bluuebunny/crossref_metadata_2025 huggingface dataset:
Diversity of receptive field changes in auditory cortex during natural sleep
Authors: Edeline Jean‐Marc , Dutrieux Gérard , Manunta Yves , Hennevin Elizabeth
Published: March 2003

Twenty years ago, the study by Livingstone and Hubel [(1981) Nature, 291, 554] was viewed as a first step toward understanding how changes in state of vigilance affect sensory processing. Since then, however, very few attempts have been made to progress in this direction. In the present study, 56 cells were recorded in the auditory cortex of adult, undrugged guinea pigs, and the frequency tuning curves were tested during continuous and stable periods of wakefulness and of slow‐wave sleep (SWS). Twelve cells were also tested during paradoxical sleep. Over the whole cell population, the reponse latency, the frequency selectivity and the size of the suprathreshold receptive field were not significantly modified during SWS compared with waking. However, this lack of global effects resulted from the heterogeneity of response changes displayed by cortical cells. During SWS, the receptive field size varied as a function of the changes in evoked responses: it was unchanged for the cells whose evoked responses were not modified (38% of the cells), reduced for the cells whose responses were decreased (48%) and enlarged for the cells whose responses were increased (14%). This profile of changes differs from the prevalent receptive field shrinkage that was observed in the auditory thalamus during SWS [Edeline et al. (2000), J. Neurophysiol.,84, 934]. It also contrasts with the receptive field enlargement that was described under anaesthesia when the EEG spontaneously shifted from a desynchronized to a synchronized pattern [Wörgötter et al. (1998), Nature,396, 165]. Reasons for these differences are discussed.
Expression of ephrin‐A2 in the superior colliculus and EphA5 in the retina following optic nerve section in adult rat
Authors: Rodger J. , Lindsey K. A. , Leaver S. G. , King C. E. , Dunlop S. A. , Beazley L. D.
Published: March 2003

The vertebrate retina projects topographically to visual brain centres. In the developing visual system, gradients of ephrins and Eph receptors play a role in defining topography. At maturity, ephrins but not Ephs are downregulated. Here we show that optic nerve section in adult rat differentially regulates the expression of ephrin‐A2 in the superior colliculus (SC) and of EphA5 in the retina. Expression was quantified immunohistochemically; ephrin‐A2 levels were also estimated by semiquantitative reverse transcriptase polymerase chain reaction. In the normal SC, ephrin‐A2 was expressed at low levels. At 1 month, levels of protein and of mRNA were upregulated across the contralateral SC giving rise to an increasing rostro‐caudal gradient. At 6 months, levels had fallen but a gradient remained. In the retina of normal animals, EphA5 was expressed as an increasing naso‐temporal gradient. By 1 month, expression was decreased in far temporal retina, resulting in a uniform expression across the naso‐temporal axis. We suggest that denervation‐induced plastic changes within the SC modify expression of these molecules.
Activity‐ and age‐dependent GABAergic synaptic plasticity in the developing rat hippocampus
Authors: Gubellini Paolo , Ben‐Ari Yehezkel , Gaïarsa Jean‐Luc
Published: March 2003

Activity‐dependent plasticity of GABAergic synaptic transmission was investigated in rat hippocampal slices obtained between postnatal day (P) 0–15 using the whole‐cell patch‐clamp recording technique. Spontaneous GABAA receptor‐mediated postsynaptic currents (sGABAA‐PSCs) were isolated in the presence of ionotropic glutamate receptor antagonists. A conditioning protocol relevant to the physiological condition, consisting of repetitive depolarizing pulses (DPs) at 0.1 Hz, was able to induce long‐lasting changes in both frequency and amplitude of sGABAA‐PSCs between P0 and P8. Starting from P12, DPs were unable to induce any form of synaptic plasticity. The effects of DPs were tightly keyed to the frequency at which they were delivered. When delivered at a lower (0.05 Hz) or higher (1 Hz) frequency, DPs failed to induce any long‐lasting change in the frequency or amplitude of sGABAA‐PSCs. In two cases, DPs were able to activate sGABAA‐PSCs in previously synaptically silent cells at P0–1. These results show that long‐term changes in GABAergic synaptic activity can be induced during a restricted period of development by a conditioning protocol relevant to the physiological condition. It is suggested that such activity‐induced modifications may represent a physiological mechanism for the functional maturation of GABAergic synaptic transmission.
Impaired fear conditioning but enhanced seizure sensitivity in rats given repeated experience of withdrawal from alcohol
Authors: Stephens D. N. , Brown G. , Duka T. , Ripley T. L.
Published: March 2003

