bluuebunny/crossref_metadata_2025 huggingface dataset:
An 83 kDa O-GlcNAc-glycoprotein is found in the axoplasm and nucleus of Aplysia neurons
Authors: Elliot SP , Schmied R , Gabel CA , Ambron RT
Published: April 2018

Glycoproteins containing O-linked N-acetylglucosamine (O-GlcNAc) are present in axons of Aplysia neurons (Gabel et al., 1989) and among transcription factors and other proteins in the nucleus of eukaryotic cells (Jackson and Tjian, 1988). A recently discovered pathway in neurons transports proteins through the axon and then into the nucleus (Ambron et al., 1992). If any of the axonal O-GlcNAc glycoproteins use this pathway, then the axon and the nucleus will have these glycoproteins in common. We addressed this issue by using galactosyltransferase and UDP-3H-galactose to label and identify the glycoproteins in three regions of Aplysia neurons: axoplasm, extruded from nerves; nuclei, isolated by manual dissection of single neurons; and cytoplasm, obtained after removal of nuclei. At least 21 glycoproteins were labeled by this procedure; several, at 200, 180, 83, 76, and 66 kDa, from the nucleus and axoplasm comigrated after SDS- PAGE. Radiolabeled galactosyl-N-acetylglucosaminitol was released from the glycoproteins by base/borohydride, thereby verifying the presence of O-GlcNAc. Comparison of the 83 kDa glycoprotein from the nucleus and axoplasm revealed that both were soluble, had multiple O-GlcNAcs, and were bound to WGA. Thus, the 83 kDa constituent is a good candidate to use the axonal transport/nuclear import pathway.
Motor innervation of dorsoventrally reversed wings in chick/quail chimeric embryos
Authors: Ferns MJ , Hollyday M
Published: April 2018

In the limb plexus, motor axons destined for limb muscles diverge along separate pathways to innervate muscles derived from either the dorsal or ventral premuscle masses. We have examined the axonal guidance cues involved in this initial, specific pathway choice at the plexus by making dorsoventral (D/V) limb bud reversals prior to innervation. Chick/quail chimeras were used to determine the proximodistal level of the reversal in tissue sections. The specificity of the projections to dorsal or ventral nerve trunks was assessed by retrograde HRP labeling at ages prior to motoneuron death. Axons corrected for the reversal when the level of the graft was proximal to the plexus, and when the reversed limb and its gross nerve pattern were normal. If all of these conditions were not satisfied, aberrant innervation patterns were observed. Axonal trajectories were analyzed within the host tissue, at the host-graft border, and within rotated tissue to determine where along the pathway guidance cues might be located. Special attention was given to cases in which axons compensated for the reversal to project in accord with the positions of their soma in the lateral motor column. In these correcting cases, after normal D/V sorting in the spinal nerves of the host, motor axons altered their trajectories upon entering rotated graft tissue as they approached and traversed the plexus. Because corrections were within rotated tissue and not proximal to it, the D/V pathway cues are unlikely to be long-range target- derived signals, but rather appear to be closely associated with positional information in the plexus region and also more proximally in the tissue surrounding the distal spinal nerves.
Associative long-term potentiation in piriform cortex slices requires GABAA blockade
Authors: Kanter ED , Haberly LB
Published: April 2018

Previous studies have demonstrated that NMDA-dependent, long-term potentiation (LTP) can be induced in both afferent and intrinsic association fiber systems in the piriform (primary olfactory) cortex. In this report we demonstrate that an associative form of LTP can be induced by coactivation of these two systems, which terminate on adjacent apical dendritic segments of pyramidal cells. Potentiating stimulus trains were delivered to either afferent or association fibers, and weak shocks, which were nonpotentiating when delivered alone, were delivered to the other pathway. Under control recording conditions where homosynaptic (single pathway) LTP is consistently evoked, coincident application of these stimuli failed to induce LTP of the weak shock response. However, after local blockade of the fast, GABAA-mediated IPSP, associative LTP was consistently produced in both directions. Induction was blocked by D-2-amino-5-phosphonovaleric acid, indicating that it is dependent on activation of NMDA receptors. It is speculated that afferent and association fibers are segregated on different dendritic segments of pyramidal cells in piriform cortex to allow regulation of associative LTP by way of centrifugal inputs that modulate the activity of GABAergic interneurons.
Muscle agrin: neural regulation and localization at nerve-induced acetylcholine receptor clusters
Authors: Lieth E , Fallon JR
Published: April 2018

The synapse-organizing protein agrin is expressed by muscle cells. In this study we begin to characterize the role of muscle agrin in synapse formation by investigating its distribution and expression during the formation of nerve-induced ACh receptor (AChR) aggregates in vitro. We have used species-specific anti-agrin antibodies to show that muscle agrin colocalizes with nerve-induced AChR clusters in chimeric nerve- muscle cocultures. Furthermore, quantitation by radioimmune assay shows that the expression of muscle agrin by cultured chick myotubes is increased more than twofold by coculture with cells from the motoneuron- rich ventral spinal cord. These data suggest a role for muscle agrin in neuromuscular junction development.
Cellular distribution of dopamine D1 and D2 receptors in rat medial prefrontal cortex
Authors: Vincent SL , Khan Y , Benes FM
Published: April 2018

