bluuebunny/crossref_metadata_2025 huggingface dataset:
Glycine transporters are differentially expressed among CNS cells
Authors: Zafra F , Aragon C , Olivares L , Danbolt NC , Gimenez C , Storm-Mathisen J
Published: April 2018

Glycine is the major inhibitory neurotransmitter in the spinal cord and brainstem and is also required for the activation of NMDA receptors. The extracellular concentration of this neuroactive amino acid is regulated by at least two glycine transporters (GLYT1 and GLYT2). To study the localization and properties of these proteins, sequence- specific antibodies against the cloned glycine transporters have been raised. Immunoblots show that the 50–70 kDa band corresponding to GLYT1 is expressed at the highest concentrations in the spinal cord, brainstem, diencephalon, and retina, and, in a lesser degree, to the olfactory bulb and brain hemispheres, whereas it is not detected in peripheral tissues. Pre-embedding light and electron microscopic immunocytochemistry show that GLYT1 is expressed in glial cells around both glycinergic and nonglycinergic neurons except in the retina, where it is expressed by amacrine neurons, but not by glia. The expression of a 90–110 kDa band corresponding to GLYT2 is restricted to the spinal cord, brain-stem, and cerebellum; in addition, very low levels occur in the diencephalon. GLYT2 is found in presynaptic elements of neurons thought to be glycinergic. However, in the cerebellum, GLYT2 is expressed both in terminal boutons and in glial elements. The physiological consequences of the regional and cellular distributions of these two proteins as well as the possibility of the existence of an unidentified neuronal form of GLYT1 are discussed.
Reactive synaptogenesis and neuron densities for neuropeptide Y, somatostatin, and glutamate decarboxylase immunoreactivity in the epileptogenic human fascia dentata
Authors: Mathern GW , Babb TL , Pretorius JK , Leite JP
Published: April 2018

This study determined differences of fascia dentata (FD) peptide and inhibitory neuroanatomy between patients with epileptogenic hippocampal sclerosis (HS), those with extrahippocampal seizure pathologies, and autopsy comparisons. Surgically treated temporal lobe epilepsy patients were clinically classified into two pathogenic categories: (1) HS with focal mesial temporal neuroimaging and histories of initial precipitating injuries to the brain (n = 18) and (2) non-HS patients with extrahippocampal mass lesions or idiopathic seizures (i.e., without lesions or HS; mass lesion/idiopathic; n = 9). The hippocampal sections were studied for (1) granule cell, hilar, CA4, and CA3 neuron densities; (2) hilar densities and the percentage of neurons immunoreactive (IR) for neuropeptide Y (NPY), somatostatin (SS), and glutamate decarboxylase (GAD); (3) densities of GAD neurons in the lower granule cell and infragranular zone (basket-like cells); (4) the semiquantitative pattern of IR peptides/GAD FD molecular layer axon sprouting; (5) IR gray values (GV) of the FD molecular layers; and (6) the thickness of the supragranular molecular layer. Results showed the following. (1) Compared to autopsies, both HS and mass lesion/idiopathic patients showed less granule cell and CA3 neuron densities, but there were no statistical differences between the latter two pathogenic categories. (2) By contrast, compared to autopsies and mass lesion/idiopathic cases, HS patients showed less hilar and CA4 neuron densities, and there were no differences between autopsies and mass lesion/idiopathic. (3) Compared to autopsies, the NPY and SS hilar neuron densities in HS patients, but not mass lesion/idiopathic cases, were less. (4) Compared to autopsies, the hilar GAD neuron densities for HS and mass lesion/idiopathic patients were not less. (5) In HS patients the averaged percentages of hilar SS neurons were less than autopsies, and no other differences of IR hilar percentages were found. (6) The densities of GAD basket-like neurons and the thickness of the supragranular molecular layer were not different between any combination of pathogenic categories and autopsies. (7) By semiquantitative visual assessments, peptides/GAD axon sprouting into the FD was greater in HS compared to mass lesion/idiopathic or autopsies. (8) Compared to mass lesion/idiopathic cases, in HS NPY outer molecular layer GVs were lower, SS GVs were not different, and GAD inner molecular layer GVs were higher. (9) Analyses comparing the two pathogenic categories and neuron densities with peptides/GAD axon sprouting found six comparisons that correlated sprouting with hilar and CA4 neuron losses, and four comparisons showing greater sprouting in HS compared to mass lesion/idiopathic.
Modulation of neural network activity in vitro by cyclothiazide, a drug that blocks desensitization of AMPA receptors
Authors: Funk GD , Smith JC , Feldman JL
Published: April 2018

To determine whether AMPA receptor channel desensitization has a physiological role in shaping network activity by modulating signal transmission through excitatory circuits, we examined the effects of cyclothiazide (CYT), the most potent known blocker of AMPA receptor desensitization, on the behavior of an endogenously active neural system in vitro, the brainstem network generating rhythmic respiratory oscillations in neonatal rat medullary slices. Bath application of 100 microM CYT significantly increased the frequency of endogenously generated rhythm and increased the amplitude of the circuit output [i.e., discharge of hypoglossal (XII) respiratory motoneurons]. Local application of CYT within the XII motor nucleus produced a concentration-dependent increase (up to 35%) in amplitude of the motoneuron population discharge as well as an increase in the peak current (23%) and total charge transfer of the rhythmic inspiratory synaptic drive (33%) to individual XII motoneurons. CYT also acted postsynaptically to increase the amplitude of spontaneous EPSCs in motoneurons. In addition, CYT produced a profound, long-term augmentation of network frequency and motor output that may be secondary to block of desensitization. These results suggest that AMPA receptor desensitization has functionally significant effects on the temporal behavior and output of the rhythmic, respiratory neural network, and, by extrapolation, on other integrative actions of the mammalian CNS.
Differential expression of an inwardly rectifying chloride conductance in rat brain neurons: a potential mechanism for cell-specific modulation of postsynaptic inhibition
Authors: Smith RL , Clayton GH , Wilcox CL , Escudero KW , Staley KJ
Published: April 2018

