bluuebunny/crossref_metadata_2025 huggingface dataset:
Acid efflux from retinal glial cells generated by sodium bicarbonate cotransport
Authors: Newman EA
Published: April 2018

Sodium bicarbonate cotransport was studied in freshly dissociated Muller cells of the salamander retina. Variations in intracellular and extracellular pH evoked extracellular potassium concentration ([K+]o were recorded. Intracellular pH was measured by standard ratio imaging of the pH-sensitive dye BCECF, whereas extracellular pH was monitored by imaging BCECF fixed to coverslips under dissociated cells. Increasing [K+]o from 2.5 to 50 mM resulted in an intracellular alkalinization. The rate of alkalinization, 0.047 pH units/min, was reduced to 42% of control when HEPES was substituted for HCO3- and was reduced to 36% of control by the addition of 0.5 mM DIDS, a Na+/HCO3- cotransport blocker. The K(+)-evoked alkalinization was Cl(-)- independent and was not substantially reduced by amiloride or bumetanide. Increasing [K+]o to 50 mM also produced a rapid extracellular acidification, 0.01 to 0.05 pH units in amplitude. HEPES substitution and addition of 0.5 mM DIDS reduced the acidification to 7– 8% of control, respectively. These results confirm the presence of a Na+/HCO3- cotransport system in salamander Muller cells and provide definitive evidence that glial cells can generate an extracellular acidification when [K+]o is increased. The K(+)-evoked extracellular acidification measured beneath cell endfeet was 304% of the amplitude of the acidification beneath cell somata, confirming that cotransporter sites are preferentially localized to the endfoot. The carbonic anhydrase inhibitor benzolamide (2 x 10(-5) M), which is poorly membrane permeant, increased the K(+)-evoked extracellular acidification to 269% of control, demonstrating that salamander Muller cells possess extracellular carbonic anhydrase.
Persistent dentate granule cell hyperexcitability after neonatal infection with lymphocytic choriomeningitis virus
Authors: Pearce BD , Steffensen SC , Paoletti AD , Henriksen SJ , Buchmeier MJ
Published: April 2018

Infection of neonatal Lewis rats with lymphocytic choriomeningitis virus (LCMV) produces distinct retinal, cerebellar, and hippocampal neuropathology. To understand the neurophysiological consequences of LCMV-induced hippocampal pathology, we studied evoked monosynaptic potentials and electro-encephalographic (EEG) activity in the dentate gyrus and CA1 and CA3 subfields of the hippocampus in vivo. Lewis rats were inoculated intracerebrally with LCMV at postnatal day 4. In rats studied 84–107 d postinfection, virus was cleared from the dentate gyrus and the number of dentate granule cells was decreased by 70%. No viral antigen or cell loss was apparent in CA1 or CA3. The hippocampal EEG of LCMV-infected rats 84–102 d postinfection was dominated by continuous theta. Although evoked potentials elicited in CA1 and CA3 by monosynaptic afferent stimulation revealed no differences between sham- and LCMV-infected rats, there was a site-specific dissociation of synaptic [population excitatory postsynaptic potential (pEPSP)] and cellular (population spike) responses and a suppression of GABA- mediated recurrent inhibition in the dentate gyrus of LCMV-infected rats. These findings indicate that GABA-mediated inhibition was markedly decreased in LCMV-infected rats. In support of this, parvalbumin-immunoreactive cell bodies and neuronal processes were decreased in LCMV-infected rats, suggesting that a subpopulation of GABA interneurons was affected. These findings indicate that abnormalities in synaptic function persist after clearance of infectious virus from the central nervous system and suggest that decreased inhibition subsequent to pathological sequence in a subpopulation of GABA interneurons may be implicated in the hyperexcitability of dentate granule cells.
Synchronization of fast (30-40 Hz) spontaneous cortical rhythms during brain activation
Authors: Steriade M , Amzica F , Contreras D
Published: April 2018

We investigated the synchronization of fast spontaneous oscillations (mainly 30–40 Hz) in anesthetized and behaving cats by means of simultaneous extra- and intracellular recordings from multiple neocortical areas. Fast Fourier transforms, auto- and cross- correlations, and spike- or wave-triggered averages were used to determine the frequency and temporal coherence of fast oscillations that outlasted the stimulation of ascending activating systems or that occurred naturally during behavioral states of waking and rapid eye movement (REM) sleep but also appeared during the depolarizing phases of slow sleep oscillations. In 90% of microelectrode tracks, the fast oscillations did not show field reversal at any depth of the cortex and were not observable in the underlying white matter. The negative field potentials of the fast oscillations were associated at all depths with neuronal firing. This field potential property of fast oscillations was in sharp contrast to the reversal of slow sleep oscillation or evoked potentials at depths of 0.25–0.5 mm. The coherence of fast spontaneous rhythms was spatially limited, being confined within a cortical column and among closely located neocortical sites, in contrast to the long- range synchronization of slow sleep rhythms. Depolarizing current pulses elicited spike-bursts (200–400 Hz) recurring at a frequency of 30–40 Hz. Our experiments demonstrate that the conventional notion of a totally desynchronized cortical activity upon arousal should be revised as fast rhythms are enhanced and synchronized within intracortical networks during brain activation. Spontaneously occurring, subthreshold membrane potential depolarizing oscillations may bias cortical and thalamic neurons to respond synchronously, at fast frequencies, to relevant stimuli in the wake state or to internally generated drives in REM sleep.
Vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, and noradrenaline induce the transcription factors CCAAT/enhancer binding protein (C/EBP)-beta and C/EBP delta in mouse cortical astrocytes: involvement in cAMP-regulated glycogen metabolism
Authors: Cardinaux JR , Magistretti PJ
Published: April 2018