The relative distribution and cellular localization of the dopamine D1 and D2 receptor subtypes were assessed in frozen sections of rat medial prefrontal cortex (mPFC). The D1 and D2 receptor binding sites were labeled with the selective high-affinity antagonists SCH 23390 and N-(p- aminophenethyl)-spiperone (NAPS), respectively, coupled to either Bodipy or Texas red fluorophores. Under the incubation conditions employed, kinetic, competition, and selectivity studies showed that these modified ligands retained pharmacological selectivity. Optimal binding fluorescence was at 100 nM of each ligand, and fluorescence increased linearly from 1 to 15 min of incubation at 2 degrees C. NAPS- Texas red binding fluorescence was inhibited with 10 nM quinpirole (D2 agonist), but not 10 nM SKF 38393 (D1 agonist), while SCH 23390-Texas red binding was inhibited with SKF 38393, but not quinpirole. The localization of dopamine receptor binding was assessed in montages constructed from low-magnification photomicrographs through the depth of the cortex, or in corresponding high-magnification photomicrographs. Cells showing D1- or D2-like receptor binding fluorescence were present in layers II-VI, with the highest density observed in layers V and VI. The addition of mianserin (100 nM, 5-HT2 antagonist) to incubated sections slightly reduced the numbers of labeled cells in each cortical layer, but retained the preferential localization to the deeper layers. Two separate observations supported the idea that the fluorescently coupled ligands were localized to neuronal cell bodies. First, receptor labeling with the fluorescently coupled ligands co-localized almost exclusively to cells in the cortical mantle showing neuron-specific enolase immunoreactivity. Second, a comparison of the cell size distribution taken from adjacent Nissl-stained sections with the size of cells showing D1- or D2-like receptor binding fluorescence revealed complete overlapping of fluorescence with neuronal cell bodies. In mPFC layer VI, the size of cells showing D1-like receptor binding fluorescence was 77.8 +/- 5.1 microns2, similar to non-pyramidal neurons, while that for D2-like receptor binding fluorescence was 108.2 +/- 4.5 microns2, consistent with both large interneurons and small pyramidal cells. Only a small percentage of cells showing D1- or D2- like receptor binding overlapped in size with glia, but this occurred almost exclusively within the white matter region below the cortical mantle. These findings are consistent with the hypothesis that the D1 and D2 receptor subtypes are found on different populations of neurons, although some overlap probably occurs.(ABSTRACT TRUNCATED AT 400 WORDS)
In vivo development of voltage-dependent ionic currents in embryonic Xenopus spinal neurons
Authors: Desarmenien MG , Clendening B , Spitzer NC
Published: April 2018

Initial evidence that electrical excitability is both an early aspect of neuronal differentiation and a developmentally regulated property was obtained from recordings of action potentials in vivo. Subsequently, the analysis of the underlying voltage-dependent currents during early stages of embryogenesis was facilitated by investigation of dissociated neurons and muscle cells differentiating in culture. Calcium and potassium currents play a major role in the differentiation of the action potential of Xenopus spinal neurons, and calcium influx triggers specific features of neuronal differentiation. However, the extent to which differentiation of currents in vitro parallels that in vivo is uncertain. We have undertaken a study of in vivo differentiation of these macroscopic currents in Xenopus embryos. Spinal cords were isolated from embryos at several early stages of neurogenesis. Neurons in these isolated spinal cords were accessible to patch-clamp electrodes. Neuronal currents were recorded within 1 hr to assure that the characteristics of the currents resulted from developmental events occurring in vivo prior to the experiment. Whole- cell voltage-clamp recordings from neurons in these acutely isolated and intact embryonic spinal cords demonstrate that both the delayed- rectifier and inactivating potassium current and a low-voltage- activated calcium current mature in a manner closely parallel to that observed in culture. The results validate those from the culture system and indicate that the spinal cord is another region of the CNS accessible to cellular analysis in an intact preparation.
Extrinsic factors influence the expression of voltage-gated K currents on neonatal rat sympathetic neurons
Authors: McFarlane S , Cooper E
Published: April 2018

Voltage-gated potassium (K) currents are important in controlling a neuron's excitability. We have shown previously (McFarlane and Cooper, 1992) that neonatal superior cervical ganglia (SCG) neurons express three voltage-gated K currents: a noninactivating delayed-rectifier type current (IK), a rapidly inactivating A-current (IAf), and a slowly inactivating A-current (IAs). When grown in culture for 4 weeks without other cell types, SCG neurons lose their expression of IAf and IAs, suggesting that an extrinsic factor(s) is involved in controlling the expression of these currents. In vivo, SCG neurons are surrounded by non-neuronal cells. Therefore, in this study we investigated whether the ganglionic non-neuronal cells provide a factor required for A- current expression. We show that postnatal day 1 (P1) SCG neurons continue to express IAf and IAs when cocultured with their ganglionic non-neuronal cells. Medium conditioned by ganglionic non-neuronal cells mimics the non-neuronal cell influence on IAf and IAs expression, suggesting that the effects of non-neuronal cells are mediated by way of a secreted factor. Ciliary neurotrophic factor, a factor present in peripheral non-neuronal cells, had similar effects to those of ganglionic cell-conditioned medium. Moreover, we find that the dependence of IAf on a non-neuronal cell factor is developmentally regulated; P14 neurons grown in culture without other cell types continue to express IAf. However, IAs on P14 neurons maintains its dependence on a factor from non-neuronal cells. Finally, in addition to extrinsic control of voltage-gated K currents, we suggest that SCG neurons use intrinsic mechanisms to coordinate their expression of IAf, IAs, and IK such that changes in one K current are compensated for by reciprocal changes in one or more of the other K currents.
Interleukin-1 induces substance P in sympathetic ganglia through the induction of leukemia inhibitory factor (LIF)
Authors: Shadiack AM , Hart RP , Carlson CD , Jonakait GM
Published: April 2018