A voltage-sensitive inwardly rectifying chloride (Cl-) conductance (GCl(V) is present in hippocampal pyramidal but not dentate gyrus neurons and has a significant role in modulation of neuronal inhibition by GABA. GCl(V) has the same activation properties as the cloned and expressed Cl- channel CIC-2. In brain, CIC-2 was detected selectively in neurons, and in hippocampus was detected in the same populations of neurons that demonstrate GCl(V). CIC-2 mRNA expression varied widely in different neuronal populations in brain but was greatest in pyramidal and other large neurons and least in interneurons. The observed differential expression of CIC-2 provides a potential molecular basis for the paradoxical excitation produced by GABAA receptor activation in selected neuronal populations.
Amphetamine redistributes dopamine from synaptic vesicles to the cytosol and promotes reverse transport
Authors: Sulzer D , Chen TK , Lau YY , Kristensen H , Rayport S , Ewing A
Published: April 2018

Whether amphetamine acts principally at the plasma membrane or at synaptic vesicles is controversial. We find that d-amphetamine injection into the Planorbis giant dopamine neuron causes robust dopamine release, demonstrating that specific amphetamine uptake is not required. Arguing for action at vesicles, whole-cell capillary electrophoresis of single Planorbis dopamine neurons shows that amphetamine reduces vesicular dopamine, while amphetamine reduces quantal dopamine release from PC12 cells by > 50% per vesicle. Intracellular injection of dopamine into the Planorbis dopamine neuron produces rapid nomifensine-sensitive release, showing that an increased substrate concentration gradient is sufficient to induce release. These experiments indicate that amphetamine acts at the vesicular level where it redistributes dopamine to the cytosol, promoting reverse transport, and dopamine release.
Monkey median nerve repaired by nerve graft or collagen nerve guide tube
Authors: Archibald SJ , Shefner J , Krarup C , Madison RD
Published: April 2018

Nerve regeneration was followed in 15 median and 1 ulnar nerve of eight Macaca fascicularis monkeys by serial electrophysiological assessments over a period of three and a half years. Nerve gaps of 5 mm at the wrist were bridged by collagen-based nerve guides, nerve autografts, or direct suture repairs. Thenar muscle reinnervation occurred between 50 and 70 d for all groups, indicating axonal elongation rates of approximately 1 mm/d. The recovery rates of the compound muscle action potential (CMAP) and the compound sensory action potential (CSAP) amplitudes were significantly slower after direct suture repair compared to the other two procedures, although the final levels of recovery were all comparable. Similar results were achieved in one median and one ulnar nerve following nerve guide repair of a 15 mm nerve gap. The functional reinnervation of Pacinian corpuscles was detected in all cases following either nerve graft or nerve guide repair, with similar amplitudes and latencies of the tactile evoked CSAP for both types of repair. Histological analysis demonstrated a significant increase in the number of myelinated axons in the median nerve distal to the nerve lesions following both nerve graft and nerve guide repairs compared to proximal and normal controls, with significant reductions of fiber diameter and corresponding increases in g-ratio. The return of a bimodal frequency distribution of myelinated axon fiber diameter was confirmed by three-dimensional surface plots which illustrate the frequency distribution of the relationship between fiber diameter and g-ratio. These combined results demonstrate that nerve regeneration after repair of a 5 mm nerve gap with a collagen nerve guide in the nonhuman primate is similar to that after graft repair, and the final level of physiological recovery for both repair procedures is comparable to direct suture repair of the median nerve.
Neurotrophins and the neuroendocrine brain: different neurotrophins sustain anatomically and functionally segregated subsets of hypothalamic dopaminergic neurons
Authors: Berg-von der Emde K , Dees WL , Hiney JK , Hill DF , Dissen GA , Costa ME , Moholt- Siebert M , Ojeda SR
Published: April 2018

