bluuebunny/crossref_metadata_2025 huggingface dataset:
NGF and NT-3 have differing effects on the growth of dorsal root axons in developing mammalian spinal cord
Authors: Zhang L , Schmidt RE , Yan Q , Snider WD
Published: April 2018

The functions of neurotrophins in relation to axon growth and branching during development of the nervous system are unknown. In order to address this question, we have investigated the influences of systemically administered mouse nerve growth factor (mNGF) and human recombinant neurotrophin-3 (hrNT-3) on dorsal root axon growth in the spinal cord of embryonic rats. As anticipated, mNGF has a marked influence on growth of dorsal root axons. In mNGF-treated animals, dorsal root axons in the developing dorsal funiculi and axon collaterals in developing gray matter are substantially longer than those of age-matched controls. Furthermore, growth cones of some dorsal root axons have more than twice the surface area of controls. These effects of NGF are highly selective. Dorsal root axons that occupy a lateral position in white matter and that normally give off collaterals to superficial dorsal horn are prominently affected. Axons that run medially in dorsal columns and that give off collaterals to laminae III and IV and the ventral horn are not demonstrably influenced by treatment with exogenous mNGF. In contrast to the striking effects of mNGF on dorsal root axon growth, the influences of hrNT-3 were considerably more complex. Administration of hrNT-3 increased the mean soma area of DRG neurons, particularly those at the larger end of the size spectrum, consistent with its hypothesized role as a growth factor for proprioceptive sensory neurons. However, in striking contrast to the actions of mNGF, hrNT-3 consistently inhibited axon collateral growth in gray matter at early developmental stages. At later stages, we could not discern a clear-cut influence of hrNT-3 on dorsal root axon growth and branching. We conclude that the ability of mNGF to stimulate axon growth in both white and gray matter is consistent with the idea that mNGF regulates the developing axonal projections of DRG neurons in vivo. In contrast, systemically administered hrNT-3 inhibits the axon collateralizations of DRG neurons in gray matter at early developmental stages. We hypothesize that this inhibitory effect may be related to disruption of a chemotropic gradient of NT-3, or to the widespread expression of the NT-3 receptor trkC, on non-neuronal cells.
Cellular and subcellular localization of NMDA-R1 subunit immunoreactivity in the visual cortex of adult and neonatal rats
Authors: Aoki C , Venkatesan C , Go CG , Mong JA , Dawson TM
Published: April 2018

NMDA receptor activation can alter synaptic strength, cause cell death, and may modulate the release of glutamate and other neurotransmitters. Using a specific and selective antiserum directed against the R1 subunit of the NMDA receptor, we examined (1) whether NMDA receptors in the adult rat visual cortex are exclusively postsynaptic or also presynaptic and (2) whether NMDA-R1 subunits are incorporated into the plasma membrane prior to, contemporaneously, or following the formation of synapses during postnatal development. By light microscopy, NMDA-R1 immunoreactivity in the adult visual cortex is easily detectable within perikarya and proximal dendrites in laminae 2–6. Many of them have the morphological features of pyramidal neurons. In addition, fine punctate labeling is evident throughout the neuropil. Electron microscopy reveals these puncta to reside at postsynaptic densities of axospinous junctions and at fine astrocytic processes and axon terminals. In the deeper laminae, the majority of labeled profiles are astrocytic. Visual cortices of animals in their first postnatal week show concentrated immunoreactivity in a few nonpyramidal neurons within laminae that have just differentiated from the cortical plate. Electron microscopy reveals diffuse labeling along the plasma membrane of dendritic shafts lacking morphologically identifiable synaptic junctions or appositions to axons. Immunoreactivity is detectable in dendritic processes by postnatal day (PND) 2, in axonal processes by PND 4, and in astrocytic profiles by PND 14. Immunoreactivity also is detectable along the postsynaptic membrane of presumably transient axosomatic junctions. At all ages, the prevalence of NMDA-R1-immunoreactive profiles is lamina 1 > 4/5 > 6/6B. These results provide the cellular basis for NMDA receptors' participation in (1) postsynaptic membrane excitability, (2) regulation of transmitter release, (3) and, in the deeper laminae, astrocyte responses. During development, NMDA-R1 subunits are associated with the plasma membrane prior to axons' arrival while clustering of receptors to junctions may be promoted by axonal contact. Finally, spatial segregation of axonal growth cones may be mediated by NMDA-R1 subunits on these axonal processes.
Differential expression of synaptic vesicle protein 2 (SV2) isoforms
Authors: Bajjalieh SM , Frantz GD , Weimann JM , McConnell SK , Scheller RH
Published: April 2018

