bluuebunny/crossref_metadata_2025 huggingface dataset:
Potent human p140-TrkA agonists derived from an anti-receptor monoclonal antibody
Authors: LeSauteur L , Maliartchouk S , Le Jeune H , Quirion R , Saragovi HU
Published: April 2018

Monoclonal antibody (mAb) 5C3 directed against human p140 TrkA is a structural and functional mimic of nerve growth factor (NGF) and an artificial receptor agonist. mAb 5C3 binds in the NGF-docking site and, like NGF, it promotes TrkA internalization, TrkA and phosphatidylinositol-3 kinase tyrosine phosphorylation, and increased transformation of TrkA-expressing fibroblasts. More important, mAb 5C3 protects human TrkA-expressing cells from apoptotic death in serum-free media. Interestingly, agonistic activity is observed with monomeric F(ab) 5C3 fragments. mAb 5C3 (Kd approximately 2 nM) was used to study features of ligand binding by TrkA and the distribution of TrkA protein in normal human brain.
Ba2+ ions evoke two kinetically distinct patterns of exocytosis in chromaffin cells, but not in neurohypophysial nerve terminals
Authors: Seward EP , Chernevskaya NI , Nowycky MC
Published: April 2018

The coupling between divalent cations and exocytosis of large dense- cored vesicles (LDCV) was studied with capacitance-detection techniques in nerve terminals of the rat neurohypophysis (NHP) and bovine chromaffin cells. Ba2+ substitution for Ca2+ produced kinetically distinct responses in the two preparations. In NHP terminals, Ba2+ ions behave as weak substitutes for Ca2+. Exocytotic events occur principally during depolarizing pulses, i.e., events are “stimulus- coupled” to Ba2+ entry through voltage-gated Ca2+ channels. Stimulus- coupled exocytosis apparently requires elevated submembrane cation concentrations that dissipate rapidly on hyperpolarization-induced Ca(2+)-channel closure. Intracellular dialysis of NHP terminals with Ba2+ does not evoke exocytosis, nor does it interfere with depolarization-evoked Ca2+ influx and exocytosis. In chromaffin cells, Ba2+ ions evoke a small quantity of stimulus-coupled secretion, but the dominant response is an additional pronounced poststimulus capacitance increase that outlasts channel closures by 20–50 sec. “Stimulus- decoupled” exocytosis is slow (approximately 25–40 fF/sec) compared with Ca(2+)-evoked stimulus-coupled exocytosis (approximately 1000 fF/sec). Decoupled secretion is not attributable to Ba2+ displacement of intracellular Ca2+ ions, because it is insensitive to 10 mM EGTA or thapsigargin. Slow exocytosis is initiated by inclusion of Ba2+ ions in the recording pipette and continues steadily for 5–12 min, producing a total increase of several thousand fF, which ultimately doubles or triples the original cell-surface area. We propose that two pathways of regulated exocytosis with distinct kinetics and divalent cation sensitivity exist in chromaffin cells. Only a single kinetic pattern is detected in NHP terminals, suggesting that mechanisms for secretion are not universally distributed in excitable cells.
Opioid desensitization: interactions with G-protein-coupled receptors in the locus coeruleus
Authors: Fiorillo CD , Williams JT
Published: April 2018

In rat locus coeruleus (LC) neurons, alpha 2 adrenoceptors, mu-opioid and somatostatin receptors all activate the same potassium conductance. Chronic treatment with morphine causes a loss of sensitivity that is specific to the mu-opioid response, with no change in the alpha 2 adrenoceptor-mediated response. Acute desensitization induced by opioid, somatostatin, and alpha 2-adrenoceptor agonists was studied in brain slices of rat LC using intracellular recording. A supramaximal concentration of the opioid agonist Met5-enkephalin induced a profound homologous desensitization but little heterologous desensitization to an alpha 2-adrenoceptor agonist (UK 14304) or somatostatin. All desensitized currents showed partial recovery. A supramaximal concentration of UK14304 caused a relatively small amount of desensitization. Although little interaction was observed among inhibitory G-protein-coupled receptors, activation of an excitatory receptor had marked effects on inhibitory responses. Muscarinic agonists, which produce an inward current in LC neurons, reduced the magnitude of agonist-induced outward currents and increased both the rate and amount of opioid desensitization. Muscarinic activation did not alter desensitization of alpha 2-adrenoceptor responses. Acute desensitization shares several characteristics with the tolerance induced by chronic morphine treatment of animals.
Defects in courtship and vision caused by amino acid substitutions in a putative RNA-binding protein encoded by the no-on-transient A (nonA) gene of Drosophila
Authors: Rendahl KG , Gaukhshteyn N , Wheeler DA , Fry TA , Hall JC
Published: April 2018

