular organelles, chemically reactive molecules like H.sup.+, superoxides, ATP, citric acid, protein albumin, as well as combinations or aggregate representations of these types of biological variables. In addition, biological variables can include therapeutic agents such anti-psychotic and neuroleptic drugs. 
     The term “biological state” is used herein to mean the result of the occurrence of a series of biological processes. As the biological processes change relative to each other, the biological state also undergoes changes. One measurement of a biological state, is the level of activity of biologic variables, parameters, and/or processes at a specified time and under specified experimental or environmental conditions. 
     The term biological attribute is used herein to mean biological characteristics of a biological state, including a disease state. For example, biological attributes of a particular disease state include clinical signs and diagnostic criteria associated with the disease. The biological attributes of a biological state, including a disease state, can be measurements of biological variables, parameters, and/or processes. For example, for the disease state of schizophrenia, the biological attributes can include measurements of DR2 or dopamine levels. 
     The term “reference activity pattern” is used herein to mean a set of biological attributes that are measured in a normal or diseased biological system. For example, the measurements may be performed on blood samples, on biopsy samples, or cell cultures derived from a normal or diseased human or animal or imaging scans of the brain. Examples of diseased biological systems include cellular or animal models of schizophrenia, including a human schizophrenia patient. 
     The term “simulation” is used herein to mean the numerical or analytical integration of a mathematical model. For example, simulation can mean the numerical integration of the mathematical model of the biological state defined by a mathematical equation. 
     A biological state can include, for example, the state of an individual cell, an organ, a tissue, and/or a multi-cellular organism. A biological state can also include the activation state of a neural circuit, the firing pattern of specific neurons or the neurotransmitter level in a specific brain region. These conditions can be imposed experimentally, or may be conditions present in a patient type. For example, a biological state of the striatum can include an elevated free dopamine level for a schziophrenic patient at a certain age and disease duration. In another example, the biological states of the prefrontal cortex can include the state in which a patient with a certain disease undergoes a specific treatment. 
     The term cognitively normal is used herein to mean a process of receiving, processing, storing, and using information that occurs in a non-diseased brain. 
     The term “disease state” is used herein to mean a biological state where one or more biological processes are related to the cause or the clinical signs of the disease. For example, a disease state can be the state of a diseased cell, a diseased organ, a diseased tissue, or a diseased multi-cellular organism. Such diseases can include, for example, schizophrenia, bipolar disorder, major depression, ADHD, autism obsessive-compulsive disorder, substance abuse, Alzheimer&#39;s disease, Mild Cognitive impairment, Parkinson&#39;s disease, stroke, vascular dementia, Huntington&#39;s disease, epilepsy and Down syndrome. A diseased state could also include, for example, a diseased protein or a diseased process, such as defects in receptor signaling, neuronal firing, and cell signaling, which may occur in several different organs. 
     The term cerebro-active drugs is used herein to mean any drug affecting the brain such as, but not limited to, neuroleptics. 
     The terms antipsychotics/neuroleptics are used herein to mean drugs used for the treatment of psychosis, such as schizophrenia. These drugs include, but are not limited to, clozapine, risperidone, aripiprazole, olanzapine, Zyprexa, quetiapine, and ziprasidone. 
     The term “computer-readable medium” is used herein to include any medium which is capable of storing or encoding a sequence of instructions for performing the methods described herein and can include, but not limited to, optical and/or magnetic storage devices and/or disks, and carrier wave signals. 
     The term circadian profiles is used herein to mean profiles of brain wave activity, hormone production, cell regeneration and other biological activities linked to a roughly-24-hour cycle in physiological processes. 
     The computer system as used here is any conventional system including a processor, a main memory and a static memory, which are coupled by bus. The computer system can further include a video display unit (e.g., a liquid crystal display (LCD) or cathode ray tube (CRT)) on which a user interface can be displayed). The computer system can also include an alpha-numeric input device (e.g., a keyboard), a cursor control device (e.g., a mouse), a disk drive unit, a signal generation device (e.g., a speaker) and a network interface device medium. The disk drive unit includes a computer-readable medium on which software can be stored. The software can also reside, completely or partially, within the main memory and/or within the processor. The software can also be transmitted or received via the network interface device. 
       FIG. 1  shows the general innovative concept of this invention. The clinical or systemic readout is a consequence of the interaction of numerous pathways and processes, the majority of which are unknown. The classical drug discovery paradigm focuses on one specific target (i.e. D2R in schizophrenia) and medicinal chemistry efforts are aimed to develop very specific modulatory agents, under the assumption that any additional pharmacology might lead to unwanted side-effects. The statistical distance between that specific target and the clinical outcome is substantial, leading to a modest correlation between the effectivity of the compound at the target and the clinical scale. In contrast, one embodiment of this invention describes a systems biology approach which takes into account the whole constellation, including both the known environment of process around the key target as well as a number of processes which are not yet full described, all of which would lead to a better correlation between the predicted outcome and the clinical reality. For instance, this invention relates to the modeling of a number of interactions with neighboring targets (i.e. D1, D3, 5HT1A, 5HT2A, etc in CNS diseases) around the specific target for which the functional relation is known to a certain extent. 
       FIG. 2  shows a simulation platform made of four conceptual dimensions. These dimensions are involved in the validation process which consists of a number of iterative steps, leading to increasingly better correlations with the clinical reality. The simulation platform described herein consists of (1) a database of knowledge on pharmacology, physiology and pathology; both preclinical and clinical databases which supports the actual development of the different simulation models; (2) a number of simulation models, which take the known pharmacology of the drug as input and outputs a system measure; (3) a statistical correlation module, which correlates the module output with actual clinical results of the same drugs at the same doses, resulting in (4) a correlation confidence measure. 
     The database, extracted from publicly available data on neurophysiology, neuron-anatomy and pharmacology in both preclinical models and clinical situations, provides the scientific basis for the development of the different simulation modules. The drug pharmacology (in terms of its affinity, plasma concentrations and functional effects) is used as input into the model to calculate a specific readout. This readout is then correlated with the actual clinical effect of the same drug-dose combination in the human patient. When this is done over all the available drug-dose combinations for which there is clinical information, a correlation coefficient can be calculated, which indicates how good the model predicts the actual clinical reality. This process is used in an iterative cycle which leads to better and better predictive models. 
     In one embodiment, the clinical database used for optimizing and validating the model consists of a detailed database of 26967 patients (taken from 87 published papers), 20 different neuroleptic drugs and 71 neuroleptic-dose combinations on the following clinical scales:
         Positive and Negative Syndrome Scale (PANSS) total, positive change, negative size effect, negative change, disorganization size effect, psychopathology change, change in anxiety/depression, fraction of patients improved on PANSS   Scale for Assessment of Negative Symptoms (SANS)   Brief Psychiatric Rating Scale (BPRS) total, size effect change in BPRS total, BPRS core items, BPRS positive, BPRS negative, BPRS activity, BPRS anergia, change in anxiety/depression, BPRS hostility, BPRS thought disturbance, fraction of BPRS responders   Clinical Global Impressions-Severity of Illness Scale (CGI-S) improvement, CGI-Global improvement.       

     The database further contains preclinical information on rodent and primate models of neuro-anatomy, neurophysiology and neuropharmacology and on clinical information from imaging, postmortem and genotype origin. 
     This information is used to develop and validate the different mathematical models as disclosed herein. During the development of the model, preclinical validation is used in a bootstrap mode, i.e. the model predicts the outcome of preclinical studies which were not used for its development. The different models use pharmacology input from different agents at well-defined concentrations and calculate the outcome. This outcome is then used in a statistical correlation module, in which these results are compared to the actual clinical results obtained in patients with the same drug at that particular concentration. The model is successively validated using the correlation coefficient as benchmark. 
       FIG. 3  shows the system simulation modules consisting of a number of interconnected brain regions, known to play a role in psychiatric or cognitive diseases. The dorsal striatum receives input from the dopaminergic neurons arising from the substantia nigra and afferent glutamate fibers arising from the Supplemental Motor Area in the cortex. The medium spiny striatal neurons project to the thalamo-cortical pathway of the motor circuit. The ventral striatum receives dopaminergic input from Ventral tegmentum Area and afferent glutamatergic fibers from prefrontal association cortices, hippocampus and amygdala. The medium spiny striatal neurons projects to the thalamic nuclei which form a closed cortico-striato-thalamic circuit. Prefrontal cortex is innervated by dopaminergic fibers arising from VTA, serotonergic fibers arising from Dorsal Raphe, noradrenerge fibers arising from Locus Coeruleus and cholinergic fibers originating from N. basalis of Meynert. There is a well-documented reciprocal feedback interaction between Dorsal Raphe and Locus Coeruleus. 
     This invention describes four mathematical models: (i) a generic receptor competition model, (ii) a model of the animal and human striatum, (iii) a model of 5HT-NE interaction and (iv) a model of the prefrontal cortex. The models are linked to each other using neuro-anatomical and neurophysiological connections. 
     A. Generic Receptor Competition Model 
       FIG. 4  shows a generic receptor competition model that can be used for different types of neurotransmitter synapses, including, but not limited to, a dopamine synapse. The model is based upon a series of ordinary differential equations, which regulates the receptor activation state in different situations and accounts for: presynaptic autoreceptor regulation of neurotransmitter release; specific presynaptic firing patterns; appropriate facilitation and depression of presynaptic vesicle release; the presence of endogenous neurotransmitter and two agents competing for the same site with appropriate kon and koff values (agonist or antagonists). Unless otherwise noted, we assume kon is limited by diffusion, in 3D or 2D for a lipophilic compound; diffusion out of the synaptic cleft; re-uptake or degradation of the neurotransmitter; and the presence of low- and high affinity binding sites. 
     For illustrative purposes a dopaminergic synapse is shown, where dopamine interacts with the presynaptic D2-R in a negative feedback cycle and with postsynaptic D2-receptors, is degraded by the Catechol-O-methyl Transferase (COMT) enzyme and is taken up by the dopamine transporter (DAT). Neuroleptics and tracer molecules are interacting with both pre- and postsynaptic D2-receptors. The tracer possibility allows studying the interaction between for instance a parent compound and its metabolite if they both have an affinity for the D2-R (as is the case for risperidone and 9-OH risperidone). Similar models can be used for studying the dynamics at the level of the postsynaptic D1, D3, D4, D5-receptors. With the appropriate changes, specific serotonergic synapses are built with the fourteen known 5HT-3 receptor subtypes. Also, specific noradrenergic, glutamatergic and GABAergic and muscarinic synapses are constructed using the same themes. We will illustrate in some detail the dopaminergic synapse. 
     Dopaminergic Synapse 
     The number of presynaptic receptors is estimated to be 30 receptors/micron 2 , while we assume a number of 300 postsynaptic receptors/micron 2 . This is derived from the following arguments. B max  of D2R in the rat striatum is about 125 fM/mg (Cai 2002), equivalent to about 8×10 16  molecules/I. Assuming a synaptic membrane area of 1 m 2 , and a volume of 1 m 3 , we arrive at about 100-200 molecules/synapse. Furthermore, 25-30% of these receptors are of the high affinity type [84]. 
     Dopamine dynamics can be determined based on the binding affinities, K d , for the different types of D2 receptors. We assume K d =10 nM for high affinity receptors while K d =5000 nM for low affinity receptors [84]. We assume that the binding rate is diffusion limited and that this depends upon the size and molecular weight of the molecules (see Table I). As a consequence, the unbinding rate is calculated by k on ×K d . For DA binding to the D1 receptor, K d  values for both low and high affinity receptors are about 4 times higher ([89], [33]). Given the concentration of free high affinity receptors, [D h ], and free low affinity receptors, [D l ], the concentration of dopamine bound to these receptors, [D hb ] and [D lb ], respectively can be calculated using classical Ordinary Differential Equations (ODE). 
     The dopamine transporter pumps dopamine out of the cleft and follows Michaelis-Menten kinetics. The maximum uptake rate, k max , of the transporter is 4.74 nM/ms (Schmitz, 2001). The dopamine concentration, K m , at which the transporter works at half its maximum, is 890 nM (Schmitz, 2001). In order for the dopamine concentration to go to zero within one second after release, the number of transporters, N, in a single cleft within the striatum is 500. 
     The COMT enzyme breaks down dopamine at a maximum rate, k max , of 200 nM/ms. The dopamine concentration, K m , at which the enzyme is half effective is 440 microM (Yan, 2002). In order for the COMT to have an effect at these low rates, many enzymes, N, must be present. In this simulation we consider 7800 enzymes within the cleft. Breakdown of dopamine by the COMT enzyme is modeled via Michaelis-Menten kinetics so that dopamine within the cleft, [dop], follows a simple ODE. The very large value of Km makes the COMT enzyme very inefficient. The density of COMT enzymes is dependent upon the brain region, i.e. in the striatum it is much less important than the DAT, while the COMT effect in the Prefrontal Cortex tends to be more important, producing more regulation than the DATs, which are less dense in the PFC. 
     Drug binding dynamics can be determined based on their binding affinity, K d , for the D2 receptors. Table I gives information on K d , k on  and k off  rates for the different drugs [81]. The binding on rate is assumed to be diffusion controlled. The k off  rate is then calculated as k on ×K d . The Stokes-Einstein equation determines that the diffusion constant is inversely related to the first power of the radius (the third root of the mass), therefore from a constant diffusion for all neuroleptics, we can determine the K on  rate based upon their molecular weight, which are in the 312-448 dalton range (dopamine MW is 189.4). Quetiapine is an exception with a molecular weight of 883 dalton. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 K on  and K off  rates for different compounds at the D2-R. 
               
