bluuebunny/crossref_metadata_2025 huggingface dataset:
The process of reinnervation in the dentate gyrus of adult rats: time course of increases in mRNA for glial fibrillary acidic protein
Authors: Steward O , Torre ER , Phillips LL , Trimmer PA
Published: April 2018

The present study evaluates the time course of increased expression of the mRNA for glial fibrillary acidic protein (GFAP) within the dentate gyrus and hippocampus after unilateral lesions of the entorhinal cortex. Levels of GFAP mRNA were evaluated by dot blot hybridization of mRNA isolated from the hippocampus and quantitative in situ hybridization. For dot blot hybridization, RNA was isolated from pooled hippocampi obtained from animals killed at 12 hr, 1, 2, 4, 6, 8, 10, 14, and 30 d postlesion. A separate set of animals killed at 2, 4, 6, 8, 10, 12, 14, and 32 d were prepared for in situ hybridization. The dot blot analyses of mRNA isolated from the hippocampus revealed that on the side ipsilateral to the lesion, the levels of GFAP mRNA increased rapidly, reaching a peak at 2 d postlesion. The increases were not evident by 12 hr postlesion, but by 24 hr, levels of GFAP mRNA were 5-fold higher than control, and by 48 hr, the levels were over 6- fold higher than control. The levels of GFAP mRNA decreased after 2 d postlesion. At 4 and 6 d postlesion the levels were about 2-fold higher than control. At later postlesion intervals, mRNA levels were comparable to the control. At 2 d postlesion, the levels of GFAP mRNA were also increased about 2-fold over control levels on the contralateral side. After 2 d, the levels of GFAP mRNA on the contralateral side were comparable to the control. In situ hybridization revealed a complex pattern of changes in the levels of GFAP. At 2 d postlesion, the levels of GFAP mRNA increased dramatically throughout the hippocampus bilaterally. The increases were most pronounced in the denervated portions of the neuropil; however, the levels of GFAP mRNA were also substantially elevated in laminae that do not receive direct projections from the entorhinal cortex. GFAP mRNA levels were also increased in other areas that receive projections from the entorhinal cortex, including the septum, lateral-dorsal thalamus, and entorhinal cortex contralateral to the lesion. In addition, GFAP mRNA levels were increased in regions bordering the ventricles throughout the brain, and over the surface of the tectum. After 2 d postlesion, the increases in the levels of GFAP mRNA were for the most part restricted to areas containing terminal degeneration. The generalized increases throughout the hippocampus were no longer apparent. Areas bordering the ventricles continued to exhibit higher labeling than in control animals, but this effect was not as prominent as at 2 d postlesion.(ABSTRACT TRUNCATED AT 400 WORDS)
Prenatal specification and target induction underlie the enrichment of calcitonin gene-related peptide in the trigeminal ganglion neurons projecting to the cerebral vasculature
Authors: Horgan K , O'Connor TP , van der Kooy D
Published: April 2018

The neurotransmitter calcitonin gene-related peptide (CGRP) is enriched in the adult rat trigeminal visceral projection to the cerebral arteries compared both to other neurotransmitters in this projection and to the percentage of CGRP-positive trigeminal cells projecting to cutaneous targets. In colchicine-treated ganglia approximately 30% of adult trigeminal ganglion cells projecting to the middle cerebral artery contain CGRP. Several possible developmental mechanisms underlying this enrichment were investigated. Some of this enrichment is accounted for by a prenatal selection of CGRP cells in the cerebrovascular projection. The remainder of the enrichment can be explained by a late (postnatal days 55–-90)target-induced expression of CGRP in some trigeminal neurons innervating cerebral arteries. Most surprisingly, the massive postnatal regression in the trigeminal projection to the cerebral arteries (between postnatal days 5 and 55, cell death and axon retraction delete 3/4 of the neurons that innervate the middle cerebral artery neonatally) has no role in the CGRP enrichment in the cells remaining at maturity in this projection. These regressive events appear to affect equally the CGRP-positive and CGRP-negative populations. However, axon retraction is involved in the postnatal loss of CGRP enrichment seen in 1 small subpopulation of the trigeminal projection. Each trigeminal cell in this population sends axon collaterals to both the cerebral artery and the forehead skin neonatally, and then later most of these dually projecting neurons retract only their artery collaterals but do not die. A low percentage of CGRP-containing neurons does not appear to predate artery collateral retraction.(ABSTRACT TRUNCATED AT 250 WORDS)
Distribution of GAP-43 mRNA in the brain stem of adult rats as evidenced by in situ hybridization: localization within monoaminergic neurons
Authors: Bendotti C , Servadio A , Samanin R
Published: April 2018