The Drosophila no-on-transient A (nonA) gene is involved in the visual behaviors and courtship song of the fly. The NONA polypeptide contains two copies of the RNA-recognition motif (RRM), a hallmark of proteins involved in RNA binding, and an adjacent conserved charged region. This 311-amino-acid region is found in four other proteins and largely overlaps the Drosophila-Behavior/Human Splicing (or DBHS) domain. The newest family member, Drosophila nAhomo, was discovered in a database search, and encodes a protein with 80% identity to NONA. In this study, three nonA mutations generated by chemical mutagenesis were sequenced and found to fall within the conserved region. Site-directed mutagenesis of the two RRMs, and within a (conserved) charged region located C-terminal to them, was performed to determine the significance of these domains with respect to whole-organismal phenotypes. Behavior and viability were assessed in transformed flies, the genetic background of which lacks the nonA locus. Point mutations of amino acid 548 in the charged region confirmed the etiology of the nonAdiss courtship-song mutation and showed that a milder substitution at this site produced intermediate singing behavior, although it failed to rescue visual defects. Mutagenesis of the RRM1 domain resulted in effects on viability, vision, and courtship song. However, amino acid substitutions in RNP-II of RRM2 led to near-normal phenotypes, and the in vivo nonA mutations located in or near RRM2 caused visual defects only. Thus, we suggest that the first RRM could be important for all functions influenced by nonA, whereas the second RRM may be required primarily for normal vision.
Context-specific multi-site cingulate cortical, limbic thalamic, and hippocampal neuronal activity during concurrent discriminative approach and avoidance training in rabbits
Authors: Freeman JH , Cuppernell C , Flannery K , Gabriel M
Published: April 2018

This study assessed the context specificity of learning-related neuronal activity: whether the same physical stimuli would elicit different neuronal responses depending on the learning situation. Neuronal activity was recorded simultaneously in six limbic areas as rabbits learned to approach a spout for water reinforcement after a tone (CS+) and to ignore the spout after a different tone (CS-). The rabbits then received avoidance training in which they learned to prevent a foot-shock by stepping in an activity wheel after one tone (CS+) and to ignore a different tone (CS-). Avoidance training sessions were alternated (1 session daily) with sessions in the well learned approach task. The tone assigned as the CS+ for approach training was the CS- for avoidance training and vice versa. The neuronal records of the anterior ventral and medial dorsal thalamic nuclei and the anterior and posterior cingulate cortices showed neuronal discrimination appropriate to the approach task during pretraining in the avoidance training apparatus with unpaired presentations of the tones and foot- shock. This finding demonstrated that the discriminative neuronal activity for approach learning was unaffected by a change in context in the pretraining session. However, context-appropriate discrimination occurred in both tasks thereafter, with the exception that medial dorsal thalamic neurons no longer showed discrimination during overtraining in the approach task. Hippocampal area CA1 neurons showed entirely context-appropriate discrimination in both tasks, with no carryover of the approach-relevant discrimination to the avoidance training apparatus. Avoidance training stage-specific peaks of training- induced excitation in different brain areas were not elicited by the same physical stimuli during concurrent approach training sessions. The results are consistent with an involvement of limbic-circuit neuronal activity in the use of context cues for mnemonic retrieval. Differential persistence of the approach-related neuronal discrimination in anterior and posterior cingulate cortex confirmed the previously hypothesized distinct mnemonic functions of these areas.
Behavioral effects mediated by the modulatory glycine site of the NMDA receptor in the anterodorsal striatum and nucleus accumbens
Authors: Kretschmer BD , Schmidt WJ
Published: April 2018

The striatum and the nucleus accumbens are the main input structures of the basal ganglia (BG). They contribute differently to motor behavior controlled by the BG in rats, e.g., stereo-typed behavior, catalepsy, and locomotion. Whereas the striatum is predominantly involved in the control of sniffing behavior and catalepsy, the nucleus accumbens contributes to control of locomotion. To test whether the allosteric glycine site of the NMDA receptor complex modulates these behavioral variables, we injected the glycine-site antagonist 7-chlorokynurenate and the glycine-site agonist D-serine into the anterodorsal striatum and the nucleus accumbens and studied their influence on stereotypical snout contacts and locomotion. Additionally, the effects of intrastriatal injections of 7-chlorokynurenate on haloperidol- and SCH 23390-induced catalepsy were investigated. 7-Chlorokynurenate enhanced stereotypical snout contacts in the anterodorsal striatum and in the nucleus accumbens but did not change spontaneous locomotion in either of these structures. Haloperidol- but not SCH 23390-induced catalepsy was attenuated by intrastriatally administered 7-chlorokynurenate. The glycine-site agonist D-serine had no effect on stereotypical snout contacts and locomotion. The results suggest that motor behavior mediated by the striatopallidal output pathway is modulated by the glycine site, whereas motor behavior mediated by the accumbopallidal and striatonigral output pathway is not.
Coarticulation of jaw movements in speech production: is context sensitivity in speech kinematics centrally planned?
Authors: Ostry DJ , Gribble PL , Gracco VL
Published: April 2018

Coarticulation in speech production is a phenomenon in which the articulator movements for a given speech sound vary systematically with the surrounding sounds and their associated movements. Although these variations may seem to be planned centrally, without explicit models of the speech articulators, the kinematic patterns that are attributable to central control cannot be distinguished from those that arise because of dynamics and are not represented in the underlying control signals. We address the origins of coarticulation by comparing the results of empirical and modeling studies of jaw motion in speech. The simulated kinematics of sagittal-plane jaw rotation and horizontal jaw translation are compared with the results of empirical studies in which subjects produce speech-like sequences at a normal rate and volume. The simulations examine both “anticipatory” and “carryover” coarticulatory effects. In both cases, the results show that even when no account is taken of context at the level of central control, kinematic patterns vary in amplitude and duration as a function of the magnitude of the preceding or following movement, in the same manner as that observed empirically in coarticulation. Because at least some coarticulatory effects may arise from muscle mechanics and jaw dynamics and not from central control, these factors must be considered before drawing inferences about control in coarticulation.
Insulin receptor in Aplysia neurons: characterization, molecular cloning, and modulation of ion currents
Authors: Jonas EA , Knox RJ , Kaczmarek LK , Schwartz JH , Solomon DH
Published: April 2018

