bluuebunny/crossref_metadata_2025 huggingface dataset:
Dystroglycan in the Cerebellum is a Laminin α2‐chain Binding Protein at the Glial‐Vascular Interface and is Expressed in Purkinje cells
Authors: Tian M. , Jacobson C. , Gee S. H. , Campbell K. P. , Carbonetto S. , Jucker M.
Published: April 2006

Dystroglycan is a core component of the dystrophin receptor complex in skeletal muscle which links the extracellular matrix to the muscle cytoskeleton. Dystrophin, dystrophin‐related protein (DRP, utrophin) and dystroglycan are present not only in muscles but also in the brain. Dystrophin is expressed in certain neuronal populations while DRP is associated with perivascular astrocytes. To gain insights into the function and molecular interactions of dystroglycan in the brain, we examined the localization of α‐and β‐dystroglycan at the cellular and subcellular levels in the rat cerebellum. In blood vessels, we find a‐dystroglycan associated with the laminin a2‐chain‐rich parenchymal vascular basement membrane and β‐dystroglycan associated with the endfeet of perivascular astrocytes. We also show that a‐dystroglycan purified from the brain binds a2‐chain‐containing laminin‐2. These observations suggest a dystroglycan‐mediated linkage between DRP in perivascular astrocytic endfeet and laminin‐2 in the parenchymal basement membrane similar to that described in skeletal muscle. This linkage of the astrocytic endfeet to the vascular basement membrane is likely to be important for blood vessel formation and stabilization and for maintaining the integrity of the blood‐brain barrier. In addition to blood vessel labelling, we show that β‐dystroglycan in the rat cerebellum is associated with the surface of Purkinje cell bodies, dendrites and dendritic spines. Dystrophin has previously been localized to the inner surface of the plasma membrane of Purkinje cells and is enriched at postsynaptic sites. Thus, the present results also support the hypothesis that dystrophin interacts with dystroglycan in cerebellar Purkinje neurons.
Muscarinic Inhibition of Glutamatergic Transmission onto Rat Magnocellular Basal Forebrain Neurons in a Thin‐slice Preparation
Authors: Sim J. A. , Griffith W. H.
Published: April 2006

We have examined excitatory and inhibitory transmission in visually identified rat magnocellular basal forebrain neurons using whole‐cell patch‐clamp recordings in a thin‐slice preparation of the rat brain. In most cells, spontaneous excitatory and inhibitory synaptic activities could be recorded from their resting membrane potential. Following focal stimulation within the basal forebrain nucleus or directly onto visualized neighbouring neurons, postsynaptic currents were elicited in magnocellular basal forebrain cells held at ‐70 mV (a value close to their resting membrane potential). The synaptic responses were complex, consisting either mainly of excitatory postsynaptic currents (EPSCs), or inhibitory postsynaptic currents (IPSCs), or an EPSC‐IPSC sequence. The EPSC component was consistent with the activation of AMPA/KA receptors, as it could be selectively blocked by CNQX. The IPSC component resulted in the activation of GABAA receptors, and could be blocked by bicuculline. Since GABA‐mediated transmissions were not frequently recorded, we focused on the glutamate‐mediated transmission. Studies using specific calcium channel blockers suggested that both ω‐conotoxin GVIA‐sensitive and ω‐agatoxin VIA‐sensitive calcium channels contribute to the glutamatergic transmission onto magnocellular basal forebrain neurons. Carbachol (0.3–30 μM) had no observable effect on holding current, but produced a dose‐dependent inhibition of the amplitude of evoked EPSCs. This cholinergic modulation was mediated by muscarinic receptors, as it could be antagonized by atropine. The inhibitory effect of carbachol on the amplitude of EPSCs could be significantly antagonized by 100 nM methoctramine, an M2‐receptor antagonist. In contrast, only a small degree of antagonism could be obtained with pirenzepine, an M1‐muscarinic receptor antagonist, when present at relatively high concentration of 1 μM. Moreover, the action of carbachol was presynaptic, since the frequency of miniature postsynaptic currents was reduced without affecting their amplitude. In conclusion, the present findings indicate that glutamate‐mediated transmission onto magnocellular basal forebrain neurons appeared to involve both N‐ and P/Q‐type calcium channels, and that muscarinic modulation of glutamatergic transmission to MBF neurons is mediated by a presynaptic M2‐muscarinic receptor subtypes.
Post‐translational Processing of the Alternative Neuropeptide Precursor Encoded by the FMRFamide Gene in the Pulmonate Snail Lymnaea stagnalis
Authors: Santama Niovi , Wan Li Ka , Geraerts Wijnand P. M. , Benjamin Paul R. , Burke Julian F.
Published: April 2006

The neuropeptide gene encoding FMRFamide‐like peptides in the pulmonate mollusc Lymnaea is subject to alternative splicing that generates cell‐specific expression of distinct sets of peptides in the CNS. In this paper, we analyse the post‐translational processing of the alternative protein precursor encoded by the exon I, III–V transcript (type 2 transcript). We raised anti‐peptide antisera specific to distinct segments of the precursor in order to address the pattern of endoproteolytic cleavages, specifically around the tetrabasic site RRKR. We first showed that not all peptides predicted by the precursor structure are generated as final steady‐state products. We then identified a novel peptide by biochemical purification, amino acid sequencing and mass spectrometry—the 35 amino acid SDPFFRFGKQQVATDDSGELDDEILSRVSDDDKNI, which we termed the acidic peptide, previously not predicted on the basis of the precursor structure. This novel peptide, abundant in the snail brain (0.7 pmol per central nervous system), includes the N‐terminal sequence SDPFFRF, which was previously considered to be a variant of the known heptapeptide SDPFLRFamide, also encoded within the same protein precursor. We showed by in situ hybridization and immunocytochemistry that the acidic peptide is produced in all cells that transcribe type 2 FMRFamide mRNA. We mapped the expression of this novel peptide in the CNS and localized it mainly in three identifiable neuronal clusters—the E, F and B groups of cells—and some additional neurons, all situated in three of the eleven central ganglia. Immunoreactive neurons included the single identifiable visceral white interneuron (VWI or VD4), a key cell of the cardiorespiratory network.
Emx1 and Emx2 Show Different Patterns of Expression During Proliferation and Differentiation of the Developing Cerebral Cortex in the Mouse
Authors: Gulisano Massimo , Broccoli Vania , Pardini Celia , Boncinelli Edoardo
Published: April 2006