               
                 Calculations are based upon diffusion controlled k on  rates and 
               
               
                 K off  rates calculated as k on  × K i . Given the concentration of free 
               
               
                 D2 receptors, [D f ], one can determine the concentration 
               
               
                 of receptors bound by the drug, [D b ]. 
               
            
           
           
               
               
               
               
               
            
               
                 Compound 
                 MW (Dalton) 
                 K d  (nM) 
                 K on  (nM/sec) 
                 K off  (sec −1 ) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Risperidone 
                 410.9 
                 3.0 
                 1.10 × 10 −4   
                 0.0003300 
               
               
                 Paliperidone 
                 426.1 
                 4.1 
                 1.09 × 10 −4   
                 0.0004469 
               
               
                 Clozapine 
                 326.8 
                 160 
                 1.19 × 10 −4   
                 0.0190400 
               
               
                 Olanzapine 
                 312.2 
                 20 
                 1.20 × 10 −4   
                 0.0024000 
               
               
                 Aripiprazole 
                 448.4 
                 1.64 
                 1.07 × 10 −4   
                 0.0001754 
               
               
                 Quetiapine 
                 883 
                 360 
                 0.85 × 10 −4   
                 0.0289000 
               
               
                 Zyprasidone 
                 467.2 
                 4 
                 1.05 × 10 −4   
                 0.000420 
               
               
                 Haloperidol 
                 530.1 
                 1.21 
                 1.01 × 10 −4   
                 0.00012 
               
               
                 Raclopride 
                 347.2 
                 90 
                 1.16 × 10 −4   
                 0.0104 
               
               
                 IBZM 
                 404.2 
                 160 
                 1.10 × 10 −4   
                 0.0176 
               
               
                 Dopamine 
                 189 
                 10 (high aff) 
                 1.42 × 10 −4   
                 0.0014200 
               
               
                   
               
            
           
         
       
     
     Tracer binding dynamics can be determined based on their binding affinity, K d , for the D2 receptors. For instance raclopride has an affinity of 95 nM and IBZM of 160 nM for the D2-R. Note that the affinity of the tracers is much lower than neuroleptics such as haloperidol, risperidone and olanzapine, however, they have a higher or equal affinity compared to compounds like clozapine and quetiapine. This can substantially influence the interpretation of the tracer PET data. Similar differential equations describe the time-dependence of the tracer binding dynamics. 
     Dopamine is released from the presynaptic membrane every time the dopaminergic nerve fires. In this model, we do not track ion movements which would take another level of complexity. Therefore, instead of using internal Ca ++  levels to determine release, we consider the facilitation and depletion of dopamine release based on the amount of time elapsed since the previous firing along the lines of Montague et al. [70]. The maximum facilitation enhancement, w f , is 10% and decays exponentially at a rate, k f , of ¼ sec −1 . The maximum depletion weight, w p , is 3% and decays exponentially at a rate, k p , of ⅕ sec −1 . 
     At time zero, the first firing occurs so that t 1 =0. Later firings are determined based on the firing frequency f so that the time of the n th  firing can be calculated. The amount of dopamine released is based upon the history of firing. 
     D2 receptor occupation at the presynaptic membrane affects the amount of dopamine released. Because the D2 receptor is a G-coupled protein, its effect is not immediate. Thus, when the time for release comes, we determine the effect based upon how many receptors were bound, B (both high and low affinity), 150 ms earlier. The typical number of bound receptors is B 0 . We use a Hill equation to model the effect of the D2 receptor with a maximal effect of 25% at full D2-R block. 
     Free dopamine vanishes from the cleft primarily via diffusion. Because we do not consider any spatial properties, we model diffusion via an exponential decay with a half-life, H, of 150 ms as a default value. This parameter can be adjusted so as to correspond to the specific DA kinetics in various brain regions (see further). 
     The simulation is initiated by first finding the equilibrium given a constant amount of free dopamine at 1000 nM. The simulation is then run for a transitory time of 5 seconds at the tonic firing rate of 5 Hz. Finally, the simulation runs for an additional 2.5 seconds during which time average binding levels are determined. When the simulation finally runs, it begins with the first 7.5 seconds in memory which helps keep all the processes determining release primed. 
     The clearance of DA is mostly determined by the half-life as a free parameter. A number of papers have given estimates for this value. In mice [17], the rate of clearance (V max /K on ) for N. accumbens core was determined to be 8 sec −1  and 4 sec −1  for N. accumbens shell. For DAT KO mice the clearance values are 0.03 and 0.04 see respectively. Cocaine reduces clearance rate to about 0.35 sec −1  in wild-type mice. Clearance of DA from medial PFC is about 8.3 times slower than from N. accumbens [102, 75], and at least two mechanisms have been identified (DAT/NET and MAO). DAT/NET accounts for about 40-70% of DA clearance [102]. In mouse dorsal striatum, uptake is 60% faster than in N. accumbens (110) giving a clearance rate of 12.8 sec −1  or a DA half-life of 54 msec. The same article suggests that clearance is enhanced 33% by full D3-agonism and reduced by 50% by full D3 antagonism in the N. accumbens. Neuroleptics with high D3-antagonism will have an effect on DA clearance. In the mice caudate putamen, clearance is V max /K m =2.65/0.21 or half-life is 72 msec [65]. 
     The processes of presynaptic habituation and facilitation are incorporated as follows (modified after [70]). When firings are close together, they facilitate the amount of release. However, when they are far apart, they depress the amount of release. If we denote the time of the n th  firing by t n , then the release amount is modified based on all previous firings as follows 
     
       
         
           
             
               release 
               new 
             
             = 
             
               release 
                
               
                 ( 
                 
                   1 
                   + 
                   
                     
                       ∑ 
                       
                         i 
                         = 
                         1 
                       
                       
                         n 
                         - 
                         1 
                       
                     
                      
                     
                         
                     
                      
                     
                       
                         w 
                         f 
                       
                        
                       
                           
                       
                        
                       
                         exp 
                          
                         
                           [ 
                           
                             - 
                             
                               
                                 k 
                                 f 
                               
                                
                               
                                 ( 
                                 
                                   
                                     t 
                                     n 
                                   
                                   - 
                                   
                                     t 
                                     i 
                                   
                                 
                                 ) 
                               
                             
                           
                           ] 
                         
                       
                     
                   
                   - 
                   
                     
                       w 
                       d 
                     
                      
                     
                         
                     
                      
                     
                       exp 
                        
                       
                         [ 
                         
                           - 
                           
                             
                               k 
                               d 
                             
                              
                             
                               ( 
                               
                                 
                                   t 
                                   n 
                                 
                                 - 
                                 
                                   t 
                                   i 
                                 
                               
                               ) 
                             
                           
                         
                         ] 
                       
                     
                   
                 
                 ) 
               
             
           
         
       
     
     where w f  is the facilitation weight (adjusted by “Relative increase due to firing facilitation”), w d  is the depression weight (adjusted by “Relative decrease due to firing depression”), k f  is the decay rate of facilitation (adjusted by the reciprocal of “Decay time of facilitation mechanism”), and k d  is the decay rate of depression (adjusted by the reciprocal of “Decay time of depression mechanism”). In order to make this mechanism meaningful the following should be satisfied: w f &gt;w d  and k f &gt;k d . The parameters can be adjusted so that they satisfy experimental results [25]. 
     Binding data for both D1-R and D2-R in the simulation are gathered from a dopaminergic cleft which fires at 4 Hz for 2 seconds, 40 Hz for half a second, 1 Hz for 5 seconds, 4 Hz for one second and at 80 Hz for ⅛ of a second. 
     Application to Other Types of Synapses 
     The following table illustrates the different parameters for other types of synapses 
     
       
         
           
               
               
               
               
               
             
               
                   
               
               
                   
                   
                   
                 Adrenergic 
                 Serotonergic 
               
               
                 Parameter 
                 DA cleft 
                 Cholinergic Cleft 
                 Cleft 
                 cleft 
               
               
                   
               
             
            
               
                 Pre- 
                 D2 
                 M2 
                 Alpha2A 
                 5HT1B 
               
               
                 synaptic 
               
               
                 Receptor 
               
               
                 subtype 
               
               
                 Post- 
                 D1, D2, D3, 
                 M1, a7 nAChR, 
                 Alpha1A, 
                 5HT1A, 
               
               
                 synaptic 
                 D4, D5 
                 a4b2 nAChR 
                 alpha2A 
                 5HT2A 
               
               
                 Receptor 
               
               
                 subtype 
               
               
                 Genotype 
                 COMT 
                   
                 COMT 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 List of different pre-and postsynaptic receptors for which the generic receptor 
               
               
                 competition model is implemented. 
               
               
                 Affinity of neurotransmitters for their relevant receptors 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 Frequency 
                 4 
                 40 
                 1 
                 4 
                 80 
               
               
                   
                 Number 
                 8 
                 10 
                 5 
                 4 
                 4 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 Neurotransmitter 
                 Receptor 
                 Affinity (nM) 
                 Ref 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Ach 
                 M1 
                 3.4 
                   
                 63 
               
               
                   
                   
                 M2 
                 370 
                   
                 55 
               
               
                   
                   
                 M3 
                 4200 
                   
                 22 
               
               
                   
                   
                 M4 
                 5600 
                   
                 22 
               
               
                   
                   
                 M5 
                 800 
                   
                 22 
               
               
                   
                 5HT 
                 5HT2C 
                 27 
                 nM 
                 48 
               
               
                   
                 DA 
                 D1 
                 40 
                 nM (high-aff) 
                 89 
               
               
                   
                   
                 D2 
                 10 
                 nM (high-aff) 
                 84 
               
               
                   
                   
                 D3 
                 0.62 
                 nM (high-aff) 
                 33 
               
               
                   
                   
               
            
           
           
               
               
            
               
                   
                 Dopaminergic Cleft 
               
               
                   
                 Total Duration = 8300 ms 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 Normal 
                 Normal 
                 Normal 
                 SZ 
                 SZ 
                 SZ 
               
               
                   
                 COMT 
                 COMT 
                 COMT 
                 COMT 
                 COMT 
                 COMT 
               
               
                 Parameters 
                 Met/Met 
                 Val/Met 
                 Val/Val 
                 Met/Met 
                 Met/Val 
                 Val/Val 
               
               
                   
               
               
                 Base Release (nM) 
                 500 
                 500 
                 500 
                 400 
                 400 
                 400 
               
               
                 Half-life (ms) 
                 1300 
                 900 
                 500 
                 1300 
                 900 
                 500 
               
               
                 Density of presyn D2 
                 330 
                 330 
                 330 
                 330 
                 330 
                 330 
               
