bluuebunny/crossref_metadata_2025 huggingface dataset:
Localization of nAChR subunit mRNAs in the brain of Macaca mulatta
Authors: Han Zhi‐Yan , Le Novère Nicolas , Zoli Michele , Hill Joseph A. , Champtiaux Nicolas , Changeux Jean‐Pierre
Published: March 2003

We present here a systematic mapping of nAChR subunit mRNAs in Macaca mulatta brain. A fragment, from the transmembrane segments MIII to MIV of Macaca neuronal nAChR subunits was cloned, and shown to exhibit high identity (around 95%) to the corresponding human subunits. Then, specific oligodeoxynucleotides were synthesized for in situ hybridization experiments. Both α4 and β2 mRNA signals were widely distributed in the brain, being stronger in the thalamus and in the dopaminergic cells of the mesencephalon. Most brain nuclei displayed both α4 and β2 signals with the exception of some basal ganglia regions and the reticular thalamic nucleus which were devoid of α4 signal. α6 and β3 mRNA signals were selectively concentrated in the substantia nigra and the medial habenula. The strongest signals for α3 or β4 mRNAs were found in the epithalamus (medial habenula and pineal gland), whereas there were no specific α3 or β4 signals in mesencephalic dopaminergic nuclei. α5 and α7 mRNA signals were found in several brain areas, including cerebral cortex, thalamus and substantia nigra, although at a lower level than α4 and β2. The distribution of α3, α4, α5, α6, α7, β2, β3 and β4 subunit mRNAs in the monkey is substantially similar to that observed in rodent brain. Surprisingly, α2 mRNA signal was largely distributed in the Macaca brain, at levels comparable with those of α4 and β2. This observation represents the main difference between rodent and Macaca subunit mRNA distribution and suggests that, besides α4β2*, α2β2* nAChRs constitute a main nAChR isoform in primate brain.
Induction of VEGF and VEGF receptors in the spinal cord after mechanical spinal injury and prostaglandin administration
Authors: Sköld Mattias , Cullheim Staffan , Hammarberg Henrik , Piehl Fredrik , Suneson Anders , Lake Staffan , Sjögren AnnMargret , Walum Erik , Risling Mårten
Published: March 2003

Vascular endothelial growth factor (VEGF) is an angiogenetic factor that promotes endothelial cell proliferation during development and after injury to various types of tissue, including the central nervous system (CNS). Using immunohistochemical and in situ hybridization methods we have here demonstrated that VEGF and its receptors Flk‐1, Flt‐1 and Neuropilin‐1 mRNAs and proteins are induced after incisions in the rat spinal cord. The inducible enzyme for prostaglandin synthesis cyclooxygenase‐2 (COX‐2) is known to be upregulated after spinal injury, cerebral ischemia and to stimulate angiogenesis. To test the hypothesis that prostaglandins may be involved in the VEGF response after lesion we investigated whether intraspinal microinjections of prostaglandin F2α (PGF2α) alters VEGF expression in the spinal cord. Such treatment was followed by a strong upregulation of VEGF mRNA and protein in the injection area. Finally, by use of an in vitro model with cell cultures of meningeal fibroblast and astrocyte origin, resembling the lesion area cellular content after spinal cord injury but devoid of inflammatory cells, we showed that VEGF is expressed in this in vitro model cell system after treatment with PGF2α and prostaglandin E2 (PGE2). These data suggest that cells within a lesion area in the spinal cord are capable of expressing VEGF and its receptors in response to mechanical injury and that prostaglandins may induce VEGF expression in such cells, even in the absence of inflammatory cells.
Transient changes in cortical distribution of S100 proteins during reorganization of somatotopy in the primary motor cortex induced by facial nerve transection in adult rats
Authors: Landgrebe Michael , Laskawi Rainer , Wolff Joachim R.
Published: March 2003

In adult rats, the primary motor cortex (MI) comprises a somatotopic map of muscle representations. This somatotopy is modified after transection of the facial nerve (N7x). Mapping with cortical stimulation revealed that the underlying cortical reorganization is biphasic. Primary changes cause a transient disinhibition of long cortico‐cortical connections in both hemispheres. While the first reaction vanishes within a few hours, short intra‐areal connections are disinhibited within MI contralateral to N7x. The resulting co‐operation between adjacent parts of MI persists as long as peripheral reinnervation is prevented. Cellular mechanisms underlying this cortical reorganization are largely unknown. Here, we utilized changes in immunoreactivity of S100 proteins (S100‐IR) known as a sensitive indicator of astroglial reactions during plastic reactions in the central nervous system. Within 1 h of N7x, zones with enhanced S100‐IR appeared in both hemispheres. Between 3.5 and 18 h, reaction patterns with changing topography were transiently prominent in many cortical areas including parts of MI which surrounded the facial muscle representation fields. After 24 h, the facial muscle representation contralateral to N7x became labelled while S100‐IR enhancement disappeared in most of the cortex. S100‐IR‐enhancement vanished completely during the next day of survival. Data presented suggest that (i) enhancement of S100‐IR labels cortical tissue during the functional reorganization that is induced by N7x, (ii) large parts of the cerebral cortex participate in the reorganization, before it is finally focused on the representation field of MI that corresponds with contralateral N7x, and (iii) temporo‐spatial patterns of astrocytic reactions apparently play a role in the underlying plasticity reaction.
Age‐dependent pre‐ and postsynaptic distribution of AMPA receptors at synapses in CA3 stratum radiatum of hippocampal slice cultures compared with intact brain
Authors: Fabian‐Fine Ruth , Volknandt Walter , Fine Alan , Stewart Michael G.
Published: March 2003

