bluuebunny/crossref_metadata_2025 huggingface dataset:
Early postnatal maturation of GABAA‐mediated inhibition in the brainstem respiratory rhythm‐generating network of the mouse
Authors: Ritter B. , Zhang W.
Published: March 2003

It is well established that GABAA‐mediated postsynaptic potentials are excitatory in many brain regions during embryonic and early postnatal life. The pre‐Bötzinger complex (PBC) in the brainstem is an essential component of the respiratory rhythm‐generating network, where GABAA‐mediated inhibition plays a critical role in generating a stable respiratory rhythm in adult animals. In the present study, using the perforated patch technique, we investigated the maturation of GABAA receptor‐mediated effects on rhythmically active PBC neurons and on the motor output in slice preparations from P0–15 neonatal mice. The reversal potential of GABAA receptor‐mediated current (EGABA‐A) switched from depolarizing to hyperpolarizing within the first postnatal week. EGABA‐A was −13.7 ± 9.8 mV at P0, then it changed to −44.8 ± 7.0 mV at P2 and −71.5 ± 6.8 mV at P4. Perfusion of bicarbonate‐free saline has no detectable influence on EGABA‐A, indicating that a lack of Cl– extrusion during P0–3 is mainly responsible for early GABAA‐ergic excitation. At the network level, blockade of GABAA receptors with bicuculline did not significantly change the frequency of rhythmic bursts recorded from hypoglossal nerve roots before P3, whereas it increased the coefficient of variation. After P3, bicuculline increased burst frequency with little effect on the coefficient of variation. Thus, chloride‐mediated inhibition, which appears in PBC neurons after P3, coincides with the appearance of GABAA‐mediated modulation of the respiratory rhythm. GABAA receptor‐activated inhibition may therefore be necessary for frequency modulation in the respiratory network beginning on the fourth postnatal day in the mouse brainstem.
Programmed cell death in the developing human telencephalon
Authors: Rakic Sonja , Zecevic Nada
Published: March 2003

Programmed cell death (PCD) in the form of apoptosis is recognized as one of the central events in the development of the central nervous system. To study the time of onset, extent and distribution of PCD in the human telencephalon, embryos and fetuses from 4.5 to 27 gestational weeks (g.w.) were examined using the TUNEL (TdT‐mediated dUTP‐biotin nick‐end labelling) in situ method. At 4.5 g.w. sparse TUNEL(+) nuclei were observed in the ventricular zone of the neural tube. With the formation of the cortical plate at 7–8 g.w., TUNEL(+) nuclei were seen in all developmental layers of the cortical anlage, as well as in the subcortical regions such as the ganglionic eminence and the internal capsule. The proliferative zones (the ventricular zone, the subventricular zone and the ganglionic eminence) contained the majority of all apoptotic nuclei observed in each specimen. However, the apoptotic index was highest in the subplate zone and in layer I. Double‐labelling experiments suggested that neuronal precursors were the main population of cells undergoing PCD in the first trimester of gestation, whereas glial cells probably start dying around midgestation. The onset of labelling of microglial cells and apoptotic nuclei were synchronous, indicating the involvement of microglia in PCD. In conclusion, two distinct types of PCD were observed during human telencephalic development: embryonic apoptosis, which was synchronous with proliferation and migration of neuronal cells and probably not related to establishment of neuronal circuitry, and fetal apoptosis, which coincided with differentiation and synaptogenesis, and therefore may be related to the development of axonal‐target connectivity.
α1‐Adrenergic receptor‐induced slow rhythmicity in nonrespiratory cervical motoneurons of neonatal rat spinal cord
Authors: Morin Didier , Bonnot Agnès , Ballion Bérangère , Viala Denise
Published: March 2003

Previous studies have reported that the α1‐adrenergic system can activate spinal rhythm generators belonging to the central respiratory network. In order to analyse α1‐adrenergic effects on both cranial and spinal motoneuronal activity, phenylephrine (1–800 μm) was applied to in vitro preparations of neonatal rat brainstem–spinal cord. High concentration of phenylephrine superfusion exerted multiple effects on spinal cervical outputs (C2–C6), consisting of a lengthening of respiratory period and an increase in inspiratory burst duration. Furthermore, in 55% of cases a slow motor rhythm recorded from the same spinal outputs was superimposed on the inspiratory activity. However, this phenylephrine‐induced slow motor rhythm generated at the spinal level was observed neither in inspiratory cranial nerves (glossopharyngeal, vagal and hypoglossal outputs) nor in phrenic nerves. Whole‐cell patch‐clamp recordings were carried out on cervical motoneurons (C4–C5), to determine first which motoneurons were involved in this slow rhythm, and secondly the cellular events underlying direct phenylephrine effects on motoneurons. In all types of motoneurons (inspiratory and nonrespiratory) phenylephrine induced a prolonged depolarization with an increase in neuronal excitability. However, only nonrespiratory motoneurons showed additional rhythmic membrane depolarizations (with spiking) occurring in phase with the slow motor rhythm recorded from the ventral root. Furthermore the tonic depolarization produced in all motoneurons results from an inward current [which persists in the presence of tetrodotoxin (TTX)] associated with a decrease in neuron input conductance, with a reversal potential varying as a Nernstian function of extracellular K+ concentration. Our results indicate that the α1‐adrenoceptor activation: (i) affects both the central respiratory command (i.e. respiratory period and inspiratory burst duration) and spinal inspiratory outputs; (ii) induces slow spinal motor rhythmicity, which is unlikely to be related to the respiratory system; and (iii), increases motoneuronal excitability, probably through a decrease in postsynaptic leak K+ conductance.
Release and elimination of dopamine in vivo in mice lacking the dopamine transporter: functional consequences
Authors: Benoit‐Marand M. , Jaber M. , Gonon F.
Published: March 2003

