bluuebunny/crossref_metadata_2025 huggingface dataset:
Voltage-Gated K+Channel β Subunits: Expression and Distribution of Kvβ1 and Kvβ2 in Adult Rat Brain
Authors: Rhodes Kenneth J. , Monaghan Michael M. , Barrezueta Nestor X. , Nawoschik Stanley , Bekele-Arcuri Zewditu , Matos Maria F. , Nakahira Kensuke , Schechter Lee E. , Trimmer James S.
Published: April 2018

Recent cloning of K+channel β subunits revealed that these cytoplasmic polypeptides can dramatically alter the kinetics of current inactivation and promote efficient glycosylation and surface expression of the channel-forming α subunits. Here, we examined the expression, distribution, and association of two of these β subunits, Kvβ1 and Kvβ2, in adult rat brain.In situhybridization using cRNA probes revealed that these β-subunit genes are heterogeneously expressed, with high densities of Kvβ1 mRNA in the striatum, CA1 subfield of the hippocampus, and cerebellar Purkinje cells, and high densities of Kvβ2 mRNA in the cerebral cortex, cerebellum, and brainstem. Immunohistochemical staining using subunit-specific monoclonal and affinity-purified polyclonal antibodies revealed that the Kvβ1 and Kvβ2 polypeptides frequently co-localize and are concentrated in neuronal perikarya, dendrites, and terminal fields, and in the juxtaparanodal region of myelinated axons. Immunoblot and reciprocal co-immunoprecipitation analyses indicated that Kvβ2 is the major β subunit present in rat brain membranes, and that most K+channel complexes containing Kvβ1 also contain Kvβ2. Taken together, these data suggest that Kvβ2 is a component of almost all K+channel complexes containing Kv1 α subunits, and that individual channels may contain two or more biochemically and functionally distinct β-subunit polypeptides.
Plasticity in GABAAReceptor Subunit mRNA Expression by Hypothalamic Magnocellular Neurons in the Adult Rat
Authors: Fénelon Valérie S. , Herbison Allan E.
Published: April 2018

The magnocellular hypothalamic neurons exhibit a substantial degree of structural and functional plasticity over the time of pregnancy, parturition, and lactation. This study has usedin situhybridization techniques to examine whether the content of α1, α2, β2, and γ2GABAAreceptor subunit mRNAs expressed by these cells fluctuates over this period. A process of regional, followed by cellular and then topographical, analyses within the supraoptic (SON) and posterior paraventricular (PVN) nuclei revealed that an increase in magnocellular α1subunit mRNA content occurred during the course of pregnancy up to day 19, after which a decline in expression was detected on the day of parturition. Significant fluctuations of this nature were observed only in the oxytocin neuron-enriched regions of the SON and PVN. The expression of α2, β2, and γ2subunit mRNAs in the SON and PVN and of all subunit mRNAs in the cingulate cortex did not change over this period. During lactation, γ2subunit mRNA content within the PVN increased significantly on day 14 of lactation as compared with day 7, and topographical analysis suggested that it involved principally magnocellular vasopressin neurons.

These results demonstrate the cell- and subunit-specific regulation of GABAAreceptor mRNA expression within the hypothalamic magnocellular system. In particular, they suggest that fluctuations in α1subunit expression may contribute to the marked variations in electrical activity exhibited by magnocellular oxytocin neurons at the time of parturition. More generally, they provide evidence in support of GABAAreceptor plasticity within a physiological context in the adult rat brain.
The Clustering of Axonal Sodium Channels during Development of the Peripheral Nervous System
Authors: Vabnick Ian , Novaković Sanja D. , Levinson S. Rock , Schachner Melitta , Shrager Peter
Published: April 2018

The distribution of Na+channels in rat peripheral nerve was measured during development by using immunofluorescence. Small segments of sciatic nerve from postnatal day 0–13 (P0–P13) pups were labeled with an antibody raised against a well conserved region of the vertebrate Na+channel. At day P0 axons contained almost no Na+channel aggregates. The number of clusters increased dramatically throughout the first week. In almost all cases Na+channels clustered in the vicinity of Schwann cell processes. At least four classes of aggregates were noted. Clusters formed singly at Schwann cell edges, in pairs or in broad regions between neighboring Schwann cells, and in more focal zones at presumptive nodes. Almost all Na+channel aggregates had reached the latter stage by the end of the first week. Histograms plotting the frequency of occurrence of each cluster type suggested a sequence of events in node formation involving the initiation of channel aggregation by Schwann cell processes. The requirement for Schwann cells during sodium channel clustering was tested by blocking proliferation of these cells with the antimitotic agent mitomycin C. Na+channel clustering was sharply reduced, whereas node formation was normal at a distal site along the same nerve. Immunocytochemical detection of myelin-associated glycoprotein (MAG) indicated that Schwann cells must begin to ensheathe axons before inducing Na+channel clustering.
Neural Influence on Protein Kinase C Isoform Expression in Skeletal Muscle
Authors: Hilgenberg Lutz , Yearwood Simone , Milstein Stuart , Miles Kathryn
Published: April 2018

