bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of the POU domain gene SCIP during cerebral cortical development
Authors: Frantz GD , Bohner AP , Akers RM , McConnell SK
Published: April 2018

The mammalian cerebral cortex is patterned into layers of neurons that share characteristic morphologies, physiological properties, and axonal connections. Neurons in the various layers are thought to acquire their lamina-specific identities shortly before the time of their final mitosis in the cortical ventricular zone. In order to investigate the molecular basis of laminar patterning in the CNS, we have performed in situ hybridization studies of the POU homeodomain gene SCIP (also known as Tst-1 or Oct-6), which is expressed in proliferating Schwann cells in the PNS and O2A progenitor cells in the developing CNS. In the CNS of adult rats, SCIP is expressed at high levels in the cerebral cortex, specifically in layer 5 pyramidal neurons that form subcortical axonal connections. SCIP is both temporally and spatially regulated during cortical development. Its initial expression in the intermediate zone and cortical plate is correlated with the early migration and differentiation of layer 5 neurons. SCIP hybridization was not, however, observed within the ventricular zone during the period of neurogenesis. SCIP is also expressed at high levels in the neurons of cortical layer 2/3, during their migration and differentiation within the cortical plate. This expression in the upper layers is apparently downregulated during postnatal periods, with the adult pattern apparent by postnatal day 30 (P30). POU domain genes are thought to play a role in cell lineage and cell fate decisions in several systems; thus, SCIP may serve a function in generating discrete laminar phenotypes in the developing cerebral cortex. In addition, since SCIP is a putative repressor of myelin gene expression, our results suggest that SCIP plays a role in regulating transcription in differentiated CNS neurons as well as in proliferating glial precursors.
GAP-43 transgenic mice: dispersed genomic sequences confer a GAP-43- like expression pattern during development and regeneration
Authors: Vanselow J , Grabczyk E , Ping J , Baetscher M , Teng S , Fishman MC
Published: April 2018

Using transgenic mice, we have examined the expression pattern conferred by regions of genomic GAP-43 coupled to beta-galactosidase. We demonstrate that gene constructions that include the GAP-43 5′- flanking region along with sufficient sequences of the first intron drive beta-galactosidase (lacZ) expression to mimic in many regards the complex spatial and temporal pattern of endogenous GAP-43 expression. Transgene expression reaches peak levels during development, and persists at high levels in particular adult brain regions, such as the hippocampus and olfactory bulb. The inclusion of a stretch of the first intron in the construction is necessary to prevent expression outside of the nervous system, indicating that some of the cell specificity of GAP-43 expression is due to suppression of expression in inappropriate tissues. Injury caused by sciatic nerve crush causes reexpression of the transgene in adult sensory and motor neurons. This genomic region of GAP-43, therefore, includes elements responsive to neuronal growth signals that regulate both development and regeneration.
Colocalization of somatostatin with GABA or glutamate in distinct afferent terminals presynaptic to the Mauthner cell
Authors: Sur C , Korn H , Triller A
Published: April 2018

The presence of somatostatin in afferent fibers impinging on the goldfish Mauthner (M-) cell was determined using immunohistochemical methods, combined with confocal and electron microscopy, and the relationship of this peptide with inhibitory and excitatory terminals was studied. Somatostatin-reactive boutons were present only on the distal part of the M-cell's lateral dendrite. Somatostatin immunoreactivity was observed in typical large myelinated club endings (LMCEs) corresponding to mixed (electrical and chemical) eighth nerve primary afferent fibers. The axoplasm of these fibers contained dense- core vesicles (DCVs) dispersed among round vesicles. We have made a novel finding that the excitatory transmitter glutamate is present in LMCEs. Colocalization of this amino acid with somatostatin was detected in 75% of these endings using postembedding staining with gold particles of various sizes. The other structures labeled by somatostatin antibody were found to be small vesicle boutons (SVBs), which establish symmetrical synapses and contain a population of pleiomorphic vesicles with DCVs scattered among them. Double labeling with antibodies against glutamic acid decarboxylase and GABA allowed the definition of three types of biochemically characterized terminals: [somatostatin-GABA], [GABA], and [somatostatin]. However, the occurrence of DCVs in SVBs stained for GABA alone suggests that neuropeptides other than somatostatin may also coexist with GABA in this class of boutons. The coexistence of somatostatin with both inhibitory and excitatory neurotransmitters acting on the same region of a postsynaptic cell is discussed in relation to the role postulated for this peptide in synaptic plasticity.
NK-1 and NK-3 type tachykinin receptor mRNA expression in the rat spinal cord dorsal horn is increased during adjuvant or formalin- induced nociception
Authors: McCarson KE , Krause JE
Published: April 2018