Hypothalamic neurons control a variety of important hormonal and behavioral functions. Little is known, however, about the neurotrophic factors that these neurons may require for survival and/or maintenance of their differentiated functions. We conducted experiments to examine this issue, utilizing a combination of immunohistochemical, in situ hybridization and cell culture approaches. We found that the low affinity receptor for nerve growth factor (p75 NGFR) is present in small subsets of hypothalamic peptidergic neurons identified as such by their content of galanin, luteinizing hormone-releasing hormone (LHRH) and vasointestinal peptide (VIP). More prominently, however, examination of hypothalamic dopaminergic (DA) neurons for the presence of p75 NGFR-like immunoreactivity revealed that the receptor was present on tyrosine hydroxylase (TH)-positive neurons of the zona incerta and periventricular region, but not on neuroendocrine DA neurons of the tuberoinfundibular region. In situ hybridization experiments using a p75 NGFR cRNA confirmed this distribution. Regardless of the presence or absence of p75 NGFR, neither DA group expresses trkA mRNA, indicating that these two major hypothalamic subsets of DNA neurons are NGF-insensitive. A substantial fraction of TH mRNA-positive cells in the zona incerta expresses trkB mRNA, which encodes the receptor for brain derived neurotrophic factor (BDNF); in turn BDNF supports the in vitro survival of hypothalamic TH neurons bearing p75-NGFR, suggesting that BDNF is trophic for DNA neurons of the zona incerta. In contrast, tuberoinfundibular DA neurons do not express trkB mRNA, but some have trkC mRNA, which encodes the receptor for neurotrophin-3 (NT-3). The in vitro survival of TH neurons devoid of p75-NGFR is supported by NT-3, implying that NT-3 may be trophic for a subset of tuberoinfundibular DA neurons. These results suggest that, in spite of expressing an identical neurotransmitter phenotype, anatomically and functionally segregated DA neurons of the neurodendocrine brain are sustained by different neurotrophic factors.
Effects of estradiol and progesterone on voltage-gated calcium and potassium conductances in rat CA1 hippocampal neurons
Authors: Joels M , Karst H
Published: April 2018

In this study we recorded voltage gated Ca and K conductances with patch electrodes in hippocampal slices from female rats, which were ovariectomized (OVX) 1 week before the experiment. One experimental group was primed with estradiol at day 3 and 4 after OVX and received progesterone 4 hr before the start of the experiment. The second group was treated with estradiol at day 3 and 4, but received vehicle at the day of the experiment. The third group was not treated with hormones (only vehicle injections). We observed that the amplitude of both sustained and transient Ca currents were significantly enhanced in CA1 pyramidal neurons recorded in tissue from estradiol primed rats receiving progesterone when compared to the nontreated OVX controls; without estradiol priming Ca current amplitudes were small. Current amplitudes in tissue from rats treated with estradiol only, were at intermediate values. The voltage for half-maximal steady state inactivation of the transient Ca current was at a less negative value for the estradiol/progesterone treated group in comparison to the OVX controls. Input resistances of the cells, and voltage or kinetic properties of the currents as recorded under these conditions were comparable for the three groups. Characteristics of two voltage gated K conductances, that is, the transient IA and the delayed rectifier, displayed no consistent differences for the three experimental groups. We conclude that long-term modulation of estradiol and progesterone levels alters the Ca but not the K currents that were tested in CA1 pyramidal neurons. These alterations may be linked to steroid-induced morphological changes in dendrites of CA1 pyramidal cells.
mu-Opioid receptor activation reduces multiple components of high- threshold calcium current in rat sensory neurons
Authors: Rusin KI , Moises HC
Published: April 2018

Whole-cell patch-clamp recordings were used to characterize calcium channel types that are modulated by mu-opioid receptor activation in rat dorsal root ganglion (DRG) neurons. Five distinct components of high-threshold calcium current were isolated on the basis of their sensitivity to the selective channel blockers omega-conotoxin GVIA, nifedipine, omega-conotoxin MVIIC, or omega-agatoxin IVA. The mu-opioid selective agonist Tyr-Pro-NMePhe-D-Pro-NH2 (PLO17) routinely suppressed high-threshold currents and this effect was always reduced by omega- conotoxin GVIA. A fraction of PLO17-sensitive current remained after omega-conotoxin GVIA that was eliminated by application of omega- agatoxin IVA alone or in combination with omega-conotoxin MVIIC. Nifedipine had no effect on mu-opioid responses nor did PLO17 affect the slow component of tail current induced by Bay K 8644. These data suggest that mu-opioid receptors are negatively coupled to three types of calcium channels in rat DRG neurons, including an omega-conotoxin GVIA-sensitive (N-type) channel, an omega-agatoxin IVA-sensitive (P- type) channel and an omega-conotoxin MVIIC-sensitive, nifedipine/GVIA/omega-Aga IVA-resistant (presumptive Q-type) channel.
Effects of proctolin on contractions, membrane resistance, and non- voltage-dependent sarcolemmal ion channels in crustacean muscle fibers
Authors: Erxleben CF , deSantis A , Rathmayer W
Published: April 2018

The neuropeptide proctolin in nanomolar concentrations enhances the contraction of crustacean muscle fibers manyfold. The cellular mechanisms underlying this potentiation were investigated in single, isolated, fast-contracting abdominal extensor muscle fibers of a small crustacean, the marine isopod Idotea baltica. Force measurements and current-clamp experiments revealed two actions of proctolin on the muscle fibers. In half of the preparations, proctolin (10(-9)-10(-6) M) increased the fiber's input resistance by up to 25%. In about one- fourth of the preparations, proctolin induced all-or-none action potentials in response to depolarizing current pulses in muscle fibers that showed graded electric responses under control conditions. In both cases, proctolin potentiated the peak force of muscle contractions (between 1.5- and 18-fold for 5 x 10(-9) M proctolin). Proctolin affected neither the membrane resting potential nor the threshold for excitation-contraction coupling. Using cell-attached patches on the sarcolemmal membrane, we identified non-voltage-dependent ion channels which contribute to the passive membrane properties of the muscle fibers. A 53 +/- 6 pS channel had its reversal potential near rest and carried outward current at depolarized potentials with physiological saline in the recording pipette. With isotonic K+ saline in the patch pipette, the reversal potential was +85 +/- 12 mV depolarized from the resting potential and single-channel conductances ranged from 36 to 166 pS. Proctolin modulated the activity of all these putative K+ channels by reducing the number of functionally active channels. The effects of proctolin on force of contraction, input resistance, and single-channel activity were mimicked by a membrane-permeating analog of cAMP. Conversely, a monothio analog of cAMP (Rp-cAMPS), a blocker of protein kinase A activity, substantially decreased the membrane input resistance of the muscle fibers. The results suggest that activation of the cAMP signal pathway and phosphorylation of non-voltage-dependent K+ channels by protein kinase A are involved in the potentiation of contractions by proctolin in the muscle fibers of this crustacean.
Activation of a nonselective cationic conductance by metabotropic glutamatergic and muscarinic agonists in CA3 pyramidal neurons of the rat hippocampus
Authors: Guerineau NC , Bossu JL , Gahwiler BH , Gerber U
Published: April 2018