Insights into the complex structure of the forebrain and its regulation have recently come from the analysis of the expression of genes that are likely to be involved in regionalization of this structure. We cloned four new homeo box genes, Emx1, Emx2, Otx1 and Otx2, and we showed that in day 10 mouse embryos their expression domains are continuous regions of the developing brain contained within each other in the sequence Emx1 < Emx2 < Otx1 < Otx2. Recently Otx1 has been found to be specifically expressed during neurogenesis of layers 5 and 6 in the developing cerebral cortex. In order to better understand the role of Emx1 and Emx2 in the maturation of the cortex we analysed by in situ hybridization their expression patterns in the developing mouse cerebral cortex, from embryonic day 12.5 to adulthood. We found that Emx2 is expressed exclusively in proliferating cells of the ventricular zone whereas Emx1 is expressed in both proliferating and differentiated neurons, throughout the cortical layers and during all the developmental stages examined. Therefore, Emx2 gene products might control some biological parameters of the proliferation of cortical neuroblasts or of the subsequent cell migration of postmitotic neurons, leaving the cortical germinal zone. Conversely, Emx1 expression, which is confined exclusively to the dorsal telencephalon, characterizes most cortical neurons during proliferation, differentiation, migration and postnatal development and maturation.
Topographically Organized Climbing Fibre Sprouting in the Adult Rat Cerebellum
Authors: Zagrebelsky Marta , Rossi Ferdinando , Hawkes Richard , Strata Piergiorgio
Published: April 2006

Adaptive recovery following brain injury requires the topography of projection maps to be restored. In the adult mammalian brain, the regeneration of severed axons does not normally occur and repair mainly relies on collateral reinnervation from uninjured neurons. Although reinnervation can be target specific at the single cell level, it is not known if the new connections are organized correctly. The normal olivocerebellar projection has a precise topography in which subnuclei of the inferior olive terminate as climbing fibres on chemically defined bands of cerebellar Purkinje cells. This precision has been exploited to determine the topography of climbing fibre sprouting following an inferior olive lesion in the adult rat. Collateral reinnervation was found to respect the boundaries between the Purkinje cell compartments. Thus, topographical cues are available in the adult during post‐lesion plasticity to guide the restoration of the olivocerebellar projection map.
Transient Colocalization of Parvalbumin and Calbindin D28k in the Postnatal Cerebral Cortex: Evidence for a Phenotypic Shift in Developing Nonpyramidal Neurons
Authors: Alcántara Soledad , de Lecea Luis , Del Río Jose. A. , Ferrer Isidro , Soriano Eduardo
Published: April 2006

In the adult rat cerebral cortex the calcium‐binding proteins parvalbumin and calbindin D28k are present in essentially non‐overlapping populations of GABAergic interneurons. These proteins follow different developmental patterns in the cortex: calbindin D28k‐immunoreactive nonpyramidal neurons are abundant until the second postnatal week and decrease markedly thereafter; it is at this time that parvalbumin immunoreactivity develops in cortical nonpyramidal neurons. To determine whether parvalbumin‐immunoreactive neurons derive from calbindin D28k‐positive cells we used double‐immunofluorescence studies for both calcium‐binding proteins, together with combined immunocytochemistry for calbindin D28k and in situ hybridization for parvalbumin mRNA during postnatal development. Double‐labelled cells were found in all cortical layers between P9 and P21, coinciding with the onset of parvalbumin expression. The percentage of colocalization of the two calcium‐binding proteins depended on the age and layer examined. Colocalization reached a peak (80–100%) during the second postnatal week in layers II–IV and VI and decreased thereafter to adult levels by the end of the third postnatal week. Double‐labelled neurons were rare in layer V at all ages studied. The present results indicate a phenotypic shift during the development of some cortical interneurons that halts the expression of calbindin D28k while parvalbumin expression starts. These findings agree with lineage analyses reporting that different types of nonpyramidal neuron arise from a common progenitor.
The Convergence of Axon Terminals from the Mediodorsal Thalamic Nucleus and Ventral Tegmental Area on Pyramidal Cells in Layer V of the Rat Prelimbic Cortex
Authors: Kuroda Masaru , Murakami Kunio , Igarashi Hiroaki , Okada Akiko
Published: April 2006