In mice lacking the dopamine transporter (DAT), the amplitude of dopamine (DA) release and the kinetics of dopamine elimination were measured in vivo using carbon fibre electrodes combined with amperometry. DA release was evoked by electrical stimulation of the medial forebrain bundle. The amplitude of DA release per pulse was lower (7% in striatum and 21% in nucleus accumbens) than in wild‐type mice. Inhibition of monoamine oxidases (MAOs) by pargyline, but not of catechol‐O‐methyltransferase (COMT) by tolcapone, slowed down DA elimination in knockout mice. As DA half‐life was two orders of magnitude higher in these mice, the DA diffusion distance was 10‐times higher than in wild‐types (100 and 10 μm, respectively). In knockout mice, α‐methyl‐p‐tyrosine induced a much faster decline of DA release and haloperidol was less effective in potentiating DA release. Therefore, DA release was more dependent on DA synthesis than in normal animals but was less influenced by D2 autoregulation. Dopaminergic neurons exhibit two kinds of discharge activity, i.e. single spikes and bursts of 2–6 action potentials. In wild‐type mice, stimuli mimicking bursts evoked significant increases in extracellular DA over its basal level sustained by tonic activity. However, in mice lacking the DAT, low frequency firing resulted in consistently high extracellular DA levels that could not be distinguished from DA levels achieved by high frequency firing. Therefore, the burst firing activity cannot be specifically translated into phasic changes in extracellular DA. This deficit might contribute to the difficulties of these mice in spatial cognitive function.
Generation and propagation of 4‐AP‐induced epileptiform activity in neonatal intact limbic structures in vitro
Authors: Luhmann H. J. , Dzhala V. I. , Ben‐Ari Y.
Published: March 2003

We examined the generation, propagation and pharmacology of 4‐aminopyridine (4‐AP)‐induced epileptiform activity (EA) in the intact interconnected limbic structure of the newborn (P0–7) rat in vitro. Whole‐cell recordings of CA3 pyramidal cells and multisite field potential recordings in CA3, CA1, dentate gyrus, and lateral and medial entorhinal cortex revealed 4‐AP‐induced EA as early as P0–1. At this age, EA was initiated in the CA3 region and propagated to CA1, but not to the entorhinal cortex. Starting from P3–4, EA propagated from CA3 to the entorhinal cortex. Along the CA3 septo‐temporal axis, EA arose predominantly from the septal pole and spread towards the temporal site. Whereas the onset of 4‐AP‐induced EA decreased with age from 21.2 ± 1.6 min at P0–1 to 4.7 ± 0.63 min at P6–7, the seizure duration increased in the same age groups from 98 ± 14 s to 269.4 ± 85.9 s, respectively. The EA was blocked by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) but not by dl‐2‐amino‐5‐phosphonovaleric acid (APV), (+)‐MK‐801 hydrogen maleate (MK‐801) or (±)‐alpha‐methyl‐4‐carboxyphenylglycine (MCPG), suggesting that they were mediated by α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA)/kainate receptor activation. We conclude that: (i) the septal pole of the hippocampal CA3 region plays a central role in the generation of EA in the neonatal limbic system; and (ii) AMPA/kainate receptor‐mediated EA can be generated in CA3 already at birth. Therefore, the recurrent collateral synapses and circuits required for the generation of EA are developed earlier than previously suggested on the basis of studies on hippocampal slices.
Regulation of the biosynthesis and processing of chromogranins in organotypic slices: influence of depolarization, forskolin and differentiating factors
Authors: Bauer Richard , Hoflehner Johannes , Doblinger Alfred , Kapeller Ingeborg , Laslop Andrea
Published: March 2003

Slices from rat hippocampus in organotypic culture were used to study the biosynthesis regulation of chromogranins A and B and secretogranin II. Additionally, we investigated the proteolytic conversion of secretogranin II and the levels of prohormone convertases putatively involved. Forskolin treatment and depolarization with potassium plus BayK 8644 led to significant increases in secretogranin II mRNA in the principal cells of the hippocampus. Enhanced expression of secretogranin II was also reflected by a rise in peptide levels. Despite this induction of biosynthesis the extensive processing to secretoneurin normally observed in brain was maintained. Both forskolin and depolarization upregulated the prohormone convertase (PC)1, but not PC2, indicating that PC1 levels are critical for secretoneurin production under stimulating conditions. Results obtained for chromogranins A and B were less consistent. For chromogranin A mRNA, changes were restricted to granule cells; for chromogranin B, a response in granule cells was observed to depolarization but not to forskolin, and effects in pyramidal neurons were weak. Accordingly, we were unable to detect alterations in chromogranin A and B protein levels. Furthermore, we tested several neurotrophic growth factors and found that only basic fibroblast growth factor raised secretogranin II expression without affecting chromogranins A and B. The hippocampal slice preparation allowed well controlled treatment with identification of neuronal subpopulations and yielded data largely matching experiments in vivo and in cell culture. The pronounced regulation of secretogranin II and its effective processing underlines the importance of the resulting peptide secretoneurin as an active neuropeptide in the nervous system.
Basal forebrain nitric oxide synthase (NOS)‐containing neurons project to microvessels and NOS neurons in the rat neocortex: cellular basis for cortical blood flow regulation
Authors: Tong X. K. , Hamel E.
Published: March 2003