It has become increasingly clear that immune cytokines perform growth and differentiation functions in the nervous system similar to those performed in the immune system. In previous studies we have shown that interleukin-1 beta (IL-1 beta) raises substance P (SP) and the mRNA coding for its preprotachykinin precursor in cultured sympathetic superior cervical ganglia (SCG) (Jonakait and Schotland, 1990; Hart et al., 1991a). The action of IL-1 is blocked both by depolarization of the ganglia and by glucocorticoid hormones (Hart et al., 1991a). In the present report, we have found that IL-1 does not act directly upon neurons to raise SP, but rather induces the production of a soluble intermediate molecule that raises both SP and the cholinergic-specific enzyme ChAT. Its induction by IL-1 is blocked by the synthetic glucocorticoid hormone dexamethasone; its action is compromised under depolarizing conditions. Because medium conditioned by IL-1 (IL-1CM) is functionally similar to leukemia inhibitory factor (LIF), we sought to determine whether this molecule might be an active constituent of IL- 1CM. Immunoprecipitation with an antiserum directed against LIF eliminated large proportions of SP-inducing activity from IL-1CM. In addition, steady-state levels of mRNA coding for LIF are increased by IL-1 treatment of SCG. These data suggest that LIF, induced by IL-1, may ultimately be responsible for the IL-1 induction of SP.
Specific high-affinity receptors for neurotrophin-3 on sympathetic neurons
Authors: Dechant G , Rodriguez-Tebar A , Kolbeck R , Barde YA
Published: April 2018

When used at concentrations allowing interactions only with its high- affinity receptors, neurotrophin-3 (NT-3) promotes the survival of sensory neurons isolated from embryonic day 8 (E8) chicks, but not the survival of E11 sympathetic neurons. These sympathetic neurons (which can be rescued by the addition of NGF) display high-affinity receptors for NT-3 (Kd of 1.6 x 10(-11) M) that cannot be distinguished from the high-affinity NT-3 receptors on sensory neurons using equilibrium binding or kinetic criteria. This represents the first example of embryonic neurons that cannot be rescued by the in vitro addition of a neurotrophin in spite of the presence of corresponding neurotrophin high-affinity receptors. At elevated concentrations, beyond the saturation of its high-affinity receptors, NT-3 supports the survival of some E11 sympathetic neurons, an effect that might be mediated by the high-affinity NGF receptor. Using E7 sympathetic neurons, about 40% of the cells initially plated can be rescued in vitro by the addition of low concentrations of NT-3 (but not of NGF) and produce profuse neurites. This indicates that NT-3 may play a role in the early development of some sympathetic neurons.
Role of calcium in astrocyte volume regulation and in the release of ions and amino acids
Authors: O'Connor ER , Kimelberg HK
Published: April 2018

Primary astrocyte cultures exposed to hypotonic media undergo a rapid initial swelling followed by a regulatory volume decrease (RVD), which is associated with the release of ions and amino acids. The Ca2+ dependence of RVD was investigated. Using a method that measures extracellular electrical resistance to measure cell volume changes in substratum-attached cells, we found that when astrocytes were exposed to hypotonic media without calcium, RVD was abolished. The addition of CaCl2 to astrocytes swollen in hypotonic media without calcium caused an almost immediate initiation of volume regulation, with an EC50 of approximately 0.1 mM CaCl2. Swelling of astrocytes in hypotonic medium caused an increased influx of 45Ca2+, which was partially blocked (60%) by 1 microM nimodipine, suggesting that voltage-gated L-type calcium channels were being opened. Previous work had shown that hypotonic media-induced swelling of astrocytes caused membrane potential depolarizations sufficient to open such channels (Kimelberg and O'Connor, 1988). By measuring intracellular free calcium with fura-2, we found that astrocytes swollen in hypotonic medium showed a rapid increase in [Ca2+]i, reaching a peak of approximately 600 nM, followed by a decrease to a sustained plateau (approximately 250 nM) mirroring the time course of volume regulation. The removal of extracellular calcium totally abolished, and the addition of 1 microM nimodipine partially abolished the elevated plateau, while neither affected the initial [Ca2+]i peak. These data suggest that the initial peak of the hypotonic-induced rise in [Ca2+]i is caused by release from intracellular stores and that the sustained elevated plateau is due to extracellular calcium influx. The removal of extracellular calcium also abolished swelling-induced K+(86Rb) and 36Cl- efflux, but did not affect the swelling-induced release of 3H-D-aspartate, or 3H-taurine (data not shown). These data indicate that hypotonic-induced aspartate and taurine release is not necessary for RVD in astrocytes swollen by exposure to hypotonic media, since RVD is completely inhibited by the omission of external Ca2+. The addition of 1 mM quinine HCl, which is known to block Ca(2+)-activated K+ channels, also abolished both volume regulation and 86Rb+ efflux in hypotonic media-swollen astrocytes in the presence of medium calcium, but did not affect 3H-D-aspartate efflux. We suggest that the swelling of astrocytes in hypotonic media which leads to a rapid membrane depolarization first opens voltage- gated calcium channels.(ABSTRACT TRUNCATED AT 400 WORDS)
Coexpression of multiple acetylcholine receptor genes in neurons: quantification of transcripts during development
Authors: Corriveau RA , Berg DK
Published: April 2018

