bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence that protein constituents of postsynaptic membrane specializations are locally synthesized: analysis of proteins synthesized within synaptosomes
Authors: Rao A , Steward O
Published: March 2018

Previous studies have led to the hypothesis that some proteins of the postsynaptic membrane are locally synthesized at postsynaptic sites. To evaluate this hypothesis, synaptosome fractions that included fragments of dendrites were allowed to incorporate labeled amino acid into protein. The labeled synaptosomes were then subfractionated to the level of the synaptic plasma membrane (SPM) and then the synaptic junctional complex (SJC). The specific activity (cpm/microgram protein) of the synaptosome fraction and its subfractions was assessed by scintillation counting and protein assay, and labeled polypeptides were characterized by SDS-PAGE and fluorography. The contribution of mitochondrial and eucaryotic protein synthesis to the overall incorporation was evaluated using cycloheximide (CYC), a eucaryotic protein synthesis inhibitor, and chloramphenicol (CAP), a mitochondrial protein synthesis inhibitor. Both the SPM and the SJC subfractions obtained from labeled synaptosomes contained labeled polypeptides. The SPM from labeled synaptosomes had a specific activity approximately equal to that of other nonmitochondrial membrane components of the synaptosome. Thus, labeling of the SPM was not due to contamination by these other labeled membrane components. The mitochondrial fraction had the highest specific activity of the membrane components of the labeled synaptosome, but the specific activity was reduced by 47% in mitochondrial fractions from CAP-treated synaptosomes, while the specific activity of the SPM was not reduced by this treatment. Thus, SPM labeling is not due to mitochondrial contamination. The specific activity of the detergent-insoluble SJC was comparable to that of the SPM from which it was derived. The possibility of labeling of SPM and SJC by contamination with soluble proteins was assessed by adding labeled soluble proteins to a cold synaptosome preparation that was then subfractionated to obtain the SPM and SJC. There was no detectable binding of labeled soluble proteins to the SPM or SJC. These results support the hypothesis that some synaptic proteins are locally synthesized. Fluorographs of SDS gels of SPM from labeled synaptosomes revealed labeled bands at approximate molecular weights of 14, 18, 26, 28, 36, 38, 42, 45, 55, 60, and 116 kDa. Six of these labeled polypeptides at 38, 42, 45, 55, 60, and 116 kDa were still evident in fluorographs of the synaptic junctional complex from labeled synaptosomes. None of these labeled bands were seen in fluorographs of SPM and SJC from CYC-treated synaptosomes, whereas they were still present in fluorographs of CAP-treated synaptosomes. These labeled polypeptides are therefore produced by eucaryotic ribosomal systems.
Axonal transport kinetics and posttranslational modification of synapsin I in mouse retinal ganglion cells
Authors: Petrucci TC , Macioce P , Paggi P
Published: March 2018

Synapsin I is a neuron-specific phosphoprotein primarily localized at the presynaptic terminals, where it is thought to play an important role in the mechanisms involved in neurotransmitter release. Its interaction with cytoskeletal proteins and with small synaptic vesicles is regulated in vitro by phosphorylation by a calcium/calmodulin- dependent kinase. Here, we present the first evidence that, in the mouse retinal ganglion cells, synapsin I, moving along the axon with the slow component of axonal transport, is phosphorylated in vivo at both the head and tail regions. In addition, our data suggest that, after synapsin I has reached the nerve endings, the relative proportion of differently phosphorylated molecules of synapsin I changes, and that these changes lead to a decrease of the overall content of phosphorus. The more basic forms, here collectively referred to as beta-forms, become predominant at the terminals after 7 d postlabeling, when the bulk of transported synapsin I has entered the superior colliculus. Along the axon, phosphorylation could be functional in preventing synapsin I from forming, with actin, a dense meshwork that would restrict organelle movement. On the other hand, at the terminals, the dephosphorylation-phosphorylation of synapsin I may regulate the clustering of small synaptic vesicles and modulate neurotransmitter release by controlling the availability of small synaptic vesicles for exocytosis.
Social deprivation of infant rhesus monkeys alters the chemoarchitecture of the brain: I. Subcortical regions
Authors: Martin LJ , Spicer DM , Lewis MH , Gluck JP , Cork LC
Published: March 2018