Organotypic slice cultures of rat hippocampus are widely used as experimental preparations for the study of synaptic plasticity, but their degree of correspondence with intact brain is not fully known. Here, using postembedding immunogold labelling, we describe the ultrastructural distribution of AMPA‐type glutamate receptors (GluR1–4) in CA3 stratum radiatum of organotypic hippocampal slice cultures at 10 days to 11 weeks in vitro and compare the labelling with intact brain of corresponding age. In both types of preparation, the 11‐week‐old samples contained the highest proportion of AMPA receptor‐like immunoreactive synapses. The incidence of labelled synapses, however, was higher in vivo (49%) than in vitro (24%). The intensity of labelling (number of gold particles per labelled synapse) also increased with age and was also higher in vivo than in vitro. In both organotypic cultures and intact brain, labelling was frequently found at presynaptic sites, often attached to vesicular structures. The specificity of these findings was supported both by light microscopic immunolabelling of GluR2/3 subunits and by electron microscopic double labelling of different epitopes of the GluR2 subunit. The vesicular localization of AMPA receptors was supported by Western blot analysis of subcellular fractions. Morphological evidence of presynaptic excitatory innervation of glutamatergic neurons supports a functional role for presynaptically located AMPA receptors. Our results therefore suggest that AMPA receptors occur in both pre‐ and postsynaptic profiles and that the distribution of AMPA receptors in cultured brain slices is fundamentally similar to intact brain, but that synaptic maturation may be retarded in vitro.
Two splice variants of the hypoxia‐inducible factor HIF‐1α as potential dimerization partners of ARNT2 in neurons
Authors: Drutel Guillaume , Kathmann Markus , Héron Anne , Gros Claude , Macé Séverine , Schwartz Jean‐Charles , Arrang Jean‐Michel
Published: March 2003

The hypoxia‐inducible factor (HIF‐1α), a basic helix‐loop‐helix transcription factor, is known to heterodimerize with ARNT1, a nuclear translocator, to trigger the overexpression in many cells of genes involved in resistance to hypoxia. Although HIF‐1α and ARNT1 are both expressed in brain, their cellular localization and function therein are unknown. Here, using in situ hybridization and immunocytochemistry, we show that HIF‐1α is expressed in normoxic cerebral neurons together with not only ARNT1 but also ARNT2, a cerebral translocator homologous to ARNT1 but displaying, unlike ARNT1, a selective neuronal expression. In contrast, other potential partners of the translocators, i.e. the aryl hydrocarbon receptor (AHR) and the single‐minded protein 2 (SIM2), are not expressed in the adult brain. We also identify two splice variants of HIF‐1α in brain, one of which dimerizes with ARNT2 even more avidly than with ARNT1. The resulting heterodimer, in contrast with the HIF‐1α/ARNT1 complex, does not recognize the HIF‐1‐binding site of the hypoxia‐induced erythropoietin (Epo) gene, suggesting that it controls transcription of a distinct set of genes. We therefore propose that HIF‐1α and ARNT2 function as preferential dimerization partners in neurons to control specific responses, some of which may not be triggered by hypoxia. In support of this proposal, in nonhypoxic PC12 cells constitutively coexpressing HIF‐1α, ARNT1 and ARNT2, downregulation of either HIF‐1α or ARNT2, obtained with selective antisense nucleotides, resulted in inhibition of [3H]thymidine incorporation.
Modulation of histamine H3 receptors in the brain of 6‐hydroxydopamine‐lesioned rats
Authors: Anichtchik Oleg V. , Huotari Marko , Peitsaro Nina , Haycock John W. , Männistö Pekka T. , Panula Pertti
Published: March 2003

Parkinson's disease is a major neurological disorder that primarily affects the nigral dopaminergic cells. Nigral histamine innervation is altered in human postmortem Parkinson's disease brains. However, it is not known if the altered innervation is a consequence of dopamine deficiency. The aim of the present study was to investigate possible changes in the H3 receptor system in a well‐characterized model of Parkinson's disease – the 6‐hydroxydopamine (6‐OHDA) lesioned rats. Histamine immunohistochemistry showed a minor increase of the fibre density index but we did not find any robust increase of histaminergic innervation in the ipsilateral substantia nigra on the lesioned side. In situ hybridization showed equal histidine decarboxylase mRNA expression on both sides in the posterior hypothalamus. H3 receptors were labelled with N‐alpha‐[3H]‐methyl histamine dihydrochloride ([3H] NAMH). Upregulation of binding to H3 receptors was found in the substantia nigra and ventral aspects of striatum on the ipsilateral side. An increase of GTP‐γ‐[35S] binding after H3 agonist activation was found in the striatum and substantia nigra on the lesioned side. In situ hybridization of H3 receptor mRNA demonstrated region‐specific mRNA expression and an increase of H3 receptor mRNA in ipsilateral striatum. Thus, the histaminergic system is involved in the pathological process after 6‐OHDA lesion of the rat brain at least through H3 receptor. On the later stages of the neurotoxic damage, less H3 receptors became functionally active. Increased H3 receptor mRNA expression and binding may, for example, modulate GABAergic neuronal activity in dopamine‐depleted striatum.
Development of NMDA and non‐NMDA receptor‐mediated excitatory synaptic transmission in geniculocortical and corticocortical connections in organotypic coculture preparations
Authors: Yamada Katsuya , Yamamoto Nobuhiko , Toyama Keisuke
Published: March 2003