The synaptic vesicle proteins SV2A and SV2B (SV2 = synaptic vesicle protein 2) are two highly related proteins belonging to a family of transporters. As a first step toward identifying the function of the SV2 proteins, we examined the expression of SV2A and SV2B in the rat brain by in situ hybridization, immunohistochemistry, and immunoprecipitation with isoform-specific antibodies. These analyses revealed that one isoform, SV2A, is expressed ubiquitously throughout the brain at varying levels. The other isoform, SV2B, has a more limited distribution with varying degrees of coexpression with SV2A. Immunoprecipitation of brain synaptic vesicles with isoform-specific antibodies followed by Western analyses suggests that both isoforms can be present on the same synaptic vesicle. The expression of the SV2 proteins did not correlate either with neurotransmitter phenotype or with the expression of other synaptic vesicle protein isoforms. SV2B expression was observed to change during development; it is more widely expressed in the immature brain and is found in cells that have yet to establish synaptic contacts. The ubiquitous and overlapping expression of the SV2s suggests that they perform a function common to all synaptic vesicles. Variable and changing coexpression of the SV2 isoforms may indicate that SV2 function is regulated by the isoform composition of synaptic vesicles. The observation that the synaptic vesicle proteins, all occurring in multiple isoforms, are differentially expressed with respect to each other indicates that up to 90 different vesicle types are possible.
State-dependent release of acetylcholine in rat thalamus measured by in vivo microdialysis
Authors: Williams JA , Comisarow J , Day J , Fibiger HC , Reiner PB
Published: April 2018

Mesopontine cholinergic neurons have long been thought to play a key role in behavioral state control. In particular, they have been implicated in the process of EEG desynchrony and in the generation of rapid eye movement (REM) sleep. However, the behavioral profile of identified mesopontine cholinergic neurons has not been unequivocally demonstrated. In an attempt to address this issue, in vivo microdialysis was used to monitor acetylcholine (ACh) release across behavioral state in the rat thalamus, a major projection site of mesopontine cholinergic neurons. Because REM periods in rats are of short duration, a method was developed to collect and accumulate sufficiently large samples from each of the individual states of wake, slow-wave sleep, and REM sleep to permit off-line analysis via (HPLC- ECD). Probe placement and the source of cholinergic innervation to the vicinity of the microdialysis probe were verified using retrograde tracing combined with ChAT immunohistochemistry. Finally, the sodium and calcium dependence of ACh measured in the thalamus were tested using TTX and calcium-free dialysates. The results showed that (1) extracellular ACh concentrations in the thalamus are high during both wake and REM sleep and significantly lower during slow-wave sleep, (2) the majority of cholinergic projections to the vicinity of the dialysis probes originate in the mesopontine tegmentum, and (3) ACh release in the thalamus is due to sodium- and calcium-dependent mechanisms. In contrast to predictions of some previous hypotheses, these results demonstrate that mesopontine cholinergic neurons are active during both wake and REM sleep.
Changes in paired-pulse facilitation suggest presynaptic involvement in long-term potentiation
Authors: Schulz PE , Cook EP , Johnston D
Published: April 2018

Long-term potentiation (LTP) is a use-dependent form of synaptic plasticity that is of great interest as a potential cellular substrate underlying memory. It is important to determine the pre- and/or postsynaptic locus of LTP expression in order to study its underlying mechanisms. Despite intensive investigation, however, its locus of expression remains uncertain. It has been hypothesized that if LTP expression includes a presynaptic locus then it may alter the expression of another presynaptically mediated form of potentiation like paired-pulse facilitation (PPF), which is an increase in a second population excitatory postsynaptic potential when it is elicited shortly after a first. Previous authors have found no change in PPF in association with LTP. We re-examined the hypothesis, however, to reconcile the negative PPF data with other data that have suggested presynaptic involvement in LTP. Extracellular recordings were made in area CA1 of the rat hippocampal slice preparation. Surprisingly, PPF both increased and decreased significantly in association with LTP. The changes in PPF occurred in a predictable way, however. They correlated inversely with initial PPF magnitude so that a larger initial PPF was associated with a decrease in PPF with LTP while a smaller initial PPF was associated with an increase. Because PPF increased or decreased in individual slices in association with LTP, the average PPF of all slices did not change, in agreement with previous studies. The changes in PPF were also specific to LTP; that is, they were input specific, were not due to changes in inhibition or nonspecific effects of high- frequency stimulation, were not due to active postsynaptic currents or their nonlinear summation, and PPF changed with the same time course as LTP. We conclude that the mechanism of early LTP expression includes at least the presynaptic locus. Two hypotheses regarding the presynaptic mechanism underlying LTP expression, which are consistent with finding both increases and decreases in PPF with LTP, are (1) that there is an increase in the number of release sites with LTP or (2) that there is an increase in both the number of release sites and the probability of neurotransmitter release. Increases in the probability of neurotransmitter release alone would not appear to account for our findings since such increases have been associated only with decreases in PPF. Our findings do not exclude additional postsynaptic involvement.(ABSTRACT TRUNCATED AT 400 WORDS)
Membrane properties underlying the firing of neurons in the avian cochlear nucleus
Authors: Reyes AD , Rubel EW , Spain WJ
Published: April 2018

Neurons of the avian nucleus magnocellularis (NM) relay auditory information from the VIIIth nerve to other parts of the auditory system. To examine the cellular properties that permit NM neurons to transmit reliably the temporal characteristics of the acoustic stimulus, we performed whole-cell recordings in neurons of the chick NM using an in vitro thin slice preparation. NM neurons exhibited strong outward rectification near resting potential; the voltage responses to depolarizing current steps were substantially smaller than to equivalent hyperpolarizing steps. Suprathreshold current steps evoked only a single action potential at the start of the step. In contrast, stimulation with trains of brief current pulses evoked repetitive firing that was phase-locked to the stimulus cycle. The number of action potentials evoked by the pulses during the train decreased with increasing stimulus rate. Voltage-clamp experiments revealed a rapidly activating, slowly inactivating, outward current with a threshold near - 65 mV. During depolarizing voltage steps, the outward current rose sigmoidally to a peak and then decayed slowly, reaching steady state within 5 sec. Application of 200 microM 4-aminopyridine (4-AP) reduced the peak of the outward current by 84%, leaving a small, persistent component. Under current clamp, application of 200 microM 4-AP reduced the outward rectification and increased the amplitude and duration of the action potentials. Moreover, NM neurons could no longer sustain firing during high rates of stimulation with the current pulses: increased temporal summation of the potentials caused sufficient depolarization to inactivate the sodium conductance underlying the action potential. These results suggest that the outward current is necessary for NM neurons to transmit well-timed events reliably for the duration of an acoustic stimulus.
Few cell lines with GABAA mRNAs have functional receptors
Authors: Hales TG , Tyndale RF
Published: April 2018