Substance P (SP) and other related tachykinins such as neurokinin B (NKB) have been studied widely as mediators of sensory information. The release of SP into the dorsal horn of the spinal cord is increased during nociception, and SP activates nociception-specific dorsal horn neurons. The tachykinin NKB has antinociceptive effects in the spinal cord and is contained in intrinsic spinal neurons; thus, NKB may also contribute to the processing of sensory information. Both neurokinin-1 (NK-1) and neurokinin-3 (NK-3) receptors have been localized in the superficial laminae of the dorsal horn. This study investigated changes in NK-1 and NK-3 receptor mRNA expression during nociception. Following injection of either formalin or complete Freund's adjuvant (CFA) into one hindpaw, the levels of expression of NK-1 and NK-3 mRNAs in the spinal cord dorsal horn and preprotachykinin (PPT) mRNA expression in the lumbar dorsal root ganglia (DRG) were quantitated using solution hybridization-nuclease protection assays. Peptide and receptor mRNA expression levels were normalized to beta-actin mRNA levels, which did not change during the treatments. Formalin (2 or 6 hr) or CFA (4 d) injection produced approximately a twofold increase in SP-encoding PPT mRNA expression in the ipsilateral lumbar DRG. Increased activity in primary afferent neurons containing SP may stimulate the production of SP precursors, providing substrate for increased SP production, release, and turnover in the dorsal horn and periphery.(ABSTRACT TRUNCATED AT 250 WORDS)
Suppression of sodium channel function in differentiating C2 muscle cells stably overexpressing rat androgen receptors
Authors: Tabb JS , Fanger GR , Wilson EM , Maue RA , Henderson LP
Published: April 2018

Differentiation of skeletal muscle and the formation of the neuromuscular junction are regulated by steroid hormones. The effects of androgens on ion channel proteins central to neuromuscular signalling have been investigated in differentiating mouse muscle C2 cells and in C2 cells that stably overexpress the rat androgen receptor (AR) cDNA. Neither the expression nor function of ACh receptors was regulated by androgenic actions in these cells. However, voltage- dependent sodium (Na) current density was decreased by androgen treatment of C2 cells and was abolished, even in the absence of androgens, in C2 cells that overexpress the AR. The decrease in functional Na current was not accompanied by concomitant decreases in Na channel mRNA, suggesting that AR influence posttranscriptional processing of Na channels in differentiating C2 cells.
Characterization of antibodies to the rat substance P (NK-1) receptor and to a chimeric substance P receptor expressed in mammalian cells
Authors: Vigna SR , Bowden JJ , McDonald DM , Fisher J , Okamoto A , McVey DC , Payan DG , Bunnett NW
Published: April 2018

Antibodies to neuropeptide receptors can be used to localize and characterize the receptors in tissues and cell lines. Two strategies were used to study the rat substance P receptor (SPR, NK-1) by immunological methods. First, a polyclonal antiserum was raised by immunizing rabbits with a peptide corresponding to the 15 amino acid residues (KTMTESSSFYSNMLA, SPR393–407) at the intracellular C-terminus of the rat SPR coupled to bovine thyroglobulin. An antiserum was obtained with a titer for half-maximal binding of 125I-SPR393–407 of 1:70,000. Nonradioactive SPR393–407 inhibited 50% of binding at a concentration of 10 pM. Binding of 125I-SPR393–407 to the antiserum was also displaced in a parallel manner by membrane proteins from tissues expressing high levels of the SPR (brain and submaxillary gland). Second, a chimeric SPR construct of a hydrophilic Flag peptide (DYKDDDDK) genetically engineered in sequence with the extracellular N- terminus of rat SPR was generated by polymerase chain reaction. The Flag-SPR chimera was expressed in rat kidney epithelial cells (KNRK) and judged to be fully functional, assessed by binding of 125I- substance P (apparent Kd of 5.63 nM) and calcium mobilization in response to substance P (EC50 of 0.66 nM). Antibodies to SPR393–407 and the Flag peptide stained the plasma membrane of KNRK cells expressing the native SPR or the Flag-SPR chimera. Staining was abolished by preincubation with SPR393–407 or the Flag peptide. Cells transfected with vector alone were unstained. The SPR antiserum recognized a broad protein band on Western blots of membranes prepared from cells expressing SPR but not from cells transfected with vector alone. The signal was quenched by preincubation of the antiserum with SPR393–407. By immunohistochemistry, the SPR antiserum was found to bind to neurons in the dorsal horn of the rat spinal cord and to ganglion cells in the myenteric plexus of the rat ileum near substance P-immunoreactive nerve fibers. Staining was abolished by preabsorption of the antiserum with SPR393–407. These antibodies can be used to localize the SPR in tissues and cells and to examine the function of the receptor in cell lines.
Emetic reflex arc revealed by expression of the immediate-early gene c- fos in the cat
Authors: Miller AD , Ruggiero DA
Published: April 2018

The organization of the central neuronal circuitry that produces vomiting was explored by mapping the distribution of c-fos protein (Fos)-like immunoreactivity (FLI) as a monitor of functional activity. The brainstem and spinal cord were examined in cats administered multiple emetic drugs (cisplatin, lobeline, protoveratrine, naloxone, apomorphine) or control saline injections. Some animals were decerebrated, paralyzed, and artificially ventilated to avoid possible Fos expression induced by sensory feedback or fluid depletion during vomiting. Fictive vomiting was identified in these animals by a characteristic pattern of respiratory muscle nerve (phrenic and abdominal) coactivation. Tissues were immunoprocessed using an antibody raised against amino acids 1–131 of Fos and the avidin-biotin peroxidase complex method. Enhanced nuclear FLI was observed in experimental animals along portions of the sensorimotor emetic reflex arc, including the nodose ganglia, area postrema, nuclei of the solitary tract (especially medial and subpostrema subnuclei), intermediate reticular zone of the lateral tegmental field, nucleus retroambiguus, C2 inspiratory propriospinal cell region, and dorsal vagal and phrenic motor nuclei. Enhanced FLI was also detected in the raphe magnus, subretrofacial nucleus, and spinal dorsal horn. Regions showing no recognizable differences in FLI between experimental and control animals included the vestibular, cochlear, spinal trigeminal, subtrigeminal, and lateral reticular nuclei. Only minor differences were observed in the distributions of FLI between intact and decerebrate animals. No unique, well-defined group of labeled neurons that might function as a “vomiting center” could be identified. Instead, the pattern of c-fos expression suggests that neurons involved in coordinating the emetic response may radiate from the area postrema and nucleus of the solitary tract to an arc in the lateral tegmental field implicated in somato-autonomic integration.
Neuronal acetylcholine receptors that bind alpha-bungarotoxin mediate neurite retraction in a calcium-dependent manner
Authors: Pugh PC , Berg DK
Published: April 2018