Rhesus monkeys (Macaca mulatta) reared during the first year of life without social contact develop persistent stereotyped movements, self- directed behaviors, and psychosocial abnormalities, but neurobiological mechanisms underlying the behaviors of socially deprived (SD) monkeys are unknown. Monkeys were reared in total social deprivation for the first 9 months of life; control monkeys were reared socially (SR) with mothers and peers. Subjects were killed at 19-24 yr of age. Because the behaviors of SD monkeys are reminiscent of changes in striatal or amygdalar function, we used immunocytochemistry for substance P (SP), leucine-enkephalin (LENK), somatostatin, calbindin, and tyrosine hydroxylase (TH) to evaluate qualitatively and quantitatively patterns of neurotransmitter marker immunoreactivity within subcortical regions. In SD monkeys, the chemoarchitecture of the striatum was altered. Neuronal cell bodies and processes immunoreactive for SP and LENK were depleted markedly in patch (striosome) and matrix regions of the caudate nucleus and putamen; the average density of SP-immunoreactive neurons was reduced 58% relative to SR monkeys. Calbindin and TH immunoreactivities were diminished in the matrix of caudate and putamen of SD monkeys. TH-immunoreactive neurons, but not cresyl violet-stained neurons, in the substantia nigra pars compacta were decreased (43%) in SD monkeys. Peptide-immunoreactive terminals were reduced in the globus pallidus and substantia nigra in SD monkeys. The nucleus accumbens was the least affected of striatal regions. Striatal somatostatin immunoreactivity wa qualitatively and quantitatively similar in SD and SR monkeys. Several regions, for example, bed nucleus of the stria terminalis, amygdala, and basal forebrain magnocellular complex, that were in the same sections and are enriched in these markers did not appear altered in SD monkeys, suggesting a regional specificity for vulnerability. The altered chemoarchitecture of some basal ganglia regions in adult monkeys that experienced social deprivation as infants suggests that the postnatal maturation of neurotransmitter phenotypes in some structures is influenced by social environment. Abnormal motor and psychosocial behaviors resulting from this form of social/sensory deprivation may result from alterations in peptidergic and dopaminergic systems within the basal ganglia.
Distribution of Ca2+ and Na+ conductances during neuronal differentiation of chick DRG precursor cells
Authors: Gottmann K , Rohrer H , Lux HD
Published: March 2018

The distribution of Ca2+ and Na+ conductances on neuronal precursor cells was investigated during differentiation. Ionic conductances on the soma or on the growth cone were isolated by superfusing all other parts of the cells with sucrose. Conductances on the neuritic shaft were detected as additional conductances after removing sucrose from the neuritis shaft. Neuronal precursor cells were isolated from chick dorsal root ganglia by selectively killing differentiated neurons. Cultured precursor cells differentiated into morphological and functional mature neurons. Functionally undifferentiated precursor cells (during the first 10 hr in culture) expressed only low-voltage- activated (LVA) Ca2+ currents. High-voltage-activated (HVA) Ca2+ and Na+ currents appeared delayed after more than 10 hr in culture. Voltage- dependent conductances, if expressed by a cell, were present on all parts of the surface membrane at all stages of differentiation. LVA Ca2+ conductances were well represented on the growth cone as well as on the soma in functionally undifferentiated precursor cells. During differentiation of precursor cells, LVA Ca2+ and HVA Ca2+ as well as Na+ conductances were expressed on the somatic membrane, on the neuritic shaft, and on the growth cone. These results demonstrate the expression of Ca2+ channels on growth cones during differentiation.
The generation of monoclonal antibodies that bind preferentially to adrenal chromaffin cells and the cells of embryonic sympathetic ganglia
Authors: Carnahan JF , Patterson PH
Published: March 2018

Adrenal chromaffin cells, sympathetic neurons, and small intensely fluorescent (SIF) cells are each derived from the neural crest, produce catecholamines, and share certain morphological features. These cell types are also partially interconvertible in cell culture (Doupe et al., 1985a,b; Anderson and Axel, 1986). Thus, these cells are said to be members of the sympathoadrenal (SA) lineage and could share a common progenitor. To investigate the origins of this lineage further, we used the cyclophosphamide immuno-suppression method (Matthew and Patterson, 1983) to generate five monoclonal antibodies (SA1-5) that bind strongly to chromaffin cells, with little or no labeling of sympathetic neurons or SIF cells in frozen sections from adult rats. Competition experiments indicate that these antibodies bind to at least three distinct epitopes in tissue sections. The SA antibodies also label most of the cells of embryonic sympathetic ganglia and adrenal primordia. Labeling of sympathetic ganglia appears as the cells initially coalesce and express high levels of tyrosine hydroxylase (TH). Not all TH+ cells in the embryo are SA 1-5+, however; carotid body SIF cells, nodose ganglion TH+ cells, and the transiently TH+ cells in the dorsal root ganglia do not display detectable SA 1-5 labeling. Thus, the expression of these markers for the SA 1-5 lineage is selective. SA antigen expression is hormonally controlled; removal of glucocorticoid and addition of NGF to cultured adrenal chromaffin cells result in the loss of SA 1-5 labeling. These results suggest that the presumed precursors for sympathetic neurons and SIF cells initially express chromaffin cell markers.
The monoclonal antibody E587 recognizes growing (new and regenerating) retinal axons in the goldfish retinotectal pathway
Authors: Vielmetter J , Lottspeich F , Stuermer CA
Published: March 2018

E587 is a new monoclonal antibody against a 200 kDa cell-surface glycoprotein in the fish retinotectal pathway. The E587 antigen probably belongs to the class of cell adhesion molecules, and more specifically, to the family of L1-like molecules. The immunopurified protein is recognized by the antibody against the HNK1/L2 sugar epitope (associated with most cell adhesion molecules) and by a polyclonal antiserum against chick G4, which is related to the cell adhesion molecule L1 in mouse. Moreover the NH2-terminal sequence of E587 shows similarity with L1 and Ng-CAM. The E587 immunostaining pattern in the fish retinotectal pathway suggests that the E587 antigen is a growth- associated molecule on fish retinal axons. In fish embryos, all retinal axons are labeled. In adult fish, however, only the young axons from newly added ganglion cells carry E587 staining. After optic nerve transection (ONS) and retinal axonal regeneration, all axons reexpress the E587 antigen into their terminal processes in the tectal retinorecipient layers. The reexpression of the E587 antigen is temporally regulated, and E587 immunoreactivity declines by 7 months and disappears at 12 months after ONS. We hypothesize that the E587 antigen may mediate axon-axon associations. In its restricted appearance on young axons in normal adult fish, it may contribute to the selective fasciculation of the newest axons with young axons and thus participate in the creation of the age-related fiber organization in the fish optic nerve.
Functional organization of human supplementary motor cortex studied by electrical stimulation
Authors: Fried I , Katz A , McCarthy G , Sass KJ , Williamson P , Spencer SS , Spencer DD
Published: March 2018

