bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential modulation of TTX-sensitive and TTX-resistant Na+ channels in spinal cord astrocytes following activation of protein kinase C
Authors: Thio CL , Sontheimer H
Published: March 2018

TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) Na+ currents are expressed in high densities (2–8 channels/microns2) in astrocytes cultured from neonatal rat spinal cord. The two Na+ current types differ up to 1000-fold in their TTX sensitivity and additionally have different steady-state activation (g-V) and inactivation (h infinity) curves. Expression of TTX-S and TTX-R Na+ currents is confined to morphologically distinguishable subtypes of astrocytes, allowing characterization of the two types of Na+ currents in isolation: stellate cells express TTX-S Na+ currents and flat pancake cells express TTX-R Na+ currents. Activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) exhibited different effects on TTX-S and TTX-R Na+ currents. PMA reduced peak TTX-S Na+ currents by 25– 60%; in contrast, PMA potentiated peak TTX-R Na+ currents by 60–150%. These effects developed within minutes, and were typically not reversible. PMA effects were voltage dependent, and shifted steady- state Na+ current activation of TTX-R and TTX-S currents by 6 and 18 mV, respectively, but without affecting their steady-state current inactivation (h infinity). PMA treatment also changed Na+ current kinetics. TTX-R current activation (tau m) was faster and current inactivation (tau h) changed from a single- to a bi-exponential after PMA exposure, suggesting that PKC phosphorylation may have activated formerly quiescent Na+ channels. In contrast, TTX-S current activation (tau m) was unchanged, and current inactivation (tau h), on average, decreased by 50% following PMA exposure.(ABSTRACT TRUNCATED AT 250 WORDS)
Preganglionic autonomic motor neurons display normal translocation patterns in slice cultures of embryonic rat spinal cord
Authors: Barber RP , Phelps PE , Vaughn JE
Published: March 2018

Phenotypic differences between somatic and autonomic motor neurons (SMNs and AMNs, respectively) may be modulated by epigenetic factors during the histogenic migrations of these cells. In order to study this problem experimentally, we have developed an in vitro, organotypic slice preparation of embryonic rat spinal cord. Our main objectives for this preparation were to determine whether in vivo patterns of motor neuronal translocations were mimicked in vitro, and, if they were, to begin to analyze such movements with experimental procedures that cannot be applied to the study of mammalian spinal cord development in vivo. Using a modification of existing organotypic slice procedures, we have shown that ChAT, an axonal surface glycoprotein and a low- molecular-weight neurofilament protein are expressed in slices cultured for up to 21 d, thus indicating that spinal neurons remained viable in vitro for relatively long periods. Most importantly, retrograde labeling and subsequent confocal microscopy have shown that the SMNs and AMNs of the slice preparations become segregated ventrodorsally into two distinct subcolumns as seen in vivo. The formation of separate AMN and SMN subcolumns appears to result from a dorsal translocation of AMNs. The fact that this cellular movement occurs in the slice preparation has allowed us to follow this phenomenon directly within the same specimen over a period of days. In addition, we have been able to observe the translocation of AMNs following the removal of their peripheral synaptic targets. The results of these experiments provide further evidence that AMNs undergo a dorsal translocation during the course of spinal cord development, and that this cellular movement may be due to an active migration. They also indicate that AMN movement is not dependent upon continual connection of these neurons with the paravertebral sympathetic ganglia.
Neurotrophin-4/5 is a mammalian-specific survival factor for distinct populations of sensory neurons
Authors: Davies AM , Horton A , Burton LE , Schmelzer C , Vandlen R , Rosenthal A
Published: March 2018

We have studied the effect of human recombinant neurotrophin-4/5 (NT- 4/5) on the survival of developing PNS neurons from embryonic mice and chickens. NT-4/5 transiently supported mouse NGF-dependent trigeminal and jugular neurons at early stages of target field innervation and mouse brain-derived neurotrophic factor (BDNF)-dependent no-dose neurons during the phase of naturally occurring cell death. NT-4/5 was as potent as BDNF in supporting the survival of these neuronal populations. Surprisingly, NT-4/5 was 3 orders of magnitude less potent than BDNF as a survival factor for early chick dorsomedial trigeminal sensory neurons and did not support the survival of chick BDNF- dependent trigeminal mesencephalic or ventrolateral trigeminal sensory neurons at any of the developmental stages tested. Thus, NT-4/5 is a survival factor for certain embryonic mouse cranial sensory neurons. It is the first species-specific neurotrophin to be identified and it can discriminate at high concentrations between different BDNF-responsive chick neurons.
Primary sensory neurons express a Shaker-like potassium channel gene
Authors: Ribera AB , Nguyen DA
Published: March 2018

