bluuebunny/crossref_metadata_2025 huggingface dataset:
Generalization to Local Remappings of the Visuomotor Coordinate Transformation
Authors: Ghahramani Zoubin , Wolpert Daniel M. , Jordan Michael I.
Published: April 2018

During visually guided movement, visual representations of target location must be transformed into coordinates appropriate for movement. To investigate the representation and plasticity of the visuomotor coordinate transformation, we examined the changes in pointing behavior after local visuomotor remappings. The visual feedback of finger position was limited to one or two locations in the workspace, at which a discrepancy was introduced between the actual and visually perceived finger position. These remappings induced changes in pointing, which were largest near the locus of remapping and decreased away from it. This pattern of spatial generalization highly constrains models of the computation of the visuomotor transformation in the CNS. A simple model, in which the transformation is computed via the population activity of a set of units with large sensory receptive fields, is shown to capture the observed pattern.
Local Opioid Withdrawal in Rat Single Periaqueductal Gray NeuronsIn Vitro
Authors: Chieng Billy , Christie MacDonald J.
Published: April 2018

Opioid dependence in ventrolateral periaqueductal gray (PAG) neurons was studied by using intracellular recordings from brain slices. In slices from morphine-dependent rats maintained in morphine (5 μm)in vitro, action potential frequencies of opioid-sensitive neurons did not differ from untreated control neurons but were greater than in control neurons maintained in morphinein vitro, indicating development of tolerance. Naloxone (100 nmor 1 μm) depolarized 25 of 51 neurons from morphine-dependent rats maintained in morphinein vitro, 19 of which previously had been classified as opioid-sensitive. Action potential frequencies in the presence of naloxone were greater than in control neurons in the absence of opioids, as well as in control neurons in the presence of both morphine and naloxone, demonstrating opioid withdrawal. In slices from control animals, opioid-induced hyperpolarizations and naloxone-induced depolarizations (in the presence of morphine) reversed polarity near expectedEK(−111 ± 3 mV and −113 ± 3 mV, respectively). In contrast, the reversal potential of naloxone-induced depolarizations was more negative than expected in neurons from dependent animals (−143 ± 9 mV), indicating that the depolarization was not attributable simply to antagonism of a K-conductance increase. Naloxone-induced depolarizations were not inhibited by tetrodotoxin (1 μm), bicuculline (30 μm), 6-cyano-7-nitroquinoxaline-2,3-dione (10 μm), or prazosin (300 nm), suggesting no involvement of major synaptic neurotransmitters. Clonidine (1 μm) and baclofen (30 μm) overcame naloxone-induced depolarizations. These results demonstrate development of both tolerance and withdrawal in PAG neurons and suggest induction of a novel opioid-sensitive current that could be involved in withdrawal behavior.
Physiological Mapping of Local Inhibitory Inputs to the Hypothalamic Paraventricular Nucleus
Authors: Boudaba Cherif , Szabó Kriszta , Tasker Jeffrey G.
Published: April 2018

Local inhibitory synaptic inputs to neurons of the rat hypothalamic paraventricular nucleus (PVN) were studied by using glutamate microstimulation and conventional intracellular and whole-cell patch-clamp recordings in coronal, horizontal, and parasagittal slices of rat hypothalamus. PVN cells were classified as magnocellular or parvocellular neurons on the basis of electrophysiological andpost hocimmunohistochemical analyses; GABA-producing neurons were localized within situhybridization. Glutamate microstimulation of different sites around the PVN evoked volleys of postsynaptic potentials in 43% of the PVN neurons tested. Some responses to stimulation at each site were blocked by bicuculline, suggesting that they were mediated by the activation of presynaptic GABA neurons. In the coronal plane, presynaptic inhibitory sites were located lateral to the PVN and ventral to the fornix, corresponding to the lateral hypothalamic area and the posterior bed nucleus of the stria terminalis (BNST). In the horizontal plane, presynaptic inhibitory sites were found rostral, lateral, and caudal to the nucleus, corresponding to parts of the anterior hypothalamic area, the posterior BNST, the medial preoptic area, and the dorsomedial hypothalamus. In the parasagittal plane, presynaptic inhibitory neurons were revealed at sites rostral and caudal to the nucleus, corresponding to the medial preoptic area and the dorsomedial hypothalamus, and in a site dorsal to the optic chiasm that included the suprachiasmatic nucleus. These presynaptic sites each contained GABA-producing neurons based onin situhybridization with a glutamic acid decarboxylase riboprobe and together formed a three-dimensional ring around the PVN. Unexpectedly, both magnocellular and parvocellular neurons received inhibitory synaptic inputs from common sites.
Methylmalonyl-CoA Mutase Induction by Cerebral Ischemia and Neurotoxicity of the Mitochondrial Toxin Methylmalonic Acid
Authors: Narasimhan Purnima , Sklar Robert , Murrell Matthew , Swanson Raymond A. , Sharp Frank R.
Published: April 2018

Differential screening of gerbil brain hippocampal cDNA libraries was used to search for genes expressed in ischemic, but not normal, brain. The methylmalonyl-CoA mutase (MCM) cDNA was highly expressed after ischemia and showed a 95% similarity to mouse and 91% similarity to the human MCM cDNAs. Transient global ischemia induced a fourfold increase in MCM mRNA on Northern blots from both hippocampus and whole forebrain. MCM protein exhibited a similar induction on Western blots of gerbil cerebral cortex 8 and 24 hr after ischemia. Treatment of primary brain astrocytes with either the branched-chain amino acid (BCAA) isoleucine or the BCAA metabolite, propionate, induced MCM mRNA fourfold. Increased concentrations of BCAAs and odd-chain fatty acids, both of which are metabolized to propionate, may contribute to inducing the MCM gene during ischemia.