A large family of genes encoding subunits of nicotinic ACh receptors (AChRs) has been identified in vertebrates and shown to be expressed in the nervous system. The multiplicity of genes raises questions about which gene products coassemble to produce native receptor subtypes and how the expression of receptor genes is regulated in neurons. We report here that five neuronal AChR genes are expressed in the chick ciliary ganglion at both early and late times in development. Quantitative RNase protection experiments demonstrated that at embryonic day 18 (E18) the ganglion contains about 1800 copies of alpha 7 transcript per neuron, 900 copies of alpha 3 transcript per neuron, and 200–300 copies each of alpha 5, beta 2, and beta 4 transcripts per neuron. The same five genes are expressed at significantly lower levels at E8 but show the same rank order of abundance in transcripts per neuron. Few, if any, transcripts were found for the alpha 2, alpha 4, alpha 8, and beta 3 AChR genes in ciliary ganglion RNA at either E8 or E18. The 6- and 13- fold increases previously reported for two classes of AChRs on the neurons between E8 and E18 approximate the 4–14-fold increases observed here in AChR gene mRNA levels per neuron over the same time period. The alpha 3, alpha 5, alpha 7, and beta 4 genes have previously been correlated with subunits of ciliary ganglion AChRs, but the beta 2 gene has not. The abundance of beta 2 transcripts raises the possibility either that the known AChRs in the ganglion have a more complex subunit composition than previously described or that additional receptor subtypes remain to be discovered. Northern blot analysis revealed no changes in transcript pattern for the alpha 3, alpha 5, and beta 4 genes between E8 and E18; a small change may occur in the transcript pattern for the alpha 7 gene. In situ hybridizations demonstrated that alpha 5 and beta 4 transcripts are expressed in essentially all ciliary ganglion neurons as has been shown previously for the more abundant alpha 3 transcript and inferred for the alpha 7 transcript. The results indicate that neurons can stably coexpress multiple AChR genes, including three of the alpha type, and that transcript levels may be rate limiting for accumulation of AChRs during development.
Cocaine-induced c-fos messenger RNA is inversely related to dynorphin expression in striatum
Authors: Steiner H , Gerfen CR
Published: April 2018

The effects of the indirect dopamine receptor agonist cocaine in the striatum on levels of mRNAs of the immediate-early gene c-fos and the neuropeptides dynorphin, substance P, and enkephalin were analyzed with quantitative in situ hybridization histochemistry. Both single (acute) and repeated (twice a day for 4 d) systemic injections of cocaine (3.75– 30 mg/kg) to rats resulted in dose-dependent, regionally specific elevations of mRNA expression in striatal neurons. A single drug treatment elevated c-fos mRNA expression, whereas repeated treatments resulted in little c-fos expression but elevated dynorphin mRNA levels. Both the regional and temporal patterns of gene expression revealed an inverse relationship between dynorphin and c-fos expression. This relationship was examined in a time course experiment in which cocaine (30 mg/kg) was administered for 1, 2, 3 or 4 d. Basal levels of dynorphin expression were relatively high in the ventral striatum, including the nucleus accumbens, a ventrolateral region, and an area along the medial bank of the striatum. A single injection of cocaine induced c-fos mRNA in striatal areas with low basal expression of dynorphin. Thus, c-fos mRNA induction was highest in the dorsal central striatum, where basal dynorphin mRNA levels were lowest. In this region, dynorphin mRNA expression increased on subsequent treatment days parallel to diminished c-fos mRNA induction. Changes in substance P. mRNA levels appeared to match directly both the temporal and regional patterns of c-fos induction. Enkephalin mRNA expression was altered, but only slightly, by these cocaine treatments. Statistical analysis of the regional patterns of basal and altered mRNA levels shows a unique inverse relationship between basal dynorphin expression and c-fos induction by cocaine. Further evidence of this relationship is provided by the dose-dependent blockade of cocaine-induced c-fos expression by spiradoline, a dynorphin agonist. Together, these results suggest that the restricted regional pattern of cocaine-induced c-fos expression is related, in part, to the basal level of dynorphin expression, and that cocaine treatment elevates dynorphin expression in striatal regions with a strong c-fos response, thereby limiting subsequent c-fos induction by cocaine. These findings lead to the hypothesis that dynorphin acts to regulate the responsiveness of striatal neurons to dopamine stimulation.
Corticospinal terminations in two new-world primates: further evidence that corticomotoneuronal connections provide part of the neural substrate for manual dexterity
Authors: Bortoff GA , Strick PL
Published: April 2018