In the preceding companion article (Tyndale et al., 1994) we used the PCR to investigate the occurrence of 13 GABAA receptor subunit mRNAs in several cell lines, including those derived from brain (B65, B104, and NB41A3), cerebellum (C17), glia (C6), pituitary (AtT-20), adrenal medulla (PC12), and the endocrine pancreas (RINm5F and beta TC3). In the present study we used the whole-cell configuration of the patch- clamp technique to determine which of these cell lines express functional GABAA receptors. All of the cell lines contain detectable levels of at least one GABAA receptor subunit mRNA (Tyndale et al., 1994); however, only RINm5F and beta TC3 cells exhibited GABA-evoked currents. GABA activated currents in all RINm5F cells, but currents were only barely detectable in 50% of beta TC3 cells tested. Many of the cell lines that failed to respond to GABA were derived from cell types with functional GABAA receptors. For example, the failure of PC12 cells to respond to GABA contrasts with the observation of GABA responses recorded from all primary cultured adrenomedullary chromaffin cells tested. GABA-evoked currents recorded from beta TC3 cells were too small (< 10 pA) to characterize pharmacologically. However, GABA activated robust currents recorded from RINm5F cells. These currents reversed at a holding potential similar to the equilibrium potential for Cl-, were blocked by the antagonist bicuculline methiodide (10 microM), and were potentiated by pentobarbital (100 microM). RINm5F cell GABAA receptors were insensitive to diazepam (10 microM) and were inhibited by Zn2+ (10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)
Peptides containing the RERMS sequence of amyloid beta/A4 protein precursor bind cell surface and promote neurite extension
Authors: Jin LW , Ninomiya H , Roch JM , Schubert D , Masliah E , Otero DA , Saitoh T
Published: April 2018

Amyloid beta/A4 protein precursor (APP) is secreted into medium by most cultured cells and can function as an autocrine factor. To study the biological function of secreted forms of APP (sAPP) on neurons, we used a clonal CNS neuronal line, B103, which does not synthesize detectable levels of APP. B103 cells transfected with APP construct developed neurites faster than the parent B103 cells when plated in a serum-free defined medium. Neurite outgrowth of B103 cells was promoted by the conditioned medium of APP-695-over-producing cells or by the bacteria- produced sAPP-695 (named KB75). A series of peptides having sequences between Ala-319 and Met-335 of APP-695 also stimulated neurite outgrowth of B103 cells. The sequence of five amino acids, RERMS (APP 328–332), within this stretch of sequence, was the shortest active peptide, although the concentration required for the neuritotropic activity was higher than that of KB75. Binding assay using 125I-labeled APP 17-mer peptide corresponding to Ala-319 to Met-335 of APP-695 as a ligand demonstrated specific and saturable cell-surface binding sites. The predicted KD value was 20 +/- 5 nM and the Bmax value was 80 +/- 8 fmol/10(6) cells. The binding could be displaced with KB75. A 17-mer peptide with reverse sequence neither induced neurite outgrowth nor competed for the binding. A bacteria-produced sAPP fragment lacking the active 17-mer sequence (named KB75 delta) did not compete with 125I- labeled 17-mer for binding or stimulate neurite extension. A peptide of sequence RMSQ (APP 330–333), which partially overlaps the active sequence RERMS, could block the neuritotropic effects of both KB75 and the 17-mer at higher concentrations. APP 17-mer was also found to induce the accumulation of inositol polyphosphates, suggesting that the APP 17-mer effects involve activation of inositol phospholipid signal transduction systems. These data indicate that sAPP induces neurite extension through cell-surface binding and that the domain containing the RERMS sequence (APP 328–332) represents the active site responsible for this function.
Gene expression in a subpopulation of luteinizing hormone-releasing hormone (LHRH) neurons prior to the preovulatory gonadotropin surge
Authors: Porkka-Heiskanen T , Urban JH , Turek FW , Levine JE
Published: April 2018

