bluuebunny/crossref_metadata_2025 huggingface dataset:
FMRFamide peptides in Helisoma: identification and physiological actions at a peripheral synapse
Authors: Bulloch AG , Price DA , Murphy AD , Lee TD , Bowes HN
Published: March 2018

Previous reports have demonstrated powerful neuromodulatory actions of the molluscan tetrapeptide FMRFamide in both the central and peripheral nervous systems of the freshwater snail Helisoma. The present study was designed to examine both the nature of the FMRFamide-like peptides in Helisoma and to define their physiological actions at a peripheral synapse. We report that, as determined by HPLC/RIA and mass spectrometry, Helisoma contains both FMRFamide and 2 of its analogs, FLRFamide and GDPFLRFamide. Whereas whole animals contain about 100 pmol/gm of these peptides, they were enriched in the nervous system (3000 pmol/gm) and in a peripheral target organ, the salivary glands (500 pmol/gm). For histochemical and physiological studies we examined the salivary glands, which are known to be innervated by neuron 4 of the buccal ganglion. We confirmed the presence of FMRFamide-like fibers on the salivary gland by immunohistochemistry using a polyclonal antiserum. These fibers appear to be largely derived from somata located in the central ring ganglia. For physiological tests we examined the neuron 4-gland synapse, at which presynaptic action potentials normally evoke a suprathreshold EPSP in gland cells. Bath application of FMRFamide, FLRFamide, or GDPFLRFamide at micromolar concentration to a buccal ganglia/salivary gland preparation completely suppressed spontaneous rhythmic activity. The sites of action of these peptides were examined by iontophoretic application of FMRFamide to neuron 4 or the salivary gland. Application of the peptide to the soma of neuron 4 caused a hyperpolarization that suppressed spontaneously generated action potentials. When applied to the salivary gland, FMRFamide caused a hyperpolarization that reduced the EPSPs evoked by neuron 4 to below spike threshold. The latter observation implies a postsynaptic locus of action for FMRFamide, and this possibility was tested by direct depolarization of the gland with iontophoresis of ACh (the putative transmitter of neuron 4). Such depolarizations were also reduced by FMRFamide. We conclude that Helisoma contains FMRFamide and 2 of its analogs, these peptides being enriched in the nervous system and salivary glands. Furthermore, these peptides can suppress activation of the salivary glands by actions both directly on gland cells and on the effector neuron.
Axonal regulation of myelin protein mRNA levels in actively myelinating Schwann cells
Authors: Trapp BD , Hauer P , Lemke G
Published: March 2018

Upon transection of a peripheral nerve, axons distal to the transection degenerate. As a consequence of this axonal degeneration, myelin- forming Schwann cells cease biosynthesis of new myelin membrane, contribute to phagocytosis of previously formed myelin, and markedly down-regulate expression of myelin-specific markers. Among the most prominent of these down-regulated markers are the major structural proteins of peripheral myelin, Po and myelin basic protein (MBP). We have used slot blot and in situ hybridization techniques to demonstrate that for actively myelinating Schwann cells, down-regulation of the Po and MBP genes occurs primarily at the level of mRNA expression. Together with other recent data, these findings strongly argue for axonal modulation of Po and MBP gene transcription during active myelination.
Topographic distribution of modality-specific amygdalar neurons in alert monkey
Authors: Nishijo H , Ono T , Nishino H
Published: March 2018

Neuronal activity in the amygdala (AM) was recorded from alert monkeys during performance of tasks that led to presentation of rewarding or aversive stimuli. The tasks had 3 phases: (1) discrimination (visual, auditory), (2) operant response (bar pressing), and (3) ingestion (reward) or avoidance (aversion). Neuronal activity was analyzed and compared during each of these phases. Of 585 AM neurons tested, 312 (53.3%) responded to at least one stimulus in one or more of 5 major groups: vision related, audition related, ingestion related, multimodal, and selective. Forty neurons (6.8%) in the anterior dorsolateral capsule of the basolateral nuclei responded exclusively to visual stimuli (vision related). Twenty-six neurons (4.4%) further posterior in the basolateral group responded only to auditory stimuli (audition related). During ingestion an additional 41 neurons (7.0%) increased their activity (ingestion related). These were in the corticomedial group and at the boundaries between the nuclei of the basolateral group. Of these, 27 responded only in the ingestion phase, 11 during ingestion and at the sight of food, and 3 during ingestion and to certain sounds. Throughout the AM other neurons (n = 117, 20.0%) responded to visual, auditory, and somesthetic stimuli and, when tested, to involuntary ingestion of liquid (multimodal). Of these, 40 responded transiently (phasic; 36 excited, 4 inhibited). The remaining 77 maintained their altered activity into the subsequent phases of the task (tonic; 69 excited, 8 inhibited). In each of these 4 categories, most cells were activated primarily by novel or unfamiliar stimuli, and their responses habituated during repeated stimulation. A small number of cells in the basolateral and the basomedial nuclei (n = 14, 2.4%) were highly selective in that they responded specifically to one biologically significant object or sound more than to any other stimuli (selective). Some of these neurons responded to both sight and ingestion of a specific food. In summary, most AM neurons responded vigorously to novel stimuli, and some of the neurons had multimodal responsiveness. These results suggest the AM is related to processing of new environmental stimuli and to those cross-modal association.
Presynaptic transmitters and depolarizing influences regulate development of the substantia nigra in culture
Authors: Friedman WJ , Dreyfus CF , McEwen B , Black IB
Published: March 2018

