bluuebunny/crossref_metadata_2025 huggingface dataset:
Serotonergic modulation of the responses to excitatory amino acids of rat dorsal horn neurons in vitro: implications for somatosensory transmission
Authors: Lopez‐Garcia
Published: March 2003

Serotonin (5‐HT) is one of the major transmitters involved in supraspinal control of somatic sensation and nociception. The aim of the present study was to investigate if the 5‐HT‐induced modulation of sensory transmission in the dorsal horn could be due to regulation of neuronal responses to excitatory amino acids. Experiments were performed in an in vitro preparation of the young rat spinal cord. Responses to dorsal root stimulation (DR‐EPSP) and to droplet application of N‐methyl‐d‐aspartic acid (NMDA) and α‐amino‐2,3‐dihydro‐5‐methyl‐3‐oxo‐4‐isoxazolepropanoic acid (AMPA) were obtained by means of intracellular recordings of dorsal horn neurons. Bath applications of 5‐HT (50 μm) generally caused reductions in amplitude and integrated area of DR‐EPSPs and of responses to NMDA but the responses to AMPA were unaltered. A linear correlation was found between the effects of 5‐HT on the DR‐EPSP and on the NMDA response measured as percentage change in amplitude (r2 = 0.45;P≤ 0.01) and integrated area (r2 = 0.77;P≤ 0.001). The NMDA receptor antagonist d‐AP5 (50 μm) completely abolished NMDA responses and caused a depression of the DR‐EPSP similar to that of 5‐HT. The 5‐HT1 receptor agonist 5‐carboxamidotryptamine (5‐CT; 1 μm) mimicked the depressant effects of 5‐HT but had a stronger depressant action on the DR‐EPSP than 5‐HT. The depression of NMDA responses induced by 5‐HT and 5‐CT was tetrodotoxin (1 μm) resistant. It is concluded that 5‐HT‐induced depression of NMDA responses explains partially the depressant action of 5‐HT on dorsal horn synaptic transmission activating a postsynaptic site sensitive to 5‐CT. The possible activation of coadjuvant mechanisms is discussed.
Hyperpolarizing after‐potentials regulate generation of long‐duration plateau depolarizations in rat paraventricular nucleus neurons
Authors: Bains Jaideep S. , Ferguson Alastair V.
Published: March 2003

Activation of N‐methyl‐d‐aspartate (NMDA) receptors in a population of neurons of the paraventricular nucleus (PVN) results in long‐duration plateau depolarizations during which the membrane rapidly depolarizes, reaching a stable plateau near −20 mV. These responses were observed in 29% of the Type II PVN neurons tested with 1 μm NMDA agonist (n = 84). The stable plateau phase is characterized by an increase in ionic conductance, from 1.19 ± 0.11 nS to 5.24 ± 2.17 nS (n = 5). Bath application of tetrodotoxin (n = 4) or alternatively inclusion of QX‐314 in the pipette solution (n = 3) prevented the generation of these events. The remaining cells tested (n = 56) also depolarized in response to NMDA agonist, but long duration plateau depolarizations were not observed.

Previous evidence from hypothalamic cultures has demonstrated synaptically driven plateau potentials following the blockade of repolarizing conductances. Pharmacological blockade of the post‐spike hyperpolarizing afterpotential with 4‐aminopyridine (200 μm), in cells that did not generate plateaux, resulted in the observance of long duration plateau depolarizations in response to a subsequent application of NMDA agonist (n = 4). Our results demonstrate that this 4‐aminopyridine‐sensitive ionic conductance plays a critical role in determining whether a cell will depolarize for a prolonged duration in response to NMDA receptor activation. As a prolonged depolarization of the postsynaptic membrane and accompanying membrane permeability changes are essential for neurotoxicity, these findings provide evidence for a potential protective mechanism that depends solely on the ability of the cell, through its ionic conductances, to control imposed changes in membrane potential.
A phenytoin‐sensitive cationic current participates in generating the afterdepolarization and burst afterdischarge in rat neocortical pyramidal cells
Authors: Kang Youngnam , Okada Takashi , Ohmori Harunori
Published: March 2003

We report here on the ionic mechanisms underlying the depolarizing afterpotential (DAP) in neocortical pyramidal cells, with special interest in those underlying the burst afterdischarge. Injections of short depolarizing current pulses under whole‐cell current clamp with a CsCl‐based internal medium generated, in most pyramidal cells, a single action potential with a plateau phase (plateau‐AP), followed by a slowly decaying DAP both in the absence and presence of TTX. Under voltage‐clamp, the same cells displayed a slow tail current (tail‐I) at the offset of depolarization. When intracellular free Ca2+ was chelated with 10 mm BAPTA or when extracellular Ca2+ was replaced with equimolar Ba2+, neither the slow DAP nor the slow tail‐I was observed. Extracellular application of Co2+ or Cd2+ reduced Ca2+ currents and the slow tail‐I. Cation substitution experiments revealed that the channel generating the slow tail‐I was permeable to K+ and Cs+ more than to Na+ (PK≈PCs > PNa > PNMDG≈PTEA). The cationic slow tail‐I was not reduced by applying antagonists of the metabotropic glutamate receptor (MCPG, 1 mm) and the muscarinic receptor (atropine, 1–10 μm). Thus, the slow DAP was produced by activation of the cationic channel whose gating is solely dependent on [Ca2+]i. An increase in [K+]o from 3 to 6 or 9 mm enhanced the slow DAP, and resulted in a generation of burst afterdischarges. An anticonvulsant, phenytoin (PT; 1–10 μm) suppressed the slow DAP while enhancing the plateau‐AP in the presence of TTX, most likely by blocking the cationic channel.
Presynaptic and postsynaptic localization of GABAB receptors in neurons of the rat retina
Authors: Koulen Peter , Malitschek Barbara , Kuhn Rainer , Bettler Bernhard , Wässle Heinz , Brandstätter Johann Helmut
Published: March 2003