We have described previously a transcription-dependent induction of glycogen resynthesis by the vasoactive intestinal peptide (VIP) or noradrenaline (NA) in astrocytes, which is mediated by cAMP. Because it has been postulated that the cAMP-mediated regulation of energy balance in hepatocytes and adipocytes is channeled at least in part through the CCAAT/enhancer binding protein (C/EBP) family of transcription factors, we tested the hypothesis that C/EBP isoforms could be expressed in mouse cortical astrocytes and that their level of expression could be regulated by VIP, by the VIP-related neuropeptide pituitary adenylate cyclase-activating peptide (PACAP), or by NA. We report in this study that in these cells, C/EBP beta and C/EBP delta are induced by VIP, PACAP, or NA via the cAMP second-messenger pathway. Induction of C/EBP beta and -delta mRNA by VIP occurs in the presence of a protein synthesis inhibitor. Thus, c/ebp beta and c/ebp delta behave as cAMP- inducible immediate-early genes in astrocytes. Moreover, transfection of astrocytes with expression vectors selectively producing the transcriptionally active form of C/EBP beta, termed liver-enriched transcriptional activator protein, or C/EBP delta enhance the glycogen resynthesis elicited by NA, whereas an expression vector producing the transcriptionally inactive form of C/EBP beta, termed liver-enriched transcriptional inhibitory protein, reduces this resynthesis. These results support the idea that C/EBP beta and -delta regulate gene expression of energy metabolism-related enzymes in astrocytes.
A G-protein-activated inwardly rectifying K+ channel (GIRK4) from human hippocampus associates with other GIRK channels
Authors: Spauschus A , Lentes KU , Wischmeyer E , Dissmann E , Karschin C , Karschin A
Published: April 2018

Transcripts of a gene, GIRK4, that encodes for a 419-amino-acid protein and shows high structural similarity to other subfamily members of G- protein-activated inwardly rectifying K+ channels (GIRK) have been identified in the human hippocampus. When expressed in Xenopus oocytes, GIRK4 yielded functional GIRK channels with activity that was enhanced by the stimulation of coexpressed serotonin 1A receptors. GIRK4 potentiated basal and agonist-induced currents mediated by other GIRK channels, possibly because of channel heteromerization. Despite the structural similarity to a putative rat KATP channel, no ATP sensitivity or KATP-typical pharmacology was observed for GIRK4 alone or GIRK4 transfected in conjunction with other GIRK channels in COS-7 cells. In rat brain, GIRK4 is expressed together with three other subfamily members, GIRK1–3, most likely in identical hippocampal neurons. Thus, heteromerization or an unknown molecular interaction may cause the physiological diversity observed within this class of K+ channels.
Modulation of calcium efflux from cultured rat dorsal root ganglion neurons
Authors: Werth JL , Usachev YM , Thayer SA
Published: April 2018

The free intracellular Ca2+ concentration ([Ca2+]i) is governed by the balance between the activation of Ca2+ channels and buffering and efflux processes. We tested the hypothesis that Ca2+ efflux pathways are susceptible to modulation. The whole-cell patch-clamp technique was used in combination with Indo-1-based microfluorometry to record Ca2+ current and [Ca2+]i simultaneously from single rat dorsal root ganglion (DRG) neurons grown in culture. Depolarizing test pulses (-80 to 0 mV, 100–300 msec) elicited [Ca2+]i transients that recovered to basal levels by a process best-fit with a single exponential (tau = 5.1 +/- 0.4 sec; n = 14) and were independent of Ca2+ load (40–500 pC) over this range of test pulses. [Ca2+]i transients recorded in whole-cell configuration were similar to those elicited by a brief train of action potentials in unclamped neurons. Inhibition of Ca2+ sequestration into intracellular stores with thapsigargin had no effect on the kinetics of recovery. Inhibition of plasma membrane Ca2+ ATPase (PMCA) function by including a peptide inhibitor (C28R2) in the patch pipette significantly slowed recovery to basal [Ca2+]i (tau = 9.9 +/- 0.8 sec; n = 4). Preincubation with calmidazolium, a calmodulin antagonist, produced modest slowing of Ca2+ efflux. Phorbol dibutyrate, an activator of protein kinase C (PKC), accelerated Ca2+ efflux only when the PMCA had been inhibited by C28R2. We conclude that in DRG neurons PMCAs are responsible for lowering [Ca2+]i after small Ca2+ loads and that PMCA-mediated Ca2+ efflux is modulated by calmodulin- and PKC- signaling pathways.
Beta-amyloid-related peptides inhibit potassium-evoked acetylcholine release from rat hippocampal slices
Authors: Kar S , Seto D , Gaudreau P , Quirion R
Published: April 2018