Recent evidence suggests that extracellular signals regulate neurotransmitter traits in brain catecholaminergic (CA) neurons as in the periphery. Development of the dopaminergic phenotype in the mouse substantia nigra (SN) was studied by monitoring tyrosine hydroxylase (TH), the rate-limiting enzyme in CA biosynthesis in vivo and in culture. Explants of SN were dissected from embryonic day 15 embryos and grown in culture for a week. To define the influence of depolarizing signals on central dopaminergic neurons, cultures were grown with the pharmacologic depolarizing agent veratridine. This treatment elicited a significant increase in TH enzyme activity, accompanied by elevated levels of enzyme protein. The increase in activity was prevented by TTX, suggesting that transmembrane Na+ influx was necessary for the rise in TH. A physiologic presynaptic agonist, substance P, also evoked a significant increase in TH activity; however, the coproduced tachykinin peptide, substance K (SK, neurokinin A) elicited a more dramatic rise. The SK effect was blocked by TTX, suggesting that the physiologic agonist was acting through the same mechanism as the pharmacologic agent veratridine. Immunoblot analysis revealed that SK elicited a parallel increase in TH enzyme protein. Our observations suggest that the novel peptide, SK, serves a physiological role in the regulation of TH in the striatonigral pathway.
Postmitotic, postmigrational expression of tyrosine hydroxylase in olfactory bulb dopaminergic neurons
Authors: McLean JH , Shipley MT
Published: March 2018

The developmental expression of tyrosine hydroxylase (TOH) was studied in a large, specific population of dopaminergic (DA) neurons in the main olfactory bulb (MOB) of the rat. These DA neurons comprise an anatomically distinctive population that has been well characterized in the adult hamster (Davis and Macrides, 1983) and rat (Halasz et al., 1981; Baker et al., 1983, 1984). We addressed a basic question in developmental neurobiology: What factors regulate the expression of neuronal transmitter phenotype during development? Olfactory bulb DA neurons are born in the ventricular and subependymal zones and migrate through all intervening layers to the most superficial layer in the bulb (Altman, 1969; Bayer, 1983). The time of TOH expression in these neurons was determined using immunohistochemistry and light microscopic image-analysis techniques. The results indicate that TOH phenotype is not expressed when the cells are born in the subependymal zone nor during their migration to the periglomerular region but only after they reached their final destination, the glomerular layer. This suggests that epigenetic factors associated with the glomeruli initiate the expression of the key transmitter synthesizing enzyme in these neurons. Primary olfactory neurons in the nasal epithelium project exclusively to glomeruli of the MOB; removal of this input in adult rats (Kawano and Margolis, 1982; Baker et al., 1983, 1984), mice (Nadi et al., 1981; Baker et al., 1983), dogs (Nadi et al., 1981), and hamsters (Kream et al., 1984) appears to down-regulate the expression of the TOH in periglomerular cells. The present results suggested that the input from the primary olfactory nerve is also necessary for the initial expression of the TOH phenotype. In support of this notion, we found that lesions of the olfactory nerve during the first postnatal week caused a significant reduction in the number of TOH-positive juxtaglomerular neurons in the following weeks. Thus, the olfactory nerve appears to be necessary for both the initiation and maintenance of TOH expression in olfactory bulb neurons. These findings suggest that specific cell-cell interactions play a key role in CNS neuronal transmitter phenotype regulation.(ABSTRACT TRUNCATED AT 400 WORDS)
Quantitative autoradiography of major neurotransmitter receptors in the monkey striate and extrastriate cortex
Authors: Rakic P , Goldman-Rakic PS , Gallager D
Published: March 2018

In vitro autoradiography was used to determine the binding properties and distribution of 9 major neurotransmitter receptors and their subtypes in the striate (area 17 of Brodmann) and extrastriate (areas 18 and 19) cortex of 1 infant and 3 adult rhesus monkeys. Differences in total labeling and nonspecific labeling, as well as Kd and Bmax values, were determined for all cortical layers and sublayers in both cytoarchitectonic areas by Scatchard analysis of autoradiograms. Area 17 differed from area 18 in the laminar pattern and density of virtually every ligand examined, i.e., 3H-clonidine, 3H-prazosin, 125I- iodopindolol, 3H-quinuclidinyl benzilate, 3H-5-hydroxytryptamine, 3H- ketanserin, 3H-muscimol, 3H-flunitrazepam, and 3H-spiperone. Kd and Bmax values for each ligand were remarkably consistent across the 3 adult monkeys analyzed quantitatively. Particularly dramatic contrasts were observed with clonidine, 5-hydroxytryptamine, and ketanserin, which have high affinity for alpha 2-adrenergic, 5-HT1-, and 5-HT2- receptors, respectively. The differences in distribution of these ligands, especially clonidine and 5-hydroxytryptamine, correlated well with specific laminae and hence exhibited distinctly different patterns in areas 17 and 18. Other ligands, such as flunitrazepam and quinuclidinyl benzilate that bind to GABAergic and cholinergic receptors, were visually less discriminating both among layers and between regions. However, layer for layer, the Bmax values for quinuclidinyl benzilate were higher in area 17 than 18, indicating the subtle differences between areas may be revealed only by quantitative measures. Some ligands were particularly dense in layer I (iodopindolol in areas 17 and 18; 5-hydroxytryptamine in area 18), and others subdivided cortical layers that are otherwise cytoarchitectonically uniform (e.g., flunitrazepam and clonidine in layer VI of area 17), indicating that areal differences in ligand binding are not a simple read-out of cell-packing density but most likely reflect a genuine difference related to the neurotransmitters of intrinsic and extrinsic afferents in each area. The presence of binding sites in every layer of both areas for all ligands examined indicates that their distribution across laminae is quantitative and not all-or-none. No layer contained less than 50% of binding sites present in any other layer. These findings reveal that visual cortical areas differ in density and lamination of neurotransmitter receptors and presumably in their sensitivity to circulating levels of endogenous neurotransmitters and pharmacologically active compounds.
Correlated electrophysiology and morphology of the ependyma in rat hypothalamus
Authors: Jarvis CR , Andrew RD
Published: March 2018