Anterograde transport of 2–10% WGA-HRP was used to examine the pattern of termination of efferents from the primary motor cortex to cervical segments of the spinal cord in cebus (Cebus apella) and squirrel (Saimiri sciureus) monkeys. We have compared the pattern of termination in these monkeys because of marked differences in their manipulative abilities. Both primates have pseudo-opposable thumbs; however, only cebus monkeys use independent finger movements to pick up small objects. We found that corticospinal terminations in cervical segments of the cebus monkey are located in three main zones: a dorsolateral region of the intermediate zone, a dorsomedial region of the intermediate zone, and the ventral horn. The projection to the ventral horn in these monkeys is particularly dense at C8-T1 segments, where terminations form a “ring” that encircles the lateral motoneuronal cell group. In contrast, there are only two main zones of terminations in the squirrel monkey: a dorsolateral region of the intermediate zone and a dorsomedial region of the intermediate zone. As others have noted, efferents from the primary motor cortex of squirrel monkeys have, at best, only sparse terminations within the ventral horn. Thus, there are marked differences between cebus and squirrel monkeys in the extent of corticospinal terminations within the ventral horn. These observations provide further support for the concept that monosynaptic projections from the primary motor cortex to motoneurons in the ventral horn provide part of the neural substrate for dexterous movements of the fingers.
cAMP-activated Na+ current of molluscan neurons is resistant to kinase inhibitors and is gated by cAMP in the isolated patch
Authors: Sudlow LC , Huang RC , Green DJ , Gillette R
Published: April 2018

The cAMP-dependent Na+ current (INa,cAMP) modulates excitability in many molluscan neurons. Rapid activation of INa,cAMP by cyclic nucleotide, its ion dependence, and its blockade by divalent cations resemble cyclic nucleotide-activated cation currents in vertebrate photoreceptors and olfactory receptors, where activation has been found to be independent of kinase activity. We tested the phosphorylation dependence of INa,cAMP in neurons of the feeding and locomotory networks of the predatory marine snail Pleurobranchaea. Identified neurons of pedal and buccal ganglia were axotomized for recording the INa,cAMP response to iontophoretic injection of cAMP under two- electrode voltage clamp. Intracellular injections of specific peptide inhibitor of protein kinase A had no blocking effects on activation of INa,cAMP by iontophoretic injection of cAMP. Inward single-channel currents were activated in excised inside-out patches during exposure to cAMP in salines without added ATP. Sodium was the major current carrying ion. Two distinct types of INa,cAMP channel activity were observed, where opening probability and open times differed, but conductance was similar, 36.7 pS. These observations suggest that INa,cAMP activation occurs by direct binding of cAMP to a regulatory site at the channel, rather than by phosphorylation.
Step-tracking movements of the wrist. III. Influence of changes in load on patterns of muscle activity
Authors: Hoffman DS , Strick PL
Published: April 2018

Human subjects performed step-tracking movements of the wrist in the radial direction. Movement amplitude, external load, and accuracy instructions were varied. We used surface electrodes to record muscle activity from an agonist, extensor carpi radialis longus, and an antagonist, extensor carpi ulnaris. When subjects performed movements “as fast as possible” that were opposed by different external loads, we observed two distinct patterns of modulation of the agonist burst. In one pattern, termed pulse-height modulation, the force of the agonist muscle was graded by varying the peak amplitude of a short-duration agonist burst. This pattern occurred when subjects performed movements of different amplitudes with a lightweight manipulandum. In the other pattern, termed pulse-width modulation, the force of the agonist muscle was graded by varying the duration of an agonist burst of nearly maximal amplitude. When the agonist burst was prolonged, the onset of antagonist activity was delayed. This pattern occurred when subjects performed movements of different amplitudes that were opposed by elastic or viscoelastic loads applied to a heavy manipulandum. The strongest subject exhibited more pulse-height modulation and less pulse- width modulation of the agonist burst than other subjects. Conversely, the weakest subject displayed more pulse-width modulation of the agonist burst than other subjects. These observations indicate that the force requirements of a task, relative to the force generating capacity of a subject's agonist muscle(s), have a significant influence on the pattern of agonist modulation. In a second experiment using three nonhuman primates, we observed that agonist bursts in wrist flexor and extensor muscles exhibited strikingly different patterns of modulation. For wrist flexion, agonist bursts in wrist flexors were brief and displayed pulse-height modulation when movement amplitude was varied. For wrist extension, agonist bursts in wrist extensors were prolonged and displayed largely pulse-width modulation when movement amplitude was varied. We suggest that the distinct patterns of modulation observed in the wrist muscles of monkeys were due to differences in the strength of wrist flexors and extensors, rather than to alterations in movement strategy. In a third experiment, we instructed human subjects to be “accurate” when they made step-tracking movements. When subjects performed movements with a lightweight manipulandum, most displayed short-duration agonist bursts that were pulse-height modulated. When subjects performed “accurate” movements that were opposed by elastic loads, they displayed pulse-width modulation of a small-amplitude agonist burst.(ABSTRACT TRUNCATED AT 400 WORDS)
Long-term potentiation and long-term depression of primary afferent neurotransmission in the rat spinal cord
Authors: Randic M , Jiang MC , Cerne R
Published: April 2018