The 4 kDa beta-amyloid (A beta) protein, a major component of cerebral and cerebrovascular plaques in Alzheimer's disease (AD), is derived from the proteolytic cleavage of a larger, membrane-bound precursor, the A beta precursor protein (APP). Until recently, it was assumed that an aberrant AD-specific proteolysis generated A beta peptides, which subsequently could initiate and/or contribute to the pathological cascade leading to plaque formation and losses of selected neuronal populations, including basal forebrain cholinergic neurons that provide major inputs to the hippocampus and neocortex. However, the recent detection of soluble A beta fragments in the plasma and CSF of normal individuals, as well as in the conditioned media of cultured brain cells, suggests a role for A beta-related peptides in normal brain functions. Taking into consideration the reported toxic properties of A beta and the preferential vulnerability of basal forebrain cholinergic neurons in AD, we investigated the possible effects of A beta-related peptides on the release of endogenous acetylcholine (ACh) from rat brain slices. A beta 1–28, in a concentration-dependent manner (10(-12)– 10(-8) M), potently inhibited K(+)-evoked ACh release from hippocampal slices. The inhibition of ACh release was fully reversible and was observed using other A beta-related peptides such as A beta 1–42, A beta 1–40, and A beta 25–35, but not with the scrambled, reverse, or all D-isomer A beta-peptide sequences, indicating that the effect of A beta on ACh release is mediated via a stereoselective mechanism. Tetrodotoxin (10 microM) failed to alter the effect of A beta 1–28 on ACh release, which suggests the lack of involvement of voltage- dependent Na+ channels. Except for the hippocampal formation, the inhibitory effect of A beta on K(+)-evoked ACh release also was observed in the frontal cortex but not in the striatum. Taken together, our results demonstrate that APP-derived A beta-related peptides can regulate the release of ACh potently by acting on cholinergic terminals. Additionally, the evidence that selected cholinergic neuronal populations are sensitive to A beta suggests a potential mechanistic link between the deposition of A beta and the preferential vulnerability of certain cholinergic projections in AD.
Fluctuations in relative levels of choline acetyltransferase mRNA in different regions of the rat basal forebrain across the estrous cycle: effects of estrogen and progesterone
Authors: Gibbs RB
Published: April 2018

Quantitative in situ hybridization techniques were used to compare relative cellular levels of choline acetyltransferase (ChAT) mRNA in different regions of the female rat basal forebrain at different stages of the estrous cycle and at different time points after the administration of physiological levels of estrogen and progesterone. Significant fluctuations in relative levels of ChAT mRNA were detected during the course of the estrous cycle. In the medial septum (MS) and striatum, the highest levels of ChAT mRNA were detected on diestrus 1. Fluctuations in the nucleus basalis magnocellularis (NBM) were highly variable, with the highest levels detected on diestrus 2. In ovariectomized animals, significant increases in ChAT mRNA were detected in the MS, NBM, and striatum within 1–3 d after a single administration of estradiol. In addition, the effects of estradiol on ChAT mRNA expression in the NBM and striatum were significantly enhanced by the subsequent administration of progesterone. The magnitude and timing of the effects of steroid replacement were consistent with the magnitude and time course of the fluctuations detected during the course of the estrous cycle. These data demonstrate that estrogen and progesterone can increase basal forebrain levels of ChAT mRNA significantly in specific regions of the rat basal forebrain, that the magnitude and time course of the effects vary between different subpopulations of cholinergic neurons, and that the effects are associated with changes in the functioning of specific basal forebrain cholinergic neurons across the estrous cycle.
Mutations in shaking-B prevent electrical synapse formation in the Drosophila giant fiber system
Authors: Phelan P , Nakagawa M , Wilkin MB , Moffat KG , O'Kane CJ , Davies JA , Bacon JP
Published: April 2018

The giant fiber system (GFS) is a simple network of neurons that mediates visually elicited escape behavior in Drosophila. The giant fiber (GF), the major component of the system, is a large, descending interneuron that relays visual stimuli to the motoneurons that innervate the tergotrochanteral jump muscle (TTM) and dorsal longitudinal flight muscles (DLMs). Mutations in the neural transcript from the shaking-B locus abolish the behavioral response by disrupting transmission at some electrical synapses in the GFS. This study focuses on the role of the gene in the development of the synaptic connections. Using an enhancer-trap line that expresses lacZ in the GFs, we show that the neurons develop during the first 30 hr of metamorphosis. Within the next 15 hr, they begin to form electrical synapses, as indicated by the transfer of intracellularly injected Lucifer yellow. The GFs dye-couple to the TTM motoneuron between 30 and 45 hr of metamorphosis, to the peripherally synapsing interneuron that drives the DLM motoneurons at approximately 48 hr, and to giant commissural interneurons in the brain at approximately 55 hr. Immunocytochemistry with shaking-B peptide antisera demonstrates that the expression of shaking-B protein in the region of GFS synapses coincides temporally with the onset of synaptogenesis; expression persists thereafter. The mutation shak-B2, which eliminates protein expression, prevents the establishment of dye coupling shaking-B, therefore, is essential for the assembly and/or maintenance of functional gap junctions at electrical synapses in the GFS.
Homogeneous development of electrical excitability via heterogeneous ion channel expression
Authors: Ribera AB
Published: April 2018

Synchronous differentiation of delayed-rectifier potassium current regulates electrical excitability and calcium entry in motor, sensory, and interneurons of the developing amphibian spinal cord. Although Kv1 and Kv2 potassium channel transcripts are detectable in these cells, it is not known which transcript contributes to functional expression. Overexpression of a Kv1 dominant-negative subunit indicates that 20% of neurons have only Kv1 potassium currents. In other neurons, non-Kv1 channels function because the dominant-negative subunit either only partially suppresses or has no effect on current. Thus, diverse embryonic neurons coordinate differentiation of excitability yet rely on heterogeneous potassium channel gene expression.
Muscimol acts in dorsomedial but not paraventricular hypothalamic nucleus to suppress cardiovascular effects of stress
Authors: Stotz-Potter EH , Willis LR , DiMicco JA
Published: April 2018