Gene expression in luteinizing hormone-releasing hormone (LHRH) neurons was analyzed during the periovulatory period to (1) characterize temporal patterns of LHRH gene expression and their relationship(s) to gonadotropin surges, and (2) determine if any such changes are uniform or dissimilar at different rostrocaudal levels of the basal forebrain. The number of neurons expressing mRNA for the decapeptide, and the relative degree of expression per cell were analyzed using in situ hybridization and quantitative image analysis. Rats were killed at 1800 hr on metestrus (Met), 0800 hr, 1200 hr, 1800 hr, and 2200 hr on proestrus (Pro), or 0200 hr, 0800 hr, and 1800 hr on estrus (E; n = 5–6 rats/group). All sections were processed for LHRH mRNA in a single in situ hybridization assay. Sections were atlas matched and divided into four rostrocaudal groups for analysis: vertical limb of the diagonal band of Broca (DBB), rostral preoptic area/organum vasculosum of the lamina terminalis (rPOA/OVLT), medial preoptic area (mPOA), and suprachiasmatic/anterior hypothalamic area (SCN/AHA). Plasma LH and FSH levels from all animals were analyzed by RIA. The labeling intensity per cell was similar among all time points at all four rostrocaudal levels. The number of cells expressing LHRH mRNA, however, varied as a function of time of death during the estrous cycle, and this temporal pattern varied among the four anatomical regions. At the level of the mPOA, the number of cells was highest at 1200 hr on Pro, and then declined and remained low throughout the morning of E. At the level of the rPOA/OVLT, the greatest number of LHRH neurons was noted later in Pro, at 1800 hr, dropping rapidly to lowest numbers at 2200 hr. No significant changes in LHRH cell number occurred at the DBB or SCN/AHA levels. At all anatomical levels, the secondary surge of FSH was unaccompanied by any change in the number of neurons expressing LHRH mRNA. These data demonstrate that (1) the number of detectable LHRH mRNA-expressing cells fluctuates during the periovulatory period and (2) peak numbers of LHRH-expressing cells are attained in the mPOA before the onset of the LH surge and before peak LHRH cell numbers are seen at more rostral levels. A model is proposed in which gene expression in this subpopulation of LHRH neurons may be activated by preovulatory estrogen secretion and acutely reduced following the proestrous surge of progesterone.
Functional comparisons of three glutamate transporter subtypes cloned from human motor cortex
Authors: Arriza JL , Fairman WA , Wadiche JI , Murdoch GH , Kavanaugh MP , Amara SG
Published: April 2018

Reuptake plays an important role in regulating synaptic and extracellular concentrations of glutamate. Three glutamate transporters expressed in human motor cortex, termed EAAT1, EAAT2, and EAAT3 (for excitatory amino acid transporter), have been characterized by their molecular cloning and functional expression. Each EAAT subtype mRNA was found in all human brain regions analyzed. The most prominent regional variation in message content was in cerebellum where EAAT1 expression predominated. EAAT1 and EAAT3 mRNAs were also expressed in various non- nervous tissues, whereas expression of EAAT2 was largely restricted to brain. The kinetic parameters and pharmacological characteristics of transport mediated by each EAAT subtype were determined in transfected mammalian cells by radio-label uptake and in microinjected oocytes by voltage-clamp measurements. The affinities of the EAAT subtypes for L- glutamate were similar, with Km determinations varying from 48 to 97 microM in the mammalian cell assay and from 18 to 28 microM in oocytes. Glutamate uptake inhibitors were used to compare the pharmacologies of the EAAT subtypes. The EAAT2 subtype was distinguishable from the EAAT1/EAAT3 subtypes by the potency of several inhibitors, but most notably by sensitivity to kainic acid (KA) and dihydrokainic acid (DHK). KA and DHK potently inhibited EAAT2 transport, but did not significantly affect transport by EAAT1/EAAT3. Using voltage-clamp measurements, most inhibitors were found to be substrates that elicited transport currents. In contrast, KA and DHK did not evoke currents and they were found to block EAAT2-mediated transport competitively. This selective interaction with the EAAT2 subtype could be a significant factor in KA neurotoxicity. These studies provide a foundation for understanding the role of glutamate transporters in human excitatory neurotransmission and in neuropathology.
Oxygen deprivation activates an ATP-inhibitable K+ channel in substantia nigra neurons
Authors: Jiang C , Sigworth FJ , Haddad GG
Published: April 2018

Depending on its severity and duration, O2 deprivation activates mechanisms that can lead to profound deleterious changes in neuronal structure and function. Hypoxia also evokes inherent adaptive mechanisms that can possibly delay injury and increase neuronal survival. One of these neuronal adaptive mechanisms is believed to be the activation of K+ channels, but direct evidence for their activation is lacking. We performed experiments to test the hypothesis that hypoxia induces activation of K+ channels via changes in cytosolic and membrane factors such as ATP, Ca2+, and membrane potential. The effect of hypoxia on single-channel currents was studied in rat substantia nigra neurons, since these have a high density of glibenclamide binding sites. In cell-attached patches, hypoxia or cyanide reversibly activated an outward current. This hypoxia-activated current in excised inside-out patches was K+ selective and voltage dependent, and had a high sensitivity to internal ATP, ADP, and AMP-PNP, a nonhydrolyzable ATP analog. Activation of this channel required the presence of free Ca2+ on the cytosolic side, but charybdotoxin or apamin did not have any effect on this channel. The effect of ATP on channel activity was not a result of Ca2+ chelation because Mg.ATP in high Mg2+ background and K2.ATP in high Ca2+ environment inhibited the channel. These results suggest that although this hypoxia-activated K+ channel shares properties with ATP-sensitive K+ (KATP) channels in other tissues, substantia nigra neurons seem to have a different subtype or isoform of KATP channels. Gating this channel by multiple factors simultaneously would allow this channel to be particularly suitable for activation during metabolic stress.
Age-associated and cell-type-specific neurofibrillary pathology in transgenic mice expressing the human midsized neurofilament subunit
Authors: Vickers JC , Morrison JH , Friedrich VL , Elder GA , Perl DP , Katz RN , Lazzarini RA
Published: April 2018