Using in situ hybridization, we examined the distribution of the mRNA encoding for the growth-associated protein GAP-43 in the brain stem of adult rats. GAP-43 was expressed at the highest level in the nucleus raphe dorsalis (NDR), nucleus centralis superior (NCS), substantia nigra compacta (SNc), ventral tegmental area (VTA), and locus coeruleus (LC). An intermediate level of signal was detected over the periaque- ductal gray, superior colliculi, and thalamic region, and no significant signal was detected in the substantia nigra pars reticulata and red nucleus. The hybridization signals of GAP-43 mRNA and tryptophan hydroxylase mRNA completely overlapped in the NDR and NCS, and signals for GAP-43 mRNA and tyrosine hydroxylase mRNA overlapped in the SNc, VTA, and LC. The disappearance of the hybridization signal for GAP-43 mRNA after intracerebroventricular injections of the neurotoxins 5,7-dihydroxytryptamine (5,7-DHT) or 6-hydroxydopamine (6-OHDA) indicated that high levels of GAP-43 are synthesized in the serotonergic neurons of the raphe nuclei and in the catecholaminergic neurons of the SNc, VTA, and LC. In light of the role of GAP-43 in axonal outgrowth, modulation of signal transduction, and release of different neurotransmitters in the adult CNS, this phosphoprotein might be involved in the functional plasticity and synaptic transmission of monoaminergic neurons.
Light paired with serotonin in vivo produces both short- and long-term enhancement of generator potentials of identified B-photoreceptors in Hermissenda
Authors: Crow T , Forrester J
Published: April 2018

An in vivo conditioning procedure consisting of light paired with the direct application of 5-HT to the exposed but otherwise intact nervous system of Hermissenda produces a long-term modification of phototactic behavior. The long-term change in phototactic behavior produced by in vivo conditioning is dependent upon pairing light with 5-HT. In this paper, we investigate neural correlates of in vivo conditioning detected in 2 different identified photoreceptors. We found that in vivo conditioning produces a short-term and long-term enhancement of light-evoked generator potentials recorded from medial and lateral B- photoreceptors. We show that short-term enhancement is not dependent upon pairing light with 5-HT, is observed in both lateral and medial B- photoreceptors, and is expressed by a larger peak and plateau phase of light-evoked generator potentials. In contrast to short-term enhancement, we found that long-term enhancement is dependent upon pairing light with 5-HT, is detected in only lateral B-photoreceptors, and is expressed by a larger steady-state plateau phase of light-evoked generator potentials. We also present evidence that the direct action of 5-HT interacting with light- and/or voltage-dependent processes is sufficient to mimic the effects of in vivo conditioning on long-term enhancement. These results suggest that long-term enhancement may contribute to modified phototactic behavior in Hermissenda produced by 1-trial in vivo conditioning.
Brain-specific polyA- transcripts are detected in polyA+ RNA: do complex polyA- brain RNAs really exist?
Authors: Fung BP , Brilliant MH , Chikaraishi DM
Published: April 2018

Transcripts encoded by 2 different rat genomic clones, rg13 and rg100, appear to be typical brain-specific polyA- RNAs, as defined by previous criteria (rare, polysomal, and postnatally expressed from single-copy genes). However, we have found by using a sensitive nuclease protection assay that low levels of these transcripts (10% and 3%, respectively) are detected in polyA+ RNA. To determine if rg transcripts that fractionate as polyA- could have resulted from nicking of polyA+ RNA, we assessed the integrity of 2 known polyA+ RNAs, those of tyrosine hydroxylase, a 2-kilobase (kb) mRNA, and sodium channel, a 9.5-kb RNA. Using RNA prepared by several different procedures, including LiCl-urea and guanidine thiocyanate followed by CsCl centrifugation, the shorter message fractionated as polyA+ after 2 cycles over oligodeoxythymidine (oligo-dT) cellulose, whereas the majority of the longer sodium channel RNA fractionated as polyA, as assayed by nuclease protection using probes from the 5′ end of the 2 genes. However, on Northern blots, the same RNA preparations showed an intact 9.5-kb sodium channel band only in polyA+ RNA, suggesting that the polyA- RNAs were randomly cleaved, resulting in a smear of sizes that could not be detected as a discrete band. These data imply that long messages may be nicked during standard isolation procedures and that this would not be detected by Northern blot analysis.(ABSTRACT TRUNCATED AT 250 WORDS)
Neuropeptides Gly-Asp-Pro-Phe-Leu-Arg-Phe-amide (GDPFLRFamide) and Ser- Asp-Pro-Phe-Leu-Arg-Phe-amide (SDPFLRFamide) are encoded by an exon 3' to Phe-Met-Arg-Phe-NH2 (FMRFamide) in the snail Lymnaea stagnalis
Authors: Saunders SE , Bright K , Kellett E , Benjamin PR , Burke JF
Published: April 2018

Biochemical analysis has shown the pond snail Lymnaea stagnalis to contain 2 main classes of Phe-Met-Arg-Phe-NH2 (FMRFamide)-like neuropeptides: the tetrapeptides FMRFamide and Phe-Leu-Arg-Phe-NH2 (FLRFamide), and the heptapeptides Gly-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (GDP- FLRFamide) and Ser-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (SDPFFRFamide). By genomic mapping and DNA sequencing, we show here that the GDP/SDPFLRFamide coding region lies 3′ to the FMRFamide coding region. The absence of an initiating start methionine and the presence of good- concensus 3′ and 5′ splice sites suggests that the GDP/SDPFLRFamide coding region makes up 1 exon of a larger gene. In addition to 7 copies of GDPFLRFamide and 6 copies of SDPFLRFamide, the exon encoding the heptapeptides also encodes 3 novel peptides, Glu-Phe-Phe-Pro-Leu-NH2 (EFFPLamide), Ser-Asp-Pro-Tyr-Leu-Phe-Arg-NH2 (SDPYLFRamide), and Ser- Asp-Pro-Phe-Phe-Arg-Phe-NH2 (SDPFFRFamide). In contrast to the tetrapeptide FMRFamide precursor protein, the GDP/SDPFLRFamide peptides are encoded contiguously, being separated only by single basic amino acids.
Modular organization of projection neurons in the matrix compartment of the primate striatum
Authors: Gimenez-Amaya JM , Graybiel AM
Published: April 2018