Both the dorsomedial hypothalamic nucleus (DMH) and the paraventricular hypothalamic nucleus (PVN) have been implicated in the neural control of the cardiovascular response to stress. We used the GABAA agonist muscimol to inhibit neuronal activation and attempted to identify hypothalamic nuclei required for the cardiovascular response to air stress. Chronically instrumented rats received bilateral injections of either 80 pmol of muscimol or 100 nl of saline vehicle into the DMH, the PVN, or an intermediate area (including the rostral edge of the DMH and the region between the two nuclei) and were placed immediately in a restraining tube and subjected to 20 min of air stress. In all rats, air stress after vehicle injections caused marked increases in heart rate (137 +/- 6 beats/min) and blood pressure (26 +/- 2 mmHg). Microinjection of muscimol into the DMH suppressed the heart rate and blood pressure response by 85 and 68%, respectively. Identical microinjection of muscimol into the intermediate area between the DMH and the PVN attenuated the increases in heart rate by only 46% and in blood pressure by 52%. In contrast, similar injections into the vicinity of the PVN failed to alter the cardiovascular response to air stress. These findings demonstrate that muscimol-induced inhibition of neuronal activity in the region of the DMH blocks air stress-induced increases in heart rate and arterial pressure, whereas similar treatment in the area of the PVN has no effect.
Potentiation of neuronal NMDA response induced by dehydroepiandrosterone and its suppression by progesterone: effects mediated via sigma receptors
Authors: Bergeron R , de Montigny C , Debonnel G
Published: April 2018

We have shown previously that low doses of selective sigma (sigma)- receptor ligands potentiate the excitatory response of pyramidal neurons to NMDA in the CA3 region of the dorsal hippocampus in the rat. Because progesterone competitively displaces the binding of the ligand N-[3H]allyl-normetazocine (SKF-10,047), the present studies were undertaken to determine in vivo the effect of neuroactive steroids on NMDA-induced excitation of rat CA3 pyramidal neurons. Low doses of dehydroepiandrosterone (DHEA) potentiated the NMDA response selectively and dose-dependently. The effect of DHEA was reversed by the selective sigma antagonist N-dipropyl-2-(4-methoxy-3- (2-phenylethoxy)phenyl)- ethylamine monohydrochloride (NE-100) and by haloperidol, but not by spiperone. Progesterone had no effect by itself but reversed, at low doses, the potentiation of the NMDA response induced by DHEA as well as those induced by nonsteroidal sigma ligands. Neither pregnenolone nor pregnenolone sulfate had any effect on the NMDA response--nor did they antagonize the potentiation of the NMDA response induced by DHEA and by nonsteroidal sigma ligands. A pertussis toxin pretreatment, which inactivates Gi/o-proteins, abolished the potentiating effects of DHEA. Ovariectomy enhanced the potentiation of the NMDA response by the nonsteroidal sigma ligand di(2-tolyl)guanidine (DTG). There was a reciprocal occlusion of the effects of DHEA and DTG; DTG did not potentiate the NMDA response further after DHEA, and DHEA did not do so after DTG. These results suggest that some neuroactive steroids modulate the NMDA response via sigma receptors.
The effect of depressing glial function in rat brain in situ on ion homeostasis, synaptic transmission, and neuron survival
Authors: Largo C , Cuevas P , Somjen GG , Martin del Rio R , Herreras O
Published: April 2018

The supporting role of glial cells in maintaining neurons and in ion homeostasis has been studied in situ by perfusing the gliotoxin fluorocitrate (FC) through a microdialysis fiber in the CA1 area of urethane-anesthetized rats. Extracellular direct current potential, extracellular potassium concentration ([K+]o) and amino acid levels, extracellular pH (pHo), and evoked field activity were studied. Histology verified the swelling of glial cells after 4 hr of FC treatment. Massive neuron damage was evident after 8 hr. FC dialysis caused the rapid decrease of glutamine, pHo became progressively more acid, and [K+]o moderately elevated. Orthodromic transmission was variably blocked within 30 min to 4 hr. After 4 hr, spreading depression (SD) waves that originated from the neocortex invaded hippocampal CA1, [K+]o increased to higher levels, pHo became very acid, and there were steep increases in taurine, glutamate, and GABA levels. Simultaneously, the antidromic population spike (a-PS) became depressed and eventually disappeared. When a shorter dialysis probe that spared cortex was used to sample CA1, no SD was seen, a-PS was not abolished, and ion homeostasis was altered less markedly. Repeated SD provoked in hippocampus in the absence of FC caused only mild depression of a-PS. Dialysis of high-K+ solution in healthy neocortex or hippocampus caused only slight elevation of [K+]o at distances of 200–400 microns from the dialysis membrane. After treatment with FC, similar high-K+ dialysis raised [K+]o much more. We conclude the following: (1) recurrent SD waves injure neurons if and only if glial function has failed; (2) neurons can regulate [K+]o, albeit imperfectly; (3) glia is required for the normal fine tuning of [K+]o and particularly for the recovery of pathologically elevated [K+]o; and (4) glia are required for the regulation of pHo. The similarities between glial poisoning by FC and the reported changes in the penumbra of ischemic infarcts suggest that the extension of neuron loss into the penumbral region might depend on failure of glial protection.
Altered inhibition of dentate granule cells during spatial learning in an exploration task
Authors: Moser EI
Published: April 2018