Methylmalonic acid, which is formed from the MCM substrate methylmalonyl-CoA and which inhibits succinate dehydrogenase (SDH), produced dose-related cell death when injected into the basal ganglia of adult rat brain. This neurotoxicity is similar to that of structurally related mitochondrial SDH inhibitors, malonate and 3-nitropropionic acid. Methylmalonic acid may contribute to neuronal injury in human conditions in which it accumulates, including MCM mutations and B12deficiency. This study shows that methylmalonyl-CoA mutase is induced by several stresses, including ischemia, and would serve to decrease the accumulation of an endogenous cellular mitochondrial inhibitor and neurotoxin, methylmalonic acid.
Intrahypothalamic Implantation of Progesterone in Castrated Male Whiptail Lizards (Cnemidophorus inornatus) Elicits Courtship and Copulatory Behavior and Affects Androgen Receptor- and Progesterone Receptor-mRNA Expression in the Brain
Authors: Crews David , Godwin John , Hartman Vesta , Grammer Michael , Prediger Ellen A. , Sheppherd Rebecca
Published: April 2018

A primary tenet of behavioral neuroendocrinology is that gonadal steroid hormones act on limbic nuclei to activate mating behavior in vertebrates. Traditionally, research has focused on the regulation of male-typical sexual behavior by testicular androgens and female-typical sexual behavior by ovarian estrogen and progesterone. Indeed, progesterone generally is regarded as an antiandrogen, acting centrally to inhibit sexual behavior in males. However, experiments with lizards, and more recently with rats, have challenged this paradigm. For example, exogenous progesterone induces mating behavior in some, but not all, castrated male whiptail lizards. The present study determined that implantation of progesterone into the anterior hypothalamus preoptic area of castrated, progesterone-sensitive males completely restored sexual behavior but failed to elicit sexual activity in castrated, progesterone-insensitive males. Further, androgen receptor- and progesterone receptor-mRNA expression in specific brain regions was significantly different in progesterone-sensitive versus progesterone-insensitive animals. Progesterone-sensitive males showed significantly higher relative abundance of androgen receptor-mRNA in the preoptic area, amygdala, and lateral septum, as compared with progesterone-insensitive animals receiving the same treatment. In contrast, progesterone receptor-mRNA abundance was lower in preoptic area of progesterone-sensitive males than in progesterone-insensitive males. No differences were found in the baseline abundance of androgen receptor- or progesterone receptor-mRNA in these nuclei between control groups of progesterone-sensitive and progesterone-insensitive males who were castrated but not implanted. This suggests that progesterone differentially regulates its own receptor as well as androgen receptor in areas of the brain involved in the control of sexual behavior of males and that the nature of this regulation shows individual variability.
Different Balance of Excitation and Inhibition in Forward and Feedback Circuits of Rat Visual Cortex
Authors: Shao Zhengwei , Burkhalter Andreas
Published: April 2018

Different cortical areas are linked reciprocally via forward and feedback connections. Forward connections are involved in the representation of retinal images, whereas feedback pathways may play a role in the selection and interpretation of visual information. To examine the synaptic mechanisms of forward and feedback connections between primary and secondary visual cortical areas directly, we have performed intracellular recordings in slices of rat visual cortex. Irrespective of stimulus intensity and membrane potential, 78% (45/58) of the cells in striate cortex activated by feedback input showed monosynaptic responses that were depolarizing only, and inhibitory inputs were evident merely as a slight acceleration in the decay of EPSPs. In contrast, in 89% (17/19) of the cells, stimulation of forward input evoked monosynaptic excitatory postsynaptic potentials (EPSPs), followed by disynaptic, hyperpolarizing inhibitory postsynaptic potentials (IPSPs). EPSPs followed by IPSPs also were recorded after stimulation of local connections within primary visual cortex (92%, 12/13) and after activation of thalamocortical input (91%, 10/11). These results suggest that the synaptic organization of feedback connections are distinct from forward, local, and thalamocortical circuits. The findings further indicate that intracortical back projections exert modulatory influences via synaptic mechanisms in which weak inhibitory input is strongly dominated by excitation.
Enhanced Amphetamine Sensitivity and Increased Expression of Dopamine D2 Receptors in Postpubertal Rats after Neonatal Excitotoxic Lesions of the Medial Prefrontal Cortex
Authors: Flores Gonzalo , Wood Graham K. , Liang Jin-Jun , Quirion Remi , Srivastava Lalit K.
Published: April 2018