Protein kinase C (PKC) is a family of enzymes involved in synapse formation and signal transduction at the neuromuscular junction. Two PKC isoforms, classical PKC α and novel PKC θ, have been shown to be enriched in skeletal muscle or localized to the endplate. We examined the role of nerve in regulating the expression of these PKC isoforms in rat skeletal muscle by denervating diaphragm muscle and measuring PKC protein expression at various postoperative times. nPKC θ protein levels decreased 65% after denervation, whereas cPKC α levels increased 80% compared with control hemidiaphragms. These results suggest that innervation regulates PKC θ and α isoform expression in skeletal muscle. To explore further how nerve regulates PKC expression, we characterized PKC isoform expression in rat myotubes deprived of neural input. Myoblast expression of nPKC θ was low, and the increase in nPKC θ expression that occurred during differentiation into myotubes resulted in levels of nPKC θ significantly below adult skeletal muscle. cPKC α expression in myoblasts increased during differentiation to levels that exceeded expression in adult skeletal muscle. Coculturing myotubes with a neuroblastoma X glioma hybrid clonal cell line (NG108-15) increased nPKC θ expression, but not cPKC α, suggesting that nPKC θ in skeletal muscle and myotubes is regulated by nerve contact or by a factor(s) provided by nerve. Treating myotubes with tetrodotoxin did not affect either basal- or NG108-15 cell-stimulated nPKC θ expression. Together these results suggest that expression of nPKC θ in skeletal muscle is regulated by a transynaptic interaction with nerve that specifically influences nPKC θ expression.
Two Alternative Promoters Direct Neuron-Specific Expression of the Rat Microtubule-Associated Protein 1B Gene
Authors: Liu Dong , Fischer Itzhak
Published: April 2018

Microtubule-associated protein 1B (MAP1B) is a major constituent of the neuronal cytoskeleton that is expressed at high levels during early brain development and plays a role in axonal growth and neuronal plasticity. Previous studies suggested that the regulation of its gene expression is primarily at the transcriptional level. Thus, the characterization of the promoter region should help to define regulatory elements that control neuron-specific and developmental expression of the MAP1B gene. We have isolated genomic clones containing up to 11 kb of the upstream region of the rat MAP1B gene, sequenced ∼1.8 kb upstream from the translation start codon, and identified several consensus sequences. These sequences include a consensus element common to several neuronal genes, a TCC repeat, a cAMP response element, and two TATA boxes that were 134 nucleotides apart from each other. S1 nuclease and RNase protection assays identified two corresponding groups of transcription initiation sites that were used selectively in distinct regions of the nervous system and during different stages of development. Transient transfection assays with neuronal and non-neuronal cell lines demonstrated that each TATA sequence and its corresponding adjacent region could independently direct neuron-specific expression of a reporter gene. Furthermore, the transcription of the reporter gene was initiated from the same sites as those of the MAP1B genein vivo. These results suggest that two alternative and overlapping promoters, one inducible and the other constitutive, regulate the temporal and tissue-specific expression of the rat MAP1B gene.
Distinct Properties of Neuronal and Astrocytic Endopeptidase 3.4.24.16: A Study on Differentiation, Subcellular Distribution, and Secretion Processes
Authors: Vincent Bruno , Beaudet Alain , Dauch Pascale , Vincent Jean-Pierre , Checler Frédéric
Published: April 2018

Endopeptidase 3.4.24.16 belongs to the zinc-containing metalloprotease family and likely participates in the physiological inactivation of neurotensin.The peptidase displays distinct features in pure primary cultured neurons and astrocytes. Neuronal maturation leads to a decrease in the proportion of endopeptidase 3.4.24.16-bearing neurons and to a concomitant increase in endopeptidase 3.4.24.16 activity and mRNA content. By contrast, there is no change with time in endopeptidase 3.4.24.16 activity or content in astrocytes. Primary cultured neurons exhibit both soluble and membrane-associated endopeptidase 3.4.24.16 activity. The latter behaves as an ectopeptidase on intact plated neurons and resists treatments with 0.2% digitonin and Na2CO3. Further evidence for an association of the enzyme with plasma membranes was provided by cryoprotection experiments and electron microscopic analysis. The membrane-associated form of endopeptidase 3.4.24.16 increased during neuronal differentiation and appears to be mainly responsible for the overall augmentation of endopeptidase 3.4.24.16 activity observed during neuronal maturation. Unlike neurons, astrocytes only contain soluble endopeptidase 3.4.24.16. Astrocytes secrete the enzyme through monensin, brefeldin A, and forskolin-independent mechanisms. This indicates that endopeptidase 3.4.24.16 is not released by classical regulated or constitutive secreting processes. However, secretion is blocked at 4°C and by 8 bromo cAMP and is enhanced at 42°C, two properties reminiscent of that of other secreted proteins lacking a classical signal peptide. By contrast, neurons appear unable to secrete endopeptidase 3.4.24.16.
Optical Recordings of the Effects of Cholinergic Ligands on Neurons in the Ganglion Cell Layer of Mammalian Retina
Authors: Baldridge William H.
Published: April 2018

Cholinergic regulation of the activity of rabbit retinal ganglion cells and displaced amacrine cells was investigated using optical recording of changes in intracellular free calcium ([Ca2+]i). Labeling of neurons in the mature retina was achieved by injecting calcium green-1 dextran (CaGD) into the isolated retina. Nicotine increased ganglion cell [Ca2+]i, affecting every loaded cell in some preparations; the pharmacology of nicotine was consistent with an action at neuronal nicotinic receptors, and specifically it was κ-(neuronal-)bungarotoxin-sensitive but α-bungarotoxin-insensitive. Muscarine also raised [Ca2+]i, but it was less potent than nicotine, affecting only a subpopulation of ganglion cells, with an M1-like muscarinic receptor pharmacology. Neither the nicotine- nor muscarine-induced increases of ganglion cell [Ca2+]iwere blocked by the glutamate receptor antagonists 6,7-dinitroquinoxaline-2,3-dione and aminophosphonopentanoic acid. Therefore, the effects of cholinergic agonists on ganglion cell [Ca2+]iwere not attributable to an indirect effect mediated by glutamatergic bipolar cells. The effects of nicotine and muscarine were abolished in calcium-free solution, indicating that the responses depend on calcium influx.