Development of N‐methyl‐ d‐aspartate (NMDA) and non‐NMDA receptor‐mediated excitatory synaptic transmission was studied in the visual cortex using organotypic slice cocultures. A slice of visual cortex (VC) dissected from newborn rats was cocultured with either a chunk of embryonic lateral geniculate nucleus (LGN) or another VC. During 7–38 days in vitro (DIV), geniculocortical monosynaptic excitatory postsynaptic potentials (EPSPs) were recorded from layer IV neurons in response to stimulation of the LGN in cocultures of the VC with the LGN. Similarly, corticocortical monosynaptic EPSPs were recorded from layers II/III and V/VI neurons in cocultures of two VCs when stimulating the partner VC. The initial slopes of the non‐NMDA and NMDA receptor‐mediated components of the EPSPs, which were dissociated pharmacologically, were assessed and compared among three different culture stages, early (7–11 DIV), middle (12–15 DIV) and late (17–38 DIV). With progression of the culture stage, the non‐NMDA component tended to increase in both the geniculocortical and corticocortical connections. In contrast, the NMDA component exhibited distinct developmental changes. The NMDA component in layer IV neurons, which receive geniculate inputs, showed a transient increase in the middle stage. In the corticocortical connection, the magnitude of the NMDA component was large in the early stage and maintained through all culture stages in layer V/VI cells, whereas in layer II/III cells it decreased sharply by the late stage. Our results suggest that glutamatergic transmission in the visual cortex develops differently in the geniculocortical and corticocortical connections.
A quantitative analysis of l‐glutamate‐regulated Na+ dynamics in mouse cortical astrocytes: implications for cellular bioenergetics
Authors: Chatton Jean‐Yves , Marquet Pierre , Magistretti Pierre J.
Published: March 2003

The mode of Na+ entry and the dynamics of intracellular Na+ concentration ([Na+]i) changes consecutive to the application of the neurotransmitter glutamate were investigated in mouse cortical astrocytes in primary culture by video fluorescence microscopy. An elevation of [Na+]i was evoked by glutamate, whose amplitude and initial rate were concentration dependent. The glutamate‐evoked Na+ increase was primarily due to Na+‐glutamate cotransport, as inhibition of non‐NMDA ionotropic receptors by 6‐cyano‐7‐nitroquinoxiline‐2,3‐dione (CNQX) only weakly diminished the response and d‐aspartate, a substrate of the glutamate transporter, produced [Na+]i elevations similar to those evoked by glutamate. Non‐NMDA receptor activation could nevertheless be demonstrated by preventing receptor desensitization using cyclothiazide. Thus, in normal conditions non‐NMDA receptors do not contribute significantly to the glutamate‐evoked Na+ response. The rate of Na+ influx decreased during glutamate application, with kinetics that correlate well with the increase in [Na+]i and which depend on the extracellular concentration of glutamate. A tight coupling between Na+ entry and Na+/K+ ATPase activity was revealed by the massive [Na+]i increase evoked by glutamate when pump activity was inhibited by ouabain. During prolonged glutamate application, [Na+]i remains elevated at a new steady‐state where Na+ influx through the transporter matches Na+ extrusion through the Na+/K+ ATPase. A mathematical model of the dynamics of [Na+]i homeostasis is presented which precisely defines the critical role of Na+ influx kinetics in the establishment of the elevated steady state and its consequences on the cellular bioenergetics. Indeed, extracellular glutamate concentrations of 10 μm already markedly increase the energetic demands of the astrocytes.
The lethal expression of the GluR2flip/GluR4flip AMPA receptor in HEK293 cells
Authors: Iizuka Masaki , Nishimura Seiichiro , Wakamori Minoru , Akiba Isamu , Imoto Keiji , Barsoumian Edward L.
Published: March 2003

α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) ‐type glutamate receptors play a critical role in excitotoxicity associated with cerebral hypoxia, ischaemia and other acute brain insults. AMPA receptors are composed of GluR1–GluR4 subunits in homomeric and heteromeric assemblies, forming nonselective cation channels. In addition, each subunit has alternative splice variants, flip and flop forms. Heterologous expression studies showed that the AMPA receptor channels exhibit diverse properties depending on subunit/variant composition. For example, the absence of the GluR2 subunit makes AMPA receptor assemblies Ca2+‐permeable. Excitotoxicity induced by activating AMPA receptor channels has been linked to excessive Ca2+ influx through the GluR2‐lacking channels. Here we demonstrate that coexpression of the AMPA receptor GluR2flip and GluR4flip subunits exerts a lethal effect on HEK293 cells, whereas no lethal activity is observed in other homomeric or heteromeric combinations of AMPA receptor subunits. Patch clamp recordings and Ca2+ imaging analyses have revealed that this GluR2flip/GluR4flip receptor exhibits a low Ca2+ permeability. This subunit combination, however, showed prolonged Na+ influx following AMPA stimulation, even in the absence of cyclothiazide, which attenuates AMPA receptor desensitization. Furthermore, the GluR2flip/GluR4flip‐mediated lethality was potentiated by the interruption of cellular Na+ extrusion mechanisms using ouabain or benzamil. These observations suggest that the GluR2flip/GluR4flip receptor‐mediated excitotoxicity is attributed to Na+ overload, but not Ca2+ influx.
Formation of mixed glycine and GABAergic synapses in cultured spinal cord neurons
Authors: Dumoulin A. , Lévi S. , Riveau B. , Gasnier B. , Triller A.
Published: March 2003