Functional and structural abnormalities in the medial prefrontal cortex (MPFC) and overactive dopamine (DA) neurotransmission are thought to be the key pathologies in schizophrenia. To understand the role of MPFC in the pre- and postpubertal development of the subcortical DA system, the effects of neonatal [postnatal day 7 (PD7)] MPFC excitotoxic lesions on locomotor behaviors and the expression of DA receptor subtypes and DA transporter were investigated in Sprague Dawley rats at PD35 and PD56, respectively. No significant differences in the novelty ord-amphetamine-induced locomotion were observed between sham-operated and ibotenic acid-lesioned rats at PD35. Postpubertally (at PD56), however, the locomotor activity of lesioned rats in the novel environment and afterd-amphetamine administration was enhanced significantly compared with controls. The expressions of DA D1, D2, D3, and D4 receptors and DA transporter were then estimated in MPFC-lesioned and sham-operated rats at PD39 and PD60. The levels of DA D2 receptors, measured using [3H]-YM-09151-2 binding, and its mRNA byin situhybridization, were observed to be significantly increased at PD60 in striatal and limbic areas of lesioned rats. Levels of other DA receptor subtypes were not significantly affected at any time points. Lesioned rats at PD39 show a small increase in DA transporter level in the shell of nucleus accumbens; however, this effect seems to wear off at PD60. The data suggest that neonatal MPFC lesions may alter the functional development and maturation of mesolimbic/nigrostriatal DA systems in that neonatally lesioned rats grow into a behavioral/neurochemical deficit.
Amyloid β Peptide of Alzheimer’s Disease Downregulates Bcl-2 and Upregulates Bax Expression in Human Neurons
Authors: Paradis Eric , Douillard Hélène , Koutroumanis Maria , Goodyer Cynthia , LeBlanc Andréa
Published: April 2018

Neuronal apoptosis is a suspected cause of neurodegeneration in Alzheimer’s disease (AD). Increased levels of amyloid β peptide (Aβ) induce neuronal apoptosisin vitroandin vivo. The underlying molecular mechanism of Aβ neurotoxicity is not clear. The normal concentration of Aβ in cerebrospinal fluid is 4 nm. We treated human neuron primary cultures with 100 nmamyloid β peptides Aβ1–40and Aβ1–42and the control reverse peptide Aβ40–1. We find that although little neuronal apoptosis is induced by either peptide after 3 d of treatment, Aβ1–42provokes a rapid and sustained downregulation of a key anti-apoptotic protein, bcl-2, whereas it increases levels of bax, a protein known to promote cell death. In contrast, the Aβ1–40downregulation of bcl-2 is gradual, although the levels are equivalent to those of Aβ1–42-treated neurons by 72 hr of treatment. Aβ1–40does not upregulate bax levels. The control, reverse peptide Aβ40–1, does not affect either bcl-2 or bax protein levels. In addition, we found that the Aβ1–40- and Aβ1–42- but not Aβ40–1-treated neurons had increased vulnerability to low levels of oxidative stress. Therefore, we propose that although high physiological amounts of Aβ are not sufficient to induce apoptosis, Aβ depletes the neurons of one of its anti-apoptotic mechanisms. We hypothesize that increased Aβ in individuals renders the neurons vulnerable to age-dependent stress and neurodegeneration.
Expression and Subunit Interaction of Voltage-Dependent Ca2+Channels in PC12 Cells
Authors: Liu Hongyan , Felix Ricardo , Gurnett Christina A. , De Waard Michel , Witcher Derrick R. , Campbell Kevin P.
Published: April 2018

Nerve growth factor (NGF)-induced differentiation in PC12 cells is accompanied by changes in the expression of voltage-dependent Ca2+channels. Ca2+channels are multimeric complexes composed of at least three subunits (α1, β, and α2δ) and are involved in neuronal migration, gene expression, and neurotransmitter release. Although attempts have been undertaken to elucidate NGF regulation of Ca2+channel expression, the changes in subunit composition of these channels during differentiation still remain uncertain. In the present study, patch-clamp recordings show that in addition to the previously documented L-type and N-type Ca2+currents, undifferentiated PC12 cells also express an ω-agatoxin-IVA-sensitive (P/Q-type) component. In addition, the corresponding mRNA encoding the pore-forming α1subunits for these channels (C, B, and A, respectively) was detected. Likewise, mRNA for three distinct auxiliary β subunits (1, 2, 3) were also found, β3protein being dominantly expressed. Immunoprecipitation experiments show that the N-type Ca2+channel is associated with either a β2or β3subunit and that NGF increases the channel expression without affecting its β subunit association. These results (1) indicate that the diversity of Ca2+currents in PC12 cells arise from the expression of three distinct α1and three different β subunit genes; (2) support a model for heterogenous β subunit association of the N-type Ca2+channel in a single cell type; and (3) suggest that the regulation of the N-type Ca2+channel during NGF-mediated differentiation involves an increase in the number of functional channels with no apparent changes in subunit composition.
Emergence of Activity-Dependent, Bidirectional Control of Microtubule-Associated Protein MAP2 Phosphorylation during Postnatal Development
Authors: Quinlan Elizabeth M. , Halpain Shelley
Published: April 2018

Pronounced changes in neuronal morphology occur as synapses mature; however, little is known about how synaptic transmission regulates the developing neuronal cytoskeleton. The postsynaptic, microtubule-associated protein MAP2 is a target of multiple, calcium-dependent signaling pathways activated by synaptic transmission. Here we demonstrate that MAP2 phosphorylation is differentially regulated across development. In32P-labeled hippocampal slices prepared from adult rats, depolarization stimulated a bidirectional change in the phosphorylation of immunoprecipitated MAP2. A transient increase was mediated by metabotropic glutamate receptors (mGluRs) and stimulation of mitogen-activated protein kinases (MAPKs), Ca2+/calmodulin-dependent protein kinases (CaMKs), and protein kinase C (PKC). This increase was followed by a persistent dephosphorylation mediated by NMDA receptors and activation of protein phosphatase 2B (PP2B or calcineurin). In contrast, depolarization of neonatal hippocampal slices stimulated exclusively a net increase in MAP2 phosphorylation, which was attenuated by inhibitors of MAPKs, but not CaMKs or PKC. Furthermore, although incubation in NMDA induced a time-dependent decrease in MAP2 phosphorylation in both adults and neonates, this effect was both less robust and less sensitive to calcineurin inhibitors in neonates than in adults. These data indicate that the mechanisms coupling glutamate release to MAP2 dephosphorylation are relatively lacking in the neonatal hippocampus. Highly phosphorylated MAP2 is impaired in its ability to stabilize microtubules and actin filament bundlesin vitro. The neonatal propensity toward glutamate-stimulated MAP2 phosphorylation may serve to reduce cytoskeletal stability and permit dendritic arborization early in postnatal development. In mature neurons, the bidirectional control of MAP2 phosphorylation may participate in activity-dependent synaptic remodeling.
Effects of Binge Pattern Cocaine Administration on Dopamine D1and D2Receptors in the Rat Brain: AnIn VivoStudy Using Positron Emission Tomography
Authors: Tsukada Hideo , Kreuter Jason , Maggos Christopher E. , Unterwald Ellen M. , Kakiuchi Takeharu , Nishiyama Shingo , Futatsubashi Masami , Kreek Mary Jeanne
Published: April 2018