To investigate the extent to which inhibitory interneurons control impulse flow through the dentate gyrus during spatial learning in an exploration task, dentate field potentials were recorded in response to paired stimulation of the perforant path while rats rested or explored. Recurrent inhibition of the granule cells was measured as the reduction of the second waveform when a population spike was present in the first. Both the population spike and the field EPSP (fEPSP) were suppressed at interstimulus intervals shorter than approximately 40 msec. Consistent differences were observed between potentials recorded at equivalent brain temperature in the exploration and resting (reference) conditions. During exploration, the fEPSP of the second (test) waveform was reduced further compared with reference potentials with a similar response to the first (conditioning) stimulus. This reduction was observed only when the first pulse elicited a population spike. The population spike of the second waveform was facilitated compared with reference potentials with similar fEPSP slopes. These observations suggest that exploration is coupled to increased inhibition on the perforant-path terminals or the dendrites of the granule cells, whereas the inhibition on the somata is decreased. The two phenomena were not correlated and followed different time courses. The suppression of the fEPSP decayed gradually, although it was still present at 15 min, whereas the facilitation of the population spike was stable. Together, these changes, which likely involve different populations of interneurons, may focus and amplify incoming signals from the entorhinal cortex.
Sequential expression of Trks A, B, and C in the regenerating olfactory neuroepithelium
Authors: Roskams AJ , Bethel MA , Hurt KJ , Ronnett GV
Published: April 2018

This study examines how the family of neurotrophin receptor tyrosine kinases (Trks) participates in the regeneration and replacement of olfactory neurons within the adult rat olfactory neuroepithelium. mRNA and protein products representing the high-affinity nerve growth factor (NGF) receptor Trk A, its family members Trk B and Trk C, and the low- affinity NGF receptor (INGFR) are all detected within both mature and regenerating olfactory neuroepithelium and within primary cultures of olfactory neurons. Cellular immunoreactivity for Trks A, B, and C and INGFR changes dramatically during the lifetime of an olfactory neuron and is demonstrated by inducing the epithelium into a coordinate rapid cycle of degeneration and regeneration in vivo by removal of the target organ, the olfactory bulb. Trk A-positive neuronal precursor basal cells undergo mitosis to produce Trk B-positive immature neurons that mature under the local influence of the olfactory neuroepithelium and the target-derived influence of the olfactory bulb to become a Trk C- positive mature neuron. Primary cultures of immature olfactory neurons demonstrate neurotrophin-induced phosphorylation of Trks A, B, and C and subsequent activation of the immediate early gene c-Fos, and they change their expression of differentiation stage-specific markers after treatment with individual and combinations of neurotrophins. This is the first population of neurons of a single lineage in which Trks A, B, and C and the INGFR have been demonstrated to be expressed sequentially during neuronal division, commitment, and differentiation and to be fully capable of transducing cellular signals causing phenotypic changes in differentiation state.
PSD-95 is associated with the postsynaptic density and not with the presynaptic membrane at forebrain synapses
Authors: Hunt CA , Schenker LJ , Kennedy MB
Published: April 2018

PSD-95, a prominent protein component of the postsynaptic density (PSD) fraction from rat forebrain, has been localized by light microscopy to dendrites of hippocampal neurons (Cho et al., 1992) and to the presynaptic plexus of cerebellar basket cells (Kistner et al., 1993). Here we extend these studies to show that an affinity-purified antibody to PSD-95 labels the dendrites of most neurons in the forebrain and of a subset of neurons in the cerebellum. To confirm that PSD-95 is associated with the PSD at forebrain synapses and to clarify whether it is also associated with the presynaptic membrane, we employed immunogold electron microscopy of forebrain synaptosomes. Gold-labeled antibodies to PSD-95 labeled postsynaptic densities in both intact and lysed forebrain synaptosomes but did not label presynaptic terminals or the presynaptic membrane. The asymmetric distribution of PSD-95 at synapses contrasts with that of its homologs, disks-large and ZO-1, which are arranged symmetrically at septate and tight junctions, respectively.
Mechanisms for the maintenance and eventual degradation of neurofilament proteins in the distal segments of severed goldfish mauthner axons
Authors: Raabe TD , Nguyen T , Archer C , Bittner GD
Published: April 2018

Cellular mechanisms that might affect the degradation of neurofilament proteins (NFPs) were examined in the distal segments of severed goldfish Mauthner axons (M-axons), which do not degenerate for more than 2 months after severance. Calpain levels, as determined by reactivity to a polyclonal antibody, remained constant for 80 d postseverance in distal segments of M-axons and then declined from 80 to 85 d postseverance. Calpain activity in rat brain, as determined by a spectrophotometric assay, was much higher than calpain activity in control and severed goldfish brain, spinal cord, muscle, or M-axons. Calpain activity was extremely low in M-axons compared with that in all other tissues and remained low for up to 80 d postseverance in distal segments of M-axons. Phosphorylated NFPs, as determined by Stains-All treatment of SDS gels, were maintained for up to 72 d postseverance and then decreased noticeably at 75 d postseverance when NFP breakdown products appeared on silver-stained gels. By 85 d post-severance, phosphorylated NFPs no longer were detected, and NFP breakdown products were the most prominent bands on silver-stained gels. These results suggest that the distal segments of M-axons survive for months after severance, because NFPs are maintained in a phosphorylated state that stabilizes and protects NFPs from degradation by low levels of calpain activity in the M-axon; the distal segments of severed M-axons degenerate eventually when NFPs no longer are maintained in a phosphorylated state and become susceptible to degradation, possibly by low levels of calpain activity in the M-axon.
Delayed clearance of transmitter and the role of glutamate transporters at synapses with multiple release sites
Authors: Otis TS , Wu YC , Trussell LO
Published: April 2018