Alterations in neurofilaments are a common occurrence in neurons of the human nervous system during aging and diseases associated with aging. Such pathologic changes may be attributed to species-specific properties of human neurofilaments as well as cell-type-specific regulation of this element of the cytoskeleton. The development of transgenic animals containing human neurofilament subunits offers an opportunity to study the effects of aging and other experimental conditions on the human-specific form of these proteins in a rodent model. The present study shows that mice from the transgenic line NF(M)27, which express the human midsized neurofilament subunit at low levels (2–25% of the endogenous NF-M), develop neurofilamentous accumulations in specific subgroups of neurons that are age dependent, affecting 78% of transgenic mice over 12 months of age. Similar accumulations do not occur in age-matched, wild-type littermates or in 3-month-old transgenic mice. In 12-month-old transgenic mice, somatic neurofilament accumulations resembling neurofibrillary tangles were present predominantly in layers III and V of the neocortex, as well as in select subpopulations of subcortical neurons. Intraperikaryal, spherical neurofilamentous accumulations were particularly abundant in cell bodies in layer II of the neocortex, and neurofilament-containing distentions of Purkinje cell proximal axons occurred in the cerebellum. These pathological accumulations contained mouse as well as human NF subunits, but could be distinguished by their content of phosphorylation-dependent NF epitopes. These cytoskeletal alterations closely resemble the cell-type-specific alterations in neurofilaments that occur during normal human aging and in diseases associated with aging, indicating that these transgenic animals may serve as models of some aspects of the pathologic features of human neurodegenerative diseases.
Amphetamine regulates gene expression in rat striatum via transcription factor CREB
Authors: Konradi C , Cole RL , Heckers S , Hyman SE
Published: April 2018

Amphetamine is a psychostimulant drug of abuse that can produce long- lived changes in behavior including sensitization and dependence. The neural substrates of these drug effects remain unknown, but based on their prolonged time course, we hypothesize that they involve drug- induced alterations in gene expression. It has recently been demonstrated that amphetamine regulates the expression of several genes, including c-fos, via dopamine D1 receptors in rat striatum. Here we report that amphetamine induces phosphorylation of transcription factor cAMP response element binding protein (CREB) in rat striatum in vivo and that dopamine D1 receptor stimulation induces phosphorylation of CREB within specific complexes bound to cAMP regulatory elements. In addition, we show by antisense injection that CREB is necessary for c- fos induction by amphetamine in vivo. Since CREB has been implicated in the activation of a number of immediate-early genes as well as several neuropeptide genes, CREB phosphorylation may be an important early nuclear event mediating long-term consequences of amphetamine administration.
Disruption of target interactions prevents the development of enkephalin immunoreactivity in sympathetic neurons
Authors: Tyrrell S , Landis SC
Published: April 2018

We compared the development of enkephalin (Enk) expression in normal rats and rats in which target contact was transiently disrupted with 6- hydroxydopamine (6-OHDA). During the first 3 postnatal weeks, there was a striking increase in Enk immunoreactivity (-IR) in superior cervical ganglia (SCG) assayed by radioimmunoassay (RIA). This increase was correlated with the appearance of Enk-IR in postganglionic neurons. In the caudal region of the SCG, the proportion of Enk-IR neurons and their immunoreactivity increased until one-third of the neurons possessed Enk-IR between postnatal days (P) 14 and 21. After P21, the number of Enk-IR neurons and their immunofluorescence decreased. By 6 weeks, only occasional neurons possessed moderate Enk-IR. The increases in Enk-IR were correlated with increased ganglionic proenkephalin A mRNA detected by in situ hybridization. The decrease in IR after P21 was not, however, paralleled by a comparable decrease in proenkephalin A mRNA. To determine whether interactions between SCG neurons and their target tissues influence Enk expression, we disrupted them by treating neonatal rats with a single dose of 6-OHDA at P0. This treatment transiently reduced sympathetic fiber density in the submandibular gland, one target of Enk-IR neurons, over 90%. Two weeks later, the fiber density in glands of treated animals was not different from control. Following 6-OHDA, the concentration of Enk-IR in SCG extracts and the number of Enk-IR neurons and their immunofluorescence intensity failed to increase. SCG from treated rats also contained fewer neurons with proenkephalin A mRNA. In contrast, the content of neuropeptide Y (NPY) and the proportion of NPY-IR neurons were not decreased by 6-OHDA treatment. Our results indicate that the developmental history of Enk expression differs from that of other neuropeptides in rat sympathetic ganglia, suggesting that distinct mechanisms regulate the expression of individual neuropeptides. Further, they provide evidence that target contact during a critical period is important for the induction of Enk.
Facilitation of transmission at the frog neuromuscular junction at O degrees C is not maximal at time zero
Authors: Van der Kloot W
Published: April 2018

Facilitation of quantal release has been thought to be maximal immediately after the first action potential in the presynaptic terminal. However, at the frog neuromuscular junction at 0 degrees C no facilitation was observed in response to direct nerve depolarization when the interval between stimulus pairs was less than 10 msec (Dudel, 1986), while at intervals of 20 msec and beyond, facilitation was increased (Molgo and Van der Kloot, 1991). In the present experiments, facilitation to pairs of nerve action potentials was measured both by the method of failures and by comparing the total inward postsynaptic current generated by the first and second action potentials. Facilitation was observed at intervals as short as 7 msec, but 20–30 msec was required for facilitation to reach a maximum. This suggests that facilitation requires a second messenger or the action of Ca2+ at a site other than that eliciting exocytosis.
Calcium permeability of the neuronal nuclear envelope: evaluation using confocal volumes and intracellular perfusion
Authors: O'Malley DM
Published: April 2018