The aim of the present study was to determine the effect of “binge” pattern cocaine administration on dopamine D1and D2receptors in the rat brain. Male Sprague Dawley rats were injected three times at 1 hr intervals with saline or cocaine (15 mg/kg) each day for 2, 7, or 14 d. Thein vivobinding of [11C]SCH23390 (dopamine D1receptor antagonist) and [11C]N-methylspiperone (NMSP; dopamine D2receptor antagonist) in the striatal region was measured by a high-resolution positron emission tomography at 1 and 3.5 hr, respectively, after the last cocaine or saline injection. Acute (2 d) binge cocaine administration did not change thein vivobinding potential of [11C]SCH23390 or the binding of [11C]NMSP in the striatum. After 7 d of binge cocaine administration, a significant decrease in the binding potential of [11C]SCH23390 was observed, whereas no change in the binding of [11C]NMSP was found. After 14 d of binge cocaine administration, thein vivobinding was significantly reduced for both [11C]SCH23390 and [11C]NMSP. Separate saturation experiments indicated that the observed alterations ofin vivobinding were attributable mainly to apparent alterations in the affinity and not the number of binding sites. These results suggest that both dopamine D1and D2receptors may have altered physiologically available binding sites after binge pattern cocaine administration.
Cortical Systems for the Recognition of Emotion in Facial Expressions
Authors: Adolphs Ralph , Damasio Hanna , Tranel Daniel , Damasio Antonio R.
Published: April 2018

This study is part of an effort to map neural systems involved in the processing of emotion, and it focuses on the possible cortical components of the process of recognizing facial expressions. We hypothesized that the cortical systems most responsible for the recognition of emotional facial expressions would draw on discrete regions of right higher-order sensory cortices and that the recognition of specific emotions would depend on partially distinct system subsets of such cortical regions. We tested these hypotheses using lesion analysis in 37 subjects with focal brain damage. Subjects were asked to recognize facial expressions of six basic emotions: happiness, surprise, fear, anger, disgust, and sadness. Data were analyzed with a novel technique, based on three-dimensional reconstruction of brain images, in which anatomical description of surface lesions and task performance scores were jointly mapped onto a standard brain-space. We found that all subjects recognized happy expressions normally but that some subjects were impaired in recognizing negative emotions, especially fear and sadness. The cortical surface regions that best correlated with impaired recognition of emotion were in the right inferior parietal cortex and in the right mesial anterior infracalcarine cortex. We did not find impairments in recognizing any emotion in subjects with lesions restricted to the left hemisphere. These data provide evidence for a neural system important to processing facial expressions of some emotions, involving discrete visual and somatosensory cortical sectors in right hemisphere.
Kinetics, Ca2+Dependence, and Biophysical Properties of Integrin-Mediated Mechanical Modulation of Transmitter Release from Frog Motor Nerve Terminals
Authors: Chen Bo-Ming , Grinnell Alan D.
Published: April 2018

Neurotransmitter release from frog motor nerve terminals is strongly modulated by change in muscle length. Over the physiological range, there is an ∼10% increase in spontaneous and evoked release per 1% muscle stretch. Because many muscle fibers do not receive suprathreshold synaptic inputs at rest length, this stretch-induced enhancement of release constitutes a strong peripheral amplifier of the spinal stretch reflex. The stretch modulation of release is inhibited by peptides that block integrin binding of natural ligands. The modulation varies linearly with length, with a delay of no more than ∼1-2 msec and is maintained constant at the new length. Moreover, the stretch modulation persists in a zero Ca2+Ringer and, hence, is not dependent on Ca2+influx through stretch activated channels. Eliminating transmembrane Ca2+gradients and buffering intraterminal Ca2+to approximately normal resting levels does not eliminate the modulation, suggesting that it is not the result of release of Ca2+from internal stores. Finally, changes in temperature have no detectable effect on the kinetics of stretch-induced changes in endplate potential (EPP) amplitude or miniature EPP (mEPP) frequency. We conclude, therefore, that stretch does not act via second messenger pathways or a chemical modification of molecules involved in the release pathway. Instead, there is direct mechanical modulation of release. We postulate that tension on integrins in the presynaptic membrane is transduced mechanically into changes in the position or conformation of one or more molecules involved in neurotransmitter release, altering sensitivity to Ca2+or the equilibrium for a critical reaction leading to vesicle fusion.
Neuronal Regulation of Glutamate Transporter Subtype Expression in Astrocytes
Authors: Swanson Raymond A. , Liu Jialing , Miller Johann W. , Rothstein Jeffrey D. , Farrell Kevin , A. Stein Becky , Longuemare Maria C.
Published: April 2018