The ependyma lines the ventricular system of the vertebrate brain and spinal cord. Although its embryology and morphology have been studied extensively, little is known of its physiological properties, particularly in mammals. Tanycytes are modified ependymal cells that are found predominantly lining the floor of the third ventricle, overlying the median eminence. Their processes accompany and enwrap neuroendocrine axons that course from hypothalamic nuclei to terminals in the median eminence, but the significance of this interaction is not yet understood. Intracellular recording and injection techniques were used to study ependymal cells and tanycytes of the rat in the hypothalamic slice preparation after differentiating their respective regions morphologically. With extracellular [K+] = 6.24 mM, the mean membrane potential (+/- SD) for common ependyma was -79.9 +/- 1.40 mV and for tanycytes, -79.5 +/- 1.77 mV. Input resistances (Rin) were very low (much less than 1 M omega). Single-cell injection of Lucifer yellow revealed dye coupling among 2–70 ependymal cells and 5–48 tanycytes. In both freeze-fractured replicas and thin sections, large numbers of gap junctions were found between adjacent ependymal cells and between adjacent tanycytes. The observations of numerous gap junctions, extensive dye coupling and low input resistance demonstrated that both populations are strongly coupled networks. Perhaps for this reason, attempts to uncouple these cells using sodium propionate or CO2 were unsuccessful. Electrical stimulation of the arcuate nucleus did not elicit any detectable synaptic response in impaled tanycytes, so that the functional significance of synaptoid contacts between neuroendocrine neurons and the postsynaptic tanycytes is not yet apparent. Ependymal cells and tanycytes demonstrated a near-Nernstian response to changes in extracellular [K+] between 3 and 20 mM. This finding, as well as their high negative resting potential, low Rin, extensive coupling and absence of spontaneous electrical excitability demonstrate that ependymal cells possess numerous glial characteristics and may therefore have similar functions. In the hypothalamus, ependyma probably take up K+ released from adjacent endocrine neurons and shunt it to the ventricular space.
Development of inward currents in chick sensory and autonomic neuronal precursor cells in culture
Authors: Gottmann K , Dietzel ID , Lux HD , Huck S , Rohrer H
Published: March 2018

The development of ionic inward currents was studied in cultured neuronal precursors from chick sensory dorsal root ganglia (DRG) and compared with neuronal precursors from the cholinergic ciliary ganglia (CG) using whole cell patch-clamp recording. Neuronal precursors devoid of neuron-specific surface markers were isolated during the period of neuronal birth, i.e., at embryonic day (E) 6 from DRG and at E4.5 from CG. All neuronal precursor cells from DRG, as well as CG, showed outward K+ currents directly after they had attached to the substrate. During the first 5 hr in culture, half of the DRG cells had no inward currents at all, whereas the other half displayed a rapidly and fully inactivating Ca2+ current, which was activated with small depolarizing pulses from a holding potential of -80 mV to a -50 mV membrane potential (low-voltage-activated current, LVA). At these early stages, no other inward currents were resolved. TTX-blockable Na+ currents and slowly inactivating classical Ca2+ currents, which were activated with larger depolarizing pulses to a -20 mV membrane potential (high-voltage- activated currents, HVA) appeared concurrently after 15–20 hr in culture. In contrast, more than half of the CG cells showed LVA currents, as well as Na+ currents, as early as during the first 5 hr in culture. The HVA Ca2+ currents from the majority of the cells could be recorded only after 10–15 hr in culture. In both types of precursor- derived neurons, the LVA Ca2+ current preceded the classical HVA Ca2+ current. However, the temporal relation of the first Na+ currents to the first HVA Ca2+ currents seemed to be different in the 2 preparations. In DRG cells, Na+ and HVA Ca2+ currents appeared at the same time, whereas in CG cells, the HVA Ca2+ current showed a time lag with respect to the Na+ current. In addition, the relative amplitudes of the currents differed in the CG and DRG cells. This shows that as early as E4–6, shortly after their terminal mitosis, neurons from distinct peripheral ganglia in chick vary in the development of their basic ionic currents.
Effect of testosterone on input received by an identified neuron type of the canary song system: a Golgi/electron microscopy/degeneration study
Authors: Canady RA , Burd GD , DeVoogd TJ , Nottebohm F
Published: March 2018