Neuronal membrane components that bind alpha-bungarotoxin with high affinity have only recently been shown unambiguously to function as nicotinic receptors. Activation of the receptors increases intracellular levels of free calcium in neurons. In the chick ciliary ganglion, where the receptors have been studied in some detail, they have been shown to have a predominantly nonsynaptic location on neurons and may be concentrated on pseudodendrites emerging from the somata. This has raised questions about the physiological significance of the receptors for the neurons. Here we show that activation of the receptors on isolated ciliary ganglion neurons in cell culture produces neurite retraction. Focal application of either nicotine or ACh at low concentrations induces the retraction, and alpha-bungarotoxin blocks the effect. The retraction requires external calcium and is confined to the individual neurite stimulated with agonist. Brief exposure to elevated concentrations of K+ also induces neurite retraction, and both the K(+)-induced and the nicotine-induced retractions can be prevented by the calcium channel blocker omega-conotoxin. The results suggest that activation of the alpha-bungarotoxin-binding nicotinic receptors on neurites triggers activation of voltage-gated calcium channels presumably by depolarizing the membrane, and that together they permit sufficient calcium to enter the neurite to prevent further outgrowth and induce retraction.
Glutamate receptor agonists stimulate diverse calcium responses in different types of cultured rat cortical glial cells
Authors: Holzwarth JA , Gibbons SJ , Brorson JR , Philipson LH , Miller RJ
Published: April 2018

We examined the effects of different types of glutamate receptor agonists on the intracellular calcium concentration, ([Ca2+]i), in cultured rat cortical glial cells. The cells in these cultures were characterized immunocytochemically using antibodies against glial fibrillary acidic protein, A2B5, and OX-42. The metabotropic glutamate receptor agonist (1S,3R)-1-aminocyclopentane-1,3- dicarboxylic acid produced Ca2+ mobilization from intracellular stores in all classes of cells. Agonists at non-NMDA glutamate receptors also produced large increases in [Ca2+]i, primarily in cells of the O-2A lineage. Disruption of intracellular Ca2+ stores with thapsigargin showed that increases in [Ca2+]i produced by activating AMPA/kainate receptors were primarily due to Ca2+ influx rather than Ca(2+)-induced Ca2+ release. Agonists at NMDA receptors were ineffective. Electrophysiological studies revealed that cells of the O-2A lineage exhibited moderate inward currents in response to kainate in Na(+)-containing solutions, but only small inward currents and outward rectification in Na(+)-free solutions. However, in the presence of cyclothiazide, the kainate- induced currents were increased in size and a rightward shift of the reversal potential with increased [Ca2+]o could be demonstrated. Activation of cells by kainate, but not by depolarizing stimuli, stimulated the uptake of Co2+. Polymerase chain reaction studies showed that the glutamate receptor subunits GluR1–4 and GluR6 were all expressed in these cultures, but GluR5 was absent. The nature of the Ca2+ uptake pathway activated by non-NMDA receptor agonists in the O-2A lineage population is discussed. It is considered most likely that the O-2A lineage cells express both non-NMDA receptors that are relatively impermeable to divalent cations, as well as a smaller population that are Ca2+ permeable.
Subplate pioneers and the formation of descending connections from cerebral cortex
Authors: McConnell SK , Ghosh A , Shatz CJ
Published: April 2018

The adult cerebral cortex extends axons to a variety of subcortical targets, including the thalamus and superior colliculus. These descending projections are pioneered during development by the axons of a transient population of subplate neurons (McConnell et al., 1989). We show here that the descending axons of cortical plate neurons appear to be delayed significantly in their outgrowth, compared with those of subplate neurons. To assess the possible role of subplate neurons in the formation of these pathways, subplate neurons were ablated during the embryonic period. In all cases, an axon pathway formed from visual cortex through the internal capsule and into the thalamus. In half of all cases, however, cortical axons failed to invade their normal subcortical targets. In the other half, targets were innervated normally. Subplate neurons are therefore likely to provide important cues that aid the process by which cortical axons grow toward, select, and invade their subcortical targets.
Neurosteroids modulate calcium currents in hippocampal CA1 neurons via a pertussis toxin-sensitive G-protein-coupled mechanism
Authors: ffrench-Mullen JM , Danks P , Spence KT
Published: April 2018