GLT-1, GLAST, and EAAC1 are high-affinity, Na+-dependent glutamate transporters identified in rat forebrain. The expression of these transporter subtypes was characterized in three preparations: undifferentiated rat cortical astrocyte cultures, astrocytes cocultured with cortical neurons, and astrocyte cultures differentiated with dibutyryl cyclic AMP (dBcAMP). The undifferentiated astrocyte monocultures expressed only the GLAST subtype. Astrocytes cocultured with neurons developed a stellate morphology and expressed both GLAST and GLT-1; neurons expressed only the EAAC1 transporter, and rare microglia in these cultures expressed GLT-1. Treatment of astrocyte cultures with dBcAMP induced expression of GLT-1 and increased expression of GLAST. These effects of dBcAMP on transporter expression were qualitatively similar to those resulting from coculture with neurons, but immunocytochemistry showed the pattern of transporter expression to be more complex in the coculture preparations. Compared with astrocytes expressing only GLAST, the dBcAMP-treated cultures expressing both GLAST and GLT-1 showed an increase in glutamate uptakeVmax, but no change in the glutamateKmand no increased sensitivity to inhibition by dihydrokainate. Pyrrolidine-2,4-dicarboxylic acid andthreo-β-hydroxyaspartic acid caused relatively less inhibition of transport in cultures expressing both GLAST and GLT-1, suggesting a weaker effect at GLT-1 than at GLAST. These studies show that astrocyte expression of glutamate transporter subtypes is influenced by neurons, and that dBcAMP can partially mimic this influence. Manipulation of transporter expression in astrocyte cultures may permit identification of factors regulating the expression and function of GLAST and GLT-1 in their native cell type.
N-Ethylmaleimide Blocks Depolarization-Induced Suppression of Inhibition and Enhances GABA Release in the Rat Hippocampal SliceIn Vitro
Authors: Morishita Wade , Kirov Sergei A. , Pitler Thomas A. , Martin Laura A. , Lenz Robert A. , Alger Bradley E.
Published: April 2018

Regulation of synaptic, GABAAreceptor-mediated inhibition is a process of critical importance to normal brain function. Recently, we have described a phenomenon in hippocampus of a transient, yet marked, decrease in spontaneous, GABAAreceptor-mediated IPSCs after depolarization activated Ca2+influx into a pyramidal cell. This process, depolarization-induced suppression of inhibition (DSI), is absent in hippocampal cells that previously had been exposed to pertussis toxinin vivo, implicating a G-protein in the DSI process. To circumvent the problem that a single cell cannot be studied before and after G-protein block using the pertussis toxin pretreatment method, we have used the sulfhydryl alkylating agentN-ethylmaleimide (NEM), which blocks pertussis toxin-sensitive G-proteins, to determine whether acute inhibition of G-proteins can eliminate DSI of spontaneous IPSCs (sIPSCs). In whole-cell recordings from CA1 pyramidal cells that were first determined to express DSI, we have found that NEM does block DSI of sIPSCs. We also report that DSI of monosynaptic, evoked IPSCs is blocked by NEM, suggesting that a similar mechanism underlies both forms of DSI. It was of interest that DSI was abolished at a time when NEM had increased, not decreased, GABA transmission. Indeed, NEM greatly increased quantal GABA release by a Ca2+-independent mechanism, an observation with potentially important implications for understanding synaptic GABA release.
Multiple Ionic Conductances of the Human Dopamine Transporter: The Actions of Dopamine and Psychostimulants
Authors: Sonders Mark S. , Zhu Si-Jia , Zahniser Nancy R. , Kavanaugh Michael P. , Amara Susan G.
Published: April 2018

Electrophysiological and pharmacological studies of a cloned human dopamine transporter (hDAT) were undertaken to investigate the mechanisms of transporter function and the actions of drugs at this target. Using two-electrode voltage-clamp techniques with hDAT-expressingXenopus laevisoocytes, we show that hDAT can be considered electrogenic by two criteria. (1) Uptake of hDAT substrates gives rise to a pharmacologically appropriate “transport-associated” current. (2) The velocity of DA uptake measured in oocytes clamped at various membrane potentials was voltage-dependent, increasing with hyperpolarization. Concurrent measurement of transport-associated current and substrate flux in individual oocytes revealed that charge movement during substrate translocation was greater than would be expected for a transport mechanism with fixed stoichiometry of 2 Na+and 1 Cl−per DA+molecule. In addition to the transport-associated current, hDAT also mediates a constitutive leak current, the voltage and ionic dependencies of which differ markedly from those of the transport-associated current. Ion substitution experiments suggest that alkali cations and protons are carried by the hDAT leak conductance. In contrast to the transport-associated functions, the leak does not require Na+or Cl−, and DAT ligands readily interact with the transporter even in the absence of these ions. The currents that hDAT mediates provide a functional assay that readily distinguishes the modes of action of amphetamine-like “DA-releasing” drugs from cocaine-like translocation blockers. In addition, the voltage dependence of DA uptake suggests a mechanism through which presynaptic DA autoreceptor activation may accelerate the termination of dopaminergic neurotransmissionin vivo.
Mediation by Protein Kinases C and A of Go-Linked Slow Responses of Enteric Neurons to 5-HT
Authors: Pan Hui , Wang Hoau-Yan , Friedman Eitan , Gershon Michael D.
Published: April 2018