Combinations of the Golgi stain, anterograde degeneration, and electron microscopy are used to further characterize the hormone-sensitive “type IV” neuron of the forebrain nucleus robustus archistriatalis (RA) of adult female canaries. Anterograde degeneration was used to “stain,” at the electron-microscopic level, the axon terminals of neurons projecting to RA from hyperstriatum ventralis, pars caudalis (HVc) and from the lateral magnocellular nucleus of the anterior neostriatum (L- MAN). The HVc neurons projecting to RA type IV cells form synapses predominantly on the dendritic spines of those cells, while L-MAN neurons that project to RA type IV cells form a 2.5:1 mixture of shaft and spine synapses. There were about 1000 synapses from HVc neurons (about 30% of all spine synapses) on typical type IV cells and about 50 synapses from L-MAN neurons. Earlier work had shown that in female canaries the dendrites of type IV neurons of the avian song control nucleus RA increase in total length after systemic testosterone treatment, and that this increase in dendritic length was accompanied by the development of malelike song. We now show that testosterone treatment also increases the number of dendritic spines present in type IV neurons. Presumably this is accompanied by an increase in the number of synaptic inputs received by type IV cells. Earlier evidence suggested that the testosterone-induced addition of extra dendritic length to type IV cells occurred at existing dendritic tips. We tested the hypothesis that these added peripheral ends received a special subset of inputs, which might then account for the change in behavior, and found it to be false. Mapping and counts of degenerating synapses resulting from lesion of HVc and L-MAN suggest that under the influence of hormone, new synapses are added throughout the dendritic tree, with no special distribution or change in ratio of inputs occurring at the tip of dendrites. Under the influence of testosterone, each type IV cell may receive only “more of the same” inputs it received before onset of treatment. We speculate on how such changes in circuitry may relate to song stability and learning.
Costorage of serotonin binding protein with serotonin in the rat CNS
Authors: Kirchgessner AL , Gershon MD , Liu KP , Tamir H
Published: March 2018

Previous studies have identified two neurectoderm-specific serotonin binding proteins (SBP), one with an apparent Mr of 45 kDa, and one of 56 kDa. The current experiments were undertaken to test the hypothesis that these proteins are specific components of serotonergic neurons. Since actin has been found to bind serotonin, the relationship of the 2 forms of SBP to actin was also investigated. Antisera against purified 45 and 56 kDa SBP were raised in rabbits and shown by analyses of immunoblots and differential absorption to be monospecific and not cross-reactive. Neither antiserum reacted with purified actin and none of 3 different anti-actin sera reacted with purified 45 or 56 kDa SBP. The antisera to 45 and 56 kDa were used for immunocytochemical localization of the proteins, which was compared to that of serotonin. SBP immunoreactivity was found in rat brain and spinal cord; however, no significant differences were observed in the pattern of distribution of 45 and 56 kDa SBP-immunoreactive structures. Immunostaining of neuronal perikarya by either SBP antiserum required pretreatment of animals with colchicine. The distribution of neurons and terminals labeled by each antiserum to SBP was similar to that of neurons and terminals labeled by anti-5-HT sera. SBP-immunoreactive neuronal perikarya were present in the nuclei raphe dorsalis, raphe centralis superior, raphe medianus, raphe magnus, raphe obscurus, raphe pallidus, dorsal to the medial lemniscus in the region of the B9 cell group, near the interpeduncular nucleus, in the area postrema, the pars compacta of the substantia nigra, the dorsomedial nucleus of the hypothalamus, and the arcuate nucleus. SBP-immunoreactive fibers and terminals were present in many additional areas of the brain, as well as the spinal cord, where they paralleled those that were immunostained with antibodies to 5-HT. When double-immunostaining was used, serotonin and 45 and 56 kDa SBP immunoreactivities were found to be colocalized in both the brain and spinal cord. Cells and fibers found to be stained by one immunoreagent were also stained by the others; therefore, serotonergic neurons of the CNS probably contain both 45 and 56 kDa SBP. Moreover, it also seems likely that nonserotonergic neurons contain neither form of SBP. These data strongly suggest that SBP is an intrinsic and specific component of serotonergic neurons that can serve as a serotonergic marker.
Effects of nicotine on local cerebral glucose utilization in the rat
Authors: London ED , Connolly RJ , Szikszay M , Wamsley JK , Dam M
Published: March 2018

We used the autoradiographic 2-deoxy-D-[1–14C]glucose (14C-DG) method of Sokoloff to identify brain areas with altered rates of local cerebral glucose utilization (LCGU) in vivo in response to peripheral I- nicotine administration (0.1, 0.3, 1.0, and 1.75 mg/kg, s.c.). Nicotine stimulated LCGU primarily in areas reported to contain nicotine binding sites, indicating that the sites are true receptors. Increases in LCGU of 100% or more over control were obtained in the medial habenula, fasciculus retroflexus, superior colliculus, and median eminence. Substantial stimulation (50–100% increases) also was obtained in the cerebellar vermis, interpeduncular nucleus, and anteroventral and interanteromedial thalamic nuclei. Moderate increases (20–50%) were observed in the reticular nucleus of the medulla, paramedian lobule, nucleus of the spinal tract of the trigeminal nerve, presubiculum, subiculum, red nucleus, ventral tegmental area, substantia nigra, nucleus ambiguus, nucleus tractus solitarius, dorsal lateral geniculate nucleus, mammillothalamic tract, and fornix. The greatest stimulation in most affected areas was obtained with 0.3 mg/kg nicotine administered at 2 min, but not longer, before 14C-DG. Effects of nicotine on LCGU were antagonized by mecamylamine. The findings indicate that the interaction of nicotine with specific binding sites is coupled to cerebral energy metabolism. The distribution of in vivo cerebral metabolic effects of nicotine implicates various brain regions in the behavioral and physiological effects of nicotine.
Dopamine-sensitive anticonvulsant site in the rat striatum
Authors: Turski L , Cavalheiro EA , Bortolotto ZA , Ikonomidou-Turski C , Kleinrok Z , Turski WA
Published: March 2018