In the spinal cord, GABA and glycine mediate inhibition at separate or mixed synapses containing glycine and/or GABAA receptors (GlyR and GABAAR, respectively). We have analysed here the sequence of events leading to inhibitory synapse formation during synaptogenesis of embryonic spinal cord neurons between 1 and 11 days in vitro (DIV). We used immunocytochemical methods to detect simultaneously an antigen specific to inhibitory terminals, the vesicular inhibitory amino acid transporter (VIAAT), and one of the following postsynaptic elements: GlyR, GABAAR or gephyrin, the anchoring protein of GlyR, which is also associated with GABAAR. Quantitative analysis revealed that until 5 DIV most gephyrin clusters were not adjacent to VIAAT‐positive profiles, but became associated with them at later stages. In contrast, GlyR and GABAAR clustered predominantly in front of VIAAT‐containing terminals at all stages. However, about 10% of receptor aggregates were detected at nonsynaptic loci. The two receptors colocalized in 66.2 ± 2.5% of the inhibitory postsynaptic domains after 11 DIV, while 30.3 ± 2.6% and 3.4 ± 0.8% of them contained only GlyR and GABAAR, respectively. Interestingly, at 3 DIV GABAAR clustered at a postsynaptic location prior to gephyrin and GlyR; GABAAR could thus be the initiating element in the construction of mixed glycine and GABAergic synapses. The late colocalization of gephyrin with GABAAR, and the demonstration by other groups that, in the absence of gephyrin, postsynaptic GABAAR is not detected, suggest that gephyrin is involved in the stabilization of GABAAR rather than in its initial accumulation at synaptic sites.
Reducing conditions significantly attenuate the neuroprotective efficacy of competitive, but not other NMDA receptor antagonists in vitro
Authors: Pringle Ashley K. , Self James , Eshak Marie , Iannotti Fausto
Published: March 2003

Inappropriate activation of NMDA receptors during a period of cerebral ischaemia is a crucial event in the pathway leading to neuronal degeneration. However, significant research has failed to deliver a clinically active NMDA receptor antagonist, and competitive NMDA antagonists are ineffective in many experimental models of ischaemia. The NMDA receptor itself has a number of modulatory sites which may affect receptor function under ischaemic conditions. Using rat organotypic hippocampal slice cultures we have investigated whether the redox modulatory site affects the neuroprotective efficacy of NMDA receptor antagonists against excitotoxicity and experimental ischaemia (OGD). NMDA toxicity was significantly enhanced in cultures pretreated with a reducing agent. The noncompetitive antagonist MK‐801 and a glycine‐site blocker were equally neuroprotective in both normal and reduced conditions, but there was a significant rightward shift in the dose–response curves of the competitive antagonists APV and CPP and the uncompetitive antagonist memantine. OGD produced neuronal damage predominantly in the CA1 region, which was prevented by MK‐801 and memantine, but not by APV or CPP. Inclusion of an oxidizing agent during the period of OGD had no effect alone, but significantly enhanced the neuroprotective potency of the competitive antagonists. These data clearly demonstrate that chemical reduction of the redox modulatory site of the NMDA receptor decreases the ability of competitive antagonists to block NMDA receptor‐mediated neuronal damage, and that the reducing conditions which occur during simulated ischaemia are sufficient to produce a similar effect. This may have important implications for the design of future neuroprotective agents.
Attenuation and augmentation of ischaemia‐related neuronal death by tumour necrosis factor‐α in vitro
Authors: Wilde Geraint J. C. , Pringle Ashley K. , Sundstrom Lars E. , Mann Derek A. , Iannotti Fausto
Published: March 2003

Upregulation of the pro‐inflammatory cytokine tumour necrosis factor‐α (TNF) occurs rapidly in the brain following ischaemia, although it is unclear whether this represents a neurotoxic or neuroprotective response. We have investigated whether TNF has different actions in the pre‐ and postischaemic periods in a tissue culture model of cerebral ischaemia. Organotypic hippocampal slice cultures were prepared from 8–10‐day‐old rats and maintained in vitro for 14 days. Neuronal damage was induced by either 1 h oxygen–glucose deprivation or 3 h exposure to NMDA or the superoxide generator duroquinone, and assessed after 24 h by propidium iodide fluorescence. TNF pretreatment was neuroprotective against both oxygen–glucose deprivation and duroquinone. This effect was associated with an activation of the transcription factor NFκB and upregulation of manganese superoxide dismutase, and was prevented by a free radical scavenger. When addition of TNF was delayed until the postinsult period, an exacerbation of neurotoxicity occurred, which was also prevented by a free radical scavenger. The actions of TNF are determined by whether TNF is present before or after an ischaemia‐related insult. Both actions are mediated through the production of free radicals, and the response to TNF is determined by whether a cell is metabolically competent to respond by synthesis of antioxidant defences.
Preservation of a functional nigrostriatal dopamine pathway by GDNF in the intrastriatal 6‐OHDA lesion model depends on the site of administration of the trophic factor
Authors: Kirik Deniz , Rosenblad Carl , Björklund Anders
Published: March 2003