In many calcium-imaging studies, the nuclear envelope appears to maintain a gradient of free calcium between the nucleus and cytosol. This issue was examined by loading amphibian sympathetic neurons with the calcium indicator fluo 3 via whole-cell patch clamping. Confocal optical sectioning allowed acquisition of independent calibration curves for the nucleus and cytoplasm. Cells were loaded with free calcium levels ranging from 10 nM to 50 microM, using 10 mM BAPTA to control free calcium. The nuclear fluorescence was usually about 130% brighter than the cytoplasmic fluorescence. Had the increased nuclear fluorescence been due to a calcium gradient, then, as fluo 3 was saturated with calcium in both compartments, the fluorescence gradient should have gradually disappeared. Instead, with free-calcium in the pipette set at 50 microM, about five times the level required to nearly saturate fluo 3, the nuclear/cytoplasmic (N/C) fluorescence ratio was not decreased but instead increased slightly. Perfusion of the patch pipette was used in conjunction with imaging to confirm that cytoplasmic fluo 3 was saturated with calcium. After loading cells with 10 nM free calcium, the patch pipette was perfused with high calcium (10 microM). Again, the N/C fluorescence ratio increased at high calcium. The effectiveness of patch-pipette perfusion in changing cellular free calcium levels was indicated by the degree of fluorescence increase--both nuclear and cytosolic compartments showed a roughly 20-fold increase in fluorescence, that is, most of the dynamic range observed in test droplets. To confirm further that cytoplasmic fluo 3 was saturated, cells were perfused with manganese, which binds with very high affinity to fluo 3. Manganese rapidly entered the cytoplasm and nucleus, causing a large increase in fluorescence, but the N/C fluorescence ratio remained relatively constant. Because free manganese in the pipette was 50,000 times the amount required to saturate fluo 3, the greater nuclear fluorescence probably results from additional fluo 3 in the nucleus rather than from calcium or manganese gradients. To gauge further the permeability of the nuclear envelope, the diffusion of calcium was visualized. Under voltage clamp, calcium channels were opened for periods ranging from 5 to 200 msec. Peak calcium levels were observed within 2 microns of the plasma membrane, and declined as calcium diffused into the cell. The nuclear fluorescence increased more than cytosolic fluorescence, but this apparent “amplification” was eliminated by correcting for autofluorescence. Use of cells cultured on glass coverslips and a high- NA microscope objective allowed a satisfactory correction.(ABSTRACT TRUNCATED AT 400 WORDS)
Sodium/calcium exchange in rat cortical astrocytes
Authors: Goldman WF , Yarowsky PJ , Juhaszova M , Krueger BK , Blaustein MP
Published: April 2018

Regulation of the cytosolic free Ca2+ concentration ([Ca2+]cyt) by an Na/Ca exchanger was studied in primary cultured rat cortical astrocytes. [Ca2+]cyt was measured by digital imaging in cells loaded with fura-2. The resting [Ca2+]cyt, approximately 150 nM, was only slightly increased by reducing the extracellular Na+ concentration ([Na+]o) to 6.2 mM, or by treating the cells with ouabain for 15 min (to raise cytosolic Na+). Following treatment with ouabain, however, lowering [Na+]o caused [Ca2+]cyt to rise rapidly to approximately 1300 nM. When Ca2+ sequestration in intracellular stores was blocked by thapsigargin, lowering [Na+]o increased [Ca2+]cyt to approximately 1500 nM in the absence of ouabain. The low-[Na+]o-stimulated rise in [Ca2+]cyt was abolished by removal of external Ca2+, but was not blocked by the Ca2+ channel blocker verapamil, or by caffeine or ryanodine, which deplete an intracellular Ca2+ store responsible for Ca(2+)-induced Ca2+ release. These data suggest that Na+ gradient reduction promotes net Ca2+ gain via Na/Ca exchange. Normally, however, a large rise in [Ca2+]cyt is prevented by sequestration of the entering Ca2+; this buffering of cytosolic Ca2+ can be circumvented by blocking sequestration with thapsigargin, or overwhelmed by enhancing net Ca2+ gain by pretreating the cells with ouabain. The presence of Na/Ca exchanger protein and mRNA in the astrocytes was confirmed by Western and Northern blot analyses, respectively. Immunohistochemistry revealed that exchanger molecules are distributed in a reticular pattern over the astrocyte surface. We suggest that the Na/Ca exchanger plays a role in regulating both [Ca2+]cyt and the intracellular stores of Ca2+ in astrocytes, and may thus contribute to the control of astrocyte responsiveness to neurotransmitters and neurotoxins.
c-Fos expression in the spinal cord and pain-related symptoms induced by chronic arthritis in the rat are prevented by pretreatment with Freund adjuvant
Authors: Abbadie C , Besson JM , Calvino B
Published: April 2018