Developmentally regulated action potentials are a hallmark of Rohon- Beard cells, a class of sensory neurons. In these neurons as well as other primary spinal neurons of Xenopus laevis, the functional differentiation of delayed-rectifier potassium current regulates the waveform of the action potential during the initial day of its appearance. Later, the acquisition of another voltage-dependent potassium current--the A current--plays a major role in regulating excitability. In order to understand the molecular basis of this functional differentiation, genes encoding voltage-dependent potassium currents expressed in the embryonic amphibian nervous system are being cloned. Here, we report the functional properties and developmental localization of a second Xenopus Shaker-like gene (Xenopus Kv 1.1; XSha1; GenBank accession numberM94258) encoding a potassium current. Homology screening with the mouse gene MBK1 led to its isolation. Functional expression in oocytes identifies it as a delayed-rectifier current when assembled as a homooligomeric structure. Specific transcripts corresponding to XSha1 and to the previously cloned gene XSha2 are both detectable by RNase protection in RNA isolated from the embryonic nervous system. However, whole-mount in situ hybridization reveals the temporal pattern and cellular localization of XSha1 but not XSha2 mRNA, suggesting that the concentration of XSha2 transcripts in individual cells is lower than the threshold for detection by this method. Of particular interest, Rohon-Beard cells express XSha1 mRNA. In addition, XSha1 mRNA is detected in several structures containing neural crest derivatives including spinal ganglia, the trigeminal ganglion, and branchial arches; its presence in motor nerves and lateral spinal tracts suggests that both CNS and PNS glia express the mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Naturally Occurring Truncated trkB Receptors Have Dominant Inhibitory Effects on Brain-Derived Neurotrophic Factor Signaling
Authors: Eide Fernette F. , Vining Ella R. , Eide Brock L. , Zang Keling , Wang Xiao-Yun , Reichardt Louis F.
Published: April 2018

trkB encodes a receptor tyrosine kinase activated by three neurotrophins—brain-derived neurotrophic factor (BDNF), neurotrophin-3, and neurotrophin-4/5.In vivo, three isoforms of the receptor are generated by differential splicing—gp145trkBor the full-length trkB receptor, and trkB.T1 and trkB.T2, two cytoplasmically truncated receptors that lack kinases, but contain unique C termini. Although the truncated receptors appear to be precisely regulated during nervous system development and regeneration, their role in neurotrophin signaling has not been directly tested. In this paper, we studied the signaling properties and interactions of gp145trkB, trkB.T1, and trkB.T2 by expressing the receptors in aXenopusoocyte microinjection assay. We found that oocytes expressing gp145trkB, but not trkB.T1 or trkB.T2, were capable of eliciting45Ca efflux responses (a phospholipase C-γ-mediated mechanism) after stimulation by BDNF. When trkB.T1 and trkB.T2 were coexpressed with gp145trkB, they acted as dominant negative receptors, inhibiting the BDNF signal by forming nonfunctional heterodimers with the full-length receptors. An ATP-binding mutant of gp145trkBhad similar dominant inhibitory effects. Our data suggest that naturally occurring truncated trkB receptors function as inhibitory modulators of neurotrophin responsiveness. Furthermore, the homodimerization of gp145trkBappears to be an essential step in activation of the BDNF signaling cascade.
BEN As a Presumptive Target Recognition Molecule during the Development of the Olivocerebellar System
Authors: Chédotal Alain , Pourquié Olivier , Ezan Frédéric , San Clemente Hélène , Sotelo Constantino
Published: April 2018

It has been shown previously that in the chick embryo the cell adhesion molecule BEN/SC1/DM-GRASP is expressed by neurons in the inferior olive (IO) and by their terminal axonal arbors in the cerebellar cortex, the climbing fibers (Pourquié et al., 1992b). Here, new information on the expression of BEN during the formation of the olivocerebellar projection adds the important notion that BEN is also expressed by the cerebellar targets of inferior olivary axons, Purkinje cells (PCs) and deep nuclear neurons. This expression is transient, starting at E7–E8 and vanishing shortly after hatching. More importantly, BEN expression is restricted to precise subsets of IO neurons and PCs. In the cerebellar cortex, BEN-immunoreactive (BEN-IR) structures are not found randomly but are distributed according to a reproducible pattern of parasagittal stripes. A maximum of four distinct sagittal stripes is found in each lobule, along the whole rostrocaudal extent of the cerebellum. Moreover, BEN-expressing stripes belong to two classes; one contains BEN-IR climbing fibers terminating on BEN-IR PCs and the other, more frequent class is solely composed of BEN-IR climbing fibers. Organotypic cultures of isolated cerebella have shown that the expression of BEN in the IO and in the cerebellum arise independently, probably because of an intrinsic developmental program. Thus, the cell adhesion molecule BEN meets all criteria for a recognition molecule involved in the formation of the olivocerebellar projection.
Neuropeptide Y Depresses GABA-Mediated Calcium Transients in Developing Suprachiasmatic Nucleus Neurons: A Novel Form of Calcium Long-Term Depression
Authors: Obrietan Karl , van den Pol Anthony N.
Published: April 2018

In contrast to its inhibitory role in mature neurons, GABA can exert excitatory actions in developing neurons, including mediation of increases in cytosolic Ca2+. Modulation of this excitatory activity has not been studied previously. We used Ca2+digital imaging with Fura-2 to test the hypothesis that neuropeptide Y (NPY) would depress GABA-mediated Ca2+rises in neurons cultured from the developing suprachiasmatic nucleus (SCN). SCN neurons were chosen as a model system for this study because SCN neurons are primarily GABAergic, they express high levels of NPY and GABA receptors, and functionally, NPY causes profound phase-shifts in SCN-generated circadian rhythms.