We investigated the ultrastructural basis of the synaptic convergence of afferent fibres from the mediodorsal thalamic nucleus (MD) and the ventral tegmental area (VTA) on the prefrontal cortical neurons of the rat by examining the synaptic relationships between thalamocortical or tegmentocortical terminals labelled with anterograde markers [lesion‐induced degeneration or transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA—HRP)] and randomly selected unlabelled apical dendrites of layer V pyramidal cells in the prelimbic cortex. WGA—HRP‐labelled terminals from the VTA ranged in diameter from 0.7 to 2.8 μm and established synaptic contacts with large dendritic profiles, i.e. proximal segments of apical dendritic shafts and spines from layer V pyramidal cells. Symmetrical synapses, i.e. inhibitory synapses, were more often seen than asymmetrical ones. Degenerating terminals from the MD formed asymmetrical synapses on dendritic spines or occasionally on small dendritic shafts of apical dendrites from layer V pyramidal cells, which received tegmentocortical synapses, mostly within layer III. Thalamocortical synapses were more distally distributed over common apical dendrites than tegmentocortical synapses, although some of them overlapped. The numerical density of direct synaptic inputs from the MD and VTA was low. These results suggest that fibres from the VTA exert their inhibitory effects directly on pyramidal cells in layer V via synaptic junctions with apical dendrites of these pyramidal cells, and that the tegmentocortical fibres are in an ideal anatomical position to modulate the reverberatory circuits between the MD and the prelimbic cortex.
Evidence for an Anuran Homologue of the Mammalian Spinocervicothalamic System: An In Vitro Tract‐tracing Study in Xenopus laevis
Authors: Muñoz A. , Muñoz M. , González A. , ten Donkelaar H. J.
Published: April 2006

Evidence is presented for an anuran homologue of the mammalian spinocervicothalamic system. In vitro tract‐tracing experiments with biotinylated dextran amine in Xenopus laevis show that ascending spinal fibres from all levels of the spinal cord, passing via the dorsolateral funiculus, terminate in a cell area ventrolateral to the dorsal column nucleus. This cell area can be considered a possible homologue of the mammalian lateral cervical nucleus. After tracer applications to the ventral thalamus or to the torus semicircularis (both targets for somatosensory projections), the anuran lateral cervical nucleus was retrogradely labelled contralateral to the application sites. Tracer applications to the dorsolateral funiculus at the obex level and rostral spinal cord resulted in labelling of the cells of origin of the spinocervical tract. These were found, mainly ipsilaterally, in the ventral part of the dorsal horn, and were rather evenly distributed throughout the spinal cord. These data suggest the presence of an anuran homologue of the mammalian spinocervicothalamic system. A brief survey of the literature shows that such a system is much more common in vertebrates than previously thought.
Expression of Heat Shock Protein‐70 and Limbic Seizure‐induced Neuronal Death in the Rat Brain
Authors: Zhang X. , Boulton A. A. , Yu P. H.
Published: April 2006

The effect of MK‐801, a non‐competitive N‐methyl‐D‐aspartate (NMDA) antagonist, on the kainic acid‐induced expression of the inducible heat shock protein 70 kDa (HSP70) and on neuronal death in the rat hippocampus was investigated. HSP70 is expressed in ˜80% of the pyramidal neurons in the CA1 field 1 day after kainic acid injection. The majority of these HSP70‐immunopositive neurons exhibited swelling and a hollow appearance in the perikaryon, indicating that they had been injured following kainic acid‐elicited limbic seizures. Four days after administration of kainic acid, 87% of the pyramidal neurons in the CA1 field were dead. When a single dose of MK‐801 was administered 1 h before kainic acid injection, the number of rats suffering with seizures was reduced, the severity of limbic seizures was attenuated and seizure onset was delayed. Neither HSP70 expression on day 1 nor neuronal loss on day 4 in the CA1 pyramidal cell layer was observed in these animals. A considerable number of HSP70‐immunopositive neurons was detected in the dentate hilus, however, and somewhat fewer in the CA3a and CA3c subfields on day 1. Severe neuronal damage in these regions followed on day 4. Interestingly, little HSP70 expression or neuronal loss was observed in the CA3b subfield in these same animals. When a single dose of MK‐801 was given 4 h after kainic acid treatment, HSP70 expression was partially blocked; 18% of neurons expressed HSP70 on day 1 and 37% on day 4 in CA1 pyramidal neurons in comparison to the kainic acid controls. About 50% neuronal death was detected in the CA1 pyramidal cell layer 4 days after kainic acid treatment followed by MK‐801. When the animals were treated with MK‐801 4 h after kainic acid treatment followed by additional daily administration for 3 days, a negligible number of pyramidal neurons expressed HSP70, and the survival of pyramidal cells was significantly increased in the CA1 field. Limbic seizure‐induced HSP70 expression not only indicates neuronal injury in the pyramidal cell layer of the hippocampus but also predicts delayed neuronal death, at least in the case of the CA1 field of animals that suffered stage IV—V seizures.
Simultaneous Recording of Spontaneous Activities and Nociceptive Responses from Neurons in the Pars Compacta of Substantia Nigra and in the Lateral Habenula
Authors: Gao D. M. , Hoffman D. , Benabid A. L.
Published: April 2006

Using simultaneous extracellular single‐unit recording in the pars compacta of the substantia nigra and in the lateral habenula of rats, 45 pairs of neurons responding to peripheral nociceptive stimulation were recorded. In 41 of these pairs, nigral dopaminergic neurons were inhibited by peripheral nociceptive stimulation, while lateral habenula neurons were excited. Moreover, in 14 pairs, when sweeps were triggered randomly by spontaneous spikes from lateral habenula neurons the spontaneous firing rate of the dopaminergic neurons during the first 250 ms after the sweep was much lower than rates after this time period. In this case, the sweep was often triggered by burst‐firing of lateral habenula neurons. Our results indicate a cross‐correlation between the spontaneous activities of these two nuclei, suggesting that the excitation of lateral habenula neurons induced by peripheral nociceptive stimulation might be directly responsible for inhibition of nigral dopaminergic neurons.
Dissociations in Hippocampal 5‐Hydroxytryptamine Release in the Rat Following Pavlovian Aversive Conditioning to Discrete and Contextual Stimuli
Authors: Wilkinson Lawrence S. , Humby Trevor , Killcross Simon , Robbins Trevor W. , Everitt Barry J.
Published: April 2006