We previously showed that pathological adjuvant-induced arthritis (AIA), a chronic pain model, is associated with an increase in the number of Fos-like immunoreactivity (Fos-LI) in lumbar spinal cord neurons; maximal Fos-LI corresponds to the peak of pathological and behavioral signs of the disease. On the other hand, AIA is prevented in rats that have previously been injected with diluted complete Freund adjuvant (CFA). Here, the effects of pretreatment with CFA were studied in parallel on both pathological parameters and pain-related tests, and on Fos-LI, 3 weeks after the inoculation of the arthritogenic solution during the acute phase of the disease, that is, when hyperalgesia is maximal. One group of rats (vaccinated group) was pretreated with diluted CFA, 3 weeks and 1 week before inoculation (AIA) (with the concentrated arthritogenic solution), while a second group (arthritic group) was pretreated with the vehicle for the CFA solution and then inoculated with the concentrated arthritogenic solution. Control groups consisted of rats treated with various combinations of either saline or vehicle solutions during the pretreatment periods or at the time of inoculation. The arthritic group developed all pathological symptoms of AIA (i.e., loss of weight, difficulties in walking, increases in paw diameter, and decreases in the vocalization thresholds to mechanical pressure of the hindpaw, and finally an increase in the vocalization response to flexion and extension). In contrast, the vaccinated group did not develop AIA symptoms.(ABSTRACT TRUNCATED AT 250 WORDS)
Rapid sprouting of filopodia in nerve terminals of chromaffin cells, PC12 cells, and dorsal root neurons induced by electrical stimulation
Authors: Manivannan S , Terakawa S
Published: April 2018

Rapid morphological changes induced by direct electrical stimulation of nerve terminals were studied by using video-enhanced differential interference contrast microscopy at a very high magnification (12,000x). We used mainly cultured bovine chromaffin cells, which developed neurite-like processes, and PC12 cells, which showed neuronal differentiation upon NGF treatment. In a few cases, primary neurons of the rat dorsal root ganglion were also examined. Brief pulse stimulation of the terminals and varicosities induced exocytosis accompanied by rapid formation of filopodia. These filopodia, 0.1–0.2 micron in diameter and up to 10 microns in length, formed within a few hundreds of milliseconds and then retracted within tens of seconds. They could also be induced by K depolarization. This rapid filopodial sprouting strongly depended on the presence of extracellular Ca2+ and could be abolished in a medium containing a Ca chelator (EGTA) or La2+. Anti-cytoskeletal agents colchicine and cytochalasin B failed to block this response completely but lidocaine fully suppressed it. Quantitative analysis of exocytosis and filopodial sprouting showed that they were independent events, not directly linked to each other, having different thresholds usually higher for filopodial formation. In PC12 cells, the extent of filopodial sprouting varied with the state of differentiation of the cells, suggesting a functional role of rapid sprouting during a particular phase of their differentiation. Filopodia could be induced with greater ease by repetitive stimulation. The same responses may occur at growth cones approaching the target cells or even at mature synapses particularly after repetitive electrical activity, possibly playing a role in use-dependent synapse formation or plasticity.
NMDA and non-NMDA glutamate receptors in auditory transmission in the barn owl inferior colliculus
Authors: Feldman DE , Knudsen EI
Published: April 2018

The pharmacology of auditory responses in the inferior colliculus (IC) of the barn owl was investigated by iontophoresis of excitatory amino acid receptor antagonists into two different functional subdivisions of the IC, the external nucleus (ICx) and the lateral shell of the central nucleus (lateral shell), both of which carry out important computations in the processing of auditory spatial information. Combined application of the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (AP5) and the non-NMDA receptor antagonist 6-cyano-5-nitroquinoxaline-2,3- dione (CNQX) significantly reduced auditory-evoked spikes at all sites in these two subdivisions, and completely eliminated responses at many locations. This suggests that excitatory amino acid receptors mediate the bulk, if not all, of auditory responses in the ICx and lateral shell. NMDA and non-NMDA receptors contributed differently to auditory responses in the two subdivisions. In the ICx, AP5 significantly reduced the number of auditory-evoked spikes at every site tested. On average, AP5 eliminated 55% of auditory-evoked spikes at multiunit sites and 64% at single-unit sites in this structure. In contrast, in the lateral shell, AP5 significantly reduced responses at less than half the sites tested, and, on average, AP5 eliminated only 19% of spikes at multiunit sites and 25% at single-unit sites. When the magnitude of response blockade produced by AP5 at individual multiunit sites was normalized to adjust for site-to-site differences in the efficacy of iontophoresed AP5 and CNQX, AP5 blockade was still significantly greater in the ICx than the lateral shell. CNQX application strongly reduced responses in both subdivisions. These data suggest that NMDA receptor currents make a major contribution to auditory responses in the ICx, while they make only a small contribution to auditory responses in the lateral shell. Non-NMDA receptor currents, on the other hand, contribute to auditory responses in both subdivisions, and mediate the bulk of auditory transmission in the lateral shell. The time course of the NMDA receptor contribution to ICx auditory responses and the dependence of this contribution on stimulus level were both examined in detail. AP5 preferentially blocked spikes late in ICx auditory responses, while CNQX blocked spikes equally throughout the responses. This pattern is consistent with a simple model in which slow NMDA receptor currents and faster non-NMDA receptor currents are both activated by auditory inputs to ICx neurons.
Behavioral, biochemical, histological, and electrophysiological effects of 192 IgG-saporin injections into the basal forebrain of rats
Authors: Wenk GL , Stoehr JD , Quintana G , Mobley S , Wiley RG
Published: April 2018