The inhibition of Ca2+ channel currents by endogenous brain steroids was examined in freshly dissociated pyramidal neurons from the adult guinea pig hippocampal CA1 region. The steady-state inhibition of the peak Ca2+ channel current evoked by depolarizing steps from -80 to -10 mV occurred in a concentration-dependent manner with the following IC50 values: pregnenolone sulfate (PES), 11 nM; pregnenolone (PE), 130 nM; and allotetrahydrocorticosterone (THCC), 298 nM. THCC, PE, and PES depressed a fraction of the Ca2+ channel current with a maximal inhibition of 60% of the total current. However, substitution of an acetate group for the sulfate group on PES resulted in a complete loss of activity. Progesterone had no effect (4% inhibition at 100 microM). Intracellular dialysis of PES had no effect on the Ca2+ current; concomitant extracellular perfusion of PES showed normal inhibitory activity, suggesting that the steroid binding site can only be accessed extracellularly. Analysis of tail currents at -80 mV demonstrated that THCC and PES slowed the rate of Ca2+ current activation and deactivation with no change in the voltage dependence of activation. Inhibition of the Ca2+ channel current by THCC and PES was voltage dependent. THCC primarily inhibits the omega-conotoxin (CgTX)-sensitive or N-type Ca2+ channel current. PE was nonselective in inhibiting both the CgTX- and the nifedipine (NIF)-sensitive Ca2+ channel current. These neurosteroids had no effect on the CgTX/NIF-insensitive current. In neurons isolated from pertussis toxin (PTX)-treated animals by chronic intracerebroventricular infusion (1000 ng/24 hr for 48 hr), the Ca2+ channel current inhibition by PES, PE, and THCC was significantly diminished. Intracellular dialysis with GDP-beta-S (500 microM) also significantly diminished the neurosteroid inhibition of the Ca2+ channel current. Intracellular dialysis with the general kinase inhibitors H-7 (100 microM), staurosporine (400 nM), and a 20 amino acid protein kinase inhibitor (1 microM) also significantly prevented the THCC and PES inhibition of the Ca2+ channel current. Intracellular dialysis with the more specific inhibitors of protein kinase C (PKC), the pseudosubstrate inhibitor (PKCI 19–36) (1–2 microM) and bisindolylmaleimide (1 microM) significantly diminished the THCC and PE inhibition of the Ca2+ channel current. Rp- cAMP (100 microM), a specific inhibitor of cAMP-dependent protein kinase (PKA), had no effect on the THCC and PE inhibition of the Ca2+ current.(ABSTRACT TRUNCATED AT 400 WORDS)
Glucocorticoid-induced impairment in declarative memory performance in adult humans
Authors: Newcomer JW , Craft S , Hershey T , Askins K , Bardgett ME
Published: April 2018

Glucocorticoids (GCs) have a variety of effects on the brain including site-preferential, inhibitory effects on hippocampal neurons. In the case of dexamethasone (DEX), extended rather than single-dose treatment in vivo may be required for binding to brain rather than peripheral (e.g., pituitary) GC receptors and for maximizing other biologic effects in hippocampus (e.g., GC receptor downregulation, inhibition of glucose transport). Based on the contributory role of hippocampal neurons in declarative memory performance, we investigated the cognitive consequences of DEX treatment in normal adult human subjects, hypothesizing a decrease in declarative memory performance after extended but not overnight treatment. Double-blind, placebo-controlled treatment with DEX was given at 2300 hr for four consecutive days (0.5, 1, 1, 1 mg, respectively). Plasma sampling (0800 and 1600 hr) and cognitive testing (1600 hr) were performed on study days 0 (baseline), 1, and 4, and 7 d posttreatment. Repeated-measures ANOVA found a significant interaction between study day and treatment condition for correct recall during a paragraph recall task [F(3,51) = 3.52, p = 0.02]. DEX (n = 10) in comparison to placebo (n = 9) treatment decreased correct paragraph recall on study day 4 [F(1,17) = 5.01, p = 0.04] and study day 11 [F(1,17) = 5.82, p = 0.03], with the lowest level of performance occurring on day 4 followed by a return toward baseline performance level by day 11. In the placebo-treated subjects, correct paragraph recall improved over the course of treatment, consistent with practice.(ABSTRACT TRUNCATED AT 250 WORDS)
Magnocellular and parvocellular contributions to the responses of neurons in macaque striate cortex
Authors: Nealey TA , Maunsell JH
Published: April 2018

Anatomical and physiological studies of the primate visual system have suggested that the signals relayed by the magnocellular and parvocellular subdivisions of the LGN remain segregated in visual cortex. It has been suggested that this segregation may account for the known differences in visual function between the parietal and temporal cortical processing streams in extrastriate visual cortex. To test directly the hypothesis that the temporal stream of processing receives predominantly parvocellular signals, we recorded visual responses from the superficial layers of V1 (striate cortex), which give rise to the temporal stream, while selectively inactivating either the magnocellular or parvocellular subdivisions of the LGN. Inactivation of the parvocellular subdivision reduced neuronal responses in the superficial layers of V1, but the effects of magnocellular blocks were generally as pronounced or slightly stronger. Individual neurons were found to receive contributions from both pathways. We furthermore found no evidence that magnocellular contributions were restricted to either the cytochrome oxidase blobs or interblobs in V1. Instead, magnocellular signals made substantial contributions to responses throughout the superficial layers. Thus, the regions within V1 that constitute the early stages of the temporal processing stream do not appear to contain isolated parvocellular signals. These results argue against a direct mapping of the subcortical magnocellular and parvocellular pathways onto the parietal and temporal streams of processing in cortex.
Dynamic behaviors of growth cones extending in the corpus callosum of living cortical brain slices observed with video microscopy
Authors: Halloran MC , Kalil K
Published: April 2018