The experiments examined the release of 5‐hydroxytryptamine using in vivo microdialysis methods in the hippocampus of freely moving rats following Pavlovian aversive conditioning to discrete and contextual stimuli. Differential conditioning was achieved by manipulating the interval between the offset of a discrete auditory ‘clicker’stimulus and the onset of a mild foot‐shock reinforcer (0.5 mA, 0.5 s). Foot‐shock occurred either simultaneously with the last second of the discrete auditory stimulus (in short‐trace subjects) or 60 s later (long‐trace subjects). In this way, subjects were preferentially conditioned to the discrete stimulus and background ‘contextual’stimuli respectively. During conditioning subjects also received two identical unpaired visual stimuli. At test, dialysates were collected and behavioural measures taken as all animals experienced (i) the aversive and two other ‘neutral’environments, and (ii) the discrete unconditioned and conditioned stimuli presented in both aversive and neutral environments. Exposure to the aversive environment, but not to either of the two neutral environments, was associated with significantly increased hippocampal 5‐hydroxytryptamine release in long‐trace subjects. There was also a small but non‐significant increase in 5‐hydroxytryptamine release in short‐trace animals. In contrast, hippocampal 5‐hydroxytryptamine release was unaffected by presentation of either of the discrete stimuli under all conditions. The last result was obtained despite robust behavioural responses (freezing) to the discrete conditioned stimulus. These data do not agree with the hypothesis that aversive cues generally activate 5‐hydroxytryptamine function in the hippocampus. Rather, they suggest a degree of specificity whereby 5‐hydroxytryptamine release in the hippocampus was determined primarily by other qualitative properties of the conditioned aversive stimulus, namely whether the aversive cue was discrete or contextual, as well as by the magnitude of conditioning.
Dopamine D1 Receptor‐mediated Facilitation of GABAergic Neurotransmission in the Rat Strioentopeduncular Pathway and its Modulation by Adenosine A1 Receptor‐mediated Mechanisms
Authors: Ferré Sergi , O'Connor William T. , Svenningsson Per , Björklund Lars , Lindberg Jakob , Tinner Barbro , Strömberg Ingrid , Goldstein Menek , Ögren Sven Ove , Ungerstedt Urban , Fredholm Bertil B. , Fuxe Kjell
Published: April 2006

By using in vivo microdialysis it was found that one of the main functions of striatal dopamine D1 receptors is to selectively facilitate GABAergic neurotransmission in the ‘direct’strioentopeduncular pathway. D1 receptors localized in the entopeduncular nucleus were also found to facilitate GABA release. However, results obtained from in vivo microdialysis, in vivo electrochemistry, immunohistochemistry and confocal laser microscopy suggested that entopeduncular D1 receptors could only be activated under pharmacological conditions. Adenosine A1 receptors were found to antagonistically modulate the D1‐mediated regulation of the strioentopeduncular pathway. Furthermore, using in situ hybridization D1 and A1 receptors were shown to be colocalized in medium‐sized striatal neurons. These results show that the strioentopeduncular neuron is a main locus for adenosine‐dopamine interactions in the brain.
Regulation of CD44 in the Regenerating Mouse Facial Motor Nucleus
Authors: Jones Leonard L. , Kreutzberg Georg W. , Raivich Gennadij
Published: April 2006

CD44 is a cell adhesion molecule which plays an important role in cell movement and adhesion, e.g. in lymphocyte homing and tumour metastasis. Here we studied the expression of CD44 mRNA and protein immunoreactivity in the facial nucleus after nerve injury and during the ensuing regeneration. Transection of the facial nerve led to a strong up‐regulation of CD44, peaking 4 days after injury on the motoneurons of the axotomized facial nucleus. Use of the polymerase chain reaction confirmed the de novo expression of CD44 and detected only the standard haematopoietic CD44 isoform. Western blotting also detected the 76 kDa protein subtype, in line with the predicted size of the haematopoietic CD44 variant. At the ultrastructural level, CD44 immunoreactivity was restricted to the surface of the neuronal perikarya, their dendrites and axons. It was not seen in the adjacent activated astrocytes, microglia or vascular endothelia. This study shows strong up‐regulation of the cell adhesion molecule CD44 on the regenerating rnotoneurons in the axotomized facial nucleus. These data suggest that CD44 may play a role in neurite outgrowth, in synaptic stripping or in the adhesion of activated glial cells to the perikaryal surface of the axotomized motoneurons.
Intraneuronal Traffickina and Distribution of Amphiphysin and Synaptojanin in thg Rat Peripheral Nervous System and the Spinal Cord
Authors: Li Jia‐Yi , De Camilli Pietro , Dahlström Annica
Published: April 2006