It is well known that the striatum has a chemical architecture dividing it into striosomes and matrix, and that these compartments have different input-output connections. However, striatal afferent-fiber systems also form vividly patchy terminal fields in the matrix, and studies in the past year have uncovered instances of nonstriosomal clustering of striatal output neurons. In the experiments reported here, we systematically investigated this output-neuron clustering in the primate, using the striatopallidal system as a model. Our goals were to determine whether the modular organization is a general characteristic of projection neurons in the striatal matrix, whether the modularity occurs independent of striosomal boundaries, and whether the output modules are systematically organized. We studied the distribution of striatopallidal projection neurons in 9 adult squirrel monkeys by centering deposits of the retrograde tracer HRP-WGA in either the external segment or the internal segment of the globus pallidus. Following injections of each type, many retrogradely labeled neurons appeared in the striatal matrix in clusters and bands having cross-sectional diameters of 0.2–0.8 mm. Comparisons with adjoining sections stained to demonstrate striosomes established that the local groups of striatal output neurons sometimes abutted striosomes but often did not. The retrogradely labeled clusters and bands appeared both in the caudate nucleus and in the putamen. Their arrangements were regular and often periodic. These findings suggest that the large matrix compartment of the primate striatum, which is the primary site of origin of striatal outputs to the pallidum and the reticular part of the substantia nigra, contains systematic mosaics of projection neurons. We propose that this output-neuron modularity of the striatal matrix in the primate could serve as the template for redistribution of the massive afferent-fiber systems of the striatum into specialized striatopallidal output channels.
Initial expression and endogenous activation of NMDA channels in early neocortical development
Authors: LoTurco JJ , Blanton MG , Kriegstein AR
Published: April 2018

We have made patch-clamp recordings from slices of fetal and postnatal rat neocortex in order to study the initial expression and activation of NMDA channels. Recordings from both whole cells and outside-out patches indicated that functional NMDA channels are expressed on neurons within the cortical plate, but not on younger cells within the ventricular zone. The NMDA channels on cortical plate neurons had a unitary conductance of approximately 40 pS, had a mean open time of approximately 6 msec, required glycine to open, and were blocked in a voltage-dependent manner by magnesium. These precocious channels were present before the appearance of functional synaptic activity, yet like NMDA channels in the mature neocortex, they were spontaneously activated by an agonist within brain slices. These results demonstrate that NMDA channels are initially expressed on neocortical neurons some time between the last mitotic division within the ventricular zone and completion of migration into the cortical plate. These early NMDA channels have properties characteristic of NMDA channels on more mature neurons and are similarly activated by an endogenous agonist in situ. Their early appearance and activation indicate that NMDA channels may play a role during early stages of cortical development.
Inhibitory effects of brain chondroitin sulfate proteoglycans on neurite outgrowth from PC12D cells
Authors: Oohira A , Matsui F , Katoh-Semba R
Published: April 2018

Soluble chondroitin sulfate proteoglycans (CSPGs), prepared from 10-d- old rat brain, were added to the culture medium of PC12D cells containing NGF to examine the effects on NGF-induced neurite outgrowth from the cells. PC12D cells, a flat-shaped variant of PC12 pheochromocytoma cells, are characteristic of prompt neurite formation in response not only to NGF, but also to cAMP-enhancing reagents such as forskolin. Brain CSPGs inhibited the neurite elongation irreversibly in a dose-dependent manner; complete inhibition was observed at a concentration of 50 nmol uronic acid/ml. Closely similar dose-dependent inhibition was observed in the forskolin-induced neurite outgrowth from PC12D cells. NGF-induced neurite outgrowth from conventional PC12 cells was also inhibited completely by 50 nmol uronic acid/ml CSPGs. Some brain CSPGs seemed to be inhibitory, but the cartilage-unique CSPG did not show any inhibitory effect. Chondroitin sulfate, a polysaccharide moiety of CSPGs, did not show any inhibitory effect even at a concentration of 250 nmol uronic acid/ml, while core proteins prepared from brain CSPGs by digestion with chondroitinase ABC exhibited inhibitory activity similar to that of intact CSPGs. This indicates that the site of the inhibitory activity exists in the core protein moiety of brain CSPGs. From these observations, it is conceivable that brain CSPGs are involved in the regulation of neuronal differentiation.
Medial-to-lateral gradient of neostriatal NGF receptors: relationship to cholinergic neurons and NGF-like immunoreactivity
Authors: Altar CA , Dugich-Djordjevic M , Armanini M , Bakhit C
Published: April 2018