During development, axons of the mammalian corpus callosum must navigate across the midline to establish connections with corresponding targets in the contralateral cerebral cortex. To gain insight into how growth cones of callosal axons respond to putative guidance cues along this CNS pathway, we have used time-lapse video microscopy to observe dynamic behaviors of individual callosal growth cones extending in living brain slices from neonatal hamster sensorimotor cortex. Crystals of the lipophilic dye 1,1′-dioctadecyl–3,3,3′,3′- tetramethylindocarbocyanine perchlorate (Dil) were inserted into the cortex in vivo to label small populations of callosal axons and their growth cones. Subsequently, 400 microns brain slices that included the injection site, the corpus callosum, and the target cortex were placed in culture and viewed under low-light-level conditions with a silicon- intensified target (SIT) camera. Time-lapse video observations revealed striking differences in growth cone behaviors in different regions of the callosal pathway. In the tract, which is defined as the region of the callosal pathway from the injection site to the corresponding target cortex, growth cones advanced rapidly, displaying continual lamellipodial shape changes and filopodial exploration. Forward advance was sometimes interrupted by brief pauses or retraction. Growth cones in the target cortex had almost uniform compact shapes that were consistently smaller than those in the tract. In cortex, axons adhered to straight radial trajectories and their growth cones extended at only half the speed of those in the tract. Growth cones in subtarget regions of the callosum beneath cortical targets displayed complex behaviors characterized by long pauses, extension of transitory branches, and repeated cycles of collapse, withdrawal, and resurgence. Video observations suggested that extension of axons into cortical targets could occur by interstitial branching from callosal axons rather than by turning behaviors of the primary growth cones. These results suggest the existence of guidance cues distinct for each of these callosal regions that elicit characteristic growth cone behaviors.
Neural correlates of feature selective memory and pop-out in extrastriate area V4
Authors: Motter BC
Published: April 2018

Neural activity in area V4 was examined to assess (1) whether the effects of attentive selection for stimulus features could be based on the memory of the feature, (2) whether dynamically changing the feature selection would cause activity associated with the newly selected stimuli to pop out, and (3) whether intrusion of more than one stimulus into the receptive field would disrupt the feature-selective activity. Rhesus monkeys were trained on several variations of a conditional orientation discrimination task. A differential activation of area V4 neurons was observed in the conditional discrimination task based on the presence of a match or a nonmatch between the conditional cue (a particular color or luminance) and the color or luminance of the receptive field stimulus. The differential activation was unchanged when the cue was removed and the animal had to remember its color (or luminance) to perform the task. When the cued feature was switched from one alternative to another in the middle of a trial the differential activation of neurons reversed over the course of 150–300 msec. If the stimulus in the receptive field contained the newly selected feature, V4 neurons became activated without a concomitant change in the stimulus in classical receptive field. Across the topographic map of V4 the activity associated with the newly selected stimuli popped out, whereas the activity of deselected stimuli faded to the background levels of other background objects. Evidence of a suppressive input from stimuli outside the classical receptive field was clear in only 3 of 24 neurons examined. Intrusion into the classical receptive field by a second stimulus resulted in a diminished difference between matching and nonmatching conditions. These physiological data suggest a major role for attentional control in the parallel processing of simple feature-selective differences.
The distribution of GABAergic cells, fibers, and terminals in the monkey amygdaloid complex: an immunohistochemical and in situ hybridization study
Authors: Pitkanen A , Amaral DG
Published: April 2018

The primate amygdaloid complex is a prominent structure in the medial temporal lobe that comprises a variety of cytoarchitectonically distinct nuclei and cortical regions. The present study employed immunohistochemical and in situ hybridization techniques to determine the distribution of GABAergic cells, fibers, and terminals in the macaque monkey amygdala. Using a monoclonal antibody to GABA, immunoreactive neurons were observed throughout the amygdaloid complex (constituting approximately 20% of the neurons in the lateral nucleus), with higher densities located in the intercalated nuclei, amygdalohippocampal area, and posterior cortical nucleus. The lowest densities of labeled cells were observed in the central and medial nuclei. Interestingly, despite the low number of GABA-immunoreactive neurons, the terminal density in the central nucleus was among the highest in the amygdala. The GABAergic neurons in the amygdala were heterogeneous in morphology, and three general classes of immunopositive aspiny cells were observed. Using mRNA probes for glutamic acid decarboxylase (GAD: GAD65 and GAD67), in situ hybridization demonstrated distributions of labeled cells that generally replicated the immunohistochemical findings. One exception was in the central nucleus where, in contrast to the low number of immunohistochemically labeled cells, both GAD probes labeled large numbers of neurons. These studies on the organization of GABAergic circuits in the primate amygdala are significant because alterations of function in the human amygdala have been implicated in the pathogenesis of temporal lobe epilepsy and in the etiology of affective and other behavioral disorders. The present study provides baseline information with which to evaluate the role of GABAergic systems in amygdala- mediated behaviors and the potential contribution of GABAergic dysfunction in amygdala-associated disorders.
Endogenous ganglioside GM1 modulates L-type calcium channel activity in N18 neuroblastoma cells
Authors: Carlson RO , Masco D , Brooker G , Spiegel S
Published: April 2018