The basal ganglia are involved in the organization of movement and function in the initiation and expression of generalized and limbic seizures. Dopamine is the principal neurotransmitter of the mesencephalic efferent pathways terminating in the mammalian striatum. No function has been ascribed to mesostriatal dopamine in the control of seizure spread in the brain. This work presents evidence that bilateral application of picomole amounts of apomorphine (a dopamine agonist) into the striatum confers protection against seizures produced by pilocarpine (a cholinergic agonist) in rats. The anticonvulsant effect of apomorphine is topographically confined to the caudate- putamen, nucleus accumbens, and olfactory tubercle. Bilateral application of nanomolar amounts of haloperidol (a dopamine antagonist) into the caudate-putamen or systemic application of haloperidol both lower the threshold for pilocarpine-induced seizures. Local application of an excitatory amino acid N-methyl-D-aspartate, into the substantia nigra pars compacta, ventral tegmental area, or retrorubral area, sites of origin of mesostriatal dopaminergic pathways, protects rats against seizures produced by pilocarpine. These results suggest that dopaminergic transmission in the striatum may be operative in complex neuronal networks modulating the seizure threshold.
Common cortical and subcortical targets of the dorsolateral prefrontal and posterior parietal cortices in the rhesus monkey: evidence for a distributed neural network subserving spatially guided behavior
Authors: Selemon LD , Goldman-Rakic PS
Published: March 2018

Common efferent projections of the dorsolateral prefrontal cortex and posterior parietal cortex were examined in 3 rhesus monkeys by placing injections of tritiated amino acids and HRP in frontal and parietal cortices, respectively, of the same hemisphere. Terminal labeling originating from both frontal and parietal injection sites was found to be in apposition in 15 ipsilateral cortical areas: the supplementary motor cortex, the dorsal premotor cortex, the ventral premotor cortex, the anterior arcuate cortex (including the frontal eye fields), the orbitofrontal cortex, the anterior and posterior cingulate cortices, the frontoparietal operculum, the insular cortex, the medial parietal cortex, the superior temporal cortex, the parahippocampal gyrus, the presubiculum, the caudomedial lobule, and the medial prestriate cortex. Convergent terminal labeling was observed in the contralateral hemisphere as well, most prominently in the principal sulcal cortex, the superior arcuate cortex, and the superior temporal cortex. In certain common target areas, as for example the cingulate cortices, frontal and parietal efferents terminate in an array of interdigitating columns, an arrangement much like that observed for callosal and associational projections to the principal sulcus (Goldman-Rakic and Schwartz, 1982). In other areas, frontal and parietal terminals exhibit a laminar complementarity: in the depths of the superior temporal sulcus, prefrontal terminals are densely distributed within laminae I, III, and V, whereas parietal terminals occupy mainly laminae IV and VI directly below the prefrontal bands. Subcortical structures also receive apposing or overlapping projections from both prefrontal and parietal cortices. The dorsolateral prefrontal and posterior parietal cortices project to adjacent, longitudinal domains of the neostriatum, as has been described previously (Selemon and Goldman-Rakic, 1985); these projections are also found in close apposition in the claustrum, the amygdala, the caudomedial lobule, and throughout the anterior medial, medial dorsal, lateral dorsal, and medial pulvinar nuclei of the thalamus. In the brain stem, both areas of association cortex project to the intermediate layers of the superior colliculus and to the midline reticular formation of the pons.(ABSTRACT TRUNCATED AT 400 WORDS)
Protein kinase C activators block specific calcium and potassium current components in isolated hippocampal neurons
Authors: Doerner D , Pitler TA , Alger BE
Published: March 2018

Whole-cell voltage-clamp techniques were used to study the effects of the protein kinase C (PKC) activators phorbol esters and OAG on Ca and K currents in differentiated neurons acutely dissociated from adult hippocampus and in tissue-cultured neurons from fetal hippocampus. PKC activators had selective depressant effects on K currents, with persistent currents (IK and IK-Ca) being reduced and transient current (IA) being unaffected. In both cell types we recorded both high-voltage- activated, noninactivating (L-type) and high-voltage-activated, rapidly inactivating (N-type) Ca current. A low-voltage-activated, rapidly inactivating (T-type) Ca current was also recorded in tissue-cultured neurons but not in acutely dissociated neurons. PKC activators markedly reduced N-type current with less effect on L-type and no effect on T- type Ca current. Effects of PKC activators could be reversed with washing or with application of PKC inhibitors H-7 or polymyxin-B, an effect that could not be attributed to inhibition of cAMP-dependent protein kinase. The Ca/calmodulin inhibitor calmidazolium was ineffective in reversing the actions of PKC activators. Using whole- cell voltage-clamp techniques, we have demonstrated that hippocampal neurons possess 3 distinguishable components of calcium current. Distinct K currents were also observed. Our data strongly support the hypothesis that both Ca and K currents are selectively regulated by PKC and that these effects occur directly on the postsynaptic neuron.
Controlled outgrowth of dissociated neurons on patterned substrates
Authors: Kleinfeld D , Kahler KH , Hockberger PE
Published: March 2018