The roles of glutamate diffusion, uptake, and channel kinetics in shaping the AMPA receptor EPSC were examined at a calyceal synapse. The EPSC decay was described by three exponential components: two matching desensitizing channel kinetics, and a third component at least 10 times slower. The slowest component had identical voltage dependence to the steady-state AMPA current and was selectively increased and prolonged by blockade of glutamate uptake, indicating that the slow EPSC represented rebinding of glutamate at partially desensitized AMPA receptors. The data were in strong agreement with the predictions of a model of transmitter diffusion from multiple release sites into a large synaptic cleft. Within the first millisecond after release, transmitter concentrations in the cleft fell below millimolar levels, causing the fastest parts of the EPSC to be shaped by channel kinetics. The slowest component was determined by the removal over tens of milliseconds of the final 10–100 microM glutamate by diffusion and uptake. The data and modeling indicate that transmitter uptake and cooperation between release sites are significant determinants of a slow “tail” of glutamate in the synaptic cleft. This slow clearance of glutamate is likely to limit postsynaptic receptor availability through desensitization.
Apoptosis of bcl-x-deficient telencephalic cells in vitro
Authors: Roth KA , Motoyama N , Loh DY
Published: April 2018

bcl-x is a member of the bcl-2 gene family, which is expressed at high levels in the embryonic brain. The targeted disruption of bcl-x results in massive cell death of immature neurons in the developing mouse brain (Motoyama et al., 1995). bcl-x-deficient mice die around embryonic day 13 (E13), probably secondary to their inability to produce mature red blood cells. To determine whether the death of immature neurons in the bcl-x-deficient brain is cell autonomous, we examined primary telencephalic cell cultures from E12.5 homozygous mutant (bcl-x-/-), heterozygous mutant (bcl-x+/-), and wild-type (bcl-x+/+) mice. bcl-x-/- telencephalic cells cultured in 0.5 or 2.0% fetal calf serum (FCS)- containing medium for 48 hr showed increased apoptosis, defined by abnormal bisbenzamide staining and terminal-deoxytransferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL), and decreased numbers of microtubule-associated protein-2-immunoreactive neurons compared with bcl-x+/- and bcl-x+/+ cultures. Cycloheximide treatment of bcl-x-/- telencephalic cell cultures failed to prevent the increased cell death observed in low FCS-containing medium, suggesting a protein synthesis-independent apoptosis. There were no significant differences among bcl-x-/-, bcl-x+/-, and bcl-x+/+ telencephalic cells grown for 48 hr in 5% FCS-containing medium or in a chemically defined serum-free medium (ITS). bcl-x-/- neurons generated in ITS showed increased susceptibility to subsequent serum deprivation. These results indicate that bcl-x is important for both neuron maturation and survival.
Cell death in the midbrain of the murine mutation weaver
Authors: Roffler-Tarlov S , Martin B , Graybiel AM , Kauer JS
Published: April 2018

The midbrain of the adult homozygous weaver (wv/wv) mouse is notable for a reduction in the numbers of dopamine-containing cells in the substantia nigra (A9) and the retrorubral nucleus (A8). We have determined that the reduction in tyrosine hydroxylase (TH)-positive neurons in the ventral midbrain of the weaver is attributable to the loss of neurons after postnatal day 7 (P7). Because the number and spatial distribution of TH-positive mesencephalic neurons in wv/wv, heterozygous weavers (+/wv), and wild-type mice are not significantly different on P7, we conclude that the early developmental steps of proliferation and migration have taken place normally in the mutant. Although numbers and distribution of cells are normal in the wv/wv on P7, the appearance of the TH-stained ventral midbrain is abnormal because of the paucity of TH-stained dendritic processes. The ventrally extending TH-positive dendrites are largely absent in the young wv/wv. The wv/wv also can be distinguished from both homozygous normal (+/+) and wv/wv littermates on P7 based on the appearance of dendrites that are more numerous than in the wv/wv but thin, disorganized, and sparse compared with +/+. Most cell death seems to take place in wv/wv before P21. However, at least one subset of dopamine-containing neurons disappears later. The zone of densely packed TH-positive neurons in the substantia nigra that is likely to be the origin of innervation to striosomes in the caudoputamen disappears between P21 and adulthood. Despite the early pathology evident in the mesencephalic dopamine- producing neurons of the +/wv, no evidence for cell death was observed there even in the oldest +/wv weavers studied.
Tissue-specific expression of a Ca(2+)-activated K+ channel is controlled by multiple upstream regulatory elements
Authors: Brenner R , Thomas TO , Becker MN , Atkinson NS
Published: April 2018