Repeated experience of withdrawal from chronic alcohol treatment increases sensitivity to seizures. It has been argued by analogy that negative affective consequences of withdrawal also sensitize, but repeated experience of withdrawal from another sedative‐hypnotic drug, diazepam, results in amelioration of withdrawal anxiety and aversiveness. We tested whether giving rats repeated experience of withdrawal from alcohol altered their ability to acquire a conditioned emotional response (CER). Male Hooded Lister rats were fed a nutritionally complete liquid diet as their only food source. Different groups received control diet, or diet containing 7% ethanol. Rats receiving ethanol diet were fed for either 24 days (Single withdrawal, SWD), or 30 days, with two periods of 3 days, starting at day 11, and 21, in which they received control diet (Repeated withdrawal, RWD). All rats were fed lab chow at the end of their liquid diet feeding period. Starting 12 days after the final withdrawal, groups of Control, SWD and RWD rats were given pentylenetetrazole (PTZ; 30 mg/kg, i.p.) three times a week, and scored for seizures. The occurrence of two successive Stage 5 seizures was taken as the criterion for full PTZ kindling. Other groups of control, SWD and RWD rats were trained to operate levers to obtain food, and were then exposed, in a fully counterbalanced design, to light and tone stimuli which predicted unavoidable footshock (CS+), or which had no consequences (CS–). Rats consumed approximately 17.5 g/kg/day of ethanol, resulting in blood alcohol levels of approximately 100 mg/dL. Repeated administration of PTZ resulted in increasing seizure scores. RWD rats achieved kindling criterion faster than either Control or SWD rats. No differences were seen in the groups in flinch threshold to footshock (0.3 mA). At a shock intensity of 0.35 mA, Control, but not RWD or SWD rats showed significant suppression to the CS+ CS– presentation did not affect response rates. The three groups differed in their response to pairing the CS+ with increasing shock levels, the Controls remaining more sensitive to the CS+. SWD rats showed significant suppression of lever pressing during CS+ presentations only at 0.45 and 0.5 mA, and RWD rats only at 0.5 mA. Giving rats repeated experience of withdrawal from chronic ethanol results in increased sensitivity to PTZ kindling, but reduces their ability to acquire a CER. Withdrawal kindling of sensitivity to anxiogenic events does not seem to occur under circumstances which give rise to kindling of seizure sensitivity.
Estrogen and exercise interact to regulate brain‐derived neurotrophic factor mRNA and protein expression in the hippocampus
Authors: Berchtold Nicole C. , Kesslak J. Patrick , Pike Christian J. , Adlard Paul A. , Cotman Carl W.
Published: March 2003

We investigated the possibility that estrogen and exercise interact in the hippocampus and regulate brain‐derived neurotrophic factor (BDNF), a molecule increasingly recognized for its role in plasticity and neuron function. An important aspect of this study is to examine the effect of different time intervals between estrogen loss and estrogen replacement intervention. We demonstrate that in the intact female rat, physical activity increases hippocampal BDNF mRNA and protein levels. However, the exercise effect on BDNF up‐regulation is reduced in the absence of estrogen, in a time‐dependent manner. In addition, voluntary activity itself is stimulated by the presence of estrogen. In exercising animals, estrogen deprivation reduced voluntary activity levels, while estrogen replacement restored activity to normal levels. In sedentary animals, estrogen deprivation (ovariectomy) decreased baseline BDNF mRNA and protein, which were restored by estrogen replacement. Despite reduced activity levels in the ovariectomized condition, exercise increased BDNF mRNA levels in the hippocampus after short‐term (3 weeks) estrogen deprivation. However, long‐term estrogen‐deprivation blunted the exercise effect. After 7 weeks of estrogen deprivation, exercise alone no longer affected either BDNF mRNA or protein levels. However, exercise in combination with long‐term estrogen replacement increased BDNF protein above the effects of estrogen replacement alone. Interestingly, protein levels across all conditions correlated most closely with mRNA levels in the dentate gyrus, suggesting that expression of mRNA in this hippocampal region may be the major contributor to the hippocampal BDNF protein pool. The interaction of estrogen, physical activity and hippocampal BDNF is likely to be an important issue for maintenance of brain health, plasticity and general well‐being, particularly in women.
The voltage‐sensitive motor protein and the Ca2+‐sensitive cytoskeleton in developing rat cochlear outer hair cells
Authors: Beurg Maryline , Bouleau Yohan , Dulon Didier
Published: March 2003

Cochlear outer hair cells (OHCs) possess a unique fast voltage‐driven motility associated with a voltage‐sensitive motor protein embedded in the basolateral membrane. This mechanism is believed to underlie the cochlear amplification in mammals. OHCs also have a Ca2+/calmodulin‐dependent mechanical pathway which involves a submembranous circumferential cytoskeleton. The purpose of this study was to compare the functional appearance of the voltage‐sensitive motor proteins with that involving the Ca2+‐sensitive cytoskeleton during postnatal development of rat OHCs. We demonstrate that whole‐cell electromotility and Ca2+‐voked mechanical responses, by ionomycin, develop concomitantly after postnatal day 5 (P5). These two mechanical properties also develop simultaneously in OHCs isolated from two‐week‐old cultures of P0‐P1 organs of Corti. This excludes the participation of neural innervation in the postnatal maturation of the OHCs' motile properties. In addition, we show that the expression of the membranous voltage‐sensitive motor protein precedes, by several days, the appearance of whole‐cell electromotility. The concomitant development of whole‐cell electromotility and Ca2+‐sensitive motility, both in vivo and in vitro, underlines the cytoskeleton as an important factor in the functional organization of the voltage‐sensitive motor proteins within the plasma membrane.
AMPA receptor activation induces association of G‐beta protein with the alpha subunit of the sodium channel in neurons
Authors: Marin Philippe , Fagni Laurent , Torrens Yvette , Alcaraz Gisèle , Couraud François , Bockaert Joël , Glowinski Jacques , Prémont Joël
Published: March 2003

