bluuebunny/crossref_metadata_2025 huggingface dataset:
Upregulation of the Enzyme Chain Hydrolyzing Extracellular ATP after Transient Forebrain Ischemia in the Rat
Authors: Braun Norbert , Zhu Yuan , Krieglstein Josef , Culmsee Carsten , Zimmermann Herbert
Published: April 2018

A short ischemic period induced by the transient occlusion of major brain arteries induces neuronal damage in selectively vulnerable regions of the hippocampus. Adenosine is considered to be one of the major neuroprotective substances produced in the ischemic brain. It can be released from damaged cells, but it also could be generated extracellularly from released ATP via a surface-located enzyme chain. Using the rat model of global forebrain ischemia, we applied a short (10 min) transient interruption of blood flow and studied the distribution of ectonucleotidase activities in the hippocampus. Northern hybridization of mRNA isolated from hippocampi of sham-operated and ischemic animals revealed an upregulation of ectoapyrase (capable of hydrolyzing nucleoside 5′-tri- and diphosphates) and ecto-5′-nucleotidase (capable of hydrolyzing nucleoside 5′-monophosphates). A histochemical analysis that used ATP, UTP, ADP, or AMP as substrates revealed a strong and selective increase in enzyme activity in the injured areas of the hippocampus. Enhanced staining could be observed first at 2 d. Staining increased within the next days and persisted at 28 d after ischemia. The spatiotemporal development of catalytic activities was identical for all substrates. It was most pronounced in the CA1 subfield and also could be detected in the dentate hilus and to a marginal extent in CA3. The histochemical staining corresponded closely to the development of markers for reactive glia, in particular of microglia. The upregulation of ectonucleotidase activities implies increased nucleotide release from the damaged tissue and could play a role in the postischemic control of nucleotide-mediated cellular responses.
Immunohistochemical and Cytochemical Localization of the Somatostatin Receptor Subtype sst1in the Somatostatinergic Parvocellular Neuronal System of the Rat Hypothalamus
Authors: Helboe Lone , Stidsen Carsten E. , Møller Morten
Published: April 2018

Somatostatin is known to mediate its actions through five G-protein-coupled receptors (sst1–sst5). We have studied the expression of the sst1receptor in the rat hypothalamus by using a subtype-specific antiserum. In Western blotting, the antiserum reacted specifically with a band with an apparent molecular weight of 80,000 in membranes prepared from hypothalamic tissue.

The localization of the sst1receptor was investigated by immunohistochemistry in hypothalamus sections. Additionally, an immunofluorescent double-labeling was performed for the sst1receptor and somatostatin. Light microscopy revealed that the sst1receptor is located in perikarya and nerve fibers in the rostral periventricular area surrounding the third ventricle as well as in nerve fibers projecting from the perikarya to the external layer of the median eminence. In these neuronal structures, sst1immunoreactivity was found to be colocalized with somatostatin. Furthermore, the location of sst1receptors was studied by immunoelectron microscopy in the median eminence. In the external layer, receptor immunoreactivity was confined to nerve terminals. Immunoreactive nerve terminals were seen to make synapse-like junctions with other both stained and unstained nerve terminals. Thus, the sst1receptor is present in the classic somatostatinergic hypothalamic parvocellular system inhibiting hormone secretion from the anterior pituitary gland. These findings indicate that the sst1receptor may act as an autoreceptor and inhibit the release of somatostatin from periventricular neurons projecting to the median eminence.
cAMP-Mediated Regulation of Neurotrophin-Induced Collapse of Nerve Growth Cones
Authors: Wang Qun , Zheng James Q.
Published: April 2018

Neurotrophins are known to promote the survival, differentiation, and neurite outgrowth of developing neurons. Here we report that acutely applied brain-derived neurotrophic factor (BDNF) induces rapid growth cone collapse and neurite retraction of embryonicXenopusspinal neurons in culture. The collapsing effect of BDNF depends on the activation of Trk receptor tyrosine kinase, requires an influx of extracellular Ca2+, and is regulated by cAMP-dependent activity. Elevation of intracellular cAMP levels ([cAMP]i) by forskolin or (Sp)-cAMP completely blocked the collapsing effect, whereas inhibition of protein kinase A (PKA) by (Rp)-cAMP potentiated the collapsing action. BDNF-induced growth cone collapse was only observed in 6 hr cultures but not in 24 hr cultures. However, inhibition of PKA by (Rp)-cAMP restored the collapsing response of these “old” neurons in 24 hr cultures, suggesting that embryonicXenopusspinal neurons may upregulate their endogenous cAMP-dependent activity during development in culture, leading to the blockade of their collapsing response to BDNF. Taken together, our results suggest the presence of cross-talk between Ca2+- and cAMP-signaling pathways involved in the collapsing action of neurotrophins, in which the cAMP-pathway regulates the Ca2+-mediated signal transduction required for BDNF-induced collapse. By modulating the cAMP-dependent activity through the intrinsic programming or interaction with other factors present in the environment, a neuron thus could respond to the same extracellular factors with different morphological and cellular changes at different stages during development.
GABAAReceptor Pharmacology and Subtype mRNA Expression in Human Neuronal NT2-N Cells
Authors: Neelands Torben R. , Greenfield L. John , Zhang Jie , Turner R. Scott , Macdonald Robert L.
Published: April 2018

