bluuebunny/crossref_metadata_2025 huggingface dataset:
Relative densities of synaptic and extrasynaptic GABAA receptors on cerebellar granule cells as determined by a quantitative immunogold method
Authors: Nusser Z , Roberts JD , Baude A , Richards JG , Somogyi P
Published: April 2018

Ion channels gated by the inhibitory neurotransmitter gamma- aminobutyric acid (GABA) are thought to be located in synaptic junctions, but they have also been found throughout the somatodendritic membrane of neurons independent of synapses. To test whether synaptic junctions are enriched in GABAA receptors, and to determine the relative densities of synaptic and extrasynaptic receptors, the alpha 1 and beta 2/3 subunits of the GABAA receptor were localized on cerebellar granule cells using a postembedding immunogold method in cats. Immunoparticle density for the alpha 1 and beta 2/3 subunits was approximately 230 and 180 times more concentrated, respectively, in the synaptic junction made by GABAergic Golgi cell terminals with granule cell dendrites than on the extrasynaptic somatic membrane. Quantification of immunoreactivity revealed one synapse population for the beta 2/3, but appeared to show two populations for the alpha 1 subunit immunoreactivity. The concentration of these subunits on somatic membrane was significantly lower than on the extrasynaptic dendritic membrane. Synaptic junctions with glutamatergic mossy fiber terminals were immunonegative. The results demonstrate that granule cells receiving GABAergic synapses at a restricted location on their distal dendrites exhibit a highly compartmentalized distribution of GABAA receptor in their plasma membrane.
ATP causes release of intracellular Ca2+ via the phospholipase C beta/IP3 pathway in astrocytes from the dorsal spinal cord
Authors: Salter MW , Hicks JL
Published: April 2018

Calcium signaling within astrocytes in the CNS may play a role comparable to that of electrical signaling within neurons. ATP is a molecule known to produce Ca2+ responses in astrocytes, and has been implicated as a mediator of intercellular Ca2+ signaling in other types of nonexcitable cells. We characterized the signal transduction pathway for ATP-evoked Ca2+ responses in cultured astrocytes from the dorsal spinal cord. Nearly 100% of these astrocytes respond to extracellularly applied ATP, which causes release of Ca2+ from an intracellular pool that is sensitive to thapsigargin and insensitive to caffeine. We found that intracellular administration of IP3 also caused release of Ca2+ from a thapsigargin-sensitive intracellular pool, and that IP3 abolished the response to ATP. The ATP-evoked Ca2+ response was blocked by the IP3 receptor antagonist heparin, applied intracellularly, but not by N-desulfated heparin, which is not an antagonist at these receptors. The Ca2+ response caused by ATP was also blocked by a phospholipase C inhibitor, U-73122, but not by its inactive analog, U- 73343. Increases in [Ca2+]i were elicited by intracellular application of activators of heterotrimeric G-proteins, GTP gamma S and AIF4-. On the other hand, [Ca2+], was unaffected by a G-protein inhibitor, GDP beta S, but it did abolish the Ca2+ response to ATP. Pretreating the cultures with pertussis toxin did not affect responses to ATP. Our results indicate that in astrocytes ATP-evoked release of intracellular Ca2+ is mediated by IP3 produced as a result of activating phospholipase C coupled to ATP receptors via a G-protein that is insensitive to pertussis toxin. ATP is known to be released under physiological and pathological circumstances, and therefore signaling via the PLC-IP3 pathway in astrocytes is a potentially important mechanism by which ATP may play a role in CNS function.
Anterograde tracing of trigeminal afferent pathways from the murine tooth pulp to cortex using herpes simplex virus type 1
Authors: Barnett EM , Evans GD , Sun N , Perlman S , Cassell MD
Published: April 2018

Due to its predominantly nociceptive innervation, viral tracing from the tooth pulp provides a potential means for tracing central pain pathways. The neural pathways from the tooth pulp to cortex were determined using in situ hybridization to detect the anterograde transneuronal spread of herpes simplex virus type 1 strain H129 following inoculation into the murine mandibular incisor pulp. Virus first appeared in the brain at day 3 in the dorsomedial region of all three subnuclei of the spinal trigeminal nucleus and the principal sensory nucleus. By days 5–6 virus had spread to the contralateral medial nucleus of the medial geniculate complex, posterior thalamus, and ventroposteromedial thalamus. At days 7–8 virus was detected in laminae IV and Va of the primary somatosensory cortex and lamina IV of the secondary somatosensory cortex in regions previously shown to receive input from the lower jaw. Several mice also showed infection of laminae II/III of the ipsilateral dysgranular insular cortex, along with labeling for virus in the ipsilateral external lateral parabrachial nucleus, posterior thalamus, and posterior basolateral amygdala. Our results are highly consistent with previous tracing and electrophysiological studies utilizing the tooth pulp and with studies implicating the infected structures in nociception. Viral spread appeared to define two separate afferent systems with infection of structures which have been implicated in the sensory-discriminative aspects of pain, such as the ventroposteromedial thalamus and somatosensory cortex, as well as in the dysgranular insular cortex and related subcortical nuclei which may have a role in the affective- motivational aspects of pain.
Neuronal regeneration enhances the expression of the immunophilin FKBP- 12
Authors: Lyons WE , Steiner JP , Snyder SH , Dawson TM
Published: April 2018