The presence of somatotopic organization in the human supplementary motor area (SMA) remains a controversial issue. In this study, subdural electrode grids were placed on the medial surface of the cerebral hemispheres in 13 patients with intractable epilepsy undergoing evaluation for surgical treatment. Electrical stimulation mapping with currents below the threshold of afterdischarges showed somatotopic organization of supplementary motor cortex with the lower extremities represented posteriorly, head and face most anteriorly, and the upper extremities between these two regions. Electrical stimulation often elicited synergistic and complex movements involving more than one joint. In transitional areas between neighboring somatotopic representations, stimulation evoked combined movements involving the body parts represented in these adjacent regions. Anterior to the supplementary motor representation of the face, vocalization and speech arrest or slowing of speech were evoked. Various sensations were elicited by electrical stimulation of SMA. In some cases a preliminary sensation of “urge” to perform a movement or anticipation that a movement was about to occur were evoked. Most responses were contralateral to the stimulated hemisphere. Ipsilateral and bilateral responses were elicited almost exclusively from the right (nondominant) hemisphere. These data suggest the presence of combined somatotopic organization and left-right specialization in human supplementary motor cortex.
Acetylcholine receptors in extrajunctional regions of innervated muscle have a slow degradation rate
Authors: Salpeter MM , Marchaterre M
Published: March 2018

Scanning EM autoradiography was used to determine the degradation rate of extrajunctional ACh receptors (AChRs) in innervated sternomastoid muscles of the mouse. We report that in innervated muscles, extrajunctional AChRs have a slow degradation rate (t1/2, approximately 8 d), similar to that seen at the neuromuscular junction. We conclude that slowly degrading AChRs (Rs) need not be localized at the specialized structure of the nerve-muscle junction. Degradation of extrajunctional as well as junctional AChRs may depend primarily on the state of innervation of the muscle.
Protons selectively induce lasting excitation and sensitization to mechanical stimulation of nociceptors in rat skin, in vitro
Authors: Steen KH , Reeh PW , Anton F , Handwerker HO
Published: March 2018

In ischemic and in inflamed tissues, pH levels down to 5.4 have been measured, and this local acidosis may contribute to pain and hyperalgesia in disease states. To evaluate the role of acid pH in nociception, we have studied identified primary afferents in a rat skin- saphenous nerve preparation in vitro where the receptive fields can be superfused at the highly permeable corium side with controlled solutions. The nerve endings were exposed to CO2-saturated synthetic interstitial fluid (SIF;pH 6.1) and to carbogen-gassed SIF phosphate buffered to different acid pH levels (5 min duration, 10 min intervals). Mechanical thresholds were repeatedly tested in a “blind” fashion by von Frey hair stimulation. Low-threshold mechanosensitive A beta- (n = 12) and A delta-fibers (n = 11) were not excited or sensitized by acid pH levels. In 24 of 96 nociceptor type C- and A delta-fibers, irregular low-frequency discharge with poor response characteristics was induced. However, a distinct subpopulation of mechanoheat sensitive, “polymodal” C-units (n = 25; 38%) showed stimulus-related responses increasing with proton concentration and encoding the time course of the pH change. Threshold levels were found to range from pH 6.9 to 6.1; mean maximum discharge was at pH 5.2. All such fibers responded to CO2 as well as to phosphate-buffered solution at the same pH 6.1. The CO2 responses, however, displayed significantly shorter latencies and more pronounced dynamic phases. The carboanhydrase blocker acetazolamide markedly delayed and reduced the CO2 responses. Prolonged application of acid pH (30 min) evoked nonadapting activity irrespective of oxygen supply. Many, but certainly not all, fibers sensitive to protons were also driven by capsaicin (10(- 6) M, 10(-5) M) and vice versa. Repeated or prolonged treatment with low pH induced a significant and lasting decrease of the mechanical (von Frey) thresholds in almost all C-fibers tested (from 35 to 16 mN, on average), and this occurred whether or not a fiber was excited by protons. The sensitizing effect was more pronounced the higher the initial von Frey thresholds (0.75 rank correlation). This sensitization to mechanical stimulation was in contrast to the combined action of other inflammatory mediators, bradykinin, 5-HT, histamine and prostaglandin E2. In conclusion, we suggest that pH sensitivity of nociceptors may be an important source of pain and hyperalgesia.
Kappa-opioids decrease excitatory transmission in the dentate gyrus of the guinea pig hippocampus
Authors: Wagner JJ , Caudle RM , Chavkin C
Published: March 2018