Digital imaging fluorescence microscopy was used to investigate the effect of the B subunit of cholera toxin on calcium homeostasis in neuroblastoma N18 cells. The B subunit, which binds specifically to ganglioside GM1 in the outer leaflet of the cell membrane, was found to induce a sustained increase of intracellular calcium concentration ([Ca2+]i). The increase in [Ca2+]i was not observed in the absence of extracellular calcium, or in the presence of the calcium chelator EGTA, and was blocked by nickel. The B subunit was also found to induce an influx of manganese ions, as indicated by a quench of the intracellular fura-2 fluorescence. These data suggest that the B subunit induces an increase in calcium influx in N18 cells. Potassium-induced depolarization also stimulated manganese influx; however, after the onset of depolarization-induced influx, the B subunit had no further effect. This occlusion suggests involvement of voltage-dependent calcium channels. Treatment with BayK8644, a dihydropyridine agonist selective for L-type calcium channels, induced manganese influx that was not altered by the B subunit and apparently blocked the effect of the B subunit itself. Furthermore, the dihydropyridine L-type channel antagonists niguldipine or nicardipine completely inhibited B subunit- induced manganese influx. Thus, the B subunit-induced manganese influx is likely due to activation of an L-type voltage-dependent calcium channel. Spontaneous influx of manganese ions was also inhibited by nicardipine or niguldipine and by exogenous gangliosides. Ganglioside GM1 was more potent than GM3, but globoside had no significant effect. The modulation of L-type calcium channels by endogenous ganglioside GM1 has important implications for its role in neural development, differentiation, and regeneration and also for its potential function in the electrical excitability of neurons.
The developmental increase in ACh current densities on rat sympathetic neurons correlates with changes in nicotinic ACh receptor alpha-subunit gene expression and occurs independent of innervation
Authors: Mandelzys A , Pie B , Deneris ES , Cooper E
Published: April 2018

Determining factors that control the expression of neurotransmitter receptors and the mechanisms by which these factors operate is essential to understand how synapses form during development and how receptor numbers change in the adult. In this study, we have investigated one such factor, the influence of innervation, on the developmental expression of nicotinic ACh receptors (nAChRs) on neonatal rat sympathetic neurons, both in terms of ACh current densities, and in terms of mRNA levels for the transcripts that encode these receptors. To date, nine genes have been cloned that encode neuronal nAChRs subunits in mammals. We demonstrate that mRNA encoding five nAChR subunits, alpha 3, alpha 5, alpha 7, beta 2, and beta 4, are present in neonatal rat sympathetic neurons. We show that mRNA levels for alpha 3 and alpha 7 subunits increase by more than threefold over the first 2 postnatal weeks, a period when most synapses are forming on the neurons; however, we observed no significant change in mRNA levels for alpha 5, beta 2, or beta 4. Using whole-cell voltage-clamp techniques, we show that the increase in alpha-subunit mRNA correlates with increases in ACh current densities, which double over the same period. To investigate the role of innervation, we cut the preganglionic nerve at birth and measured subunit mRNA levels and ACh current densities in denervated neurons 1–2 weeks later. Our results indicate that the preganglionic nerve differentially affects the mRNA level for the five nAChR transcripts, yet it has little influence on the developmental increase in ACh current densities.
Role of dynorphin and GABA in the inhibitory regulation of NMDA-induced dopamine release in striosome- and matrix-enriched areas of the rat striatum
Authors: Krebs MO , Gauchy C , Desban M , Glowinski J , Kemel ML
Published: April 2018

Using a new superfusion procedure in vitro, we have previously reported that the NMDA-evoked release of newly synthesized 3H-dopamine (DA) was higher in matrix- than in striosome-enriched areas of the rat striatum. In addition, GABAergic medium-sized spiny neurons were shown to be indirectly involved in this regulation. Since dynorphin and GABA are colocalized in a population of medium-sized spiny neurons, the role of dynorphin-containing neurons in the NMDA-evoked release of 3H-DA has been investigated using the same superfusion procedure on rat striatal slices. (1) The NMDA (50 microM, 25 min application)-evoked release of 3H-DA was increased in the presence of naloxone (1 microM, continuously delivered) in both striatal compartments, the overall response being more elevated in the striosome-enriched area. (2) The TTX (1 microM, continuously delivered)-resistant NMDA-evoked responses were also enhanced in the presence of naloxone, but in this case, the disinhibitory effects of naloxone were similar in striosome- and matrix- enriched areas. (3) The selective kappa-agonist U-50488 (1 microM) totally reversed the naloxone-disinhibitory effect on the NMDA-evoked response in the matrix-enriched area, but only partially in the striosome-enriched area. It also completely prevented the disinhibitory effect of naloxone on the TTX-resistant NMDA-evoked release of 3H-DA in both compartments. (4) The bicuculline (5 microM)- and naloxone (1 microM)-disinhibitory effects on the NMDA-evoked release of 3H-DA were additive in the matrix- but not in the striosome-enriched areas.(ABSTRACT TRUNCATED AT 250 WORDS)
Blue cones and cone bipolar cells share transcriptional specificity as determined by expression of human blue visual pigment-derived transgenes
Authors: Chiu MI , Nathans J
Published: April 2018