Stimulation of basal forebrain neurons results in local increases in cortical cerebral blood flow that are dependent upon cholinergic and nitrergic mechanisms. In the present study, we investigated the possibility that basal forebrain nitric oxide synthase (NOS)‐containing neurons project to microvessels and NOS interneurons in the rat cerebral cortex. We performed quisqualic (QUIS) acid lesions of the basal forebrain and evaluated their effects on cortical NOS immunostained nerve terminals, with emphasis on those associated with microvessels and NOS interneurons, both at the light and/or electron microscopic levels. The results show that basal forebrain NOS neurons provide about one third of the overall cortical NOS innervation. Further, the data indicate that basalocortical NOS fibres establish privileged associations with microvessels and NOS neurons, as respective denervations of 60 and 45% were observed following lesion. At the electron microscopic level, most perivascular NOS neuronal elements corresponded to nerve terminals and a majority (∼ 25%) of these were located in the immediate vicinity of the blood vessels, similar to the perivascular distribution reported previously for classic neurotransmitters/neuromediators. NOS terminals abutting on cortical NOS neurons were primarily nonjunctional. Altogether, these results raise the possibility that not only cholinergic but also nitrergic basal forebrain neurons are involved in the flow response observed following stimulation of the basal forebrain. Further, they suggest interactions between basalocortical and intracortical NOS neurons. We conclude that these interactions are involved in the spatial and temporal regulation of cortical perfusion following basal forebrain activation, and that they may become dysfunctional in pathologies such as Alzheimer's disease which affects both the basal forebrain and the cortical NOS neurons.
Rescue of γ2 subunit‐deficient mice by transgenic overexpression of the GABAA receptor γ2S or γ2L subunit isoforms
Authors: Baer Kristin , Essrich Christian , Balsiger Sylvia , Wick Marilee J. , Harris R. Adron , Fritschy Jean‐Marc , Lüscher Bernhard
Published: March 2003

The γ2 subunit is an important functional determinant of GABAA receptors and is essential for formation of high‐affinity benzodiazepine binding sites and for synaptic clustering of major GABAA receptor subtypes along with gephyrin. There are two splice variants of the γ2 subunit, γ2 short (γ2S) and γ2 long (γ2L), the latter carrying in the cytoplasmic domain an additional eight amino acids with a putative phosphorylation site. Here, we show that transgenic mice expressing either the γ2S or γ2L subunit on a γ2 subunit‐deficient background are phenotypically indistinguishable from wild‐type. They express nearly normal levels of γ2 subunit protein and [3H]flumazenil binding sites. Likewise, the distribution, number and size of GABAA receptor clusters colocalized with gephyrin are similar to wild‐type in both juvenile and adult mice. Our results indicate that the two γ2 subunit splice variants can substitute for each other and fulfil the basic functions of GABAA receptors, allowing in vivo studies that address isoform‐specific roles in phosphorylation‐dependent regulatory mechanisms.
Behavioural changes produced by transgenic overexpression of γ2L and γ2S subunits of the GABAA receptor
Authors: Wick Marilee J. , Radcliffe Richard A. , Bowers Barbara J. , Mascia Maria Paola , Lüscher Bernhard , Harris R. Adron , Wehner Jeanne M.
Published: March 2003

Transgenic mice overexpressing either the mouse γ2L or γ2S subunit of the GABAA receptor were generated in a C57BL/6 J × DBA/2 J mixed background and expanded into transgenic lines. Transgenic mice and littermate controls were analysed with respect to altered behaviour indicative of anxiety, motor activity and acute effects of benzodiazepines and alcohol, as well as with regard to altered responses to alcohol withdrawal and acute functional tolerance to alcohol. Biochemical tests assessed flunitrazepam‐ and ethanol‐enhanced 36Cl– flux stimulated by muscimol in cerebellar and cortical microsacs and [3H]‐flunitrazepam binding to cerebellar membranes. There were no significant differences in any of these measures between the transgenic and control mice, except in tests of acute functional tolerance to acute injection of ethanol. Compared to controls, mice carrying either the γ2L or γ2S transgene developed significantly less tolerance to the ataxic effects of ethanol. We conclude that acute functional tolerance to ethanol is very sensitive to the amount of GABAA receptor γ2 subunit available (regardless of whether it is γ2L or γ2S) but overexpression of neither subunit isoform alters other behavioural and biochemical phenotypes.
Afferent ingrowth and onset of activity in the rat trigeminal nucleus
Authors: Waite P. M. E. , Ho S. M. , Henderson T. A.
Published: March 2003

A novel in vitro preparation, consisting of the rat brainstem with the trigeminal ganglion attached, has been used to study the anatomical and functional development of the trigeminal nucleus from embryonic day (E)13 to postnatal day (P)6. Neurobiotin injections into the trigeminal ganglion showed that primary afferents had reached the trigeminal tract by E13 and had grown simple, mainly unbranched, collaterals into all levels of the nucleus by E15. By E17, these collaterals were extensively branched, with occasional boutons present. Patches of intense neurobiotin‐labelled terminals, corresponding to whisker‐related patterns, were first seen at E20 and became clearer over the next few days. Terminal arbours at this stage were relatively localized and densely branched, with many boutons. Responses from the trigeminal nucleus were recorded with suction electrodes, following stimulation of the trigeminal ganglion. Recordings from the main sensory nucleus showed a postsynaptic response was first present at E15. At E16, bath application of AP5 and DNQX showed that the response contained both NMDA and AMPA components, with NMDA predominating (75%). The NMDA : AMPA ratio remained high until P1, then gradually declined to 50% by P6. The postsynaptic response was also reduced by bath application of bicuculline, indicating the presence of a GABAA‐mediated excitatory component. GABAergic excitation was present at all ages but was maximal from E20 to P1, the age at which whisker‐related patterns are developing. It is hypothesized that both GABAergic excitation and NMDA receptor activation play a role in the consolidation of trigeminal connections, and are thus important in the development of whisker‐related patterns.
Connexin43 phosphorylation state and intercellular communication in cultured astrocytes following hypoxia and protein phosphatase inhibition
Authors: Li W. E. I. , Nagy J. I.
Published: March 2003