The recently cloned GABAB receptors were localized in rat retina using specific antisera. Immunolabelling was detected in the inner and outer plexiform layers (IPL, OPL), and in a number of cells in the inner nuclear layer and the ganglion cell layer. Double‐labelling experiments for GABA (γ‐aminobutyric acid) and GABAB receptors, respectively, demonstrated a co‐localization in horizontal cells and amacrine cells. Electron microscopy showed that GABAB receptors of the OPL were localized presynaptically in horizontal cell processes invaginating into photoreceptor terminals. In the IPL, GABAB receptors were present presynaptically in amacrine cells, as well as postsynaptically in amacrine and ganglion cells. The postnatal development of GABAB receptors was also studied, and immunoreactivity was observed well before morphological and synaptic differentiation of retinal neurons. The present results suggest a presynaptic (autoreceptor) as well as postsynaptic role for GABAB receptors. In addition, the extrasynaptic localization of GABAB receptors could indicate a paracrine function of GABA in the retina.
NT‐4/5 and LIF, but not NT‐3 and BDNF, promote NPY mRNA expression in cortical neurons in the absence of spontaneous bioelectrical activity
Authors: Wirth Marcus J. , Obst Kirstin , Wahle Petra
Published: March 2003

Epigenetic factors are known to influence the differentiation of neocortical neurons. The present study analyses the role of spontaneous bioelectrical activity (SBA) and neurotrophic factors on the expression of neuropeptide Y (NPY) in rat visual cortical neurons using organotypic monocultures prepared from newborn animals and in situ hybridization to detect the NPY messenger ribonucleic acid (mRNA).

Spontaneously active cortex cultures display NPY mRNA expression in about 7% of all cortical neurons from 10 days in vitro (DIV) on. Blocking the SBA by chronic application of 10 mm Mg2+ for 3–30 DIV reduces the percentage of NPY neurons to about 2%. Allowing an initial phase of SBA (1–20 DIV) followed by an SBA blockade (for 21–50 DIV) results in 2% labelled neurons, indicating a dramatic reduction of NPY mRNA expression in the absence of SBA. Surprisingly, the reverse experiment (a period of SBA blockade for 1–20 DIV followed by a period of SBA recovery for 21–40 DIV) does not cause an upregulation of NPY mRNA expression. However, allowing cultures to differentiate as spontaneously active cultures, then applying a transient period of SBA blockade which is followed by a second period of SBA, does rescue the NPY mRNA expression in 7% of the cortical neurons. We conclude that SBA is a main trigger for NPY mRNA expression and it is particularly important during an early postnatal period of differentiation.

We then analysed whether neurotrophic factors known to modulate cortical neuropeptide expression are able to do so in the absence of SBA. Supplementing chronically blocked cultures with the neurotrophins, brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3), neurotrophin‐4/5 (NT‐4/5) and the cytokine, leukaemia inhibitory factor (LIF), reveals that BDNF and NT‐3 are unable to increase the percentage of NPY neurons. In contrast, LIF and NT‐4/5 increase the percentage of NPY neurons to 4 and 6–7%, respectively. Moreover, neurons treated with NT‐4/5 display a very high level of NPY mRNA expression in somata and in the dendritic trees. The data suggest a complex interplay and a hierarchy of epigenetic factors in regulating the neurochemical architecture of the developing neocortex.
Region‐specific expression of thyrotrophin‐releasing hormone‐degrading ectoenzyme in the rat central nervous system and pituitary gland
Authors: Heuer Heike , Ehrchen Jan , Bauer Karl , Schäfer Martin K. H.
Published: March 2003

Thyrotrophin‐releasing hormone (TRH), a hypothalamic neuropeptide hormone and a putative neuromodulator/neurotransmitter in the central nervous system is inactivated by the TRH‐degrading ectoenzyme (TRH‐DE), a TRH‐specific metallopeptidase localized on the surface of neuronal brain cells in culture and on lactotrophic cells of the pituitary. After succeeding in cloning the cDNA of TRH‐DE we now report on the cellular distribution pattern of this enzyme in rat brain, spinal cord and pituitary gland using in situ hybridization histochemistry. In the pituitary, TRH‐DE mRNA was found both in the anterior and the neural lobe but not in the intermediate lobe. After treatment with triiodothyronine (T3) a dramatic increase in the mRNA levels of the TRH‐DE and a decrease in the intensity of the TRH receptor could be observed in the anterior lobe of the pituitary. In brain, TRH‐DE transcripts were predominantly found in neo‐ and allocortical regions with strongest signals in the olfactory bulb, the piriform cortex, the cerebral cortex, the granular layer of the cerebellar cortex and the pyramidal cells of the Ammon's horn. In the diencephalon, the highest TRH‐DE mRNA levels were observed in the medial habenulae followed by several hypothalamic subregions. In the mesencephalon and brainstem, moderate signals were present in the superior colliculi, substantia nigra, dorsal raphe and in the periolivar region. In the spinal cord, TRH‐DE mRNA positive neurons were present in all layers. The very distinct distribution of TRH‐DE in the brain and the hormonal regulation of the adenohypophyseal enzyme support the concept that this peptidase serves very specialized functions.
Expression of the mitotic motor protein CHO1/MKLP1 in postmitotic neurons
Authors: Ferhat Lotfi , Kuriyama Ryoko , Lyons Gary E. , Micales Bruce , Baas Peter W.
Published: March 2003