Displaced (Cb) cholinergic amacrine cells were also loaded with CaGD and were identified by selective labeling with the nuclear dye 4′,6-diamidino-2-phenyl-indole. Cbamacrine cells did not respond to either nicotine or muscarine, but responded vigorously to the glutamate receptor agonist kainic acid. There is anatomical evidence indicating that cholinergic amacrine cells make synaptic contact with each other, but the present results do not support the hypothesis that communication between these cells is cholinergic.
Role of Olf-1 and Pax-6 Transcription Factors in Neurodevelopment
Authors: Davis Janine A. , Reed Randall R.
Published: April 2018

The Olf-1 transcription factor is expressed in olfactory sensory neurons where it regulates the expression of genes that encode components of the odorant signal transduction cascade and contributes to the terminal phenotype of these sensory neurons. We examined the pattern of expression of Olf-1 protein during mouse embryogenesis and observed Olf-1 expression transiently in a subset of neural precursor cells in the CNS and peripheral nervous system. The expression of Olf-1 protein was enriched in sensory components and coincided with postmitotic cells and the initiation of overt differentiation within the nervous system. The spatial and temporal patterns of Olf-1 expression during development suggest a role in neurogenesis that is common among different neural cell types. In parallel, the expression pattern of Pax-6, a transcription factor that is widely expressed in the developing nervous system, including the visual and olfactory systems, was examined with a C-terminal antibody. In the retina, Pax-6 protein is detected in the lens, the cornea, and the neural and pigmented retinas. In the olfactory epithelium, Pax-6 protein is expressed exclusively in cells of non-neuronal lineage, including sustentacular cells, basal cells, and Bowman’s glands. The nonoverlapping, cellular localization patterns of Pax-6 and Olf-1 demarcate distinct cell lineages within the developing olfactory epithelium.
Onset of Electrical Excitability during a Period of Circus Plasma Membrane Movements in DifferentiatingXenopusNeurons
Authors: Olson Eric C. E.
Published: April 2018

Living neurons are usually first identifiable in primary cultures at the time of neurite initiation, and studies of excitability have been restricted largely to the subsequent period. A morphological early marker is described that identifies neurons for whole-cell voltage-clamp recordings before neurite initiation. Video time-lapse recordings of cultured cells dissociated from neurectoderm ofXenopusneural plate stage embryos reveal cells demonstrating circus movements, in which blebs of plasma membrane propagate around the cell circumference within a period of several minutes. All neurons demonstrate circus movements before morphological differentiation; the fraction of cells exhibiting circus movements that differentiate morphologically depends on the substrate on which they are cultured. Blockade of circus activity with cytochalasin B does not prevent neuronal differentiation. Circus movements are not neurectoderm-specific because they similarly predict differentiation of myocytes developing in mesodermal cultures.

Initially inexcitable, neurons develop voltage-dependent K+, Na+, and Ca2+currents during the period of several hours in which they exhibit circus movements. The early development of depolarization-induced elevations of [Ca2+]iseveral hours before morphological differentiation corresponds to the previously described onset of functionally significant spontaneous elevations of [Ca2+]iin these neurons and demonstrates a role for early expression of voltage-dependent ion channels.
Haltere Afferents Provide Direct, Electrotonic Input to a Steering Motor Neuron in the Blowfly,Calliphora
Authors: Fayyazuddin Amir , Dickinson Michael H.
Published: April 2018

The first basalar muscle (b1) is one of 17 small muscles in flies that control changes in wing stroke kinematics during steering maneuvers. The b1 is unique, however, in that it fires a single phase-locked spike during each wingbeat cycle. The phase-locked firing of the b1’s motor neuron (mnb1) is thought to result from wingbeat-synchronous mechanosensory input, such as that originating from the campaniform sensilla at the base of the halteres. Halteres are sophisticated equilibrium organs of flies that function to detect angular rotations of the body during flight. We have developed a new preparation to determine whether the campaniform sensilla at the base of the halteres are responsible for the phasic activity of b1. Using intracellular recording and mechanical stimulation, we have found one identified haltere campaniform field (dF2) that provides strong synaptic input to the mnb1. This haltere to mnb1 connection consists of a fast and a slow component. The fast component is monosynaptic, mediated by an electrical synapse, and thus can follow haltere stimulation at high frequencies. The slow component is possibly polysynaptic, mediated by a chemical synapse, and fatigues at high stimulus frequencies. Thus, the fast monosynaptic electrical pathway between haltere afferents and mnb1 may be responsible in part for the phase-locked firing of b1 during flight.
Three Cases of Enduring Memory Impairment after Bilateral Damage Limited to the Hippocampal Formation
Authors: Rempel-Clower Nancy L. , Zola Stuart M. , Squire Larry R. , Amaral David G.
Published: April 2018