The effects of hypoxia and phosphatase inhibitors on connexin43 (Cx43) phosphorylation state, gap junctional intercellular communication (GJIC) and immunolabelling with anti‐Cx43 antibodies were investigated in cultured astrocytes. Astrocytes contained predominantly phosphorylated forms of Cx43 and these underwent dephosphorylation 30 min after hypoxia. This was preceded by a 77% reduction in GJIC 15 min after hypoxia, indicating that reduced GJIC occurs prior to Cx43 dephosphorylation. Hypoxia caused a reduction in punctate immunostaining (epitope masking) at cell–cell contacts with one anti‐Cx43 antibody, and increased labelling with another antibody (13–8300) that detects only a dephosphorylated form of Cx43. Inhibition of protein phosphatase (PP)‐1 and PP‐2A with okadaic acid or calyculin A had little effect on hypoxia‐induced Cx43 dephosphorylation. Inhibition of PP‐2B (calcineurin) with cyclosporin A or FK506 reduced Cx43 dephosphorylation and junctional uncoupling seen after hypoxia. These results demonstrate that responses of astrocytic Cx43 to hypoxia in vitro are similar to those seen after ischaemia in vivo, and that inhibition of protein phosphatase protects astrocytes from hypoxia‐induced Cx43 dephosphorylation and junctional uncoupling. In addition, calcineurin may play a direct role in the regulation of astrocytic GJIC and Cx43 phosphorylation state.
A quantitative study of gamma‐band activity in human intracranial recordings triggered by visual stimuli
Authors: Lachaux Jean‐Philippe , Rodriguez Eugenio , Martinerie Jacques , Adam Claude , Hasboun Dominique , Varela Francisco J.
Published: March 2003

This paper studies gamma‐band responses from two implanted epileptic patients during a simple visual discrimination task. Our main aim was to ascertain, in a reliable manner, whether evoked (stimulus‐locked) and induced (triggered by, but not locked to, stimuli) responses are present in intracranial recordings. For this purpose, we introduce new methods adapted to detect the presence of gamma responses at this level of recording, intermediary between EEG‐scalp and unicellular responses. The analysis relies on a trial‐by‐trial time–frequency analysis and on the use of surrogate data for statistical testing. We report that visual stimulation reliably elicits evoked and induced responses in human intracranial recordings. Induced intracranial gamma activity is significantly present in short oscillatory bursts (a few cycles) following visual stimulation. These responses are highly variable from trial to trial, beginning after 200 ms and lasting up to 500 ms. In contrast, intracranial‐evoked gamma responses concentrate around 100 ms latencies corresponding to evoked responses observed on the scalp. We discuss our results in relation to scalp gamma response in a similar protocol [Tallon‐Baudry et al. (1996) J. Neurosci., 16, 4240–4249] and draw some conclusions for bridging the gap between gamma oscillations observed on the scalp surface and their possible cortical sources.
β‐Amyloid neurotoxicity is mediated by a glutamate‐triggered excitotoxic cascade in rat nucleus basalis
Authors: Harkany T. , Ábrahám I. , Timmerman W. , Laskay G. , Tóth B. , Sasvári M. , Kónya C. , Sebens J. B. , Korf J. , Nyakas C. , Zarándi M. , Soós K. , Penke B. , Luiten P. G. M.
Published: March 2003

Whereas a cardinal role for β‐amyloid protein (Aβ) has been postulated as a major trigger of neuronal injury in Alzheimer's disease, the pathogenic mechanism by which Aβ deranges nerve cells remains largely elusive. Here we report correlative in vitro and in vivo evidence that an excitotoxic cascade mediates Aβ neurotoxicity in the rat magnocellular nucleus basalis (MBN). In vitro application of Aβ to astrocytes elicits rapid depolarization of astroglial membranes with a concomitant inhibition of glutamate uptake. In vivo Aβ infusion by way of microdialysis in the MBN revealed peak extracellular concentrations of excitatory amino acid neurotransmitters within 20–30 min. Aβ‐triggered extracellular elevation of excitatory amino acids coincided with a significantly enhanced intracellular accumulation of Ca2+ in the Aβ injection area, as was demonstrated by 45Ca2+ autoradiography. In consequence of these acute processes delayed cell death in the MBN and persistent loss of cholinergic fibre projections to the neocortex appear as early as 3 days following the Aβ‐induced toxic insult. Such a sequence of Aβ toxicity was effectively antagonized by the N‐methyl‐d‐aspartate (NMDA) receptor ligand dizocilpine maleate (MK‐801). Moreover, Aβ toxicity in the MBN decreases with advancing age that may be associated with the age‐related loss of NMDA receptor expression in rats. In summary, the present results indicate that Aβ compromises neurons of the rat MBN via an excitotoxic pathway including astroglial depolarization, extracellular glutamate accumulation, NMDA receptor activation and an intracellular Ca2+ overload leading to cell death.
Spatio‐temporal distribution of cellular retinoid binding protein gene transcripts in the developing and the adult cochlea. Morphological and functional consequences in CRABP‐ and CRBPI‐null mutant mice
Authors: Romand R. , Sapin V. , Ghyselinck N. B. , Avan P. , Le Calvez S. , Dollé P. , Chambon P. , Mark M.
Published: March 2003