The behavioral, biochemical, histological, and electrophysiological effects of a basal forebrain injection of saporin, a ribosome- inactivating protein, coupled to a monoclonal antibody against the low- affinity NGF receptor (192 IgG) were investigated in adult rats. Within the basal forebrain region, the low-affinity NGF receptor is exclusively expressed by cholinergic neurons in the medial septal area, diagonal band, and nucleus basalis magnocellularis (NBM). The presence of this receptor upon these cells confers a degree of specificity to the 192 IgG-saporin that could not previously be achieved by previous lesioning techniques, such as excitatory amino acids. Rats with unilateral injections of different amounts of 192 IgG-saporin were prepared to determine the optimal conditions in order to produce a lesion restricted to the NBM that would not destroy cholinergic afferents to hippocampus or nearby regions. Electroencephalographic (EEG) recordings were taken from these lesioned rats before and during treatment with scopolamine (1 mg/kg, i.p.). Another group of rats received bilateral NBM injections of 192 IgG-saporin and were behaviorally tested using a rewarded, delayed-alternation task on a T- maze and a passive avoidance task. Finally, histological and biochemical investigations confirmed the effectiveness and specificity of the 192 IgG-saporin. The results showed that the 192 IgG-saporin did not destroy neurotensin, galanin, somatostatin, NADPH-diaphorase, or neuropeptide Y neurons within the NBM. Also, biomarkers of cholinergic function were significantly decreased throughout the neocortex and within the NBM, but not in the olfactory bulbs, hippocampus, or dorsal caudate nucleus. Intraperitoneal injections of scopolamine, but not NBM injections of 192 IgG-saporin, increased total power across all frequency bands; however, slow-wave frequencies showed a greater increase in power as compared to fast-wave frequencies. Acquisition, and performance of the delayed-alternation or passive avoidance tasks were not impaired by the lesions. These data confirm the effectiveness and specificity of this novel lesioning tool and suggest that selective loss of NBM cholinergic cells is not sufficient to impair performance in these behavioral tasks.
Locus coeruleus stimulation by corticotropin-releasing hormone suppresses in vitro cellular immune responses
Authors: Rassnick S , Sved AF , Rabin BS
Published: April 2018

Previous studies have demonstrated that stressors alter cellular immune system function, and increase the activity of locus coeruleus neurons. Furthermore, stressors increase the release of corticotropin-releasing hormone (CRH) and locus coeruleus neurons are activated by CRH. Thus, the present study examined whether activation of the locus coeruleus by infusion of CRH modulates the function of blood and spleen lymphocytes assessed in vitro. CRH (100 ng) was administered into the region of the locus coeruleus in awake rats 1 hr before spleen and peripheral blood lymphocytes were collected for culture with nonspecific mitogens. Unilateral or bilateral microinfusion of CRH into the locus coeruleus produced a decrease in blood and spleen T-lymphocyte mitogenic responses to phytohemagglutinin, ConA, and an antibody to the T- lymphocyte antigen receptor. In contrast, infusion of saline into the locus coeruleus or CRH into the surrounding region of the dorsal pons did not alter spleen or blood lymphocyte responses. Plasma concentrations of adrenocorticotropic hormone, corticosterone, and IL-6 were increased by CRH infusion into the locus coeruleus. These results suggest that CRH-evoked activation of the locus coeruleus stimulates the hypophysial adrenal axis, possibly activates the sympathetic nervous system, and results in immunosuppression. Comparable changes in lymphocyte and hormone responses are produced by an aversive stimulus or a conditioned stressor, suggesting that activation of the locus coeruleus may be a component of stressor-induced immune alterations.
The NMDA receptor subunits NR2A and NR2B show histological and ultrastructural localization patterns similar to those of NR1
Authors: Petralia RS , Wang YX , Wenthold RJ
Published: April 2018

Neuronal plasticity associated with learning, memory and development is controlled, in part, by NMDA receptors, which are complexes consisting of the subunit NMDAR1 (NR1) and one or more NMDAR2 subunits (NR2A- NR2D). We made a polyclonal antibody to a C-terminus peptide of NR2A. In analysis of transfected cell membranes, this antibody recognizes NR2A and NR2B, and to a slight extent, NR2C and NR2D. In Western blots of rat brain, the antibody labeled a single band that comigrated with NR2A and NR2B. This antibody (NR2A/B) did not cross-react with extracts from transfected cells expressing other glutamate receptor subunits, nor did it label non-neuronal tissues. Immunostained sections of rat brain showed significant staining throughout the nervous system, including olfactory bulb, cerebral cortex, hippocampus, caudate- putamen, and many brainstem nuclei, as well as in neurons of spinal cord and sensory ganglia. This widespread distribution of staining was similar to that found with an antibody to NR1, supporting the presence of functional NR1/NR2 complexes throughout the nervous system. In the cerebellum, in contrast to staining with NR1 antibody, Purkinje cell staining with NR2A/B antibody was low, indicating that these neurons may lack functional NMDA receptors. EM examination revealed dense staining in dendrites and postsynaptic densities in cerebral cortex and hippocampus, similar to those seen with antibody to NR1. Since functional NMDA receptor complexes at synapses appear to require both NR1 and NR2 subunit proteins for full function, this study provides structural evidence for functional NR1/NR2 receptors in vivo in the nervous system.