Sequences 5′ of the human blue visual pigment gene have been assayed in transgenic mice for their ability to direct cell-type-specific expression of linked beta-galactosidase (lacZ) and placental alkaline phosphatase (P ALP1) reporters. Constructs containing either 5.4 kilobases (kb) or 0.47 kb of 5′ flanking DNA direct expression exclusively to the retina. Within the retina, transgene expression is confined to blue cones and cone bipolar cells, as determined, respectively, by double labeling with anti-cone pigment antibodies and by morphologic analyses. These results imply that blue cones and cone bipolar cells have partially overlapping transcriptional specificities.
VIP modulates neuronal nicotinic acetylcholine receptor function by a cyclic AMP-dependent mechanism
Authors: Gurantz D , Harootunian AT , Tsien RY , Dionne VE , Margiotta JF
Published: April 2018

Neuronal nicotinic ACh receptors (AChRs) mediate synaptic transmission throughout the nervous system, and are regulated by cellular processes and interactions that include second messenger signaling pathways. In the case of chick ciliary ganglion neurons, activation of the cAMP- dependent signaling pathway with cAMP analogs enhances ACh sensitivity in a manner consistent with an increase in the number of functional nicotinic receptors. We have now identified vasoactive intestinal peptide (VIP) as a neuromodulator or “first messenger” in the cAMP- mediated pathway that regulates neuronal AChRs. Using cAMP imaging and biochemical detection assays, we find that bath application of VIP elevates intracellular cAMP in freshly isolated ciliary ganglion neurons within minutes. The VIP treatment also enhances neuronal ACh sensitivity assessed with whole-cell recording. The enhanced ACh sensitivity produced by VIP appears with a short latency, similar to that associated with the increase in cAMP, and is not additive with the enhanced ACh sensitivity produced by bath application of a cAMP analog. In contrast, calcitonin gene-related peptide (CGRP), known to regulate muscle nicotinic AChRs via a cAMP-dependent pathway, has no detectable effect on levels of either cAMP or ACh sensitivity in the neurons. The results indicate that VIP enhances the ACh sensitivity of ciliary ganglion neurons via a cAMP-dependent signaling pathway, presumably by interaction with a specific receptor. Since VIP-like immunoreactivity is present in the presynaptic nerve terminals of avian ciliary ganglia, a VIP-like peptide could modulate AChRs in vivo.
Spinal cord neuroblasts proliferate in response to basic fibroblast growth factor
Authors: Ray J , Gage FH
Published: April 2018

Trophic factors may function as one of the epigenic signals responsible for the proliferation, growth, migration, and differentiation of neurons and glia during embryogenesis. The present study reports that basic fibroblast growth factor (bFGF) at high concentrations (10–100 ng/ml) is a mitogen for embryonic spinal cord cells that have already committed to a neuronal pathway and are expressing neuronal phenotypes (neuroblasts). Neuroblasts proliferate with a doubling time of 2.5 d. To characterize the nature of cells proliferating in response to bFGF, we have established long-term cultures of neuroblasts that can be passaged, freeze thawed, and recultured. In cultures the proportion of astrocytes remained the same, indicating limited survival and proliferation of these cells in response to bFGF. These results indicate that bFGF has mitogenic effects preferably on neuroblasts. The morphological and biochemical characterizations of the neuronal populations present in the long-term neuroblast cultures are presented here. The presence of cholinergic and GABAergic neurons in the cultures was established by immunocytochemical analysis. The cultures contain a small number of motoneurons as judged by their immunostaining with ChAT, low-affinity NGF receptor (LNGFR), and large size. Among all other growth factors tested for their mitogenic effects on embryonic spinal cord cells in culture, only epidermal growth factor (EGF) showed such effects, but to a lesser degree. The proliferative nature of neuroblasts has made it possible to transduce the Escherichia coli beta- galactosidase (LacZ) gene stably into these cells in vitro using a retroviral vector. The transfected cells expressing the foreign gene can be passaged, freeze thawed, and recultured without the loss of transgenes. The ability to transduce foreign genes stably into these cells permits implantation of these cells in the spinal cord to study cellular and biochemical behaviors and gene expression in defined neuronal populations in in vivo environments.
Neuronal expression of glypican, a cell-surface glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan, in the adult rat nervous system
Authors: Litwack ED , Stipp CS , Kumbasar A , Lander AD
Published: April 2018