Amphiphysin and synaptojanin are two nerve terminal proteins with a putative role in synaptic vesicle endocytosis and recycling. We have investigated the intraneuronal dynamics and distribution of these two proteins, using nerve crush techniques in combination with immunofluorescence, cytofluorimetric scanning (CFS), confocal laser scanning microscopy and immuno‐electron microscopy (EM). Accumulations of amphiphysin and synaptojanin immunoreactivities at the crush site were detected as short as 1 h after the lesion, indicating that a pool of these two partially cytosolic proteins moves along the axon by fast axoplasmic transport. The amount of proximal accumulation increased linearly between 1 and 8 h. CFS analysis demonstrated that only 30% of fast anterogradely transported amphiphysin and synaptojanin was returned by fast retrograde transport, in contrast to the 70% value observed for synaptophysin, a transmembrane protein. This indicates that the majority of amphiphysin and synaptojanin is degraded/metabolized in the nerve terminals. Immuno‐EM showed that both amphiphysin and synaptojanin are primarily associated with heterogeneous membrane profiles in the crushed sciatic nerve and the immunoperoxidase reaction product is concentrated in the nerve terminal cytomatrix of the spinal cord. Both proteins were differentially distributed in subsets of nerve terminals, indicating heterogeneous expression in neurons.
A Role for Neurotrophins in the Survival of Murine Embryonic Thalamic Neurons
Authors: Lotto R. Beau , Clausen Julia A. , Price David J.
Published: April 2006

The mechanisms that determine whether developing CNS neurons live or die are poorly understood. We studied the role of the neurotrophins and fibroblast growth factors in the survival of embryonic thalamic neurons in culture. Dissociated embryonic dorsal thalamic neurons cultured at high density in defined serum‐free medium survived and grew neurites. As in vivo, they expressed all the neurotrophins, fibroblast growth factor‐1 and their high‐affinity tyrosine kinase receptors. The survival of these cells was reduced by the addition of the protein kinase inhibitor K252a at concentrations that block neurotrophin receptor activity but not the activity of other tyrosine kinase receptors. In low‐density cultures, most dorsal thalamic neurons died, but their survival was increased by co‐culture with thalamic explants or with most of the neurotrophins and fibroblast growth factor‐1 added singly. These results indicate that thalamic neurons have remarkably promiscuous trophic responses to a battery of neurotrophins and fibroblast growth factors. They suggest that neurotrophins endogenous to the early embryonic thalamus may be required to promote the survival of its neurons.
BDNF is Expressed at the Crush Site after Spinal Cord Lesion in Newborn Opossum (Monodelphis domestica)
Authors: Vischer Heinrich A.
Published: April 2006

Brain‐derived neurotrophic factor (BDNF) supports the regeneration of axotomized motoneurons in vitro. To study the role of BDNF in vivo, a partial cDNA encoding the BDNF of the opossum (Monodelphis dornestica) was cloned by reverse transcription‐polymerase chain reaction. The sequence was highly homologous to the BDNF of several mammals and chick. The spinal cord of newborn opossums was lesioned and allowed to regenerate in its entirety in a culture system for 3 days. In response to lesion, cells within the lesion and the ventrorostral wound margin showed strong expression of opossum BDNF mRNA as detected by in situ hybridization. In contrast, no BDNF mRNA expression was detected in unlesioned CNS preparations. This suggests that BDNF plays a role in the repair of lesioned neonatal spinal cord.
Connexins, Gap Junctions and Cell‐Cell Signalling in the Nervous System
Authors: Bruzzone Roberto , Ressot Catherine
Published: April 2006

Connexins form a multigene family of polytopic membrane proteins that, in vertebrates, are the constitutive subunits of intercellular channels and provide the structural basis for electrical coupling. The appearance of electrical coupling in the nervous system is developmentally regulated and restricted to distinct cell types. Electrical coupling between neurons persists after the establishment of chemical transmission, thus suggesting that this form of cell‐cell signalling may be functionally interrelated with, rather than alternative to chemical transmission. Furthermore, evidence for the possible role of gap junctions in human neurological diseases is also mounting, following the discovery that the X‐linked form of Charcot‐Marie‐Tooth syndrome, a demyelinating neuropathy of the peripheral nervous system, is associated with mutations in a connexin gene. These findings raise new questions on the significance of connexin diversity and on their functional role in the nervous system.
Analysis of Cutaneous Sensory Neurons in Transgenic Mice Lacking the Low Affinity Neurotrophin Receptor p75
Authors: Bergmann Ilka , Priestley John V. , McMahon Stephen B. , Bröcker Eva‐B. , Toyka Klaus V. , Koltzenburg Martin
Published: April 2006

Mice with a targeted mutation of the p75 low affinity neurotrophin receptor display smaller peripheral nerves and dorsal root ganglia. Here we show that transgenic mice have a significant elevation of thresholds to noxious mechanical and heat stimuli compared with p75+/+ control mice. Immunocytochemical analysis using antibodies against PGP 9.5 (a panaxonal marker) and calcitonin gene related peptide (CGRP, which labels peptidergic neurons) showed a reduction to 73% and 54%, respectively, of the epidermal innervation density. However, analysis of the cell size distribution of toluidine blue‐stained dorsal root ganglia showed no selective loss of neurons of particular diameters. Moreover, the neurochemical profile of dorsal root ganglia cells as defined by trkA, CGRP, I84 and RT97 immunostaining revealed no significant differences in comparison with p75+/+ animals. Staining of the dorsal horn of the spinal cord for CGRP and 164 was also normal in p75‐/‐animals. Taking into account a previously reported loss of ‐50% dorsal root ganglion neurons, we conclude that all types of sensory neurons are equally depleted in p75‐/‐ mice and that the absence of p75 impedes the development of more than one neuronal subtype.
Electron Microscopic Distribution of Mu Opioid Receptors on Noradrenergic Neurons of the Locus Coeruleus
Authors: Moyse Emmanuel , Marcel Dominique , Leonard Kathleen , Beaudet Alain
Published: April 2006