In the guinea pig hippocampus, kappa 1-opioid binding sites were primarily localized in the molecular layer of the dentate gyrus as shown by autoradiography using either the kappa 1-selective radioligand 3H-U69,593 or the nonselective radioligand 3H-diprenorphine in the presence of unlabeled mu- and delta-blocking ligands. In this region, the electrophysiological effects of kappa 1-receptor activation were identified using extracellular and intracellular recordings of dentate granule cell responses. The amplitude of the extracellularly recorded population spike was reduced by U69,593 with an EC50 of 26 nM; this effect was reversible and blocked by the opioid antagonist naloxone. The kappa 1-selective antagonist norbinaltorphimine also blocked the effect of U69,593 with an apparent equilibrium dissociation constant (Ki) of 0.26 nM determined by Schild analysis in the physiologic assay. This value agreed well with the Ki for norbinaltorphimine at kappa 1- binding sites measured by radioligand binding displacement (0.24 nM). These results indicate that the electrophysiologic response observed was likely mediated by kappa 1-receptors. As seen with U69,593, dynorphin B, an endogenous opioid peptide that is present in the dentate gyrus, also inhibited the population spike response. mu- and delta-selective opioid agonists had no effect on the amplitude of the maximally evoked response. Intracellular recordings of dentate granule cells showed no direct effects of U69,593 on the granule cells themselves. However, analysis of synaptic potentials revealed that U69,593 significantly reduced the amplitude of glutaminergic EPSPs evoked by afferent stimulation without affecting IPSP amplitudes. The specific effect of U69,593 application on granule cell EPSPs indicates that presynaptic kappa 1-receptor activation inhibits glutamate release from perforant path terminals in the molecular layer of the dentate gyrus. These results suggest that endogenous dynorphins present in the granule cells may act as feedback inhibitors of the major excitatory input to the dentate gyrus.
Plasticity in the barrel cortex of the adult mouse: effects of chronic stimulation upon deoxyglucose uptake in the behaving animal
Authors: Welker E , Rao SB , Dorfl J , Melzer P , van der Loos H
Published: March 2018

We investigated experience-dependent regulation of neuronal activity in the whisker-to-barrel pathway of the adult mouse using the autoradiographic deoxyglucose (DG) method. Animals were placed in the Lausanne whisker stimulator, and three of their whisker follicles were passively stimulated for a period of 1, 2, or 4 d. After this period, mice received a dose of DG and were placed in a cage containing a pile of wooden sticks. Mice that underwent the same procedure except the passive stimulation served as controls. Patterns of stimulus-dependent DG uptake were studied in the somatosensory cortex and in the trigeminal sensory brainstem complex. DG uptake in the barrels corresponding to the passively stimulated whiskers was lower than in controls. This decrease was present throughout the radial extent of a barrel column and was observed in all passively stimulated animals. Quantitative analysis confirmed these observations and, furthermore, showed a statistically significant decrease in DG uptake in barrels neighboring the passively stimulated ones. In half of the animals, the brainstem nuclei showed a decreased DG uptake in the representation of the passively stimulated whiskers, whereas in the other animals the pattern of DG uptake was as in controls. We propose that the signs of cortical plasticity are due to a mechanism that operates in layer IV and functions as a gate for peripheral sensory activity to enter cortical circuitry.
Characterization of the integration time for the stabilization of long- term potentiation in area CA1 of the hippocampus
Authors: Colino A , Huang YY , Malenka RC
Published: March 2018

In area CA1 of the hippocampus, synaptic activation of NMDA receptors during postsynaptic depolarization can generate either a decremental synaptic potentiation termed short-term potentiation (STP) or stable, long-term potentiation (LTP). Examining the relationship between these two forms of synaptic enhancement should provide information about the intracellular processes responsible for the stabilization of LTP. Using the hippocampal slice preparation, initial experiments confirmed that STP can be generated either by a weak tetanus or by pairing a single EPSP with postsynaptic depolarization. Following the generation of submaximal LTP, application of a weak, STP-inducing tetanus resulted in STP (not LTP), suggesting that the processes responsible for stabilizing LTP must be activated during induction and cannot be accessed at later times. To determine the interval over which processes activated during STP can be integrated and result in stable LTP (the “integration time” for the stabilization of LTP), a fixed number of afferent stimuli were given at varying intervals (5–60 sec) during postsynaptic depolarization. Using either extracellular or whole-cell recording, LTP was rarely (11% of experiments) elicited at 1 min intervals and frequently (76% of experiments) elicited at 10 sec intervals. These results indicate that following a single EPSP during postsynaptic depolarization, the processes responsible for the stabilization of LTP decay significantly within approximately 1 min, although this value may depend on the level of activation of the requisite intracellular processes.
Cervicothalamic tract terminals are enriched in glutamate-like immunoreactivity: an electron microscopic double-labeling study in the cat
Authors: Broman J , Ottersen OP
Published: March 2018