Immunophilins are a group of proteins that serve as receptors for the immunosuppressant drugs cyclosporin A and FK506. The immunophilin designated FK-506 binding protein-12 (FKBP-12) is concentrated more than 10 times higher in the brain than in immune tissues. The complex of FK506 and FKBP-12 inhibits the calcium activated phosphatase, calcineurin, increasing phosphorylated levels of calcineurin substrates with growth associated protein-43 (GAP-43), being most prominent in the brain. We now demonstrate an association of FKBP-12 with neuronal regeneration and GAP-43 disposition. Facial nerve crush markedly augments expression of FKBP-12 mRNA in the facial nucleus with a time course paralleling changes in GAP-43 mRNA. Following sciatic nerve lesions, similar increases in FKBP-12 mRNA occur in lumbar motor neurons and dorsal root ganglia neuronal cells. Increased FKBP-12 expression appears linked to regeneration rather than degeneration as facial nerve lesions elicited by ricin injection, which produce neuronal death without regeneration, fail to augment FKBP-12 expression in the facial nucleus. The time course for accumulation of FKBP-12 in sciatic nerve segments following nerve crush indicates rapid axonal transport at a rate similar to GAP-43.
Pharmacological dissection of multiple types of Ca2+ channel currents in rat cerebellar granule neurons
Authors: Randall A , Tsien RW
Published: April 2018

The diversity of Ca2+ channel types in rat cerebellar granule neurons was investigated with whole-cell recordings (5 mM external Ba2+). Contributions of five different high-voltage-activated Ca2+ channel current components were distinguished pharmacologically. Nimodipine- sensitive L-type current and omega-CTx-GVIA-sensitive N-type current contributed 15 and 20% of the total current, respectively. The bulk of the remaining current (46%) was inhibited by omega-Aga-IVA. The current blocked by this toxin was further subdivided into two components, P- type and Q-type, on the basis of differences in their inactivation kinetics and sensitivity to omega-Aga-IVA. P-Type current was noninactivating during 0.1 sec depolarizations, half-blocked at about 1- 3 nM omega-Aga-IVA, and contributed approximately 11% of the total current; Q-type current was prominently inactivating, half-blocked at approximately 90 nM omega-Aga-IVA, and comprised 35% of the total current. Both P- and Q-type currents were potently inhibited by the Conus magus toxin omega-CTx-MVIIC. A current component resistant to all of the aforementioned blockers (R-type) displayed more rapid inactivation than the other components and constituted 19% of the total current. The Q-type current, the largest of the current components in the granule neurons, resembles currents that can be generated in Xenopus oocytes by expression of cloned alpha 1A subunits.
The activation of protein kinase A pathway selectively inhibits anterograde axonal transport of vesicles but not mitochondria transport or retrograde transport in vivo
Authors: Okada Y , Sato-Yoshitake R , Hirokawa N
Published: April 2018

To shed light on how axonal transport is regulated, we examined the possible roles of protein kinase A (PKA) in vivo suggested by our previous work (Sato-Yoshitake et al., 1992). Pharmacological probes or the purified catalytic subunit of PKA were applied to the permeabilized- reactivated model of crayfish walking leg giant axon, and the effect was monitored by the quantitative video-enhanced light microscopy and the quantitative electron microscopy. Dibutyryl cyclic AMP caused concentration-dependent transient reduction in the number of anterogradely transported small vesicles, while the retrogradely transported organelles and anterogradely transported mitochondria showed no decrease. This transient selective inhibition of anterograde vesicle transport was reversed by the application of a specific inhibitor of PKA (KT5720) in a concentration-dependent manner, and was reproduced by the application of the purified catalytic subunit of PKA and augmented by the application of adenosine 5′-O-(3-thiotriphosphate) (ATP gamma S). Corresponding biochemical study showed that several axoplasmic proteins including kinesin were specifically phosphorylated by the activation of the PKA pathway. These findings suggest the possible roles of PKA in the regulation of the direction of the axonal transport in vivo. The finding that only vesicle transport but not mitochondria transport was inhibited also suggests that the transport of vesicles and that of mitochondria are differently regulated and might be supported by different motors.
The transfer of rats from a familiar to a novel environment prolongs the increase of extracellular dopamine efflux induced by CCK8 in the posterior nucleus accumbens
Authors: Ladurelle N , Roques BP , Dauge V
Published: April 2018

The effects of cholecystokinin octapeptide (CCK8) on extracellular dopamine (DA) and its metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, were measured in the nucleus accumbens (N. Acc.). The experiments were carried out using in vivo microdialysis in awake rats submitted, or not, to a novel environment, the four-hole box. The exploratory behavior of the animals was studied concurrently in these boxes and in the elevated plus maze. Without CCK8 treatment, the transfer of animals from their home cages to the four-hole box induced a transient increase in DA efflux and also tended to increase its metabolites in the posterior N. Acc. In rats placed in a familiar environment, the administration of 25 pmol of CCK8 in this region immediately enhanced DA release, with levels rapidly returning to normal at the end of the perfusion. In contrast, this treatment produced a longer effect in rats transferred to the four-hole box, since the DA efflux was still increased 80 min after the removal of CCK8. The intra-accumbal administration of CCK8 induced a hypoexploration in the four-hole box. Moreover, an anxiogenic-like effect of CCK8 was found in the elevated plus maze, only in rats submitted to a novel environment (four-hole box). These data show that (1) the postero-accumbens DA neurons can be activated by environmental changes and (2) that the intensity of the CCK8 effects on extracellular DA levels and on anxiety-like responses seems to depend on the activity of these neurons previous to CCK8 treatment. The prolonged DA release induced by CCK8 in animals placed in a new situation could correspond to a biochemical anticipation preparing them to react when faced by another stimulus.
Recordings from slices indicate that octopus cells of the cochlear nucleus detect coincident firing of auditory nerve fibers with temporal precision
Authors: Golding NL , Robertson D , Oertel D
Published: April 2018