The cytoarchitecture of nervous tissue is lost during the dissociation procedures used to form primary cell cultures. As a first step toward reestablishing an ordered arrangement of these cells in vitro, we developed a set of procedures for patterning the outgrowth of cells cultured on 2-dimensional substrates. These procedures used a combination of surface chemistry and photolithographic techniques. The adhesive properties of either silicon or silicon dioxide (quartz) surfaces were controlled by covalently binding small organic molecules to the surface with silane coupling agents. The attachment and growth of either embryonic mouse spinal cells or perinatal rat cerebellar cells were found to be promoted by binding certain amine derivatives to the surface. In particular, cells grown on surfaces bound with diamines and triamines, but not with monoamines, formed cultures whose morphology was similar to that of cells cultured on conventional substrates, i.e., glass coated with poly(D-lysine). The attachment of cells to a substrate was inhibited by binding alkane chains (e.g., n- tetradecane) to the surface and plating the cells in media containing 5– 10% (vol/vol) serum. Patterns of selected adhesivity were formed using photochemical resist materials and lithographic masking techniques compatible with the silane chemistry. Cultures of either spinal cord cells or cerebellar cells could be confined to square regions on the scale of 50 micron. Cerebellar cells could be confined to grow on lines with widths less than 10 micron. This width is comparable to the diameter of granule cell somata. The patterned growth of cerebellar cells was maintained up to 12 d in vitro. Over this time period the granule cells were observed to develop electrical excitability and immunoreactivity for neuron-specific enolase. Purkinje neurons also developed electrical excitability when grown on the chemically modified surfaces. Immunochemical reactivity of the patterned cultures for glial fibrillary acid protein (GFAP) showed that glia are patterned along with the associated granule cells. Interestingly, the GFAP-positive glia that proliferated on surfaces bound with amine derivatives attained primarily a tile-shaped, fibroblast-like morphology, while those proliferating on glass coated with poly(D-lysine) developed primarily a spindle-shaped, process-bearing morphology. Granule cells preferentially associated with the spindle-shaped glia.
The alpha-2 adrenergic agonist guanfacine improves memory in aged monkeys without sedative or hypotensive side effects: evidence for alpha-2 receptor subtypes
Authors: Arnsten AF , Cai JX , Goldman-Rakic PS
Published: March 2018

The present study attempted to identify an alpha-2 agonist that could improve working memory in aged nonhuman primates without the marked hypotensive and sedative side effects produced by clonidine. Toward this end, the hypotensive, sedative, and memory-altering properties of the alpha-2 adrenergic agonists, B-HT920 and guanfacine, were compared with clonidine's effects in 9 aged rhesus monkeys. Memory capacity was assessed by a variable delay, spatial delayed response paradigm that requires the animal to remember information over short temporal intervals and to update this information on every trial. B-HT920 was found to produce a dose-response profile qualitatively similar to, but weaker than, clonidine: low doses impaired memory and began to lower blood pressure and produce sedation, while high doses improved memory. In contrast, guanfacine produced a dose-response profile opposite to that seen with clonidine: low doses improved memory without inducing hypotension or sedation, while the memory-impairing, hypotensive, and sedating properties of the drug were observed at higher doses. The potency of the 3 agonists to lower blood pressure was clonidine = B- HT920 greater than guanfacine; sedation was affected in the order clonidine greater than B-HT920 greater than guanfacine; for memory impairment, as measured by performance on the delayed response task, the rank order potency was clonidine greater than B-HT920 greater than guanfacine, while for memory improvement it was guanfacine greater than clonidine greater than B-HT920. These differences in rank order potency are consistent with the recent proposal of alpha-2 receptor subtypes, a rauwolscine-sensitive site (Rs) that binds clonidine greater than B- HT920 greater than guanfacine and a rauwolscine-insensitive site (Ri) that binds guanfacine greater than clonidine greater than B-HT920 (Boyajian and Leslie, 1987). The data suggest that the hypotensive, sedating, and memory-impairing effects of alpha-2 agonists may be due to actions at one subtype of receptor (Rs), while the memory-enhancing effects of these drugs may result from actions at another alpha-2 receptor subtype, the Ri site. The ability of low doses of guanfacine to improve memory without inducing hypotension or sedation indicates that this agonist may be an excellent candidate for treating memory disorders in man.
Antibody to galactocerebroside alters organization of oligodendroglial membrane sheets in culture
Authors: Dyer CA , Benjamins JA
Published: March 2018