5-HT activates the peristaltic reflex and is the neurotransmitter of a subset of myenteric interneurons. Hyperpolarizing afterpotential (AH)/type 2 neurons respond to 5-HT with a long-lived depolarization that is caused by the inhibition of a Ca2+-activated K+conductance (gKCa). This effect is mediated by a G-protein-coupled receptor, 5-HT1P. 5-HT1Pagonists specifically activate Gαo, the immunoreactivity of which was found to be highly abundant and membrane-associated in almost all enteric neurons. Responses of hyperpolarizing AH/type 2 neurons to 5-HT were inhibited by intracellular injection of GDPβS or anti-GαoFab fragments but were potentiated and prolonged by intracellular GTPγS. Responses to 5-HT were antagonized by pertussis toxin, downregulation of protein kinase C (PKC) and inhibitors of phosphatidylcholine phospholipase C (PC-PLC), PKC (including pseudosubstrate peptides, chelerythrine, and the α/β isoform-specific inhibitor Gö6976), protein kinase A (PKA), and adenylate cyclase. Responses to 5-HT were mimicked by activators of PKC, and 5-HT induced a concentration-dependent increase in the membrane-associated PKC activity in isolated myenteric ganglia. Immunocytochemical studies suggested that the most abundant isoforms of PKC in enteric neurons are α and δ. These data suggest that signal transduction of the 5-HT1P-mediated slow response to 5-HT involves activation of PC-PLC by Gαoto liberate diacylglycerol, which stimulates PKC (most likely α). PKC probably activates adenylate cyclase, which through cAMP, activates PKA. Activation of both PKA and PKC lead to closure of gKCa.
Growth Cone Form Is Behavior-Specific and, Consequently, Position-Specific along the Retinal Axon Pathway
Authors: Mason Carol A. , Wang Li-Chong
Published: April 2018

Video time-lapse microscopy has made it possible to document growth cone motility during axon navigation in the intact brain. This approach prompted us to reanalyze the hypothesis, originally derived from observations of fixed tissue, that growth cone form is position-specific. The behaviors of DiI-labeled retinal axon growth cones were tracked from retina through the optic tract in mouse brain at embryonic day (E) 15–17, and these behaviors were matched with different growth cone forms. Patterns of behavior were then analyzed in the different locales from the retina through the optic tract.

Throughout the pathway, episodes of advance were punctuated by pauses in extension. Irrespective of locale, elongated streamlined growth cones mediated advance and complex forms developed during pauses. The rate of advance and the duration of pauses were surprisingly similar in different parts of the pathway. In contrast, the duration of periods of advance was more brief in the chiasm compared to those in the optic nerve and tract. Consequently, in the chiasm, growth cones spent relatively more time pausing and less time advancing than in the optic nerve or tract. Thus, because growth cone form is behavior-specific and certain behaviors predominate in particular loci, growth cone form appears to be position-specific in static preparations, due to the fraction of time spent in a given state in different locales.
G1/S Cell Cycle Blockers and Inhibitors of Cyclin-Dependent Kinases Suppress Camptothecin-Induced Neuronal Apoptosis
Authors: Park David S. , Morris Erick J. , Greene Lloyd A. , Geller Herbert M.
Published: April 2018

Previous studies have demonstrated that G1/S cell cycle blockers and inhibitors of cyclin-dependent kinases (CDKs) prevent the death of nerve growth factor (NGF)-deprived PC12 cells and sympathetic neurons, suggesting that proteins normally involved in the cell cycle may also serve to regulate neuronal apoptosis. Past findings additionally demonstrate that DNA-damaging agents, such as the DNA topoisomerase (topo-I) inhibitor camptothecin, also induce neuronal apoptosis. In the present study, we show that camptothecin-induced apoptosis of PC12 cells, sympathetic neurons, and cerebral cortical neurons is suppressed by the G1/S blockers deferoxamine and mimosine, as well as by the CDK-inhibitors flavopiridol and olomoucine. In each case, the IC50values were similar to those reported for inhibition of death induced by NGF-deprivation. In contrast, other agents that arrest DNA synthesis, such as aphidicolin andN-acetylcysteine, failed to block death. This suggests that the inhibition of DNA synthesis per se is insufficient to provide protection from camptothecin. We find additionally that the cysteine aspartase family protease inhibitor zVAD-fmk inhibits apoptosis evoked by NGF-deprivation but not camptothecin treatment. Thus, despite their shared sensitivity to G1/S blockers and CDK inhibitors, the apoptotic pathways triggered by these two causes of death diverge at the level of the cysteine aspartase. In summary, neuronal apoptosis induced by the DNA-damaging agent camptothecin appears to involve signaling pathways that normally control the cell cycle. The consequent death signals of such deregulation, however, are different from those that result from trophic factor deprivation.
Retrograde Transport and Steady-State Distribution of125I-Nerve Growth Factor in Rat Sympathetic Neurons in Compartmented Cultures
Authors: Ure Daren R. , Campenot Robert B.
Published: April 2018

