bluuebunny/crossref_metadata_2025 huggingface dataset:
Cellular and subcellular morphological localization of normal prion protein in rodent cerebellum
Authors: Lainé Jeanne , Marc Marie‐Elizabeth , Sy Man‐Sun , Axelrad Herbert
Published: March 2003

Normal cellular prion protein, a necessary protagonist in fatal neurodegenerative prion diseases, was mapped in rodent cerebellum to establish its cellular and ultrastuctural localization. Existing morphological data about native prion protein distribution in brain tissues remain, indeed, contradictory and do not fit with biochemical and cell biological results. Using ultrastructural preembedding immunocytochemistry and a monoclonal anti‐mouse prion protein antibody, this report shows that cellular prion protein is present in all cortico‐cerebellar and deep nuclei neuronal cell types, as well as in all glial cell types. The heaviest expression appears on parallel fibres and astrocytic processes. The protein is exclusively located on the outer cell membrane and in Golgi and endosomal intracytoplasmic organelles, with no cytoplasmic or synaptic vesicle labelling. Most important, and in contrast with previous ultrastructural data, cellular prion protein is shown to be distributed on all portions of neurons, without any preferential synaptic targeting. The present morphological report shows, for the first time in vivo, that the cellular prion protein is present on the entire cell surface membrane of all neuronal and glial cell types of the rat cerebellum. This ubiquitous presence supports the notion that prion protein has a generalized cellular function in brain tissue rather than a specialized role restricted to synaptic transmission.
Purification of Purkinje cells by fluorescence‐activated cell sorting from transgenic mice that express green fluorescent protein
Authors: Tomomura Mineko , Rice Dennis S. , Morgan James I. , Yuzaki Michisuke
Published: March 2003

The cerebellar Purkinje cell has been the focus of numerous studies involving the analysis of development and information processing in the nervous system. Purkinje cells represent less than 0.1% of the total cell content of the cerebellum. To facilitate studies of molecules that are expressed in such a small proportion of neurons, we have established procedures for the purification of these cells. Transgenic mice were developed in which the expression of green fluorescent protein (GFP) was controlled by the L7 promoter. In adult cerebellum, GFP fluorescence was only detected in Purkinje cells, where it filled dendrites, soma and axons. GFP fluorescence was detected in Purkinje cells as early as embryonic day 17 and increased during development in vivo and in dissociated cerebellar culture. Mirroring endogenous L7 expression, high levels of GFP were observed in retinal rod bipolar cells. Lower levels of GFP were seen in olfactory periglomerular cells, neurons in the interpeduncular nucleus, and superior colliculus neurons. Cerebella from transgenic mice were dissociated by mild enzymatic treatment and Purkinje cells were isolated by fluorescence‐activated cell sorting (FACS). By selecting optimal parameters, a fraction of viable Purkinje cells that was 94% pure was obtained. These results indicate that FACS is a powerful tool for isolating Purkinje cells from postnatal L7‐GFP transgenic mice. GFP‐positive neurons will also be useful in the real‐time observation of dendritic morphogenesis and axonal outgrowth during development, or after neuronal activity in vitro.
Fos imaging reveals differential neuronal activation of areas of rat temporal cortex by novel and familiar sounds
Authors: Wan H. , Warburton E. C. , Kuśmierek P. , Aggleton J. P. , Kowalska D. M. , Brown M. W.
Published: March 2003