Glutamatergic transmission is mediated by ionotropic receptors that directly gate cationic channels and metabotropic receptors that are coupled to second messenger generating systems and to ionic channels via heterotrimeric guanine‐nucleotide binding‐ (G) proteins. This distinction cannot be made for the ionotropic receptor subclass activated by α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA), which has been shown to be physically associated with the α‐subunit of Gi1 protein and activates this G‐protein. Here, we report that, in addition to a Ca2+ influx, AMPA induces the mobilization of Ca2+ from the mitochondrial pool by reversing the mitochondrial Na+/Ca2+ exchanger in mouse neurons in primary culture. Both processes required the activation of tetrodotoxin‐sensitive Na+ channels. AMPA receptor activation modified the gating properties of the Na+ channel, independently of the AMPA current, suggesting a G‐protein‐mediated process. Indeed, co‐immunoprecipitation experiments indicated that AMPA receptor activation induced the association of Gβ with the α‐subunit of the Na+ channel. These results suggest that, in addition to its ionic channel function, the AMPA receptor is coupled to Na+ channels through G‐proteins and that this novel metabotropic function is involved in the control of neuronal excitability.
Stabilization of mutant Cu/Zn superoxide dismutase (SOD1) protein by coexpressed wild SOD1 protein accelerates the disease progression in familial amyotrophic lateral sclerosis mice
Authors: Fukada Kei , Nagano Seiichi , Satoh Masahiko , Tohyama Chiharu , Nakanishi Toyofumi , Shimizu Akira , Yanagihara Takehiko , Sakoda Saburo
Published: March 2003

Transgenic mice carrying familial amyotrophic lateral sclerosis (FALS)‐linked mutant Cu/Zn superoxide dismutase (SOD1) genes such as G93A (G93A‐mice) and G85R (G85R‐mice) genes develop limb paresis. Introduction of human wild type SOD1 (hWT‐SOD1) gene, which does not cause motor impairment by itself, into different FALS mice resulted in different effects on their clinical courses, from no effect in G85R‐mice to acceleration of disease progression in G93A‐mice. However, the molecular mechanism which causes the observed difference, has not been clarified. We hypothesized that the difference might be caused by the stability of mutant SOD1 proteins. Using a combination of mass spectrometry and enzyme‐linked immunosorbent assay, we found that the concentration of G93A‐SOD1 protein was markedly elevated in tissues of transgenic mice carrying both G93A‐ and hWT‐SOD1 genes (G93A/hWT‐mice) compared to that in G93A‐mice, and also found that the concentration of G93A‐SOD1 protein had a close relation to the disease duration. The concentration of metallothionein‐I/II in the spinal cord, reflecting the degree of copper‐mediated oxidative stress, was highest in G93A/hWT‐mice, second in G93A‐mice, and normal in the mice carrying hWT‐SOD1 gene. These results indicated that the increase of G93A‐SOD1 protein was responsible for the increase of oxidative stress and disease acceleration in G93A/hWT‐mice. We speculate that coexpression of hWT‐SOD1 protein is deleterious to transgenic mice carrying a stable mutant such as G93A‐SOD1, because this mutant protein is stabilized by hWT‐SOD1 protein, but not to transgenic mice carrying an unstable mutant such as G85R‐SOD1, because this mutant protein is not stabilized by hWT‐SOD1.
Substance P in the descending cholinergic projection to REM sleep‐induction regions of the rat pontine reticular formation: anatomical and electrophysiological analyses
Authors: Kohlmeier Kristi A. , Burns Joan , Reiner Peter B. , Semba Kazue
Published: March 2003

Release of acetylcholine within the pontine reticular formation (PRF) from the axon terminals of mesopontine cholinergic neurons has long been hypothesized to play an important role in rapid eye movement (REM) sleep generation. As some of these cholinergic neurons are known to contain substance P (SP), we used anatomical, electrophysiological and pharmacological techniques to characterize this projection in the rat. Double immunofluorescence demonstrated that 16% of all cholinergic neurons within the mesopontine tegmentum contained SP; this percentage increased to 27% in its caudal regions. When double immunofluorescence was combined with retrograde tracing techniques, it was observed that up to 11% of all SP‐containing cholinergic neurons project to the PRF. Whole‐cell patch‐clamp recordings from in vitro brainstem slices revealed that SP administration depolarized or evoked an inward current in a dose‐dependent manner in all PRF neurons examined, and that these effects were antagonized by a SP antagonist. The amplitude of the SP‐induced inward current varied with changes in the Na+ concentration, did not reverse at the calculated K+ or Cl– equilibrium potentials, and was not attenuated in the presence of tetrodotoxin, low Ca2+ concentration or caesium ions. These data suggest that activation of a tetrodotoxin‐insensitive cation channel(s) permeable to Na+ is responsible for a SP‐induced inward current at resting membrane potentials. The depolarizing actions of SP appeared to be primarily due to activation of the adenylate cyclase pathway, and were additive with cholinergic receptor activation even at maximal concentrations. These data indicate that SP is colocalized in a subpopulation of mesopontine tegmental cholinergic neurons projecting to REM sleep‐induction regions of the PRF, and that actions of these two neuroactive substances on PRF neurons are additive. If SP is coreleased with acetylcholine, the additive actions of the two neurotransmitters might heighten the excitability of postsynaptic PRF neurons and ensure the initiation and maintenance of REM sleep.
The neural substrate of orientation short‐term memory and resistance to distractor items
Authors: Cornette L. , Dupont P. , Orban G. A.
Published: March 2003