Vigorous GABA-mediated Ca2+activity was found in young SCN neurons that were maintainedin vitrofor 4–14 d. NPY showed a dose-dependent rapid depression of the amplitude of Ca2+rises generated by GABA released from presynaptic SCN axons. NPY exerted a long-term depression of cytosolic Ca2+in the majority of neurons tested, which lasted more than 1 hr after NPY washout. The magnitude of the NPY depression was dose-dependent. NPY did not affect Ca2+levels when GABAAreceptor activity was blocked by bicuculline; however, when bicuculline and NPY were withdrawn from the perfusion solution, the subsequent Ca2+rise was either significantly reduced or completely absent, suggesting that the NPY receptor was activated in the absence of elevated intracellular Ca2+and GABAAreceptor activity, and that the latent effect of NPY was revealed only after depolarizing GABA stimulation was renewed. Pretreating neurons with pertussis toxin greatly reduced the ability of NPY to depress GABAergic Ca2+rises, suggesting that the NPY modulation of the GABA activity was based largely on a mechanism involving pertussis toxin-sensitive Gi/Goproteins.

NPY receptor stimulation depressed (<30%) postsynaptic Ca2+rises evoked by GABA (20 μm) application in the presence of tetrodotoxin (TTX). The effects of NPY were mimicked by the NPY Y1 receptor agonist [Pro34,Leu31] NPY and the Y2 receptor agonist NPY 13–36 and by peptide YY (PYY). Together, our data suggest that the Y1 and Y2 type NPY receptors act both presynaptically and postsynaptically to depress GABA-mediated Ca2+rises. If related mechanisms exist in peptide modulation of inhibitory GABA activity in mature neurons, this could underlie long-term changes in the behavior of neurons of the SCN necessary for phase-shifting the circadian clock by NPY. NPY also modulated GABA responses in neuroendocrine neurons from the hypothalamic arcuate nucleus. NPY thus can play an important role in evoking long-term depression of GABA-mediated Ca2+activity in these developing neurons, allowing NPY-secreting cells to modulate the effects of GABA on neurite outgrowth, gene expression, and physiological stimulation. This is the first example of such a cellular memory: that is, long-term Ca2+depression based on modulation of depolarizing GABA activity.
Specific Involvement of Human Parietal Systems and the Amygdala in the Perception of Biological Motion
Authors: Bonda Eva , Petrides Michael , Ostry David , Evans Alan
Published: April 2018

To explore the extent to which functional systems within the human posterior parietal cortex and the superior temporal sulcus are involved in the perception of action, we measured cerebral metabolic activity in human subjects by positron emission tomography during the perception of simulations of biological motion with point-light displays. The experimental design involved comparisons of activity during the perception of goal-directed hand action, whole body motion, object motion, and random motion. The results demonstrated that the perception of scripts of goal-directed hand action implicates the cortex in the intraparietal sulcus and the caudal part of the superior temporal sulcus, both in the left hemisphere. By contrast, the rostrocaudal part of the right superior temporal sulcus and adjacent temporal cortex, and limbic structures such as the amygdala, are involved in the perception of signs conveyed by expressive body movements.
Nerve Growth Factor (NGF)-Mediated Protection of Neural Crest Cells from Antimitotic Agent-Induced Apoptosis: The Role of the Low-Affinity NGF Receptor
Authors: Cortazzo Megan H. , Kassis Edmund S. , Sproul Kari A. , Schor Nina Felice
Published: April 2018

Prevention by nerve growth factor (NGF) of apoptotic death in neural cells has been variously ascribed to binding of NGF to its low-affinity (p75) or high-affinity (trkA) receptor or to a cooperative interaction between the two. In a series of studies using, in turn, neuroblastoma cell lines that express only p75, mutant NGF species that bind selectively to either p75 or trkA, and a polyclonal antibody that binds to the NGF-binding domain of p75, we demonstrate that NGF binding to p75 is both necessary and sufficient for the abrogation of apoptosis in neuroblastoma cells treated with antimitotic agents.
Systemic NMDA Receptor Antagonist CGP-40116 Does Not Impair Memory Acquisition but Protects against NMDA Neurotoxicity in Rhesus Monkeys
Authors: Gutnikov Sergei A. , Gaffan David
Published: April 2018

A widely accepted hypothesis is that long-term potentiation (LTP) is a synaptic mechanism of memory. NMDA receptors are critically involved in induction but not maintenance of LTP; therefore, their blockade should impair memory acquisition but not retrieval. In Experiment 1, we investigated the effect of a systemic NMDA receptor antagonist, CGP-40116 [d-isomer of CGP-37849: (E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid (6 mg/kg, i.m.) 60 min before the testing session] on memory acquisition and retrieval by monkeys in the “object-in-place” visual memory task, an analog of human episodic memory. Only a small increase in error rate was produced (<3%), and this increase was observed in both retention and acquisition tests. This deficit is substantially smaller than the previously reported deficit after fornix transection in the same task, and is not specific to memory acquisition. In Experiment 2, we investigated the neuroprotective effect of CGP-40116. NMDA (68 nmol) was injected into the right hippocampus, then CGP-40116 (6 mg/kg) was given intramuscularly, and then NMDA was injected into the left hippocampus. The area of cell loss in CA1 and CA3 fields was smaller in both hemispheres compared with unprotected monkeys (without CGP-40116). Thus, CGP-40116 provides both retrograde and anterograde protection against NMDA neurotoxicity. These data (1) demonstrate that acquisition of episodic memories remains almost intact when an NMDA receptor antagonist is given in a dose sufficient to block NMDA receptors in the hippocampus, and (2) indirectly oppose the hypothesis that NMDA receptor-dependent LTP plays the key role in memory.
Morphology and Physiology of Cortical Neurons in Layer I
Authors: Hestrin Shaul , Armstrong William E.
Published: April 2018

The electrophysiological and morphological properties of layer I neurons were studied in visual cortex slices from 7- to 19-d-old rats using whole-cell recording and biocytin labeling. A heterogeneous population of small, nonpyramidal neurons was found. Approximately one third of the cells we recorded were neurogliaform cells; another third were multipolar neurons with axons descending out of layer I. The remaining cells were heterogeneous and were not classified. In slices from 7- to 10-d-old animals only, we identified Cajal-Retzius cells.