               
                 Receptors 
               
               
                 Fraction of high affinity D2-R 
                 0.25 
                 0.25 
                 0.25 
                 0.25 
                 0.25 
                 0.25 
               
               
                 Maximum relative change for 
                 0.45 
                 0.45 
                 0.45 
                 0.45 
                 0.45 
                 0.45 
               
               
                 release 
               
               
                 Normal presyn binding 
                 4160 
                 4160 
                 4160 
                 4160 
                 4160 
                 4160 
               
               
                 density of postsyn D1 
                 300 
                 300 
                 300 
                 300 
                 300 
                 300 
               
               
                 Receptors 
               
               
                 Fraction of high affinity D1-R 
                 0.25 
                 0.25 
                 0.25 
                 0.2 
                 0.2 
                 0.2 
               
               
                   
               
            
           
           
               
               
            
               
                   
                 Serotonergic Clefts 
               
               
                   
                 Total Duration = 9200 ms 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 Frequency 
                 1.3 
                 60 
                 0.8 
                 40 
                 1 
               
               
                   
                 Number 
                 1 
                 3 
                 5 
                 2 
                 2 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 5HT2A synapse Parameters 
                 Normal 
                 SZ 
               
               
                   
                   
               
               
                   
                 Base Release (nM) 
                 1000 
                 1000 
               
               
                   
                 Half-life (ms) 
                 500 
                 500 
               
               
                   
                 Density of presynaptic 5HT1B-R 
                 30 
                 30 
               
               
                   
                 Fraction of high affinity receptors 
                 0.25 
                 0.25 
               
               
                   
                 Maximum relative change for release 
                 0.45 
                 0.45 
               
               
                   
                 Normal presyn binding 
                 1170 
                 1170 
               
               
                   
                 Density of postsynaptic 5HT2A-R 
                 300 
                 255 
               
               
                   
                 Fraction of high affinity receptors 
                 0.25 
                 0.25 
               
               
                   
                 5HT1A synapse Parameters 
                 Normal 
                 SZ 
               
               
                   
                 Base Release (nM) 
                 1000 
                 1000 
               
               
                   
                 Half-life (ms) 
                 500 
                 500 
               
               
                   
                 Density of presynaptic 5HT1B 
                 30 
                 30 
               
               
                   
                 Fraction of high affinity receptors 
                 0.25 
                 0.25 
               
               
                   
                 Maximum relative change for release 
                 0.45 
                 0.45 
               
               
                   
                 Normal presyn binding 
                 1170 
                 1170 
               
               
                   
                 Density of postsynaptic 5HT1A-R 
                 350 
                 385 
               
               
                   
                 Fraction of high affinity receptors 
                 0.25 
                 0.25 
               
               
                   
                   
               
            
           
           
               
               
            
               
                   
                 Noradrenergic Cleft 
               
               
                   
                 Total Duration = 9600 ms 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 Frequency 
                 2 
                 60 
                 1.5 
                 40 
                 1.67 
               
               
                   
                 Number 
                 1 
                 3 
                 9 
                 2 
                 5 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 Normal 
                 Normal 
                 Normal 
                 SZ 
                 SZ 
                 SZ 
               
               
                   
                 COMT 
                 COMT 
                 COMT 
                 COMT 
                 COMT 
                 COMT 
               
               
                 Parameters 
                 Met/Met 
                 Val/Met 
                 Val/Val 
                 Met/Met 
                 Met/Val 
                 Val/Val 
               
               
                   
               
               
                 Base Release (nM) 
                 1000 
                 1000 
                 1000 
                 1000 
                 1000 
                 1000 
               
               
                 Half-life (ms) 
                 910 
                 630 
                 350 
                 910 
                 630 
                 350 
               
               
                 Presynaptic alpha2A density 
                 50 
                 50 
                 50 
                 50 
                 50 
                 50 
               
               
                 Fraction of high affinity 
                 0.25 
                 0.25 
                 0.25 
                 0.25 
                 0.25 
                 0.25 
               
               
                 receptors 
               
               
                 Maximum relative change for 
                 0.45 
                 0.45 
                 0.45 
                 0.45 
                 0.45 
                 0.45 
               
               
                 release 
               
               
                 Normal presyn binding 
                 1030 
                 1030 
                 1030 
                 1030 
                 1030 
                 1030 
               
               
                 Postsynaptic alpha2A density 
                 300 
                 300 
                 300 
                 300 
                 300 
                 300 
               
               
                 Fraction of high affinity 
                 0.25 
                 0.25 
                 0.25 
                 0.25 
                 0.25 
                 0.25 
               
               
                 receptors 
               
               
                   
               
            
           
         
       
     
     Estimating Functional Brain Concentration of Pharmacological Agents 
     The generic receptor model can incorporate tracer data, available in human volunteers or patients, to determine effective functional brain concentrations of pharmacological agents. In this case, we would run the simulation first in the presence of the endogeneous neurotransmitter being targeted (for example dopamine) and the radio-active tracer (for example,  11 C raclopride), as a baseline occupancy. Next we would run the simulation in the presence of the endogenous neurotransmitter (i.e. dopamine), the drug (for example a neuroleptic binding to D2R) and the radio-active tracer. The concentration of the drug would be varied systematically until we get the appropriate displacement seen in the radio-active study. This is the most likely effective drug concentration which we then use in our further models. 
     B. The Dorsal Striatum Model for Extra-Pyramidal Symptoms 
       FIG. 5  illustrates the various processes used in the simulation of the Extra-Pyramidal Symptoms Module. The GABA spiny neuron in the dorsal striatum is the key processing unit and is driven by both glutamatergic and dopaminergic (D1) excitatory input. D2-R are presumed to act on glutamatergic synapses, modulating excitatory input into the spiny neuron. D3 and 5HT2C receptor modulate DA kinetics. M1-receptors directly modulate GABAergic neuron excitability (Ksi=slow inward K-channel, Kir2=inward-rectifier K-channel, L-Ca, L-type Ca channel, L-Cl, Chloride channel). 
     In the normal state, the putamen receives excitatory afferents from the motor and somatosensory cortical areas and communicates with the globus pallidus/substantia nigra pars reticulata (GP i /SN r ) through a direct inhibitory pathway and though a multisynaptic (GP e , STN=subthalamic Nucleus) indirect pathway. Both pathways pass through the ventral lateral thalamus (VL) to close the loop back to the cortex. 
     The direct output pathway links the striatum to the inhibitory internal segment of the globus pallidus (GP i ) and the inhibitory substantia nigra pars reticulate (SN r ). Activation of the direct pathway decreases the inhibitory output from the basal ganglia and disinhibits the motor thalamus, such that movement is facilitated. 
     In contrast, in the indirect output pathway, the medium spiny projection neurons inhibit the external segment of the globus pallidus (GP e ), which then sends less inhibition to the subthalamic nucleus. Disinhibiting the STN excites the internal segment of the globus pallidus (GP i ), leading to an enhanced inhibitory output from the basal ganglia to the motor thalamus so that movement is disfacilitated. 
     Dopamine is believed to modulate striatal activity, mainly by inhibiting the indirect and facilitating the direct pathways. The disinhibition hypothesis [23] suggests that in Parkinson&#39;s disease like syndromes, dopamine deficiency leads to increased inhibitory activity from the putamen onto the GPe and disinhibition of the STN. In turns, STN hyperactivity by virtue of its glutamatergic action produces excessive excitation of the GP i /SN i  neurons, which overinhibit the thalamocortical and brain stem motor centers. This process is assumed to be the basis of D2-R block mediated Parkinsonian symptoms (24). Blocking the D2-R, the activation of which has been shown to negatively modulate glutamnate release from cortical afferents [4] increases the GABAergic inhibition of the GPe, leading to less inhibition of the subthalamic nucleus in the indirect pathway. This disrupts the regulation of the thalamocortical connection finally leading to less excitatory control from the cerebral cortex to the brain stem and spinal cord centers involved in movement control. 
     Recent studies [95] have extended this model considerably and have shown that normal activity patterns in the indirect pathway are of an irregular nature. Increased (dorsal) striatal input in the indirect pathway and weakened intrapallidal inhibition drives the GP e -subthalamic regions into a repetitive and rhythmic spiking pattern, the frequency of which is consistent with the movement tremor frequency. In Parkinson&#39;s patients treated for deep brain stimulation, subthalamic Nucleus frequency increased from 20 Hz to 70 Hz when patients went on L-dopa therapy and resolved their motor problems [12]. 
     The major computational unit of the striatum (both ventral and dorsal) is the medium spiny GABA-ergic neuron (modified and extended considerably after [40]), in which dopaminergic, glutamatergic and cholinergic input fully characterizes the spike-train readout ( FIG. 4 ). 
     Ach Effect on Medium Spiny Neuron 
     In the striatum, expression of muscarinic subtypes is mostly restricted to M1 and M4 subtypes [106], mostly on striatal medium spiny neurons [49]. M4 and D1 receptors often colocalize on spiny neurons in the indirect output pathway [106] and they have opposing actions, suggesting that this interaction might fine-tune the modulatory control of the spiny projection neurons. Muscarinic agonists have been documented to induce an inward current by reducing outward K +  currents, therefore leading to depolarization and increased excitability [86]. Stimulation of the muscarinic receptors by the general agonist muscarine, as in the case of normal cholinergic stimulation, significantly reduces the inhibitory postsynaptic potentials (IPSP) in the GABAergic interneurons [54]. Blocking the muscarinic receptors with atropine restores the IPSP, leading to enhanced negative feedback on the spiny GABAergic neurons. The effect of Ach on the excitability of medium spiny GABA-ergic neurons in the striatum is complex, as two opposite effects are carried respectively by nicotinic and muscarinic receptors [54]. However, as none of the neuroleptics studied is documented to modulate any nicotinic receptor, but some of them are prominent anti-muscarinic receptors antagonist, we focus on the effect of muscarinic receptor activation on the excitability of the medium spiny neuron. This effect is carried probably by M1-R, as pirenzepine blocks this effect of muscarine in the striatum [46]. As a further confirmation, in dementia with Lewy Body disease (DLB), densities of M1-R in the striatum are significantly reduced compared to AD and normal controls and this  3 H pirenzepine binding is inversely correlated with cortical DLB pathology [74]. Lower M1-R densities reduce the chances of muscarinic stimulation to exacerbate the EPS. 
     The disinhibitory effect mediated by mAchR activation is translated into a change in g Cl  conductance according to the following sigmoid equation. 
         g   Cl =4*(1+0.3* M 1_block) 
     Where g Cl  is the chloride conductance (maximal value is 4 pS). Maximal effect of muscarinic block is 30% [54], and the M1 block is calculated with the competition model of acetylcholine and neuroleptic at the appropriate concentration. 
     Role of 5HT2C Receptor in Dopamine Regulation 
     Many neuroleptics also antagonize the 5HT2C receptor to a variable degree. This receptor subtype is particularly interesting in the striatal homeostasis of dopamine. It has been shown indeed that 5HT2C receptor activation by a specific agonist Ro-60-0175 can actually reduce free DA as measured by microdialysis with a maximal effect of 30%, suggesting that this serotonergic circuit has a negative feedback effect on dopamine release and clearance (di Matteo 2002). The affinity of 5HT for the 5HT2C receptor, at least for GTP-γ-S metabolism is 27 nM (Im et al 2003). Using this value we can simulate the competition between endogenous serotonin and the neuroleptics using their appropriate affinity values. The amount of block at the 5HT2C receptor (5HT2C-block) was determined by simulating the competition at the 5HT2C receptor using the appropriate affinity of the compound and the above mentioned affinity of 5HT for the 5HT2C receptor. Because maximal stimulation of 5HT2C gives rise to a 30% decrease in free DA (Di Matteo 2002), the amount of free DA was then calculated as 
       Free_DA=Basal_DA(1+0.3*5HT2C-block) 
     Due of the relative affinities of the neuroleptics under consideration in relation to the affinities for 5HT, it turns out that the above formula can be simplified to 
       Free_DA=Basal_DA{1+0.3*Conc/(Conc+ K   i )} 
     where K i  is the affinity of the neuroleptic against the 5HT2C receptor. 
     Activity at the D3-receptor also influences DA [110]. Basically, full D3-R activation increases clearance of dopamine more in the ventral striatum than in the dorsal striatum. This effect is modeled as a linear interpolation with a maximum of 30%. 
       FIG. 6  shows how the EPS liability index is determined. First the effect of drugs on spike train frequencies is calculated in 4 different conditions. A mathematical formula (see below) combines these outcomes together with a convolution based mupno prefrontal serotonin receptor activation to yield the final EPS Liability Index. 
     The EPS Liability Index ( FIG. 5 ) is finally calculated as the 
       EPS LI=(dorsal striatum firing) 2 ×(1+PFC_Thr̂2)/[ventral striatum firing].
 