Antibodies to galactocerebroside (GalC) cause major changes in the organization of the membrane sheets elaborated by murine oligodendroglia in culture. Exposure of oligodendroglia to rabbit anti- GalC IgG for 15 min followed by fluoresceinated second antibodies results in patches of surface GalC staining; when second antibodies are applied after 2 hr of anti-GalC, the pattern of staining on membrane sheets is solid and wrinkled. Anti-GalC exposure for 24 hr results in contracted membrane sheets. No membrane contraction is detected in cultures treated with anti-sulfatide IgM or anti-proteolipid protein IgG. In cultures exposed to anti-GalC continuously for 4–7 d, there is a marked decrease in numbers of extended membrane sheets with an accompanying increase in contracted sheets. This effect is reversible upon removal of anti-GalC from the culture media. By scanning electron microscopy, normally flat membrane sheets appear ruffled after 2 hr of anti-GalC treatment; by 24 hr, contracted membrane sheets consist entirely of bulbous protrusions. Oligodendrocyte membranes exposed to anti-sulfatide for 24 hr are not contracted but are covered with bulbous protrusions. The organization of underlying membrane structures was examined in relation to membrane patching and sheet contraction. In membranes with patching induced by exposure to anti-GalC for 15 min, the anti-GalC: GalC complexes are localized over cytoplasmic MBP domains, with the unstained areas located above cytoplasmic microtubular structures. Membrane sheets that are contracted in response to anti-GalC exposure for 6–24 hr show intense GalC staining over microtubular structures. Anti-GalC exposure does not change metabolism of GalC; in cultures incubated with 3H-galactose and anti- GalC for 24 hr, there are no alterations in GalC labeling compared with control cultures. In summary, these results provide direct evidence that interaction between surface glycolipids and external antibodies can initiate a sequence of events leading to dramatic changes within the oligodendrocyte.
Comparison of effects of D-1 and D-2 dopamine receptor agonists on neurons in the rat caudate putamen: an electrophysiological study
Authors: Hu XT , Wang RY
Published: March 2018

Extracellular single-unit recording and microiontophoretic techniques were used to characterize the pharmacological properties of dopamine (DA) receptor subtypes within the rat caudate putamen (CPu), a striatal structure that receives a dense innervation from DA neurons originating from the substantia nigra pars compacta (A9 DA neurons). Similar to the action of DA, the DA D-1 receptor agonist (+)SKF-38393 generally potentiated the activation produced by glutamate (GLU) at low ejection currents (less than or equal to 5 nA); at higher ejection currents, it depressed 97% of the CPu neurons tested. By contrast, the D-2 receptor agonist LY-171555 (quinpirole) was much less effective in affecting the firing rate of CPu cells. The selective D-1 antagonist SCH-23390, administered either intravenously or iontophoretically, completely blocked the (+)SKF-38393-induced effects on CPu cells but failed to change the depressant effects produced by either quinpirole or 5-HT. On the other hand, the selective D-2 antagonist I-sulpiride, blocked the effects induced by quinpirole but not (+)SKF-38393. These observations suggest that the D-1 and D-2 DA receptor agonists elicit their effects via distinct DA receptor subtypes. A comparison of these results with our previous results obtained from the nucleus accumbens (NAc) indicates that NAc cells are more responsive to DA D-2 agonist, whereas CPu cells are more sensitive to D-1 agonist. Therefore, D-1 receptors in the CPu may have a critical role in mediating the effect produced by DA.(ABSTRACT TRUNCATED AT 400 WORDS)
Control of contractile properties within adaptive ranges by patterns of impulse activity in the rat
Authors: Westgaard RH , Lomo T
Published: March 2018

These experiments explore the relationship between patterned impulse activity and contractile properties of skeletal muscles. Soleus (SOL) and extensor digitorum longus (EDL) muscles of adult rats were denervated and stimulated directly from 4 to 15 weeks with the same number of pulse trains at different intratrain pulse frequencies (1–500 Hz), with different numbers of pulse trains (864–4,320,000 pulses/d) at the same intratrain pulse frequencies, or with different combinations of pulse trains at 10 and 100 Hz. Chronic stimulation of the denervated SOL resulted in twitch times-to-peak and half-relaxation times that varied in a graded manner between values longer than those in the normal SOL to values as fast as those in the normal EDL, depending upon the pattern used. Increasing pulse frequencies (constant number) resulted in faster twitches, lower twitch/tetanus ratios, increasing post-tetanic potentiations, and larger tetanic tensions. Increasing pulse numbers (constant frequencies) resulted in slower twitches, lower twitch/tetanus ratios, post-tetanic depressions, and higher fatigue indices. The effect of varying the pulse number on twitch parameters was greater at low frequencies (10–20 Hz) than at high frequencies (100 Hz). SOL muscles receiving pulse trains at both 10 and 100 Hz became much faster than muscles receiving pulse trains at 10 Hz only, even in the experiments where the stimulation pattern contained 9 times as many pulses at 10 as at 100 Hz. Chronic stimulation of both the denervated and the innervated EDL with large numbers of pulses at 10 or 15 Hz resulted in twitches that were only half as slow as those induced in the SOL by the same “slow” patterns. In addition, these patterns led to a marked decrease in maximum tetanic tension and a marked increase in twitch/tetanus ratio. During stimulation with a small number of pulses at 150 Hz, on the other hand, twitch speed, twitch/tetanus ratio, and maximum tetanic tension remained normal or almost normal. We conclude that the isometric twitch and related properties of the rat SOL muscle can be graded within wide “adaptive ranges” by varying either the number or the frequency of pulses. In the EDL, the corresponding adaptive ranges appear much narrower, suggesting that the EDL and the SOL contain intrinsically different muscle fibers.
Changes in rapidly transported proteins in developing hamster retinofugal axons
Authors: Moya KL , Benowitz LI , Jhaveri S , Schneider GE
Published: March 2018