High-affinity binding sites for recombinant human NGF (rhNGF) were studied in the caudate-putamen of the adult rat and rabbit. Displaceable 125I-rhNGF binding sites were densely distributed throughout the caudate-putamen and were 2–3-fold more prevalant in the ventrolateral and lateral than in the medial caudate-putamen. The amount of nondisplaceable binding did not vary throughout the caudate- putamen. The medial-to-lateral receptor gradient was correlated (r = +0.99) with a 2–3-fold medial-to-lateral increase in ChAT activity. In contrast, NGF-like immunoreactivity (NGF-LI) was prevalent but uniformly distributed in the caudate-putamen. Lesions of intrinsic cholinergic neurons by quinolinic acid produced extensive gliosis in the medial, central, and lateral caudate-putamen, yet 125I-rhNGF binding was decreased in each of these regions. The activity of ChAT and 125I-rhNGF binding throughout the caudate-putamen were each decreased by 40% following quinolinic acid. Binding was not changed after 70–77% dopamine nerve terminal depletions induced by 6- hydroxydopamine, demonstrating a nonglial, nondopaminergic locus for striatal NGF binding sites. The cholinergiclike topography of NGF binding sites throughout the intact caudate-putamen, the parallel decreases of cholinergic neurons and NGF binding sites following intrinsic neuronal loss, and the uniform neostriatal gradient of NGF-LI are consistent with the trophic role of endogenous NGF for cholinergic interneurons of the caudate-putamen.
Endogenous opioids modulate neuronal survival in the developing avian ciliary ganglion
Authors: Meriney SD , Ford MJ , Oliva D , Pilar G
Published: April 2018

Most studies on the trophic regulation of the normal neuronal competition for survival have focused on interactions between neurons and their target environment. However, it is also likely that trophic modulators are released from premotor inputs onto motoneurons. We have examined the developmental distribution of endogenous enkephalin-like immunoreactivity and the role that these endogenous opioid peptides play in normal neuronal degeneration. During the early portion of the normal cell death period, enkephalin-like immunoreactivity is highest within preganglionic cell bodies in the midbrain and their nerve terminals in the ciliary ganglion. Exogenous daily morphine administration to the chick embryo has previously been shown to delay most of the normal neuronal death in the ciliary ganglion (see Meriney et al., 1985). We hypothesized that opiate receptor activation increases the probability that ciliary ganglion neurons will survive their developmental competition and, further, that the endogenous opioid peptides in the ciliary ganglion normally modulate this competition. However, in our previous report (Meriney et al., 1985), we noted that daily administration of the antagonist naloxone to the chorioallantoic membrane did not significantly alter neuronal survival, as would have been expected if endogenous opioids were involved in regulating cell death. In contrast, in this report we show that three times daily application of naltrexone (a long-lasting opiate antagonist) significantly decreased neuronal survival among the ciliary ganglion cells, and that the surviving cells were not ultrastructurally different than neurons from controls of the same developmental stage. To control for toxic effects of naltrexone, we performed cell counts following naltrexone, we performed cell counts following naltrexone treatment in another population of cholinergic motoneurons (lumbar spinal motoneurons). In this population of cells, the total number of motoneurons remains unchanged following naltrexone treatment. To test for a specific toxic effect on the neurons of the ciliary ganglion, we generated a dose-response curve for toxicity in vitro and determined that naltrexone was not toxic over concentration ranges that are likely to exist in vivo. It appears, therefore, that a multiple daily antagonist application protocol blocks opiate receptors sufficiently in the ciliary ganglion to decrease an endogenous opiate influence significantly. We tested the possibility that endogenous opioids exert their effect by modifying transmission at peripheral and ganglionic synapses. In the generally accepted hypothesis, paralysis at the peripheral nerve-striated muscle synapse would rescue cells, while paralysis of ganglionic synapses would decrease survival. Iris neuromuscular junctions onto striated muscle cells were not blocked by opioids, but neuromuscular transmission in the smooth muscle of the choroid coat was blocked.(ABSTRACT TRUNCATED AT 400 WORDS)
Developmental expression of muscarinic cholinergic receptors and coupling to phospholipase C in rat sweat glands are independent of innervation
Authors: Grant MP , Landis SC
Published: April 2018

During development, the innervation of rat sweat glands undergoes a striking change from noradrenergic to cholinergic function. The acquisition of secretory responsiveness by the glands is temporally correlated with the appearance of cholinergic properties. In addition, responsiveness fails to appear in the absence of innervation. To investigate the basis of the onset of functional transmission and secretory responsiveness and its possible relationship to innervation, we analyzed the development of muscarinic cholinergic receptors in sweat glands, examined their expression in the glands of adult rats sympathectomized at birth, and assayed the ability of muscarinic agonists to increase phosphoinositide (PI) turnover. Autoradiographic and in situ hybridization analysis revealed that muscarinic ligand binding sites were first detectable as glands begin to form on postnatal day 4 (P4). Between P4 and P14, receptor concentration increased in parallel with mRNA for the m3 receptor subtype. On P14, the concentration of ligand binding sites approached adult levels, although only a small proportion of glands at this age secrete in response to nerve stimulation or cholinergic agonists. When the pharmacological properties of muscarinic receptors in sweat glands of adult rats sympathectomized at birth were compared to those of normal glands, the concentration and affinity determined with [N-methyl-3H]- scopolamine and the Ki values determined with the subtype-selective muscarinic antagonists 4-DAMP, pirenzepine, and AF DX-116 were similar. In addition, the molecular subtype was unchanged as was the level of m3 message. Studies of PI turnover in response to muscarinic stimulation indicated that the receptors expressed in sweat glands isolated from sympathectomized and acutely denervated, as well as control, rats were functionally coupled to phospholipase C. The absence of sympathetic innervation therefore does not appear to influence either the development of muscarinic receptors or their coupling to PI turnover. Our results suggest that functional sympathetic cholinergic innervation plays a central role in the development and maintenance of secretory function at a step distal to signal transduction across the cell membrane.
Normally unused positional cues guide ectopic afferents in the leech CNS
Authors: Passani MB , Peinado A , Engelman H , Baptista CA , Macagno ER
Published: April 2018