Cell-surface proteoglycans have been implicated in cell responses to growth factors, extracellular matrix, and cell adhesion molecules. M12, one of the most abundant membrane-associated proteoglycans in the adult rat brain, is a approximately 65 kDa glycosylphosphatidylinositol- linked protein that bears heparan sulfate chains (Herndon and Lander, 1990). To assess its identity, M12 was purified and internal peptide sequences obtained. Comparison of the results with protein sequence predicted by a cDNA cloned from PC12 cells indicated that M12 is rat glypican, a proteoglycan first cloned from human fibroblasts. In addition, antibodies raised against a rat glypican fusion protein specifically detected the 65 kDa brain proteoglycan core protein, both by immunoprecipitation and by Western blotting. Northern blot analysis using a rat glypican probe also detected glypican message in the adult, as well as the developing rat brain. In situ hybridization with glypican RNA probes showed that glypican is expressed in a subset of structures in the adult rat nervous system. These include the hippocampus, dorsal thalamus, amygdala, cerebral cortex, piriform cortex, olfactory tubercle, several cranial nerve nuclei, the ventral horn of the spinal cord, and the dorsal root ganglia. Several other brain regions exhibited little or no hybridization over background. In most cases where glypican hybridization was observed, the signal could be localized specifically to the cell bodies of identifiable neurons, for example, spinal motoneurons, hippocampal pyramidal cells. In the cerebral cortex, glypican hybridization was found in layers 2/3, 5, and 6, but was missing from 1 and 4. The data suggest that glypican is expressed primarily by subpopulations of projection neurons in the adult rat nervous system.
Neuronal coupling in the developing mammalian retina
Authors: Penn AA , Wong RO , Shatz CJ
Published: April 2018

During the first 3 weeks of postnatal development in the ferret retina, cells in the ganglion cell layer spontaneously generate waves of electrical activity that travel across the retina in the absence of mature photoreceptors (Meister et al., 1991; Wong et al., 1993). Since few chemical synapses are present at the earliest stages when waves are present, we have explored whether gap junctions could act to correlate the activity of cells in the immature ganglion cell layer. Retinal ganglion cells in a living in vitro preparation from postnatal day 1 (P1) to P45 were intracellularly injected with the tracer Neurobiotin and the fluorescent dye Lucifer yellow, molecules that are known to pass through gap junctions. Lucifer yellow consistently filled only the injected cell, whereas Neurobiotin filled not only the injected cell but also passed to a constellation of neighboring cells. Coupling revealed by Neurobiotin is seen as early as P1, but, at this stage, it was not possible to identify the various morphological types of cells that were coupled. Thereafter, alpha ganglion cells showed homologous coupling to other alpha cells and to both conventionally placed and displaced amacrine cells. Likewise, gamma ganglion cells appeared coupled to other gamma cells and to amacrine cells. However, beta ganglion cells never showed tracer coupling in the neonatal or in adult retinas. The percentage of alpha and gamma cells that were coupled to other cells increased progressively with age. By the end of the third postnatal week, the pattern of Neurobiotin coupling in the ferret retina was adult-like, with virtually every injected alpha cell showing tracer coupling. Our observations suggest that intercellular junctions able to pass Neurobiotin are present in the inner plexiform layer during the period when the firing of retinal ganglion cells is highly correlated. Such junctions could contribute to synchronization of the activity of subsets of neighboring ganglion cells during development, but it cannot be the sole mediator of this activity because beta cells, which also participate in the correlated activity, showed no coupling at any stage. In addition, the continued presence of coupling in the adult retina implies that other changes in retinal circuitry are likely to contribute to the disappearance of the waves.
Willardiines differentiate agonist binding sites for kainate- versus AMPA-preferring glutamate receptors in DRG and hippocampal neurons
Authors: Wong LA , Mayer ML , Jane DE , Watkins JC
Published: April 2018

Concentration jump responses to 5-substituted (S)-willardiines were recorded from dorsal root ganglion (DRG) and hippocampal neurons under voltage clamp. After block of desensitization by concanavalin-A, dose- response analysis for activation of kainate-preferring receptors in DRG neurons gave the potency sequence trifluoromethyl > iodo > bromo approximately chloro > nitro approximately cyano > kainate > methyl > fluoro > (R,S)-AMPA >> willardiine; EC50 values for the most and least potent willardiine derivatives, 5-trifluoromethyl (70 nM) and 5-fluoro (69 microM), differed 1000-fold. The potency sequence for equilibrium responses at AMPA-preferring receptors in hippocampal neurons was strikingly different from that obtained in DRG neurons: fluoro > cyano approximately trifluoromethyl approximately nitro > chloro approximately bromo > (R,S)-AMPA > iodo > willardiine > kainate > methyl. In hippocampal neurons EC50 values for the most and least potent willardiine derivatives, 5-fluoro (1.5 microM) and 5-methyl (251 microM), differed only 170-fold. Consistent with equilibrium potency measurements, in DRG neurons the kinetics of deactivation for willardiines, recorded following a return to agonist-free solution, were rapid for 5-fluoro (tau off = 43 msec) but slow for 5-iodo (tau off = 4.2 sec), while the opposite sequence was observed for hippocampal neurons, slow for 5-fluoro (tau off = 2.1 sec) and rapid for 5-iodo (tau off = 188 msec). The kinetics of recovery from desensitization showed comparable agonist- and cell-dependent differences. Structure-activity analysis for agonist responses recorded from DRG and hippocampal neurons suggests that for both kainate- preferring and AMPA-preferring receptors the binding of willardiines involves interactions with polar groups such that potency is related to ionization of the uracil ring, and hence the electron-withdrawing ability of the 5-position substituent. However, kainate-preferring receptors differ from AMPA-preferring receptors in possessing a lipophilic pocket that further enhances agonist potency by hydrophobic bonding of the 5-substituent. In contrast, AMPA-preferring receptors lack such a lipophilic site, and for 5-position substituents of the same electron-withdrawing ability, potency decreases with increase in size.