Proteins synthesized in retinal ganglion cells and conveyed to the terminals of optic tract axons in the rapid phase of axonal transport were analyzed at different developmental stages in the hamster. Animals between 2 d of age and adulthood were labeled intraocularly with 35S- methionine, and after a 4 hr survival time, the superior colliculus was dissected out, subjected to subcellular fractionation, and radiolabeled proteins in the particulate fraction analyzed by 2-dimensional gel electrophoresis and fluorography. The previously identified growth- associated phosphoprotein, GAP-43 (GAP-48, B-50, F1, pp46), was synthesized and transported at high levels in the neonate, but these levels declined precipitously after the second postnatal week. Immunohistochemical studies using a monospecific antibody showed that GAP-43 was localized along the entire length of retinal axons in the optic tract and target areas in P2 animals but was virtually absent in the adult visual pathway. By metabolic labeling, 2 proteins with molecular weights of about 230 kDa also showed a sharp decrease during development. In contrast, acidic proteins of 27 and 64 kDa, which were barely detectable in the neonate, increased steadily to become the most heavily labeled proteins of rapid axonal transport by the second postnatal week. Another group of proteins, of about 94–110 kDa, also rose to peak levels after birth but then declined. Temporal correlations between the molecular changes described here and the known anatomical events in optic tract development suggest that the synthesis and transport of particular membrane proteins may be directly related to the sequence of morphological changes.
A behavioral analysis of habituation and sensitization of shortening in the semi-intact leech
Authors: Boulis NM , Sahley CL
Published: March 2018

We have previously demonstrated that the shortening reflex of the leech Hirudo medicinalis displays habituation, dishabituation, and sensitization. In this paper we demonstrate that the shortening reflex of the semi-intact animal also displays these phenomena. In the first experiment we found that the magnitude of the touch-elicited shortening reflex decreased as a result of repeated stimulations of the skin every 2 min. The second experiment examined the change in this reflex as a function of the interstimulus interval (ISI). The reflex failed to decrease when the ISI was 10 sec, but decreased significantly when the ISI was either 45 or 360 sec. Finally, in a third experiment we found that the presentation of noxious stimuli prior to habituation training prevented habituation. Thus, as we have observed previously in the intact animal, the semi-intact animal displays habituation, dishabituation, and sensitization.
Secretory function of the vestibular nerve calyx suggested by presence of vesicles, synapsin I, and synaptophysin
Authors: Scarfone E , Dememes D , Jahn R , De Camilli P , Sans A
Published: March 2018

Type I sensory hair cells of the vestibular epithelium are nearly completely ensheathed by an afferent nerve ending, the vestibular nerve calyx. We have recently reported that the nerve calyx, and, in particular, its apical portions surrounding the neck of the hair cell, are immunoreactive for synapsin I (Favre et al., 1986), a major membrane component of small synaptic vesicles of axonal endings. We have now found, by electron microscopy, that the same region of the calyx is densely populated by microvesicles morphologically similar to typical presynaptic small synaptic vesicles. Furthermore, we have established by light microscopy immunocytochemistry that this region of the calyx also contains a high concentration of synaptophysin, another well-characterized major component of small synaptic vesicle membranes. These results suggest that the upper portion of the calyx is equipped with the machinery that in presynaptic terminals is involved in the release of neurotransmitters and raise the possibility that the calyx, via secretion of neurotransmitterlike substances, might modulate the function of type I hair cells.
Immunocytochemical localization of protein kinase C isozymes in rat brain
Authors: Huang FL , Yoshida Y , Nakabayashi H , Young WS , Huang KP
Published: March 2018

Recently, we isolated 3 protein kinase C (PKC) isozymes from rat brain (Huang et al., 1986a). Using isozyme-specific antibodies for immunoblot, we have determined the relative levels of each isozyme in various regions of the rat brain (Huang et al., 1987b). The present paper describes the cellular distributions of PKC isozymes in rat brain as determined by light microscopic immunocytochemistry. Staining with PKC antibodies revealed strong immunoreactivities in neuronal somata and their dendrites and weak to no reaction in axon and the astroglial structures. In the cerebellum, the type I PKC antibodies stained the Purkinje cell bodies and dendrites; the type II PKC antibodies stained the granule cells; and the type III PKC antibody stained both Purkinje and granule cells. In the cerebral cortex, all antibodies stained neurons resembling pyramidal cells and their apical dendrites in layers II to VI, while layer I was nearly devoid of staining. However, the various isozyme-specific antibodies revealed distinct laminar distribution patterns of the positively stained neurons, and the type III PKC-positive neurons exhibited a higher density than those of type I or II PKC-positive ones, especially in layer II of cingulate (retrosplenial) and piriform cortices. In the hippocampal formation, both pyramidal cells of the hippocampus and granule cells of the dentate gyrus were stained by all PKC antibodies. Subcellularly, type III PKC appeared mostly in the cytoplasm of these neurons, whereas type I and II PKC seemed to associate with the nucleus as well. In the olfactory bulb, both type II and III PKC antibodies stained the periglomerular and granular cells, and the latter also stained the mitral cells. The distinct cellular and subcellular distribution of PKC isozymes suggests that each isozyme plays a unique role in the various neural functions.