Here we studied whether glial cell line‐derived neurotrophic factor (GDNF), given as a single bolus injection before an intrastriatal 6‐hydroxydopamine (6‐OHDA) lesion, can protect the nigrostriatal dopamine neurons against the toxin‐induced damage and preserve normal motor functions in the lesioned animals. GDNF or vehicle was injected in the striatum (25 μg), substantia nigra (25 μg) or lateral ventricle (50 μg) 6 h before the 6‐OHDA lesion (20 μg/3 μL). Motor function was evaluated by the stepping and drug‐induced motor asymmetry tests. Lesioned animals given vehicle alone showed a clear ipsilateral‐side bias in response to amphetamine (13 turns/min), a moderate contralateral‐side bias to apomorphine (4.5 turns/min) and a moderate to severe stepping deficit on the contralateral forepaw (three to four steps, as compared with 11–13 steps on the unimpaired side). Injection of GDNF into the striatum had a significant protective effect both on nigrostriatal function (1–2 turns/min in the rotation tests and seven to eight steps in the stepping test), and the integrity of the nigrostriatal pathway, seen as a protection of both the cell bodies in the substantia nigra and the dopamine innervation in the striatum. Injection of GDNF in the nigra had a protective effect on the nigral cell bodies, but not the striatal innervation, and failed to provide any functional benefit. In contrast, intranigral GDNF had deleterious effects on both the striatal TH‐positive fibre density and on drug‐induced rotation tests. Intraventricular injection had no effect. We conclude that preservation of normal motor functions in the intrastriatal 6‐OHDA lesion model requires protection of striatal terminal innervation, and that this can be achieved by intrastriatal, but not nigral or intraventricular, administration of GDNF.
Low‐threshold heat response antagonized by capsazepine in chick sensory neurons, which are capsaicin‐insensitive
Authors: Marín‐Burgin Antonia , Reppenhagen Stephan , Klusch Andreas , Wendland Jens R. , Petersen Marlen
Published: March 2003

The heat‐transducing receptor VR1 cloned from rat sensory neurons can be activated by both noxious heat and capsaicin. As the response of sensory neurons to capsaicin is species dependent, it is conceivable that the responses to noxious heat and to capsaicin are transduced by distinct receptors across different species. Therefore, we investigated responses to noxious heat from a capsaicin‐insensitive (chick) and a capsaicin‐sensitive (rat) species. In chick, whole‐cell patch‐clamp experiments in isolated dorsal root ganglion neurons revealed two populations of neurons with different thresholds to noxious heat, activated at ≈ 43 °C and ≈ 53 °C. In cobalt uptake experiments, the proportion of neurons showing a heat‐induced response increased with increasing heat stimuli. Application of capsaicin (1–10 μm) did not result in inward currents or cobalt uptake. Rat neurons yielded comparable results in heat experiments, but were capsaicin‐sensitive. Although chick neurons are insensitive to capsaicin, the competitive capsaicin antagonist capsazepine (1–10 μm) was effective in blocking heat‐induced responses, verified by patch‐clamp and cobalt uptake methods. The noncompetitive capsaicin antagonist ruthenium red (10 μm) reduced to almost nil the proportion of heat‐responsive neurons identified with the cobalt uptake method. These findings suggest that chick DRG neurons express a low‐threshold heat‐transducing receptor with a pharmacological profile distinct from the low‐threshold heat receptor VR1 cloned from rat DRG neurons. The data support the idea that there might be heat receptor subtypes with differences in the capsaicin binding site.
The Calliphora rpa mutant lacks the PDZ domain‐assembled INAD signalling complex
Authors: Huber Armin , Belusic Gregor , Da Silva Noel , Bähner Monika , Gerdon Gabriele , Draslar Kazimir , Paulsen Reinhard
Published: March 2003

The visual transduction cascade of fly photoreceptors is a G protein‐coupled phospholipase C‐signalling pathway which is assembled into a supramolecular signalling complex by the PDZ (postsynaptic density protein‐95, discs large, Z0–1) domain protein INAD (inactivation no afterpotential D). The norpA‐encoded phospholipase Cβ, the light‐activated transient receptor potential (TRP) Ca2+ channel and an eye‐specific protein kinase C are bound to INAD and together form the core of the signalling complex. In the present study we show that the Calliphora rpa mutant, which has previously been hypothesized to represent an equivalent of Drosophila norpA mutants, has normal amounts of norpA mRNA but fails to express inaD mRNA. Electrophysiological recordings from the eyes of the rpa mutant reveal that the electroretinogram is reduced (about 12% of wild type) but not completely absent, and that it exhibits markedly prolonged deactivation kinetics. Furthermore, rpa mutants display a slow, light‐dependent degeneration of the photoreceptor cells. With respect to the INAD signalling complex, the rpa mutant is similar to the Drosophila inaD null mutant: not only INAD itself, but also the other core components of the INAD signalling complex, are reduced or absent in photoreceptor membranes of rpa flies. Residual TRP is localized throughout the plasma membrane of the photoreceptor cell, rather than being restricted to the microvillar photoreceptor membrane. [35S]methionine‐labelling of newly synthesized retinal proteins reveals that TRP is synthesized in the rpa mutant at wild‐type level, but is transported to or incorporated into the microvillar photoreceptor membrane at a much lower rate. We thus suggest, that the formation of the INAD signalling complex is required for specifically targeting its components to the photoreceptor membrane.
AMPA/kainate receptor‐triggered Zn2+ entry into cortical neurons induces mitochondrial Zn2+ uptake and persistent mitochondrial dysfunction
Authors: Sensi Stefano L. , Yin Hong Z. , Weiss John H.
Published: March 2003