The distribution of mu opioid receptors was examined by light and electron microscopic autoradiography in the locus coeruleus of the rat following in vitro labelling with the iodinated agonist [125I]FK‐33824. At the light microscopic level, specific mu opioid binding sites were concentrated over the perikarya and dendrites of neurons that were tyrosine hydroxylase‐immunopositive in adjacent sections. Accordingly, both the number of tyrosine hydroxylase‐immunoreactive neurons and the density of labelled mu receptors decreased markedly throughout the rostrocaudal extent of the nucleus following treatment with the catecholaminergic neurotoxin 6‐hydroxydopamine. By electron microscopy, specifically labelled receptors were detected both inside and on the surface of locus coeruleus neurons. Intracellular sites were found by resolution circle analysis to be highly concentrated within the endoplasmic reticulum and Golgi apparatus, suggesting that the ligand recognizes both glycosylated and preglycosylated forms of receptor. The remainder were found mainly over the cytoplasmic matrix or intracytoplasmic vesicles, and were attributed to newly synthesized or recycled receptors in transit. Cell surface receptors were present over both dendritic and perikaryal membranes of noradrenergic cells. These were most highly concentrated opposite abutting axon terminals, suggesting the existence of receptor ‘hot spots’ at sites of putative endogenous ligand release. However, only a small proportion of these sites was associated with synaptic specializations. Furthermore, an important contingent was detected opposite non‐axonal elements, such as dendrites and glial cells, suggesting that mu opioid ligands act mainly parasynaptically on locus coeruleus neurons. Finally, ˜5% of labelled receptors were associated with axoglial interfaces, indicating that a minor action of mu opioids in the locus may be presynaptic and/or glial.
Oligodendrocyte‐type 2 Astrocyte Progenitors Use a Metalloendoprotease to Spread and Migrate on CNS Myelin
Authors: Amberger Verena R. , Avellana‐Adalid Virginia , Hensel Tamara , Evercooren Anne Baron‐Van , Schwab Martin E.
Published: April 2006

abstract

Oligodendrocyte‐type 2 astrocyte (O‐2A) progenitors are highly motile cells which migrate in the developing and adult central nervous system (CNS). Adult CNS myelin, however, contains inhibitory proteins, the neurite growth inhibitors NI 35/250, which block neurite outgrowth and spreading of many different cell types, such as astrocytes and fibroblasts. In the present study we investigated the spreading of dissociated cells and migration out of aggregates (‘spheres’) of primary O‐2A cultures and of a glial precursor cell line (CG‐4) on purified CNS myelin and on CNS tissue. Primary O‐2A progenitors and CG‐4 cells quickly attached to and spread on CNS myelin‐coated culture dishes, showing no inhibition by the neurite growth inhibitors. CG‐4 cells migrated with a velocity of 30 pm/h on a CNS myelin protein extract and at 5.7 μm/h on adult spinal cord tissue. Both cell spreading and migration on a CNS substrate could be specifically blocked by metalloprotease blockers like o‐phenanthroline and the tetrapeptide carbobenzoxy‐phe‐ala‐phe‐tyr‐amide, whereas blockers of the serine, aspartyl and cysteine proteases had no effect. On differentiation to astrocytes, the O‐2A progenitors lost their ability to spread on CNS myelin. These results suggest the crucial involvement of a metalloprotease in the mechanism of migration on a CNS substrate. In vivo, migration of oligodendrocyte progenitors may be an important aspect of myelin repair following local traumatic, inflammatory or toxin‐induced myelin loss.
Morphology, Electrophysiology and Pathophysiology of Supragranular Neurons in Rat Primary Somatosensory Cortex
Authors: Schröder Rita , Luhmann Heiko J.
Published: April 2006

Intracellularly biocytin‐labelled neurons in layers 11/111 of adult rat primary somatosensory cortex were analysed for their morphological and electrophysiological properties and studied for their response pattern to transient hypoxia under in vitro conditions. The largest dendritic region is formed by the basal dendrites, which constitute an average area of 0.06 mm2 and which can receive synaptic inputs over horizontal distances of more than 300 μm. The dendritic territories formed by the oblique dendrites situated on the apical trunk and by the apical tuft are much smaller. The spine density is highest on the apical trunk, suggesting that large numbers of excitatory synapses are present in this region of the cell. All neurons revealed intrinsic membrane properties of typical regular spiking cells and received an excitatory and a strong biphasic inhibitory input. Whereas a significant correlation could be detected between the cell's input resistance and soma area, no correlation existed between the cell's total dendritic length and input resistance or membrane time constant/input resistance. Neurons responded to transient hypoxia either with an anoxic hyperpolarization with an apparent reversal potential of ‐82.4 mV, or with a gradual anoxic depolarization which reversed at ‐56 mV. Oxygen deprivation caused a significant reduction in the extent of axonal collaterals, whereas dendritic proportions and spine density were unaffected. The present study indicates that the dendritic tree is well preserved under in vitro conditions, whereas axonal connections are diminished by oxygen deprivation. Our results further suggest that certain structural properties correlate with the cellular physiology, but that the cell's morphology does not determine its responsiveness to hypoxia.
Spontaneous Openings of NMDA Receptor Channels in Cultured Rat Hippocampal Neurons
Authors: Tureček Rostislav , Vlachová Viktorie , Vyklický Ladislav
Published: April 2006

Spontaneous and N‐methyl‐D‐aspartate (NMDA)‐evoked single‐channel currents were studied in outside‐out patches isolated from cultured rat hippocampal neurons. Both spontaneous and NMDA‐evoked single‐channel currents reversed at potentials close to 0 mV and exhibited multiple amplitude levels of similar amplitude. Both spontaneous and NMDA‐evoked single‐channel currents were inhibited by Mg2+ in a voltage‐dependent manner and by 7‐chlorokynurenic acid. The activity of spontaneous single‐channel currents was reduced by the competitive NMDA receptor antagonists, but by one to three orders of magnitude less than expected assuming that the spontaneous activity is due to an ambient NMDA receptor agonist present in the extracellular solution. Our results suggest that, similar to other ligand‐gated ion channels, NMDA receptor channels have a dual mode of activation ‐ spontaneous and agonist induced.
Presynaptic Modulation of Glutamate Release Targets Different Calcium Channels in Rat Cerebrocortical Nerve Terminals
Authors: Vázquez Elena , Sánchez‐Prieto José
Published: April 2006