We have isolated the cDNA for a tyrosine kinase receptor that is expressed in the nervous system of Aplysia californica and that is similar to the vertebrate insulin receptor. Binding studies and immunocytochemical staining show that the receptor is abundant in the bag cell neurons. Application of vertebrate insulin to clusters of bag cell neurons stimulates the phosphorylation of the receptor on tyrosine residues, and exposure of isolated bag cell neurons to insulin produces an increase in height and a decrease in duration of the action potentials that can be detected within 15–30 min. These effects were not seen with insulin-like growth factor-1. In voltage-clamped neurons, insulin produces an increase in the amplitude of the voltage-dependent Ca2+ current that can be blocked by preincubation with herbimycin A, an inhibitor of tyrosine kinases. Insulin also enhances a delayed K+ current. We suggest that insulin-like peptides regulate the excitability of the bag cell neurons.
Lobster shal: comparison with Drosophila shal and native potassium currents in identified neurons
Authors: Baro DJ , Coniglio LM , Cole CL , Rodriguez HE , Lubell JK , Kim MT , Harris- Warrick RM
Published: April 2018

The transient potassium (K+) current, or A-current (IA), plays an essential role in shaping the firing properties of identified neurons in the 14-cell pyloric network in the stomatogastric ganglion of the spiny lobster, Panulirus interruptus. The different cells in the pyloric network have distinct IAs. To begin to understand the molecular basis for IA heterogeneity, we examined the relationship between the Panulirus shal current, the IAs in the lateral pyloric (LP) and pyloric dilator (PY) cells, and the Drosophila shal current. After isolating a complete open reading frame for lobster shal 1, which shows significant sequence homology to the fly, mouse, and rat shal homologs, we used a single-cell reverse transcription polymerase chain reaction method to demonstrate that the shal 1 gene was expressed in the LP and PY cells. Next, we compared the lobster shal 1 current generated in a Xenopus oocyte expression system to the IAs in the LP and PY neurons as well as to the Drosophila shal current in Xenopus oocytes. While the transient K+ lobster shal 1 current was similar to the IAs in pyloric neurons, a detailed comparison shows that they are not identical and differ in kinetic and voltage-dependent parameters. The highly homologous lobster and fly shal genes also produce currents with some significant similarities and differences in an oocyt.
The effects of growth factors on the survival and differentiation of cultured dentate gyrus neurons
Authors: Lowenstein DH , Arsenault L
Published: April 2018

Dentate granule cells (DGCs) are the principal cell population of the hippocampal dentate gyrus, and granule cells provide the main excitation to the hippocampus proper via their mossy fibers axons. Although it is well established that granule cells express various growth factors and growth factor receptors, the functional effects of growth factors on the normal development and response to injury of granule cells are relatively unknown. To address this question, primary cultures enriched in DGCs were prepared by microdissecting hippocampal slices from neonatal rats and growing dissociated cells in defined media with added nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT4/5), ciliary neurotrophic factor, basic fibroblast growth factor (bFGF), or vehicle. The effects on cell survival and morphology were quantified by studying neuron-specific enolase-immunostained cells at various time points, plating densities, host ages, and growth factor concentrations. BDNF or bFGF significantly increased both neuronal survival and differentiation by 30–80% compared with control cultures. Maximal effects were observed at relatively longer time points (5–12 d), with younger cells (postnatal day 3–5) and at lowest plating densities. Addition of a trkB- IgG fusion protein that blocks the activity of BDNF or NT4/5 inhibited the effects of BDNF and attenuated the differentiation of cells cultured at high plating densities. Furthermore, treatment of cultures with the kinase inhibitor K252b specifically blocked the effects of BDNF, suggesting involvement of trkB (the high-affinity BDNF receptor) in BDNF-induced differentiation. These results show that growth properties of cultured neonatal DGCs are influenced by exogenously applied BDNF or bFGF in a time-, age-, and density-dependent manner. The effect of plating density suggests an endogenous expression of growth factors in these culture conditions, and this is mediated in part by endogenous BDNF acting via a tyrosine kinase receptor. Combined with previous work showing that various growth factors and their receptors are expressed by DGCs, these findings provide strong support for the hypothesis that BDNF and bFGF influence both the growth and development of DGCs in vivo.
GABA stimulates chemotaxis and chemokinesis of embryonic cortical neurons via calcium-dependent mechanisms
Authors: Behar TN , Li YX , Tran HT , Ma W , Dunlap V , Scott C , Barker JL
Published: April 2018