Acoustic information in auditory nerve discharges is integrated in the cochlear nuclei, and ascends through several parallel pathways to higher centers. Octopus cells of the posteroventral cochlear nucleus form a pathway known to carry information in the timing of action potentials. Octopus cells have dendrites oriented to receive converging input from many auditory nerve fibers. In all 34 intracellular recordings from anatomically identified octopus cells in slices, shocks to the auditory nerve evoked brief, consistent, graded EPSPs. EPSPs were about 1 msec in duration. At all but the lowest shock strengths, the delays between shocks and the peaks of resultant EPSPs had SDs of 0.02 msec. Polysynaptic excitation, perhaps arising from the axon collaterals of octopus cells, was observed. No detectable glycinergic or GABAergic inhibition was evoked with shocks. The input resistances were low, around 10 M omega, voltage changes were rapid, with time constants of about 1 msec, and action potentials were small. The low input resistance resulted in part from a Cs(+)-sensitive conductance. In the presence of 10 or 15 mM extracellular Cs+ the time constants increased 20-fold in the hyperpolarizing voltage range. As several subthreshold inputs were required to produce suprathreshold responses, octopus cells detect the coincident firing of auditory nerve fibers. Under physiological conditions the low input resistance and resulting short time constant limit the time over which temporal summation of excitation from auditory nerve fibers can occur and thus provide temporal precision to electrical signaling.
Ethanol inhibits kainate responses of glutamate receptors expressed in Xenopus oocytes: role of calcium and protein kinase C
Authors: Dildy-Mayfield JE , Harris RA
Published: April 2018

Recombinant alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptors expressed in oocytes are inhibited by ethanol and the sensitivity to ethanol depends on the kainate concentration and the subunit(s) expressed. For example, GluR3 kainate channels are more sensitive to inhibition by ethanol than GluR6 channels in the presence of maximally effective kainate concentrations. To determine if the ethanol inhibition was influenced by the cation permeability (Na+ vs Na+ and Ca2+) of the channels expressed, we compared ethanol inhibition of Ca(2+)-permeable glutamate receptors (GluRs) in oocytes perfused with normal- and high-Ca2+ buffers. The ethanol inhibition was much greater when Ca2+ was the only permeant cation. When Ba2+ was substituted for Ca2+, the ethanol inhibition was reduced, although it was still greater than with normal buffer. The enhanced ethanol inhibition of kainate-stimulated Ca2+ currents was reduced in oocytes injected with the Ca2+ chelator BAPTA, suggesting a role for intracellular Ca2+ in mediating enhanced ethanol sensitivity of kainate channels. The enhanced ethanol inhibition of Ca2+ currents was not due to a direct ethanol inhibition of Ca(2+)-stimulated Cl- currents in the oocyte because ethanol produced no effect on Ca(2+)-stimulated Cl- currents induced by injection of myo-inositol-1,4,5-trisphosphate. Because Ca2+ activates protein kinase C (PKC) and because we found that the PKC activator phorbol 12-myristate 13-acetate inhibits kainate responses (Dildy-Mayfield and Harris, 1994), we examined the role of PKC in mediating the enhanced ethanol inhibition of kainate responses produced by increased Ca2+. Inhibition of PKC by injection of the PKC inhibitor peptide or calphostin C prevented the enhanced ethanol inhibition of kainate-induced Ca2+ responses without altering ethanol inhibition in normal buffer. Thus, ethanol inhibition of kainate channels may involve two mechanisms, one that is independent of PKC and a second type that is due to activation of PKC under conditions of elevated Ca2+, resulting in enhanced inhibition of kainate responses.
Modulation of the glutamate-evoked release of arachidonic acid from mouse cortical neurons: involvement of a pH-sensitive membrane phospholipase A2
Authors: Stella N , Pellerin L , Magistretti PJ
Published: April 2018

Excitatory synaptic transmission is associated with changes in both extracellular and intracellular pH. Using mouse cortical neurons in primary cultures, we studied the sensitivity of glutamate-evoked release of 3H-arachidonic acid (3H-AA) to changes in extracellular pH (pHo) and related intracellular pH (pHi). As pHo was shifted from 7.2 to 7.8, the glutamate-evoked release of 3H-AA was enhanced by approximately threefold. The effect of alkaline pHo on the glutamate response was rapid, becoming significant within 2 min. 3H-AA release, evoked by both NMDA and kainate, was also enhanced by pHo alkalinization. NMDA- and kainate-induced increase in free intracellular Ca2+ was unaffected by changing pHo from 7.2 to 7.8, indicating that the receptor-induced Ca2+ influx is not responsible for the pHo sensitivity of the glutamate-evoked release of 3H-AA. Alkalinization of pHi obtained by incubating neurons in the presence of HCO3- or NH4 enhanced the glutamate-evoked release of 3H-AA, while pHi acidification obtained by blockade of Na+/H+ and Cl-/HCO3- exchangers decreased the glutamate response. Membrane-bound phospholipase A2 (mPLA2) activity was stimulated by Ca2+ in a pH-dependent manner, increasing its activity as pH was shifted from 7.2 to 7.8. This pH profile corresponds to the pH profile of the glutamate-, NMDA- and kainate-evoked release of 3H-AA. Taken together, these results indicate that the glutamate-evoked release of 3H-AA may be mediated by the pH- sensitive mPLA2. Since excitatory neurotransmission mediated by glutamate results in both pHo and pHi changes and since AA enhances glutamatergic neurotransmission at both pre- and postsynaptic levels, the data reported here reveals a possible molecular mechanism whereby glutamate can modulate its own signalling efficacy in a pH-dependent manner by regulating the release of AA.
Differential expression of protein phosphatase 1 isoforms in mammalian brain
Authors: da Cruz e Silva EF , Fox CA , Ouimet CC , Gustafson E , Watson SJ , Greengard P
Published: April 2018