     Correlation Between Outcome of the Model and Actual Clinical Scales 
     We used the clinical database to gather information on various clinical scales related to motor side-effects. For instance, the parameter ‘fraction of patients needing anticholinergic medication’ was a readout of motor side-effects during clinical trials of schizophrenia (see  FIG. 7 ). We compared the outcome of 51 neuroleptic-dose combinations covering 15 different neuroleptics and showed that the outcome of our EPS module had a correlation r 2  of 0.64 (r=0.8) compared to a correlation r 2  of 0.045 (r=0.21) for the relation with the simple D2R occupancy, which has been used sofar. This table 3 gives the comparison between the correlations for various other clinical scales related to motor side-effects. 
                     TABLE 3                  Performance of the EPS module in predicting the known clinical       effects. For each clinical scale, the correlation and p-value is       given for the D2R occupancy rule and for the outcome of our       simulation modules.                                     D2R   D2R   EPS   EPS       Scale   corr(r 2 )   p-value   corr(r 2 )   pval                                         Fraction of patients on   0.02   0.34   0.65   0.00001       anticholinergic medication       Change SAS vs baseline   0.02   0.326   0.41   0.00007       Fraction of patients worsening   0.00   0.8482   0.57   0.0014       on SAS       Anticholin medication (dose)   0.41   0.0209   0.14   0.2258       EPS syndrome (fraction)   0.06   0.0734   0.12   0.0130       Hypertonia (fraction)   0.01   0.7393   0.00   0.8709       dystonia (fraction)   0.03   0.3153   0.28   0.0023       Tremor (fraction)   0.00   0.9192   0.27   0.0027       Parkinsonism (fraction)   0.06   0.2917   0.06   0.2665       Fraction of patients with   0.17   0.1240   0.53   0.0018       hypotonia       Fraction of patients with   0.00   0.9492   0.15   0.0862       dyskinesia       Change on Barnes Akathisa   0.01   0.5988   0.19   0.0144       scale       Fraction of patients with   0.01   0.5908   0.00   0.8428       akathisa       AIMS change from baseline   0.06   0.2668   0.14   0.0956       AMS Final change   0.00   0.8719   0.03   0.4128       Change on total ESRS   0.06   0.4758   0.09   0.3536       Change on ESRS   0.20   0.1256   0.13   0.2270       Parkinsonism       Change in ESRS hyperkinesia   0.00   0.8823   0.16   0.3207       Change on ESRS dystonia   0.00   0.9523   0.01   0.8014       Change on ESRS Dyskinesia   0.19   0.2631   0.01   0.8030                       # of       # of               significant       significant               Average   0.07   1   0.19   6                    
C. The Ventral Striatum, Prefontal Cortex, Hippocampus Model with Output for Predicting Clinical Efficacy of Drugs
 
     This model is constructed using a model of the GABAergic spiny neuron, which is the major neuron subtype in both the dorsal and ventral striatum.  FIG. 8  shows the major processes contributing to the medium spiny neuron model. The model readout is a function reflecting information content change between incoming and outgoing action potential train. Different receptor subtypes can be modulated by neuroleptics. We implement in a quantitative fashion the ideas about signal-to-noise changes observed in schizophenic patients and their unaffected siblings [105]. 
     The GABA spiny neuron originates in the striatum and is stimulated in large part due to Glu neurons from the PFC. In this simulation, we allow the following currents to affect the neuron: slowly inactivating potassium (Ksi), inward rectifying potassium (Kir2), L-type calcium (L-Ca), a general passive leak (L) and a specific passive CI leak (L-Cl). A spike train is generated based on time and the neuron&#39;s membrane potential. The Signal is the amount of action potentials coming out of the medium spiny neuron in the pathological state over a period of five seconds with the neuroleptics present at the appropriate concentration. The GABA spiny neuron has many Glu connections, with a baseline synaptic conductance density of 10.5 uS/cm̂2. This translates to a maximum conductance of 10.5 pS given the dimensions of our neuron. Due to the great innervation, we allow firing to occur every 3 ms. In order to reach the initial density, we also assume that the decay in signal is 3 ms. In the simulations, the background signal begins 100 ms after the simulation begins to allow the cell to be close to equilibrium before being stimulated. Gating input (originating from various brain regions, such as hippocampus and amygdala) is provided at frequencies of 330 Hz. 
     Glu Signal 
     We assume that there are 20 Glu neurons responsible for getting the medium spiny neuron to fire correctly. Under the “noise” condition, we assume that these neurons behave like the ones described above firing at 6 Hz with a strength of 4.8e-6. Under the “signal” condition, we assume that the frequency goes to 30 Hz and they fire simultaneously in two groups. This translates to 60 Hz input, but with a strength of 5*4.8e-6. In addition to the background Glu signal, there is a stimulating Glu signal which tries to make the GABA spiny neuron fire in response. Once again, due to the high Glu innervation, we assume a stimulation every 3 ms with a decay of 3 ms. The critical conductance which changes behavior of the cell is 2.8 pS. Thus, in order to get rich dynamics, we assume a conductance of 3.35 pS. We assume that there is always some noisy input from Glu inputs. We assume that it consists of 40 neurons firing at 6 Hz which means that if they are evenly spread out, we have an input of 240 Hz. The strength of the gating signal is 4.8e-6. In the simulations, the stimulating signal begins at 250 ms to allow the background stimulation to affect the cell. This stimulation then continues to the end (at 5 seconds). The glutamatergic signal input from the PFC is a 50 Hz signal. Activation of postsynaptic D2R on glutamatergic afferents (which decreases glutamatergic activation of the spiny neuron) is modulated by means of the receptor competition model (see above) and is dependent upon the particular concentration of neuroleptics. D2 activation decreases this stimulating Glu signal by reducing the signal&#39;s conductance. We assume a simple linear relation which allows ventral spiking in the control case, but not as much in the SZ case. If there is no D2 activation, then the conductance is 3.35 pS. However, if the activity is at the very high percentage of 60%, the conductance drops to 1.85 pS. Activation of postsynaptic D1R on the spiny neuron affects Kir2 and L-Ca currents and modulates the glutamatergic drive such that concomitant stimulation drives the spiny neuron in the upstate, allowing it to fire. Kir2 and L-Ca currents are both multiplied by a dopaminergic factor called u. u is determined by D1 activation for both tonic firing and burst firing. Both patterns repeat at 4 Hz. However, tonic firing is depicted by a relatively slow increase to u_max (rate ˜25 ms) and an almost immediate decrease from the max, but once again slowly (rate ˜50 ms). Burst firing on the other hand is depicted by a rapid increase to u_max (rate ˜1 ms), followed by a long duration at this maximum (˜100 ms) finally followed by an even slower decay (rate ˜100 ms). In the case of tonic firing, u_max is determined from the average D1 receptor activation for the first firing pattern (@ 4 Hz), third firing pattern (@ 1 Hz) and fourth firing pattern (@ 4 Hz). In the case of burst firing, u_max is determined from the average D1 receptor activation for the final firing pattern (@ 80 Hz). u_max is 0.8 plus 0.006 times the D1 receptor activation. (Note: D1 receptor activation is the percentage of D1 receptors bound by DA and as such varies between 0 and 100.) This dopaminergic signal begins 500 ms into the simulation (to allow the signal to establish itself) and stops 500 ms before the end. High D1 activation amplifies strong signals and diminishes weak signals. We assume that the “noise” condition has tonic DA activity while the “signal” condition has burst DA activity. 
     The Hippocpampus and Amygdala 
     The gating signal from the hippocampus and amygdala is a high frequency, low strength glutamatergic input. The gating signal from the amygdala is under the influence of adrenergic pharmacology [10], so that alpha1A-R activation tends to strengthen the gating signal. The model allows to flexibly adapting the gating signal input from the hippocampus, so that network simulations similar to the prefrontal cortex network can be developed as front-end input modules to the SN module. It would be envisageble at some point to incorporate concepts related to the SOCRATES (Sequences of Condensed Representations, Autocorrected, Theta Gamma Coded in Context) model [59]. 
     The muscarinic M1R couples to the chloride channel, regulating the membrane potential. The model calculates the timing of action potential, when the membrane potential exceeds a certain membrane threshold. Blocking of the M1 receptor causes a modest increase in the conductance of the leak current. A maximum block of 100% would cause the conductance to increase by 30%. However, because ACh has such a strong affinity for M1-R, the maximum block would more practically be ˜20% which would have a maximum increase on the conductance of ˜6%. 
     The output is captured as an information content parameter (see below). 
     Basically, this parameter describes the quantity of encoded bits in the action potential train, which is proportional to the amount of information a network can handle. Therefore the difference between input and output is a useful readout as it is proportional to the information processing capacity in the ventral striatum, and represents a type of signal to noise readout. 
     We introduce a schizophrenia pathology as follows. PET imaging studies indicate that certain parameters are changed between the healthy state and the schizophrenia pathology. As an example, using alpha-methyl-para-tyrosine induced acute dopamine depletion, PET imaging with  11 C raclopride in normal controls and schizophrenia patients indicated that free dopamine in patients was essentially double the value for normals controls [2]. Such experiments also provide us with degrees of variability for the patient population. For a population of already treated schizophrenic patients, the values are 
                     TABLE 4                  Parameters used for defining a pathological state of the       simulation modules.                             Parameter   Change vs. healthy   Variation   Reference               DA released   +100%    CV = 20%    [2]       D1-R   No change   CV = 35%    [1]       D1-R high affinity   −20%   N/A   [53]       D2-R   +30%   CV = 50%   [85]       D3-R   No change   CV = 30%   [42]       DAT   −30%   CV = 35%   [57]       M1-R   No change   CV = 40%   [82]       M2-R   No change   CV = 40%   [82]       5HT2C-R   −20%   CV = 40%   [98]                    
In addition, we assume that the Noisy Glu Signal and the Glu Signal are both affected by D2-R activation. In the case of schizophrenia, the signal strength is reduced by 20%.
 