Central projections from peripheral sensory neurons segregate into distinct, ventrally positioned longitudinal tracts within the segmental ganglia of the leech Hirudo medicinalis. As documented here, there is an additional tract in the neuropils of the fifth and sixth body ganglia, located at the lateral margin and formed by afferent axons (the “sex afferents”) originating from sensory neurons located in the male and female genitalia. Ablation of the genitalia results in the complete absence of this additional tract. We asked (1) whether segmental differences exist in the distribution of pathway cues available to the sex afferents, and (2) whether central pathway selection by these axons is specific. We transplanted the primordia of the male genitalia to several ectopic positions posterior to the sixth body segment and labeled the ectopic sex afferents in order to examine their paths in the CNS. In about 50% of the experimental animals, afferent axons originating in the transplanted tissue segregated into a distinct lateral fiber bundle within the neuropil of a nearby ganglion, in a position corresponding to their normal one in the sex ganglia. The sex afferents therefore find their normal pathways even in the ganglia of inappropriate segments, although these pathways are not used by any other afferents in these ganglia. We propose, therefore, that the positional cues employed by afferent axons to select appropriate pathways in the ganglionic neuropil are expressed in all segments of the leech CNS, regardless of whether such cues are normally used by afferent axons in each segment.
Proliferative and degenerative changes in striatal spiny neurons in Huntington's disease: a combined study using the section-Golgi method and calbindin D28k immunocytochemistry
Authors: Ferrante RJ , Kowall NW , Richardson EP
Published: April 2018

Dysmorphic alterations of dendritic arbors and spines in spiny striatal neurons were identified in section-Golgi impregnations of moderate and severe grades of Huntington's disease (HD). These alterations could be characterized as either proliferative or degenerative changes. Proliferative changes included prominent recurving of distal dendritic segments, short-segment branching along dendrites, and increased numbers and size of dendritic spines. Degenerative alterations consisted of truncated dendritic arborizations, occasional focal dendritic swellings, and marked spine loss. Proliferative changes were found primarily in moderate grades of HD, while degenerative changes were predominantly found in severe grades. Cytopathologic changes increased with neuropathologic severity. Similar morphologic alterations were observed in calbindin D28k (Calb) stained neurons in HD striatum. The immunoreactive intensity of Calb staining was increased in the distal dendrites of positive neurons in HD striatum. The present findings provide morphologic and quantitative evidence that confirms an early and marked involvement of spiny striatal neurons in HD and suggest that neuronal growth, rather than degeneration, may be the harbinger of cell death in this disorder.
Behavioral analysis of internal memory states using cooling-induced retrograde amnesia in Limax flavus
Authors: Yamada A , Sekiguchi T , Suzuki H , Mizukami A
Published: April 2018

Temporal evolution of internal memory states in a terrestrial mollusk, Limax flavus, was studied using cooling-induced retrograde amnesia. The slug was conditioned to avoid carrot odor by temporally correlated presentation of carrot juice and a bitter-taste stimulus of quinidine sulfate. We could induce retrograde amnesia by cooling of the conditioned slug immediately after the training trial. Thus, we studied the memory states in the slug using the retrograde amnesia according to strategies used in the studies of memory states in mammals or insects. In the early process of memory acquisition, at least two distinctive memory states were observed, short-term memory and long-term memory (LTM). For LTM, two states were also observed. One was a reactivated state of LTM, which was sensitive to the cooling used to induce the amnesia. The other was a so-called resting state of LTM, which was insensitive to cooling. A few days after memory acquisition, further evolution was observed in that the amnesia could not be induced even if the memory trace was reactivated. The results obtained in Limax flavus was comparable with those obtained in a variety of animals.
Expression of tenascin in the developing and adult cerebellar cortex
Authors: Bartsch S , Bartsch U , Dorries U , Faissner A , Weller A , Ekblom P , Schachner M
Published: April 2018