Neurogliaform neurons had a very dense local axonal field, which was largely contained within layer I. Cells with descending axons had a relatively sparse local axonal arbor and projected at least to layer II and sometimes deeper. Spiking in neurogliaform neurons was followed by an afterdepolarizing potential, whereas spiking in cells with descending axons was followed by a slow after-hyperpolarizing potential (AHP). In addition, neurogliaform cells exhibited less spike broadening and a larger fast AHP after single spikes than did cells with descending axons. Generally, cells in layer I received synaptic inputs characterized as either GABA- or glutamate-mediated, suggesting the presence of excitatory and inhibitory inputs.

With their output largely limited to layer I, neurogliaform cells could synapse with other layer I neurons, the most distal dendritic branches of pyramidal cells, or the dendrites of layer II/III interneurons, which invade layer I. Cells with descending axons could contact a wide variety of cortical cells throughout their vertical projection.
Calcium Influx via the NMDA Receptor Induces Immediate Early Gene Transcription by a MAP Kinase/ERK-Dependent Mechanism
Authors: Xia Zhengui , Dudek Henryk , Miranti Cindy K. , Greenberg Michael E.
Published: April 2018

The regulation of gene expression by neurotransmitters is likely to play a key role in neuroplasticity both during development and in the adult animal. Therefore, it is important to determine the mechanisms of neuronal gene regulation to understand fully the mechanisms of learning, memory, and other long-term adaptive changes in neurons. The neurotransmitter glutamate stimulates rapid and transient induction of many genes, including the c-fosproto-oncogene. The c-fospromoter contains several critical regulatory elements, including the serum response element (SRE), that mediate glutamate-induced transcription in neurons; however, the mechanism by which the SRE functions in neurons has not been defined. In this study, we sought to identify transcription factors that mediate glutamate induction of transcription through the SRE in cortical neurons and to elucidate the mechanism(s) of transcriptional activation by these factors. To facilitate this analysis, we developed an improved calcium phosphate coprecipitation procedure to transiently introduce DNA into primary neurons, both efficiently and consistently. Using this protocol, we demonstrate that the transcription factors serum response factor (SRF) and Elk-1 can mediate glutamate induction of transcription through the SRE in cortical neurons. There are at least two distinct pathways by which glutamate signals through the SRE: an SRF-dependent pathway that can operate in the absence of Elk and an Elk-dependent pathway. Activation of the Elk-dependent pathway of transcription seems to require phosphorylation of Elk-1 by extracellular signal-regulated kinases (ERKs), providing evidence for a physiological function of ERKs in glutamate signaling in neurons. Taken together, these findings suggest that SRF, Elk, and ERKs may have important roles in neuroplasticity.
A Diurnal Rhythm of Stimulatory Input to the Hypothalamo–Pituitary–Adrenal System as Revealed by Timed Intrahypothalamic Administration of the Vasopressin V1Antagonist
Authors: Kalsbeek Andries , van Heerikhuize Joop J. , Wortel Joke , Buijs Ruud M.
Published: April 2018

The mammalian suprachiasmatic nuclei (SCN) contain an endogenous pacemaker that generates daily rhythms in behavior and secretion of hormones. We hypothesized that the SCN imposes its circadian rhythm on the rest of the brain via a rhythmic release of its transmitters in its target areas. Previously, we demonstrated a pronounced inhibitory effect of vasopressin (VP), released from SCN terminals in the dorsomedial hypothalamus, on the release of the adrenal hormone corticosterone. In the present study, microdialysis-mediated intracerebral administration of the VP V1-receptor antagonist was used to pursue the study of the mechanisms underlying the circadian control of basal corticosterone release. Using timed administrations of the VP antagonist divided equally over the day/night cycle, we were able to uncover the existence of an additional stimulatory input from the SCN to the hypothalamo–pituitary–adrenal (HPA) axis. Peak activity of this stimulatory SCN input takes place during the second half of the light period, after the daily peak of VP secretion, with a delay of ∼4–6 hr. In all likelihood, the inhibitory and stimulatory circadian input via separate mechanisms affects corticosterone release. Together, these two opposing circadian control mechanisms of the HPA axis enable a precise timing of the circadian peak in corticosterone release.
The Regulation of Heme Turnover and Carbon Monoxide Biosynthesis in Cultured Primary Rat Olfactory Receptor Neurons
Authors: Ingi Tatsuya , Chiang George , Ronnett Gabriele V.
Published: April 2018