The distribution of glutamate-like immunoreactivity (Glu-LI) in the thalamic ventral posterolateral nucleus (VPL) of cats was studied with the EM immunogold technique in order to identify nerve terminal populations that may use glutamate as a neurotransmitter. The investigation was focused on cervicothalamic tract (CTT) terminals, which were labeled by WGA-HRP transported anterogradely from injection sites in the lateral cervical nucleus (LCN). The amount of Glu-LI in different profiles was evaluated quantitatively by counting the number of gold particles and then calculating the areal density of gold particles over different profile types. The highest density of gold particles was found over terminals with morphologic characteristics of terminals of cortical origin (RS terminals), a finding that further supports the glutamatergic nature of these terminals suggested by previous studies. Enrichment of Glu-LI was also found in CTT terminals and in non-peroxidase-labeled terminals with the same morphologic characteristics as CTT terminals (RL terminals). The labeling density over these terminals was about twice the average tissue density of gold particles. The labeling density over large VPL neuronal cell bodies was on average 127%, and that over vesicle-containing dendritic appendages and truncs (presynaptic dendrites) about 80%, of the average tissue density of gold particles. Immunogold labeling with antiserum against glutamine (Gln) indicated low levels of Gln-like immunoreactivity in CTT terminals and a high Glu:Gln ratio as compared to astrocytes and the average Glu:Gln ratio in the VPL. The present findings provide further support for a transmitter role of glutamate in terminals of ascending somatosensory afferents to the VPL, including the CTT. Taken together with previous findings of an enrichment of Glu-LI in terminals of the spinocervical tract (Broman et al., 1990), our results suggest that synaptic transmission in the spinocervicothalamic pathway is dependent on the release of glutamate both at the levels of the LCN and the VPL.
Cellular mechanisms underlying cholinergic and noradrenergic modulation of neuronal firing mode in the cat and guinea pig dorsal lateral geniculate nucleus
Authors: McCormick DA
Published: March 2018

The functional properties, ionic basis, and possible convergence and interaction of postsynaptic actions mediated by muscarinic and alpha 1- adrenergic receptors were examined in cat and guinea pig dorsal lateral geniculate (LGNd) neurons maintained in thalamic slices in vitro. The possible involvement of GTP-binding proteins was also examined. Extracellular recordings from cat LGNd revealed the presence of two subpopulations of neurons. The most prevalent generated rhythmic high- frequency (300–500 Hz) bursts of two to six action potentials each, with an interburst frequency of 1–3 Hz. Intracellular recordings revealed that this activity is typical of thalamocortical relay cells in the apparent absence of neuromodulatory input. Application of ACh or noradrenaline (NA) to rhythmically oscillating neurons in the cat LGNd resulted in cessation of this activity followed by the appearance of single spike firing. Intracellular recordings revealed that this change in firing mode was associated with a depolarization of the neuron out of the range of intrinsic rhythmic oscillation and into or near the single spike firing mode. The voltage characteristics of the current underlying the cholinergic and noradrenergic slow depolarization were investigated in guinea pig LGNd neurons. Application of the muscarinic agonist acetyl-beta-methylcholine (MCh) to presumed relay neurons resulted in a hyperpolarization due to the activation of an outward K+ current. This response was followed by a slow depolarization due to reduction of a relatively non-voltage-dependent potassium current distinct from IM and IAHP. Application of NA resulted in a slow depolarization that was also associated with reduction of this relatively linear K+ current. The MCh- and NA-induced slow depolarizations displayed the property of occlusion, indicating convergence of action. However, these responses were mediated by pharmacologically distinct receptors since the MCh-induced reduction in K+ current was blocked by scopolamine while that induced by NA was blocked by the alpha 1-adrenoceptor antagonist prazosin. Intracellular diffusion of GTP-gamma-S resulted in the inward current responses to NA and MCh being irreversible, suggesting the possible involvement of a G- protein. Prior exposure to pertussis toxin did not affect the inward current response to NA and MCh, while the outward K+ current responses induced by application of MCh or the GABAB agonist baclofen were blocked. These results reveal that activation of muscarinic or alpha 1- adrenergic postsynaptic receptors in the LGNd result in a shift in firing mode from rhythmic oscillation to tonic single spike activity through a decrease in a relatively linear K+ current mediated through a pertussis toxin-insensitive G-protein.(ABSTRACT TRUNCATED AT 400 WORDS)
Presynaptic calcium signals and transmitter release are modulated by calcium-activated potassium channels
Authors: Robitaille R , Charlton MP
Published: March 2018

The regulation of synaptic transmission by Ca(2+)-activated potassium (gKca) channels was investigated at the frog neuromuscular junction (nmj). Charybdotoxin (CTX), a blocker of certain types of gKca channels, induced a twofold increase of transmitter release. Similar results were obtained with purified natural toxin, synthetic toxin, and recombinant toxin. Apamin, a blocker of a different type of gKca channel, did not alter transmitter release. CTX was ineffective after intraterminal Ca2+ buffering was increased by application of the membrane-permeant Ca2+ buffer dimethyl-BAPTA-AM. By itself, the permeant buffer first caused a slight increase in transmitter release before release was eventually decreased. This increase of release did not occur when the buffer was applied in the presence of CTX or Ba2+, another gKca channel blocker. Stimulus-evoked entry of Ca2+ in nerve terminals, detected with the fluorescent Ca2+ indicator FLUO-3, was increased after blockade of gKca channels by CTX. CTX had no effect on the amount or the time course of synaptic depression. The results are consistent with the hypothesis that CTX-sensitive gKca channels normally narrow the presynaptic action potential and thus, by indirectly regulating Ca2+ entry, can serve as powerful modulators of evoked transmitter release. In order to affect presynaptic action potentials, the gKca channels must be located close to Ca2+ channels.
Correlation between intrinsic firing patterns and thalamocortical synaptic responses of neurons in mouse barrel cortex
Authors: Agmon A , Connors BW
Published: March 2018