We have characterized a cationic membrane conductance activated by metabotropic glutamatergic and muscarinic cholinergic agonists in CA3 neurons in hippocampal slice cultures using the patch-clamp technique. When the potassium concentration in the superfusing fluid was raised above 5 mM, a biphasic current was observed in cells held at -60 mV in response to stimulation of postsynaptic metabotropic glutamate receptors (mGluRs) with 1S,3R-ACPD (50 microM) or muscarinic receptors with methacholine (MCh, 5 microM). The initial inward component was due to an increase in a cationic membrane conductance as determined by its reversal potential and its sensitivity to changes in extracellular K+ or Na+. The conductance underlying this current displayed no apparent voltage sensitivity over the range -120 to -50 mV. The response was reduced by extracellular application of Ba2+, Cd2+, Mg2+, or TEA, whereas extracellular Cs+ or loading cells with BAPTA or Cs+ did not affect the current. The effects of 1S,3R-ACPD were reversibly inhibited by bath-applied MCPG, an antagonist at mGluRs. Experiments with atropine and pirenzepine indicated that non-M1 muscarinic receptors mediated the MCh-induced current. A decrease in a resting leak potassium conductance (IK,leak) was responsible for the late component of the 1S,3R-ACPD- and MCh-induced response, seen as an outward current in the bathing solution with high K+ concentration. Loading cells with GDP beta S, GTP gamma S, or GTP did not alter the cationic current, while, in the same cells, the reduction in IKleak was abolished or irreversibly activated. Single-channel recordings of cationic channel activity in the cell-attached configuration provided evidence for the requirement of second messengers in coupling these receptors to the cationic channels. The data indicate that in addition to the previously described reduction of IK,leak, IM, and IAHP, both 1S,3R-ACPD and MCh activate a nonselective cationic conductance that is clearly revealed upon elevating external K+ concentration. This current is mediated by activation of metabotropic receptors, although no evidence could be obtained to show an involvement of G-proteins.
Expression of molecules associated with neuronal plasticity in the striatum after aspiration and thermocoagulatory lesions of the cerebral cortex in adult rats
Authors: Szele FG , Alexander C , Chesselet MF
Published: April 2018

Like the hippocampus, the striatum receives excitatory afferents from the cerebral cortex but, in the case of the striatum, very little is known about the molecular events associated with plasticity after lesions of this pathway. Using immunohistochemical techniques, we have examined the effects of cortical lesions induced either by aspiration of the frontoparietal cortex or by thermocoagulation of pial blood vessels on axonal and glial molecules associated with neuronal plasticity in the striatum. The growth associated protein GAP-43, a molecule present in axons and growth cones, decreased in the dorsolateral striatum after aspiration but not after thermocoagulatory lesions. In contrast, synaptophysin, a marker of synaptic vesicles, remained unchanged in the denervated striatum after both types of lesions. Immunostaining for basic fibroblast growth factor (bFGF) markedly decreased in striatal astrocytes after both lesions, despite an increased staining for glial fibrillary acidic protein (GFAP). The adhesion molecules tenascin, chondroitin sulfate proteoglycans, highly polysialylated neural cell adhesion molecule (PSA-NCAM), and laminin did not change significantly in the gray matter of the dorsolateral striatum after either type of lesion. These effects differed from those observed after partial denervation of the hippocampus and spinal cord, revealing marked regional differences in the response of axonal and glial proteins to afferent lesions. In addition, the results further indicate that cortical lesions have both similar and distinct consequences, depending on the procedure by which the lesions are induced, suggesting that cortical lesions associated with different types of pathology may differentially affect subcortical structures.
Topography of visual cortex connections with frontal eye field in macaque: convergence and segregation of processing streams
Authors: Schall JD , Morel A , King DJ , Bullier J
Published: April 2018