Heme oxygenase (HO) converts heme to carbon monoxide (CO) and biliverdin, which is metabolized rapidly to bilirubin. CO is implicated as an intercellular messenger, whereas bilirubin could function as an antioxidant. These cellular functions differ significantly from those of HO in peripheral tissues, in which it degrades heme from senescent erythrocytes, suggesting that the regulation of HO may differ in neurons from that in other tissues. Among neurons, olfactory receptor neurons have the highest level of HO activity. Metabolic labeling with [2-14C]glycine or δ-[3H]aminolevulinic acid ([3H]ALA) was used to investigate heme metabolic turnover and CO biosynthesis in primary cultures of olfactory receptor neurons. The production rates of heme precursors and metabolites from [14C]glycine over 6 hr were (in pmol/mg protein): 100 for ALA, 8.2 for heme, and 2.9 for CO. Taking into account endogenous heme content, the amount of total CO production was determined to be 1.6 nmol/mg protein per 6 hr. Heme biosynthesis usually is subject to end-product negative feedback at the level of ALA synthase. However, metabolic control in these neurons is different. Both heme concentration (heme formation) and HO activity (heme degradation) were enhanced significantly during immature stage of neuronal differentiation in culture. Neuronal maturation, which is accelerated by transforming growth factor-β2 (TGF-β2), suppressed the activities of both heme biosynthesis and degradation. To explore the physiological importance of this endogenous production of CO, we examined the potency of CO as a soluble guanylyl cyclase activator. Exogenous CO (10–30 μm), comparable to endogenous CO production, significantly activated guanylyl cyclase, suggesting that HO activity may regulate cGMP levels in the nervous system.
Comparison of Neurodegenerative Pathology in Transgenic Mice Overexpressing V717F β-Amyloid Precursor Protein and Alzheimer’s Disease
Authors: Masliah Eliezer , Sisk Abbyann , Mallory Margaret , Mucke Lennart , Schenk Dale , Games Dora
Published: April 2018

Overexpression of mutated human amyloid precursor protein (hAPP717V→F) under control of platelet-derived growth factor promoter (PDAPP minigene) in transgenic (tg) mice results in neurodegenerative changes similar to Alzheimer’s disease (AD). To clarify the pathology of these mice, we studied images derived from laser scanning confocal and electron microscopy and performed comparisons between PDAPP tg mice and AD. Similar to AD, neuritic plaques in PDAPP tg mouse contained a dense amyloid core surrounded by anti-hAPP- and anti-neurofilament-immunoreactive dystrophic neurites and astroglial cells. Neurons were found in close proximity to plaques in PDAPP tg mice and, to a lesser extent, in AD. In PDAPP tg mice, and occasionally in AD, neuronal processes contained fine intracellular amyloid fibrils in close proximity to the rough endoplasmic reticulum, coated vesicles, and electron-dense material. Extracellular amyloid fibrils (9–11 nm in diameter) were abundant in PDAPP tg and were strikingly similar to those observed in AD. Dystrophic neurites in plaques of PDAPP tg mouse and AD formed synapses and contained many dense multilaminar bodies and neurofilaments (10 nm). Apoptotic-like figures were present in the tg mice. No paired helical filaments have yet been observed in the heterozygote PDAPP tg mice. In summary, this study shows that PDAPP tg mice develop massive neuritic plaque formation and neuronal degeneration similar to AD. These findings show that overproduction of hAPP717V→F in tg mice is sufficient to cause not only amyloid deposition, but also many of the complex subcellular degenerative changes associated with AD.
Mobility of Synaptic Vesicles in Nerve Endings Monitored by Recovery from Photobleaching of Synaptic Vesicle-Associated Fluorescence
Authors: Kraszewski Kajetan , Daniell Laurie , Mundigl Olaf , De Camilli Pietro
Published: April 2018

In nerve terminals, synaptic vesicles form large clusters anchored to the presynaptic plasmalemma. Recently, FM1-43 photobleaching experiments carried out at frog motor endplates demonstrated lack of lateral intermixing of synaptic vesicles within clusters, even during sustained nerve terminal stimulation (Henkel and Betz, 1995; Henkel et al., 1996b). We now have investigated the mobility of synaptic vesicle membranes during the endocytic limb of their exo-endocytic cycle. To this aim, we have carried out photobleaching experiments on nerve terminals of hippocampal neurons prelabeled with CY3-conjugated antibodies directed against lumenal epitopes of synaptotagmin I. This conjugate is taken up specifically by synaptic vesicle membranes during endocytosis and then is recovered in newly formed synaptic vesicles. Using this method, we show that synaptic vesicle membranes intermix after endocytosis. Staurosporine, which at hippocampal synapses partially inhibits unloading of FM1-43, but does not block uptake of antibody probes, prevents this intermixing. Our results indicate that synaptic vesicle docking and/or fusion with the plasmalemma correlate with the release of their membranes from a restraining matrix that hinders their lateral mobility. They suggest that membrane intermediates involved in synaptic vesicle reformation interact with a distinct, highly dynamic cytoskeleton and that newly formed synaptic vesicles are recaptured at random within vesicle clusters. Staurosporine, by inhibiting mobility within the terminal, may favor recapture of new vesicles near sites of endocytosis.
Glutathione Is Involved in the Granular Storage of Dopamine in Rat PC12 Pheochromocytoma Cells: Implications for the Pathogenesis of Parkinson’s Disease
Authors: Drukarch Benjamin , Jongenelen Cornelis A. M. , Schepens Erik , Langeveld Cornelis H. , Stoof Johannes C.
Published: April 2018