Patient RB (Human amnesia and the medial temporal region: enduring memory impairment following a bilaterial lesion limited to field CA1 of the hippocampus, S. Zola-Morgan, L. R. Squire, and D. G. Amaral, 1986, J Neurosci 6:2950–2967) was the first reported case of human amnesia in which detailed neuropsychological analyses and detailed postmortem neuropathological analyses demonstrated that damage limited to the hippocampal formation was sufficient to produce anterograde memory impairment. Neuropsychological and postmortem neuropathological findings are described here for three additional amnesic patients with bilateral damage limited to the hippocampal formation. Findings from these patients, taken together with the findings from patient RB and other amnesic patients, make three important points about memory. (1) Bilateral damage limited primarily to the CA1 region of the hippocampal formation is sufficient to produce moderately severe anterograde memory impairment. (2) Bilateral damage beyond the CA1 region, but still limited to the hippocampal formation, can produce more severe anterograde memory impairment. (3) Extensive, temporally graded retrograde amnesia covering 15 years or more can occur after damage limited to the hippocampal formation. Findings from studies with experimental animals are consistent with the findings from amnesic patients. The present results substantiate the idea that severity of memory impairment is dependent on locus and extent of damage within the hippocampal formation and that damage to the hippocampal formation can cause temporally graded retrograde amnesia.
An Identified Interneuron Contributes to Aspects of Six Different Behaviors inAplysia
Authors: Xin Yuanpei , Weiss Klaudiusz R. , Kupfermann Irving
Published: April 2018

Previous results have indicated that the bilateral cerebral interneuron CC5 mediates the pedal artery shortening that is a component of defensive withdrawal responses involving the head. Current studies suggest that CC5 contributes to aspects of at least six different behaviors: locomotion, head turning, defensive head withdrawal, local tentacular withdrawal, rhythmic feeding, and head lifting. In addition to receiving input from mechanoreceptors in the head, CC5 receives synaptic input during fictive locomotor and feeding programs. Firing of CC5 produces widespread monosynaptic or polysynaptic actions in all ganglia in the animal. CC5 excites presumptive motor neurons for the neck, and its activity can contract neck muscles. The pedal artery shortener motor neuron (PAS), a key excitatory follower cell of CC5, fires during ipsilateral head turning, head withdrawal, tentacle withdrawal, feeding, and locomotion. For all behaviors, except locomotion and biting, responses of PAS were eliminated by cutting the ipsilateral-pleural connective, which interrupts the only direct connection of CC5 to the ipsilateral PAS. The data suggest that CC5 is a multifunctional interneuron that plays different roles during different behaviors. The neuron appears to be involved in producing coordinated movements of the head, involving both somatic and visceral muscles. For some behaviors, or for certain aspects of behaviors, CC5 appears to act as an individual command-like neuron; for other behaviors, CC5 appears to act more as an element of a distributed circuit and is neither necessary nor sufficient for any aspects of the behavior.
Shaping of IPSCs by Endogenous Calcineurin Activity
Authors: Jones Mathew V. , Westbrook Gary L.
Published: April 2018

Synaptic inhibition, mediated by GABAAreceptors, regulates neuronal firing, influences coincidence detection (König et al., 1996), and can synchronize the output of neural circuits (Cobb et al., 1995). Although GABAAreceptors can be modulated by phosphorylation, few studies have directly addressed the role of such modulation at synapses, where the nonequilibrium conditions of receptor activation are quite different from those often used to study GABAAreceptorsin vitro. Here we promoted endogenous phosphorylation by inhibiting specific phosphatases in rat hippocampal neurons and compared the effects on IPSCs with GABAAchannel responses in outside-out patches. Brief and saturating GABA pulses (5 msec; 10 mm) activated patch currents resembling the IPSC. Inhibition of calcineurin (protein phosphatase 2B), but not phosphatases 1 or 2A, produced a similar shortening of IPSC and patch responses, as did nonspecific inhibition of dephosphorylation using ATPγS or high concentrations of intracellular phosphate. Calcineurin inhibition increased the microscopic ligand unbinding rate, which was measured using the competitive antagonist 2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium bromide, suggesting that the IPSC shortening was partly caused by destabilization of the ligand binding site. Calcineurin inhibition also increased the rate and extent of macroscopic receptor desensitization. These results show that endogenous regulation by kinases and calcineurin can produce substantial changes in the IPSC duration by altering the unbinding and gating kinetics of the GABAAreceptor. Dynamic regulation of synaptic inhibition may thus allow for the tuning of circuit behavior at the level of individual inhibitory synapses.
A Role for TrkA during Maturation of Striatal and Basal Forebrain Cholinergic NeuronsIn Vivo
Authors: Fagan Anne M. , Garber Melinda , Barbacid Mariano , Silos-Santiago Inmaculada , Holtzman David M.
Published: April 2018