Rapid Zn2+ influx through Ca2+‐permeable AMPA/kainate (Ca‐A/K) channels triggers reactive oxygen species (ROS) generation and is potently neurotoxic. The first aim of this study was to determine whether these effects might result from direct mitochondrial Zn2+ uptake. Adapting the mitochondrially sequestered divalent cation sensitive probe, rhod‐2, to visualize mitochondrial Zn2+, present studies indicate that Zn2+ is taken up into these organelles. The specificity of the signal for Zn2+ was indicated by its reversal by Zn2+ chelation, and its mitochondrial origin indicated by its speckled extranuclear appearance and by its elimination upon pretreatment with the mitochondrial protonophore, carbonyl cyanide p‐(trifluoromethoxy)phenylhydrazone (FCCP). Consistent with inhibition of mitochondrial Zn2+ uptake, FCCP also slowed the recovery of cytosolic Zn2+ elevations in Ca‐A/K(+) neurons. Further studies sought clues to the high toxic potency of intracellular Zn2+. In experiments using the mitochondrial membrane polarization (ΔΨm)‐sensitive probe tetramethylrhodamine ethyl ester and the ROS‐sensitive probe hydroethidine, brief kainate exposures in the presence of 300 μm Zn2+ (with or without Ca2+) resulted in prolonged loss of ΔΨm and corresponding prolonged ROS generation in Ca‐A/K(+) neurons, in comparison to the more rapid recovery from loss of ΔΨm and transient ROS generation after kainate/1.8 mm Ca2+ exposures.
The spatial organization of cholinergic mosaics in the adult mouse retina
Authors: Galli‐Resta Lucia , Novelli Elena , Volpini Maila , Strettoi Enrica
Published: March 2003

We analysed the spatial organization of the cholinergic amacrine cell mosaics in the mouse retina, as part of a general study of the major mouse retinal arrays, aiming at providing intrinsic cellular reference grids to monitor anomalies in retinal growth and/or functional organization in mouse models of retinal degeneration. The spatial organization of the cells was analysed by means of the nearest neighbour distance analysis, as well as by the analysis of Voronoi and Delaunay tesselations. We found non random cell spacing in both cholinergic arrays, although the mosaic in the ganglion cell layer tiles the retina scarcely better than a random distribution. Autocorrelation analysis revealed no detectable pattern in cell positioning, but there was a tendency towards a minimal spacing between array elements. Finally, we found no correlation in the spatial organization of the two arrays.
Auditory fear conditioning increases CS‐elicited spike firing in lateral amygdala neurons even after extensive overtraining
Authors: Maren Stephen
Published: March 2003

We examined the influence of extensive overtraining (75 trials) of auditory fear conditioning on the expression of conditional stimulus (CS)‐elicited spike firing in lateral amygdala (LA) neurons. Single units were recorded from chronic multichannel electrodes implanted in the LA of conscious and freely moving rats. In sequential training sessions, the rats received either five or 70 fear conditioning trials, which consisted of a white‐noise CS and a coterminating footshock unconditional stimulus (US). Unpaired (sensitization) controls received the same number of trials, but the CS and US were explicitly unpaired. Paired CS–US presentations yielded robust increases in CS‐elicited spike firing in LA neurons after both five and 70 conditioning trials, and the magnitude of the spike firing increases was correlated with the expression of conditional freezing to the CS. After 75 training trials, maximal conditioning‐related increases in LA firing were exhibited within 20 ms of CS onset, indicating that this increase is mediated by direct thalamo‐amygdala projections. There was no significant increase in CS‐elicited spike firing or freezing behaviour in the unpaired group. These results complement amygdala lesion studies [e.g. Maren, S. (1999a) J. Neurosci., 19, 8696–8703] and support the view that the basolateral complex of the amygdala is involved in the encoding and storage of fear memories even after extensive overtraining.
α‐Latrotoxin forms calcium‐permeable membrane pores via interactions with latrophilin or neurexin
Authors: Van Renterghem Catherine , Iborra Cécile , Martin‐Moutot Nicole , Lelianova Vera , Ushkaryov Yuri , Seagar Michael
Published: March 2003

In order to explore the mechanisms by which α‐latrotoxin activates neurotransmitter release, we have characterized its effects by patch‐clamp methods on cells heterologously expressing its receptors, latrophilin‐1 or neurexin‐Iα. Application of α‐latrotoxin (1 n m) to cells expressing rat latrophilin or neurexin, but not mock‐transfected cells, induced a cationic conductance. In cells expressing latrophilin, current development was slow in the absence of divalent cations, but was accelerated by Ca2+ or Mg2+. In cells expressing neurexin, α‐latrotoxin did not elicit currents in the absence of Ca2+. The toxin‐induced conductance was rectifying, persistent, permeable to monovalent and divalent cations, but blocked by La3+. Single‐channel recording revealed a permanently open state, with the same unitary conductance irrespective of whether cells expressed latrophilin or neurexin. Therefore, while pore formation displayed differences consistent with the reported properties of α‐latrotoxin binding to latrophilin and neurexin, the pores induced by α‐latrotoxin had identical properties. These results suggest that after anchoring to either of its nerve terminal receptors, α‐latrotoxin inserts into the membrane and constitutes a single type of transmembrane ion pore.
Distribution of spinocerebellar Purkinje cell responses to passive forelimb movements in the rat
Authors: Bosco Gianfranco , Giaquinta Genoveffa , Valle Maria Stella , Caserta Cinzia , Casabona Antonino , Perciavalle Vincenzo
Published: March 2003

We recorded Purkinje cell activity throughout the spinocerebellum of anaesthetized rats while imposing circular passive movements to the unrestrained forelimb. The aim was to understand the type of processing of sensory information occurring at the level of the cerebellar cortex, on the basis that precerebellar sensory neurons have been shown to represent whole limb movement parameters better than single joint movements. We observed that neurons representing sensory aspects of arm movements were scattered throughout the spinocerebellar cortex without a distinct segregation from those that did not respond, albeit the relative density of responsive and unresponsive neurons was quite variable and depended on the area of the cortex. Furthermore, Purkinje cells that responded significantly to the arm movement cycles all showed the same response pattern consisting of a firing rate increase during the downward extension of the arm. These results are discussed as suggesting a coordinate framework for the representation of proprioceptive information in the cerebellum congruent to that observed for encoding motor parameters.
Coupled intra‐ and extracellular Ca2+ dynamics in recurrent seizure‐like events
Authors: Szilágyi Nóra , Kovács Richárd , Kardos Julianna
Published: March 2003