Rat cDNAs encoding neuronal isoforms of protein phosphatase 1 (PP1) were isolated and their primary structures elucidated. The derived amino acid sequences allowed us to design synthetic C-terminal peptides that were used to raise antibodies. Isoform-specific anti-peptide antibodies against PP1 alpha and PP1 gamma 1 were used to investigate the tissue distribution of PP1 isoforms by immunoblotting. Both isoforms were ubiquitously expressed in mammalian tissues, with the highest levels being observed in brain. Of all neuronal tissues examined, PP1 alpha and PP1 gamma 1 were found to be most abundantly expressed in the striatum. Lesion experiments with kainic acid indicated that both the alpha and the gamma 1 isoforms of protein phosphatase 1 were relatively enriched in the medium-size spiny neurons of the striatum. “In situ” hybridization to rat brain slices using highly sensitive riboprobes also showed PP1 alpha, PP1 beta, and PP1 gamma 1 to be widely expressed in mammalian brain. However, some interesting differences were observed. For example, PP1 alpha and PP1 gamma 1 were found to be expressed in the striatum, where DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, M(r) = 32,000 Da) is also known to be highly expressed. PP1 beta appeared to be relatively less abundant in the same cells, as judged both by “in situ” hybridization and by the apparent absence of PP1 beta clones from the striatal cDNA libraries used.
Exocytosis in peptidergic nerve terminals exhibits two calcium- sensitive phases during pulsatile calcium entry
Authors: Seward EP , Chernevskaya NI , Nowycky MC
Published: April 2018

The link between electrical activity, Ca2+ entry through voltage-gated channels, and transmitter or hormone secretion is a central issue in neurobiology. In peptidergic nerve terminals of the mammalian neurohypophysis (NHP), secretion is elicited by patterned bursts of action potentials (APs). All parameters of the bursts are important to elicit efficient secretion, including AP frequency, AP broadening, burst duration, and interburst interval (Leng, 1988). We have studied Ca(2+)-secretion coupling of peptide-containing large dense-core vesicles (LDCV) in isolated rat NHP terminals. Ca2+ influx through voltage-gated Ca2+ channels was elicited and recorded by the whole-cell patch-clamp technique. Exocytosis was monitored on line with high temporal resolution by the capacitance detection technique (Neher and Marty, 1982). AP bursts were simulated by depolarizing pulse trains that mimic pulsatile submembrane Ca2+ elevations predicted for physiological stimuli. The characteristic capacitance response (delta Cm) to a train of depolarizing pulses was triphasic. It consisted of a threshold phase during which early pulses did not elicit secretion, a subsequent secretory phase during which Cm increases were coupled to depolarizing pulses, and a fatigued or inactivated state during which additional Ca2+ entry was ineffective. Both the threshold phase and secretory phase were correlated with the integrals of Ca2+ current. Ca2+ chelators affect both the threshold and secretory phase at submillimolar concentrations. Thus, a “shell” rather than “microdomain” model of Ca2+ elevation is appropriate for analyzing Ca(2+)-secretion coupling in NHP terminals (Nowycky and Pinter, 1993). We propose a two- step model, with a ca(2+)-dependent preparatory step followed by a final exocytotic step that is coupled to active Ca2+ influx. The results suggest that under physiological conditions, APs early in a burst prepare an NHP terminal for secretion, but later APs actually trigger exocytosis. Since NHP terminals do not possess a readily releasable pool of vesicles that require only a single Ca2+ step for exocytosis as seen in chromaffin cells (Neher and Zucker, 1993) and melanotrophs (Thomas et al, 1993a), Ca(2+)-secretion coupling mechanisms may be heterologous even within a single class of vesicles.
Conditional rescue of the dunce learning/memory and female fertility defects with Drosophila or rat transgenes
Authors: Dauwalder B , Davis RL
Published: April 2018

Mutations in the Drosophila dunce gene, the structural gene for a cAMP- specific phosphodiesterase (PDE), disrupt normal learning and memory and lead to female sterility. Our experiments address a long-standing question in the genetic dissection of learning and memory of whether dnc is involved in physiological processes underlying learning. Conditional expression of dunce transgenes in dnc adults shortly before training significantly improves learning over nontransgenic controls. Remarkably, behavioral rescue was also observed after induction of a transgene carrying a rat counterpart of dunce. Induction of the transgenes in adult dnc females confers partial rescue of the female sterility phenotype. These data are consistent with a major physiological requirement for the gene's activity in the learning process and show that a rat counterpart can substitute functionally for the Drosophila gene.
Time course of Fos-like immunoreactivity associated with cholinergically induced REM sleep
Authors: Shiromani PJ , Malik M , Winston S , McCarley RW
Published: April 2018