Human NT2 teratocarcinoma cells differentiate into neuron-like NT2-N cells when treated with retinoic acid. GABA evoked concentration-dependent whole-cell currents in NT2-N cells with an EC50of 21.8 μmand a Hill slope of 1.2. GABAAreceptor (GABAR) currents reversed atECl−and did not display voltage-dependent rectification. GABAR single channels opened in bursts to a 23 pS main conductance level and a 19 pS subconductance level, with infrequent openings to a 27 pS conductance level. Kinetic properties of the main conductance level were similar to other native and recombinant GABAR channels. Diazepam and zolpidem enhanced GABAR currents with moderate affinity, whereas methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate inhibited GABAR currents. Loreclezole enhanced GABAR currents with high affinity, but furosemide antagonized GABAR currents with low affinity. The neurosteroids alphaxalone and pregnenolone sulfate appropriately modulated GABAR currents. Zinc blocked GABAR currents with low affinity, but lanthanum did not significantly alter NT2-N GABAR currents. Reverse transcription PCR (RT-PCR) performed on RNA from NT2-N cells clearly detected transcripts encoding human α2, α3, α5, β3, γ3, and π subtypes. The combined pharmacological and RT-PCR results are most consistent with a single or predominant GABAR isoform composed of an α2 and/or α3 subtype combined with the β3 and γ3 subtypes. The data do not rule out receptors containing combinations of α2 and/or α3 subtypes with the α5 subtype or receptors with both β1 and β3 subtypes. The presence or absence or the π subunit in functionally expressed receptors could not be determined.
Frequency Regulation of a Slow Rhythm by a Fast Periodic Input
Authors: Nadim Farzan , Manor Yair , Nusbaum Michael P. , Marder Eve
Published: April 2018

Many nervous systems contain rhythmically active subnetworks that interact despite oscillating at widely different frequencies. The stomatogastric nervous system of the crabCancer borealisproduces a rapid pyloric rhythm and a considerably slower gastric mill rhythm. We construct and analyze a conductance-based compartmental model to explore the activation of the gastric mill rhythm by the modulatory commissural neuron 1 (MCN1). This model demonstrates that the period of the MCN1-activated gastric mill rhythm, which was thought to be determined entirely by the interaction of neurons in the gastric mill network, can be strongly influenced by inhibitory synaptic input from the pacemaker neuron of the fast pyloric rhythm, the anterior burster (AB) neuron. Surprisingly, the change of the gastric mill period produced by the pyloric input to the gastric mill system can be many times larger than the period of the pyloric rhythm itself. This model illustrates several mechanisms by which a fast oscillatory neuron may control the frequency of a much slower oscillatory network. These findings suggest that it is possible to modify the slow rhythm either by direct modulation or indirectly by modulating the faster rhythm.
Striatal Extracellular Dopamine Levels in Rats with Haloperidol-Induced Depolarization Block of Substantia Nigra Dopamine Neurons
Authors: Moore Holly , Todd Christopher L. , Grace Anthony A.
Published: April 2018

Correlations between substantia nigra (SN) dopamine (DA) cell activity and striatal extracellular DA were examined using simultaneous extracellular single-unit recordings andin vivomicrodialysis performed in drug-naive rats and in rats treated repeatedly with haloperidol (HAL). Intact rats treated with HAL for 21–28 d exhibited significantly fewer active DA cells, indicating the presence of depolarization block (DB) in these cells. However, in rats that received surgical implantation of the microdialysis probe followed by a 24 hr recovery period, HAL-induced DA cell DB was reversed, as evidenced by a number of active DA neurons that was significantly higher than that in HAL-treated intact rats and similar to that of drug-naive rats. In contrast, using a modified probe implantation procedure that did not reverse SN DA neuron DB, we found striatal DA efflux to be significantly lower than in controls and significantly correlated with the reduction in DA neuron spike activity. Furthermore, although basal striatal DA efflux was independent of SN DA cell burst-firing activity in control rats, these variables were significantly correlated in rats with HAL-induced DA cell DB. Therefore, HAL-induced DB of SN DA neurons is disrupted by implantation of a microdialysis probe into the striatum using standard procedures. However, a modified microdialysis method that allowed reinstatement of DA neuron DB revealed that the HAL-induced inactivation of SN DA neurons was associated with significantly lower extracellular DA levels in the striatum. Moreover, the residual extracellular DA maintained in the presence of DB may, in part, depend on the burst-firing pattern of the noninactivated DA neurons in the SN.
Electrophysiology of the Hippocampal and Amygdaloid Projections to the Nucleus Accumbens of the Rat: Convergence, Segregation, and Interaction of Inputs
Authors: Mulder Antonius B. , Hodenpijl Martijn Gijsberti , Lopes da Silva Fernando H.
Published: April 2018

The nucleus accumbens (Nacb) receives inputs from hippocampus and amygdala but it is still unclear how these inputs are functionally organized and may interact. The interplay between these input pathways was examined using electrophysiological tools in the rat,in vivo, under halothane anesthesia. After fornix/fimbria stimulation (Fo/Fi, subicular projection fibers to the Nacb), mono- and polysynaptically driven single units were recorded in the medial shell/core regions of the Nacb and in the ventromedial caudate putamen. Monosynaptically driven neurons by basolateral amygdala (BLA) stimulation were found in the medial shell/core and in the ventrolateral shell/core regions. In the areas of convergence (medial shell/core), paired activation of BLA followed by that of Fo/Fi resulted in an enhancement of the Fo/Fi response, whereas stimulation in the reverse order, Fo/Fi followed by BLA, led to a depression of the BLA response. In addition to these patterns of interactions, the tetanization of the Fo/Fi to Nacb pathway caused a homosynaptic decremental (long-term) potentiation in the Nacb, accompanied by a heterosynaptic (long-term) depression of the nontetanized BLA to Nacb pathway. We postulate that the hippocampal inputs may close a “gate” for the amygdala inputs, whereas the gate is opened for the hippocampus inputs by previous amygdalar activity. These opposite effects on the Nacb neuronal populations should be taken into account when interpreting behavioral phenomena, particularly with respect to the contrasting effects of the amygdala and the hippocampus in locomotion and place learning.
Neuroprotective Actions of Dipyridamole on Cultured CNS Neurons
Authors: Farinelli Stephen E. , Greene Lloyd A. , Friedman Wilma J.
Published: April 2018