The primate visual system consists of at least two processing streams, one passing ventrally into temporal cortex that is responsible for object vision, and the other running dorsally into parietal cortex that is responsible for spatial vision. How information from these two streams is combined for perception and action is not understood. Visually guided eye movements require information about both feature identity and location, so we investigated the topographic organization of visual cortex connections with frontal eye field (FEF), the final stage of cortical processing for saccadic eye movements. Multiple anatomical tracers were placed either in parietal and temporal cortex or in different parts of FEF in individual macaque monkeys. Convergence from the dorsal and ventral processing streams occurred in lateral FEF but not in medial FEF. Certain extrastriate areas with retinotopic visual field organizations projected topographically onto FEF. The dorsal bank of the superior temporal sulcus projected to medial FEF; the ventral bank, to lateral FEF, and the fundus, throughout FEF. Thus, lateral FEF, which is responsible for generating short saccades, receives visual afferents from the foveal representation in retinotopically organized areas, from areas that represent central vision in inferotemporal cortex and from other areas having no retinotopic order. In contrast, medial FEF, which is responsible for generating longer saccades, is innervated by the peripheral representation of retinotopically organized areas, from areas that emphasize peripheral vision or are multimodal and from other areas that have no retinotopic order or are auditory.
AMPA receptor desensitization predicts the selective vulnerability of cerebellar Purkinje cells to excitotoxicity
Authors: Brorson JR , Manzolillo PA , Gibbons SJ , Miller RJ
Published: April 2018

Cerebellar Purkinje cells are selectively vulnerable to ischemia, although the reasons for this are unknown. In cultured embryonic rat cerebellar neurons, the steady state responses to the desensitizing agonist AMPA relative to responses to the nondesensitizing agonist kainate were greater in Purkinje cells compared to other cells, as measured by whole cell voltage clamp studies. Fluorimetric [Ca2+]i imaging experiments similarly found greater responses to AMPA relative to kainate in Purkinje cells than in other cerebellar neurons. In toxicity experiments measuring cell survival 24 hr following agonist exposure, AMPA and glutamate produced Ca(2+)-dependent toxicity which was selective for the Purkinje cell fraction of the neurons, whereas kainate produced nonselective toxicity, and NMDA selectively spared the mature Purkinje cells. Cyclothiazide, which inhibits AMPA receptor desensitization, enhanced steady state current responses to AMPA and increased the toxicity of AMPA. We conclude that the vulnerability of cerebellar neurons in culture to glutamate agonist-induced toxicity parallels the magnitude of the steady state currents produced, and that Purkinje cells may be selectively vulnerable because they express AMPA receptors which undergo less complete desensitization.
Dopamine-modulated potassium channels on rat striatal neurons: specific activation and cellular expression
Authors: Greif GJ , Lin YJ , Liu JC , Freedman JE
Published: April 2018

We have used cell-attached patch-clamp electrophysiology to characterize the activation and distribution of an 85 pS K+ channel on freshly dissociated rat striatal (caudate-putamen) neurons. In recordings from 643 cells, openings of this channel showed an absolute dependence on the presence of dopamine or the D2-like dopamine receptor agonist quinpirole in the cell-attached patch pipette, but were never seen when the D2 antagonist domperidone was applied along with quinpirole, or in the absence of drug. This channel displayed inward rectification at depolarized membrane potentials, but its activation was otherwise voltage insensitive. It was largely restricted to a subset of dissociated cells with diameters > or = 10 microns, with channel openings seen in about 25% of patches. When present, there were typically multiple channels per patch. Cells of this size were immunocytochemically stained for neuron-specific enolase but not glial fibrillary acidic protein; about 40% were also labeled for gamma-amino butyric acid (GABA) and about 60% for NADPH diaphorase, with GABAergic cells displaying a shape most similar to that of cells expressing the channel. A large number of distinct types of other channels were also present, comprising inwardly rectifying channels of 5–35 pS conductance and voltage-activated channels of 100–250 pS, but the frequencies of occurrence and fractional open times of these channels were independent of the presence or absence of dopaminergic agonists. Thus, the 85 pS K+ channel uniquely requires activation by a D2-like dopamine receptor on rat striatal neurons, and is selectively expressed by a subset of these cells, which are most likely to be GABAergic neurons.
Kinase and phosphatase activities intimately associated with a reconstituted calcium-dependent potassium channel
Authors: Reinhart PH , Levitan IB
Published: April 2018

Type-2 calcium-dependent potassium (KCa) channels from mammalian brain, reconstituted into planar phospholipid bilayers, are modulated by ATP or ATP analogs via an endogenous protein kinase activity intimately associated with the channel (Chung et al., 1991). We show here that the endogenous protein kinase activity is protein kinase C (PKC)-like because (1) modulation by ATP can be mimicked by exogenous PKC, and (2) the effects of ATP can be blocked by PKC(19–36), a specific peptide inhibitor of PKC. Furthermore, adding the PKC inhibitor peptide after the addition of ATP reverses the modulation produced by ATP, suggesting that there is a phosphoprotein phosphatase activity closely associated with type-2 KCa channels. Consistent with this idea is the finding that microcystin, a non-specific phosphatase inhibitor, enhances the modulation of KCa channel activity by ATP. Inhibitor-1, a specific protein inhibitor of phosphoprotein phosphatase-1, also enhances the effect of ATP, suggesting that the endogenous phosphatase activity is phosphatase-1-like. The results imply that type-2 KCa channels exist as part of a regulatory complex that includes a PKC-like protein kinase and a phosphatase-1-like phosphoprotein phosphatase, both of which participate in the modulation of channel function.
Differential regulation of sympathetic neuron neuropeptide Y and catecholamine content and secretion
Authors: May V , Brandenburg CA , Braas KM
Published: April 2018