The kinesin‐related motor protein CHO1/MKLP1 was initially thought to be expressed only in mitotic cells, where it presumably transports oppositely oriented microtubules relative to one another in the spindle mid‐zone. We have recently shown that CHO1/MKLP1 is also expressed in cultured neuronal cells, where it is enriched in developing dendrites (Sharp et al. 1997a) J. Cell Biol., 138, 833–843]. The putative function of CHO1/MKLP1 in these postmitotic cells is to intercalate minus‐end‐distal microtubules among oppositely oriented microtubules within developing dendrites, thereby establishing their non‐uniform microtubule polarity pattern. Here we used in situ hybridization to determine whether CHO1/MKLP1 is expressed in a variety of rodent neurons both in vivo and in vitro. These analyses revealed that CHO1/MKLP1 is expressed within various neuronal populations of the brain including those in the cerebral cortex, hippocampus, olfactory bulb and cerebellum. The messenger ribonucleic acid (mRNA) levels are high within these neurons well after the completion of their terminal mitotic division and throughout the development of their dendrites. After this, the levels decrease and are relatively low within the adult brain. Parallel analyses on developing hippocampal neurons in culture indicate that the levels of expression increase dramatically just prior to dendritic development, and then decrease somewhat after the dendrites have differentiated. Dorsal root ganglion neurons, which generate axons but not dendrites, express significantly lower levels of mRNA for CHO1/MKLP1 than hippocampal or sympathetic neurons. These results are consistent with the proposed role of CHO1/MKLP1 in establishing the dendritic microtubule array.
UV‐sensitive input to horizontal cells in the turtle retina
Authors: Ammermüller J. , Itzhaki A. , Weiler R. , Perlman I.
Published: March 2003

Microspectrophotometry, electroretinography and behavioural studies have indicated that ultraviolet (UV) light contributes to functional vision in various vertebrate species. Based on behavioural evidence, this was also suggested for turtle vision. In order to reveal the interactions underlying detection of UV light in the distal retina, we recorded intracellularly the photoresponses of cones and horizontal cells in retinas of
Pseudemys scripta elegans and Mauremys caspica and calculated the action spectra of these cells under different conditions of adaptation. In the dark‐adapted retina, all three types of horizontal cells; luminosity‐type, red/green chromaticity‐type and yellow/blue chromaticity‐type exhibited increased sensitivity in the UV region of the spectrum. However, chromatic adaptation indicated that only the yellow/blue chromaticity‐type horizontal cells received excitatory input from UV‐sensitive cones with peak sensitivity ≈ 360 nm. The enhanced UV sensitivity of luminosity‐type horizontal cells probably reflected the β‐band of the long‐wavelength sensitive visual pigment as indicated by the action spectra of dark‐adapted L‐cones. It is suggested that the enhanced UV sensitivity of red/green chromaticity‐type horizontal cells reflects the β‐band of the medium‐wavelength sensitive visual pigment. Transmission measurements of the optical media (cornea, lens and vitreous) indicated that UV vision can be functional under normal circumstances.
Characterization of a directional selective inhibitory input from the medial terminal nucleus to the pretectal nuclear complex in the rat
Authors: Schmidt Matthias , Van Der Togt Chris , Wahle Petra , Hoffmann Klaus‐Peter
Published: March 2003

The receptive field properties of neurons in the medial terminal nucleus of the accessory optic system (MTN) that project to the ipsilateral nucleus of the optic tract (NOT) and dorsal terminal nucleus (DTN), as identified by antidromic electrical activation, were analysed in the anaesthetized rat. The great majority (88%) of MTN neurons that were antidromically activated from NOT and DTN preferred downward directed movement of large visual stimuli while the remaining cells preferred upward directed stimulus movement. Distinct retrograde tracer injections into the NOT/DTN and the ipsilateral inferior olive (IO) revealed that no MTN neurons project to both targets. MTN neurons projecting to the ipsilateral NOT/DTN were predominantly found in the ventral part of the MTN, whereas those projecting to the IO were found in the dorsal part of the MTN. In situ hybridization for glutamic acid decarboxylase (GAD) mRNA was used as a marker for GABAergic neurons. Up to 98% of MTN neurons retrogradely labelled from the ipsilateral NOT/DTN also expressed GAD mRNA. Earlier studies have shown that MTN neurons that prefer upward directed stimulus movements are segregated from MTN neurons that prefer downward directed stimulus movements. It also has been demonstrated that directionally selective neurons in the NOT/DTN prefer horizontal stimulus movements and receive an inhibitory input from ipsilateral MTN. Our results indicate that this input is mediated by GABAergic cells in the ventral part of MTN, which to a large extent prefer downward directed stimulus movements, and that the great majority of MTN neurons that prefer upward directed stimulus movements project to other targets one of which possibly is the IO.
Comparison of the electrophysiology and morphology of layers III and II neurons of the rat medial entorhinal cortex in vitro
Authors: van der Linden Solange , da Silva Fernando H. Lopes
Published: March 2003

The basic membrane characteristics of neurons in layers II and III of the medial entorhinal cortex (MEA) were recorded using the intracellular current clamp technique in in vitro slices of the rat brain. Two types of cells were distinguished according to the presence of a time‐dependent inward rectification (SAG current) with hyperpolarizing current pulses. The cells in which this inward rectification was not observed (No‐SAG cells) had a larger input resistance, a more negative resting membrane potential and a more depolarized firing threshold. They more often displayed a strongly adapting firing pattern, and their action potentials had a slower decay rate and lacked a depolarizing afterpotential, compared with the SAG cells. SAG cells typically had a prominent rebound depolarization at the end of a hyperpolarizing current and membrane potential oscillations (7 Hz) upon subthreshold depolarizations. Cs+ blocked the time‐dependent inward rectification. The rebound depolarization persisted, even in the presence of tetrodotoxin. Biocytin labelling showed that layer III consisted mainly of pyramidal‐shaped cells. Most layer III cells were of the No‐SAG type. All cells in layer II, stellate and pyramidal cells, were classified as SAG cells. We conclude that the cells in MEA layers II and III display different electroresponsiveness, but that this appears to be more related to the layer where they are located than to a specific morphology. As layer III consisted mainly of cells of the No‐SAG type, we suggest that layer III cells are less excitable than the SAG type layer II cells.
Cloning of a new gap junction gene (Cx36) highly expressed in mammalian brain neurons
Authors: Condorelli Daniele Filippo , Parenti Rosalba , Spinella Francesca , Salinaro Angela Trovato , Belluardo Natale , Cardile Venera , Cicirata Federico
Published: March 2003