The expression patterns of the mouse cellular retinoid binding protein genes were investigated by in situ hybridization analysis in the inner ear from 10.5 days post coïtum (dpc) up to the adult stage. The cellular retinoic acid binding protein II (CRABPII) and cellular retinol binding protein I (CRBPI) were present in a widespread and abundant pattern in cochlear structures during embryogenesis. Expression of the cellular retinoic acid binding protein I (CRABPI) is restricted during development in Kölliker's organ whilst cellular retinol binding protein II (CRBPII) is only visible after birth with a ubiquitous distribution in most regions of the cochlea including nervous components. No CRABP or CRBP transcripts were observed in the auditory receptors. Morphological observations of CRBPI‐ and CRABPI/CRABPII‐null mutant fetus at 18.5 dpc do not show any structural modification at the level of the organ of Corti. Furthermore, electrophysiological tests performed by measuring distorsion‐product otoacoustic emissions and auditory brainstem evoked responses did not present significant alteration of the auditory function for the different types of mutants. The expression of retinoid binding proteins in cochlear structures during embryogenesis could suggest important roles for these proteins during ontogenesis and morphogenesis of the inner ear. Despite these observations, morphological and functional data from mutant mice did not present obvious modifications of the cochlear structures and auditory thresholds. It is therefore unlikely that CRABPs and CRBPI are directly involved in development of the cochlea and hair cell differentiation.
Basic properties of an inositol 1,4,5‐trisphosphate‐gated channel in carp olfactory cilia
Authors: Cadiou Hervé , Sienaert Ilse , Vanlingen Sara , Parys Jan B. , Molle Gérard , Duclohier Hervé
Published: March 2003

In addition to the activation of cAMP‐dependent pathways, odorant binding to its receptor can lead to inositol 1,4,5‐trisphosphate (InsP3) production that may induce the opening of plasma membrane channels. We therefore investigated the presence and nature of such channels in carp olfactory cilia. Functional analysis was performed by reconstitution of the olfactory cilia in planar lipid bilayers (tip‐dip method). In the presence of InsP3 (10 μm) and Ca2+ (100 nm), a current of 1.6 ± 0.1 pA (mean ± SEM, n = 4) was measured, using Ba2+ as charge carrier. The I/V curve displayed a slope conductance of 45 ± 5 pS and a reversal potential of −29 mV indicating a higher selectivity for divalent cations. This current was characterized by two mean open times (3.0 ± 0.4 ms and 42.0 ± 2.6 ms, n = 4) and was strongly inhibited by ruthenium red (30 μm) or heparin (10 μg/mL). Importantly, the channel activity was closely dependent on the Ca2+ concentration, with the highest open probability (Po) at 100 nm Ca2+ (Po = 0.50 ± 0.02, n = 4). Po is lower at both higher and lower Ca2+ concentrations. A structural identification of the channel was attempted by using a large panel of antibodies, raised against several InsP3 receptor (InsP3R)/Ca2+ release channel isoforms. The type 1 InsP3R was detected in carp cerebellum and whole brain, while a lower molecular mass InsP3R, which may correspond to type 2 or 3, was detected in heart, whole brain and the soma of the olfactory neurons. None of the antibodies, however, cross‐reacted with olfactory cilia. Taken together, these results indicate that in carp olfactory cilia an InsP3‐dependent channel is present, distinct from the classical InsP3Rs localized on intracellular membranes.
A high GluR1 : GluR2 expression ratio is correlated with expression of Ca2+‐binding proteins in rat forebrain neurons
Authors: Kondo Masahiro , Okabe Shigeo , Sumino Rhyuji , Okado Haruo
Published: March 2003

α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazle propionic acid (AMPA) receptors are ubiquitously expressed; however, their subtypes and abundance vary from region to region. We classified the neurons in various forebrain regions (hippocampus, striatum, amygdala, piriform cortex and somatosensory cortex) into six types: [R1+/R2+], [R1–/R2+], [R1+/R2–], [R1–/R2–], [R1++/R2+] and [R1++/R2–], and analysed the expression of Ca2+‐binding proteins, such as parvalbumin and calbindin‐D28k, using a triple‐staining method. The neurons showing a high GluR1 : GluR2 expression ratio, [R1+/R2–], [R1++/R2+] and [R1++/R2–] neurons, comprised 13–30% of the total neuronal population. In addition, the expression of Ca2+‐binding proteins was mainly observed in these three types of neurons. The results suggest that Ca2+‐binding protein‐positive neurons express Ca2+‐permeable AMPA receptors, because the Ca2+‐permeability of AMPA receptors is enhanced by the relative scarcity of the GluR2 subunit. To directly test the possibility that Ca2+‐binding protein‐positive neurons express Ca2+‐permeable AMPA receptors, we performed Ca2+‐imaging experiments in cultured cortical neurons. Ca2+ influx through AMPA receptors was measured selectively by addition of AMPA together with cyclothiazide in the presence of blockers of other Ca2+ influx routes. More than half of the calbindin‐D28k‐positive neurons showed a large increase in the intracellular Ca2+ concentration ([Ca2+]i), whilst most of the calbindin‐D28k‐undetectable neurons exhibited only a slight rise in [Ca2+]i after AMPA addition. These results suggest that the expression of calbindin‐D28k is related to the expression of Ca2+‐permeable AMPA receptors.
Scorpion α and α‐like toxins differentially interact with sodium channels in mammalian CNS and periphery
Authors: Gilles Nicolas , Chen Haijun , Wilson Harry , Le Gall Frédéric , Montoya Gonzalo , Molgo Jordi , Schönherr Roland , Nicholson Graham , Heinemann Stefan H. , Gordon Dalia
Published: March 2003