     The model parameters can be adapted so as to improve correlations with specific clinical scales, leading to slightly different models for the different scales (i.e. PANSS positive or PANSS negative). This suggests differential weights to specific neurotransmitter circuits or pathways which are involved with specific clinical scales. 
     Performance of the Schizophrenia (SN) Module in Correlation Studies 
       FIG. 9  illustrates the correlation between the outcome of the computational SN module and the PANSS positive scale for 35 neuroleptic-dose combinations, covering 12 different neuroleptics. In this particular case, correlation with simple D2R occupancy yields a correlation coefficient r 2  of 0.22, suggesting a reasonable contribution of DA pharmacology to the clinical outcome. However, the correlation between PANSS positive and the outcome of the SN computer model is 0.46, suggesting that incorporation of additional physiology is able to substantially improve the correlation. 
     The following table shows the correlations between performance of drugs on clinical scales derived form the clinical database and their outcome either in (i) a D2R occupancy rule and (ii) the S/N module. The seven key clinical scales are denoted with an asterix. On average, the computer model achieves an 80% increase in correlation values, because it takes into account the physiological effects at more than 10 receptors. 
                     TABLE 5                  Performance of the SN module in comparison to the D2R       occupancy rule. For each clinical scale, the correlations       and p-value for both models are given.                             correlation   correlation                                 Change in Clinical Scale   D2R(r 2 )   p-val   S/N (r 2 )   p-val                                         *PANSS total   0.183   0.0178   0.625   0.0000       *PANSS positive   0.222   0.0062   0.461   0.0000       PANSS negative size effect   0.429   0.0666   0.886   0.0001       *PANSS negative   0.190   0.0138   0.725   0.0000       Size effect in PANSS disorg   0.125   0.3818   0.023   0.7162       PANSS psychopatholgy   0.188   0.0549   0.402   0.0024       PANSS Anxiety/depression   0.308   0.1114   0.480   0.0316       *SANS   0.200   0.0213   0.366   0.0011       *BPRS total   0.375   0.0000   0.382   0.0000       Size effect in BPRS total   0.089   0.2776   0.226   0.0109       BPRS core items   0.256   0.1549   0.629   0.0016       *BPRS positive   0.313   0.0078   0.474   0.0008       *BPRS negative   0.673   0.0023   0.802   0.0000       BPRS activity   0.060   0.3257   0.162   0.0868       BPRS anergia   0.136   0.1178   0.214   0.0371       BRPS anxiety/depression   0.007   0.7417   0.010   0.6923       BPRS hostility   0.024   0.6307   0.104   0.0773       BPRS Thought disturbance   0.065   0.3784   0.182   0.1245       CGI-S improvement   0.061   0.1599   0.078   0.0594       CGI-Global improvement   0.038   0.6681   0.164   0.0145                       #of               significant               Summary   0.20   7   0.358   14           0.67   2   0.864   9                    
The average correlation coefficient for the SN module is 0.358 compared to 0.20 for the D2R occupancy rule.
 
     D. Serotonin-Norepinephrine Interaction 
       FIG. 10  shows a flow chart of the interactions between Dorsal Raphe Nucleus, source of serotonergic neurons; Locus Coeuruleus, source of noradrenegric neurons and efferent projections to the PFC or hippocampus. The activity of the Dorsal Raphe Nucleus is regulated by afferent DA fibers from VTA (D2R), serotonergic 5Ht1 A autoreceptors and alpha1A receptors from Locus Coeruleus. The activity of LC is regulated by afferent 5HT2 receptors from DR and noradrenergic alpha2A autoreceptors. This gives rise to a highly non-linear circuit. 
     This computer model for serotonin-norepinephrine interaction simulates the effect of neuroleptic drugs on the serotonin and norepinephrine free level in the prefrontal cortex, based on the interaction between serotonergic neurons in the Dorsal Raphe Nucleus and noradrenergic neurons in the Locus Coeruleus. The pharmacology of drugs is modeled at three synapse levels: a serotonergic synapse onto noradrenergic cell bodies in the LC; an adrenergic synapse onto serotonergic cell bodies in the DR and a serotonergic presynaptic terminal ending in the PFC or hippocampus. 
     Serotonergic Synapse onto NE Cell Bodies in LC 
     The serotonergic synapse in the Locus Coeruleus originates in the Dorsal Raphe Nucleus. Serotonin released here activates postsynaptic 5HT2A receptors on the cell bodies of noradrenergic neurons and negatively influences the action potential firing frequency. Notably, full elimination of 5HT innervation by lesioning the Dorsal Raphe increases NE firing frequency from 2.0 to 3.5 Hz, i.e. by 75% [43]. This effect is very likely mediated by 5HT2A receptors [91]. If S(t) is the concentration of serotonin in the cleft at time t, we model the activation A(t) of the 5HT2A receptor by 
         A ( t )= cDS ( t )/[ K   i   +S ( t )] 
     where K i  (Ki=20  M), is the concentration of serotonin in which 50% of the 5HT2A receptors are occupied (McAllister 1992), c is a user controlled parameter adjusting the relative amount of 5HT2A receptors being used, (c=1). Furthermore, D is the amount of inhibition (e.g. due to drugs) at the 5HT2A receptor (a value between 0=full inhibition and 1=no inhibition). 
     We define t n  as the global time at which the n th  firing takes place, T n  as the time period of the n th  firing of the Locus Coeruleus, and T o  as the base period between firings. Because the normal firing frequency is 2 Hz, T o =1000/2=500 ms. The accumulation of 5HT2A receptor activation during the period of the (n−1) firing, can be defined as 
         I   n =∫ t     n-1     t     n-1     +T     n-1     A ( t ) dt  
 
     I o  is the average accumulation of 5HT2A activity in the case where all neurons are firing monotonically. Having determined, I n  we can calculate the time period for the nth firing as follows: 
     
       
         
           
             
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     where s is the sensitivity to change for the 5HT2A receptor. s ranges between 0 and 1 where 0 means no change and 1 means maximal sensitivity to change. (s=0.5 by default).
 
When the 5HT2A receptor is fully inhibited (A=0=&gt;I n =0), the firing frequency is increased by 75%. The time period is calculated every time the Dorsal Raphe fires. Future models may take a stochastic approach and rely on a history of 5HT2A activity.
 
     Serotonin can disappear from the cleft through diffusion or its interactions with enzymes and receptors which we do not explicitly include. For simplicity, we assume that the rate of dissipation is proportional to the amount of serotonin in the cleft. 
     
       
         
           
             
               
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     S(t) is the concentration of serotonin in the cleft at time t, whereas k is the rate of exponential decay. Studies on diffusion of DA show that it has a half-life of about 500 ms [26]. We assume the diffusion of 5HT is similar. Thus, we let k=ln(2)/500.
 
Noradrenergic Synapse into 5HT Cell Bodies in DR
 
     Whenever an action potential arrives at the presynaptic membrane of a Locus Coeruleus neuron synapsing on a serotonergic neuronal cell body in the Dorsal Raphe Nucleus, norepinephrine is released into the noradrenergic cleft. 
     The concentration N(t) of Norepinephrine in the synaptic cleft at time t is 
         N ( T )= N ( T−dt )+ b   N    
     where T is the time an action potential arrives at the Locus Coeruleus, dt is the step size in time and b N  is the concentration of norepinephrine released into the cleft. Although there are no data out for NE release in neuronal cells, we assume that the amount of neurotransmitter released is similar to the amount of released serotonin (see below), i.e. about 5000 molecules. 
     Given the concentration of norepinephrine in the cleft and the concentration of postsynaptic ✓ □  receptors, we can determine the occupancy a 1 (t) of the α 1  adrenergic receptors based on the K i  values (the concentration of norepinephrine in which 50% of the α1 receptors are occupied) which describe the affinity of NE for ✓ □  An estimated value of K i  is 4.5  M [71]. 
         a   1 ( t )= A   1   *{N ( t )/[ N ( t )+ K   i ]} 
     A 1  is the concentration of α 1  receptors. An estimation for this parameter can be deduced from autoradiography data of specific ✓ 1  adrenoceptor ligands. Using [ 125 I]HEAT ([ 125 I]iodo-2-[beta-(4-hydroxyphenyl)ethylamino methyl]), two sites with different affinities for the alpha 1-adrenoreceptor were found in normal rat brain in vivo: a high-affinity site with K d  (half-saturation constant) of 3.6+/−0.7 nM, and a low-affinity site with K d  of 668+/−552 nM. The density (B max ) of the high-affinity site in different brain regions varied from 2.2+/−0.8 to 14.6+/−0.6 pmole/g, while the low-affinity range was 149+/−44 to 577+/−30 pmole/g [36]. In a human study using biopsies from epileptic and non-epileptic foci, receptor-binding assays were performed on isolated cortical membranes using [ 3 H]prazosin. Values in nonspiking regions (i.e. non-affected regions) for B max , are 218.8+/−15.6 fmol/mg protein with a corresponding affinity of 0.17+/−0.04 nM [11]. To go form this value to a density of synaptic receptors, we acknowledge that 10 fM/mg tissue is 6×10 9  molecules/mg tissue. Assuming the density of biological material is around 1, 1 mg tissue is 1 mm 3  or (1000μ) 3 . The density of specific labels is then around 6 molecules per μ 3 . 
     The extracellular fraction (lambda) [26] is usually low (0.2), so that most of the volume is taken up by brain material. Even when neurons are outnumbered largely by glia and astrocytes, because of their long projections, they take up a substantial fraction of the volume. Suppose they take up 80% of the brain material, this would suggest that neurons contribute to about 64% of total material. Synapses are usually located on synaptic boutons, which is a central unit. If boutons account for 50% of neuronal material, then 32% of total material consists of synapses. The diameter of those boutons range from 0.5 to 3.5μ [50]. Suppose the mean radius is 1μ, then the volume is about 4μ 3 . If all receptors would be concentrated in bouton material, we get 4×6×3=72 receptors per bouton, and per synapse. A certain fraction of receptors (50%) might be located intracellularly, so we end up with 30-36 receptors/μ 2 . This is in the range of presynaptic Ca-channel density [50], which is about 45 channels/μ 2 . 
     Similarly, if A 2  is the concentration of the presynaptic ✓ 2  receptors, we can write 
         a   2 ( t )= A   2   *{N ( t )/[ N ( t )+ K   i ]} 
     where K i  is the concentration of norepinephrine which occupies 50% of the α 2  receptors and a 2 (t) is the concentration of a 2  receptors bound by norepinephrine at time t. An experimental estimate of K i  for NE at the α 2 -R is 996 nM and at the α 2 d-R is 1031 nM [72]. We take a value of 1 μM. 
     An estimation for A 2  can be deduced as follows. The specific binding of the alpha 2-adrenoceptor agonist [ 3 H]clonidine was measured in the postmortem brain of ethanol intoxicated nonalcoholic subjects and matched controls [62]. In the frontal cortex, the density of binding sites for [ 3 H]clonidine (B max =58+/−7 fmol/mg protein) and [ 3 H]bromoxidine (UK 14304) (B max =49+/−7 fmol/mg protein) in ethanol intoxicated subjects did not differ from those in the control groups (B max =68+/−4 and 56+/−8 fmol/mg protein for the respective radioligand). We can therefore calculate a B max  of 60 fM/mg protein for ✓ 2  adrenoceptors. Using a similar argument as outlined above, we can then estimate the density of presynaptic α receptors. 
     The α 2  adrenergic receptors at the presynaptic membrane are autoreceptors, which limit the release of subsequent NE. Full stimulation of these receptors (above the basal level by NE) by clonidine (an ✓ 2  adrenoceptor agonist) reduces 5HT firing (and subsequent PFC 5HT release) by 45% [45]. If R is the amount of norepinephrine released, a 2  is the ✓ 2  activity (a value between 0=no activity and 1=fully active) and b is the maximum amount of norepinephrine released. (b=i.e. 5000 molecules/action potential or 150 μM) 
         R=b (1− pa   2 )
 
     where p is a free parameter chosen so that when there is maximum activity, the release will only decrease up to 45%. (p=0.63) 
     The α 2  adrenergic autoreceptors are G-protein coupled receptors which a negative feedback on the subsequent NE release. Usually, there is a time-delay of about 40 minutes before the full effect becomes available [45]. However, we allow for an immediate effect in our current version of the simulation. Since the effect is continuous, the delay will eventually not matter. 
     The Norepinephrine Transporter (NET) uptake kinetics usually follows a Michaelis-Menten scheme and is energy- and sodium dependent. In the rat jejunum [90] two NET populations have been found. The high-affinity component (uptake 1) exhibited a Michaelis constant (K m ) of 1.72 μM and a maximum velocity (V max ) of 1.19 nmol·g-1.10 min-1. The low-affinity component (uptake 2) exhibited a K m  of 111.1  M and a V max  of 37.1 nmol·g-1.10 min-1. We use the parameters for the high-affinity uptake component. Furthermore, blocking the NET by venlafaxine, leads to a robust NE increase of 498% in cortex [9]. We let n o  be the number of working NET present and n t  be a user controlled parameter adjusting the relative amount of NET. (n t =1). The value of was n o  chosen so that when the alfa-2 receptor was completely blocked, the resulting release would increase by 50% over the normal case. An estimate of the number of NET is given by the observation that in the striatal nucleus of rhesus monkey, the  3 H-nisoxetine density is between 20 and 35 fM/mg tissue. The estimate for the density n o  is right in the experimentally determined density. 
     