Cultured principal neurons of the superior cervical ganglion (SCG), which coexpress high levels of catecholamines and neuropeptide Y (NPY), were used as a model to simultaneously examine whether sympathetic neuronal peptide and transmitter content or secretion are differentially regulated. Accumulation of NPY immunoreactivity and the dopamine metabolites DOPAC and HVA in SCG neuronal conditioned culture medium was used as an index of NPY and catecholamine secretion, respectively. Release of NPY and catecholamines was linear with time; SCG neurons exhibited a basal NPY secretory rate of approximately 0.9–3 fmol NPY immunoreactivity/10(4) cells/hr, and basal DOPAC plus HVA accumulation was about 10–20 pmol total metabolites/10(4) cells/hr. While sympathetic neuronal NPY and total catecholamine cell content increased more than 6–10-fold by 14 d of culture, secretion remained constant. Depolarization stimulated the rate of NPY secretion 18-fold, whereas medium catecholamine metabolite levels increased 3-fold. Activation of intracellular signaling pathways was shown to be an important point of regulation of sympathetic neuron peptide and transmitter content and secretion. Differential regulation of SCG neuron NPY and catecholamine expression was second messenger system specific. Activation of the protein kinase A pathway with the cAMP analog dibutyryl cAMP, or the adenylyl cyclase activator forskolin, produced a concentration-dependent, sustained stimulation of NPY secretion; maximal stimulation resulted in decreased cellular NPY content. Parallel stimulated neuronal catecholamine release was observed, but in contrast to NPY, total cellular catecholamine content was also increased. Regulation of the protein kinase C pathway with phorbol myristate acetate (PMA) stimulated SCG neuronal NPY secretion to a lesser degree than activation of protein kinase A, but did not alter cellular NPY levels. PMA minimally stimulated catecholamine release and content. NPY secretion induced by the calcium ionophore A23187 was paralleled by a concomitant decrease in cellular NPY. A23187 decreased catecholamine release, but did not change cellular total catecholamine levels. The magnitude of the secretory responses of sympathetic neurons to these regulators was far greater than changes in NPY or catecholamine content, biosynthesis or mRNA levels, suggesting that release is a primary site of regulation. The independent regulation of sympathetic neuronal NPY and catecholamine content and release is consistent with the fundamental differences in the biosynthetic pathways, vesicular compartmentalization, uptake and metabolism of neuropeptides and neurotransmitters.
The mechanism of action for the block of NMDA receptor channels by the opioid peptide dynorphin
Authors: Chen L , Gu Y , Huang LY
Published: April 2018

Dynorphin is one of the endogenous opioids that modulates the excitability of nociceptive (pain-sensing) neurons. We have shown recently that dynorphin blocks NMDA-activated currents directly without the participation of kappa-opioid receptors. In order to understand the mechanism underlying this novel action of dynorphin, we examined, in detail, the interactions between dynorphin and NMDA receptors in isolated trigeminal neurons. Dynorphin reversibly blocks NMDA-activated current (INMDA). The onset and recovery of the block were determined with concentration jump experiments. The association rate (k+) of dynorphin(1–17) is 4.9 x 10(6) sec-1 M-1 and the dissociation rate (k-) is 7.5 sec-1. The apparent dissociation constant (KD) of dynorphin, calculated from these rate constants, is 1.6 microM. Dynorphin does not change the EC50 of NMDA, nor the potentiating action of glycine. The binding site for dynorphin is distinct from that of Zn2+ or H+. Upon treatment with the disulfide reducing agent dithiothreitol (DTT), NMDA receptors become less susceptible to dynorphin block. The affinity of dynorphin for the modified NMDA receptors is reduced by 2.7-fold. In analyses of single NMDA channels in cell-free patches, we found that dynorphin shortens the mean open time, decreases the probability of opening of NMDA channels, but has no effect on the single channel conductance. These results suggest that dynorphin interacts with a site conformationally linked with the redox site(s) on the NMDA receptor, thus altering the gating properties of the channel.
Developmental changes in presynaptic calcium channels coupled to glutamate release in cultured rat hippocampal neurons
Authors: Scholz KP , Miller RJ
Published: April 2018

Excitatory synaptic transmission in the hippocampus involves the participation of at least two types of presynaptic Ca2+ channels, N- type channels sensitive to omega-conotoxin GVIA (omega-CTx GVIA) and Q- type channels sensitive to omega-agatoxin IVA (omega-Aga IVA). Hippocampal pyramidal neurons in cell culture were used to examine the participation of these two classes of channels at different stages of synapse development. Specific Ca2+ channel toxins were used to block presynaptic Ca2+ channels while whole-cell voltage-clamp recordings were used to record evoked EPSCs in postsynaptic neurons. At immature synapses (cells in culture for 10–15 d), omega-CTx GVIA (1–5 microM) blocked transmission by more than 80% while omega-Aga IVA (1 microM) was less effective. In older cultures, however, omega-Aga IVA (1 microM) was more effective than omega-CTx GVIA (1–5 microM) in blocking synaptic transmission. The pharmacological properties of the omega-Aga IVA sensitive component of synaptic transmission were examined in more detail using omega-Aga IVA and omega-conotoxin MVIIC (omega-CTx MVIIC). The properties of this component of transmitter release indicated that a Q-type Ca2+ channel was involved in presynaptic Ca2+ entry. The results suggest that different classes of presynaptic Ca2+ channels begin to participate in transmitter release at different times during synapse development and maturation.
Cell cycle progression in gentamicin-damaged avian cochleas
Authors: Bhave SA , Stone JS , Rubel EW , Coltrera MD
Published: April 2018