Scorpion α‐toxins from Leiurus quinquestriatus hebraeus, LqhII and LqhIII, are similarly toxic to mice when administered by a subcutaneous route, but in mouse brain LqhII is 25‐fold more toxic. Examination of the two toxins effects in central nervous system (CNS), peripheral preparations and expressed sodium channels revealed the basis for their differential toxicity. In rat brain synaptosomes, LqhII binds with high affinity, whereas LqhIII competes only at high concentration for LqhII‐binding sites in a voltage‐dependent manner. LqhII strongly inhibits sodium current inactivation of brain rBII subtype expressed in HEK293 cells, whereas LqhIII is weakly active at 2 μm, suggesting that LqhIII affects sodium channel subtypes other than rBII in the brain. In the periphery, both toxins inhibit tetrodotoxin‐sensitive sodium current inactivation in dorsal root ganglion neurons, and are strongly active directly on the muscle and on expressed μI channels. Only LqhII, however, induced repetitive end‐plate potentials in mouse phrenic nerve–hemidiaphragm muscle preparation by direct effect on the motor nerve. Thus, rBII and sodium channel subtypes expressed in peripheral nervous system (PNS) serve as the main targets for LqhII but are mostly not sensitive to LqhIII. Toxicity of both toxins in periphery may be attributed to the direct effect on muscle. Our data elucidate, for the first time, how different toxins affect mammalian central and peripheral excitable cells, and reveal unexpected subtype specificity of toxins that interact with receptor site 3.
Coupling between lumbar and sacral motor networks in the neonatal rat spinal cord
Authors: Cazalets Jean‐René , Bertrand Sandrine
Published: March 2003

We have studied the rhythm‐generating capabilities of the lumbar, sacral and coccygeal (Co) areas using an isolated spinal cord preparation of the newborn rat. The bath‐application of a mixture of N‐methyl‐ d‐l‐aspartate (NMA) and serotonin (5‐HT) on the whole spinal cord induced a coordinated rhythmic activity that could be recorded from the lumbar to the coccygeal ventral roots. The phase relationships and mean burst duration between the activity in the rostral lumbar segments and the activity in the sacral segments was analysed. The direct activation of the sacral network, by using sections or by selective pharmacological activation, showed that these caudal segments possess their own rhythmogenic capability. By combining section experiments and compartmentation of the spinal cord, we demonstrated that a strong coupling exists between the lumbar and sacral motor networks. In addition, we found that in an intact spinal cord the activity of the sacral networks is driven by the lumbar networks. We have found that different modes of coordination between the lumbar and the sacral activity may occur. Finally, we have shown that the coupling between the lumbar and sacral networks can be modified by sensory inputs, suggesting that the spinal machinery could modulate and adapt the coupling of these two spinal networks.
Synchronization of circadian firing rhythms in cultured rat suprachiasmatic neurons
Authors: Shirakawa Tetsuo , Honma Sato , Katsuno Yumiko , Oguchi Haruhisa , Honma Ken‐ichi
Published: March 2003

The circadian clock in mammals is located in the suprachiasmatic nucleus (SCN) which consists of multiple oscillating neurons. Integration of the cellular oscillations is essential for the generation of a single circadian period in the SCN. By using a multielectrode dish (MED), we measured circadian firing rhythms in individual SCN neurons for more than 2 weeks continuously, and examined the involvement of synaptic communication in the synchronization of circadian rhythms. Cross‐correlation analysis of spontaneous action potentials revealed that a neuron pair was functionally connected by synapses when their circadian rhythms were synchronized. No correlation was found between the paired neurons whose circadian rhythms were not synchronized. Calcium (Ca2+)‐dependent synaptic transmission in the cellular communication was indicated by dose‐dependent lengthening of an intercellular spike interval and loss of spike correlation with a Ca2+ channel blocker. Approximately 60% of the SCN neurons in culture were immunoreactive to antibodies against γ‐aminobutyric acid (GABA) or glutamic acid decarboxylase (GAD). Spontaneous firing of all the neurons tested was either increased or decreased by bicuculline, the GABAA receptor antagonist. These findings indicate that synaptic communication plays a critical role in the synchronization of circadian rhythms in individual SCN neurons and the GABAergic transmission is involved in the synchronization mechanism.
Cellular uptake and spread of the cell‐permeable peptide penetratin in adult rat brain
Authors: Bolton Sarah J. , Jones Declan N. C. , Darker John G. , Eggleston Drake S. , Hunter A. Jacqueline , Walsh Frank S.
Published: March 2003

Investigation of normal and pathological diseases of the central nervous system (CNS) has been hampered by the inability to effectively manipulate protein function in vivo. In order to address this important topic, we have evaluated the ability of penetratin, a novel cell‐permeable peptide consisting of a 16‐amino acid sequence derived from a Drosophila homeodomain protein, to act as a carrier system to introduce a cargo into brain cells. Fluorescently tagged penetratin was injected directly into rat brain, either into the striatum or the lateral ventricles, and rats were perfusion‐fixed 24 h later in order to assess the brain response to the peptide. Immunohistochemistry following intrastriatal injection showed that injection of 10 μg penetratin caused neurotoxic cell death and triggered recruitment of inflammatory cells in a dose‐dependent fashion. Doses of 1 μg or less resulted in reduced toxicity and recruitment of inflammatory cells, but interestingly, there was some spread of the penetratin. Injections of an inactive peptide sequence, derived from the same homeodomain, caused little toxicity but could still, however, trigger an inflammatory response. Intraventricular injections showed extensive inflammatory cell recruitment but minimal spread of either peptide. These results suggest that a dose of 1 μg of penetratin peptide is suitable for directing agents to small, discrete areas of the brain and as such is an interesting new system for analysing CNS function.
The HIV‐1 coat protein gp120 and some of its fragments potently activate native cerebral NMDA receptors mediating neuropeptide release
Authors: Gemignani Anita , Paudice Paolo , Pittaluga Anna , Raiteri Maurizio
Published: March 2003