The electrical properties of a cell are produced by the complement of ion channels that it expresses. To understand how ion-channel gene expression is regulated, we are studying the tissue-specific regulation of the slowpoke (slo) Ca(2+)-activated K+ channel gene. This gene is expressed in the central and peripheral nervous system, in midgut and tracheal cells, and in the musculature of Drosophila melanogaster. The entire transcriptional control region has been cloned previously and shown to reproduce the tissue and developmental expression pattern of the endogenous gene. Here we demonstrate that s/o has at least four promoters distributed over approximately 4.5 kb of DNA. Promoter C1 and C1c display a TATA box-like sequence at the appropriate distance from the transcription start site. Promoters C1b and C2, however, are TATA- less promoters. C1, C1b, and C1c transcripts differ in their leader sequence but share a common translation start site. C2 transcripts incorporate a new translation start site that appends 17 amino acids to the N terminus of the encoded protein. Deletion analysis was used to identify sequences important for tissue-specific expression. We used a transgenic in vivo expression system in which all tissues and developmental stages can be assayed easily. Six nested deletions were transformed into Drosophila, and the expression pattern was determined using a lacZ reporter in both dissected tissues and sectioned animals. We have identified different sequences required for expression in the CNS, midgut, tracheal cells, and muscle.
Developmental influence of glycinergic transmission: regulation of NMDA receptor-mediated EPSPs
Authors: Kotak VC , Sanes DH
Published: April 2018

The influence of excitatory transmission on postsynaptic structure is well established in developing animals, but little is known about the role of synaptic inhibition. We addressed this issue in developing gerbils with two manipulations designed to decrease glycinergic transmission in an auditory nucleus, the lateral superior olive (LSO), before the onset of sound-evoked activity. First, contralateral cochlear ablation functionally denervated the glycinergic pathway from the medial nucleus of the trapezoid body (MNTB) to the LSO, while leaving the excitatory pathway intact. Second, continuous release of a glycine receptor antagonist, strychnine (SN), was used to decrease transmission. The strength of excitatory and inhibitory synapses was examined with whole-cell recordings from LSO neurons in a brain-slice preparation. The percentage of LSO neurons exhibiting MNTB-evoked IPSPs was reduced in both ablated and SN-treated animals. In those neurons displaying IPSPs, the amplitude was significantly reduced. This decrease was accompanied by an 8 mV depolarization in the IPSP equilibrium potential. In contrast, the ipsilaterally evoked EPSPs were of unusually long duration in experimental animals. These long-duration EPSPs were significantly shortened by hyperpolarizing the neuron to -90 mV or exposing them to aminophosphonopentanoic acid (AP-5), an NMDA receptor antagonist. Membrane hyperpolarization and AP-5 had little effect in control neurons. In addition, LSO neurons from ablated or SN- treated animals displayed broad rebound depolarizations after membrane hyperpolarization, and these were abolished in the presence of Ni2+. Because both cochlear ablation and SN-rearing were initiated before the onset of sound-evoked activity, the results suggest that spontaneous glycinergic transmission influences the development of postsynaptic properties, including the IPSP reversal potential, NMDA receptor function, and a Ca2+ conductance.
Intraseptal procaine abolishes hypothalamic stimulation-induced wheel- running and hippocampal theta field activity in rats
Authors: Oddie SD , Stefanek W , Kirk IJ , Bland BH
Published: April 2018

Rats were implanted chronically with hippocampal recording electrodes, a microinfusion guide cannula aimed at the medial septal nucleus, and an electrode for electrical stimulation of the posterior hypothalamic nucleus (PH). PH stimulation elicited running in rats placed in a wheel and simultaneously occurring hippocampal theta field activity (HPC- theta). In the preprocaine (PRE) testing condition, a positive linear relationship was demonstrated among the intensity of electrical stimulation of the PH, wheel-running speed, and the peak frequency of HPC-theta. HPC-theta amplitude reached an asymptote at the lowest levels of electrical stimulation of the PH. Procaine hydrochloride (1.5 microliters, 20% solution), a local anesthetic, was then infused into the medial septal nucleus (MS). Five minutes after the infusion, PH stimulation no longer induced wheel-running behavior or HPC-theta, and the remaining irregular field activity was significantly reduced in amplitude. Fifteen minutes after the procaine infusion, PH stimulation still did not elicit HPC-theta or running behavior in the majority of animals but did evoke large-amplitude sharp-waves. Thirty minutes after the procaine infusion, PH stimulation again elicited HPC-theta and running behavior, but HPC-theta peak frequency and running speeds were both significantly reduced compared with PRE values. Forty-five minutes after the infusion, HPC-theta amplitude had recovered to PRE values, but HPC-theta frequency and running speeds elicited by PH stimulation were still significantly reduced. By 60 min after procaine administration, the amplitude and frequency of HPC-theta and the running speeds elicited by PH stimulation recovered to PRE values. Multiple regression analysis revealed that the recovery pattern of running behavior reflected the frequency rather than the amplitude of HPC-theta. Neither saline control infusions into the MS nor procaine infusions into the lateral septum and paraventricular thalamic nucleus affected HPC-theta or running behavior. These findings are consistent with the notion that both the locomotor activity and “movement-related” HPC-theta frequency induced by electrically stimulating the PH were attributable to ascending activation of a hypothalamo-septal pathway and not to activation of descending brainstem or peripheral motor systems.
Decreased binding of dopamine D3 receptors in limbic subregions after neonatal bilateral lesion of rat hippocampus
Authors: Flores G , Barbeau D , Quirion R , Srivastava LK
Published: April 2018