Now that the pharmacology and neuronal connectivity underlying REM sleep is beginning to be understood, it is important to begin investigations that elucidate the transcriptional response related to the REM sleep process. The present study focuses on determining the temporal development of Fos-like immunoreactivity (Fos-LI) in the dorsolateral pons following cholinergically induced, sustained rapid eye movement (REMc) sleep in cats. Microinjections (0.25 microliter) of vehicle (n = 3) or carbachol (0.2–4.0 micrograms/0.25 microliters) were made into the medial pontine reticular formation. Carbachol produced a state with all the signs of natural REM sleep, and with durations from 0 min to 120 min. Animals were killed either immediately or at various intervals after the end of REMc. Compared to vehicle- and carbachol- treated animals without REMc, the animals with REMc showed a significantly higher number of Fos-LI cells in pontine regions that have been implicated in REM sleep generation. Regions with REMc- associated Fos-LI increases included the lateral dorsal tegmental (LDT) and pedunculopontine tegmental (PPT) nuclei; the locus coeruleus; the dorsal raphe; and the medial pontine reticular formation. More Fos-LI cells were found with longer REMc bouts than with shorter-duration REMc bouts. However, with 2 hr long REMc bouts the number of Fos-LI cells returned to control levels, suggesting that the c-fos transcriptional cascade is turned off once a threshold of REMc has been reached. These findings indicate that pontine neuronal populations implicated in REM sleep express more c-fos in the course of REMc, and that the extent of expression is related to the duration of the state.
A POU homeo domain protein related to dPOU-19/pdm-1 binds to the regulatory DNA necessary for vital expression of the Drosophila choline acetyltransferase gene
Authors: Kitamoto T , Salvaterra PM
Published: April 2018

Expression of the choline acetyltransferase (ChAT) gene in Drosophila melanogaster is responsible for production of the neurotransmitter acetylcholine and is necessary for viability. In previous studies, we have shown that the regulatory region for normal ChAT expression is large and composed of multiple regulatory elements (Kitamoto et al., 1992; Kitamoto and Salvaterra, 1993). In this study, using various lengths of 5′ flanking DNA fused to wild type ChAT cDNA, we have defined a 0.3 kilobase (kb) region of the cis-regulatory DNA, which is essential for restoring viability of Cha lethal mutants. DNase I footprinting analysis of this 0.3 kb DNA revealed a protected 22 bp sequence that contains an octamer-like motif (ATTCAAAT) with one base difference from the consensus octamer motif (ATGCAAAT). Electrophoretic mobility shift assays and Southwestern blot analysis confirmed the presence of specific binding factor(s) for the 22 bp sequence in embryo nuclear extracts, and competition studies established the importance of the octamer-like motif for high-affinity binding. Using the 22-mer as a probe, we have isolated a cDNA clone encoding the Drosophila POU homeo domain protein, dPOU-19/pdm-1, whose target genes and specific binding sequences have not been identified. We propose that vital expression of the Drosophila ChAT gene is regulated by a member of the dPOU-19/pdm-1 putative transcription factor family.
Long-term structural remodeling in Aplysia sensory neurons requires de novo protein synthesis during a critical time period
Authors: O'Leary FA , Byrne JH , Cleary LJ
Published: April 2018

Long-term sensitization training induces persistent changes in both electrophysiological properties and specific structural features of sensory neurons in Aplysia californica. Previously, we found that transient elevation of intracellular cAMP could also modify these features in sensory neurons located in the pleural ganglion. In the present study we examined the role of protein synthesis in structural remodeling induced by cAMP. When applied during the intracellular injection of cAMP, anisomycin blocked increases in both the number of varicosities and the number of branch points in single sensory neurons. Exposure to anisomycin during different time periods, from as early as 12 hr prior to cAMP injection to periods as late as 15 hr after, indicated that the requirement for protein synthesis starts at the time of cAMP injection and extends for at least seven hours afterwards. Because it is metabolized rapidly, cAMP probably triggers a cascade of protein synthesis whose products continue to be synthesized for several hours after cAMP levels have returned to baseline. Thus, the present results suggest that the induction of long-term structural changes in sensory neurons has an extended but finite requirement for protein synthesis.
Activity-dependent reduction in voltage-dependent calcium current in a crayfish motoneuron
Authors: Hong SJ , Lnenicka GA
Published: April 2018

The effect of increased impulse activity upon voltage-dependent Ca2+ currents was studied in the cell body of a crayfish phasic motoneuron using two-electrode voltage-clamp technique. Increased electrical activity in this relatively inactive motoneuron produces a short-term and long-term reduction in the voltage-dependent Ca2+ current. Both forms of activity-dependent reduction in Ca2+ current are Ca2+ dependent. The short-term reduction in Ca2+ current appears to involve the Ca(2+)-dependent inactivation of Ca2+ channels, previously described in a variety of neurons. The long-term reduction in Ca2+ current is produced by prolonged Ca2+ influx and persists for days: in vivo stimulation of the phasic motor axon at 5 Hz for 1 hr results in a 30% reduction in Ca2+ current density, which persists for at least 3 d. Both the short-term and long-term reductions in Ca2+ current appear to result from changes in a single type of high-voltage-activated (HVA) Ca2+ channel. Inhibition of protein synthesis attenuates the long-term reduction in Ca2+ current and has no effect upon the short-term Ca2+ current reduction. During the long-term reduction in Ca2+ current, it appears that Ca2+ channels located distant to the site of Ca2+ influx are affected. The relationship of these results to a previously described Ca(2+)-dependent reduction in transmitter release is discussed.
Swelling-induced chloride currents in neuroblastoma cells are calcium dependent
Authors: Basavappa S , Chartouni V , Kirk K , Prpic V , Ellory JC , Mangel AW
Published: April 2018