Parkinson’s disease (PD) is characterized by degeneration of dopamine (DA)-containing nigro-striatal neurons. Loss of the antioxidant glutathione (GSH) has been implicated in the pathogenesis of PD. Previously, we showed that the oxidant hydrogen peroxide inhibits vesicular uptake of DA in nigro-striatal neurons. Hydrogen peroxide is scavenged by GSH and, therefore, we investigated a possible link between the process of vesicular storage of DA and GSH metabolism. For this purpose, we used rat pheochromocytoma-derived PC12 cells, a model system applied extensively for studying monoamine storage mechanisms. We show that depletion of endogenous DA stores with reserpine was accompanied in PC12 cells by a long-lasting, significant increase in GSH content the extent of which appeared to be inversely related to the rate of GSH synthesis. A similar increase in GSH content was observed after depletion of DA stores with the tyrosine hydroxylase inhibitor α-methyl-p-tyrosine. In the presence of α-methyl-p-tyrosine, refilling of the DA stores by exogenous DA reduced GSH content back to control level. Lowering of PC12 GSH content, via blockade of its synthesis with buthionine sulfoximine, however, led to a significantly decreased accumulation of exogenous [3H]DA without affecting uptake of the acetylcholine precursor [14C]choline. These data suggest that GSH is involved in the granular storage of DA in PC12 cells and that, considering the molecular characteristics of the granular transport system, it is likely that GSH is used to protect susceptible parts of this system against (possibly DA-induced) oxidative damage.
Dynamic Organization of Endocytic Pathways in Axons of Cultured Sympathetic Neurons
Authors: Overly Caroline C. , Hollenbeck Peter J.
Published: April 2018

Despite the wealth of information about endocytic pathways in non-neuronal cells, little is known about these crucial sorting, recycling, and degradative pathways in neurons. In this report, we analyzed in detail the dynamic steady-state organization of endocytically derived organelles as they progress through the endosomal–lysosomal pathway in axons of live cultured sympathetic neurons. By ratiometric imaging of neurons endocytically labeled with the pH indicator 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS), we demonstrate a trimodal frequency distribution of endocytic organelle pH in axon shafts, indicating two rapid acidification steps in the progression from endocytosis to the lysosome. Axonal branch points display a unimodal organelle pH distribution (mean 6.4), implicating them as meeting places for endocytic organelles and Golgi-derived vesicles or as sorting sites. By following endocytic organelle traffic retrogradely from growth cone to soma, we identified significant transition points in the pathway. Growth cones exhibit a unimodal pH distribution comprised mainly of acidified recycling/sorting endosomes (mean 6.3). However, organelles in the axon shaft immediately adjacent to the growth cone display the distinct trimodal pH distribution of the axon, suggesting that important sorting events occur between these domains. An abrupt increase in organelle acidification occurs in the distal axon 50–150 μm from the growth cone, demonstrating a discontinuous spatial gradient of acidification along axons. Immunofluorescence microscopy reveals that the lysosomal glycoprotein LEP100 is present in axons and is concentrated in two important regions: the proximal axon where the endocytic organelle population is largely acidified, and the same region of the distal axon where substantial acidification occurs.
Adaptive Increase in D3Dopamine Receptors in the Brain Reward Circuits of Human Cocaine Fatalities
Authors: Staley Julie K. , Mash Deborah C.
Published: April 2018

The mesolimbic dopaminergic system plays a primary role in mediating the euphoric and rewarding effects of most abused drugs. Chronic cocaine use is associated with an increase in dopamine neurotransmission resulting from the blockade of dopamine uptake and is mediated by the activation of dopamine receptors. Recent studies have suggested that the D3receptor subtype plays a pivotal role in the reinforcing effects of cocaine. The D3receptor-preferring agonist 7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT) is a reinforcer in rhesus monkeys trained to self-administer cocaine, but not in cocaine-naive monkeys.In vitroautoradiographic localization of [3H]-(+)-7-OH-DPAT binding in the human brain demonstrated that D3receptors were prevalent and highly localized over the ventromedial sectors of the striatum. Pharmacological characterization of [3H]-(+)-7-OH-DPAT binding to the human nucleus accumbens demonstrated a rank order of potency similar to that observed for binding to the cloned D3receptor expressed in transfected cell lines. Region-of-interest analysis of [3H]-(+)-7-OH-DPAT binding to the D3receptor demonstrated a one- to threefold elevation in the number of binding sites over particular sectors of the striatum and substantia nigra in cocaine overdose victims as compared with age-matched and drug-free control subjects. The elevated number of [3H]-(+)-7-OH-DPAT binding sites demonstrates that adaptive changes in the D3receptor in the reward circuitry of the brain are associated with chronic cocaine abuse. These results suggest that the D3receptor may be a useful target for drug development of anti-cocaine medications.
Axonal Action-Potential Initiation and Na+Channel Densities in the Soma and Axon Initial Segment of Subicular Pyramidal Neurons
Authors: Colbert Costa M. , Johnston Daniel
Published: April 2018

A long-standing hypothesis is that action potentials initiate first in the axon hillock/initial segment (AH–IS) region because of a locally high density of Na+channels. We tested this idea in subicular pyramidal neurons by using patch-clamp recordings in hippocampal slices. Simultaneous recordings from the soma and IS confirmed that orthodromic action potentials initiated in the axon and then invaded the soma. However, blocking Na+channels in the AH–IS with locally applied tetrodotoxin (TTX) did not raise the somatic threshold membrane potential for orthodromic spikes. TTX applied to the axon beyond the AH–IS (30–60 μm from the soma) raised the apparent somatic threshold by ∼8 mV. We estimated the Na+current density in the AH–IS and somatic membranes by using cell-attached patch-clamp recordings and found similar magnitudes (3–4 pA/μm2). Thus, the present results suggest that orthodromic action potentials initiate in the axon beyond the AH–IS and that the minimum threshold for spike initiation of the neuron is not determined by a high density of Na+channels in the AH–IS region.
Functional Analysis of Dynactin and Cytoplasmic Dynein in Slow Axonal Transport
Authors: Dillman III James F. , Dabney Lewis P. , Karki Sher , Paschal Bryce M. , Holzbaur Erika L. F. , Pfister K. Kevin
Published: April 2018