Since tenascin may influence neuronal cell development, we studied its expression pattern using immunocytochemistry, in situ hybridization, Northern blot analysis, and immunochemistry in the developing and adult mouse cerebellar cortex. Tenascin immunoreactivity was detectable in all layers of the developing cerebellar cortex. In the external granular layer, only the radially oriented processes of Golgi epithelial cells were immunoreactive, whereas the densely packed cell bodies were immunonegative. Tenascin was hardly detectable at contact sites between migrating granule cells and processes of Golgi epithelial cells. Axons of granule cells in the molecular layer were immunoreactive, whereas their cell bodies in the internal granular layer lacked detectable levels of tenascin. By in situ hybridization, only Golgi epithelial cells and astrocytes of the internal granular layer and prospective white matter, but not nerve cells, could be shown to synthesize detectable levels of tenascin mRNA in the developing mouse cerebellar cortex. Thus, tenascin in the cerebellar cortex seems to be a glia-derived molecule that becomes adsorbed to neuronal surfaces in a topographically restricted pattern in situ. Levels of tenascin protein and mRNA decreased significantly with increasing age. In the adult, tenascin immunoreactivity was weak and mainly restricted to the molecular layer and tenascin mRNA was confined to Golgi epithelial cells, indicative for a functional heterogeneity in differentiated cerebellar astrocytes. Quantitative immunoblot analysis revealed that the 225 and 240 kDa components of tenascin were developmentally downregulated at a faster rate than the 190 and 200 kDa components, corresponding to the faster downregulation of the 8 kilobase (kb) mRNA species compared to the 6 kb mRNA species as revealed by Northern blot analysis. These observations indicate a differentially regulated expression of the tenascin components. We hypothesize that glia-derived tenascin modifies the functional properties of nerve cell surfaces and that tenascin is involved in such different morphogenetic events as neurite growth and oligodendrocyte distribution.
Demonstration of local protein synthesis within dendrites using a new cell culture system that permits the isolation of living axons and dendrites from their cell bodies
Authors: Torre ER , Steward O
Published: April 2018

The presence of polyribosomes within dendrites suggests a capability for local dendritic protein synthesis. However, local synthesis is difficult to evaluate because of rapid somatodendritic protein transport. The present study describes a two-surfaced culture system that allowed the separation of living axons and dendrites from their cell bodies of origin. Because this system eliminates the transport of proteins produced in the cell body, it was possible to study the extent of dendritic protein synthesis directly. Hippocampal neurons were plated on a Nucleopore polycarbonate membrane that was mounted on a thick matrix of proteins (Matrigel) fixed on a coverslip. As the neurons grew, axons and dendrites grew through the membrane into the Matrigel. To evaluate local protein synthesis within dendrites, the membrane with the cell bodies was removed, leaving a dense array of transected dendrites and axons on the coverslip with few contaminant cell bodies. Absence of cell bodies was confirmed by staining with the nuclear stain Hoechst 33258. Coverslips with isolated neurites were pulse labeled with 3H-leucine for 30 min, and fixed for autoradiography to identify sites of protein synthesis. Autoradiographic analyses revealed that isolated dendrites (immunochemically identified using antibodies against MAP2) became heavily labeled, whereas axons exhibited little if any labeling. The labeling was essentially eliminated when the neurites were pulse labeled with 3H-leucine in the presence of puromycin, whereas labeling was affected only minimally by chloramphenicol. The puromycin-sensitive incorporation of 3H-leucine in dendrites demonstrates that the polyribosomes previously described are active in protein synthesis. This system will allow a characterization of synthetic activity within isolated neurites and provide a new approach to identifying proteins that are produced within dendrites.
Effects of excitotoxin exposure on metabolic rate of primary hippocampal cultures: application of silicon microphysiometry to neurobiology
Authors: Raley-Susman KM , Miller KR , Owicki JC , Sapolsky RM
Published: April 2018

Increasing evidence implicates glutamate receptor over-stimulation in the neurotoxicity associated with a host of metabolic insults, including seizures and hypoxia-ischemia. To begin to understand more completely the role of energy metabolism in the mechanism of neuron death following excitatory amino acid exposure, we investigated the effects of kainic acid exposure on metabolic rate in cultured hippocampal cells using a recently developed silicon microphysiometer. The device gives a continual real-time measure of metabolism in relatively small numbers of cells, as assessed by efflux of protons generated at least in part by ATP hydrolysis and lactic acid production. In the first half of this report, we characterize the feasibility of using this device for measuring cellular metabolism in hippocampal cultures. Metabolic rate in both astrocytes and neurons was readily detectable, with a high signal-to-noise ratio. The rate was proportional to the number of cells and was sensitive to metabolic enhancement or depression. We then utilized this device to study metabolic responses to the excitotoxin kainic acid. We observed a receptor-mediated, dose-dependent increase in metabolic rate upon stimulation by kainic acid, with an EC50 of approximately 100 microM. Exposure to toxic levels of kainic acid for 10 min produced an initial elevation (for 2 hr) in metabolic rate and then a gradual decline in metabolism over the next 8 hr that preceded a measurable loss of cell viability. This study further delineates a time window for the onset of kainic acid-induced damage. The results clearly show the feasibility of using silicon microphysiometry for assessing metabolism of brain cultures and for exploring the relationship between metabolism and synaptic activation.
Cholinergic inhibition of short (outer) hair cells of the chick's cochlea
Authors: Fuchs PA , Murrow BW
Published: April 2018