We used Positron Emission Tomography to map the neural substrate of human short‐term memory for orientation, defined as retaining a single orientation in memory over a long delay, by comparing a successive discrimination task with a 6‐s delay to the same task with a brief 0.3 s delay and to an identification control task. Short‐term memory engaged the superior parietal lobe bilaterally, the middle occipital gyrus bilaterally and the left dorsolateral prefrontal cortex. In addition, we studied the resistance to a distractor item by comparing the successive discrimination task with long delay, with and without an intervening distractor stimulus. This manipulative process engaged left ventral premotor cortex and left dorsolateral prefrontal cortex. The activation of left dorsolateral prefrontal cortex is interpreted as reflecting co‐ordination between task components. These results, combined with those of two previous studies using an identical reduction strategy, underscore the functional heterogeneity in the prefrontal cortex during short‐term and working memory.
Clonidine injections into the lateral nucleus of the amygdala block acquisition and expression of fear‐potentiated startle
Authors: Schulz Brigitte , Fendt Markus , Schnitzler Hans‐Ulrich
Published: March 2003

Numerous studies of aversive learning with different animal models have shown that the noradrenergic system has an important role in the acquisition, consolidation and expression of aversive learning. We used intracerebral clonidine injections to investigate the role of the noradrenergic amygdaloid system in the fear‐potentiated startle paradigm. Clonidine is a noradrenergic α2‐receptor agonist which can decrease noradrenergic transmission by stimulating presynaptic α2‐receptors. Rats received injections of 0, 2.5, 5 and 10 nmol clonidine into the lateral amygdala (i) before fear‐conditioning, (ii) immediately after fear‐conditioning, (iii) before testing and (iv) before both fear‐conditioning and the testing of conditioned fear. Clonidine injections blocked the acquisition and expression of conditioned fear. The effect on acquisition was not caused by state‐dependency or possible side‐effects of clonidine on consolidation. Given that clonidine decreases amygdaloid noradrenaline release, these results show a crucial role of noradrenergic transmission within the amygdala in classical fear‐conditioning. Surprisingly, both the acquisition and the expression of conditioned fear were blocked after amygdaloid injections of clonidine, suggesting that amygdaloid noradrenaline is necessary to induce both unconditioned and conditioned fear.
Hippocampal granule neuron production and population size are regulated by levels of bFGF
Authors: Cheng Yinghong , Black Ira B. , DiCicco‐Bloom Emanuel
Published: March 2003

Numerous studies of the proliferative effects of basic fibroblast growth factor (bFGF) in culture, including neonatal and adult hippocampal precursors, suggest that the factor plays a ubiquitous and life‐long role in neurogenesis. In contrast, in vivo, bFGF is devoid of effects on neurons in mature hippocampus, raising the possibility that bFGF exhibits developmental stage‐specific activity in the complex animal environment. To define neurogenetic effects in the newborn, a single subcutaneous injection of bFGF (20 ng/gm) was administered to postnatal day 1 (P1) rats, and hippocampal DNA content was quantified: bFGF elicited an increase in total DNA throughout adulthood, by 48% at P4, 25% at P22, and 17% at P180, suggesting that bFGF increases hippocampal cell number. To define mechanisms, bromodeoxyuridine (BrdU) was injected at P1 and mitotically labelled cells were assessed at P22: there was a twofold increase in BrdU‐positive cells in the dentate granule cell layer (GCL), indicating that bFGF enhanced the generation of neurons, or neuronogenesis, from a cohort of precursors. Moreover, enhanced mitosis and survival led to a 33% increase in absolute GCL neuron number, suggesting that neuron production depends on environmental levels of bFGF. To evaluate this possibility, bFGF‐knockout mice were analyzed: hippocampal DNA content was decreased at all ages examined (P3, −42%; P21, −28%; P360, −18%), and total GCL neuron and glial fibrillary acidic protein (GFAP)‐positive cell number were decreased by 30%, indicating that bFGF is necessary for normal hippocampal neurogenesis. We conclude that environmental levels of bFGF regulate neonatal hippocampal neurogenesis. As adult hippocampal neuronogenesis was unresponsive to bFGF manipulation in our previous study [Wagner, J.P., Black, I.B. & DiCicco‐Bloom, E. (1999) J. Neurosci., 19, 6006], these observations suggest distinct, stage‐specific roles of bFGF in the dentate gyrus granule cell lineage.
Participation of the homing pigeon thalamofugal visual pathway in sun‐compass associative learning
Authors: Budzynski Cheri A. , Gagliardo Anna , Ioalé Paolo , Bingman Verner P.
Published: March 2003

The ascending thalamofugal visual pathway in pigeons (Columba livia) terminates in the telencephalic wulst. Characterizing the role of this pathway in visually guided behaviour has remained a challenge. To determine whether this pathway, and in particular the wulst, may participate in sun‐compass‐guided behaviour in homing pigeons, intact, ectostriatum‐lesioned or wulst‐lesioned pigeons were trained to use their sun compass to locate the direction of a food reward in an outdoor, octagonal arena. Control and ectostriatum‐lesioned pigeons learned the task well, and orientated appropriately during the first trial of the last three training sessions and after a phase‐shift manipulation. In contrast, the wulst‐lesioned pigeons learned the task but they took more sessions to learn, and their directional choices were more scattered during the first trial of the last three training sessions and after the phase‐shift manipulation. A subsequent regression analysis indicated that deeper layers of the wulst might have made more of a contribution to the observed behavioural impairments. The data indicate that the homing pigeon wulst participates in visually guided behaviour when the sun compass is used to learn the directional location of a goal.
Macrophage‐stimulating protein is a neurotrophic factor for embryonic chicken hypoglossal motoneurons
Authors: Schmidt Oliver , Doxakis Epaminondas , Davies Alun M.
Published: March 2003