We used a thalamocortical slice preparation to record both spike trains and synaptically evoked responses from neurons of mouse barrel cortex. Cells were classified as regular spiking (RS), intrinsically bursting (IB), or fast spiking (FS) according to their temporal firing patterns when injected with current. RS cells were further separated into two subtypes, RS1 and RS2 cells, the latter encountered only in the infragranular layers. Synaptic responses were elicited by focal electrical stimuli in the ventrobasal nucleus of the thalamus (VB) while holding the cells at different membrane potentials. Postsynaptic potentials were classified as excitatory (EPSPs) or inhibitory (IPSPs), and their latencies were measured from the onset of the extracellularly recorded fiber volley in layer IV. EPSPs fell into three groups, according to latency. Those in the early cluster had latencies shorter than 1 msec and were coincident with the postsynaptic layer IV population response; they were considered monosynaptic. A second group, with latencies between 1.3 and 2.5 msec, were coincident with all IPSPs and were classified as disynaptic. The rest had latencies longer than 5 msec and were considered polysynaptic. The synaptic order of a cell was correlated with its laminar position and its electrophysiological class. Specifically, monosynaptic responses were restricted to infragranular RS cells and to FS cells, while disynaptic EPSPs were found in supragranular RS cells and in IB cells. Disynaptic IPSPs were found in both deep and superficial layers; in the deep layers they nearly always followed monosynaptic EPSPs, while in the superficial layers they were mostly found in isolation. We conclude that the intrinsic spiking characteristics of a neuron are an important determinant of its position in the cortical circuit and may have a substantial role in determining its response properties.
A distinct type of GD3+, flat astrocyte in rat CNS cultures
Authors: Vaysse PJ , Goldman JE
Published: March 2018

We have identified what is apparently a distinct type of astrocyte in primary cultures from several regions of the neonatal rat CNS. These cells express GD3 ganglioside for long periods in vitro, and are GFAP+, but do not express the oligodendrocyte antigens O4 or galactocerebroside (GC). The majority, but not all, are A2B5+. The cells grow in a flat, highly spread morphology with many thin cytoplasmic processes. Gene transfer with a replication-deficient retrovirus combined with immunostaining for astro- and oligodendroglial markers (antibodies to GFAP, GD3 ganglioside, GC, and the A2B5 and O4 antibodies) demonstrated that in the neonatal rat CNS cultures these cells are clonally separate from oligodendrocytes and from the majority of (GD3-) astrocytes. The clonal analysis suggests a distinct progenitor cell and a distinct developmental sequence for these astrocytes.
Modulation of la EPSP amplitude: the effects of chronic synaptic inactivity
Authors: Webb CB , Cope TC
Published: March 2018

In this study, we test the hypothesis that monosynaptic connections between la afferents and spinal motoneurons are strengthened by chronic disuse. Impulse activity along the medial gastrocnemius (MG) nerve was blocked for 2 weeks using TTX delivered by an osmotic minipump to a Silastic cuff placed around the nerve. The duration and specificity of this block were confirmed by chronic EMG recordings from several triceps surae muscles. The effect of TTX-induced inactivity of presynaptic elements on EPSP amplitude was distinguished from the effect of treating the postsynaptic target by comparing the results from heteronymous synaptic connections, where only one or the other element was treated. After 2 weeks of synaptic inactivity, the heteronymous EPSPs generated by MG la afferents in lateral gastrocnemius/soleus (LG-Sol) motoneurons were significantly (p less than 0.005) larger than control values (48%). Sample differences in rheobase current and half-afterhyperpolarization, both of which may covary with EPSP amplitude, did not account for the differences between groups. Segregation of the two samples of motoneurons by rheobase current identified the increase as being confined to those LG-Sol cells whose rheobase fell below 10 nA. In addition, EPSPs generated by untreated LG-Sol la afferents in treated MG motoneurons were significantly enhanced (39%, p less than 0.05). Thus, TTX treatment of either presynaptic or postsynaptic elements increases synaptic strength. This increase in monosynaptic EPSP amplitude following TTX- induced inactivity may reflect an alteration intrinsic to the la afferent to motoneuron synapse, but influences from extrinsic sources cannot be discounted.
Color selectivity of neurons in the inferior temporal cortex of the awake macaque monkey
Authors: Komatsu H , Ideura Y , Kaji S , Yamane S
Published: March 2018

We tested the color selectivity of neurons in the inferior temporal (IT) cortex of two awake macaque monkeys. A color stimulus was presented at the center of the visual field while the animal performed a fixation task. The responses of single units to various colors were recorded and were plotted in a color space. The color space was based on the CIE (Commission Internationale de l'Eclairage) chromaticity diagram. Quantitative analysis of the color selectivity was performed using a standard set of colors that were evenly distributed in the color space. Sixty-five neurons recorded from eight guide tubes implanted in anterior part of IT cortex were tested quantitatively, and their color selectivity was statistically evaluated. Forty-six of them (71%) were classified as color selective. Color-selective cells responded to some colors but not to others, and we called the responsive region in the color space the “color field.” The location and the size of a cell's color field were largely independent of the luminance of the stimulus. About 80% of the color-selective neurons had color fields consisting of a single responsive region that were restricted in some part of the color space, and the remaining cells had more than one responsive region within the color space. Preferred hues of the color-selective neurons differed from cell to cell. The population of cells together covered nearly all of the color space. There was a tendency for more color-selective cells to be less sensitive to white and desaturated cyans. Some of the cells with color fields that consisted of more than one responsive region were more sensitive to saturation of the stimulus than to its hue. Although previous electrophysiological studies in IT cortex emphasized the pattern selectivity of the neurons, our results suggest that color is an important factor in the role that IT cortex plays in the object recognition.
Dynorphin increases extracellular levels of excitatory amino acids in the brain through a non-opioid mechanism
Authors: Faden AI
Published: March 2018