We considered the evolution of Ca2+ oscillation dynamics in recurrent seizure‐like events. Dynamic system behaviour was characterized in the state space reconstructed from intra‐ and extracellular [Ca2+] fluctuations simultaneously measured in cultured rat hippocampal slices under low‐[Mg2+] conditions. When associated in the seizure‐like event, these fluctuations occurred on a restricted set, the attractor, embedded in the full state space with less than five degrees of freedom. Instantaneous relative phase differences indicated field potential‐driven phase jumps locked onto seizure‐like events. To account for recurrent dynamics, calculations were performed on different extensions of a model for Ca2+ oscillation. These identified bidirectional, asymmetrical coupling of extracellular with intracellular (cytosolic, Ca2+ store, mitochondrial) Ca2+ dynamics as critical in its development.
Novel mode of nitric oxide neurotransmission mediated via S‐nitroso‐cysteinyl‐glycine
Authors: Salt T. E. , Zhang H. , Mayer B. , Benz B. , Binns K. E. , Do K. Q.
Published: March 2003

S‐nitroso‐cysteinyl‐glycine, a novel nitric oxide‐adduct thiol compound, can be detected in the brain (2.3 ± 0.6 pmol/mg protein), and released following stimulation of sensory afferents to the rat ventrobasal thalamus in vivo (resting conditions 17 n m; stimulation: 186 nm). Iontophoretic application of CysNOGly (20–80 nA) onto thalamic neurons in vivo resulted in enhancements of excitatory responses to either NMDA or AMPA (182 ± 13.6% and 244 ± 27.8% of control values, n = 15). CysNOGly enhanced responses to stimulation of vibrissal afferents to 132 ± 2.2% (n = 7) of control values. In contrast, the dipeptide CysGly reduced responses of ventrobasal neurons to NMDA and AMPA (54 ± 8.4% and 55 ± 10.8% of control, n = 5). CysNOGly was also a potent activator of soluble guanylate cyclase in vitro. Moreover, we found that NMDA elevated CysNOGly levels in vitro and this stimulatory effect was reduced by inhibitors of the neuronal NO synthase and of the γ‐glutamyl transpeptidase, suggesting that production of NO and CysGly is a prelude to CysNOGly synthesis. These findings suggest that the nitrosothiol CysNOGly plays a role in synaptic transmission in the ventrobasal thalamus. We propose a novel synaptic buffering mechanism where S‐nitroso‐cysteinyl‐glycine serves to restrict the locus of action of nitric oxide and so increase its local availability for target delivery. This could lead to a change in neuronal responses favouring sensory transmission similar to that seen in wakefulness or arousal in order to locally enhance transmission of persistent sensory stimuli.
A novel brain receptor is expressed in a distinct population of olfactory sensory neurons
Authors: Conzelmann Sidonie , Levai Olga , Bode Barbara , Eisel Ulrich , Raming Klaus , Breer Heinz , Strotmann Jörg
Published: March 2003

Three novel G‐protein‐coupled receptor genes related to the previously described RA1c gene have been isolated from the mouse genome. Expression of these genes has been detected in distinct areas of the brain and also in the olfactory epithelium of the nose. Developmental studies revealed a differential onset of expression: in the brain at embryonic stage 17, in the olfactory system at stage E12. In order to determine which cell type in the olfactory epithelium expresses this unique receptor type, a transgenic approach was employed which allowed a coexpression of histological markers together with the receptor and thus visualization of the appropriate cell population. It was found that the receptor‐expressing cells were located very close to the basal membrane of the epithelium; however, the cells extended a dendritic process to the epithelial surface and their axons projected into the main olfactory bulb where they converged onto two or three glomeruli in the dorsal and posterior region of the bulb. Thus, these data provide evidence that this unique type of receptor is expressed in mature olfactory neurons and suggests that it may be involved in the detection of special odour molecules.
Cocaine, but not morphine, induces conditioned place preference and sensitization to locomotor responses in CB1 knockout mice
Authors: Martin Miquel , Ledent Catherine , Parmentier Marc , Maldonado Rafael , Valverde Olga
Published: March 2003

The involvement of cannabinoid CB1 receptors in morphine and cocaine motivational effects was investigated using CB1 knockout mice. For this purpose, we evaluated the rewarding effects in the place conditioning paradigm and the sensitization to the locomotor responses induced by these drugs. The hyperlocomotion induced by acute morphine administration (15 mg/kg, s.c.) was preserved, but the sensitization to this locomotor response induced by chronic morphine treatment was abolished in CB1 mutant mice. Morphine (5 mg/kg, s.c.) induced conditioned place preference in wild‐type mice but failed to produce any response in knockout mice, indicating the inability of morphine to induce rewarding effects in the absence of CB1 cannabinoid receptors. When the aversive effects of morphine withdrawal were investigated using the place aversion paradigm, no differences between genotypes were observed. Acute cocaine (10 mg/kg, i.p.) induced hyperlocomotor responses in wild‐type and knockout mice and a chronic cocaine treatment produced a similar sensitization to this response in both genotypes. In the conditioning place preference paradigm, cocaine (20 mg/kg, i.p.) produced rewarding responses in both wild‐type and knockout mice. These results demonstrate that CB1 receptors are essential for adaptive responses produced by chronic morphine but not by chronic cocaine treatment.
Electrophysiological and behavioural evidence for an antagonistic modulatory role of adenosine A2A receptors in dopamine D2 receptor regulation in the rat dopamine‐denervated striatum
Authors: Strömberg Ingrid , Popoli Patrizia , Müller Christa E. , Ferré Sergi , Fuxe Kjell
Published: March 2003