We report that dipyridamole is neuroprotective for a variety of rat embryonic CNS neurons cultured in serum-free basal medium lacking trophic factors or other additives. We also describe the mechanism underlying this action. Neurons died rapidly in basal medium but were rescued in large measure by 10 μmdipyridamole. The protective action of dipyridamole seems to be attributable to its antioxidant property. Vitamin E andN-acetylcysteine provided comparable neuroprotection in basal medium, whereas an array of compounds that mimic other actions of dipyridamole (inhibition of phosphodiesterases, blockade of nucleoside and chloride transport, interference with the multidrug resistance protein, and enhancement of prostacyclin synthesis) failed to promote survival. Thus, a major cause of neuronal death in this system seems to be oxidative stress that is relieved by dipyridamole. Iron plays a significant role in generation of such stress, as indicated by the observations that addition of apotransferrin or iron chelators to basal medium or use of iron-free medium also afforded protection. Although oxidative stress was a major determinant of neuronal death, it was not the only factor. Dipyridamole or other antioxidant measures did not provide sustained neuroprotection. However, provision of insulin, which was not protective alone in basal medium, along with dipyridamole significantly enhanced long-term neuronal survival. Hence, optimal protection requires both trophic support and relief from oxidative stress. These findings lend credence to the potential use of dipyridamole or its derivatives in prevention and/or treatment of CNS injuries and degenerative disorders in which oxidative stress is a significant component.
Membrane Current Induced by Protein Kinase C Activators in Rhabdomeric Photoreceptors: Implications for Visual Excitation
Authors: Gomez Maria del Pilar , Nasi Enrico
Published: April 2018

Visual excitation in rhabdomeric photoreceptors is thought to be mediated by activation of a light-regulated phospholipase C (PLC) and the consequent hydrolysis of phosphatidylinositol bisphosphate. Whereas much attention has been devoted to inositol trisphosphate (IP3) production and intracellular Ca2+release, little is known about the possible role of the DAG branch in the generation of the light response. We have tested the effect of chemically distinct surrogates of DAG on isolatedLimaphotoreceptors. Application of the phorbol ester PMA (0.5–10 μm) or the alkaloid (−)-indolactam (20–100 μm) from a holding potential of −50 mV elicited an inward current, several hundred picoamperes in amplitude, accompanied by a pronounced increase in membrane conductance. The stereoisomers 4α-PMA and (+)-indolactam were both inactive, arguing for the specificity of the effects. Elevation of cytosolic Ca2+by intracellular dialysis accelerated this current, whereas chelerythrine antagonized it, suggesting the involvement of PKC. The reversal potential of the membrane current induced by PKC activators was approximately +10 mV; replacement of extracellular Na with impermeantN-methyl-d-glucamine decreased its amplitude and shifted the reversal potential in the negative direction. Stimulation by PMA and (−)-indolactam was accompanied by a pronounced depression of light responsiveness; conversely, steady illumination reduced the size of the current elicited by these PKC activators. Taken together, these results support the notion that the DAG branch of the PLC cascade, in addition to its suggested participation in visual adaptation, may play a role in the activation of the photoresponse or a component thereof, probably in synergy with IP3-mediated Ca2+release.
Withdrawal from 3α-OH-5α-Pregnan-20-One Using a Pseudopregnancy Model Alters the Kinetics of Hippocampal GABAA-Gated Current and Increases the GABAAReceptor α4 Subunit in Association with Increased Anxiety
Authors: Smith Sheryl S. , Gong Qi H. , Li Xinshe , Moran Maria H. , Bitran Daniel , Frye Cheryl A. , Hsu Fu-Chun
Published: April 2018

In the present study, we have characterized properties of steroid withdrawal using a pseudopregnant rat model. This paradigm results in increased production of endogenous progesterone from ovarian sources and as such is a useful physiological model. “Withdrawal” from progesterone induced by ovariectomy on day 12 of pseudopregnancy resulted in increased anxiety, as determined by a decrease in open arm entries on the elevated plus maze compared to control rats and pseudopregnant animals not undergoing withdrawal. Similar findings were obtained 24 hr after administration of a 5α-reductase blocker to a pseudopregnant animal, suggesting that it is the GABAA-modulatory 3α-OH-5α-pregnan-20-one (3α,5α-THP) that produces anxiogenic withdrawal symptoms. Twenty-four hours after steroid withdrawal, the time constant for decay of GABAA-gated current was also reduced sixfold, assessed using whole- cell patch-clamp procedures on pyramidal neurons acutely dissociated from CA1 hippocampus. Thus, 3α,5α-THP withdrawal results in a marked decrease in total GABAAcurrent, a possible mechanism for its anxiogenic, proconvulsant sequelae. In addition, 3α,5α-THP withdrawal resulted in insensitivity to the normally potentiating effect of the benzodiazepine lorazepam (LZM) on GABAA-gated Cl−current. This withdrawal profile is similar to that reported for other GABAA-modulatory drugs such as the benzodiazepines (BDZs), barbiturates, and ethanol. These changes were also associated with significant two and threefold increases in both the mRNA and protein for the α4 subunit of the GABAAreceptor, respectively, in hippocampus. The pseudopregnancy paradigm may be a useful model for periods of endogenous 3α,5α-THP withdrawal such as premenstrual syndrome and postpartum or postmenopausal dysphoria, when increased emotional lability and BDZ insensitivity have been reported.
The Neural Cell Adhesion Molecule L1 Interacts with the AP-2 Adaptor and Is Endocytosed via the Clathrin-Mediated Pathway
Authors: Kamiguchi Hiroyuki , Long Kristin E. , Pendergast Maryanne , Schaefer Andrew W. , Rapoport Iris , Kirchhausen Tomas , Lemmon Vance
Published: April 2018