Macrophage‐stimulating protein (MSP) exerts a variety of biological actions on many cell types, but has no known functions in the brain. MSP is structurally related to hepatocyte growth factor (HGF), another pleiotropic factor whose many functions include promoting neuronal survival and growth. To investigate whether MSP is also capable of acting as a neurotrophic factor, we purified hypoglossal motoneurons from the embryonic chicken hindbrain because these neurons are known to express the MSP receptor tyrosine kinase RON. MSP promoted the in vitro survival of these neurons during the period of naturally occurring neuronal death and enhanced the growth of neurites from these neurons. MSP mRNA was detected in the developing tongue whose musculature is innervated by hypoglossal neurons. Our study demonstrates that MSP is a neurotrophic factor for a population of developing motoneurons.
Characterization of retinal guanylate cyclase‐activating protein 3 (GCAP3) from zebrafish to man
Authors: Imanishi Yoshikazu , Li Ning , Sokal Izabela , Sowa Mathew E. , Lichtarge Olivier , Wensel Theodore G. , Saperstein David A. , Baehr Wolfgang , Palczewski Krzysztof
Published: March 2003

Calmodulin‐like neuronal Ca2+‐binding proteins (NCBPs) are expressed primarily in neurons and contain a combination of four functional and nonfunctional EF‐hand Ca2+‐binding motifs. The guanylate cyclase‐activating proteins 1–3 (GCAP1–3), the best characterized subgroup of NCBPs, function in the regulation of transmembrane guanylate cyclases 1–2 (GC1–2). The pairing of GCAPs and GCs in vivo depends on cell expression. Therefore, we investigated the expression of these genes in retina using in situ hybridization and immunocytochemistry. Our results demonstrate that GCAP1, GCAP2, GC1 and GC2 are expressed in human rod and cone photoreceptors, while GCAP3 is expressed exclusively in cones. As a consequence of extensive modification, the GCAP3 gene is not expressed in mouse retina. However, this lack of evolutionary conservation appears to be restricted to only some species as we cloned all three GCAPs from teleost (zebrafish) retina and localized them to rod cells, short single cones (GCAP1–2), and all subtypes of cones (GCAP3). Furthermore, sequence comparisons and evolutionary trace analysis coupled with functional testing of the different GCAPs allowed us to identify the key conserved residues that are critical for GCAP structure and function, and to define class‐specific residues for the NCBP subfamilies.
Characterizing CGI‐94 (comparative gene identification‐94) which is down‐regulated in the hippocampus of early stage Alzheimer's disease brain
Authors: Heese Klaus , Nakayama Takahiro , Hata Ryuji , Masumura Makoto , Akatsu Hiroyasu , Li Feng , Nagai Yasuo , Yamamoto Takayuki , Kosaka Kenji , Suemoto Takahiro , Sawada Tohru
Published: March 2003

The treatment of Alzheimer's disease (AD) remains a major challenge because of the incomplete understanding of the triggering events that lead to the selective neurodegeneration characteristic of AD brains. Here we describe a new protein, CGI‐94, that is down‐regulated at the mRNA level in the hippocampus of early stage AD brain. Transfection experiments with CGI‐94 as a green fluorescent protein (GFP)‐fusion‐protein show that this protein is translocated into the nucleus of the cell. The finding that this protein, which has a bipartite nuclear localization signal, is also observed in the cytoplasm and extracellular space points to a multifunctional protein. Immunohistochemical analyses reveal that CGI‐94 is mainly expressed in neurons of the hippocampal formation and the cortex but not in the cerebellar nucleus. In conclusion, the expression of the nucleolar phosphoprotein CGI‐94 appears to be disturbed in early processes of neuronal degeneration.
Transgenic mice expressing a pH and Cl– sensing yellow‐fluorescent protein under the control of a potassium channel promoter
Authors: Metzger Friedrich , Repunte‐Canonigo Vez , Matsushita Shinichi , Akemann Walther , Diez‐Garcia Javier , Ho Chi Shun , Iwasato Takuji , Grandes Pedro , Itohara Shigeyoshi , Joho Rolf H. , Knöpfel Thomas
Published: March 2003

During the last few years a variety of genetically encodable optical probes that monitor physiological parameters such as local pH, Ca2+, Cl–, or transmembrane voltage have been developed. These sensors are based on variants of green‐fluorescent protein (GFP) and can be synthesized by mammalian cells after transfection with cDNA. To use these sensor proteins in intact brain tissue, specific promoters are needed that drive protein expression at a sufficiently high expression level in distinct neuronal subpopulations. Here we investigated whether the promoter sequence of a particular potassium channel may be useful for this purpose. We produced transgenic mouse lines carrying the gene for enhanced yellow‐fluorescent protein (EYFP), a yellow‐green pH‐ and Cl– sensitive variant of GFP, under control of the Kv3.1 K+ channel promoter (pKv3.1). Transgenic mouse lines displayed high levels of EYFP expression, identified by confocal microscopy, in adult cerebellar granule cells, interneurons of the cerebral cortex, and in neurons of hippocampus and thalamus. Furthermore, using living cerebellar slices we demonstrate that expression levels of EYFP are sufficient to report intracellular pH and Cl– concentration using imaging techniques and conditions analogous to those used with conventional ion‐sensitive dyes. We conclude that transgenic mice expressing GFP‐derived sensors under the control of cell‐type specific promoters, provide a unique opportunity for functional characterization of defined subsets of neurons.
Gelatinase B and TIMP‐1 are regulated in a cell‐ and time‐dependent manner in association with neuronal death and glial reactivity after global forebrain ischemia
Authors: Rivera Santiago , Ogier Crystel , Jourquin Jérôme , Timsit Serge , Szklarczyk Arkadius W. , Miller Karen , Gearing Andrew J. H. , Kaczmarek Leszek , Khrestchatisky Michel
Published: March 2003