We have studied which type/s of Ca2+ ‐channel/s support glutamate exocytosis and its modulation by presynaptic receptors in cerebrocortical nerve terminals. Depolarization of nerve terminals with 30 mM KCI induced a Ca2+ ‐dependent release of 3.64 ± 0.25 nmol/mg of protein. The addition of either 2 μM ω‐conotoxin‐GVIA or 200 nM ω‐agatoxin‐IVA reduced the KCI‐evoked release by 47.7 ± 3.5% and 70.4 ± 8.9% respectively, and by 85.7 ± 4.1% when both toxins were co‐applied. The activation of adenosine A1 receptors with N6‐cyclohexyladenosine or the activation of rnetabotropìc glutamate receptors with L(+)‐2‐amino‐4‐phosphonobutyrate inhibited the KCI‐evoked release by 41.0 ± 5.9 and 54.3 ± 10% respectively. The extent of these inhibitions was not altered by the prior addition of 2 μM ω‐conotoxin‐GVIA but they were significantly enhanced when ω‐agatoxin‐IVA was added together with the adenosine A1 receptor agonist or the metabotropic glutamate receptor agonist, suggesting that ω‐conotoxin‐GVIA‐sensitive and not ω‐agatoxin‐IVA‐sensitive Ca2+‐channels are ínvolved in the action of these inhibitory receptors. By contrast, the facilitation of glutamate release that follows the activation of the protein kinase C, either with phorbol esters or with the stimulation of phospholipase C‐linked metabotropic receptors, was expressed by both ω‐conotoxin‐GVIA‐sensitive and ω‐agatoxin‐sensitive Ca2+‐channels. It is concluded that different Ca2+‐channels support the modulation of glutamate release by presynaptic receptors.
Adhesion Molecules Involved in Macrophage Responses to Wallerian Degeneration in the Murine Peripheral Nervous System
Authors: Brown H. C. , Castaño A. , Fearn S. , Townsend M. , Edwards G. , Streuli C. , Perry V. H.
Published: April 2006

When a peripheral nerve is damaged the severed axon undergoes Wallerian degeneration. The distal nerve is infiltrated by large numbers of monocyte‐derived macrophages which participate in the phagocytosis of degenerating myelin. In other tissues, adhesion molecules play a crucial role in leukocyte recruitment during inflammation. Blood‐borne cells enter damaged tissue by interacting with adhesion molecules expressed on activated endothelium. Having crossed the endothelium, leukocytes must adhere and migrate within the tissue. We investigated the adhesion molecules involved in both stages of the macrophage response to transection of one sciatic nerve of BALB/c mice. By injecting monoclonal antibodies in vivo, before and after peripheral nerve injury, we showed that intercellular adhesion molecule‐1 (ICAM‐1) and integrins α4β1 (VLA‐4) and αMβ2 (type 3 complement receptor) are unlikely to be involved in the transendothelial migration of monocytes responding to peripheral nerve degeneration. We also studied the adhesion of macrophages within the endoneurium, using an in vitro adhesion assay. Macrophages showed much greater levels of adhesion to cryostat sections of transected nerves than to control nerves. This increased adhesion was partially inhibited by antibodies to the β1 ‐integrin chain, and more strongly inhibited by the extracellular matrix molecules fibronectin and collagen. Adhesion was unaffected by laminin‐I and by antibodies to other adhesion molecules, including α4β1‐ and α5β1‐integrins. Thus we conclude that monocyte entry into a degenerating peripheral nerve is independent of αLβ2/αMβ2‐ICAM‐1 or α4β1/NCAM‐1 interactions, and that adhesion within the endoneurium is mediated in part by a β1 ‐integrin other than α4β1 or α5β1.
Substance P Modulates Sensory Action Potentials in the Lamprey Via a Protein Kinase C‐Mediated Reduction of a 4‐Aminopyridine‐Sensitive Potassium Conductance
Authors: Parker David , Svensson Erik , Grillner Sten
Published: April 2006

We have examined the effects of the tachykinin substance P on the action potential of lamprey mechanosensory dorsal cells. Substance P increased the spike duration and reduced the afterhyperpolarization. These effects were mimicked by stimulation of the dorsal root, which contains tachykinin‐like immunoreactive fibres. The tachykinin antagonist spantide II blocked the effects of both substance P and dorsal root stimulation. The spike broadening was voltage‐dependent, and was due to the reduction of a 4‐aminopyridine‐sensitive potassium conductance. The spike broadening was mimicked by G‐protein activators and blocked by the G‐protein inhibitor GDPβS. Pertussis toxin did not block the effects of substance P. The spike broadening was blocked by the protein kinase C and CAMP‐dependent protein kinase inhibitor H7, and by the specific protein kinase C antagonist chelerythrine, but not by the cAMP and cGMP‐dependent protein kinase inhibitor H8. The phorbol ester phorbol 12,13‐dibutyrate mimicked and blocked the effects of substance P, supporting the role of protein kinase C in the spike modulation. The adenylate cyclase activator forskolin and the cAMP agonist SpcAMPs mimicked but did not block the effects of substance P on the spike duration, suggesting that protein kinase A also modulates the dorsal cell action potential, but that substance P acts independently of this pathway. Substance P also increased the excitability of the dorsal cells. This effect was blocked by 4‐AP, PDBu and chelerythrine, but not by H8, suggesting that the increase in excitability shares the same intracellular and effector pathways as the spike broadening.
Bilateral Lesions of the Subthalamic Nucleus Induce Multiple Deficits in an Attentional Task in Rats
Authors: Baunez Christelle , Robbins Trevor W.
Published: April 2006