During rat cortical development, when neurons migrate from the ventricular zone to the cortical plate, GABA localizes within the target destinations of migratory neurons. At this time, cells in germinal zones and along migratory pathways express GABA receptor subunit transcripts, implying that in vivo, GABA may be a chemoattractant. We used an in vitro strategy to study putative chemotropic effects of GABA on embryonic rat cortical cells. GABA stimulated neuronal migration in vitro at embryonic day 15 (E15). From E16 onward, two concentration ranges (fM and microM) induced motility. Femtomolar GABA primarily stimulated chemotaxis (migration along a chemical gradient), whereas micromolar GABA predominantly initiated chemokinesis (increased random movement). These effects were mimicked by structural analogs of GABA with relative specificity at GABAA (muscimol), GABAB (R-baclofen), and GABAC (trans- or cis-4- aminocrotonic acid) receptors. Antagonists of GABAB (saclofen) and GABAC (picrotoxin) receptors partially inhibited responses to both femto- and micromolar GABA; however, only responses to femtomolar GABA were partially blocked by bicuculline, a well established antagonist of GABA at GABAA receptors. Hence, chemotactic responses to femtomolar GABA seem to involve all three classes of GABA receptor proteins, whereas chemokinetic responses to micromolar GABA involve GABAB and GABAC receptor proteins. GABA-induced motility was blocked by loading the cells with the Ca(2+)-chelating molecule bis(2-aminophenoxy)ethane- N,N,N′,N′-tetra-acetic acid, suggesting that intracellular Ca2+ mediates GABA-induced cell movement. Optical recordings of cells loaded with Ca2+ indicator dye revealed that both femto- and micromolar GABA evoked increases in intracellular Ca2+. Thus, GABA-stimulated increases in intracellular Ca2+ may mediate both chemotactic and chemokinetic responses in embryonic cortical cells.
The effects of ovariectomy and estrogen replacement on trkA and choline acetyltransferase mRNA expression in the basal forebrain of the adult female Sprague-Dawley rat
Authors: McMillan PJ , Singer CA , Dorsa DM
Published: April 2018

Cognitive deficits associated with aging and with neurodegenerative diseases such as Alzheimer's disease have been attributed to degeneration of cholinergic neurons in the basal forebrain. Estrogen is known to provide trophic support to cholinergic neurons, although the mechanisms underlying the actions of estrogen have yet to be determined. Because cholinergic neurons require neurotrophic growth factors for their survival, it is possible that the trophic effects of estrogen on basal forebrain systems are caused by enhanced expression of neurotrophins or their receptors. To begin to examine this hypothesis, we used in situ hybridization analysis to determine the effects of ovariectomy (ovx) and estrogen replacement on trkA mRNA levels in the rat basal forebrain. Ten days of estrogen deprivation after ovx resulted in significant decreases in trkA mRNA levels in the horizontal limb of the diagonal band of Broca and the nucleus basalis of Meynert. Short-term estrogen replacement therapy restored trkA mRNA expression to a level comparable with ovary-intact animals. No changes in trkA mRNA levels were observed in the vertical limb of the diagonal band of Broca after ovx or estrogen replacement. To assess the functional status of cholinergic neurons in the absence and presence of estrogen, the effects of ovx and estrogen replacement on ChAT mRNA levels were also examined and found to reflect the changes observed in trkA mRNA expression. These studies suggest that the trophic effects of estrogen on basal forebrain cholinergic systems may be mediated, in part, through the signaling of neurotrophic growth factors through their receptors.
The inhibitory effect of testosterone on hypothalamic-pituitary-adrenal responses to stress is mediated by the medial preoptic area
Authors: Viau V , Meaney MJ
Published: April 2018

In gonadectomized (GDX) animals replaced with subcutaneous steroid implants supplying physiological levels of testosterone (T; 1–10 ng/ml), the magnitude of adrenocorticotropic hormone (ACTH) and corticosterone (B) responses to restraint was negatively correlated with the level of T replacement, reflecting the inhibitory influence of T on hypothalamic-pituitary-adrenal (HPA) responses to stress. Although T had no effect on resting-state levels of corticotropin-releasing hormone (CRH) in the median eminence, arginine vasopressin (AVP) levels were significantly lower in GDX animals replaced with higher T levels, and the magnitude of the ACTH response to restraint was strongly correlated with median eminence levels of AVP. High physiological levels of T increased glucocorticoid receptor binding in the medial preoptic area (MPOA), with no effect on mineralocorticoid receptor binding or on glucocorticoid receptor binding in other regions. Crystalline T or B implants in the MPOA significantly reduced plasma ACTH and B responses to 10 min of restraint stress compared with cholesterol-implanted controls. Moreover, B or T MPOA implants also decreased resting-state levels of AVP but not CRH in the median eminence, and these effects were correlated with ACTH responses to restraint. Finally, lesioning the MPOA blocked the inhibitory effects of high peripheral T levels on ACTH and B responses to restraint. Thus, variations in the magnitude of HPA responses to stress among males are explained in part by individual differences in circulating T levels, and the MPOA is a critical site for this effect via the inhibition of hypophysial AVP.
Selective expression of the immediate early gene c-jun in axotomized rat medial septal neurons is not related to neuronal degeneration
Authors: Haas CA , Deller T , Naumann T , Frotscher M
Published: April 2018