Neonatal, bilateral lesion of the ventral hippocampus (VH) in rats recently has been proposed as a model of schizophrenia because these animals show postpubertal hypersensitivity to stress and to dopamine (DA) agonists that can be reversed by neuroleptic treatment. In search of the mechanisms of postpubertal emergence of hyperdopaminergic behavior in this model, we investigated developmental expressions of DA D1, D2, and D3 receptors in various striatal and limbic subregions of rats that had received bilateral ibotenic acid lesion of the VH at postnatal day 7 (PD7). D-Amphetamine-, apomorphine-, and stress-induced changes in locomotor activity were measured and, in accordance with previous reports, we observed an increased locomotor activity at PD56 in the hippocampal-lesioned group. The expression of DA D1, D2, and D3 receptors was then estimated in these rats by ligand autoradiography at PD41 and PD62. We observed that the levels of DA D3 receptors, as measured by tritiated 7-hydroxy-N,N-di-n-propyl-2-amino-tetralin ([3H]7- OH-DPAT) binding, are markedly reduced at PD62 in the limbic areas of lesioned rats compared with sham controls particularly in the nucleus accumbens, olfactory tubercles, and islands of Calleja. A small but significant increase in D1 receptors was also seen in the caudate- putamen of the lesioned animals at PD62, whereas no significant change in the overall expression of D2 receptors ([3H]spiperone binding) was noted. In view of the inhibitory role of D3 receptors on locomotion and, presumably, other DA-mediated behaviors, it is suggested that behavioral changes in the neonatally hippocampal-lesioned rats may be mediated by altered D3 receptor levels.
Ocular dominance columns in New World monkeys
Authors: Livingstone MS
Published: April 2018

Squirrel monkeys normally lack ocular dominance columns in V1. This study shows that squirrel monkeys can exhibit clear ocular dominance columns if they are made strabismic within a few weeks of birth. Columns were seen only in layer 4C beta and were coarser than the overlying blob pattern in the same animal. In physiological recordings from layer 4C of a normal squirrel monkey, single units were mostly monocular, but units driven by the two eyes were intermixed. These results suggest that in squirrel monkeys activity-dependent mechanisms do normally segregate geniculate inputs from the two eyes, but on a much finer scale than in Old World primates. Strabismic owl monkeys also showed ocular dominance columns; normal owl monkeys showed variable expression. Because ocular dominance columns, when present in New World monkeys, tend to occur in later-maturing parts of layer 4C, I hypothesize that a difference in the relative timing of the maturation of geniculocortical inputs and intracortical lateral connectivity explains the variability of ocular dominance column expression in New World monkeys.
Differential serotonergic innervation of the suprachiasmatic nucleus and the intergeniculate leaflet and its role in circadian rhythm modulation
Authors: Meyer-Bernstein EL , Morin LP
Published: April 2018

Serotonergic innervation is believed to inhibit the effects of light on the mammalian circadian timing system. Two anatomical components of this system, the suprachiasmatic nucleus (SCN) and the intergeniculate leaflet (IGL), receive serotonergic input from midbrain raphe nuclei. The present studies use retrograde and anterograde tracing as well as neurotoxic lesion techniques to demonstrate that serotonergic cells in the median raphe nucleus (MR) project to the SCN and that serotonergic cells in the dorsal raphe nucleus (DR) project to the IGL. Neurotoxic lesions were also used to investigate the effects of selective serotonin (5-HT) neuron loss in the MR or DR on circadian rhythm parameters of animals entrained to a light/dark cycle or housed in constant light. 5-HT depletion in the MR, but not in the DR, induces an advance in onset, a delay in offset, and a longer duration of the nocturnal running-wheel activity phase. Circadian rhythm disruption in constant light is also more frequent in hamsters with MR lesions. A second experiment was designed to investigate the relationship between lesion location, 5-HT-immunoreactive (5-HT-IR) fiber loss, and behavioral changes. Destruction of 5-HT neurons in the MR causes 5-HT- IR fiber loss in the SCN, which may account for the observed changes in circadian parameters. DR lesions result in 5-HT-IR fiber depletion of the IGL, with no associated changes in the entrained rhythm. The anatomical and behavioral results support the view that a 5-HT projection from the MR mediates 5-HT effects on circadian rhythm regulation in hamsters.
Selective clustering of GABA(A) and glycine receptors in the mammalian retina
Authors: Koulen P , Sassoe-Pognetto M , Grunert U , Wassle H
Published: April 2018

Molecular cloning has revealed a multiplicity of neurotransmitter receptor isoforms with different subunit compositions. Additionally, there is growing evidence that such receptors are clustered at postsynaptic sites of neurons. Thus, the questions arise whether individual neurons express different receptor isoforms and, if so, whether different isoforms are present within the same cluster or are aggregated at distinct postsynaptic sites. We have studied with immunofluorescence methods and antibodies that recognize specific subunits the distribution of glycine and GABA(A) receptors in mammalian retinae. Alpha ganglion cells were injected in rat or rabbit retinae with a fluorescent marker and then immunostained for receptor localization. Clusters of glycine receptors and clusters of the alpha1, and alpha2, alpha3, and gamma2 subunits of the GABA(A) receptor were found on the somatodendritic membranes of Alpha ganglion cells. Double- immunofluorescence experiments with different combinations of the subunit-specific antibodies showed that the alpha1, alpha2, and alpha3 subunits of the GABA(A) receptor are not colocalized within the same clusters. These results indicate that an individual neuron can express several isoforms of the GABA(A) receptor and that these different isoforms are aggregated at distinct postsynaptic sites. This suggests individual sorting mechanisms of GABAa receptors at GABAergic synapses.