Cochlear hair cells are thought to be inhibited by the release of ACh from efferent neurons. Several studies have implicated Ca2+ as a postsynaptic intermediary in hair cell inhibition, but its role remains unproven. We have made whole-cell, tight-seal recordings from single short hair cells (the avian analog of outer hair cells in the mammalian cochlea), isolated from the chick's cochlea, to determine the mechanism of cholinergic inhibition. These cells hyperpolarized upon exposure to ACh, although a brief depolarization preceded the much larger, longer- lasting hyperpolarization. In voltage clamp ACh evoked an outward current that reversed in sign near the K+ equilibrium potential. A small, transient inward current preceded the predominant outward current. The ACh-evoked K+ current depended on Ca2+ in the external saline, or could be prevented when the cell was dialyzed with the rapid Ca2+ buffer BAPTA. In BAPTA-loaded cells a residual inward current was seen. This activated with very little delay upon exposure of the cell to ACh and reversed near 0 mV membrane potential. Thus, the hair cell ACh receptor appears to be a nonspecific cation channel through which Ca2+ enters and triggers the opening of nearby Ca(2+)-activated K+ channels. However, the ACh-evoked K+ channels are not the same as the “maxi” K+ channels activated by Ca2+ influx through voltage-gated Ca2+ channels in these same cells.
Demonstration of coexisting catecholamine (dopamine), amino acid (GABA), and peptide (NPY) involved in inhibition of melanotrope cell activity in Xenopus laevis: a quantitative ultrastructural, freeze- substitution immunocytochemical study
Authors: de Rijk EP , van Strien FJ , Roubos EW
Published: April 2018

This quantitative ultrastructural immunocytochemical study demonstrates the coexistence of a catecholamine [dopamine (DA)], an amino acid (GABA), and a neuropeptide [neuropeptide Y (NPY)] in axon varicosities innervating the pars intermedia of Xenopus laevis. The varicosities are assumed to control the pars intermedia melanotrope cells, which regulate skin color during the physiological process of background adaptation. Varicosity profiles appear to abut melanotrope cells and folliculostellate cells, star-shaped cells that intimately contact the melanotropes. All varicosity profiles contain two morphological types of vesicle. Monolabeling studies on routinely fixed and freeze- substituted tissues showed that the small, electron-lucent vesicles store GABA, whereas DA and NPY occur in larger, electron-dense ones. Double and triple labeling experiments, in which the degree of immunoreactivity was quantified per varicosity profile and per vesicle, led to the conclusion that (1) DA, GABA, and NPY coexist within almost all varicosity profiles and (2) DA and NPY are costored within electron- dense vesicles. Varicosity profiles that about melanotrope cells show a much higher ratio between the numbers of electron-lucent and electron- dense vesicles than varicosities contacting folliculostellate cells (15.8 and 3.3, respectively). This differential distribution is in line with the previous demonstration that, in contrast to GABA, NPY does not act directly on the melanotrope cells but indirectly, by controlling the activity of the folliculostellate cells.
Aberrant projection induced by otocyst removal maintains normal tonotopic organization in the chick cochlear nucleus
Authors: Lippe WR , Fuhrmann DS , Yang W , Rubel EW
Published: April 2018

Nucleus magnocellularis (NM), a second-order nucleus in the chick auditory system, is topographically and tonotopically organized. The basilar papilla (cochlea) projects onto the ipsilateral NM via the auditory nerve. The anteromedial region of NM is innervated by the proximal end of the basilar papilla and responds to high-frequency sounds; more posterolateral regions receive input from more distal locations along the papilla and respond to progressively lower frequencies. NM projects exclusively to the third-order neurons of nucleus laminaris (NL). Otocyst removal prevents the formation of the ipsilateral cochlea and cochlear nerve and results in the development of an aberrant functional projection from the contralateral NM to the “deafferented” NM on the operated side of the brain (Jackson and Parks, 1988). In the present experiment, the otocyst was removed unilaterally and the tonotopic organization of the deafferented NM was physiologically mapped at 17–18 d of embryonic age (E17-E18). Quantitative analyses revealed that the frequency organization of the deafferented NM is almost identical to that in normal embryos. Progressively higher characteristic frequencies were recorded at successively more anterior and more medial locations in the nucleus, and the orientation of the tonotopic axis was indistinguishable from normal. Furthermore, the correlation between characteristic frequency and anatomical location is comparable in the deafferented (r = 0.91) and normal (r = 0.87) NM. The only noticeable discrepancy is that characteristic frequencies in NM on both sides of the brain of operated embryos are higher than the frequencies observed previously at comparable regions of the nucleus in unoperated controls.(ABSTRACT TRUNCATED AT 250 WORDS)
Cell-specific alternative RNA splicing of an FMRFamide gene transcript in the brain
Authors: Saunders SE , Kellett E , Bright K , Benjamin PR , Burke JF
Published: April 2018

Individual neurons synthesize different peptide neurotransmitters and neuromodulators. In general, specificity is achieved by transcriptional regulation of neuropeptide-encoding genes. In Lymnaea, the FMRFamide and GDP/SDPFLRFamide neuropeptides are encoded by separate exons. Here we provide evidence that the two exons are part of the same gene and that in neurons expressing the gene the two exons are spliced onto a common upstream exon encoding a hydrophobic leader sequence. In addition, in situ hybridization data show that there is mutually exclusive cytoplasmic expression of each of the neuropeptide-encoding exons. Thus, differential neuropeptide synthesis is likely to be regulated by an alternative splicing mechanism. The cellular specificity of these splicing events is remarkable and suggests that cell-specific alternative splicing may be of major importance in establishing neuronal diversity in this system.
Postmitotic expression of ankyrinR and beta R-spectrin in discrete neuronal populations of the rat brain
Authors: Lambert S , Bennett V
Published: April 2018