Nerve growth factor (NGF), acting via the TrkA receptor, has been shown to regulate the survival and maturation of specific neurons of the peripheral nervous system. Furthermore, exogenous NGF has potent actions on TrkA-expressing cholinergic neurons of the basal forebrain (BFCNs) and striatum. However, initial analysis of mice lacking NGF or TrkA revealed that forebrain cholinergic neurons were present in these animals through the fourth postnatal week. Because of the potential effects of NGF/TrkA interactions on these developing neurons, we have analyzed quantitatively the striatal and basal forebrain cholinergic neurons intrkA knock-out mice. By postnatal day (P) 7/8, forebrain cholinergic neurons are smaller intrkA (−/−) mice than those in wild-type littermate controls. However, cholinergic neuron number and fiber density in the hippocampus, a target region of BFCNs, are grossly intact. Interestingly, by P20–P25trkA knock-outs contain significantly fewer (20–36%) and smaller cholinergic neurons in both the striatum and septal regions, as compared with controls. Cholinergic fiber density within the hippocampus also is depleted in knock-outs by the end of the second postnatal week. Contrary to some predictions, despite expression of p75NTRin the absence oftrkA in BFCNs of these knock-out mice, many cells, although smaller, are still alive at P25. Our data suggest that, in the absence of NGF/TrkA signaling, striatal cholinergic neurons and BFCNs do not mature fully and that BFCNs begin to atrophy and/or die surrounding the time of target innervation.
Long-Lasting GABA-Mediated Depolarization Evoked by High-Frequency Stimulation in Pyramidal Neurons of Rat Hippocampal Slice Is Attributable to a Network-Driven, Bicarbonate-Dependent K+Transient
Authors: Kaila Kai , Lamsa Karri , Smirnov Sergei , Taira Tomi , Voipio Juha
Published: April 2018

Biphasic GABAA-mediated postsynaptic responses can be readily evoked in CA1 pyramidal neurons of rat hippocampal slices by high-frequency stimulus (HFS) trains in the presence of ionotropic glutamate receptor antagonists. In the present experiments with sharp microelectrodes, whole-cell techniques, and K+-selective microelectrodes, an HFS train (40 pulses at 100 Hz) applied in stratum radiatum close to the recording site evoked a brief hyperpolarizing IPSP (hIPSP), which turned into a prolonged (2–3 sec) depolarization (GABA-mediateddepolarizingpostsynapticpotential; GDPSP). TheI–Vrelationships of the postsynaptic currents (hIPSC and GDPSC) had distinct characteristics: the hIPSC and the early GDPSC showed outward rectification, whereas the late GDPSC was reduced with positive voltage steps to zero or beyond (inward rectification), but often no clear reversal was seen. That two distinct currents contribute to the generation of the GDPSP was also evident from the finding that a second HFS train at peak or late GDPSP induced a prompt GABAA-mediated hyperpolarization. The GDPSP/C was dependent on the availability of bicarbonate, but not on interstitial or intrapyramidal carbonic anhydrase activity.

The HFS train evoked a rapid GABAA-mediated bicarbonate-dependent increase in the extracellular K+concentration ([K+]o), and the GDPSP followed the K+transient in a sub-Nernstian manner. The spatial and pharmacological characteristics of the [K+]oshift indicated that it is generated by a local network of GABAergic interneurons. The brief ascending phase of the GDPSP is linked to a K+-dependent accumulation of intracellular Cl−. Thereafter, a nonsynaptic mechanism, a direct depolarizing effect of the [K+]oshift, is responsible for the most conspicuous characteristics of the GDPSP: its large amplitude and prolonged duration.
Removal of Spike Frequency Adaptation via Neuromodulation Intrinsic to theTritoniaEscape Swim Central Pattern Generator
Authors: Katz Paul S. , Frost William N.
Published: April 2018

For the molluscTritonia diomedeato generate its escape swim motor pattern, interneuron C2, a crucial member of the central pattern generator (CPG) for this rhythmic behavior, must fire repetitive bursts of action potentials. Yet, before swimming, repeated depolarizing current pulses injected into C2 at periods similar those in the swim motor program are incapable of mimicking the firing rate attained by C2 on each cycle of a swim motor program. This resting level of C2 inexcitability is attributable to its own inherent spike frequency adaptation (SFA). Clearly, this property must be altered for the swim behavior to occur. The pathway for initiation of the swimming behavior involves activation of the serotonergic dorsal swim interneurons (DSIs), which are also intrinsic members of the swim CPG. Physiologically appropriate DSI stimulation transiently decreases C2 SFA, allowing C2 to fire at higher rates even when repeatedly depolarized at short intervals. The increased C2 excitability caused by DSI stimulation is mimicked and occluded by serotonin application. Furthermore, the change in excitability is not caused by the depolarization associated with DSI stimulation or serotonin application but is correlated with a decrease in C2 spike afterhyperpolarization. This suggests that the DSIs use serotonin to evoke a neuromodulatory action on a conductance in C2 that regulates its firing rate. This modulatory action of one CPG neuron on another is likely to play a role in configuring the swim circuit into its rhythmic pattern-generating mode and maintaining it in that state.
Pathfinding by Identified Zebrafish Motoneurons in the Absence of Muscle Pioneers
Authors: Melançon Ellie , Liu Dennis W. C. , Westerfield Monte , Eisen Judith S.
Published: April 2018