The effects of osmotic stress on chloride (CI-) currents in the human neuroblastoma cell line CHP-100 were evaluated. Following exposure to hypoosmotic solution, an increase in whole-cell CI- current was observed. This current was blocked by the CI- channel blocker 5-nitro-2- (3-phenylpropylamino)-benzoic acid (NPPB). In cells loaded with the CI- permeability marker 125I, exposure to hypoosmotic solution increased 125I efflux by 197 +/- 14% (n = 41, p < 0.05) over controls. This increase was sensitive to NPPB. Hypoosmotic stress also increased cytosolic calcium levels (Ca2+) in fura-2-loaded cells. Pretreatment with EGTA inhibited the increase in cytosolic Ca2+, 125I efflux, and whole-cell CI- current produced by hypoosmotic solution. Antagonists of N-, L-, and T-type Ca2+ channels did not alter stimulation in 125I efflux or cytosolic Ca2+ levels during osmotic stress. However, omega- conotoxin MVIIC, a P-type Ca2+ channel blocker, inhibited hypoosmotically activated whole-cell CI- currents and increases in cytosolic Ca2+. It is concluded that a Ca(2+)-dependent change in CI- permeability is activated in CHP-100 cells in response to osmotic stress.
Retinal axon divergence in the optic chiasm: uncrossed axons diverge from crossed axons within a midline glial specialization
Authors: Marcus RC , Blazeski R , Godement P , Mason CA
Published: April 2018

A long-standing question is how fiber pathways in the mammalian CNS project to both sides of the brain. Static and real-time analyses of dye-labeled retinal axons (Godement et al., 1990, 1994) have demonstrated that at embryonic day 15–17 in the mouse, crossed and uncrossed axons from each eye diverge in a zone 100–200 microns proximal to the midline of the optic chiasm. In this study, we identify cellular specializations in this zone that might serve as cues for retinal axon divergence. Second, using growth cone morphology as an indicator of growth cone destination, we analyzed how crossed and uncrossed retinal growth cones related to these cellular components. Monoclonal antibody RC2, a marker for radial glia in embryonic mouse CNS, revealed a palisade of radial glia straddling the midline. At the midline, a thin raphe of cells that appear morphologically distinct from the radial glia express a free carbohydrate epitope, stage- specific embryonic antigen 1 (SSEA-1). Sections containing Dil-labeled axons and immunolabeled cells indicated that all axons enter the radial glial palisade. Uncrossed axons turn within the palisade, but never beyond the raphe of SSEA-1-positive cells. In addition, ultrastructural analysis indicated that all growth cones contact radial glia, with projections of the growth cone interdigitating with glial fibers. These results demonstrate that retinal axons diverge within a cellular specialization centered around the midline of the developing optic chiasm, consistent with the hypothesis that cues for divergence are located in this zone.
A glutamate-activated chloride current in cone-driven ON bipolar cells of the white perch retina
Authors: Grant GB , Dowling JE
Published: April 2018

Cone-driven ON-type bipolar cells were patch clamped in white perch retinal slices. Application of glutamate activated a current (IGlu) that was mediated by a conductance increase. The reversal potential for IGlu followed ECl closely when the intracellular chloride concentration was varied. IGlu was not blocked by 100 microM picrotoxin or 1 microM strychnine, indicating that it was not caused by inhibitory input. IGlu is not mediated by a typical ionotropic glutamate receptor since it was not activated by kainate, AMPA, or NMDA, or blocked by kynurenic acid, CNQX, DNQX, or AP-V. Further, IGlu is not mediated by a known metabotropic glutamate receptor since it was not activated by quisqualic acid, AP-4, ACPD, or ibotenate. IGlu required the presence of extracellular sodium and could be partially inhibited by the glutamate uptake inhibitors THA and tPDC. This is suggestive of sodium- dependent glutamate transport. However, when intracellular sodium was greatly increased, neither the magnitude nor reversal potential of IGlu was substantively affected. Thus, IGlu appears to involve a chloride channel activated by a glutamate receptor with transporter-like pharmacology. IGlu is localized to the dendrites of the bipolar cell, where bipolar cells receive an endogenous glutamatergic input from photoreceptors. Further, the reversal potential of the light response in these cells is the same as that of IGlu. Thus, it seems likely that IGlu is the current responsible for the cone component of the ON bipolar cell light response in the teleost retina.
Contributing factors to poor functional recovery after delayed nerve repair: prolonged denervation
Authors: Fu SY , Gordon T
Published: April 2018