The neuron moves protein and membrane from the cell body to the synapse and back via fast and slow axonal transport. Little is known about the mechanism of microtubule movement in slow axonal transport, although cytoplasmic dynein, the motor for retrograde fast axonal transport of membranous organelles, has been proposed to also slide microtubules down the axon. We previously showed that most of the cytoplasmic dynein moving in the anterograde direction in the axon is associated with the microfilaments and other proteins of the slow component b (SCb) transport complex. The dynactin complex binds dynein, and it has been suggested that dynactin also associates with microfilaments. We therefore examined the role of dynein and dynactin in slow axonal transport. We find that most of the dynactin is also transported in SCb, including dynactin, which contains the neuron-specific splice variant p135Glued, which binds dynein but not microtubules. Furthermore, SCb dynein binds dynactinin vitro. SCb dynein, like dynein from brain, binds microtubules in an ATP-sensitive manner, whereas brain dynactin binds microtubules in a salt-dependent manner. Dynactin from SCb does not bind microtubules, indicating that the binding of dynactin to microtubules is regulated and suggesting that the role of SCb dynactin is to bind dynein, not microtubules. These data support a model in which dynactin links the cytoplasmic dynein to the SCb transport complex. Dynein then may interact transiently with microtubules to slide them down the axon at the slower rate of SCa.
Damage, Repair, and Mutagenesis in Nuclear Genes after Mouse Forebrain Ischemia–Reperfusion
Authors: Liu Philip K. , Hsu Chung Y. , Dizdaroglu Miral , Floyd Robert A. , Kow Yoke W. , Karakaya Asuman , Rabow Lois E. , Cui Jian-K.
Published: April 2018

To determine whether oxidative stress after cerebral ischemia–reperfusion affects genetic stability in the brain, we studied mutagenesis after forebrain ischemia–reperfusion in Big Blue transgenic mice (male C57BL/6 strain) containing a reporterlacI gene, which allows detection of mutation frequency. The frequency of mutation in this reporterlacI gene increased from 1.5 to 7.7 (per 100,000) in cortical DNA after 30 min of forebrain ischemia and 8 hr of reperfusion and remained elevated at 24 hr reperfusion. Eight DNA lesions that are characteristic of DNA damage mediated by free radicals were detected. Four mutagenic lesions (2,6-diamino-4-hydroxy-5-formamidopyrimidine, 8-hydroxyadenine, 5-hydroxycytosine, and 8-hydroxyguanine) examined by gas chromatography/mass spectrometry and one corresponding 8-hydroxy-2′-deoxyguanosine by a method of HPLC with electrochemical detection increased in cortical DNA two- to fourfold (p < 0.05) during 10–20 min of reperfusion. The damage to γ-actin and DNA polymerase-β genes was detected within 20 min of reperfusion based on the presence of formamidopyrimidine DNAN-glycosylase-sensitive sites. These genes became resistant to the glycosylase within 4–6 hr of reperfusion, suggesting a reduction in DNA damage and presence of DNA repair in nuclear genes. These results suggest that nuclear genes could be targets of free radicals.
Density-Dependent Feedback Inhibition of Oligodendrocyte Precursor Expansion
Authors: Zhang Hong , Miller Robert H.
Published: April 2018

The myelin sheath in the vertebrate CNS is formed by oligodendrocytes. The number of oligodendrocytes in a mature axon tract must be sufficient to myelinate all appropriate axons. How the number of oligodendrocytes is matched to axonal requirements and whether such matching involves axon–oligodendrocyte signaling or intrinsic oligodendrocyte self-regulation are not clear.

Using a combination ofin vitroanalyses, we demonstrate that oligodendrocyte precursors closely regulate their numbers through interactions between adjacent precursors. In low-density rat spinal cord cultures, the number of oligodendrocyte lineage cells increases rapidly. The addition of large numbers of oligodendrocyte precursors substantially reduces precursor expansion and results in a normalization of oligodendrocyte lineage cell numbers in the cultures over time. Thus, the number of oligodendrocyte lineage cells that develop appears dependent on the density of oligodendrocyte lineage cells. This normalization of cell number is reflected in assays of clonal potential and proliferation. For example, precursors gave rise to fewer progeny and proliferated less at high density. Reduced precursor expansion at high density was not attributable to the depletion of growth factors. Cocultures of high and low densities did not inhibit precursor expansion in low-density cultures, suggesting the requirement for local cell–cell interactions. The inhibition of precursor expansion was cell-type-specific and dependent on the presence of oligodendrocyte lineage cells. We propose that this density-dependent feedback inhibition of oligodendrocyte precursor expansion may play a primary role in regulating the number of oligodendrocytes in the developing spinal cord.
Prominent Expression of Two Forms of Glutamate Decarboxylase in the Embryonic and Early Postnatal Rat Hippocampal Formation
Authors: Dupuy Shannon T. , Houser Carolyn R.
Published: April 2018