To identify the cellular cues that guide zebrafish neuronal growth cones to their targets, we examined interactions between identified motor growth cones and identified muscle fibers and tested whether these fibers were required for growth cone navigation. Caudal primary motoneurons (CaPs) and middle primary motoneurons (MiPs) are identified motoneurons that innervate cell-specific regions of the myotome. Growth cones of both cells initially extend along a common pathway and then pause at a set of identified muscle fibers, called muscle pioneers, before diverging along cell-specific pathways. Muscle pioneers are intermediate targets of both CaP and MiP (Westerfield et al., 1986; Liu and Westerfield, 1990); both motoneurons extend their growth cones directly to the muscle pioneers on which the first functional neuromuscular contacts form, suggesting that muscle pioneers may provide guidance information to these growth cones. We tested this idea by ablating muscle pioneers and observing the resulting motor axonal trajectories. Both CaP and MiP ultimately formed normal axonal arbors after muscle pioneer ablation, showing that muscle pioneers are unnecessary for formation of correct axonal trajectories; however, although final cellular morphology was correct in the absence of muscle pioneers, MiP growth cones branched abnormally or extended ventrally beyond the common pathway. Ablation of CaP and the muscle pioneers together increased the aberrant behavior of the MiP growth cone. Our results provide evidence that an intermediate target, the muscle pioneers, affects motor axonal extension without altering target choice, suggesting that other cues also contribute to proper pathway navigation.
Expression Patterns of Developmental Control Genes in Normal andEngrailed-1Mutant Mouse Spinal Cord Reveal Early Diversity in Developing Interneurons
Authors: Matise Michael P. , Joyner Alexandra L.
Published: April 2018

The vertebrate spinal cord has long served as a useful system for studying the pattern of cell differentiation along the dorsoventral (d/v) axis. In this paper, we have defined the expression of several classes of genes expressed in restricted d/v domains in the intermediate region (IR) of the mouse spinal cord, in which most interneurons are generated. From this analysis, we have found that spinal cord interneurons and their precursors express unique combinations of transcription factors and Notch ligands at the onset of their differentiation. The domains of expression of a number of different classes of genes share similar boundaries, indicating that there could be a basic subdivision of the ventral IR into four distinct regions. This differential gene expression suggests that spinal cord interneurons acquire unique identities early in their development and that Notch signaling mechanisms may participate in the determination of cell fate along the d/v axis. Gene expression studies inEngrailed-1(En-1) mutants showed thatEn-1-expressing and other closely positioned classes of neurons do not require the homeodomain protein En-1 for their early pattern of differentiation. Rather, it is suggested thatEn-1may function to distinguish a subset of interneurons during the later maturation of the spinal cord.
D1–D2 Interaction in Feedback Control of Midbrain Dopamine Neurons
Authors: Shi Wei-Xing , Smith Paula L. , Pun Chen-Lun , Millet Barbara , Bunney Benjamin S.
Published: April 2018

Dopamine (DA) D1-like receptors are present in pathways implicated in feedback control of midbrain DA neurons. However, stimulation of these receptors either produces no effect on DA cells, or the effect is inconsistent. It is possible that the expression of a D1 feedback effect requires co-activation of D2-like receptors. To test this hypothesis, we recorded extracellularly the spontaneous activity of nigral DA cells in a low cerveau isolé rat preparation. SKF38393 and dyhydrexidine, two D1 agonists, were administered systemically to animals pretreated with different doses of the D2 agonist quinpirole. Supporting the hypothesis, the two D1 agonists consistently inhibited DA cells in animals given high doses of quinpirole (≥40 μg/kg, i.v.). However, no significant D1 effect was observed in animals pretreated with only low doses (≤20 μg/kg) of quinpirole. Because low doses of D2 agonists preferentially act on DA autoreceptors, and because the D1 inhibition persisted in animals whose DA autoreceptors were blocked by intranigral application of raclopride, our results suggest that the expression of D1 feedback inhibition requires co-activation of D2-like receptors on DA target neurons, instead of DA neurons themselves. These results, together with the finding that chloral hydrate completely blocked the D1 inhibition, may explain why previous studies have failed to show a consistent D1 effect on DA cells and suggest that drugs designed to act specifically on one subtype of DA receptor may, via feedback pathways, influence the action of endogenous DA on other DA receptor subtypes as well.
Specific C-Receptors for Itch in Human Skin
Authors: Schmelz Martin , Schmidt Roland , Bickel Andreas , Handwerker Hermann O. , Torebjörk H. Erik
Published: April 2018

In microneurography experiments 56 unmyelinated nerve fibers were studied in the cutaneous branch of the peroneal nerve of healthy volunteers. Units were identified with the “marking” technique as mechanically and heat-responsive (CMH;n= 30), heat-responsive (CH;n= 13), or unresponsive to mechanical and heat stimulation (CMiHi;n= 13). None of the units showed spontaneous activity.

These units were tested for responsiveness to iontophoresis of histamine (1 mA, 20 sec) from a small probe (diameter, 6 mm), which induced itch sensations lasting several minutes. Twenty-three units were unresponsive to histamine, and 25 units responded weakly with a few spike discharges after iontophoresis.

Eight units, however, responded with sustained discharges to histamine, and their discharge patterns were matching the time course of the itch sensations. All C-units in this group were mechanically insensitive, and five of them were heat-responsive. They had very low conduction velocities of only 0.5 m/sec, on average, which is significantly lower than conduction velocities of the “polymodal” CMH units. This slow conduction velocities attributable to small axon diameters may be one reason why these units have not been encountered in previous studies. Histamine-sensitive C-units had very large innervation territories extending up to a diameter of 85 mm on the lower leg.