Cell–cell interactions mediated via cell adhesion molecules (CAMs) are dynamically regulated during nervous system development. One mechanism to control the amount of cell surface CAMs is to regulate their recycling from the plasma membrane. The L1 subfamily of CAMs has a highly conserved cytoplasmic domain that contains a tyrosine, followed by the alternatively spliced RSLE (Arg-Ser-Leu-Glu) sequence. The resulting sequence ofYRSLconforms to a tyrosine-based sorting signal that mediates clathrin-dependent endocytosis of signal-bearing proteins. The present study shows that L1 associates in rat brain with AP-2, a clathrin adaptor that captures plasma membrane proteins with tyrosine-based signals for endocytosis by coated pits.In vitroassays demonstrate that this interaction occurs via the YRSL sequence of L1 and the μ2 chain of AP-2. In L1-transfected 3T3 cells, L1 endocytosis is blocked by dominant-negative dynamin that specifically disrupts clathrin-mediated internalization. Furthermore, endocytosed L1 colocalizes with the transferrin receptor (TfR), a marker for clathrin-mediated internalization. Mutant forms of L1 that lack the YRSL do not colocalize with TfR, indicating that the YRSL mediates endocytosis of L1. In neurons, L1 is endocytosed preferentially at the rear of axonal growth cones, colocalizing with Eps15, another marker for the clathrin endocytic pathway. These results establish a mechanism by which L1 can be internalized from the cell surface and suggest that an active region of L1 endocytosis at the rear of growth cones is important in L1-dependent axon growth.
Neurotrophin-3 and Brain-Derived Neurotrophic Factor Induce Oligodendrocyte Proliferation and Myelination of Regenerating Axons in the Contused Adult Rat Spinal Cord
Authors: McTigue Dana M. , Horner Philip J. , Stokes Bradford T. , Gage Fred H.
Published: April 2018

Functional loss after spinal cord injury (SCI) is caused, in part, by demyelination of axons surviving the trauma. Neurotrophins have been shown to induce oligodendrogliagenesisin vitro, but stimulation of oligodendrocyte proliferation and myelination by these factorsin vivohas not been examined. We sought to determine whether neurotrophins can induce the formation of new oligodendrocytes and myelination of regenerating axons after SCI in adult rats. In this study, fibroblasts producing neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor, nerve growth factor, basic fibroblast growth factor, or β-galactosidase (control grafts) were transplanted subacutely into the contused adult rat spinal cord. At 10 weeks after injury, all transplants contained axons. NT-3 and BDNF grafts, however, contained significantly more axons than control or other growth factor-producing grafts. In addition, significantly more myelin basic protein-positive profiles were detected in NT-3 and BDNF transplants, suggesting enhanced myelination of ingrowing axons within these neurotrophin-producing grafts. To determine whether augmented myelinogenesis was associated with increased proliferation of oligodendrocyte lineage cells, bromodeoxyuridine (BrdU) was used to label dividing cells. NT-3 and BDNF grafts contained significantly more BrdU-positive oligodendrocytes than controls. The association of these new oligodendrocytes with ingrowing myelinated axons suggests that NT-3- and BDNF-induced myelinogenesis resulted, at least in part, from expansion of oligodendrocyte lineage cells, most likely the endogenous oligodendrocyte progenitors. These findings may have significant implications for chronic demyelinating diseases or CNS injuries.
Role of the Nucleus Raphe Magnus in Antinociception Produced by ABT-594: Immediate Early Gene Responses Possibly Linked to Neuronal Nicotinic Acetylcholine Receptors on Serotonergic Neurons
Authors: Bitner R. Scott , Nikkel Arthur L. , Curzon Peter , Arneric Stephen P. , Bannon Anthony W. , Decker Michael W.
Published: April 2018