In the present study, we use the anatomically well defined septohippocampal projection to study the molecular events involved in the reaction of neurons to axotomy. The expression of three immediate early genes (c-fos, c-jun, and jun B) was investigated in rat septohippocampal neurons after axotomy by bilateral fimbria-fornix transection (FFT). Moreover, the extent of retrograde degeneration in the septal complex was assessed by analyzing DNA fragmentation. In a postoperative time course analysis, a strong increase of c-jun immunoreactivity (IR) was observed in the nuclei of neurons in the medial septum/diagonal band complex (MSDB) 2 and 6 d postaxotomy, which was followed by a decline after 12 d and 3 weeks, respectively. Nine weeks after FFT, c-jun IR had disappeared. The c-jun-positive MS neurons were identified as former septohippocampal projection cells by double-labeling with the retrogradely transported tracer Fluoro-Gold injected into the hippocampus before axotomy. In line with the immunocytochemical data, there was a massive induction of c-jun mRNA in the axotomized MS neurons as visualized by in situ hybridization histochemistry. c-fos mRNA and c-fos or jun B IR were not detectable in either unoperated or lesioned medial septal neurons. Experiments using the TdT-mediated deoxyuridine triphosphate nick-end-labeling technique, designed to detect nuclear DNA fragmentation in degenerating neurons, complemented this study. During the postoperative time range studied, MS neurons did not exhibit DNA fragmentation. We conclude that MSDB neurons survive axotomy by FFT and display characteristic changes in gene expression.
Relapse to heroin-seeking in rats under opioid maintenance: the effects of stress, heroin priming, and withdrawal
Authors: Shaham Y , Rajabi H , Stewart J
Published: April 2018

It is widely believed that opioid withdrawal symptoms contribute to relapse to opioid use, but relapse is highly probable in experienced users even after prolonged abstinence and during opioid maintenance therapy. We have found using an animal model of relapse, the reinstatement procedure, that the two events that reliably reinstate heroin-seeking behavior are reexposure to heroin, and brief exposure to footshock stress. Contrary to expectation, opioid antagonist-induced withdrawal does not reinstate heroin-seeking. We now report on reinstatement of heroin-seeking in rats trained to self-administer heroin and subsequently exposed to a maintenance dose of heroin via minipump and allowed to self-administer saline. With the minipump in, naloxone-induced withdrawal did not reinstate drug-seeking, a priming injection on heroin was only mildly effective, and footshock was highly effective. Twenty-four hours after removal of the minipump (spontaneous withdrawal), animals reinitiated heroin-seeking and, subsequently, both heroin and footshock reinstated heroin-seeking. In summary, brief exposure to stress reinstated heroin-seeking in both heroin-maintained and withdrawn animals. The heroin prime reliably reinstated drug- seeking only in the absence of the minipump; opioid “withdrawal,” as such, did not reinstate drug-seeking behavior. Naloxone given to heroin- maintained animals induced withdrawal symptoms, caused a mild depression in the levels of dopamine and its metabolites in the nucleus accumbens septi (NAS), but did not reinstate drug-seeking. Reinstatement of heroin-seeking during spontaneous withdrawal was not accompanied by reductions in basal dopamine and its metabolites in NAS.
Localization of synaptotagmin-binding domains on syntaxin
Authors: Kee Y , Scheller RH
Published: April 2018

Synaptotagmin, an abundant calcium- and phospholipid-binding protein of synaptic vesicles, has been proposed to regulate neurotransmitter release at the nerve terminal. To understand better the biochemical mechanism of neurotransmitter release, we have investigated the calcium- dependent and -independent protein-protein interactions between synaptotagmin I and syntaxin 1a, a subunit of the receptor for synaptic vesicles on the presynaptic plasma membrane. Soluble syntaxin 1a binds to synaptotagmin glutathione S-transferase (GST) fusion protein, and the binding was decreased in the presence of calcium. A synaptotagmin fragment containing the second C2 repeat (Syt3–5) had the same binding profile as the whole cytoplasmic domain; however, fragments containing the first C2 repeat (Syt1–3 and Syt2–3) showed calcium-dependent binding to syntaxin. In addition, the soluble full-length cytoplasmic domain of synaptotagmin binds to a syntaxin GST fusion protein in a calcium-dependent manner. Syntaxin domains required for calcium- dependent and -independent synaptotagmin-binding were localized using syntaxin deletion mutants. Amino acids 241–266 of the syntaxin C terminus were required for calcium-independent binding of synaptotagmin. The minimal domain required for calcium-dependent binding of synaptotagmin to syntaxin was localized to amino acids 220– 266. The syntaxin domains required for synaptotagmin binding overlap with the domains for vesicle-associated membrane protein (or VAMP) and alpha-soluble N-ethyl-maleimide-sensitive fusion protein attachment protein (or alphaSNAP) interactions. The data suggest both calcium- dependent and -independent roles of synaptotagmin in regulating synaptic vesicle release and/or recycling.
G-protein-gated inward rectifier K+ channel proteins (GIRK1) are present in the soma and dendrites as well as in nerve terminals of specific neurons in the brain
Authors: Ponce A , Bueno E , Kentros C , Vega-Saenz de Miera E , Chow A , Hillman D , Chen S , Zhu L , Wu MB , Wu X , Rudy B , Thornhill WB
Published: April 2018