The objective of this study was to investigate the effects of the HIV‐1 envelope protein gp120 and its peptide fragments on the function of N‐methyl‐ d‐aspartate (NMDA) receptors mediating release of cholecystokinin (CCK) and somatostatin (SRIF). These are nonconventional NMDA receptors recently found to be activated by glycine or d‐serine ‘only’. The release of cholecystokinin‐like immunoreactivity (CCK‐LI) and of somatostatin‐like immunoreactivity (SRIF‐LI) elicited by 12 mm K+ from superfused rat neocortex synaptosomes was potently increased by gp120, its cyclic V3 loop and the linear V3 sequence BRU‐C‐34‐A, but not by RP‐135 (a central portion of BRU‐C‐34‐A). The EC50 values of gp120 were 0.02 nm (CCK‐LI release) and 0.01 nm (SRIF‐LI release). The releasing effect of gp120 was prevented by blocking the glycine site or the ion channel of NMDA receptors, but not the glutamate recognition site; in addition, the gp120 effect was strongly inhibited by nanomolar concentrations of Zn2+ ions and by low micromolar concentrations of ifenprodil. It is concluded that gp120 acts as a very potent agonist at the glycine site of NMDA receptors sited on CCK‐ and SRIF‐releasing nerve endings; the protein is able to activate the receptor channel in the absence of glutamate. Gp120 activates the receptors through its V3 loop as peptide fragments related to V3 retain near‐maximal activity. The sensitivity of the gp120 effect to both Zn2+ and ifenprodil would not be incompatible with the idea that these NMDA receptors contain the triple subunit combination NR1/NR2A/NR2B.
The circadian cycle of mPER clock gene products in the suprachiasmatic nucleus of the Siberian hamster encodes both daily and seasonal time
Authors: Nuesslein‐Hildesheim Barbara , O'brien John A. , Ebling Francis J. P. , Maywood Elizabeth S. , Hastings Michael H.
Published: March 2003

The circadian clock in the hypothalamic suprachiasmatic nuclei (SCN) regulates the pattern of melatonin secretion from the pineal gland such that the duration of release reflects the length of the night. This seasonally specific endocrine cue mediates annual timing in photoperiodic mammals. The aim of this study was to investigate how changes in photoperiod influence the cyclic expression of recently identified clock gene products (mPER and mTIM) in the SCN of a highly seasonal mammal, the Siberian hamster (Phodopus sungorus). Immunocytochemical studies indicate that the abundance of both mPER1 and mPER2 (but not mTIM) in the SCN exhibits very pronounced, synchronous daily cycles, peaking approximately 12 h after lights‐on. These rhythms are circadian in nature as they continue approximately under free‐running conditions. Their circadian waveform is modulated by photoperiod such that the phase of peak mPER expression is prolonged under long photoperiods. mPER1 protein is also expressed in the pars tuberalis of Siberian hamsters. In hamsters adapted to long days, the expression of mPER1 is elevated at the start of the light phase. In contrast, there is no clear elevation in mPER1 levels in the pars tuberalis of hamsters held on short photoperiods. These results indicate that core elements of the circadian clockwork are sensitive to seasonal time, and that encoding and decoding of seasonal information may be mediated by the actions of these transcriptional modulators.
Photoperiod differentially regulates the expression of Per1 and ICER in the pars tuberalis and the suprachiasmatic nucleus of the Siberian hamster
Authors: Messager Sophie , Hazlerigg David G. , Mercer Julian G. , Morgan Peter J.
Published: March 2003

Previous studies demonstrated that the clock gene Per1 and the transcription factor ICER are expressed rhythmically in the suprachiasmatic nucleus (SCN) and in the pars tuberalis (PT). In the Syrian hamster the duration of photoperiod affects the amplitude of gene expression in the PT, and melatonin administered before lights‐on suppressed the peak of Per1/ICER expression; these effects were not seen in the SCN. It was speculated that the inefficacy of melatonin was due to the low density of melatonin receptors in the SCN of this species. The aim of the present study was to determine whether this phenomenon also occurs in the Siberian hamster, which expresses a higher density of melatonin receptors in the SCN. Male Siberian hamsters were housed in long days (16 h light : 8 h dark) or short days (8 h light : 16 h dark) and expression of Per1 and ICER mRNA was studied by in situ hybridization. The expression of Per1 and ICER mRNA in the PT peaked 3 h following lights‐on (ZT3) under both photoperiods. The amplitudes of these peaks were greatly attenuated under short photoperiod. In the SCN, the duration of Per1 gene expression was proportional to the length of the light phase, but only a modest amplitude effect was observed. Injections of melatonin (25 μg) 1 h before lights‐on significantly reduced the expression of both genes in the PT at ZT3, but had no effect in the SCN. These data demonstrate that photoperiod‐dependent amplitude modulation of Per1 and ICER gene expression in the PT is conserved across species, and reinforce the argument that this phenomenon is driven by melatonin.
Differential modulation of AMPA receptors by cyclothiazide in two types of striatal neurons
Authors: Vorobjev Vladimir S. , Sharonova Irina N. , Haas Helmut L. , Sergeeva Olga A.
Published: March 2003