We have used compartmented cultures of rat sympathetic neurons to quantitatively examine the retrograde transport of125I-nerve growth factor (NGF) supplied to distal axons and to characterize the cellular events that maintain steady-state levels of NGF in cell bodies. In cultures allowed to reach steady-state125I-NGF transport, cell bodies contained only 5–30% of the total neuron-associated125I-NGF, whereas 70–95% remained associated with the distal axons. This was true over an 8 pmto 1.5 nm125I-NGF concentration range, indicating that saturation of high affinity receptors could not account for the large fraction of125I-NGF remaining in axons. Dissociation assays indicated that 85% of125I-NGF associated with distal axons was surface-bound. At steady-state, only 2–25% of the distal axon-associated125I-NGF was retrogradely transported each hour, with higher transport rates associated with younger cultures and lower125I-NGF concentrations. The velocity of125I-NGF retrograde transport was estimated at 10–20 mm/hr. However, as in a previous report, almost no125I-NGF transport was observed during the first hour after125I-NGF administration, indicating a significant lag between receptor binding and loading onto the retrograde transport system. During125I-NGF transport through axons spanning an intermediate compartment in five-compartment cultures, little or no125I-NGF was degraded or released from the axons. After transport,125I-NGF was degraded with a half-life of 3 hr. In summary, although some cellular events promoted NGF accumulation in cell bodies, distal axons represented by far the principal site of NGF-receptor interaction at steady-state as a result of a low retrograde transport rate.
The Intracellular Loop between Domains I and II of the B-Type Calcium Channel Confers Aspects of G-Protein Sensitivity to the E-Type Calcium Channel
Authors: Page Karen M. , Stephens Gary J. , Berrow Nicholas S. , Dolphin Annette C.
Published: April 2018

Neuronal voltage-dependent calcium channels undergo inhibitory modulation by G-protein activation, generally involving both kinetic slowing and steady-state inhibition. We have shown previously that the β-subunit of neuronal calcium channels plays an important role in this process, because when it is absent, greater receptor-mediated inhibition is observed (Campbell et al., 1995b). We therefore hypothesized that the calcium channel β-subunits normally may occlude G-protein-mediated inhibition. Calcium channel β-subunits bind to the cytoplasmic loop between transmembrane domains I and II of the α1-subunits (Pragnell et al., 1994). We have examined the hypothesis that this loop is involved in G-protein-mediated inhibition by making chimeras containing the I–II loop of α1B or α1A inserted into α1E (α1EBE and α1EAE, respectively). This strategy was adopted because α1B (the molecular counterpart of N-type channels) and, to a lesser extent, α1A (P/Q-type) are G-protein-modulated, whereas this has not been observed to any great extent for α1E. Although α1B, coexpressed with α2-δ and β1b transiently expressed in COS-7 cells, showed both kinetic slowing and steady-state inhibition when recorded with GTPγS in the patch pipette, both of which were reversed with a depolarizing prepulse, the chimera α1EBE (and, to a smaller extent, α1EAE) showed only kinetic slowing in the presence of GTPγS, and this also was reversed by a depolarizing prepulse. These results indicate that the I–II loop may be the molecular substrate of kinetic slowing but that the steady-state inhibition shown by α1B may involve a separate site on this calcium channel.
Expression and Distribution of IGF-1 Receptors Containing a β-Subunit Variant (βgc) in Developing Neurons
Authors: Mascotti Faustino , Cáceres Alfredo , Pfenninger Karl H. , Quiroga Santiago
Published: April 2018

βgcis a β-subunit variant of the insulin-like growth factor-1 (IGF-1) receptor highly enriched in growth cone membranes prepared by subcellular fractionation of fetal rat brain (Quiroga et al., 1995). The present study is focused on the expression and on the cellular and subcellular distribution of βgcin developing neurons and differentiating PC12 cells. In the developing cerebral cortex and, at least at early stages, in cultured primary neurons, βgcexpression was found to be correlated with neurite outgrowth. In PC12 cells βgcexpression was nerve growth factor (NGF)-dependent and also paralleled neurite outgrowth. In contrast, β-subunits of the insulin receptor and/or of other IGF-1 receptors (“βP5”; detected with antibody AbP5) were downregulated as βgcexpression increased. Immunofluorescence studies confirmed the enrichment of βgcat growth cones and demonstrated morphologically its spatial separation from βP5, which is confined to the perikaryon. At the growth cone, βgccolocalizes and associates in a proximal region with microtubules, but it seems independent of the more peripheral microfilaments. Some βgcimmunoreactivity is detected in the perinuclear region of PC12 cells, most likely the Golgi complex and its vicinity. βgcseems to emerge from the periphery of this structure in an apparently vesicular compartment distinct from that carrying synaptophysin to the growth cones. The facts that (1) βgcexpression is correlated closely with neurite outgrowth, that (2) it is regulated in PC12 cells by a neurotrophin, NGF, and that (3) βgcis concentrated in the proximal growth cone region raise new questions regarding a possible role of IGF-1 receptors containing βgcin the regulation of neurite growth.
The Role of Internal Models in Motion Planning and Control: Evidence from Grip Force Adjustments during Movements of Hand-Held Loads
Authors: Flanagan J. Randall , Wing Alan M.
Published: April 2018

We investigated the issue of whether or not the CNS makes use of an internal model of the motor apparatus in planning and controlling arm movements. In particular, we tested the ability of subjects to predict different hand-held loads by examining grip force adjustments used to stabilize the load in the hand during arm movements.

Subjects grasped a manipulandum using a precision grip with the tips of the thumb and index finger on either side. The grip force (normal to the contact surfaces) and the load force (tangential to the surfaces) were measured, along with the trajectory of the hand. The manipulandum was attached to two servo-controlled linear motors used to create inertial and viscous loads as well as a composite load, including inertial, viscous, and elastic components.