Matrix metalloproteinases (MMPs) belong to a large family of endopeptidases that regulate the pericellular environment through the cleavage of protein components of the extracellular matrix, membrane receptors and cytokines. MMP activity is controlled by the multifunctional tissue inhibitors of metalloproteinases (TIMPs). Proteases and their inhibitors are critically involved in developmental and pathological processes in numerous organs, including the brain. Global transient cerebral ischemia induces selective delayed neuronal death and neuroinflammation. We compared, in discrete vulnerable and resistant areas of the ischemic rat hippocampus, the kinetics and cellular distribution of gelatinase B and its principal inhibitor TIMP‐1 and we assessed byin situzymography, the net gelatinolytic activity at the cellular level. We show that gelatinases are expressed and active in neurons, suggesting that MMPs play a role in maintaining neural homeostasis. In the ischemic rat brain, expression and activity of gelatinase B, and expression of TIMP‐1 are altered in a time‐, region‐ and cell‐dependent manner. Gelatinase B is induced first in reactive microglia and subsequently in reactive astrocytes.In situ, increases in gelatinase activity accompanied the progression of neuronal death and glial reactivity. Our results suggest that MMPs and TIMPs are involved in cell viability and tissue remodelling in the ischemic brain, and reinforces the idea that the MMP/TIMP system contributes both to neuronal demise and tissue repair in the context of glial reactivity.
Neurotransmitter release from tottering mice nerve terminals with reduced expression of mutated P‐ and Q‐type Ca2+‐channels
Authors: Leenders A. G. Miriam , Van Den Maagdenberg Arn M. J. M. , Da Silva Fernando H. Lopes , Sheng Zu‐Hang , Molenaar Peter C. , Ghijsen Wim E. J. M.
Published: March 2003

Neurotransmitter release is triggered by Ca2+‐influx through multiple sub‐types of high voltage‐activated Ca2+‐channels. Tottering mice have a mutation in the α1A pore‐forming subunit of P‐ and Q‐type Ca2+‐channels, two prominent sub‐types that regulate transmitter release from central nerve terminals. Immunoblotting analysis of purified forebrain terminals from tottering mice revealed an 85% reduction in the protein expression level of the mutated α1A subunit compared to expression of the α1A subunit in wild‐type terminals. In contrast, the expression of the α1B subunit of the N‐type Ca2+‐channels was unchanged. Release of the amino acids glutamate and GABA and of the neuropeptide cholecystokinin (CCK) induced by a short (100 ms) depolarization pulse was unchanged in the terminals of tottering mice. Studies using specific blockers of Ca2+‐channels however, revealed a reduced contribution of P‐ and Q‐type Ca2+‐channels to glutamate and cholecystokinin release, whereas a greater reliance on N‐type Ca2+‐channels for release of these transmitters was observed. In contrast, the contribution of the P‐, Q‐ and N‐type Ca2+‐channels to the release of GABA was not altered in tottering mice. These results indicate that the expression of the α1A subunit was decreased in terminals from tottering mice, and that a decreased contribution of P‐ and Q‐type Ca2+‐channels to the release of glutamate and cholecystokinin was functionally compensated by an increased contribution of N‐type Ca2+‐channels.
Dopamine D1/5 receptor stimulation induces c‐fos expression in the subthalamic nucleus: possible involvement of local D5 receptors
Authors: Svenningsson Per , Le Moine Catherine
Published: March 2003

The activity of neurons in the subthalamic nucleus controls various aspects of movement. The present study examined the action of dopamine receptor agonists on c‐fos gene expression in the subthalamic nucleus in normal rats. We found that systemic administration of the dopamine D1/5 receptor agonist, SKF 82958 (1 mg/kg), induces c‐fos expression in the subthalamic nucleus. In contrast, systemic administration of the dopamine D2/3 receptor agonist, quinelorane (2 mg/kg) had no effect. When combined, SKF 82958 and quinelorane induced c‐fos expression in subthalamic neurons that was similar to that found following administration of SKF 82958 alone. We also examined c‐fos expression in the substantia nigra pars reticulata, the major projection area for subthalamic neurons, and found that SKF 82958, but not quinelorane, caused an induction of c‐fos expression in this area. In order to clarify the mechanisms underlying the SKF 82958‐mediated induction of c‐fos expression in the subthalamic nucleus and substantia nigra pars reticulata, in situ hybridization for the dopamine D1, D2, D3 and D5 receptor mRNAs was performed. The only significant observation was that D5 receptor mRNA is expressed in subthalamic neurons.