Synaptic transmission between dorsal root afferents and neurons in the superficial laminae of the spinal dorsal horn (laminae I-III) was examined by intracellular recording in a transverse slice preparation of rat spinal cord. Brief high-frequency electrical stimulation (300 pulses at 100 Hz) of primary afferent fibers produced a long-term potentiation (LTP) or a long-term depression (LTD) of fast (monosynaptic and polysynaptic) EPSPs in a high proportion of dorsal horn neurons. Both the AMPA and the NMDA receptor-mediated components of synaptic transmission at the primary afferent synapses with neurons in the dorsal horn can exhibit LTP and LTD of the synaptic responses. In normal and neonatally capsaicin-treated rats, the induction of LTP requires the activation of NMDA receptor-gated conductances. The induction of LTP or LTD, however, was not abolished in the presence of bicuculline, a GABAA receptor antagonist. The results demonstrate that distinct and long-lasting modulation in synaptic efficiency can be induced at primary afferent synapses with neurons in the superficial laminae of spinal dorsal horn by high-frequency stimulation of dorsal root afferents and that these changes may be physiologically relevant for transmission and integration of sensory information, including pain.
Marked increase in beta-tubulin mRNA expression during regeneration of axotomized retinal ganglion cells in adult mammals
Authors: McKerracher L , Essagian C , Aguayo AJ
Published: April 2018

Changes in gene expression were investigated in axotomized CNS neurons under conditions that inhibit or permit regrowth of their damaged axons. Levels of mRNA encoding beta-tubulin, the 150 kDa neurofilament subunit (NF-M), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were examined by quantitative in situ hybridization of adult rat retinal ganglion cells (RGCs) after axotomy in the optic nerve or during regeneration in a peripheral nerve (PN) graft. Soon after optic nerve section beta-tubulin, NF-M, and GAPDH mRNA levels decreased and remained low during the 1 month studied. In these retinas beta-tubulin mRNA fell to approximately 50% of normal controls. However, in the PN- grafted retinas, where approximately 20% of the surviving axotomized RGCs regenerate their axons, there were “hot spots” of beta-tubulin mRNAs where neuronal levels were nearly 300% higher than in controls. By retrograde neuronal labeling these hot spots were shown to correspond to the injured RGCs that regrew their axons into the PN graft; beta-tubulin mRNA levels in nonregenerating RGCs of the same retinas averaged 63% of controls. We suggest that interactions of RBC axons and components of the grafts' non-neuronal environment play a key role in the over fourfold differences in beta-tubulin mRNA levels observed between injured and regenerating RGCs.
The mosaic of midget ganglion cells in the human retina
Authors: Dacey DM
Published: April 2018

To study their detailed morphology, ganglion cells of the human retina were stained by intracellular tracer injection, in an in vitro, whole- mount preparation. This report focuses on the dendritic morphology and mosaic organization of the major, presumed color-opponent, ganglion cell class, the midget cells. Midget cells in the central retina were recognized by their extremely small dendritic trees, approximately 5–10 microns in diameter. Between 2 and 6 mm eccentricity, midget cells showed a steep, 10-fold increase in dendritic field size, followed by a more shallow, three- to fourfold increase in the retinal periphery, attaining a maximum diameter of approximately 225 microns. Despite large local variation in dendritic field size, midget cells formed one morphologically distinctive class at all retinal eccentricities. Two midget cell types were distinguished by their dendritic stratification in either the inner or outer portion of the inner plexiform layer (IPL), and presumably correspond to ON- and OFF-center cells respectively. The mosaic organization of the midget cells was examined by intracellularly filling neighboring cells in small patches of retina. For both the inner and outer midget populations, adjacent dendritic trees apposed one another but did not overlap, establishing a coverage of no greater than 1. The two mosaics differed in spatial scale, however: the outer midget cells showed smaller dendritic fields and higher cell density than the inner midget cells. An outer:inner cell density ratio of 1.7:1 was found in the retinal periphery. An estimate of total midget cell density suggested that the proportion of midget cells increases from about 45% of total ganglion cell density in the retinal periphery to about 95% in the central retina. Nyquist frequencies calculated from midget cell spacing closely match a recent measure of human achromatic spatial acuity (Anderson et al., 1991), from approximately 6 degrees to 55 degrees eccentricity. Outside the central retina, midget cell dendrites arborized in clusters within the overall dendritic field. With increasing eccentricity, the dendritic clusters increased in number and remained small (approximately 10–20 microns diameter) relative to the size of the dendritic field. Because neighboring midget cell dendritic trees do not overlap, the mosaic as a whole showed a pattern of clusters and holes. We hypothesize that midget cell dendritic trees may contact individual axon terminals of some midget bipolar cells and avoid contacting others, providing a basis for the formation of cone-specific connections in the IPL.
Differential expression of the 61 kDa and 63 kDa calmodulin-dependent phosphodiesterases in the mouse brain
Authors: Yan C , Bentley JK , Sonnenburg WK , Beavo JA
Published: April 2018