The effects of prolonged denervation, independent from those of prolonged axotomy, on the recovery of muscle function were examined in a nerve cross-anastomosis paradigm. The tibialis anterior muscle was denervated for various durations by cutting the common peroneal nerve before a freshly cut tibial nerve was cross-sutured to its distal stump. Nerve regeneration and muscle reinnervation were quantified by means of electrophysiological and histochemical methods. Progressively fewer axons reinnervated the muscle with prolonged denervation; for example, beyond 6 months the mean (+/- SE) motor unit number was 15 +/- 4, which was far fewer than that after immediate nerve suture (137 +/- 21). The poor regeneration after prolonged denervation is not due to inability of the long-term denervated muscle to accept reinnervation because each regenerated axon reinnervated three- to fivefold more muscle fibers than normal. Rather, it is due to progressive deterioration of the intramuscular nerve sheaths because the effects of prolonged denervation were simulated by forcing regenerating axons to grow outside the sheaths. Fewer regenerated axons account for reinnervation of less than 50% of the muscle fibers in each muscle and contribute to the progressive decline in muscle force. Reinnervated muscle fibers failed to fully recover from denervation atrophy: muscle fiber cross-sectional area being 1171 +/- 84 microns2 as compared to 2700 +/- 47 microns2 after immediate nerve suture. Thus, the primary cause of the poor recovery after long-term denervation is a profound reduction in the number of axons that successfully regenerate through the deteriorating intramuscular nerve sheaths. Muscle force capacity is further compromised by the incomplete recovery of muscle fibers from denervation atrophy.
Electrical stimulation of the prefrontal cortex increases dopamine release in the nucleus accumbens of the rat: modulation by metabotropic glutamate receptors
Authors: Taber MT , Fibiger HC
Published: April 2018

In vivo microdialysis was used to assess the effects of electrical stimulation of the prefrontal cortex (PFC) on dopamine (DA) release in the nucleus accumbens (NAC) of awake, unrestrained rats. The PFC was stimulated bilaterally for 20 min at parameters previously shown to support intracranial self-stimulation in this structure. Stimulation at 50 microA evoked a 38% increase in DA release while 100 microA produced a 69% increase. Thus, phasic activation of the PFC increases DA release in the NAC. Additional experiments were performed to establish whether glutamate receptors in the NAC mediated these effects. The noncompetitive NMDA antagonist dizocilpine maleate (MK-801) and the broad spectrum competitive antagonist kynurenic acid were each applied locally to the NAC via reverse dialysis alone or in combination with electrical stimulation of the PFC (100 microA). Both MK-801 (10 microM) and kynurenic acid (5 mM) increased DA release when administered alone. When a “subthreshold” concentration (i.e., the highest concentration employed that did not itself increase DA release) of either compound was administered together with PFC stimulation, neither kynurenic acid (1 mM) nor MK-801 (1 microM) attenuated the effect of stimulation on DA release, thereby indicating that this effect is not mediated by ionotropic glutamate receptors located within the NAC. To examine the possible role of metabotropic glutamate receptors in regulating DA release, the metabotropic glutamate agonist trans(1S,3R)-1- aminocyclopentane-1,3-dicarboxylic acid (ACPD) was employed. When applied locally to the NAC, ACPD had a dose-dependent effect on DA release with a high concentration (1 mM) causing an increase and a lower concentration (100 microM) causing a small decrease.
Sodium current density correlates with expression of specific alternatively spliced sodium channel mRNAs in single neurons
Authors: O'Dowd DK , Gee JR , Smith MA
Published: April 2018

Elements within the first cytoplasmic loop of voltage-gated sodium channels have been implicated in regulating channel function. We have examined the role of alternative splicing within the first cytoplasmic loop of the Drosophila sodium channel gene para in regulating sodium current expression, using single-cell RT-PCR. In addition to a previously described exon (a), we identified a second exon in this region, designated exon i. Alternative splicing of exons a and i results in the expression of four para transcripts that are present individually or in combination within single neurons. Analysis of sodium current density and the pattern of para mRNA expression suggested that the presence of exon a was necessary though not sufficient for expression of sodium currents in cultured embryonic neurons. A similar pattern of alternative splicing of para mRNA was also evident in RNA isolated from whole embryos. Combined with our observation that the patterns of alternative splicing of para mRNA change during development, these findings suggest that neuronal sodium current expression in vivo, is also modulated by alternative splicing.
Individual variation in and androgen-modulation of the sodium current in electric organ
Authors: Ferrari MB , McAnelly ML , Zakon HH
Published: April 2018

Electric fish of the genus Sternopygus produce a sinusoidal electric organ discharge (EOD) of low frequencies in males, high frequencies in females, and overlapping and intermediate frequencies in juveniles. Correspondingly, the cells of the electric organ, the electrocytes, generate action potentials which are of long duration in mature males, short duration in females, and intermediate duration in immatures. The androgen dihydrotestosterone (DHT) lowers EOD frequency and increases electrocyte action potential duration. We examined the electrocytes under voltage clamp to determine whether variations in the kinetic properties of the Na+ current might underlie these phenomena. We found that the fast inactivation time constants of the peak Na+ current (0 mV) ranged from 0.5 to 4.7 msec and varied systematically with EOD frequency and action potential duration. Voltage dependence of steady- state inactivation also varied with EOD frequency with the midpoint of inactivation being more positive in fish with low EOD frequencies. There was no correlation between the voltage at which the Na+ current activates, voltage at peak current, reversal potential, rate of recovery from inactivation, or TTX sensitivity and EOD frequency. We tested whether DHT influenced Na+ current inactivation by recording from electrocytes before and after juvenile fish of both sexes were implanted with a DHT-containing or empty capsule. We found that inactivation time constants were significantly slower in DHT implanted, but not control, fish. This is the first observation of functionally relevant individual variation in the kinetics of a Na+ current and the first demonstration that the kinetics of a Na+ current may be modulated by an androgen.
Effects of excitatory amino acid antagonists on the phasic depolarizing events that occur in lumbar motoneurons during REM periods of active sleep
Authors: Soja PJ , Lopez-Rodriguez F , Morales FR , Chase MH
Published: April 2018