The modulation of α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazol‐propionate (AMPA) receptor‐mediated currents by cyclothiazide was investigated in acutely isolated cells from rat striatum with whole‐cell patch‐clamp recording. Single‐cell reverse transcriptase‐polymerase chain reaction (RT‐PCR) was used to identify medium spiny and giant aspiny neurons and to determine their AMPA receptor subunit composition mostly in separate experiments. After pretreatment with cyclothiazide, kainate‐induced AMPA responses were more strongly potentiated in medium spiny than in giant aspiny neurons; cyclothiazide induced a ninefold leftward shift in the kainate concentration–response curve for medium spiny neurons (not giant aspiny neurons). The EC50s for the cyclothiazide potentiation did not differ substantially between medium spiny neurons and giant aspiny neurons. The recovery of kainate‐activated currents from modulation by cyclothiazide was slower for medium spiny neurons than for giant aspiny neurons. Medium spiny neurons expressed GluR‐A, GluR‐B and GluR‐C, but not GluR‐D subunits in both flip and flop splice variants. All giant aspiny neurons expressed GluR‐A and GluR‐D, exclusively in the flop form, half of them also expressed GluR‐B and GluR‐C. This is in keeping with slow and fast desensitization kinetics in medium spiny neurons and giant aspiny neurons, respectively, and differences in cyclothiazide modulation. The rate of cyclothiazide dissociation from the AMPA receptor, activated by glutamate, was ∼ 90 times slower in medium spiny neurons than in giant aspiny neurons. In giant aspiny neurons (not medium spiny neurons) this rate was strongly dependent on the presence of an agonist; 1 mm glutamate increased it 30‐fold. Thus, two major cell groups in the striatum display distinct AMPA receptor compositions carrying specific properties of glutamate responses. Excitatory transmission will thus be differentially affected by cyclothiazide‐type compounds.
Postnatal handling alters the activation of stress‐related neuronal circuitries
Authors: Ábrahám István M. , Kovács Krisztina J.
Published: March 2003

Postnatal handling, as a crucial early life experience, plays an essential role in the development of hypothalamo‐pituitary–adrenal axis responses to stress. The impact of postnatal handling on the reactivity of stress‐related neuronal circuitries was investigated in animals that were handled for the first 21 days of life and as adults they were exposed to physical (ether) or emotional (restraint) challenge. To assess neuronal activation we relied on the induction of immediate‐early gene product c‐Fos and analysed its spatial and temporal distribution at various time intervals after stress. Ether and restraint commonly activated parvocellular neurons in the hypothalamic paraventricular nucleus, and resulted in activation of brain areas providing stress‐related information to the hypothalamic effector neurons and/or in regions governing autonomic and behavioural responses to stress. Beyond these areas, the strength and timing of c‐Fos induction showed stressor specificity in olfactory and septal region, basal ganglia, hypothalamus, hippocampal formation, amygdala and brainstem. Handled rats displayed a lower number of c‐Fos‐positive cell nuclei and weaker staining intensity than non‐handled controls in the hypothalamic paraventricular nucleus, bed nucleus of stria terminalis, central nucleus of amygdala, hippocampus, piriform cortex and posterior division of the cingulum. Significant differences were revealed in timing of c‐Fos induction as a function of stressor and early life experience. Together, these data provide functional anatomical evidence that environmental enrichment in the early postnatal period attenuates the reactivity of stress‐related neuronal circuitries in the adult rat brain.
Human area V5 and motion in the ipsilateral visual field
Authors: Ffytche D. H. , Howseman A. , Edwards R. , Sandeman D. R. , Zeki S.
Published: March 2003

We have studied area V5 of the human brain with visually‐evoked potential (VEP) and functional magnetic resonance imaging (fMRI) methods, using hemifield motion stimuli. Our results confirmed the presence of an ipsilateral field representation in V5 and found: (i) a delay in the ipsilateral response in V5, irrespective of the hemifield stimulated; (ii) a longer ipsilateral delay for left hemifield than for right hemifield stimulation; and (iii) in a patient with a section of the splenium, an absent ipsilateral response for right but not left hemifield stimulation. Together with neurophysiological and anatomical evidence in the monkey, our non‐invasive spatial and temporal imaging studies in man reveal that ipsilateral V5 is activated by motion signals transferred from contralateral V5. The asymmetry of ipsilateral delay in normal subjects and the asymmetrical loss of ipsilateral response following splenial section imply that signals related to visual motion are transferred from one V5 to the other through two segregated pathways.
A modulatory role for protein phosphatase 2B (calcineurin) in the regulation of Ca2+ entry
Authors: Burley J. Russell , Sihra Talvinder S.
Published: March 2003

The Ca2+/calmodulin‐dependent protein phosphatase 2B (PP2B) also known as calcineurin (CN) has been implicated in the Ca2+‐dependent inactivation of Ca2+ channels in several cell types. To study the role of calcineurin in the regulation of Ca2+‐channel activity, phosphatase expression was altered in NG108‐15 cells by transfection of sense and antisense plasmid constructs carrying the catalytic subunit of human PP2Bβ3. Relative to mock‐transfected (wild‐type) controls, cells overexpressing calcineurin showed dramatically reduced high‐voltage‐activated Ca2+ currents which were recoverable by the inclusion of 1 μm FK506 in the patch pipette. Conversely, in cells with reduced calcineurin expression, high‐voltage‐activated Ca2+ currents were larger relative to controls. Additionally in these cells, low‐voltage‐activated currents were significantly reduced. Analysis of high‐voltage‐activated Ca2+ currents revealed that the kinetics of inactivation were significantly accelerated in cells overexpressing calcineurin. Following the delivery of a train of depolarizing pulses in experiments designed to produce large‐scale Ca2+ influx across the cell membrane, Ca2+‐dependent inactivation of high‐voltage‐activated Ca2+ currents was increased in sense cells, and this increase could be reduced by intracellular application of 1 mm BAPTA or 1 μm FK506. These data support a role of calcineurin in the negative feedback regulation of Ca2+ entry through voltage‐operated Ca2+ channels.