We conclude that these C-fibers represent a new class of afferent nerve fibers with particularly thin axons but excessive terminal branching. This type of C-fiber probably represents the afferent units long searched for mediating itch sensations.
Subpopulations of Gastric Myenteric Neurons Are Differentially Activated via Distinct Serotonin Receptors: Projection, Neurochemical Coding, and Functional Implications
Authors: Michel Klaus , Sann Holger , Schaaf Cornelia , Schemann Michael
Published: April 2018

The enteric nervous system coordinates various gut functions. Functional studies suggested that neurotransmitters and neuromodulators, one of the most prominent among them being 5-HT, may act through a specific modulation of ascending and descending enteric pathways. However, it is still mostly unknown how particular components of enteric reflex circuits are controlled. This report describes experiments aimed at identifying a differential activation of enteric pathways by 5-HT. Electrophysiological and immunohistochemical methods were combined to investigate the projection pattern and the transmitter phenotype of 5-HT-sensitive gastric myenteric neurons. Of 294 intracellularly labeled neurons, 60.5% showed responses mediated via 5-HT3receptors, 11.3% were 5-HT1P-responsive, 3.7% exhibited both 5-HT3and 5-HT1Preceptor-mediated depolarization, and 24.5% were not responding to 5-HT. The 5-HT3-responsive cells were mainly cholinergic (79%) and had ascending projections, whereas the 5-HT1P-responsive cells had primarily descending projections and were nitrergic (67%). Substance P-positive neurons were cholinergic; most of the cells (75%) exhibited 5-HT3mediated responses and had ascending projections. Muscle strip recordings supported the functional significance of the differential location of 5-HT receptor subtypes. Thus, contractile responses of gastric circular muscle strips were dose-dependently increased by a 5-HT3and decreased by a 5-HT1Pagonist. Results indicated that excitatory ascending enteric pathways consisting of cholinergic, substance Pergic neurons were activated by 5-HT3receptors, whereas 5-HT1Preceptors were involved in activation of inhibitory descending pathways using nitrergic neurons. This suggested that different effects of 5-HT on gastric functions are related to specific activation of receptors located on different subsets of enteric neurons.
Different Mechanisms Mediate Development and Expression of Tolerance and Dependence for Peripheral μ-Opioid Antinociception in Rat
Authors: Aley Kochuvelikakam O. , Levine Jon D.
Published: April 2018

The μ-opioid [d-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO) exerts a peripheral antinociceptive effect against prostaglandin E2(PGE2)-induced mechanical hyperalgesia in the hindpaw of the rat. Tolerance and dependence develop to this effect. We have shown previously that tolerance and dependence can be dissociated and are mediated by different second messenger systems. In the present study, we evaluated whether the same or different second messenger systems mediate thedevelopmentof this peripheral opioid tolerance or dependence compared with theexpressionof the loss of antinociceptive effect or rebound opioid antagonist hyperalgesia (i.e., expression of tolerance and dependence). DAMGO-induced tolerance was prevented by pretreatment with the nitric oxide synthase inhibitorNG-methyl-l-arginine (NMLA) but not by the protein kinase C (PKC) inhibitor chelerythrine, the adenylyl cyclase inhibitor 2′,5′-dideoxyadenosine (ddA), or the calcium chelators 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester (TMB-8) and 2-[(2-bis-[carboxymethyl]amino-5-methylphenoxy)-methyl]-6-methoxy-8-bis[carboxymethyl]aminoquinoline (Quin-2). Once established, however, expression of DAMGO tolerance was acutely reversed by TMB-8 or Quin-2 but not by chelerythrine or NMLA. In contrast, naloxone-precipitated hyperalgesia in DAMGO-tolerant paws, a measure of dependence, was blocked by pretreatment with chelerythrine but not by NMLA, ddA, TMB-8, or Quin-2. Naloxone-precipitated hyperalgesia in DAMGO-tolerant paws was acutely reversed by chelerythrine, ddA, TMB-8, or Quin-2 but not by NMLA. Taken together, these results provide the first evidence that different mechanisms mediate the development and expression of both tolerance and dependence to the peripheral antinociceptive effect of DAMGO. However, although the development of tolerance and dependence are entirely separable, the expression of tolerance and dependence shares common calcium-dependent mechanisms.
Interactions of Calmodulin and α-Actinin with the NR1 Subunit Modulate Ca2+-Dependent Inactivation of NMDA Receptors
Authors: Krupp Johannes J. , Vissel Bryce , Thomas Christopher G. , Heinemann Stephen F. , Westbrook Gary L.
Published: April 2018

Glutamate receptors are associated with various regulatory and cytoskeletal proteins. However, an understanding of the functional significance of these interactions is still rudimentary. Studies in hippocampal neurons suggest that such interactions may be involved in calcium-induced reduction in the open probability of NMDA receptors (inactivation). Thus we examined the role of the intracellular domains of the NR1 subunit and two of its binding partners, calmodulin and α-actinin, on this process using NR1/NR2A heteromers expressed in human embryonic kidney (HEK) 293 cells. The presence of the first 30 residues of the intracellular C terminus of NR1 (C0 domain) was required for inactivation. Mutations in the last five residues of C0 reduced inactivation and produced parallel shifts in binding of α-actinin and Ca2+/calmodulin to the respective C0-derived peptides. Although calmodulin reduced channel activity in excised patches, calmodulin inhibitors did not block inactivation in whole-cell recording, suggesting that inactivation in the intact cell is more complex than binding of calmodulin to C0. Overexpression of putative Ca2+-insensitive, but not Ca2+-sensitive, forms of α-actinin reduced inactivation, an effect that was overcome by inclusion of calmodulin in the whole-cell pipette. The C0 domain also directly affects channel gating because NR1 subunits with truncated C0 domains that lacked calmodulin or α-actinin binding sites had a low open probability. We propose that inactivation can occur after C0 dissociates from α-actinin by two distinct but converging calcium-dependent processes: competitive displacement of α-actinin by calmodulin and reduction in the affinity of α-actinin for C0 after binding of calcium to α-actinin.
Cellular Localization of Huntingtin in Striatal and Cortical Neurons in Rats: Lack of Correlation with Neuronal Vulnerability in Huntington’s Disease
Authors: Fusco Francesca R. , Chen Quan , Lamoreaux William J. , Figueredo-Cardenas Griselle , Jiao Yun , Coffman Jonathan A. , Surmeier D. James , Honig Marcia G. , Carlock Leon R. , Reiner Anton
Published: April 2018