Hair cells, the sensory receptors of the auditory, vestibular, and lateral-line organs, may be damaged by a number of agents including aminoglycoside antibiotics and severe overstimulation. In the avian cochlea, lost hair cells can be replaced by regeneration. These new hair cells appear to be derived from a support cell precursor which is stimulated to divide by events associated with hair cell loss. Little is known about the timing and sequencing of events leading to new hair cell production. In this study cell cycle-associated events in the avian cochlea were analyzed at early and late time intervals following a single high dose of gentamicin. This single dose protocol has been shown to consistently result in extensive morphological damage and hair cell loss in the proximal region of the cochlea while sparing a morphologically undamaged distal cochlear region. This allowed for the differential analysis of the underlying support cell populations with respect to local hair cell loss. Three cell cycle associated markers were used to evaluate which cells entered and progressed through the cell cycle: statin, a G0 associated nuclear marker; proliferating cell nuclear antigen (PCNA), a G1, S and G2 associated marker; and 5- bromodeoxyuridine (BrdU), an S phase associated marker. Using these markers we found evidence for reversible changes in cell cycle status throughout the cochlea, while progression through S phase and mitosis was restricted to the region of the cochlea which sustained hair cell loss.
Inputs to combination-sensitive neurons in the medial geniculate body of the mustached bat: the missing fundamental
Authors: Wenstrup JJ , Grose CD
Published: April 2018

This study examined projections to combination-sensitive neurons in the medial geniculate body of the mustached bat. These specialized neurons respond to the combination of two temporally and spectrally distinct components of the bat's sonar pulse and echo, encoding target information. Combination-sensitive neurons respond to the bat's sonar fundamental, between 24–31 kHz, in conjunction with a higher harmonic signal. They are thought to be formed in the medial geniculate body (MGB) by convergent input from inferior colliculus representations of 24–31 kHz and higher frequencies. This study used anterograde and retrograde tract-tracing methods in conjunction with physiological recording to test this MGB convergence hypothesis. In anterograde tracing experiments, multiple deposits of two different tracers were placed in the central nucleus of the inferior colliculus (ICC), one tracer in the 24–31 kHz region and another in an ICC representation responding to a higher sonar harmonic. We found only limited overlap in the MGB labeling patterns of the two tracers, and little in many areas where combination-sensitive neurons are common. In retrograde tracing experiments, a single deposit of tracer was placed at a combination- sensitive recording site in the MGB. With the deposit mostly limited to combination-sensitive MGB areas, labeling in 24–31 kHz representations of the ICC was absent or minor. These results suggest that many combination-sensitive neurons in the MGB do not receive 24–31 kHz ICC input. The strongest inputs to combination-sensitive MGB regions originate in high-frequency representations of the ICC and combination- sensitive regions of auditory cortex. Additional projections arrive from the thalamic reticular nucleus, external nucleus of the inferior colliculus, and pericollicular tegmentum. Each projection may contribute to the 24–31 kHz sensitivity of combination-sensitive neurons in the medial geniculate body.
The Brn-3 family of POU-domain factors: primary structure, binding specificity, and expression in subsets of retinal ganglion cells and somatosensory neurons
Authors: Xiang M , Zhou L , Macke JP , Yoshioka T , Hendry SH , Eddy RL , Shows TB , Nathans J
Published: April 2018

A search for POU domain sequences expressed in the human retina has led to the identification of three closely related genes: Brn-3a, Brn-3b, and Brn-3c. The structure and expression pattern of Brn-3b was reported earlier (Xiang et al., 1993); we report here the structures and expression patterns of Brn-3a and Brn-3c. Antibodies specific for each Brn-3 protein were generated and shown to label only ganglion cells in a variety of vertebrate retinas. A complex pattern of strongly and weakly immunolabeled ganglion cells was observed in mouse, cat, and monkey retinae. In mouse and cat retinae, Brn-3a and Brn-3b proteins are found in a large fraction of ganglion cells, whereas Brn-3c is present in fewer ganglion cells. In the cat retina, anti-Brn-3a immunoreactivity was strong in the small ganglion cells (gamma cells) and weak in the remaining ganglion cells (alpha and beta cells); anti- Brn-3b immunoreactivity was present in all ganglion cells; and anti- Brn3c immunoreactivity was confined to the small ganglion cells. Immunolabeling of macaque retinae following retrograde labeling from the lateral geniculate nucleus revealed strong anti-Brn-3a immunoreactivity in a minority of retrogradely labeled P-type ganglion cells, and weak Brn-3a immunoreactivity in all of the remaining P- and M-type ganglion cells. In the same retinae, strong anti-Brn-3b immunoreactivity was seen in nearly all P-type ganglion cells and weak immunoreactivity in nearly all M-type ganglion cells. Each of the Brn-3- specific antibodies also labeled subsets of neurons in the dorsal root and trigeminal ganglia, suggesting that primary somatosensory neurons and retinal ganglion cells share genetic regulatory hierarchies. In vitro selection of an optimal DNA binding site using the Brn-3b POU domain has revealed a consensus [(A/G)CTCATTAA(T/C)] that is recognized by each of the Brn-3 POU domains and is distinct from binding sites previously described for other POU domain proteins.
Changes in c-fos mRNA expression in rat brain during odor discrimination learning: differential involvement of hippocampal subfields CA1 and CA3
Authors: Hess US , Lynch G , Gall CM
Published: April 2018