       
         
           
             
               
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     Norepinephrine can also disappear from the cleft through diffusion. For simplicity, we assume that the rate of dissipation is proportional to the amount of norepinephrine in the cleft (k is the rate of exponential decay). We use the same value as for serotonin (see above), i.e. k=ln(2)/500. 
     
       
         
           
             
               
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     Presynaptic Serotonergic Terminal in PFC 
     The Basal Firing rate of serotonergic neurons is 1.3 Hz [29]. However about half of the neurons in the Dorsal Raphe Nucleus display a higher firing frequency of 1.9+/−0.1 Hz. We let t n  be the global time at which the n th  firing takes place, T n  is the time period of the n th  firing of the Dorsal Raphe and T o  is the base period between firings. (Because the normal firing frequency is 1.3 Hz, T o =1000/1.3=770 ms). The accumulation of activity can be defined as 
     
       
         
           
             
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     where a 1 (t) is the activation of the ✓ 1  receptor at time t (a value between 0=no activation and 1=full activation). I o  is the average accumulation of α 1  activity in the case where all neurons are firing monotonically. The accumulation of α 1  activity is used to determine the time of the next firing as follows 
     
       
         
           
             
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     where s is the sensitivity to change for the ✓ 1  receptor. s ranges between 0 and 1 where 0 means no change and 1 means sensitive to change. We take s=0.33 as default. As a consequence, when the α 1  activity is normal, I n =I o  which causes T n =T o  and when the α 1  activity is completely inhibited, I n =0 which means that T n =2 T o . (A doubling of the period means that the frequency is halved.) 
     A detailed study in serotonergic cultured Retzius cells [16] yields two populations of serotonergic containing vesicles both with a constant serotonin concentration of 270 mM. Amperometric analysis shows that these two types of vesicles (diameter 35 and 78 nm) release their content at once during an action potential. This calculates back to about 5000 and 80,000 molecules respectively [15]. In addition, most boutons release a single quantum at an action potential [76] with only 13-17% exhibiting multiple quanta release. Translated into the space of the small synaptic cleft, this is in the range of 100 μM. Although this has never been measured, it is in the same range as measures for ACh in the case of a cholinergic cleft. Therefore, if S(t) is the concentration of serotonin in the cleft at time t, then 
         S ( T )= S ( T−dt )+ b    
     where T is the time the Dorsal Raphe fires, dt is the step size in time, and b is the concentration of serotonin released into the cleft. 
     Serotonin binds to 5HT1b/d autoreceptors inhibiting subsequent 5HT release. Maximally blocking the 5HT1b/d autoreceptor by isamoltane increases free 5HT by 50%, whereas maximal stimulation by either CP93,129 or CP135,807 decreases free 5HT by 45% [45]. This allows us to derive the mathematical equation linking free serotonin concentration to activity at the 5HT1 b/d receptor. In contrast to the 5HT2 or 5HT3 receptors, receptors of the 5HT1 class display a high-affinity binding for serotonin, in the range of 15 nM [61]. The functional affinity of serotonin for the 5HT1b/1d receptor is probably in the submicromolar range, as 10  M fully activates the 5HT1b/1d receptor in situ [66]. If a is the occupation/activation of the 5HT1 b/d receptor by serotonin and S(t) is the concentration of serotonin in the cleft at time t. 
         a=r*D*S ( t )/[ K   i   +S ( t )] 
     where K i  is the concentration of serotonin in which 50% of the 5HT1b/d receptors are occupied, r is a user controlled parameter adjusting the relative amount of receptors present (r=1) and D is the amount of inhibition (e.g. due to drugs) at the 5HT1 b/d receptor (a value between 0=full inhibition and 1=no inhibition). The concentration of serotonin R to be released can then be written: 
         R=b *(1− pa )
 
     where b is the maximum amount of serotonin that can be released and p is a free parameter chosen so that when there is maximum activity, the release will only decrease up to 45%. (p=0.65). 
     The 5HT1b serotonergic autoreceptors are G-protein coupled receptors which exert a negative feedback on the subsequent 5HT release. Usually, there is a time-delay of about 40 minutes before the full effect becomes available [109]. In this version of the model we do not explicitly introduce this time-delay. 
     Finally, the 5HT transporter uptakes serotonin from the cleft. When it is blocked, the 5HT levels rise within the cleft. The rise in 5HT causes an increase in the activation of the 5HT1 b/d receptor. Maximal inhibition by fluoxetin essentially doubles the amount of free 5HT in the prefrontal cortex (from 4.3 to 8.6 fM/fraction) [6]. The 5HT-Transporter essentially follows Michaelis-Menten kinetics with a K m  of 0.34 μM [80]. Therefore if S(t) is the concentration of serotonin in the cleft at time t, we can write 
       ∂ S/∂t=−s   f   s   o   V   max   *S ( t )/[ S ( t )+ K   i ]
 
     where s t  is a user controlled parameter adjusting the relative amount of 5HT-T (s f =1) and s o  is the number of working 5HT-T (s o =40). V max  is the maximum rate at which serotonin is taken up by 5HT-T (V max =2.5). K i  is the concentration of serotonin at which the 5HT-T “pump” works at half its maximal speed (K i =340 nM). 
     Serotonin can disappear from the cleft through diffusion or its interactions with enzymes and receptors which we do not explicitly include. For simplicity, we assume that the rate of dissipation is proportional to the amount of cleft serotonin. 
     
       
         
           
             
               
                 ∂ 
                 
                   S 
                    
                   
                     ( 
                     t 
                     ) 
                   
                 
               
               
                 ∂ 
                 t 
               
             
             = 
             
               - 
               
                 kS 
                  
                 
                   ( 
                   t 
                   ) 
                 
               
             
           
         
       
     
     where k is the rate of exponential decay. We assume a similar value for k (see above), i.e. k=ln 2/500. 
     The results of this simulation yield activation levels of postsyanypic 5HT1A, 5HT2A, alpha1A receptors in hippocampus and prefrontal cortex, which are then used in the calculations of cortical SMA serotonin effects in the EPS module and serotonergic and noradrenergic effects on working memory performance (see below). 
     E. Statistical Prediction of the Model Outcome. 
       FIG. 11  presents all of the interacting entities with their geometric relationships used in predicting the performance of a new untested drug or combination on specific clinical scales. The model takes into account both G-protein coupled receptors and neurotransmitter transporters. Based upon the linear regression equation describing the correlation between the outcome of known drugs in the mathematical models and their clinical performance, we determine the values for the slope a and the intercept b. Based upon the pharmacology of the new untested drug, we calculate its outcome in the mathematical models and using the linear equation, determine the anticipated effect size on the clinical scale, together with a 95% confidence interval. 
     For each neuroleptic-dose combination used in the clinical trials, the outcome of the EPS or Signal-to-Noise module is correlated with the clinical outcome, yielding the best linear equation Y=aX+b, where Y is the expected clinical outcome, X is the outcome of the simulation and a and b are parameters determined by the correlation analysis. The correlation coefficient indicates how good the fit is, with values close to 1 for better results. In addition, a Student&#39;s t-test determines the probability level that the hypothesis “the data are described by a linear relation Y=aX+b” is true. 
     Suppose the linear correlation yields an intercept a* and a slope b*, then the expected clinical scale outcome y for a new investigational drug with a dose corresponding to a Signal-to-Noise value of x* is ( FIG. 11 ) 
     
       
      
       y=a*+b*x*  
      
     
     with a prediction interval Pi, which can be calculated using statistical techniques. As a con sequence, 95% of the cases the clinical scale will be between y−Pi and y+Pi, where 
     
       
         
           
             
               Pi 
               = 
               
                 
                   
                     t 
                      
                     
                       ( 
                       
                         α 
                          
                         
                           / 
                         
                          
                         2 
                       
                       ) 
                     
                   
                   × 
                   S 
                   × 
                   
                     
                       ( 
                       
                         1 
                         + 
                         
                           1 
                           n 
                         
                       
                     
                   
                 
                 + 
                 
                   
                     
                       ( 
                       
                         
                           x 
                           * 
                         
                         - 
                         
                           〈 
                           x 
                           〉 
                         
                       
                       ) 
                     
                      
                     2 
                   
                   
                     n 
                     × 
                     Sxx 
                   
                 
               
             
             ) 
           
         
       
     
     F. Working Memory Circuit 
       FIG. 12  shows a schematic diagram of the PFC neuronal network of 20 pyramidal cells and 10 interneurons. The pyramidal cells synapse upon each other and drive 50% of the inhibitory network in a random fashion. Ten of the pyramidal cells form the attractor pattern, while the other 10 are part of the distractor pattern. All glutamatergic synapses have NMDA and AMPA receptors, while all GABAerge synapses have GABA-A receptor types. The pyramidal neurons consist of four compartments, the GABAergic neurons of two compartments. 
     In order to ensure a biophysically realistic model, Hodgin-Huxley type equations are implemented on Ca 2+   dyn  (first order model of calcium dynamics), H va  (high voltage activated Ca 2+  current), iC (fast Ca 2+ /voltage-dependent K +  current), I ks  (slowly inactivating K +  channel), K +   dr  (delayed rectifier K+ channel), K +   dri  (delayed rectifier K +  channel for interneuron), K +   dyn  (simple first order model of K +  dynamics), Na +   f  (fast Na +  channel), Na +   nt  (fast Na +  channel for interneurons), Na +   p (persistent Na +  channel) and on NMDA, AMPA and GABA-A receptors [30]. 
     This model has been extended considerable to include the effect of pharmacological manipulation (see below). In addition, the original model yielded a proportional relationship between pyramidal cell activity and interneuron activity over the whole range of parameter settings, which is at odds with experimental data. Therefore, the model has been significantly adapted to account for the described asymmetric distribution of pyramidal-pyramidal and pyramidal-interneuron synapse. Rather than assuming a 1:1 ratio, we have allowed our model to have the number of pyramidal-interneuron connections randomly at 50% of the number of pyramidal-pyramidal interactions. This ensured the model to have an inverse relation ship between pyramidal cell firing and inhibitory cell firing. 
     In addition, more recent data on the difference between NMDA sensitivities between inhibitory and excitatory [5] have been implemented. This allows to refine the predictions. 
       FIG. 13  shows an illustration of the concept of working memory span. The x-axis is time in msec; every line represents the action potential activity of a single neuron. The first ten are the attractor pattern, cell 11-20 are the distractor pyramidal neurons and cell 21-30 are the GABA interneurons. At 2000 msec, a single current is injected in the attractor pattern, which remains active for a certain period (4-7 sec) without further stimulation. 
     Working memory in this circuit is simulated by a single injection of a current in the Target pattern which triggers a continuous reverberant synchronous activity for 6-8 seconds without any further stimulation, until the activity in the distractor pattern takes over. The length of this continuous active pattern (see  FIG. 13 ) is the major readout of the model and is anticipated to be proportional to working memory performance. For each experiment, 140 simulations are run with random number seed. 
     An entropy factor can be calculated to assess quantitatively the ‘complexity’ and richness of the signal. The information content is derived using the approximate formulas for a finite spike train proposed by Strong [87]. Briefly, the information from the simulation consists of the timing of action potential for each of the 20 pyramidal and the 10 GABAergic neurons. An input module calculates the average firing frequency of pyramidal neurons and the total number of action potentials in the 10 GABAergic neurons. The activity of the 20 pyramidal neurons is then projected upon one time-axis, i.e. the identity of the neuronal cell firing is lost. A burst event is defined as the number of bins where adjacent activity is observed for the time bin (see below). 
     For the analysis of information content, the full spike train with a certain size is divided into time bins ΔT, where ΔT is 5 msec. A bin with an action potential is given the value 1, otherwise it is 0. In this way, words can be defined with maximal length T/ΔT. T is 4, 8, 12, 16, so that maximal word length spans a time delay of 90 msec. 
     If p i  is the normalized count of i-th word p i , then a naïve estimate of the entropy is given by 
     
       
         
           
             
               
                 S 
                 naive 
               
                
               
                 ( 
                 
                   T 
                   , 
                   
                     
                       Δ 
                        
                       
                           