G-protein-gated inward rectifier potassium (GIRK) channels are coupled to numerous neurotransmitter receptors in the brain and can play important roles in modulating neuronal function, depending on their localization in a given neuron. Site-directed antibodies to the extreme C terminus of GIRK1 (or KGA1), a recently cloned component of GIRK channels, have been used to determine the relative expression levels and distribution of the protein in different regions of the rat brain by immunoblot and immunohistochemical techniques. We report that the GIRK1 protein is expressed prominently in the olfactory bulb, hippocampus, dentate gyrus, neocortex, thalamus, cerebellar cortex, and several brain stem nuclei. In addition to the expected localization in somas and dendrites, where GIRK channels may mediate postsynaptic inhibition, GIRK1 proteins were also found in axons and their terminal fields, suggesting that GIRK channels can also modulate presynaptic events. Furthermore, the distribution of the protein to either somatodendritic or axonal-terminal regions of neurons varied in different brain regions, which would imply distinct functions of these channels in different neuronal populations. Particularly prominent staining of the cortical barrels of layer IV of the neocortex, and the absence of this staining with unilateral kainate lesions of the thalamus, suggest that the GIRK1 protein is expressed in thalamocortical nerve terminals in which GIRK channels may mediate the actions of mu opiate receptors.
Reduced nicotinamide adenine dinucleotide-selective stimulation of inositol 1,4,5-trisphosphate receptors mediates hypoxic mobilization of calcium
Authors: Kaplin AI , Snyder SH , Linden DJ
Published: April 2018

To evaluate the relationship of inositol 1,4,5-trisphosphate (IP3) receptor-mediated signal transduction and cellular energy dynamics, we have characterized effects of nucleotides on IP3 receptor (IP3R)- mediated calcium (Ca2+) flux in purified IP3 receptors reconstituted in lipid vesicles (IP3RV) and examined hypoxia-induced augmentation of intracellular Ca2+ in intact cells. Reduced nicotinamide adenine dinucleotide (NADH) increases IP3-mediated Ca2+ flux in IP3RV. This effect is highly specific for NADH. Hypoxia elicited by brief exposure of nerve growth factor-differentiated PC12 cells or cerebellar Purkinje cells to cyanide elicits rapid increased in internal [Ca2+], which derives from IP3-sensitive stores. Blockade of this effect by 2- deoxyglucose and inhibition of glyceraldehyde-3-phosphate dehydrogenase implicates enhanced glycolytic production of NADH in the Ca2+ stimulation. Internal [Ca2+] is markedly and specifically increased by direct intracellular injection of NADH, and this effect is blocked by heparin, further implicating IP3R stores. These findings indicate that direct regulation of IP3R by NADH is responsible for elevated cytoplasmic [Ca2+] occurring in the earliest phase of hypoxia. This link of IP3R activity with cellular energy dynamics may be relevant to both hypoxic damage and metabolic regulation of IP3 signaling processes.
Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation
Authors: Kuhn HG , Dickinson-Anson H , Gage FH
Published: April 2018

The hippocampus is one of the few areas of the rodent brain that continues to produce neurons postnatally. Neurogenesis reportedly persists in rats up to 11 months of age. Using bromodeoxyuridine (BrdU) labeling, the present study confirms that in the adult rat brain, neuronal progenitor cells divide at the border between the hilus and the granule cell layer (GCL). In adult rats, the progeny of these cells migrate into the GCL and express the neuronal markers NeuN and calbindin-D28k. However, neurogenesis was drastically reduced in aged rats. Six-to 27-month-old Fischer rats were injected intraperitoneally with BrdU to detect newborn cells in vivo and to follow their fate in the dentate gyrus. When killed 4–6 weeks after BrdU labeling, 12- to 27- month-old rats exhibited a significant decline in the density of BrdU- positive cells in the granule cell layer compared with 6-month-old controls. Decreased neurogenesis in aging rats was accompanied by reduced immunoreactivity for poly-sialylated neural cell adhesion molecule, a molecule that is involved in migration and process elongation of developing neurons. When animals were killed immediately (12 hr) after BrdU injection, significantly fewer labeled cells were observed in the GCL and adjacent subgranular zone of aged rats, indicative of a decrease in mitotic activity of neuronal precursor cells. The reduced proliferation was not attributable to a general aged- related metabolic impairment, because the density of BrdU-positive cells was not altered in other brain regions with known mitotic activity (e.g., hilus and lateral ventricle wall). The decline in neurogenesis that occurs throughout the lifespan of an animal can thus be related to a decreasing proliferation of granule cell precursors.
Interaction between the C terminus of NMDA receptor subunits and multiple members of the PSD-95 family of membrane-associated guanylate kinases
Authors: Niethammer M , Kim E , Sheng M
Published: April 2018

Selective concentration and anchoring of ionotropic receptors at the synapse is essential for neuronal signaling. Little is known about the molecules that mediate receptor clustering in the CNS. With use of the yeast two-hybrid system to screen a rat brain cDNA library and by in vitro binding assays, we have identified an interaction between NMDA receptor subunits 2A and 2B (NR2A and NR2B) and three distinct members of the PSD-95/SAP90 family of membrane-associated putative guanylate kinases. The interaction is mediated by binding of the C terminus of the NMDA receptor subunits to the first two PDZ (also known as GLGF or DHR) domains of PSD-95/SAP90, an abundant synaptic protein associated with the membrane cytoskeleton. PSD-95 is also known to bind and cluster Shaker-type voltage-gated K+ channels. Similarities between the C-termini of NR2 subunits and K+ channels suggest a common C-terminal binding motif for PDZ domains. These data suggest that PDZ domains can function as modules for protein-protein interactions. Members of the PSD-95 family might serve to anchor NMDA receptors to the submembrane cytoskeleton and aid in the assembly of signal transduction complexes at postsynaptic sites.
Light and dark cause a shift in the spatial expression of a neuropeptide-processing enzyme in the rat retina
Authors: Schlamp CL , Nickells RW
Published: April 2018