Based on their relative abundance and regulation by Ca2+ and by phosphorylation in vitro, it is thought that the Ca2+/calmodulin- dependent phosphodiesterases (CaM-PDEs) are important modulators of cyclic nucleotide function in the brain. Two of the most abundant CaM- PDEs in the brain are the 61 kDa and 63 kDa isozymes. In this study, the regional and cellular expression of mRNA encoding these two different isoforms in mouse brain has been determined by in situ hybridization. The 63 kDa CaM-PDE mRNA has a wide-spread but uneven distribution. Very strong hybridization signals are present in the caudate-putamen, nucleus accumbens, olfactory tubercle, and dentate gyrus of the hippocampus. Somewhat lesser amounts of 63 kDa CaM-PDE mRNA are present in the olfactory bulb and piriform cortex. Weaker but still easily discernible hybridization signals are seen in several layers of the cerebral cortex, CA1 and CA3 regions of the hippocampus, amygdaloid nuclear complex, thalamus, hypothalamus, midbrain, brainstem, cerebellum, and spinal cord. A weak hybridization signal was detected in the globus pallidus of the basal ganglia. In general, the distribution of the 63 kDa CaM-PDE is very similar to that of dopamine receptors, suggesting that it may modulate dopamine function. In contrast, the 61 kDa CaM-PDE mRNA has a more limited and much different distribution, with the highest level of expression in the cerebral cortex and in the pyramidal cells of the hippocampus. A moderate hybridization signal was detected in the medial habenula and amygdaloid nuclear complex. In addition, small subsets of neurons in several other regions showed specific hybridization. Both PDE mRNAs appear to be localized exclusively in neuronal cell bodies. Their distinct distribution suggests important but different physiological roles for these two isozymes in the regional regulation of cyclic nucleotides in the CNS. Since these two isozymes are differentially phosphorylated by cAMP-dependent and Ca2+/CaM-dependent protein kinases, the differential expression also provides a potential mechanism by which these PDEs can differentially regulate cAMP and cGMP in different brain areas. The high expression levels in specific subsets of neurons also suggest that agents increasing Ca2+ in these neurons will increase the rate of cyclic nucleotide degradation.
Growth cone "collapse" in vivo: are inhibitory interactions mediated by gap junctions?
Authors: Wolszon LR , Gao WQ , Passani MB , Macagno ER
Published: April 2018

In the leech embryo, oppositely directed axons of homologous anterior pagoda (AP) neurons overlap with each other extensively within the nerves that link adjacent ganglia, and inhibit each other's further growth (Gao and Macagno, 1987b). During this 5–8 d period of inhibition, the axons begin to grow thin, and eventually they retract completely. However, deletion of one overlapping AP cell results in the renewed growth of the remaining AP cell's axon, which then innervates territory vacated by the killed cell. Thus, each neuron can detect the presence of the other, and adjust its branching pattern accordingly. To begin to explore how these neurons detect and inhibit each other, we tested for direct communication between them. Dye fills with fluorescent chromophores suggested direct contact between their axons at the light level, and this was confirmed by serial-section electron microscopic analysis. Morphological features resembling aspects of gap junctions were observed where the projections were closely apposed, and subsequent electrophysiological recordings demonstrated electrical coupling between the mutually inhibited axons. Confirmation that these projections communicate via gap junctions was obtained using intracellular injection of 5-HT as a tracer, followed by anti-5-HT immunohistochemistry. The tracer passed selectively between AP neurons. We propose that the gap junctions formed between the transient projections of the developing AP neurons may mediate the exchange of the signals that permit homologs to recognize each other and to inhibit the further forward progress of these projections.
Mossy fiber growth and synaptogenesis in rat hippocampal slices in vitro
Authors: Dailey ME , Buchanan J , Bergles DE , Smith SJ
Published: April 2018

Hippocampal slices from early postnatal rat were used to study mossy fiber (MF) growth and synaptogenesis. The ability of MFs to form new giant synapses within isolated tissue slices was established by a series of experiments involving synapsin I immunohistochemistry, electron microscopy, and whole-cell recordings. When hippocampal slices from immature rats were cultured for up to 2 weeks, the distribution of giant MF terminals was similar to that found in vivo. Using a lesioning procedure, we determined that MFs in slices extend and form appropriate synaptic connections with normal target CA3 pyramidal cells. MF terminals were dispersed more widely than normal within the CA3 pyramidal layer after a lesion, but electron microscopy indicated that synaptic junctions were still primarily associated with pyramidal cell dendrites and not the somata. Establishment of functional synaptic input in vitro was confirmed by whole-cell recordings of MF-driven excitatory postsynaptic currents (50 pA to 1 nA) in pyramidal cells. The results establish for the first time that an MF projection with appropriate and functional synaptic connections can be formed de novo and not just maintained in excised hippocampal slices. The cellular dynamics underlying MF growth and synaptogenesis were examined directly by time-lapse confocal imaging of fibers selectively stained with a fluorescent membrane dye (Dil or DiO). MFs growing deep within isolated tissue slices were tipped by small (5–10 microns), active growth cones that advanced at variable rates (5–25 microns/hr). Furthermore, dynamic filopodial structures were seen at small varicosities along the length of developing MFs, which may identify nascent en passant synaptic contacts. The hippocampal slice preparations are shown to support normal development of MF connections and allow for direct visualization of the cellular dynamics of synapse formation in a mammalian CNS tissue environment.
Phosphorylation of AMPA-type glutamate receptors by calcium/calmodulin- dependent protein kinase II and protein kinase C in cultured hippocampal neurons
Authors: Tan SE , Wenthold RJ , Soderling TR
Published: April 2018