The connexins are the protein subunits of the gap junction intercellular channels. In the present study a new rat connexin was cloned by degenerate reverse transcription‐polymerase chain reaction and its gene isolated from a mouse genomic library. The nucleotide sequence encodes a protein of 321 amino acids (called Cx36) with highly significant homology to the members of the connexin family. In situ hybridization analysis of rat brain and retina showed the strongest expression in neurons of the inferior olive, the olfactory bulb, the CA3/CA4 hippocampal subfields and several brain‐stem nuclei. An intense expression was also found in the pineal gland and in the retinal ganglion cell and inner nuclear layers. Experiments with neurotoxins, locally injected in the hippocampus or specifically acting on inferior olivary neurons, confirmed the neuronal localization of Cx36. It is the first connexin to be expressed predominantly in mammalian neurons and its identification paves the way for a molecular approach in the study of the role played by gap junctions in the physiology and the pathology of the mammalian brain.
The NMDA receptor subunit NR2B of neonatal rat brain: complex formation and enrichment in axonal growth cones
Authors: Herkert Matthias , Röttger Sabine , Becker Cord‐Michael
Published: March 2003

The N‐methyl‐d‐aspartate (NMDA) subtype of ionotropic glutamate receptors comprises a family of highly homologous subunits which assemble into oligomeric protein complexes. Alterations in subunit composition are developmentally regulated, leading to functionally distinct receptor populations. Here, the contribution of the subunit NR2B to NMDA receptor complex formation was analysed in neonatal rat brain, employing polyclonal antibodies raised against NR2B‐specific synthetic peptides. By hydrodynamic size fractionation of the solubilized receptor protein and chemical cross‐linking, NR2B antigen was found to be associated with several protein species of up to 690 kDa molecular weight. These observations show NR2B to be part of a multimeric receptor complex. Fractionation of cortex homogenates from E18 rat embryos on sucrose density gradients revealed NR2B polypeptide to be highly enriched in axonal growth cones. A similar distribution was found by fluorescence microscopy of immature hippocampal neurons, showing a preferential accumulation of NR2B antigen in axonal growth cones and varicosities. In mature cells, NR2B antigen displayed a punctated distribution pattern with redistribution to somato‐dendritic spheres. The association of NR2B with axonal growth cones and processes of immature neurons suggests a role of NMDA receptors in the regulation of neurite outgrowth and migration.
Accumulation of acetylcholine receptors is a necessary condition for normal accumulation of acetylcholinesterase during in vitro neuromuscular synaptogenesis
Authors: De S. , Porte La , Chaubourt E. , Fabre F. , Poulas K. , Chapron J. , Eymard B. , Tzartos S. , Koenig J.
Published: March 2003

To study a step of the very complex processes of the formation of the neuromuscular junction (NMJ), we have analysed the clustering of acetylcholine receptors (AChR) and acetylcholinesterase (AChE) in myotubes cultured in various conditions. On the surface of rat myotubes cultured in the presence of spinal cord cells from embryonic rat, numerous AChE clusters appeared. Such clusters are always co‐localized with AChR clusters, but the reverse is not true: the number of AChR clusters largely exceeds that of AChE clusters. Very few AChE clusters formed when such co‐cultures were treated with monoclonal antibodies (mAbs) against the main immunogenic region (MIR) of the AChR, which provoke internalization and degradation of the AChRs of the muscular membrane. The total levels of AChE and proportions of molecular forms were unaffected. We also used non‐innervated myotubes in which addition of agrin, a protein normally synthesized by motoneurons, transported to nerve terminals and inserted into the synaptic basal lamina, induces the formation of small clusters of AChE. When added to rat myotubes devoid of membrane AChR, agrin‐induced AChE clusters did not form. Finally, we analysed the capacity of the variant of the C2 mouse muscle cell line deficient in AChR (1R‐) to form clusters of AChE in co‐cultures with spinal cord cells from rat: no formation of AChE clusters could be observed.

 In all these different systems of cultures, the conditions which prevented clustering of AChR (anti‐AChR antibodies, deficiency of the variant C2 cell line) also suppressed AChE clustering. We concluded that clustering of AChR is a prerequisite for clustering of AChE, so that NMJ formation implies the sequential accumulation of these two components.
Transcripts for secreted and GPI‐anchored brevican are differentially distributed in rat brain
Authors: Seidenbecher Constanze I. , Gundelfinger Eckart D. , Böckers Tobias M. , Trotter Jacqueline , Kreutz Michael R.
Published: March 2003