Carboxypeptidase E (CPE, EC 3.4.17.10) is an essential enzyme in the post-translational processing of most neuroactive peptides. We have studied how the expression of CPE in the rat retina is modified in response to the light/dark cycle. Retinal CPE mRNA levels increase immediately after a change in lighting condition. After prolonged exposure to the dark or the light, however, CPE mRNA levels drop to comparable amounts. The increase CPE mRNA is most dramatic during the dark-to-light transition. Interestingly, during the same interval, CPE protein levels transiently decrease. This apparently contradictory change in mRNA and protein levels is attributable to a change in the spatial pattern of CPE expression in the retina in response to light and dark. Both in situ hybridization and indirect immunofluorescence studies indicate that CPE is expressed in photoreceptors in the dark. During light onset, CPE expression is rapidly induced in retinal ganglion cells, whereas expression in photoreceptors is reduced. This pattern is reversed when the animals are exposed to the dark. In contrast, CPE is apparently constitutively expressed in a subpopulation of cells in the inner nuclear layer in both the light and the dark. These changes in CPE expression are similar to light-induced changes in c-fos expression, suggesting that CPE may be a downstream target for Fos activation.
Changes in action potential duration alter reliance of excitatory synaptic transmission on multiple types of Ca2+ channels in rat hippocampus
Authors: Wheeler DB , Randall A , Tsien RW
Published: April 2018

It has been established that multiple types of Ca2+ channels participate in triggering neurotransmitter release at central synapses, but there is uncertainty about the nature of their combined actions. We investigated synaptic transmission at CA3-CA1 synapses of rat hippocampal slices and asked whether the dependence on omega-CTx-GVIA- sensitive N-type channels and omega-Aga-IVA-sensitive P/Q-type Ca2+ channels can be altered by physiological mechanisms. The reliance on multiple types of Ca2+ channels was not absolute but depended strongly on the amount of Ca2+ influx through individual channels, which was manipulated by prolonging the presynaptic action potential with the K+ channel blocker 4-aminopyridine (4-AP) and by varying the extracellular Ca2+ concentration ([Ca2+]o). We quantified the influence of spike broadening on Ca2+ influx through various Ca2+ channels by imposing mock action potentials on voltage-clamped cerebellar granule neurons. In field recordings of the EPSP in hippocampal slices, action potential prolongation increased the EPSP slope by 2-fold and decreased its reliance on either N-type or P/Q-type Ca2+ channels. The inhibition of synaptic transmission by N-type channel blockade was virtually eliminated in the presence of 4-AP, but it could be restored by lowering [Ca2+]o. These results rule out a scenario in which a significant fraction of presynaptic terminals rely solely on N-type channels to trigger transmission. The change in sensitivity to the neurotoxins with 4-AP could be explained in terms of a nonlinear relationship between Ca2+ entry and synaptic strength, which rises steeply at low [Ca2+]o, but approaches saturation at high [Ca2+]o. This relationship was evaluated experimentally by varying [CA2+]o in the absence and presence of 4-AP. One consequence of this relationship is that down-modulation of presynaptic Ca2+ channels by various modulators would increase the relative impact of spike broadening greatly.
Brain cell type specificity and gliosis-induced activation of the human cytomegalovirus immediate-early promoter in transgenic mice
Authors: Fritschy JM , Brandner S , Aguzzi A , Koedood M , Luscher B , Mitchell PJ
Published: April 2018

Human cytomegalovirus (HCMV) can cause debilitating, sometimes fatal, opportunistic infections in congenitally infected infants and in immunodeficient individuals such as patients with the acquired immunodeficiency syndrome (AIDS). Molecular mechanisms that determine cell type specificity of HCMV infection and latency are poorly understood. We recently described a transgenic mouse model for analysis of HCMV major immediate-early (IE) promoter regulation and showed that sites of IE promoter activity during murine embryogenesis correlate with known target tissues of congenital HCMV infection in human fetuses (Koedood et al., 1995). Among various permissive human tissues, the brain is a site where HCMV infections can be devastating. Here, we have used immunohistochemical double-labeling analysis to identify specific cell types with HCMV-IE promoter activity in brains of transgenic mice at several postnatal stages. IE promoter activity was restricted to some endothelial cells, ependymal cells, choroid plexus epithelia, and neurons at discrete locations in the forebrain, brainstem, and cerebellum. Endothelial cells and neurons with activity were proportionately more abundant in neonatal than in adult brains. Although the IE promoter was normally silent in most astrocytes, activity was strongly induced in reactive astrocytes in response to a neocortical stab lesion. The findings support a model, consistent with clinical literature on HCMV encephalitis, whereby tissue damage and gliosis caused by HCMV infection of endothelial and ependymal cells progressively renders adjacent permissive neurons and reactive astrocytes accessible to infection. This transgenic model system should facilitate identification of factors that regulate the HCMV IE promoter with regard to infection permissivity and reactivation from latency.
Role of neuron-glial junctional domain proteins in the maintenance and termination of neuronal migration across the embryonic cerebral wall
Authors: Anton ES , Cameron RS , Rakic P
Published: April 2018