Levels of c-fos mRNA were measured with in situ hybridization to test for behaviorally dependent changes in neuronal activity in three subdivisions of hippocampus and in components of the olfactory and visual systems. In rats that performed a well-learned nose-poke response for water reward, c-fos mRNA levels were broadly increased, relative to values in home cage-control rats, in visual cortex, superior colliculus, olfactory bulb, and, to comparable levels, regions CA3 and CA1 of hippocampus; hybridization was not increased in the dentate gyrus. In rats first trained on the nose-poke behavior and then required to discriminate between two odors for water reward, the increase in c-fos mRNA was generally not as great and was more regionally differentiated. Thus, in olfactory bulb, hybridization was more greatly elevated in lateral than medial fields, thereby exhibiting regional activation corresponding to the topographic representation of the predominant odor sampled in the discrimination task. In hippocampus of odor-discrimination rats, c-fos mRNA levels were far greater in the region CA3 than region CA1, but remained at cage control values in stratum granulosum. Interestingly, c-fos mRNA levels in each hippocampal subdivision were highly correlated with levels in other regions (e.g., visual cortex) for home cage controls but not for rats in the two behavioral groups. Thus, c-fos mRNA levels in cage-control rats appeared to be regulated by some generalized factor acting throughout much of the brain (e.g., arousal), while odor-discrimination performance changed the pattern of expression within hippocampus, and allowed for a differentiated response by olfactory regions to emerge. These findings suggest that hippocampus possesses multiple modes of functioning and makes contributions to behavior that vary according to task demands.
Characterization of the human 5-HT2A receptor gene promoter
Authors: Zhu QS , Chen K , Shih JC
Published: April 2018

The regulation of 5-HT2A receptor (5-HT2AR) expression has been implicated in a variety of pathological processes and has been shown to be extremely complicated and controversial. In order to understand the mechanisms of regulation of this receptor, it is important to characterize its promoter. In this report, the 5′ end of the human 5- HT2AR gene was cloned and characterized. Anchored PCR mapped multiple transcription initiation sites at nucleotides -1157, -1137, -1127, and - 496. Transfection of chimeric growth hormone plasmids containing various DNA fragments into 5-HT2AR-positive human cell lines (SHSY-5Y, neuroblastoma; HeLa, cervix carcinoma) showed that the 0.74 kb HaeIII/PvuII fragment, which encompasses the initiation sites between - 1157 and -1127 and 5′ of the downstream initiation site (at -496), exhibited significant promoter activity. This promoter activity was not affected by the sequence upstream of the 0.74 kb fragment. The sequence downstream (the 0.45 kb PvuII/SmaI fragment) strongly repressed this promoter activity, suggesting the presence of a silencer. Sequence analysis combined with gel retardation and Dnase 1 footprinting assay identified multiple cis and trans elements for this fragment, including Sp1, PEA3, cyclic AMP response element (CRE)-like sequence, and E- boxes. Two novel transcription factors have been detected by gel retardation and DNase 1 footprinting assay; one of them may be specific for human. The transcription factors and promoter activities were low in the negative cell line NCI-H460 (human lung large cell carcinoma). Interestingly, the 0.39 kb fragment, isolated from the 3′ end of the 0.74 kb fragment, exhibited the highest promoter activity. The possibility that this 0.39 kb fragment may be an alternative promoter is discussed. These new data are essential for further study of the regulation of 5-HT2AR gene expression.
Developmental analysis of murine Promyelocyte Leukemia Zinc Finger (PLZF) gene expression: implications for the neuromeric model of the forebrain organization
Authors: Avantaggiato V , Pandolfi PP , Ruthardt M , Hawe N , Acampora D , Pelicci PG , Simeone A
Published: April 2018

Promyelocyte Leukemia Zinc Finger (PLZF) is a Kruppel-like zinc finger gene previously identified in a unique case of acute promyelocytic leukemia (APL) as the counterpart of a reciprocal chromosomal translocation involving the retinoic acid receptor alpha gene (RAR alpha). PLZF is highly conserved throughout evolution from yeast to mammals. To elucidate its role, we isolated the murine PLZF gene and studied its expression during embryogenesis. PLZF is expressed in an extremely dynamic pattern with transcripts appearing at E 7.5 in the anterior neuroepithelium and quickly spreading to the entire neuroectoderm until E 10. At E 8.5, PLZF is transcribed in most of the endoderm. During mid to late gestation PLZF is expressed in restricted domains of the developing CNS as well as in specific organs and body structures. We have focused our attention on the developing forebrain where PLZF is transcribed in a transverse, segment-like domain corresponding to the anterior pretectum, in the alarmost part of the dorsal thalamus, in the epithalamus, and in the hypothalamus along a defined longitudinal subdomain. Furthermore, PLZF is expressed in several segmentary boundaries, among them, the zona limitans intrathalamica. Combined analysis with other regionally restricted genes, such as Orthopedia and Dlx1, indicates that in the hypothalamus the PLZF domain is contained within that of Orthopedia and both are complementary to that of Dlx1. Our data suggest a role for PLZF in the establishment and maintenance of transverse identities, longitudinal subdomains, and interneuromeric boundaries, providing additional evidences in favor of the neuromeric organization of the forebrain.