Recently, a novel cholinergic channel modulator, (R)-5-(2-azetidinylmethoxy)-2-chloropyridine (ABT-594), was shown to produce potent analgesia in a variety of rodent pain models when administered either systemically or centrally into the nucleus raphe magnus (NRM). The purpose of the present study was to investigate the possible supraspinal contribution of ABT-594 by assessing its ability to induce expression of the immediate early gene c-fos, a biochemical marker of neuronal activation, in the NRM of rats. Putative serotonergic neurons in the NRM, a medullary nucleus proposed to be involved in descending antinociceptive pathways, were identified immunohistochemically using a monoclonal antibody (mAb) against tryptophan hydroxylase. ABT-594 (0.03–0.3 μmol/kg, i.p.) produced a dose-dependent induction of Fos protein that was blocked by the central nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine (5 μmol/kg, i.p.) but not by the peripheral nAChR antagonist hexamethonium (15 μmol/kg, i.p.). Immunohistological studies using mAb 299 revealed the expression of α4-containing nAChRs in the NRM. The α4 immunostaining was dramatically reduced by pretreating (30 d) animals with the serotonin neurotoxin 5,7-dihydroxytryptamine (5,7-DHT), which was previously shown to substantially attenuate the antinociceptive actions of ABT-594. In a double immunohistochemical labeling experiment, coexpression of the serotonin marker tryptophan hxdroxylase and the α4 nAChR subunit in NRM neurons was observed. These results suggest that the analgesic mechanism of ABT-594 may in part involve the activation of the NRM, a site where α4-containing nAChRs are expressed by serotonergic neurons.
Expression and Characterization of the Neuropeptide Y Y5Receptor Subtype in the Rat Brain
Authors: Dumont Yvan , Fournier Alain , Quirion Rémi
Published: April 2018

The neuropeptide Y Y5receptor subtype has generated great interest, especially regarding its possible involvement in feeding behaviors. However, its distribution and sites of expression in the mammalian brain are, in large part, unknown because of the lack of selective tools. We demonstrate in this study that specific [125I][Leu31,Pro34]PYY binding is competed in a biphasic manner by BIBP3226, a Y1receptor antagonist, demonstrating the existence of sensitive and insensitive sites to BIBP3226. Assays performed by using [125I][Leu31,Pro34]PYY in the presence of 1 μmBIBP3226 to block the Y1receptor subtype revealed a pharmacological profile highly similar to the cloned Y5receptor. Moreover, results obtained with GW1229 suggest that the Y4subtype represents only a very small proportion of the total population of NPY receptors in the rat brain. Quantitative receptor autoradiographic data revealed the discrete distribution of [125I][Leu31,Pro34]PYY/BIBP3226-insensitive Y5sites in the rat brain, with the external plexiform layer of the olfactory bulb, the lateral septum, the anteroventral thalamic nucleus, the CA3 subfield of the ventral hippocampus, the nucleus tractus solitarius, and the area postrema being most enriched. Rather surprisingly, in the hypothalamus, a key structure modulating food intake, only low densities of Y5binding sites were detected as well as in most other regions of the rat brain. These data suggest that the Y5receptor protein is expressed and translated by a small percentage of hypothalamic neurons and that the effect of NPY on feeding behaviors likely is mediated by more than one class of NPY receptors. It also indicates that the Y5receptor may be involved in other biological actions induced by NPY. Taken together, these data represent the first pharmacological demonstration of the expression and discrete localization of the Y5receptor protein in the rat brain.
Retinal Ganglion Cell Axon Progression from the Optic Chiasm to Initiate Optic Tract Development Requires Cell Autonomous Function of GAP-43
Authors: Kruger Kelly , Tam Angie S. , Lu Cynthia , Sretavan David W.
Published: April 2018

Pathfinding mechanisms underlying retinal ganglion cell (RGC) axon growth from the optic chiasm into the optic tract are unknown. Previous work has shown that mouse embryos deficient in GAP-43 have an enlarged optic chiasm within which RGC axons were reportedly stalled. Here we have found that the enlarged chiasm of GAP-43 null mouse embryos appears subsequent to a failure of the earliest RGC axons to progress laterally through the chiasm–tract transition zone to form the optic tract. Previous work has shown that ventral diencephalon CD44/stage-specific embryonic antigen (SSEA) neurons provide guidance information for RGC axons during chiasm formation. Here we found that in the chiasm–tract transition zone, axons of CD44/SSEA neurons precede RGC axons into the lateral diencephalic wall and like RGC axons also express GAP-43. However unlike RGC axons, CD44/SSEA axon trajectories are unaffected in GAP-43 null embryos, indicating that GAP-43–dependent guidance at this site is RGC axon specific or occurs only at specific developmental times. To determine whether the phenotype results from loss of GAP-43 in RGCs or in diencephalon components such as CD44/SSEA axons, wild-type, heterozygous, or homozygous GAP-43 null donor retinal tissues were grafted onto host diencephalons of all three genotypes, and graft axon growth into the optic tract region was assessed. Results show that optic tract development requires cell autonomous GAP-43 function in RGC axons and not in cellular elements of the ventral diencephalon or transition zone.
The Role of the First Postmitotic Cortical Cells in the Development of Thalamocortical Innervation in theReelerMouse
Authors: Molnár Zoltán , Adams Richard , Goffinet AndréM. , Blakemore Colin
Published: April 2018

In the mutant mousereeler, the tangential distribution of thalamocortical fibers is essentially normal, even though neurons of the cortical plate accumulate below the entire early-born preplate population (Caviness et al., 1988). This seems incompatible with the hypothesis that cells of the subplate (the lower component of the preplate in normal mammals) form an axonal scaffold that guides thalamic fibers and act as temporary targets for them (Blakemore and Molnár, 1990, Shatz et al., 1990).