To identify glial membrane proteins that contribute to the process of neuronal migration in the developing brain, we developed a polyclonal antiserum (D4) and a monoclonal antibody (NJPA1: neuron-glial junctional polypeptide antibody) that recognize membrane proteins localized to the plasmalemmal junction between migrating neurons and adjacent radial glial fibers (Cameron and Rakic, 1994). Here, we show that in the developing cerebral cortex, immunoreactivity for these junctional polypeptides is present throughout the neuronal migratory pathway but becomes minimal or absent where radial glial cell processes enter the marginal zone region, the barrier at which newly arrived neurons normally stop their migration and detach from their glial fiber substrate. We thus tested, using imprints of embryonic cerebral wall and slice preparations, whether the junctional membrane proteins detected by our antibodies contribute to the regulation of neuronal migration in the cerebral cortex. The rate of neuronal migration on glial cell substrates was reduced significantly in the presence of D4 or NJPA1 antibodies. Antibody exposure typically led to the withdrawal of leading processes, changes in microtubular organization and, in some instances, to detachment of neurons from their glial cell substrates. These results suggest that the polypeptides recognized by the D4 and NJPA1 antibodies are essential for the maintenance of normal neuronal migration. Dismantling of neuron-glial cell junctional domains formed by these membrane proteins may underlie neuronal cell detachment from glial migratory substrates at the interface between cortical plate and marginal zone in the developing cerebral wall.
An intrinsic time limit between genesis and death of individual neurons in the developing retinal ganglion cell layer
Authors: Galli-Resta L , Ensini M
Published: April 2018

We tested the possibility that a temporal relationship exists between genesis and death of individual neurons dying during development. For this purpose, we labeled neurons born in limited time intervals and determined when they die in the ganglion cell layer (GCL) of the rat retina. We found that most neurons that die do so within a maximal interval of 5 d after their birth, irrespective of the age of genesis or of the cell type. These findings suggest the existence of a cellular clock regulating neuronal death during development. We found also that neurons migrate in no less than 3 d to the GCL, where a majority of cells that die remain a maximum of 2 d. This fast cellular turnover implies that the magnitude of neuronal death is far greater than previously believed.
Nitric oxide delays the death of trophic factor-deprived PC12 cells and sympathetic neurons by a cGMP-mediated mechanism
Authors: Farinelli SE , Park DS , Greene LA
Published: April 2018

We have used cultured PC12 cells and rat sympathetic neurons as model systems to examine the regulation of neuronal cell death and survival. Because nitric oxide (NO) may be involved in nerve growth factor (NGF) signaling in PC12 cells, we tested NO-generating compounds for their ability to protect PC12 cells and sympathetic neurons from death after withdrawal of trophic support. Three such agents, S-nitroso-N- acetylpenicillamine (SNAP), diethylenetriamine NO adduct (DETA-NO), and sodium nitroprusside provide (SNP), were found to promote complete short-term survival after removal of serum from naive PC12 cells and of NGF from neuronally differentiated PC12 cells and sympathetic neurons. One major target of NO action is guanylate cyclase, which is activated by nitrosylation of its heme prosthetic group. We observed that inhibition of guanylate cyclase blocks the protective effects of the NO generators on trophic factor-deprived PC12 cells and sympathetic neurons without preventing NGF-induced survival. We also found that permeant cGMP analogs and an inhibitor of cGMP-specific phosphodiesterase enhance cell survival, suggesting that the protective effects of NO are mediated by activation of guanylate cyclase and increased intracellular cGMP. N-Nitro-L-arginine methyl ester, a NO synthase inhibitor, did not block NGF-promoted PC12 cell or sympathetic neuron survival. These findings indicate that like NGF, NO has survival- promoting actions on neurons but that the two agents work by initially independent mechanisms.
GDNF slows loss of motoneurons but not axonal degeneration or premature death of pmn/pmn mice
Authors: Sagot Y , Tan SA , Hammang JP , Aebischer P , Kato AC
Published: April 2018

Glial cell line-derived neurotrophic factor (GDNF), a member of the TG F-beta superfamily, has been shown to be a highly potent neurotrophic factor that enhances survival of various neuronal cell types including motoneurons. To assess its therapeutic potential in treating neurodegenerative diseases such as amyotrophic lateral sclerosis, we treated mutant mice displaying motoneuron degeneration (progressive motor neuropathy; pmn) with encapsulated GDNF-secreting cells. Effects of GDNF treatment on pmn/pmn mice were compared with previous results obtained with ciliary neurotrophic factor (CNTF) [Sagot Y, Tan SA, Baetge E, Schmalbruch H, Kato AC, Aebischer P (1995) Eur J Neurosci 7:1313–1322]. In contrast to CNTF, GDNF did not increase the lifespan of pmn/pmn mice. However, GDNF significantly reduced the loss of facial motoneurons by 50%, a value similar to what was observed when CNTF was administered to the pmn/pmn mice. Surprisingly, myelinated axon counts revealed that GDNF had no effect on nerve degeneration. Therefore, despite its potential in rescuing motoneuron cell bodies, the inability of GDNF to prevent nerve degeneration in pmn/pmn mice suggests that its usefulness in the treatment of motor neuron diseases may be restricted to cotreatment with other factors that act on the nerve process.