The present data show that dopamine, via D1/D5 receptors, upregulates c‐fos expression in subthalamic neurons, and that the high expression of D5 receptors in this area might be involved. Taken together, these data suggest that dopamine D1/5 receptors are more important for the action of dopamine in the so‐called indirect pathway of the basal ganglia circuitry than what is recognized in current models of basal ganglia organization.
Control of kinetic properties of GluR2 flop AMPA‐type channels: impact of R/G nuclear editing
Authors: Krampfl Klaus , Schlesinger Friedrich , Zörner Antje , Kappler Martin , Dengler Reinhard , Bufler Johannes
Published: March 2003

The GluR2 flop subunit of α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA)‐type glutamate receptors greatly determines calcium permeability and kinetic properties of heteromeric AMPA subunit assemblies. Post‐transcriptional editing of this subunit at the Q/R/N site controls calcium permeability whereas editing at the R/G site is involved in the regulation of biophysical properties. We used patch‐clamp techniques with ultrafast solution exchange to examine the kinetics of recombinant human homomeric GluR2 flop channels transiently expressed in HEK293 cells [edited at the R/G site and Q/R/N site (GR), and unedited (RN) and edited (GN) at the R/G site both with asparagine (N) at the Q/R/N site]. The time constant of desensitization after application of 10 mm glutamate was 1.38 ± 0.05 ms (n = 10), 5.53 ± 0.57 ms (n = 7) and 1.33 ± 0.06 ms (n = 12) for the GluR2 flop GR, RN and GN channels, respectively. The time constant of resensitization was 75 ms for the GluR2 flop RN and 30 ms for the GN channels. The dose‐dependence of the peak current amplitude, kinetics of activation and deactivation, and peak open probability did not differ between RN and GN channels. The study shows that desensitization and resensitization kinetics of homomeric GluR2 flop channels are controlled by a single amino acid exchange (glycine by arginine) at the R/G site. Quantitative analysis by computer simulation using a circular kinetic scheme allows the prediction of the main experimental results.
Absence of dopamine D4 receptors results in enhanced reactivity to unconditioned, but not conditioned, fear
Authors: Falzone Tomás L. , Gelman Diego M. , Young Juan I. , Grandy David K. , Low Malcolm J. , Rubinstein Marcelo
Published: March 2003

The prefrontal cortex receives a major dopaminergic input from the ventral tegmental area, which plays an important role in the integration of neuronal signals influencing behavioural responses to stressful environmental stimuli. The dopamine D4 receptor (D4R) is expressed at highest levels in the prefrontal cortex and is the predominant D2‐like receptor localized in this brain area. To investigate the functional significance of D4Rs in dopamine‐mediated responses we have analysed a strain of mice lacking this receptor subtype (Drd4–/–). Wild‐type and Drd4–/– mice were challenged in two different approach/avoidance conflict paradigms: the elevated plus maze and the light/dark preference exploration test. By these behavioural measures Drd4–/– mice showed heightened avoidance to the more fear‐provoking areas of each maze as demonstrated by reduced exploration of the open arms of the plus maze and longer latencies to explore the illuminated compartment of the light/dark shuttle box. These exaggerated avoidance behaviours were further enhanced by an additional handling stress but completely prevented by anxiolytic agents such as the benzodiazepine midazolam and ethanol. Although Drd4–/– mice displayed heightened anxiety, they exhibited normal ethanol preference and consumption in a two‐bottle choice test. Learned fear responses evaluated by contextual, cued and instrumental fear‐conditioning tests showed no difference between wild‐type and Drd4–/– mice. Taken together these results indicate that the absence of D4Rs increases avoidance behaviour to unconditioned stimuli and does not impair behavioural reactions to Pavlovian fear‐conditioning, suggesting that the D4R could play a key role in the dopaminergic modulation of cortical signals triggered by environmental stimuli.
Pharmacological validation of behavioural measures of akinesia and dyskinesia in a rat model of Parkinson's disease
Authors: Lundblad M. , Andersson M. , Winkler C. , Kirik D. , Wierup N. , Cenci M. A.
Published: March 2003

In an attempt to define clinically relevant models of akinesia and dyskinesia in 6‐hydroxydopamine (6‐OHDA)‐lesioned rats, we have examined the effects of drugs with high (l‐DOPA) vs. low (bromocriptine) dyskinesiogenic potential in Parkinson's disease on three types of motor performance, namely: (i) abnormal involuntary movements (AIMs) (ii) rotational behaviour, and (iii) spontaneous forelimb use (cylinder test). Rats with unilateral 6‐OHDA lesions received single daily i.p. injections of l‐DOPA or bromocriptine at therapeutic doses. During 3 weeks of treatment, l‐DOPA but not bromocriptine induced increasingly severe AIMs affecting the limb, trunk and orofacial region. Rotational behaviour was induced to a much higher extent by bromocriptine than l‐DOPA. In the cylinder test, the two drugs initially improved the performance of the parkinsonian limb to a similar extent. However, l‐DOPA‐treated animals showed declining levels of performance in this test because the drug‐induced AIMs interfered with physiological limb use, and gradually replaced all normal motor activities. l‐DOPA‐induced axial, limb and orolingual AIM scores were significantly reduced by the acute administration of compounds that have antidyskinetic efficacy in parkinsonian patients and/or nonhuman primates (−91%, yohimbine 10 mg/kg; −19%, naloxone 4–8 mg/kg; −37%, 5‐methoxy 5‐N,N‐dimethyl‐tryptamine 2 mg/kg; −30%, clozapine 8 mg/kg; −50%, amantadine 40 mg/kg). l‐DOPA‐induced rotation was, however, not affected. The present results demonstrate that 6‐OHDA‐lesioned rats do exhibit motor deficits that share essential functional similarities with parkinsonian akinesia or dyskinesia. Such deficits can be quantified using novel and relatively simple testing procedures, whereas rotometry cannot discriminate between dyskinetic and antiakinetic effects of antiparkinsonian treatments.
Time‐dependent inhibition of hippocampal LTP in vitro following contextual fear conditioning in the rat
Authors: Sacchetti Benedetto , Lorenzini Carlo Ambrogi , Baldi Elisabetta , Bucherelli Corrado , Roberto Marisa , Tassoni Giovanna , Brunelli Marcello
Published: March 2003