Immunohistochemistry and single-cell RT-PCR were used to characterize the localization of huntingtin and/or its mRNA in the major types of striatal neurons and in corticostriatal projection neurons in rats. Single-label immunohistochemical studies revealed that striatum contains scattered large neurons rich in huntingtin and more numerous medium-sized neurons moderate in huntingtin. Double-label immunohistochemical studies showed that the large huntingtin-rich striatal neurons include nearly all cholinergic interneurons and some parvalbuminergic interneurons. Somatostatinergic striatal interneurons, which are medium in size, rarely contained huntingtin. Calbindin immunolabeling showed that the vast majority of the medium-sized striatal neurons that contain huntingtin are projection neurons, but only ∼65% of calbindin-labeled projection neurons (localized to the matrix compartment of striatum) were labeled for huntingtin. Calbindin-containing projection neurons of the matrix compartment and calbindin-negative projection neurons of the striatal patch compartment contained huntingtin with comparable frequency. Single-cell RT-PCR confirmed that striatal cholinergic interneurons contain huntingtin, but only ∼65% of projection neurons contained detectable huntingtin message. The finding that huntingtin is not consistently found in striatal projection neurons [which die in Huntington’s disease (HD)] but is abundant in striatal cholinergic interneurons (which survive in Huntington’s disease) suggests that the mutation in huntingtin that causes HD may not directly kill neurons. In contrast to the heterogeneous expression of huntingtin in the different striatal neuron types, we found all corticostriatal neurons to be rich in huntingtin protein and mRNA. One possibility raised by our findings is that the HD mutation may render corticostriatal neurons destructive rather than render striatal neurons vulnerable.
Reg1ulatory Role and Molecular Interactions of a Cell-Surface Heparan Sulfate Proteoglycan (N-syndecan) in Hippocampal Long-Term Potentiation
Authors: Lauri S. E. , Kaukinen S. , Kinnunen T. , Ylinen A. , Imai S. , Kaila K. , Taira T. , Rauvala H.
Published: April 2018

The cellular mechanisms responsible for synaptic plasticity involve interactions between neurons and the extracellular matrix. Heparan sulfates (HSs) constitute a group of glycosaminoglycans that accumulate in the β-amyloid deposits in Alzheimer’s disease and influence the development of neuron–target contacts by interacting with other cell surface and matrix molecules. However, the contribution of HSs to brain function is unknown. We found that HSs play a crucial role in long-term potentiation (LTP), a finding that is consistent with the idea that converging molecular mechanisms are used in the development of neuron–target contacts and in activity-induced synaptic plasticity in adults. Enzymatic cleavage of HS by heparitinase as well as addition of soluble heparin-type carbohydrates prevented expression of LTP in response to 100 Hz/1 sec stimulation of Schaffer collaterals in rat hippocampal slices. A prominent carrier protein for the type of glycans implicated in LTP regulation in the adult hippocampus was identified asN-syndecan (syndecan-3), a transmembrane proteoglycan that was expressed at the processes of the CA1 pyramidal neurons in an activity-dependent manner. Addition of solubleN-syndecan into the CA1 dendritic area prevented tetanus-induced LTP. A major substrate of src-type kinases, cortactin (p80/85), and the tyrosine kinase fyn copurified withN-syndecan from hippocampus. Moreover, association of both cortactin and fyn toN-syndecan was rapidly increased after induction of LTP.N-syndecan may thus act as an important regulator in the activity-dependent modulation of neuronal connectivity by transmitting signals between extracellular heparin-binding factors and the fyn signaling pathway.
Hippocampal Long-Term Potentiation Preserves the Fidelity of Postsynaptic Responses to Presynaptic Bursts
Authors: Selig David K. , Nicoll Roger A. , Malenka Robert C.
Published: April 2018

Hippocampal cells often fire prolonged bursts of action potentials, resulting in dynamic modulation of postsynaptic responses; yet long-term potentiation (LTP) has routinely been studied using only single presynaptic stimuli given at low frequency. Recent work on neocortical synapses has suggested that LTP may cause a “redistribution of synaptic strength” in which synaptic responses to the first stimulus of a presynaptic burst of action potentials are potentiated with later responses depressed. We have examined whether this redistribution occurs at hippocampal synapses during LTP. Using prolonged bursts that result in maximal short-term depression of later responses within the burst, we found that LTP resulted in a uniform potentiation of individual responses throughout the burst rather than a redistribution of synaptic strength. This occurred both at Schaffer collateral–CA1 synapses and at CA3–CA3 synapses, the latter being activated and monitored using paired recordings. Thus in the hippocampus, LTP preserves the fidelity of postsynaptic responses to presynaptic bursts by a uniform increase rather than a redistribution of synaptic strength, a finding that suggests there are important differences between neocortex and hippocampus in how long-term changes in synaptic strength are used to encode new information.