Brevican is a member of the aggrecan/versican family of proteoglycans. In contrast to the other family members, brevican occurs both as soluble isoforms secreted into the extracellular space and membrane‐bound isoforms which are anchored to the cell surface via a glycosylphosphatidylinositol (GPI) moiety. Expression of both variants, which are encoded by two differentially processed transcripts from the same gene, is confined to the nervous system. In the current study, we have used in situ hybridization to examine the cellular sites of synthesis for both mRNAs during postnatal development of the rat brain. Whereas the 3.6‐kb transcript encoding secreted brevican displays a widespread distribution in grey matter structures, including cerebellar and cerebral cortex, hippocampus and thalamic nuclei with silver grains accumulating over neuronal cell bodies, the smaller transcript (3.3 kb) encoding GPI‐anchored isoforms appears to be largely confined to white matter tracts and diffusely distributed glial cells. This expression pattern is further confirmed by reverse transcriptase‐polymerase chain reaction (RT‐PCR) experiments with RNA from different glial cell cultures, and by biochemical data demonstrating that the crude membrane fraction from isolated optic nerve contains high amounts of phosphatidylinositol‐specific phospholipase C (PI‐PLC)‐sensitive brevican immunoreactivity. During ontogenetic development, both brevican transcripts are generally up‐regulated. However, the expression of glypiated brevican is delayed by about 1 week, compared with the expression of the secreted isoform. This late appearance of GPI‐linked brevican, its predominant expression in glial cells and its tight association with brain myelin fractions suggest a functional role in neuroglia.
No‐go dominant brain activity in human inferior prefrontal cortex revealed by functional magnetic resonance imaging
Authors: Konishi Seiki , Nakajima Kyoichi , Uchida Idai , Sekihara Kensuke , Miyashita Yasushi
Published: March 2003

We investigated the response inhibition function of the prefrontal cortex associated with the go/no‐go task using functional magnetic resonance imaging in five human subjects. The go/no‐go task consisted of go and no‐go trials given randomly with roughly equal probability. In go trials a green square was presented and the subjects had to respond by promptly pushing a button using their right or left thumbs, but in no‐go trials a red square was presented and subjects were instructed not to respond. When brain activity in no‐go trials is dominant over that in go trials in areas in the prefrontal cortex, this no‐go dominant brain activity would reflect the neural processes for inhibiting inherent response tendency. We used a new strategy of image data analysis by which transient brain activity in go or no‐go trials can be analysed separately, and looked for the prefrontal areas in which the brain activity in no‐go trials is dominant over that in go trials. We found the no‐go dominant foci in the posterior part of the right inferior frontal sulcus reproducibly among the subjects. This was true whether the right or left hand was used. These results suggest that this region in the prefrontal cortex is related to the neural mechanisms underlying the response inhibition function.
Microglial NO induces delayed neuronal death following acute injury in the striatum
Authors: Takeuchi Akihide , Isobe Ken‐ichi , Miyaishi Osamu , Sawada Makoto , Fan Zou‐Heng , Nakashima Izumi , Kiuchi Kazutoshi
Published: March 2003

We have established a novel injury model in the central nervous system by a stereotaxic injection of ethanol into rat striatum to induce necrosis. With this model, we clarify a function of inducible nitric oxide synthase (iNOS) in a healing mechanism around a necrotic lesion. A semiquantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR) revealed that the iNOS mRNA arose at 6 h, peaked at 24 h, and declined to a lower level 48 h after an intrastriatal 5‐μL ethanol injection. From in situ hybridization, this iNOS mRNA was expressed in the area surrounding the injury. By immunohistochemistry, mononuclear cells at this boundary area of necrosis were stained with anti‐iNOS antibody on the first day after the injury. These cells turned out to be reactive microglia from the positive staining of GSA‐I‐B4, ED‐1 and OX‐42. Haematoxylin‐eosin (HE) staining showed that neurons in this boundary area gradually disappear up to 5 days after the injury with an increment of microglial cells, and this area became cavernous. Nuclei of neurons in this area were stained positive by the terminal deoxynucleotidyl‐transferase‐mediated dUTP‐biotin nick end‐labelling (TUNEL) assay on the first day after the injury. These TUNEL‐positive neurons gradually disappeared toward the third day, while microglial cells increased. L‐Ng‐nitro‐arginine methylester (L‐NAME), a competitive NOS inhibitor, administration diminished the elimination of neurons by microglia in this boundary area surrounding necrosis. Microglial NO may act as a neurotoxic agent to eliminate damaged neurons near the necrosis in the form of delayed neuronal death, and may reintegrate the neuronal circuits with functionally intact neurons.
Developmental changes in localization of NMDA receptor subunits in primary cultures of cortical neurons
Authors: Li Jin Hong , Wang Yue Hua , Wolfe Barry B. , Krueger Karl. E. , Corsi Lorenzo , Stocca Gabriella , Vicini Stefano
Published: March 2003

Immunoblot analysis, using antibodies against distinct N‐methyl‐d‐aspartic acid (NMDA) receptor subunits, illustrated that the NR2A and NR2B subunit proteins have developmental profiles in cultured cortical neurons similar to those seen in vivo. NR1 and NR2B subunits display high levels of expression within the first week. In contrast, the NR2A subunit is barely detectable at 7 days in vitro (DIV) and then gradually increased to mature levels at DIV21. Immunocytochemical analysis indicated that NMDA receptor subunits cluster in the dendrites and soma of cortical neurons. Clusters of NR1 and NR2B subunits were observed as early as DIV3, while NR2A clusters were rarely observed before DIV10. At DIV18, NR2B clusters partially co‐localize with those of NR2A subunits, but NR2B clusters always co‐localize with those of NR1 subunits. Synapse formation, as indicated by the presence of presynaptic synaptophysin staining, was observed as early as 48–72 h after plating. However, in several neurons at ages less than DIV5 where synapses were scarce, NR2B and NR1 clusters were abundant. Furthermore, while NR2B subunit clusters were seen both at synaptic and extrasynaptic sites, NR2A clusters occurred almost exclusively in front of synaptophysin‐labelled boutons. This result was supported by electrophysiological recording of NMDA‐mediated synaptic activity [NMDA‐excitatory postsynaptic currents (EPSCs)] in developing neurons. At DIV6, but not at DIV12, CP101, 606, a NR1/NR2B receptor antagonist, antagonized spontaneously occurring NMDA‐EPSCs. Our data indicate that excitatory synapse formation occurs when NMDA receptors comprise NR1 and NR2B subunits, and that NR2A subunits cluster preferentially at synaptic sites.
G‐proteins and G‐protein subunits mediating cholinergic inhibition of N‐type calcium currents in sympathetic neurons
Authors: Delmas Patrick , Abogadie Fe C. , Dayrell Mariza , Haley Jane E. , Milligan Graeme , Caulfield Malcolm P. , Brown David A. , Buckley Noel J.
Published: March 2003