Lesioning the subthalamic nucleus (STN) has been suggested as possible therapy for the treatment of parkinsonism. Previous experiments investigating this hypothesis in rats confirmed that excitotoxic STN lesions alleviate the motor impairment induced by striatal dopamine depletion, which reproduced the degeneration observed in parkinsonism, but elicited presumed non‐motor deficits such as premature rationresponding, suggesting that the STN could be involved in other aspects of response control. The aim of the present study was to extend this analysis to choice paradigms. We thus investigated the behavioural effects of bilateral excitotoxic lesions of the STN in rats performing a five‐choice test of divided and sustained visual attention, modelled on the human continuous performance task. This task required the animals to detect a brief visual stimulus presented in one of five possible locations and respond by a nose‐poke in this illuminated hole within a fixed delay, for food reinforcement. Bilateral lesions of the STN severely impaired several aspects of performance, including discriminative accuracy, but also increased premature, anticipatory ralresponding as well as perseverative panel pushes and nose‐poke responses. While increasing the stimulus duration and reducing the waiting period for the stimulus partially alleviated the accuracy deficit and the premature responding deficit respectively, other deficits, such as perseverative panel pushes and nose‐poke responses were sustained under these conditions. Systemic injection of the mixed dopaminergic D1/D2 receptor antagonist, α‐flupenthixol (0.03–0.18 mg/kg), reduced premature responses and perseverative panel pushing without affecting the perseverative nose‐poke responses, suggesting that some of the deficits were independent of striatal dopaminergic transmission. These results suggest that STN lesions have multiple, dissociable effects on attentional performance, including discriminative deficits, impulsivity and perseverative behaviour They are consistent in part with a hypothesized role of the STN in recent models of basal ganglia function in areaction selection and inhibition. The results also show that other aspects of behaviour should be monitored when examining the capacity of STN lesions to reverse the parkinsonian deficit induced by striatal dopamine depletion.
Anatomical Substrate of Amphibian Basal Ganglia Involvement in Visuomotor Behaviour
Authors: Marin Oscar , González Agustín , Smeets Wilhelmus J.A.J.
Published: April 2006

The optic tectum of amphibians is known to play a major role in the control of visually elicited orienting movements, such as prey‐catching and avoidance behaviours. The recent finding of a direct striato‐tectal connection in the frog Rana perezi prompted us to study in detail the anatomical substrate by which the basal ganglia of amphibians may modulate visuomotor behaviour. Injections of anterograde tracers into the striatum were combined with applications of retrograde tracers in the mid‐brain tectum. Apart from a direct striato‐tectal connection, at least three indirect pathways were observed, viz. a striato‐anterior entopeduncular‐tectal pathway, a striato‐pretectal‐tectal pathway and a striato–tegmento–tectal pathway. The basal ganglia–tectal connections of anurans largely resemble those described for amniotes, but appear to be more extensive. However, a pallio–tectal connection comparable to the cortico–tectal pathways of mammals was not observed in Rana perezi. Therefore, the striatum of anurans, which receives multimodal sensory information, seems to be the sole telencephalic structure that influences the mesencephalic tectum via a direct pathway.
Expression and Subcellular Localization of p42IP4/ Centaurin‐α, a Brain‐specific, High‐affinity Receptor for lnositol 1,3,4,5‐Tetrakisphosphate and Phosphatidylinositol 3,4,5‐Trisphosphate in Rat Brain
Authors: Kreutz M. R. , Böckers T. M. , Sabel B. A. , Hülser E. , Stricker R. , Reiser G.
Published: April 2006

Recently emerging evidence suggests important roles for inositol polyphosphates and inositol phospholipids in neuronal Ca2+ signalling, membrane vesicle trafficking and cytoskeletal rearrangement. A prerequisite for a detailed physiological characterization of the signalling of both potential second messengers inositol‐(1,3,4,5)‐tetrakisphosphate (InsP4) and phosphatidylinositol‐3,4,5‐trisphosphate (PtdlnsP3) in the nervous system is the precise cellular localization of their receptors. Based on the cDNA sequence of a recently cloned brain‐specific receptor with high affinity for both InsP4 and PtdlnsP3 (InsP4–PtdlnsP3R), p42IP4/centaurin‐α, we localized the mRNA and the protein in rat brain. In situ hybridization revealed a widespread expression of the InsP4–PtdlnsP3R with prominent labelling in cerebellum, hippocampus, cortex and thalamus, which moreover is developmentally regulated. Using peptide‐specific antibodies, the immunoreactivity was localized in the adult brain in the vast majority of neuronal cell types and probably also in some glial cells. Prominent immunoreactivity was found in axonal processes and in cell types characterized by extensive neurites. In the hypothalamus a subpopulation of parvocellular neurons in the peri‐ and paraventricular nuclei was most heavily labelled. This was confined by strong immunoreactivity in the lamina externa of the median eminence in close proximity to portal plexus blood vessels. Electron microscopy revealed that the InsP4‐PtdlnsP3R was frequently associated with presynaptic vesicular structures. Further studies should identify the role of the InsP4‐PtdlnsP3R in cellular neural processes.