Immunohistochemical methods were used to determine the earliest times of detection for two forms of glutamate decarboxylase (GAD67 and GAD65) in the embryonic and early postnatal rat hippocampal formation and to determine whether their distribution patterns differed from each other and from those of the adult. Both GAD67- and GAD65-containing neurons were observed as early as embryonic day 17 (E17)–E18 in the hippocampus and E19 in the dentate gyrus, and this was substantially earlier than GAD had been detected previously in the hippocampal formation. The two GAD isoforms displayed very similar distribution patterns, but these patterns were distinctly different from those of the adult. From E17 to E20, GAD67 and GAD65 were expressed in neuronal cell bodies throughout the hippocampal and dentate marginal zones (future dendritic layers), and relatively few existed within the principal cell body layers, where GAD-positive neurons are frequently concentrated in the adult. At E21 to postnatal day 1 (P1), there was a sudden shift from a predominance of GAD-containing cell bodies within the developing dendritic regions to a meshwork of GAD-positive processes with terminal-like varicosities in these same regions. This pattern also contrasted with that of the adult, in which GAD-labeled terminals are highly concentrated in the principal cell layers. Electron microscopic observations of the GAD-labeled processes at P1 confirmed their axon-like appearance and demonstrated that the immunoreactivity was consistently localized in vesicle-filled regions that were often closely apposed to and, in some instances, established synaptic contacts with dendritic profiles.

The present identification of an early abundance of GAD-containing structures in the hippocampal formation and the marked change in their distribution during development complement recent observations of developmental changes in the functioning of the GABA system and provide additional support for the early involvement of this neurotransmitter system in hippocampal development.
Characterization of Antisera Specific to NK1, NK2, and NK3 Neurokinin Receptors and their Utilization to Localize Receptors in the Rat Gastrointestinal Tract
Authors: Grady Eileen F. , Baluk Peter , Böhm Stephan , Gamp Patrick D. , Wong Helen , Payan Donald G. , Ansel John , Portbury Andrea L. , Furness John B. , McDonald Donald M. , Bunnett Nigel W.
Published: April 2018

Understanding the physiological role of tachykinins requires precise cellular and subcellular localization of their receptors. We raised antisera by immunizing rabbits with peptides corresponding to portions of the intracellular tails of the rat neurokinin 1, 2, and 3 receptors (NK1-R, NK2-R, NK3-R). Receptors were localized by immunofluorescence and confocal microscopy. NK1-R, NK2-R, and NK3-R were detected at the plasma membrane of transfected cells with minimal intracellular stores. Staining was abolished by preabsorption of the antisera with the peptides used for immunization. Nontransfected cells were unstained. Each antiserum only stained cells transfected with the appropriate receptor and did not stain cells transfected with the other receptors. Therefore, the antisera are specific and do not cross-react with other neurokinin receptors. We examined the distribution of the neurokinin receptors in the gastrointestinal tract of the rat. NK1-R was detected in myenteric and submucosal neurons and in interstitial cells of Cajal. NK2-R was localized to circular and longitudinal muscle cells and to nerve endings in the plexuses. NK3-R was detected in numerous myenteric and submucosal neurons. Some neurons expressed both NK1-R and NK3-R. Receptors were detected at the plasma membrane and in endosomes. Cells expressing the receptors were closely associated with tachykinin-containing nerve fibers. Thus, NK1-R and NK3-R mediate neurotransmission by tachykinins within enteric nerve plexuses, and NK1-R and NK2-R mediate the effects of tachykinins on interstitial and smooth muscle cells, respectively.
Localization of Rhythmogenic Networks Responsible for Spontaneous Bursts Induced by Strychnine and Bicuculline in the Rat Isolated Spinal Cord
Authors: Bracci Enrico , Ballerini Laura , Nistri Andrea
Published: April 2018

Spontaneous rhythmic bursting induced by coapplication of strychnine (1 μm) and bicuculline (20 μm) was observed with electrophysiological recording from pairs of lumbar ventral roots (usually L5) in an isolated preparation of the neonatal rat spinal cord. Bursting was insensitive to exogenously applied GABA or glycine, confirming that it was attributable to block of glycine and GABAAreceptor-mediated inhibition. NMDA accelerated bursting in a dose-dependent manner. Complete coronal spinal transection at L3 or L6 level did not block bursting recorded from L5 or L2 roots, respectively. Gradual cutting of the cord along the midline through a sagittal plane preserved bursting activity in both disconnected sides but led to loss of synchronicity. Once the spinal cord was fully separated into left and right halves, regular bursting persisted on each side with no phase-coupling between the two preparations. Section along a frontal plane to remove dorsal horns and much of the central canal area did not affect burst frequency or left-to-right synchronicity, whereas it reduced burst duration. A quadrant preparation containing mainly a single ventral horn displayed enhanced burst frequency while bursts became very short events. Bath application of 5-hydroxytryptamine (30 μm) or NMDA (5 μm) increased burst frequency and decreased burst duration in all types of preparation except the isolated quadrants, in which brief bursts were accelerated but not shortened by these chemical agents. These results suggest that bursting induced by strychnine and bicuculline apparently relied on distinct mechanisms for burst triggering and intraburst structure. The first required a relatively smaller neuronal network that was confined to a ventral quadrant. Intraburst structure was dependent on a larger circuitry comprising either both ventral horns or one side of the spinal cord including more than two segments.