Isoforms of ankyrin (ankyrinR) are expressed in both the erythrocyte and the brain. Four cDNAs representing regulatory domains of ankyrinR expressed in the rat spleen and brain were cloned and sequenced. These different cDNAs were found to result from tissue-specific alternative mRNA processing of the ankyrinR regulatory domain. One of the isolated cDNAs was used to develop an antibody to brain isoforms of ankyrinR, and this antibody was used to study the localization of ankyrinR in the rat brain. The protein was found to be widely expressed in neurons of the metencephalon but limited to a discrete subset of neurons in the rat forebrain. In the thalamus and areas of the basal ganglia, these neurons were grouped in defined nuclei, whereas in the cortex, hippocampus, and caudate putamen they appeared as isolated cells distributed randomly throughout these structures. A similar study using an antibody raised against erythrocyte spectrin (beta R) showed a comparable localization to that of ankyrinR. Both proteins were expressed late in the developing rat brain, as part of the maturation stage of neural development. These data suggest a specific role for these erythrocyte structural proteins in the postmitotic development of a subset of neurons in the rat brain.
A role for postsynaptic neurons in determining presynaptic release properties in the cricket CNS: evidence for retrograde control of facilitation
Authors: Davis GW , Murphey RK
Published: April 2018

Intracellular recording sin the cricket cercal system show that the synaptic terminals of a single sensory neuron can facilitate at one target, the medial giant interneuron (MGI), and simultaneously depress at another target, interneuron 10–3. A quantal analysis of transmission at these synapses demonstrates that facilitation and depression are properties of the presynaptic cell. For facilitating synapses contacting MGI, the mean quantal content (m), determined from the probability of the failures, increases for the second EPSP, while the quantal size (q) remains constant. Similarly, an analysis of depression for those synapses contacting 10–3 supports a presynaptic mechanism for depression. Since facilitation and depression are presynaptic and their expression at the synapses of a single, identified sensory neuron are correlated with the target interneuron, we conclude that these properties are regulated locally, at the synapse, possibly by an interaction with the postsynaptic cell.
Acetylcholine synthesis and release following continuous intracerebral administration of NGF in adult and aged Fischer-344 rats
Authors: Rylett RJ , Goddard S , Schmidt BM , Williams LR
Published: April 2018

NGF promotes the survival and enhances the neurotransmitter phenotype of basal forebrain and striatal cholinergic neurons in brain of rat. The objective of the present study was to determine whether stimulations of the cholinergic neuronal markers ChAT and high-affinity choline uptake are reflected in enhanced synthesis and release of ACh. Enhancement of ACh release in brain of adult and aged rats could result in increased cholinergic neurotransmission, and altered animal behavior. NGF (1.2 micrograms/d) was administered intracerebroventricularly for 2 weeks by osmotic minipump to male Fischer-344 rats aged 4 and 24 months. Cholinergic neuronal functional parameters were measured in frontal cortex, hippocampus, and striatum. In young adult rats, increased ChAT and choline uptake activities were accompanied by enhanced ACh synthesis, basal and depolarization-induced release of both endogenous and newly synthesized transmitter, with the largest effect generally being observed in striatum. In aged animals, the responses to NGF were less uniform. Whereas the pattern for changes in ChAT activity was similar to that seen in younger animals, choline uptake activity was increased only in frontal cortex and striatum. Coincidentally, ACh synthesis was also increased only in these two brain regions. ACh content of synaptosomes was not affected by age or NGF treatment, and the ACh levels in microwaved samples of striatum and basal forebrain were not affected by NGF treatment. However, profound deficits in both basal and evoked release of newly synthesized ACh were observed in the aged rats. NGF treatment had no significant effect on the basal release of newly synthesized ACh in aged rats.(ABSTRACT TRUNCATED AT 250 WORDS)
Unexpected presence of neurofilaments in axon-bearing horizontal cells of the mammalian retina
Authors: Peichl L , Gonzalez-Soriano J
Published: April 2018

In several mammals only one of the two types of retinal horizontal cell, the axonless A-type, appears to express neurofilaments. Neurofilament immunostaining of rodent retinas reveals a horizontal cell plexus that has previously been interpreted as belonging to A-type cells. Our intracellular Lucifer yellow injections strongly suggest that there are no A-type horizontal cells in rat and gerbil. Counterstaining of dye-injected cellular structures with a neurofilament antibody directly shows that the axon terminal systems of the axon-bearing B-type horizontal cells contain neurofilaments. These unexpected findings explain and reinterpret the neurofilament plexus in rodent retinas. In contrast, Lucifer yellow injections in guinea pig retina reveal both A- and B-type horizontal cells, showing that horizontal cell types are not uniform among rodents. In the guinea pig retina both A-type cells and B-type axon terminal systems contain neurofilaments.