Administration of dynorphin A-(1–17) (Dyn 1–17), through a microdialysis probe stereotaxically placed into rat hippocampus, caused marked increases in the extracellular levels of glutamate and aspartate. The degree and duration of elevation of these excitatory amino acids (EAA) induced by Dyn 1–17 were dose dependent but were not modified by the centrally active opioid receptor antagonist nalmefene. At comparable doses, Dyn 2–17, which is inactive at the opioid receptor, produced similar alterations in EAA as Dyn 1–17, whereas Dyn 1–8 caused significantly smaller changes of glutamate. Dynorphin and EAAs have each been implicated as pathophysiological factors in brain or spinal cord injuries, with dynorphin's actions shown to involve both opioid and non-opioid components. The present observations indicate a direct potential linkage between dynorphin and excitotoxin mechanisms of CNS injury and provide further support for the concept that dynorphin's pathophysiologic effects may include non-opioid actions of this peptide.
Autoradiographic localization of voltage-dependent sodium channels on the mouse neuromuscular junction using 125I-alpha scorpion toxin. II. Sodium distribution on postsynaptic membranes
Authors: Boudier JL , Le Treut T , Jover E
Published: March 2018

A radioiodinated alpha-scorpion toxin (toxin II from Androctonus australis Hector) (alpha ScTx) was used as a probe for EM autoradiography to study the distribution of voltage-dependent sodium channels (Na+ channel) on the postsynaptic side of the mouse neuromuscular junction. Silver grain distribution was analyzed by the cross-fire method to assess the relative Na+ channel density in each membrane domain measured by stereology. This analysis showed that the maximum Na+ channel density was located on the edge of the synaptic gutter, where it reached about twice the mean density in the postsynaptic fold membrane. Na+ channel densities have been calculated using ACh receptor (AChR) density in fold crests as reference. Sodium channel density on the edge of the synaptic gutter was estimated at about 5000/microns 2. Sodium channel distribution in the postsynaptic folds was compared to AChR distribution using density distribution analysis (Fertuck and Salpeter, 1976). The results confirmed that, as already observed by immunogold labeling (Flucher and Daniels, 1989), there are no Na+ channels on fold crests. Na+ channels are located in the rest of the fold membrane (bottom) and may be distributed according to two possible models. In the first, density would be uniformly high, although lower than on the gutter edge. In the second, density would decrease from the crest border, where the value was that of the gutter edge, to the fold end, where the value would be 50% lower. Based on the latter model, which was the “best-fit model,” we propose that the postsynaptic membrane includes two domains. The first is the fold crest, which contains almost exclusively AChRs. This domain is devoted to reception-transduction of the chemical signal. The second includes both the fold bottom membrane and the perisynaptic membrane. Sodium channel density is highest along the crest border and decreases moving away. Its functions are the integration of postsynaptic potentials and generation-conduction of the muscle action potential.
Axonogenesis and morphogenesis in the embryonic zebrafish brain
Authors: Ross LS , Parrett T , Easter SS
Published: March 2018

We have examined early neuronal differentiation and axonogenesis in the fore- and midbrain of zebrafish embryos to address general issues of early vertebrate brain development. AChE expression and HNK-1 antibody immunoreactivity were used as markers for differentiated neurons and axons, respectively. The pattern of neuronal differentiation followed a stereotyped sequence. AChE-positive cells first appeared between 14 and 16 hr in three small, isolated, bilaterally symmetrical clusters on the surface of the brain. The three clusters--the dorsorostral, ventrorostral, and ventrocaudal clusters--proved to be the progenitors of the telencephalon, ventral diencephalon, and mesencephalic tegmentum, respectively. With further development, more cells were added to these three clusters, and new clusters appeared in the anlage of the epiphysis (18 hr) and in the pituitary and dorsal mesencephalon (by 24 hr). Subsequently, as more neurons differentiated, the gaps of unlabeled cells were reduced; by 48 hr, the cluster boundaries were indistinguishable. Axonogenesis also followed a stereotyped sequence. The first HNK-1-labeled processes arose from the first three clusters of AChE-positive cells and connected the clusters. The earliest axonal growth cones appeared at 16 hr, directed caudally from two to three neurons of the ventrocaudal cluster and pioneering the ventral longitudinal tract. By 18 hr, the tract of the postoptic commissure was initiated by growth cones directed caudally from the ventrorostral cluster toward the ventrocaudal cluster. By 20 hr, axons from the dorsorostral cluster projected ventrally to form the supraoptic tract. The other dorsoventral tracts (the dorsoventral diencephalic tract and the tract of the posterior commissure) became evident between 20 and 24 hr. These observations provide a continuous record of the topological distortions involved in the conversion of the tubular embryonic brain into the contorted adult form. The telencephalon, ventral diencephalon, and hypothalamus originate from the same rostrocaudal level of the neural tube. The pattern of differentiation demonstrated that the early development of the rostral neural tube occurs simultaneously in several independent centers, similar to the overtly segmental development of the hindbrain.
Ca2+ stores in Purkinje neurons: endoplasmic reticulum subcompartments demonstrated by the heterogeneous distribution of the InsP3 receptor, Ca(2+)-ATPase, and calsequestrin
Authors: Takei K , Stukenbrok H , Metcalf A , Mignery GA , Sudhof TC , Volpe P , De Camilli P
Published: March 2018