It has been shown that striatal adenosine A2A receptors can antagonistically interact with dopamine D2 receptors at the membrane level leading to a decrease in the affinity and efficacy of D2 receptors. Extracellular recordings and rotational behaviour were employed to obtain a correlate to these findings in an animal model of Parkinson's disease (PD). The recordings were performed in rats with unilateral 6‐hydroxydopamine (6‐OHDA)‐induced catecholamine depletion. While recording in the dopamine‐depleted striatum, local applications of the dopamine D2 agonist quinpirole reduced neuronal activity. However, when the adenosine A2A antagonist MSX‐3 was applied simultaneously with quinpirole, the inhibition of neuronal firing seen after quinpirole alone was significantly potentiated (P < 0.001, n = 11). In contrast, local application of CGS 21680 attenuated the effect of quinpirole. The doses of MSX‐3 and CGS 21680 used to achieve the modulation of quinpirole action had no effect per se on striatal neuronal firing. Furthermore, rotational behaviour revealed that MSX‐3 dose‐dependently increased the number of turns when administrated together with a threshold dose of quinpirole while no enhancement was achieved when MSX‐3 was combined with SKF 38393. MSX‐3 alone did not induce rotational behaviour. In conclusion, this study shows that low ineffective doses of MSX‐3 enhance the effect of quinpirole on striatal firing rate, while the A2A agonist exerts the opposite action. This mechanism gives a therapeutic potential to A2A antagonists in the treatment of PD by enhancing D2 receptor function.
Subcellular localization of m2 muscarinic receptors in GABAergic interneurons of the olfactory bulb
Authors: Crespo Carlos , Blasco‐Ibáñez José Miguel , Briñón Jesús G. , Alonso José Ramón , Domínguez María Isabel , Martínez‐Guijarro Francisco José
Published: March 2003

We analysed the ultrastructural distribution of the m2 muscarinic receptor (m2R) in the rat olfactory bulb (OB) using immunohistochemical techniques and light and electron microscopy. m2R was differentially distributed within the cellular compartments of γ‐aminobutyric acid (GABA)ergic bulbar interneurons. It is located in the gemmules of granule cells and in the synaptic loci of the interneurons of the external plexiform layer, suggesting that m2R activation could modulate the release of GABA from these interneurons onto principal cells by a presynaptic mechanism. By contrast, the receptor appears in the somata and dendritic trunks of second‐order short‐axon interneurons located in the inframitral layers, suggesting that postsynaptic muscarinic activation in these cells could elicit the inhibition of granule cells, leading to a disinhibition of principal cells. We also detail the anatomical substrate for a new putative muscarinic modulation that has not been previously described, and that could influence the reception of sensory information within the olfactory glomeruli. m2R appears in a subset of GABAergic/dopaminergic juxtaglomerular cells innervated by olfactory axons but is absent in juxtaglomerular cells that do not receive sensory inputs. This finding suggests that m2R activation could modify, through dopaminergic local circuits, the strength of olfactory nerve inputs onto principal cells. Activation of the muscarinic receptor may modulate the olfactory information encoding within olfactory glomeruli and may facilitate the bulbar transmission to superior centres influencing the GABA release by presynaptic and postsynaptic mechanisms. Taken together, our data provide the neuroanatomical basis for a complex action of m2R at different levels in the mammalian OB.
Oxytocin receptor agonists enhance inhibitory synaptic transmission in the rat hippocampus by activating interneurons in stratum pyramidale
Authors: Zaninetti Marc , Raggenbass Mario
Published: March 2003

Oxytocin probably plays a role as a neurotransmitter/neuromodulator in the hippocampus of the rat. Oxytocin binding sites are present in the subiculum and CA1 region and oxytocin can excite a class of CA1 nonpyramidal neurons. In the present work we characterized the effect of oxytocin on hippocampal synaptic transmission. Whole‐cell recordings were obtained from pyramidal neurons, in conditions of nearly symmetrical chloride concentrations. The selective oxytocin receptor agonist, [Thr4,Gly7]‐oxytocin (TGOT), caused an increase in the frequency and amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) in virtually all neurons. These peptide‐enhanced IPSCs were blocked by bicuculline, but not by strychnine, and reversed near 0 mV, indicating that they were mediated by γ‐aminobutyric acid (GABA)A receptors. On average, TGOT caused a nearly threefold increase in the frequency and almost a doubling in the amplitude of spontaneous IPSCs. TGOT did not influence the frequency and the amplitude of miniature IPSCs or spontaneous excitatory postsynaptic currents (EPSCs), and had no effect on evoked IPSCs. The peptide did not affect the basic membrane properties of pyramidal neurons or their GABA sensitivity. Thus, TGOT facilitated inhibitory transmission by exerting an excitatory action on the soma and/or dendrites of GABAergic interneurons. Extracellular recordings were performed in interneurons located in various hippocampal strata. Their sensitivity to TGOT was compared to that of substance P (SP). Interneurons in stratum pyramidale were excited both by TGOT and by SP. By contrast, stratum radiatum interneurons responded to SP but not to TGOT. In stratum oriens, half of the interneurons responded to SP, but only a minority to TGOT. Thus, oxytocin‐responsive interneurons appear to be preferentially located in close vicinity of pyramidal neurons.