We used carbocyanine dyes to trace projections in wild-type andreelermice between embryonic day 13 and postnatal day 3. Preplate formation and early extension of corticofugal fibers to form a topographic array are indistinguishable in the two phenotypes. So too are the emergence of thalamic axons in topographic order through the primitive internal capsule, their meeting with preplate axons, and their distribution over the preplate scaffold. Distinctive differences appear after the cortical plate begins to accumulate below the preplate ofreeler, causing the preplate axons to form oblique fascicles, running through the cortical plate. Thalamic axons then pass through the plate within the same fascicles and accumulate in the “superplate” layer for ∼2–3 d, before defasciculating and plunging down to terminate deep in the cortical plate, creating the curious “looping” pattern seen in the adult. Thus, thalamocortical innervation inreelerfollows the same algorithm of development but in relation to the misplaced population of early-born neurons. Far from challenging the theory that preplate fibers guide thalamic axons,reelerprovides strong evidence for it.
The Effect of the Mouse MutationClaw Pawon Myelination and Nodal Frequency in Sciatic Nerves
Authors: Koszowski Adam G. , Owens Geoffrey C. , Levinson S. Rock
Published: April 2018

Despite the biophysical and clinical importance of differentiating nodal and internodal axolemma, very little is known about the process. We chose to study myelination and node of Ranvier formation in the hypomyelinating mouse mutantclaw paw(clp). The phenotype ofclpis delayed myelination in the peripheral nervous system. The specific defect is unknown but is thought to arise from a breakdown in the complex signaling mechanism between axon and Schwann cell. Myelination was assessed in sciatic nerve cross sections from adult and postnatal day 14 (P14) heterozygous and homozygousclpmice. Antibodies to P0, myelin-associated glycoprotein (MAG), and neural cell adhesion molecule were used to assess the stage of myelination. P14 homozygousclpmice showed an atypical staining pattern of immature myelin, which resolved into a relatively normal pattern by adulthood. Sodium channel clustering and node of Ranvier frequency were studied in whole-mount sciatic nerves with sodium channel and MAG antibodies. P14 homozygousclpnerves again showed an atypical, immature pattern with diffuse sodium channel clusters suggesting nodal formation was delayed. In the adult, homozygousclpsciatic nerves displayed dramatically shortened internodal distances. The data from this study support the hypotheses that node of Ranvier formation begins with the onset of myelination and that the number and location of nodes of Ranvier in the sciatic nerve are determined by myelinating Schwann cells.
Increased Production of Amyloid Precursor Protein Provides a Substrate for Caspase-3 in Dying Motoneurons
Authors: Barnes Natalie Y. , Li Ling , Yoshikawa Kazuaki , Schwartz Lawrence M. , Oppenheim Ronald W. , Milligan Carolanne E.
Published: April 2018

Biochemical and molecular mechanisms of neuronal cell death are currently an area of intense research. It is well documented that the lumbar spinal motoneurons of the chick embryo undergo a period of naturally occurring programmed cell death (PCD) requiring new gene expression and activation of caspases. To identify genes that exhibit changed expression levels in dying motoneurons, we used a PCR-based subtractive hybridization protocol to identify messages uniquely expressed in motoneurons deprived of trophic support as compared with their healthy counterparts. We report that one upregulated message in developing motoneurons undergoing cell death is the mRNA for amyloid precursor protein (APP). Increased levels of APP and β-amyloid protein are also detected within dying motoneurons. The predicted peptide sequence of APP indicates two potential cleavage sites for caspase-3 (CPP-32), a caspase activated in dying motoneurons. When peptide inhibitors of caspase-3 are administered to motoneurons destined to undergo PCD, decreased levels of APP protein and greatly reduced β-amyloid production are observed. Furthermore, we show that APP is cleaved by caspase-3. Our results suggest that differential gene expression results in increased levels of APP, providing a potential substrate for one of the cell death-activated caspases that may ultimately cause the demise of the cell. These results, combined with information on the toxic role of APP and its proteolytic by-product β-amyloid, in the neurodegenerative disease Alzheimer’s, suggest that events of developmental PCD may be reactivated in early stages of pathological neurodegeneration.
Heat Shock Protein 27: Developmental Regulation and Expression after Peripheral Nerve Injury
Authors: Costigan Michael , Mannion Richard J. , Kendall Giles , Lewis Susan E. , Campagna Jason A. , Coggeshall Richard E. , Meridith-Middleton Jacqueta , Tate Simon , Woolf Clifford J.
Published: April 2018

The heat shock protein (HSP) 27 is constitutively expressed at low levels in medium-sized lumbar dorsal root ganglion (DRG) cells in adult rats. Transection of the sciatic nerve results in a ninefold upregulation of HSP27 mRNA and protein in axotomized neurons in the ipsilateral DRG at 48 hr, without equivalent changes in the mRNAs encoding HSP56, HSP60, HSP70, and HSP90. Dorsal rhizotomy, injuring the central axon of the DRG neuron, does not upregulate HSP27 mRNA levels. After peripheral axotomy, HSP27 mRNA and protein are present in small, medium, and large DRG neurons, and HSP27 protein is transported anterogradely, accumulating in the dorsal horn and dorsal columns of the spinal cord, where it persists for several months. Axotomized motor neurons also upregulate HSP27. Only a minority of cultured adult DRG neurons are HSP27-immunoreactive soon after dissociation, but all express HSP27 after 24 hr in culture with prominent label throughout the neuron, including the growth cone. HSP27 differs from most axonal injury-regulated and growth-associated genes, which are typically present at high levels in early development and downregulated on innervation of their targets, in that its mRNA is first detectable in the DRG late in development and only approaches adult levels by postnatal day 21. In non-neuronal cells, HSP27 has been shown to be involved both in actin filament dynamics and in protection against necrotic and apoptotic cell death. Therefore, its upregulation after adult peripheral nerve injury may both promote survival of the injured neurons and contribute to alterations in the cytoskeleton associated with axonal growth.
Differential Distribution of α2Aand α2CAdrenergic Receptor Immunoreactivity in the Rat Spinal Cord
Authors: Stone Laura S. , Broberger Christian , Vulchanova Lucy , Wilcox George L. , Hökfelt Tomas , Riedl Maureen S. , Elde Robert
Published: April 2018