                       
                        
                       T 
                     
                     ; 
                     size 
                   
                 
                 ) 
               
             
             = 
             
               - 
               
                 
                   ∑ 
                   i 
                 
                  
                 
                   
                     p 
                     i 
                   
                    
                   
                       
                   
                    
                   
                     log 
                     2 
                   
                    
                   
                       
                   
                    
                   
                     p 
                     i 
                   
                 
               
             
           
         
       
     
     True entropy is 
         S ( T,ΔT )= lim   size→∞   S   naive ( T;ΔT ;size) 
     We are interested in the entropy rate 
         S ( T )= lim   T→∞   S ( T,ΔT )/ T    
     We calculate the values for S naive (T,ΔT; size′), where size′=total spike train/i, where i=1.4. 
     We get a parabolic estimate of S(T,ΔT) by considering 
     
       
         
           
             
               
                 S 
                 naive 
               
                
               
                 ( 
                 
                   T 
                   , 
                   
                     
                       Δ 
                        
                       
                           
                       
                        
                       T 
                     
                     ; 
                     size 
                   
                 
                 ) 
               
             
             = 
             
               
                 S 
                  
                 
                   ( 
                   
                     T 
                     , 
                     
                       Δ 
                        
                       
                           
                       
                        
                       T 
                     
                   
                   ) 
                 
               
               + 
               
                 
                   
                     S 
                     1 
                   
                    
                   
                     ( 
                     
                       T 
                       , 
                       
                         Δ 
                          
                         
                             
                         
                          
                         T 
                       
                     
                     ) 
                   
                 
                 size 
               
               + 
               
                 
                   S 
                    
                   
                       
                   
                    
                   2 
                    
                   
                     ( 
                     
                       T 
                       , 
                       
                         Δ 
                          
                         
                             
                         
                          
                         T 
                       
                     
                     ) 
                   
                 
                 
                   size 
                   2 
                 
               
             
           
         
       
     
     If the correlations in the spike train have finite range then the leading subextensive contribution to the entropy will be 
     
       
         
           
             
               
                 S 
                  
                 
                   ( 
                   
                     T 
                     , 
                     
                       Δ 
                        
                       
                           
                       
                        
                       T 
                     
                   
                   ) 
                 
               
               T 
             
             = 
             
               
                 S 
                  
                 
                   ( 
                   
                     Δ 
                      
                     
                         
                     
                      
                     T 
                   
                   ) 
                 
               
               + 
               
                 
                   C 
                    
                   
                     ( 
                     
                       Δ 
                        
                       
                           
                       
                        
                       T 
                     
                     ) 
                   
                 
                 T 
               
             
           
         
       
     
     Such that a linear correlation of S(T,ΔT)/T vs 1/T enables one to determine the S(ΔT) as the intercept with the y-axis. This is usually expressed as bits/sec. 
     Another possible readout is an anticipated size of the fMRI BOLD signal, which is readily measurable in clinical setting. The bold signal reflects the total synaptic activity in the brain (Attwell 2002). 
     
       
         
           
             
               b 
                
               
                 ( 
                 t 
                 ) 
               
             
             = 
             
               
                 ∫ 
                 o 
                 t 
               
                
               
                 
                   h 
                    
                   
                     ( 
                     
                       t 
                       - 
                       
                         t 
                         ′ 
                       
                     
                     ) 
                   
                 
                 × 
                 
                   
                     I 
                     sym 
                   
                    
                   
                     ( 
                     
                       t 
                       ′ 
                     
                     ) 
                   
                 
                  
                 
                     
                 
                  
                 
                    
                   
                     t 
                     ′ 
                   
                 
               
             
           
         
       
     
     Where I syn (t) is total synaptic activity in the network model and 
     
       
         
           
             
               h 
                
               
                 ( 
                 t 
                 ) 
               
             
             = 
             
               
                 
                   
                     
                       λ 
                       1 
                       
                         s 
                         1 
                       
                     
                     × 
                     
                       t 
                       
                         
                           s 
                           1 
                         
                         - 
                         1 
                       
                     
                   
                   
                     
                       ( 
                       
                         
                           s 
                           1 
                         
                         - 
                         1 
                       
                       ) 
                     
                     ! 
                   
                 
                 × 
                 
                    
                   
                     
                       - 
                       
                         λ 
                         1 
                       
                     
                      
                     t 
                   
                 
               
               - 
               
                 
                   
                     
                       λ 
                       2 
                       
                         s 
                         2 
                       
                     
                     × 
                     
                       t 
                       
                         
                           s 
                           2 
                         
                         - 
                         1 
                       
                     
                   
                   
                     
                       r 
                        
                       
                         ( 
                         
                           
                             s 
                             2 
                           
                           - 
                           1 
                         
                         ) 
                       
                     
                     ! 
                   
                 
                 × 
                 
                    
                   
                     
                       - 
                       
                         λ 
                         2 
                       
                     
                      
                     t 
                   
                 
               
             
           
         
       
     
     Where s 1 =6; s 2 =10; λ 1 =1.25, λ 2 =⅛; r=6 [33] is a hemodynamic response function 
     Pharmacology in Working Memory Circuit 
     We introduce the functional effects of receptor modulation using the outcome of our generic receptor competition models and published data on the link between receptor activation, intracellular signaling and phosphihorylation mediated effects on ion-conductances. 
     Dopamine interacts through direct D1 receptor activation which increases NMDA receptor density [52] reduces AMPA currents [58], reduces Ca 2+  current [12], facilitates Na current [107] and facilitate GABA currents [111]. D1R activation is modulated at the level of dopaminergic neurons itself by Glutamatergic and serotonergic afferents in the Ventral Tegmentum Area. 
     Serotonin receptor through 5-HT1A and 5-HT2AR reduces conductances of Na and Ca 2+  channels in different compartments of pyramidal neuron (distal compartment for 5HT2A, all compartments for 5HT1A). The specific localized distribution of serotonergic and dopaminergic receptors has been documented in rodents and primates [68, 7]. A saturating dose of 5HT (1  M) decreases the maximum conductance of the fast Na +  channel, the persistent Na +  channel, and the L-type Ca ++  channel by 20% [19]. 
     Norepinephrine acts through alpha2A-R on GABA interneurons; stimulation of alpha2A-R decreases GABA neuron excitability, relieving inhibition in the network and increasing working memory performance. 
     Acetylcholine acts through postsynaptic M1R on pyramidal neurons via phasic and tonic effects, while presynaptic M2R modulates free Ach, which in turns activates α7 nAChR on glutamatergic neurons and α4β2 nAChR on GABA interneurons [35]. 
     The 5-HT3 receptor has been shown to enhance GABAergic firing patterns in prefrontal cortex [78] in vivo. The GABA conductance allowing CI ions to flow into the cells is associated with a hyperpolarization. This leads to a decrease in GABAergic interneuron firing, a decrease in inhibition and subsequently an increase in pyramidal neuron excitability. 
     In the following table, Act is the level of activation of the specific receptor 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Definitions of parameter values used in subsequent tables. 
               
            
           
           
               
               
               
               
               
            
               
                 Receptor 
                 D1 
                 5HT1A 
                 5HT2 
                 alfa2A 
               
               
                   
               
               
                 Effect 
                 Activ/100 
                 0.3 * Act/100 
                 0.2 * Act/100 
                 0.04 * Act/100 
               
               
                 g-NMDA 
                 g′ = g * (1 + 0.4 * Effec 
               
               
                 g-AMPA- 
                 g′ = g * (1− 
               
               
                 g-GABA 
                 g′ = g * (1 + 0.2 * Effec 
               
               
                   
               
            
           
         
       
     
     M1-R activation by transient high Ach release inhibit pyramidal neuron excitation (induces hyperpolarization) through IP3-mediated release of intracellular Ca 2+  and subsequent activation of apamin-sensitive, Ca 2+ -activated K +  conductance [41]. Fast M1R activation by bursts hyperpolarizes −4 mV (makes them less excitable), while M1-R activation by low tonic Ach depolarizes pyramidal neurons by +5 mV and makes them more excitable. If both processes work via the same mechanisms (i.e. effect on same K-conductance), we can calculate the net effect as follows 
     
       
         
           
             
               Δ 
                
               
                   
               
                
               MembPot 
             
             = 
             
               
                 
                   - 
                   5 
                 
                 × 
                 
                   
                     ( 
                     
                       
                         
                           M 
                           1 
                           act 
                         
                         - 
                         
                           M 
                           1 
                           rest 
                         
                       
                       
                         M 
                         1 
                         rest 
                       
                     
                     ) 
                   
                   tonic 
                 
               
               + 
               
                 4 
                 × 
                 
                   
                     ( 
                     
                       
                         
                           M 
                           1 
                           act 
                         
                         - 
                         
                           M 
                           1 
                           rest 
                         
                       
                       
                         M 
                         1 
                         rest 
                       
                     
                     ) 
                   
                   phasic 
                 
               
             
           
         
       
     
     Full antagonism of presynaptic M2R increases free Ach by 251% in a neuromuscular junction [73]. Using the generic receptor competition model, the amount of M2R inhibition can be calculated for a given drug at a certain dose. Using the generic receptor competition model with α4β2nAChR postsynaptic receptors, the effect of such an increase of free Ach on the amount of receptor activation can be calculated as A-α4β2. 
     Using experimental data between galantamine concentration, nAChR activation and increase in Glu current (Santos 2002, Markus 2003), we arrive at the following relationship 
       ΔGlu current =0.103×Δ n AChR−0.00269
 
     This allows us to calculate changes in gAMPA, gNMDA. 
     Similarly for GABA mediated IPSC 
       ΔIPSC GABA =0.798 ×Δn AChR+0.326
 
     In the following table, Actrecdrug is the amount of nACHR activation in the presence of drug (possibly downstream of Acetylcholinesterase inhibition or presynaptic M2 blockade) and Actreccon is the activation level in no drug conditions. 
                     TABLE 7                  Effect of cholinergic and 5-HT3 receptor modulation on key conductances in the       working memory model                                     Receptor   M1   M2   a4b2nAChR   A7 nACHR   5-HT3               Effect   Δ MP =   Δ MP =   dIPSC =   d-Glucurr =   (100 −           −5 * tonic   −5 * tonic inh +   0.326 + 0.798 *   −0.00269 + 0.103 *   Act)/2.5           inh +   4 * phasic   (Actrecdrug −   (Actrecdrug −           4 * phasic   inh;   Actreccon)/   Actreccon)/           inh;       Acrreccon;   Acrreccon;       g-NMDA               gNMDA = 0.00346 + 0.181                       d-Glucurr;       g-AMPA-               gAMPA = 0.0058 +   g′ = g * (1 + Effect                       0.6913 * d-Glucurr       g-GABA           gGABA = 0.33 + 0.08                   d-IPSC                   (interneuron)       Pyr-gK   =0.3 * D   =0.3 * D           MP   MP                    
Individual changes of all ion-channels in the apical distal compartment of the pyramidal cell are given in the following table
 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Effect of dopaminergic, serotonergic and adrenergic G-PCR modulation on key 
               
               
                 conductances in the pyramidal apical-distal compartment in the working memory circuit. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 Default 
                   
                   
                   
                   
               
               
                 Regio 
                 Receptor 
                 values 
                 D1 
                 5HT1A 
                 5HT2A 
                 alfa2A 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Apical- 
                 gNaf 
                 0.028 
                   
                 Naf′ = Naf ** Eff 
                 Naf′ = 
                   
               
               
                 Distal 
                   
                   
                   
                   
                 Naf * effect 
               
               
                   
                 gNap 
                 0 
                   
                 Nap′ = 
                 Nap′ = 
               
               
                   
                   
                   
                   
                 Nap * Effect 
                 Nap * Effect 
               
               
                   
                 gHva 
                 0.00034 
                 g′ = g * (1 − 
                 gHVa′ = 
               
               
                   
                   
                   
                 0.5 * Effect) 
                 gHVa * Effect 
               
               
                   
                 gKdr 
                 0.0092 
               
               
                   
                 glks 
                 0.00024 
                 g′ = g * (1 − 
               
               
                   