One postsynaptic action of the transmitter acetylcholine in sympathetic ganglia is to inhibit somatic N‐type Ca2+currents: this reduces Ca2+‐activated K+currents and facilitates high‐frequency spiking. Previous experiments on rat superior cervical ganglion neurons have revealed two distinct pathways for this inhibitory action: a rapid, voltage‐dependent inhibition through activation of M4muscarinic acetylcholine receptors (mAChRs), and a slower, voltage‐independent inhibition via M1mAChRs (Hille (1994)Trends in Neurosci.,17,531–536]. We have analysed the mechanistic basis for this divergence at the level of the individual G‐proteins and their α and βγ subunits, using a combination of site‐directed antibody injection, plasmid‐driven antisense RNA expression, over‐expression of selected constitutively active subunits, and antagonism of endogenously liberated βγ subunits by over‐expression of βγ‐binding β‐adrenergic receptor kinase 1 (βARK1) peptide. The results indicate that: (i) M4mAChR‐induced inhibition is mediated by GoA;(ii) α and βγ subunits released from the activated GoAheterotrimer produce separate voltage‐insensitive and voltage‐sensitive components of inhibition, respectively; and (iii) voltage‐insensitive M1mAChR‐induced inhibition is likely to be mediated by the α subunit of Gq. Hence, Ca2+current inhibition results from the concerted, but independent actions of three different G‐protein subunits.
Glutamate receptor subunits GluR1 and GluR2/3 distribution shows reorganization in the human epileptogenic hippocampus
Authors: De Lanerolle Nihal C. , Eid Tore , Von Campe Gord , Kovacs Ilona , Spencer Dennis D. , Brines Michael
Published: March 2003

The AMPA‐type glutamate receptor subunits GluR1 and GluR2/3 were localized by immunohistochemistry with subunit‐specific antibodies in hippocampi removed surgically from patients with temporal lobe epilepsy for the control of seizures. The flip and flop splice variants of the subunits were localized by in situ hybridization histochemistry with specific oligoprobes. In patient hippocampi that were not the seizure focus, the GluR1 subunit proteins were diffusely expressed on the dendrites of neurons in all regions. In contrast, in these same hippocampi, the GluR2/3 subunit proteins were expressed strongly on the soma and proximal dendrites of principal neurons in all regions. The flip variant of these subunits was localized in the hilus and fields of Ammon's Horn (CA), while the flop variants were prominent on the dentate granule cells. In the epileptogenic hippocampus, while immunoreactivity was decreased in all fields that showed neuronal loss, there was an increased expression of GluR1 on the dendritic excrescences on the proximal dendrites of hilar neurons and CA3 pyramidal neurons, as well as expression of GluR2/3 in hilar neuron excrescences. Electron microscopic examination confirmed that the GluR1 immunoreactivity was only in dendritic processes, particularly dense at the postsynaptic membranes. Such expression of GluR1 may provide for an enhanced glutamatergic response by these neurons. GluR2/3 was also significantly increased on the dendrites of dentate granule cells in the epileptogenic hippocampus and may provide some protection against excitotoxic injury by reducing calcium flux into neurons.
Vasoactive intestinal peptide shortens both G1 and S phases of neural cell cycle in whole postimplantation cultured mouse embryos
Authors: Gressens Pierre , Paindaveine Bénédicte , Hill Joanna M. , Evrard Philippe , Brenneman Douglas E.
Published: March 2003

Vasoactive intestinal peptide, a trophic and mitogenic factor, stimulates growth in whole cultured mouse embryos. Inhibition of this growth function between embryonic days 9 and 11 induces growth retardation accompanied by severe microcephaly. In the present study, to determine the effects of this peptide on the different phases of the cell cycle of neural cells, embryonic day 9.5 cultured mouse embryos were cumulatively labelled with bromodeoxyuridine. Vasoactive intestinal peptide (10–7m) shortened S phase and G1 phase of neuroepithelial cells by 50% (4.8–2.4 h) and 58% (1.9–0.8 h), respectively, compared with controls. G2 and M phases were not modified by vasoactive intestinal peptide treatment. Total cell cycle length was consequently reduced by 43% (8.2–4.7 h) in vasoactive intestinal peptide treated embryos, compared with controls. In contrast, vasoactive intestinal peptide did not modify the rate of neuroepithelial cell death as assessed by the proportion of nuclei containing fragmented DNA. These data suggest that vasoactive intestinal peptide stimulates growth in premigratory stages of nervous system development by shortening S and G1 phases of the cell cycle and that S phase duration can be regulated by a physiological peptide.
Coexpression of dopamine D1and D3receptors in islands of Calleja and shell of nucleus accumbens of the rat: opposite and synergistic functional interactions
Authors: Ridray Sophie , Griffon Nathalie , Mignon Virginie , Souil Evelyne , Carboni Suzanna , Diaz Jorge , Schwartz Jean‐Charles , Sokoloff Pierre
Published: March 2003