The membrane potential of lumbar motoneurons is dominated during the tonic periods of active sleep by glycine-mediated inhibitory postsynaptic potentials (IPSPs). During the phasic rapid eye movement (REM) periods of active sleep there are also IPSPs but, in addition, the membrane potential exhibits depolarizing shifts and action potentials that occur in conjunction with the phasic activation of the somatic musculature. The present study was designed to provide evidence that an excitatory amino acid (EAA) neurotransmitter is responsible for these patterns of motoneuronal activation. It was found that juxtacellular microiontophoretic applications of kynurenic acid a non- NMDA antagonist of EAA neurotransmission, blocked the depolarizing potentials of motoneurons that arise during the REM periods of active sleep. In contrast, the selective NMDA receptor antagonist APV did not block these depolarizations. The conclusion is drawn that the myoclonic twitches and jerks that characterize that REM periods of active sleep are dependent upon the excitation of motoneurons that occurs as a result of EAAs acting at non-NMDA receptors.
Critical role of the capsaicin-sensitive nerve fibers in the development of the causalgic symptoms produced by transecting some but not all of the nerves innervating the rat tail
Authors: Kim YI , Na HS , Han JS , Hong SK
Published: April 2018

We investigated the role of capsaicin-sensitive small diameter fibers in the development of the thermal and mechanical allodynia in a new rat model for neuropathic pain, produced by transecting some but not all of the nerves innervating the tail. Capsaicin (50 mg/kg, s.c.) injected neonatally prior to the nerve injury produced thermal hypoalgesia in the tail the degree of which was variable across individual rats, presumably as a result of variable degeneration of the small diameter fibers. When subjected to the nerve injury, the animals with moderate thermal hypoalgesia exhibited signs of pain (e.g., tail flick) to normally innocuous mechanical stimuli applied to the tail with von Frey hairs (4.9 mN or 19.6 mN bending force), but not to thermal stimuli given by immersion of the tail into cold (4 degrees C) or warm (40 degrees C) water. The animals with marked thermal hypoalgesia, on the other hand, exhibited no signs of pain either to the mechanical or to the thermal stimuli. These results suggest that the capsaicin-sensitive fibers are critical in the development of both the mechanical and thermal allodynia. It is hypothesized that the destruction of A delta- and C-nociceptive fibers by capsaicin prevented activities induced in these fibers by the nerve injury from producing a central sensitization and thus allodynia.
R-cadherin expression during nucleus formation in chicken forebrain neuromeres
Authors: Ganzler SI , Redies C
Published: April 2018

The primordial neuroepithelium of the vertebrate forebrain consists of transverse and longitudinal morphogenetic compartments (“neuromeres”). During development, neurons born in the ventricular zone of each neuromere migrate outward to the mantle zone. Here, neuroblasts gradually accumulate and aggregate either into sheets (“laminae”) or into roundish structures (“nuclei”). As brain architecture matures, sets of nuclei and laminae derived from several neuromeres become connected by fiber tracts to form functional circuits. We show by immunostaining and in situ hybridization techniques that, in the E3-E5 chicken embryo, the cell adhesion molecule R-cadherin is expressed in several stripes and patches in the forebrain neuroepithelium. This expression pattern reflects, at least in part, the neuromeric organization of the forebrain. For example, in both the ventral and dorsal thalamus, R-cadherin expression has a sharp border at the respective caudal neuromere boundary. Moreover, focusing on the mid- hypothalamic region, we demonstrate that a subset of postmitotic neuroblasts in the ventricular zone express R-cadherin during their migration to the mantle zone, where they aggregate into particular nuclei. In the mantle zone, R-cadherin-expressing neuroblasts accumulate in parallel with neuroblasts expressing another cadherin, N- cadherin. The two types of cells segregate from each other to form adjacent nuclei. Some of the R- and the N-cadherin-positive nuclei form parts of particular functional circuits in the mature brain. In conclusion, our results suggest that cadherins play a role in the formation of brain nuclei and in the developmental transformation from neuromeric to functional organization in the vertebrate forebrain.
Changes in expression and distribution of Aplysia cell adhesion molecules can influence synapse formation and elimination in vitro
Authors: Zhu H , Wu F , Schacher S
Published: April 2018

5-HT or FMRFamide evoke long-lasting changes in the efficacy of sensorimotor (SN-L7) synapses of Aplysia, structural alterations of the presynaptic sensory cell, and cell-specific downregulation in the distribution of the adhesion molecule apCAM. We examined how the cell- specific changes in apCAM might contribute to the formation of new presynaptic varicosities by 5-HT or the elimination of existing presynaptic varicosities by FMRFamide. We report that the formation of new sensory varicosities is directed by the presence of preexisting zones on the motor axon that are enriched for apCAM. Moreover, there was a further enrichment of apCAM levels at existing sensory varicosities contacting the motor axon beginning at 1 hr and lasting 24 hr after treatment with 5-HT. As was found for synapse formation during the early stages of cell-cell interaction, incubation with anti-apCAM mAb blocked the 5-HT-induced long-term changes in synaptic efficacy and the accompanying changes in sensory neuron structure. Long-term synaptic depression with FMRFamide was accompanied by an overall decline of apCAM levels. Treatment with FMRFamide evoked an even greater decline in apCAM levels at sites of sensory varicosities that preceded the structural changes and persisted especially at sites where sensory varicosities are eliminated. These results suggest that neurotransmitters evoke both cell- and site-specific changes in the levels of adhesion molecules that can influence either the formation or the elimination of presynaptic varicosities that accompany long-term heterosynaptic modulation of a behaviorally relevant synaptic connection.