α2-Adrenergic receptors (α2-ARs) mediate a number of physiological phenomena, including spinal analgesia. We have developed subtype-selective antisera against the C termini of the α2A-AR and α2C-AR to investigate the relative distribution and cellular source or sources of these receptor subtypes in the rat spinal cord. Immunoreactivity (IR) for both receptor subtypes was observed in the superficial layers of the dorsal horn of the spinal cord. Our results suggest that the primary localization of the α2A-AR in the rat spinal cord is on the terminals of capsaicin-sensitive, substance P (SP)-containing primary afferent fibers. In contrast, the majority of α2C-AR-IR was not of primary afferent origin, not strongly colocalized with SP-IR, and not sensitive to neonatal capsaicin treatment. Spinal α2C-AR-IR does not appear to colocalize with the neurokinin-1 receptor, nor is it localized on astrocytes, as evidenced by a lack of costaining with the glial marker GFAP. However, some colocalization was observed between α2C-AR-IR and enkephalin-IR, suggesting that the α2C-AR may be expressed by a subset of spinal interneurons. Interestingly, neither subtype was detected on descending noradrenergic terminals. These results indicate that the α2-AR subtypes investigated are likely expressed by different subpopulations of neurons and may therefore subserve different physiological functions in the spinal cord, with the α2A-AR being more likely to play a role in the modulation of nociceptive information.
Emulating the Visual Receptive-Field Properties of MST Neurons with a Template Model of Heading Estimation
Authors: Perrone John A. , Stone Leland S.
Published: April 2018

We have proposed previously a computational neural-network model by which the complex patterns of retinal image motion generated during locomotion (optic flow) can be processed by specialized detectors acting as templates for specific instances of self-motion. The detectors in this template model respond to global optic flow by sampling image motion over a large portion of the visual field through networks of local motion sensors with properties similar to those of neurons found in the middle temporal (MT) area of primate extrastriate visual cortex. These detectors, arranged within cortical-like maps, were designed to extract self-translation (heading) and self-rotation, as well as the scene layout (relative distances) ahead of a moving observer. We then postulated that heading from optic flow is directly encoded by individual neurons acting as heading detectors within the medial superior temporal (MST) area. Others have questioned whether individual MST neurons can perform this function because some of their receptive-field properties seem inconsistent with this role. To resolve this issue, we systematically compared MST responses with those of detectors from two different configurations of the model under matched stimulus conditions. We found that the characteristic physiological properties of MST neurons can be explained by the template model. We conclude that MST neurons are well suited to support self-motion estimation via a direct encoding of heading and that the template model provides an explicit set of testable hypotheses that can guide future exploration of MST and adjacent areas within the superior temporal sulcus.
Convergent Input from Brainstem Coincidence Detectors onto Delay-Sensitive Neurons in the Inferior Colliculus
Authors: McAlpine David , Jiang Dan , Shackleton Trevor M. , Palmer Alan R.
Published: April 2018

Responses of low-frequency neurons in the inferior colliculus (IC) of anesthetized guinea pigs were studied with binaural beats to assess their mean best interaural phase (BP) to a range of stimulating frequencies. Phase plots (stimulating frequency vs BP) were produced, from which measures of characteristic delay (CD) and characteristic phase (CP) for each neuron were obtained. The CD provides an estimate of the difference in travel time from each ear to coincidence-detector neurons in the brainstem. The CP indicates the mechanism underpinning the coincidence detector responses. A linear phase plot indicates a single, constant delay between the coincidence-detector inputs from the two ears. In more than half (54 of 90) of the neurons, the phase plot was not linear. We hypothesized that neurons with nonlinear phase plots received convergent input from brainstem coincidence detectors with different CDs. Presentation of a second tone with a fixed, unfavorable delay suppressed the response of one input, linearizing the phase plot and revealing other inputs to be relatively simple coincidence detectors. For some neurons with highly complex phase plots, the suppressor tone altered BP values, but did not resolve the nature of the inputs. For neurons with linear phase plots, the suppressor tone either completely abolished their responses or reduced their discharge rate with no change in BP. By selectively suppressing inputs with a second tone, we are able to reveal the nature of underlying binaural inputs to IC neurons, confirming the hypothesis that the complex phase plots of many IC neurons are a result of convergence from simple brainstem coincidence detectors.
A Brainstem Network Mediating Apneic Reflexes in the Rat
Authors: Chamberlin Nancy L. , Saper Clifford B.
Published: April 2018