Using doublein situhybridization, we found extensive coexpression of dopamine D1and D3receptor (D1R and D3R) mRNAs in neurons of the island of Calleja major (ICjM) and ventromedial shell of nucleus accumbens (ShV), respectively. Thus, at least 79 and 63% of D3R mRNA‐expressing neurons in ICjM and ShV also expressed the D1R mRNA. Coexpression of D1R and D3R mRNAs was found to occur in substance P (SP) mRNA‐expressing neurons in both areas, suggesting SP mRNA as a marker of the activity of coexpressing neurons. Administration of SKF 38393, a D1R receptor agonist, increased c‐fos mRNA in ICjM, whereas administration of quinpirole, a D2R/D3R agonist, decreased it; SCH 23390, a D1R antagonist and nafadotride, a preferential D3R antagonist, given alone, had effects opposite to those of the corresponding agonists. These data indicate that basal c‐fos expression in ICjM is maintained by endogenous dopamine acting tonically upon two receptor subtypes subserving opposite effects on the same cell. However, in ShV, whereas SKF 38393 also increased c‐fosmRNA, quinpirole had no effect, a difference presumably reflecting the lower fraction of neurons coexpressing D1R and D3R in this area. In contrast, in ShV from reserpine‐treated rats, SKF 38393 increased SP mRNA and quinpirole potentiated this effect. These contrasting interactions of D1R‐ and D3R‐mediated signalling events, i.e. in either opposite or synergistic directions, most likely occurring at the single cell level, may serve to increase the dopamine response threshold of the target cells in ICjM and to maintain a strong tonic activity of ShV neurons.
Differential regulation of FAK+ and PYK2/Cakβ, two related tyrosine kinases, in rat hippocampal slices: effects of LPA, carbachol, depolarization and hyperosmolarity
Authors: Derkinderen Pascal , Siciliano Julio , Toutant Madeleine , Girault Jean‐Antoine
Published: March 2003

FAK+, an isoform of focal adhesion kinase preferentially expressed in brain and PYK2/Cakβ (proline‐rich tyrosine kinase 2/cell adhesion kinaseβ) are two related cytoplasmic tyrosine kinases. They are candidates for coupling electrical activity and stimulation of neurotransmitter receptors to short and long‐term changes in synaptic properties, cytoskeletal organization and gene expression in neurons. As the same set of stimuli appear capable of stimulating FAK and/or PYK2 in non‐neuronal cells and in cell lines with neuronal characteristics, we investigated the selectivity of regulation of these two kinases in mature nervous tissue. Using rat hippocampal slices, we compared the regulation of FAK+ and PYK2 by stimuli known to be active on one or the other of these two kinases in other cell types: lysophosphatidic acid (LPA), carbachol, depolarization, and hyperosmolarity. Phosphorylation of FAK+ was markedly increased by carbachol and LPA. Carbachol effects occurred via activation of M1 muscarinic receptors and nicotinic receptors. The effects of carbachol and LPA were prevented by protein kinase C inhibitors, whereas 8‐Br‐cAMP attenuated the effects of carbachol but not of LPA. Tyrosine phosphorylation of PYK2 but not of FAK+ was very strongly enhanced by depolarization and hyperosmolarity. This study and our previous results show that FAK+ and PYK2 are regulated differentially in hippocampal slices: FAK+ is phosphorylated on tyrosine in response to stimulation of G protein‐coupled receptors, whereas PYK2 is mainly sensitive to depolarization and hyperosmolarity. Thus, FAK+ and PYK2 may provide specific and separate links between activation of neurotransmitters receptors, depolarization and tyrosine phosphorylation in mature hippocampus.
Δ9‐Tetrahydrocannabinol increases sequence‐specific AP‐1 DNA‐binding activity and Fos‐related antigens in the rat brain
Authors: Porcella Anna , Gessa Gian Luigi , Pani Luca
Published: March 2003

Delta‐9‐tetrahydrocannabinol (Δ9‐THC), the psychoactive principle of marijuana, has been shown to upregulate the mRNA levels of immediate‐early genes in the rat brain. Using electrophoretic mobility‐shift assay and one‐dimensional Western blot, we here report that Δ9‐THC increases Activator protein‐1 (AP‐1) DNA‐binding and Fos‐related antigen activity in discrete areas of the rat brain. One hour after the intraperitoneal administration of Δ9‐THC at a dose of 10 or 15 mg/kg, AP‐1 DNA‐binding activity in the nucleus accumbens increased by 33 and 49%, respectively, while Western blot showed an increase in both c‐Fos, FosB, Fra‐1 (Fos‐related antigen) and Fra‐2. In the cingulate cortex and caudate‐putamen, Δ9‐THC significantly increased AP‐1 DNA‐binding activity only at the highest dose used (57 and 71%, respectively). While in the caudate‐putamen the increase in AP‐1 DNA binding was mainly due to an elevation of the c‐Fos and FosB proteins, the same phenomenon depended on the FosB, Fra‐1 and Fra‐2 peptides in the cingulate cortex. The effect of Δ9‐THC on the AP‐1 DNA binding and the Fos‐related antigens in the nucleus accumbens was blocked by the specific cannabinoid antagonist SR141716 A (3 mg/kg i.p.). Δ9‐THC failed to modify Specificity protein 1 (Sp1) DNA‐binding activity. The results indicate that Δ9‐THC activates gene coding for AP‐1 DNA‐binding proteins by acting on cannabinoid receptors, and induces a different transcriptional program on the early‐immediate gene of the Fos family, in different areas in the rat brain, suggesting that this mechanism might be involved in the central actions of cannabinoids.
Evidence for a striatal NMDA receptor modulation of nigral glutamate release. A dual probe microdialysis study in the awake freely moving rat
Authors: Morari Michele , Sbrenna Simone , Marti Matteo , O'Connor William T. , Bianchi Clementina , Fuxe Kjell , Beani Lorenzo
Published: March 2003