The effects of contextual fear‐learning on hippocampal synaptic excitability were investigated by means of high frequency tetanic stimulation (HFS) in rat brain slices (hippocampal CA1 region), prepared at different intervals (immediately, 24 h or 7 days) after a one‐trial contextual fear conditioning paradigm session. In the latter, rats that had previously received aversive electrical footshocks in the experimental apparatus exhibited freezing (the conditioned response) when placed again in the same apparatus (retrieval test). It was shown that contextual fear‐learning affects the hippocampal synaptic response. In fact, the HFS produced a decrease in the amplitude of short‐term (STP) and long‐term potentiation (LTP) when compared to control ‘naïve’ subject values. This decrease in STP amplitude could be observed only in slices prepared immediately after the training session. A decrease in the amplitude of long‐term but not short‐term potentiation was also observed at 24 h. At 7 days, no decreases in amplitude were observed. These modifications may be thought of as specifically associated with the learning process as they were not recorded in brain preparations from ‘shock‐only’ rats (i.e. those that received the same number of aversive stimuli of equal intensity as the conditioned group but with the shocks compressed temporally so that the shocked subjects could not associate nociceptive stimulation and surroundings – no conditioned freezing during retention testing). In ‘exploration’ preparations (brain slices from rats having only freely explored the experimental apparatus without receiving any adverse stimulation) a decrease in LTP amplitude was recorded only immediately after the training session, and STP was never modified. The synaptic response modifications do not appear to be due to presynaptic events, as they are not associated with paired‐pulse facilitation curve (PPF) modifications. The present results show that contextual fear conditioning and exploration of a novel environment (i) reduce the ability to induce synaptic plasticity; (ii) differentially influence STP and LTP and that (iii) the persistence of synaptic modifications depends on an animal's prior experience.
Voltage‐gated ionic currents in an identified modulatory cell type controlling molluscan feeding
Authors: Staras Kevin , Győri János , Kemenes György
Published: March 2003

An important modulatory cell type, found in all molluscan feeding networks, was investigated using two‐electrode voltage‐ and current‐clamp methods. In the cerebral giant cells of Lymnaea, a transient inward Na+ current was identified with activation at −58 ± 2 mV. It was sensitive to tetrodotoxin only in high concentrations (≈ 50% block at 100 µm), a characteristic of Na+ channels in many molluscan neurons. A much smaller low‐threshold persistent Na+ current (activation at < −90 mV) was also identified. Two purely voltage‐sensitive outward K+ currents were also found: (i) a transient A‐current type which was activated at −59 ± 4 mV and blocked by 4‐aminopyridine; (ii) a sustained tetraethylammonium‐sensitive delayed rectifier current which was activated at −47 ± 2 mV. There was also evidence that a third, Ca2+‐activated, K+ channel made a contribution to the total outward current. No inwardly rectifying currents were found. Two Ca2+ currents were characterized: (i) a transient low‐voltage (−65 ± 2 mV) activated T‐type current, which was blocked in NiCl2 (2 mm) and was completely inactivated at ≈ −50 mV; (ii) A sustained high voltage (−40 ± 1 mV) activated current, which was blocked in CdCl2 (100 µm) but not in ω‐conotoxin GVIA (10 µm), ω‐agatoxin IVA (500 nm) or nifedipine (10 µm). This current was enhanced in Ba2+ saline. Current‐clamp experiments revealed how these different current types could define the membrane potential and firing properties of the cerebral giant cells, which are important in shaping the wide‐acting modulatory influence of this neuron on the rest of the feeding network.
Neuronal expression and regulation of rat inhibitor of apoptosis protein‐2 by kainic acid in the rat brain
Authors: Belluardo Natale , Korhonen Laura , Mudo Giuseppa , Lindholm Dan
Published: March 2003

Inhibitors of apoptosis proteins (IAPs) define a protein family with the ability to counteract cell death by the inhibition of different caspases activated during apoptosis. These proteins are present in different cells, however, the function and roles of IAPs in brain tissue are not fully understood. We report here that RIAP‐2, the rat homologue of human cIAP‐1/HIAP‐2, is expressed in different areas of rat brain as shown by in situ hybridization and immunohistochemistry. Brain regions with relatively high expression of RIAP‐2 mRNA included cortex, cerebellum and different subregions of rat hippocampus. Double labelling using a specific anti‐RIAP antibody and markers for neurons and glial cells, showed that RIAP‐2 is predominantly expressed by nerve cells. Kainic acid treatment, which induces seizures, transiently up‐regulated RIAP‐2 mRNA levels in cerebral cortex, in the CA1 and dentate gyrus regions of hippocampus, which returned to normal levels at 24 h. However in the CA3 region, RIAP‐2 mRNA was decreased at 6 h following an early up‐regulation. This region contains neurons particularly vulnerable to kainic acid induced cell degeneration. The decrease in RIAP‐2 following kainic acid was also observed using immunohistochemistry. RIAP‐2 protein did not colocalize with TUNEL labelling present in cells undergoing cell death. The results show that in the adult rat brain RIAP‐2 is expressed mainly by neurons, and that the levels are regulated by kainic acid, which activates glutamate receptors. The decrease in RIAP‐2 in specific neuronal populations may contribute to cell degeneration in vulnerable brain regions observed after kainic acid treatment.