Apnea is an important protective response to upper airway irritation, but the central mechanisms responsible for eliciting sensory-induced apnea are not well understood. Recent studies have emphasized the Kölliker-Fuse nucleus in producing apnea and proposed a trigeminoparabrachial pathway for mediating these reflexes. However, in our earlier study of apneic responses produced by glutamate stimulation in the dorsolateral pons, we found that apnea was elicited from the area just ventral to the Kölliker-Fuse nucleus, rather than within it. Because this region was not known to be involved in respiratory control, we combined chemical microstimulation with both anterograde and retrograde axonal tracing to characterize the sites in the pons that produce apneic responses. We found that apneic sites were consistently associated with the intertrigeminal region, between the principal sensory and motor trigeminal nuclei. Injections of anterograde tracer at these sites labeled terminals in the ventral respiratory group, in the ventrolateral medulla. Injection of retrograde tracer into this target region in the ventrolateral medulla disclosed a previously unrecognized population of neurons among the trigeminal motor rootlets. Injection of retrograde tracer into this intertrigeminal region demonstrated inputs from portions of the spinal trigeminal nucleus and the nucleus of the solitary tract that have been associated with producing sensory apnea. Our observations suggest that the intertrigeminal region receives a convergence of sensory inputs capable of driving apneic responses and that it may represent a common link between input from different portions of the airway and the respiratory neurons that mediate apneic reflexes.
Identification of a Survival-Promoting Peptide in Medium Conditioned by Oxidatively Stressed Cell Lines of Nervous System Origin
Authors: Cunningham Timothy J. , Hodge Lisa , Speicher David , Reim Dave , Tyler-Polsz Carla , Levitt Pat , Eagleson Kathie , Kennedy Sarah , Wang Ying
Published: April 2018

A survival-promoting peptide has been purified from medium conditioned by Y79 human retinoblastoma cells and a mouse hippocampal cell line (HN 33.1) exposed to H2O2. A 30 residue synthetic peptide was made on the basis of N-terminal sequences obtained during purification, and it was found to exhibit gel mobility and staining properties similar to the purified molecules. The peptide maintains cells and their processesin vitrofor the HN 33.1 cell line treated with H2O2, andin vivofor cortical neurons after lesions of the cerebral cortex. It has weak homology with a fragment of a putative bacterial antigen and, like that molecule, binds IgG. The peptide also contains a motif reminiscent of a critical sequence in the catalytic region of calcineurin-type phosphatases; surprisingly, like several members of this family, the peptide catalyzes the hydrolysis ofpara-nitrophenylphosphate in the presence of Mn2+. Application of the peptide to one side of bilateral cerebral cortex lesions centered on area 2 in rats results in an increase in IgG immunoreactivity in the vicinity of the lesions 7 d after surgery. Microglia immunopositive for IgG and ED-1 are, however, dramatically reduced around the lesions in the treated hemisphere. Furthermore, pyramidal neurons that would normally shrink, die, or disintegrate were maintained, as determined by MAP2 immunocytochemistry and Nissl staining. These survival effects were often found in both hemispheres. The results suggest that this peptide operates by diffusion to regulate the immune response and thereby rescue neurons that would usually degenerate after cortical lesions. The phosphatase activity of this molecule also suggests the potential for direct neuron survival-promoting effects.
Postsynaptic Complex Spike Bursting Enables the Induction of LTP by Theta Frequency Synaptic Stimulation
Authors: Thomas Mark J. , Watabe Ayako M. , Moody Teena D. , Makhinson Michael , O’Dell Thomas J.
Published: April 2018

Long-term potentiation (LTP), a persistent enhancement of synaptic transmission that may be involved in some forms of learning and memory, is induced at excitatory synapses in the CA1 region of the hippocampus by coincident presynaptic and postsynaptic activity. Although action potentials back-propagating into dendrites of hippocampal pyramidal cells provide sufficient postsynaptic activity to induce LTP under somein vitroconditions, it is not known whether LTP can be induced by patterns of postsynaptic action potential firing that occur in these cellsin vivo. Here we report that a characteristicin vivopattern of action potential generation in CA1 pyramidal cells known as the complex spike burst enables the induction of LTP during theta frequency synaptic stimulation in the CA1 region of hippocampal slices maintainedin vitro. Our results suggest that complex spike bursting may have an important role in synaptic processes involved in learning and memory formation, perhaps by producing a highly sensitive postsynaptic state during which even low frequencies of presynaptic activity can induce LTP.
Central P2X4and P2X6Channel Subunits Coassemble into a Novel Heteromeric ATP Receptor
Authors: Lê Khanh-Tuoc , Babinski Kazimierz , Séguéla Philippe
Published: April 2018

Ionotropic ATP receptors are widely expressed in mammalian CNS. Despite extensive functional characterization of neuronal homomeric P2X receptors in heterologous expression systems, the subunit composition of native central P2X ATP-gated channels remains to be elucidated. P2X4and P2X6are major central subunits with highly overlapping mRNA distribution at both regional and cellular levels. When expressed alone inXenopusoocytes, P2X6subunits do not assemble into surface receptors responsive to ATP applications. On the other hand, P2X4subunits assemble into bona fide ATP-gated channels, slowly desensitizing and weakly sensitive to the partial agonist α,β-methylene ATP and to noncompetitive antagonists suramin and pyridoxal-5-phosphate-6-azophenyl-2′,4′-disulfonic acid. We demonstrate here that the coexpression of P2X4and P2X6subunits inXenopusoocytes leads to the generation of a novel pharmacological phenotype of ionotropic ATP receptors. Heteromeric P2X4+6receptors are activated by low-micromolar α,β-methylene ATP (EC50= 12 μm) and are blocked by suramin and by Reactive Blue 2, which has the property, at low concentrations, to potentiate homomeric P2X4receptors. The assembly of P2X4with P2X6subunits results from subunit-dependent interactions, as shown by their specific copurification from HEK-293 cells transiently transfected with various epitope-tagged P2X channel subunits. Our data strongly suggest that the numerous cases of neuronal colocalizations of P2X4and P2X6subunits observed in mammalian CNS reflect the native expression of heteromeric P2X4+6channels with unique functional properties.