The nature of second messenger-responsive intracellular Ca2+ stores in neurons remains open for discussion. Here, we demonstrate the existence in Purkinje cells (PCs) of endoplastic reticulum (ER) subcompartments characterized by an uneven distribution of three proteins involved in Ca2+ storage and release: the inositol 1,4,5-trisphosphate (InsP3) receptor, Ca(2+)-ATPase, and calsequestrin. Ca(2+)-ATPase and the InsP3 receptor have a widespread, although not identical, distribution throughout the ER. Calsequestrin is localized throughout the smooth ER and is particularly concentrated in pleiomorphic vesicles with a moderately electron-dense core, which appear to represent a subcompartment of the smooth ER. In double-labeling experiments many of these vesicles were unlabeled by InsP3 receptor antibodies. These results suggest a key role of the ER as an intracellular Ca2+ store and demonstrate a possible structural basis for distinct intracellular Ca2+ pools regulated by different second messengers.
Long-term increases in excitability in the CA1 region of rat hippocampus induced by beta-adrenergic stimulation: possible mediation by cAMP
Authors: Dunwiddie TV , Taylor M , Heginbotham LR , Proctor WR
Published: March 2018

The cellular mechanisms underlying beta-adrenergic potentiation in the CA1 region of the rat hippocampus were examined. A 10 min treatment with isoproterenol (ISO) induced a long-term depolarization of the pyramidal neurons that persisted for at least 30 min of washout; the ISO-induced decrease in the calcium-activated potassium conductance (afterhyperpolarization, or AHP) was similarly prolonged. The long-term excitability changes induced by ISO did not depend upon the calcium concentration of the medium and could be elicited in medium containing as little as 240 microM calcium. The persistent increase in population spike induced by ISO was mimicked by superfusion with several cAMP analogs and by forskolin (which directly activates adenylate cyclase), but not by the inactive dideoxyforskolin. Forskolin and cAMP analogs also induced decreases in AHPs that could be quite prolonged, but did not depolarize pyramidal neurons as consistently as did ISO. We hypothesize that activation of beta-adrenergic receptors in the CA1 region of hippocampus may induce an alteration of the hippocampal “state” that can persist for as long as several hours, during which the induction of other forms of plasticity may be enhanced.
Modulatory effects of FMRF-NH2 on outward currents and oscillatory activity in heart interneurons of the medicinal leech
Authors: Simon TW , Opdyke CA , Calabrese RL
Published: March 2018

Using single-electrode voltage clamp, heart interneurons of the medicinal leech were shown to possess both a rapidly inactivating outward current, IA, and a more slowly inactivating outward current, IK. IA and IK could be separated by their voltage sensitivity and kinetic properties. FMRF-NH2 (Phe-Met-Arg-Phe-NH2) modulates IK by shifting both steady state activation and inactivation to more hyperpolarized potentials, but it does not affect the time constants. IA and IK appear to use K+ as a charge carrier; a change in the external [K+] produced a shift in the apparent reversal potential in the direction predicted with potassium as the charge carrier. Both IA and IK are sensitive to tetraethylammonium (TEA) and 4-aminopyridine (4- AP), and TEA and 4-AP both interfere with the effects of FMRF-NH2 on IK. The biophysical properties of IA and of IK in the presence and absence of FMRF-NH2 were incorporated into a Hodgkin-Huxley model of these currents that could reproduce voltage-clamp data. FMRF-NH2 produces two apparently dissimilar effects on the heartbeat rhythm-- acceleration and disruption. We suggest that both effects could result from the hyperpolarizing shifts in steady state activation and inactivation of IK.
Synaptic plasticity in Drosophila memory and hyperexcitable mutants: role of cAMP cascade
Authors: Zhong Y , Budnik V , Wu CF
Published: March 2018

Activity-dependent synaptic plasticity has been implicated in the refinement and modification of neural circuits during development and learning. Previous studies show that activity-induced facilitation and potentiation are disrupted at larval neuromuscular junctions in the memory mutants dunce (dnc) and rutabaga (rut) of Drosophila. The diminished learning-memory capacity and synaptic transmission plasticity have been associated with altered cAMP levels since dnc affects the cAMP-specific phosphodiesterase and rut affects adenylate cyclase. In this study, the morphology of larval motor axon terminals was examined by anti-HRP immunohistochemistry. It was found that the numbers of terminal varicosities and branches were increased in dnc mutants, which have elevated cAMP concentrations. Such increase was suppressed in dnc rut double mutants by rut mutations, which reduce cAMP synthesis. More profuse projections of larval motor axons have also been reported in double-mutant combinations of ether a go-go (eag) and Shaker (Sh) alleles, which display greatly enhanced nerve activity as a result of reduction in different K+ currents. Therefore, we examined combinations of dnc and rut with eag and Sh mutations to explore the possible relation between activity- and cAMP-induced morphological changes. We found that the expanded projections in dnc were further enhanced in double mutants of dnc with either eag or Sh, an effect that could again be suppressed by rut. The results provide evidence for altered plasticity of synaptic morphology in memory mutants dnc and rut and suggest a role of cAMP cascade in mediating activity-dependent synaptic plasticity.