Dual probe microdialysis was employed to characterize dialysate glutamate levels from the substantia nigra pars reticulata of awake freely moving rats, and to test its sensitivity to alterations in striatal neurotransmission including striatal N‐methyl‐d‐aspartic acid (NMDA) receptor stimulation and blockade. Intranigral perfusion with low (0.1 mm) Ca2+ medium (60 min) did not affect nigral glutamate levels, whereas intranigral perfusion with tetrodotoxin (10 μm, 60 min) increased nigral glutamate levels. Perfusion of KCl (100 mm, 10 min) in the dorsolateral striatum transiently stimulated nigral glutamate levels (maximal increase + 60%), whereas intrastriatal perfusion (60 min) with low Ca2+ medium and tetrodotoxin gradually increased nigral glutamate levels. Intrastriatal perfusion with NMDA (0.1–100 μm, 10 min) dose‐dependently stimulated glutamate levels in the substantia nigra pars reticulata. The NMDA (1 μm)‐induced increase in nigral glutamate release was transient and maximal (+60% within 20 min), whereas that for NMDA (10 μm) had a slow onset but was long lasting (+35% after 60 min). Lower (0.1 μm) and higher (100 μm) NMDA concentrations were ineffective. The effect of intrastriatal NMDA (1 μm) was prevented by coperfusion with MK‐801 (1 μm). Intrastriatal MK‐801 (10 μm) alone gradually increased glutamate levels up to +50% after 60 min of perfusion. The present results suggest that glutamate levels in the substantia nigra pars reticulata are sensitive to changes in neuronal transmission in the dorsolateral striatum, and that striatal NMDA receptors regulate nigral glutamate release in both a tonic and phasic fashion.
Study of subcellular localization of membrane‐bound choline acetyltransferase in Drosophila central nervous system and its association with membranes
Authors: Pahud Gabrielle , Salem Natalie , Van De Goor Jana , Medilanski Jana , Pellegrinelli Nathalie , Eder‐Colli Lorenza
Published: March 2003

Choline acetyltransferase (ChAT), the enzyme which catalyses the biosynthesis of the neurotransmitter acetylcholine, exists in a soluble and membrane‐bound form in cholinergic nerve terminals of different animal species. This study was performed on the enzyme present in Drosophila central nervous system. We show that the two forms of the enzyme have the same apparent molecular weight (75 kDa) when analysed by immunoblotting using an antibody we raised against the recombinant enzyme. According to different authors, membrane‐bound enzyme might be associated with synaptic vesicles or plasma membrane. Subfractionation of Drosophila head homogenates in linear glycerol gradients showed that ChAT does not associate with synaptic vesicles. Analysis of ChAT activity and immunoreactivity showed that two peaks of ChAT were produced. One peak was present in fractions containing soluble components and the other was associated with rapidly sedimenting membranes containing plasma membranes. ChAT in the first peak was mainly hydrophilic. A large proportion of ChAT associated with rapidly sedimenting membranes was amphiphilic. Further fractionation of these membranes by flotation in sucrose gradients showed that membrane‐associated ChAT sedimented in fractions containing plasma membrane marker. Membrane‐bound ChAT was neither solubilized nor converted to hydrophilic enzyme after membrane treatment with 1 m hydroxylamine, suggesting that the enzyme is not palmitoylated and therefore not anchored to membrane through thioester‐linked long chain fatty acid. Partial solubilization of ChAT present on membranes with urea and carbonate suggests that this form of ChAT is a peripheral membrane protein. Carbonate solubilization of membrane‐bound ChAT converted the enzyme from hydrophobic to hydrophilic protein.
Antagonists‐resistant calcium currents in rat embryo motoneurons
Authors: Magnelli Valeria , Baldelli Pietro , Carbone Emilio
Published: March 2003

Ca2+channels diversity of cultured rat embryo motoneurons was investigated with whole‐cell current recordings. In 5–20 mmBa2+, the whole‐cell currents were separated in low‐ (LVA) and high‐voltage‐activated (HVA) current. The LVA current was evident since the first day in culture, while the HVA component was small and increased with time. Recordings after 4 days revealed ≈ 20% L‐, ≈ 45% N‐ and ≈ 35% P‐ and R‐type currents. P‐type currents were revealed only in 40% of motoneurons, in which 20–200 nmω‐Aga‐IVA caused 20% irreversible block of total current. The remaining 60% of cells were insensitive even to higher doses of the toxin (500 nmin 5 mmBa2+), suggesting weak expression and heterogeneous distribution of P‐type channels compensated by high densities of HVA Ca2+channels resistant to all the antagonists (R‐type). A significant residual current could also be resolved after prolonged applications of 5 μmω‐CTx‐MVIIC, which allowed separation of N‐ and P‐type currents by the distinct onset of toxin block. The antagonists‐resistant current reveals biophysical characteristics typical of HVA channels, but distinct from the α1Echannel. The current activates around −20 mV in 20 mmBa2+; inactivates slowly and independently of Ca2+; is blocked by low [Cd2+] and high [Ni2+]; and is larger with Ba2+than Ca2+. The uncovered R‐type calcium current can account for part of the presynaptic Ca2+current controlling neurotransmitter release at the mammalian neuromuscular junction whose activity is resistant to DHP‐ and ω‐CTx‐GVIA, and displays anomalous sensitivity to ω‐Aga‐IVA and ω‐CTx‐MVIIC (Hong & Chang (1995)J. Physiol. (Lond.),482,283–290;Lin & Lin‐Shiau (1997)Eur. J. Neurosci.,9,817–823].