Phosphorylation of glutamate receptors (GluRs) is emerging as an important regulatory mechanism. In this study 32P labeling of non-NMDA GluRs was investigated in cultured hippocampal neurons stimulated 2–15 min with agonists that selectively stimulate either Ca2+/calmodulin- dependent protein kinase II (CaM-kinase II), Ca2+/phospholipid- dependent protein kinase C (PKC), or cAMP-dependent protein kinase A (PKA). Treatment of hippocampal neurons with glutamate/glycine (Glu/Gly), ionomycin, or 12-O-tetradecanoylphorbol 13-acetate (TPA) increased 32P labeling of immunoprecipitated alpha-amino-3-hydroxy-5- methyl-4-isoxazoleproprionate (AMPA)-type GluRs by 145%, 180%, and 227%, respectively, of control values. This increased phosphorylation of GluRs was predominantly 32P-Ser with little 32P-Thr and no detectable 32P-Tyr. Glu/Gly and ionomycin, but not TPA, also increased 32P labeling of CaM-kinase II by 175% and 195%, respectively, of control values. Of these three agonists, only TPA stimulated phosphorylation of MARCKS (225% of control), a specific substrate of PKC. Forskolin treatment gave a three- to fourfold increase in the active catalytic subunit of PKA but did not result in the 32P labeling of AMPA-type GluRs, CaM-kinase II, or MARCKS. Phosphorylation of GluRs in response to Glu/Gly was blocked by a specific NMDA receptor/ion channel antagonist (DL-2-amino-5-phosphonovaleric acid) or by a cell- permeable inhibitor of CaM-kinase II (1-[N,O-bis(1,5- isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4- phenylpiperazine, KN-62). These results are consistent with the hypothesis that Ca2+ influx through the NMDA-type ion channel can activate CaM-kinase II, which in turn can phosphorylate and regulate AMPA-type GluR ion channels (McGlade-McCulloh et al., 1993).(ABSTRACT TRUNCATED AT 250 WORDS)
Localization and alternative splicing of agrin mRNA in adult rat brain: transcripts encoding isoforms that aggregate acetylcholine receptors are not restricted to cholinergic regions
Authors: O'Connor LT , Lauterborn JC , Gall CM , Smith MA
Published: April 2018

Agrin is a protein implicated in the formation and maintenance of the neuromuscular junction. In addition to motor neurons, agrin mRNA has been detected in the brains of embryonic rat and chick and adult marine ray, suggesting that this molecule may also be involved in the formation of synapses between neurons. As a step toward understanding agrin's role in the CNS, we utilized Northern blot and in situ hybridization techniques to analyze the regional distribution and cellular localization of agrin mRNA in the spinal cord and brain of adult rats. The results of these studies indicate that the agrin mRNA is expressed predominantly by neurons broadly distributed throughout the adult CNS. Moreover, expression of agrin mRNA is not restricted to cholinergic structures or regions of the brain receiving cholinergic input. Recently, RNA isolated from rat embryonic spinal cord was shown to contain four alternatively spliced agrin mRNAs, referred to as agrin0, agrin8, agrin11, and agrin19, each of which encodes agrin proteins that are active in acetylcholine receptor aggregating assays (Ferns et al., 1992). Using the polymerase chain reaction we demonstrate that all four of these agrin transcripts are expressed within the adult CNS. Agrin0, agrin8, and agrin19 were present in all regions analyzed. In contrast, agrin11 was detected only in forebrain. Results of these studies indicate that both the level of expression and pattern of alternative splicing of agrin mRNA are differentially regulated in the brain. The broad and predominantly neuronal distribution of agrin mRNA in the adult brain suggests that, in addition to its role at the neuromuscular junction, agrin may play a role in formation and maintenance of synapses between neurons in the CNS.
Enhancement of type B and A photoreceptor inhibitory synaptic connections in conditioned Hermissenda
Authors: Frysztak RJ , Crow T
Published: April 2018

Intrinsic changes have been identified in isolated and intact type A and type B photoreceptors following classical conditioning of the nudibranch mollusk Hermissenda. Aspects of various intrinsic, nonsynaptic modifications are expressed by alterations in the excitability of identified photoreceptors in response to the conditioned stimulus. In addition to changes in cellular excitability, changes in synaptic strength between identified neurons have been proposed as a possible mechanism of associative learning in several invertebrate preparations. Here we report that classical conditioning produces differential effects upon the strength of inhibitory monosynaptic connections between identified pairs of type B and type A photoreceptors. The amplitude of IPSPs elicited by an action potential in the medial type B photoreceptor and recorded from medical type A photoreceptors was significantly enhanced in conditioned animals as compared to pseudorandom controls. In contrast, the amplitude of IPSPs elicited by an action potential in the lateral type B photoreceptor and recorded from lateral type A photoreceptors did not show significant synaptic enhancement following conditioning. These results provide additional evidence for differential effects of conditioning upon cellular modifications in identified type A and type B photoreceptors, and further indicate that multiple sites of cellular plasticity exist in the visual system of conditioned Hermissenda.