The form of the hand trajectory was independent of load for some subjects but varied systematically across load conditions in others. Nevertheless, under all load conditions and in all subjects, grip force was modulated in parallel with, and thus anticipated, fluctuations in load force despite the marked variation in the form of the load function. This indicates that the CNS is able to predict the load force and the kinematics of hand movement on which the load depends. We suggest this prediction is based on an internal model of the motor apparatus and external load and is used to determine the grip forces required to stabilize the load.
The Role of Calcium in the Desensitization of Capsaicin Responses in Rat Dorsal Root Ganglion Neurons
Authors: Koplas Patricia A. , Rosenberg Robert L. , Oxford Gerry S.
Published: April 2018

Capsaicin (Cap) is a pungent extract of theCapsicumpepper family, which activates nociceptive primary sensory neurons. Inward current and membrane potential responses of cultured neonatal rat dorsal root ganglion neurons to capsaicin were examined using whole-cell and perforated patch recording methods. The responses exhibited strong desensitization operationally classified as acute (diminished response during constant Cap exposure) and tachyphylaxis (diminished response to successive applications of Cap). Both acute desensitization and tachyphylaxis were greatly diminished by reductions in external Ca2+concentration. Furthermore, chelation of intracellular Ca2+by addition of either EGTA or bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid to the patch pipette attenuated both forms of desensitization even in normal Ca2+. Release of intracellular Ca2+by caffeine triggered acute desensitization in the absence of extracellular Ca2+, and barium was found to effectively substitute for calcium in supporting desensitization. Cap activated inward current at an ED50of 728 nm, exhibiting cooperativity (Hill coefficient, 2.2); however, both forms of desensitization were only weakly dependent on [Cap], suggesting a dissociation between activation of Cap-sensitive channels and desensitization. Removal of ATP and GTP from the intracellular solutions resulted in nearly complete tachyphylaxis even with intracellular Ca2+buffered to low levels, whereas changes in nucleotide levels did not significantly alter the acute form of desensitization. These data suggest a key role for intracellular Ca2+in desensitization of Cap responses, perhaps through Ca2+-dependent dephosphorylation at a locus that normally sustains Cap responsiveness via ATP-dependent phosphorylation. It also seems that the signaling mechanisms underlying the two forms of desensitization are not identical in detail.
Proliferation in the Rat Olfactory Epithelium: Age-Dependent Changes
Authors: Weiler Elke , Farbman Albert I.
Published: April 2018

Vertebrate olfactory sensory neurons are replaced continuously throughout life. We studied the effect of age on proliferation in olfactory epithelium in postnatal rats ranging in age from birth (P1) until P333. Using BrdU to label dividing cells, we determined the proliferation density of basal cells, i.e., the number of labeled nuclei/unit length (240 μm) of olfactory epithelium in coronal sections from six different anterior–posterior levels from each animal. A total length of >1 m of olfactory epithelium was counted in each age group. We observed a dramatic decrease of proliferation density from P1 through P333. At P1, proliferation density is 151 cells/mm; it decreases to approximately half at P21 (70 cells/mm), and half again at P40 (37 cells/mm). At P333 the proliferation density was only 8/mm, ∼5% of that seen at P1. The changes were clearly related to age and not to body weight, because the values were essentially identical for males and females of the same age but of different body weight. Proliferating cells appear in patches that, after P40, become more separated from one another and contain fewer cells. In 6- and 11-month-old rats, 30 and 45% of all units contained no labeled cells. We confirmed the data of others that the olfactory surface area continuously increases with age; we showed that there is a reciprocal relationship between proliferation density and surface area. The proliferating cells provide neurons to sustain growth as well as to replace dying cells.
Astrogliosis in the Neonatal and Adult Murine Brain Post-Trauma: Elevation of Inflammatory Cytokines and the Lack of Requirement for Endogenous Interferon-γ
Authors: Rostworowski Maria , Balasingam Vijayabalan , Chabot Sophie , Owens Trevor , Yong Voon Wee
Published: April 2018

The relevance of astrogliosis remains controversial, especially with respect to the beneficial or detrimental influence of reactive astrocytes on CNS recovery. This dichotomy can be resolved if the mediators of astrogliosis are identified. We have measured the levels of transcripts encoding inflammatory cytokines in injury systems in which the presence or absence of astrogliosis could be produced selectively. A stab injury to the adult mouse brain using a piece of nitrocellulose (NC) membrane elicited a prompt and marked increase in levels of transcripts for interleukin (IL)-1α, IL-1β, and tumor necrosis factor (TNF)-α, which are considered to be microglia/macrophage cytokines. The elevations preceded, or occurred concomitantly with, the rise in glial fibrillary acidic protein mRNA, an early manifestation of astrogliosis. In neonatal mice, IL-1 and TNF-α mRNA were elevated to a greater extent by an NC-implant injury, which produced astrogliosis, than after an NC-stab, with minimal astrogliosis. We determined whether endogenous interferon (IFN)-γ could be responsible for the observed increases in IL-1 and TNF-α, because IFN-γ is a potent microglia/macrophage activator, and because its exogenous administration to rodents enhanced astrogliosis after adult or neonatal insults. A lack of requirement for endogenous IFN-γ was demonstrated by three lines of evidence. First, no increase in IFN-γ transcripts could be found at injury. Second, the administration of a neutralizing antibody to IFN-γ did not attenuate astrogliosis. Third, in IFN-γ knockout adult mice, astrogliosis and increases in levels of IL-1α and TNF-α were induced rapidly by injury. The marked elevation of inflammatory cytokines is discussed in the context of astrogliosis and general CNS recovery.