                   
                   
                 0.5 * Effect) 
               
               
                   
                 gCa 
                 0.0022 
                   
                 gCa′ = gCa * Effect 
                 gCa′ = gCa * Effect 
               
               
                   
               
            
           
         
       
     
     Individual changes of all ion-channels in the apical proximal compartment of the pyramidal cell are given in the following table: 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Effect of dopaminergic, serotonergic and adrenergic G-PCR modulation on key 
               
               
                 conductances in the pyramidal apical-proximalz compartment in the working memory circuit. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 Default 
                   
                   
                   
                   
               
               
                 Regio 
                 Receptor 
                 values 
                 D1 
                 5HT1A 
                 5HT2A 
                 alfa2A 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Apical- 
                 gNaf 
                 0.028 
                   
                 Naf′ = Naf ** Eff 
                   
                   
               
               
                 proximal 
               
               
                   
                 gNap 
                 0.001 
                   
                 Nap′ = Nap * Effect 
               
               
                   
                 gHva 
                 0.0007 
                 g′ = g * (1 − 
                 gHVa′ = gHVa * Effect 
               
               
                   
                   
                   
                 0.5 * Effect) 
               
               
                   
                 gKdr 
                 0.0092 
               
               
                   
                 glks 
                 0.00024 
                 g′ = g * (1 − 
               
               
                   
                   
                   
                 0.5 * Effect) 
               
               
                   
                 gCa 
                 0.0038 
                   
                 gCa′ = gCa * Effect 
               
               
                   
               
            
           
         
       
     
     Individual changes of all ion-channels in the basal compartment of the pyramidal cell are given in the following table: 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Effect of dopaminergic, serotonergic and adrenergic G-PCR modulation on key 
               
               
                 conductances in the pyramidal basal-pyramidal compartment in the working memory circuit. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 Default 
                   
                   
                   
                   
               
               
                 Regio 
                 Receptor 
                 values 
                 D1 
                 5HT1A 
                 5HT2A 
                 alfa2A 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Basal- 
                 gNaf 
                 0.028 
                   
                 Naf′ = Naf ** Eff 
                   
                   
               
               
                 pyramidal 
               
               
                   
                 gNap 
                 0.001 
                   
                 Nap′ = Nap * Effect 
               
               
                   
                 gHva 
                 0.0007 
                 g′ = g * (1 − 
                 gHVa′ = gHVa * Effect 
               
               
                   
                   
                   
                 0.5 * Effect) 
               
               
                   
                 gKdr 
                 0.0092 
               
               
                   
                 glks 
                 0.00024 
                 g′ = g * (1 − 
               
               
                   
                   
                   
                 0.5 * Effect) 
               
               
                   
                 gCa 
                 0.0038 
                   
                 gCa′ = gCa * Effect 
               
               
                 Basal- 
                 gNaf 
                 0.028 
                   
                 Naf′ = Naf ** Eff 
               
               
                 pyramidal 
               
               
                   
               
            
           
         
       
     
     Individual changes of all ion-channels in the soma compartment of the pyramidal cell are given in the following table: 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Effect of dopaminergic, serotonergic and adrenergic G-PCR modulation on key 
               
               
                 conductances in the pyramidal somatic compartment in the working memory circuit. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 Default 
                   
                   
                   
                   
               
               
                 Regio 
                 Receptor 
                 values 
                 D1 
                 5HT1A 
                 5HT2A 
                 alfa2A 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Soma- 
                 gNaf 
                 0.086 
                   
                 Naf′ = Naf ** Eff 
                   
                   
               
               
                 pyramidal 
               
               
                   
                 gNap 
                 0.0022 
                   
                 Nap′ = Nap * Effect 
               
               
                   
                 gHva 
                 0.00034 
                 g′ = g * (1 − 
                 gHVa′ = gHVa * Effect 
               
               
                   
                   
                   
                 0.5 * Effect) 
               
               
                   
                 gKdr 
                 0.0338 
               
               
                   
                 glks 
                 0.00014 
                 g′ = g * (1 − 
               
               
                   
                   
                   
                 0.5 * Effect) 
               
               
                   
                 gCa 
                 0.0022 
                   
                 gCa′ = gCa * Effect 
               
               
                   
                 gNaf 
                 0.086 
                   
                 Naf′ = Naf ** Eff 
               
               
                   
               
            
           
         
       
     
     Similarly, conductance changes in the two GABA compartments are given in the following table: 
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 Effect of dopaminergic, serotonergic and adrenergic G-PCR modulation 
               
               
                 on key conductances in the two GABAergic compartment in the working 
               
               
                 memory circuit. 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 Default 
                   
                   
                   
                   
               
               
                 Regio 
                 Receptor 
                 values 
                 D1 
                 5HT1A 
                 5HT2A 
                 alfa2A 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 GABA 
                 gCa 
                 0 
                   
                   
                   
                   
               
               
                 dendrite 
               
               
                   
                 gNa 
                 0.02 
               
               
                   
                 gK 
                 .0.008 
               
               
                 GABA 
                 gCa 
                 0 
               
               
                 soma 
                 gNa 
                 0.1 
               
               
                   
                 gK 
                 0.04 
                   
                   
                   
                 gK′ = gK(1 − 
               
               
                   
                   
                   
                   
                   
                   
                 Effect) 
               
               
                 GABA 
                 gCa 
                 0 
               
               
                 dendrite 
               
               
                   
               
            
           
         
       
     
     Validation of the Working Memory Model 
     The model is validated using the correlation between the outcome of amisulpride, haldol, olanzapine, guanfacine and risperidone on Continuous Performance Test (CPT) test reaction time and their respective outcomes in the mathematical model of working memory ( FIG. 14 ). AcCorrelation between clinical outcome on a CPT test for four different neuroleptics (7 points) and their respective effects on working memory in the Working Memory circuit is shown. The drugs used are amisulpride, haldol, olanzapine, risperidone and guanfacine. The p-value is 0.027. This shows that a reasonable correlation can be achieved between the outcome of the computer model and the clinical scales. 
     G. Applications of the Simulations 
     Target Identification 
       FIG. 15  shows the conceptual approach to use the reverse approach in order to identify a pharmacological profile which results in a much better outcome on clinical scales than the state-of-the art current treatment. Reverse application of the model allows for identifying the pharmacological profile resulting in a substantially better clinical outcome than current standard of care. After validating the model by achieving a strong correlation between computer model outcome and clinical scales (1), a systematic search over all dimensions of receptor activations is performed to find the best clinical outcome (2). The profiles are tested against EPS side-effects and are ranked according to their performance (3). Then a search for affinity constants and functional properties reveal the actual profile of the ideal drug (4), which will be confirmed using the generic receptor competition model followed by the different modules (5). 
     A systematic probing of the parameter space of receptor activation levels enables to rank order putative receptor profiles in order of their impact on anticipated clinical scales. The EPS module is used as a filter to eliminate candidate profiles which are anticipated to show adverse motor-effects. Using this approach we identified four receptors which contribute most to the beneficial effect and which therefore constitute an ideal profile. Such a profile can then be used in Drug discovery efforts to search for small molecules, whose effect on the receptors resembles very closely this ideal profile. Obviously there will be different possibilities and the simulation platform will enable them to rank order them in terms of clinical benefit and possible motor side-effects. This kind of evaluation will be helpful to select the best candidate drug(s) for further development. 
     Comedication 
     Another possible application of the simulation platform is the issue of comedications, i.e. what combination of drugs gives the best results. This is helpful in supporting decision aspects of polypharmacy, which can be a public medical health problem. As an example, we studied the possible combination therapy of a glycine-transporter T1 inhibitor with an existing neuroleptic. Briefly, the model then incorporates the pharmacological effects of the combination therapy at all levels, i.e. receptor competition model, EPS, SN module and working memory circuit. The results showed that the best order would be ziprasidone 90 mg&gt;paliperidone 12 mg&gt;risperidone 6 mg&gt;quetiapine 600 mg&gt;olanzapine 15 mg&gt;clozapine 500 mg in terms of additional gains on the PANSS total. With regard to the PANSS negative, the rank order is rsiperidone 6 mg&gt;paliperidone 12 mg&gt;ziprasidone 90 mg&gt;quetiapine 600 mg&gt;olanzapine 15 mg&gt;clozapine 500 mg. 
     Power Analysis and Virtual Patient Trial 
     A virtual patient trial can be simulated using the variability of the drug plasma levels, obtained in human volunteers or patients, combined with the inherent variability of the brain configuration as outlined in Table 4. Briefly, a virtual patient is defined from a sample taken from the probability curve of plasma variability (for example the olanzapine PK profile has been described in Callaghan [18]) and independently from the probability functions of all the receptor densities involved in the simulation modules. The effect of a particular antipyschotic is calculated and the combined outcomes for all the virtual patients constitute a database from which using classical statistical analyses, power estimates of the number of patients needed to show an effect of p&lt;α with a power of β can be derived. As an example, using the data on the variability of an investigative glycine transporter inhibitor combined with olanzapine, it has been shown that it will take 328 patients to show a difference between olanzapine and the combination treatment with a p-value&lt;0.05 at a power of 0.80. 
     Effect of Genotype on Clinical Effect 
     In some cases, the functional effect of a certain genotype is known. An example in point is the Val159Met polymorphism of the human Catechol-O-methyl transferase (COMT) gene, which leads to an twofold increase in stability at body temperature for the Val/Val form compared to the Met/Met isoform (for a review see Weinberger [104]). As this enzyme is implicated in the degradation of catecholamines, notably dopamine and norepineprhine in the prefrontal cortex, but not in the striatum, it has a substantial effect on the half-life or residence time of these neurotransmitters, especially in the working memory circuit. The simulation allows incorporating these effects by changing the half-life of dopamine and norepinephrine in the generic receptor competition model, which leads to a change in postsynaptic receptor activation. As an example we have studied the effect of the COMT Val158/Met polymorphism on the working memory performance of olanzapine. The information content, a measure of the complexity of the signal (see above) and proportional to working memory span, increases from 246.6+/−8.6 bit/sec (average+/−SE) in the VV genotype to 260.8+/−8.9 bit/sec in the MV genotype and to 263.8+/−8.7 bit/sec in the MM genotype. These results are in line with clinical results phrenia patients [8]. 
     Chronopharmacodynamics 
     In certain conditions, the circadian rhythms of the properties (such as receptor densities and neurotransmitter dynamics) are known in preclinical models. In such cases, this information can be incorporated into the parameter set of the various simulation modules, to assess the effect of these circadian rhythms on the effect of pharmacological agents. Such information can be used together with the known pharmacokinetic profile of the investigative agent to obtain the best time for drug application and clinical assessment. 
     Circadian rhythms have been described for the density of striatal D2R which follows a 24 hr cycle and for density of the vesicle Glutamate Transporter 1 (VGLUT), which follows a 12 hr cycle [108]. For the D2R fluctuation, the mRNA is about 30% higher during the wake phase than during the sleep phase [103]. Also, clinical data suggest that patients on a steady Haldol therapy have higher Parkinsonian tremor and EPS when probed in the morning [96], while dyskinesia was worse in the afternoon [47]. In case of the VGLUT, the fluctuation with a maximal change of 25% is dependent upon the clock gene Period2, as the circadian rhythm is profoundly disturbed in a Period2 KO mouse model. 
     When applying this information to the known pharmacokinetic profile of risperidone, it turns out that the dynamic range of estimated PANSS total clinical scale, is only one point. The best effects in terms of benefit-risk (PANSS-EPS) ratio are seen when the drug is given in the evening and the clinical assessment is done in the late morning. In contrast, with olanzapine the dynamic range over a 24 hour period is much bigger (4 points on the PANSS total scale). 
     While the invention has been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and/or claimed. The present invention is directed to each individual feature, system, material and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials and/or methods, if such features, systems, articles, materials and/or methods are not mutually inconsistent, is included within the scope of the present invention. 
     All definitions as used herein are solely for the purposes of this disclosure. These definitions should not necessarily be imputed to other commonly-owned patents and/or patent applications, whether related or unrelated to this disclosure. The definitions, as used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms. 
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