To provide information about the possible regions involved in auditory recognition memory, this study employed an imaging technique that has proved valuable in the study of visual recognition memory. The technique was used to image populations of neurons that are differentially activated by novel and familiar auditory stimuli, thereby paralleling previous studies of visual familiarity discrimination. Differences evoked by novel and familiar sounds in the activation of neurons were measured in different parts of the rat auditory pathway by immunohistochemistry for the protein product (Fos) of the immediate early gene c‐fos. Significantly higher counts of stained neuronal nuclei (266 ± 21/mm2) were evoked by novel than by familiar sounds (192 ± 17/mm2) in the auditory association cortex (area Te3; AudA). No such significant differences were found for the inferior colliculus, primary auditory cortex, postrhinal cortex, perirhinal cortex (PRH), entorhinal cortex, amygdala or hippocampus. These findings are discussed in relation to the results of lesion studies and what is known of areas involved in familiarity discrimination for visual stimuli. Differential activation is produced by novel and familiar individual stimuli in sensory association cortex for both auditory and visual stimuli, whereas the PRH is differentially activated by visual but not auditory stimuli. It is suggested that this latter difference is related to the nature of the particular auditory and visual stimuli used.
Receptive field organization of disparity‐sensitive cells in Macaque medial superior temporal cortex
Authors: Gonzalez Francisco , Perez Rogelio , Justo Maria S. , Bermudez Maria A.
Published: March 2003

Binocular disparities are crucial for building an accurate three‐dimensional representation of the peripersonal environment in a viewer‐centred frame of reference. Previous studies have shown that visual cells of the medial superior temporal cortex (MST) have large receptive fields and that they are sensitive to disparities present in large surfaces. By using a reverse cross‐correlation technique in this study we tested 175 disparity‐sensitive units recorded from MST in the awake Macaca mulatta monkey to determine if these large receptive fields are homogeneous in terms of disparity sensitivity. We found that the receptive fields of 50 cells (50 out of 175, 29%) showed subregions with specific disparity sensitivity. These subregions presented eccentricities from 0.8 to 22.3° and their sizes varied from 1.6 to 15.3°2. This particular receptive field organization represents a suitable mechanism for encoding the location of small objects within our peripersonal space.
Analysis of cell signalling in the rodent pineal gland deciphers regulators of dynamic transcription in neural/endocrine cells*
Authors: Stehle Jörg H. , Von Gall Charlotte , Korf Horst‐Werner
Published: March 2003

In neurons, a temporally restricted expression of cAMP‐inducible genes is part of many developmental and adaptive processes. To understand such dynamics, the neuroendocrine rodent pineal gland provides an excellent model system as it has a clearly defined input, the neurotransmitter norepinephrine, and a measurable output, the hormone melatonin. In this system, a regulatory scenario has been deciphered, wherein cAMP‐inducible genes are rapidly activated via the transcription factor phosphoCREB to induce transcriptional events necessary for an increase in hormone synthesis. However, among the activated genes is also the inhibitory transcription factor ICER. The increasing amount in ICER protein leads ultimately to the termination of mRNA accumulation of cAMP‐inducible genes, including the gene for the Aa‐nat that controls melatonin production. This shift in ratio of phosphoCREB and ICER levels that depends on the duration of stimulation can be interpreted as a self‐restriction of cellular responses in neurons and has also been demonstrated to interfere with cellular plasticity in many non‐neuronal systems.
Expression and regulation of leptin receptor proteins in afferent and efferent neurons of the vagus nerve
Authors: Buyse Marion , Ovesjö Marie‐Louise , Goïot Hélène , Guilmeau Sandra , Péranzi Gabriel , Moizo Laurent , Walker Francine , Lewin Miguel J. M. , Meister Björn , Bado André
Published: March 2003

Leptin, the product of the ob gene, plays a key role in the regulation of food intake via a cross‐talk between hypothalamic leptin receptors and neuropeptides that affect feeding behaviour. Recent studies have shown a synergistic interaction between leptin and cholecystokinin (CCK) leading to suppression of food intake, which involves CCK‐1 receptors and capsaicin‐sensitive vagal fibres. In this study, we have investigated the presence of leptin receptors in afferent and efferent neurons of the vagus nerve. By using reverse transcription‐polymerase chain reaction, mRNAs encoding long (Ob‐Rb) and short (Ob‐Ra) leptin receptor isoforms were detected in the rat nodose ganglion, which contains the cell bodies of the vagal afferent neurons. Western blot analysis confirmed the presence of leptin receptor‐immunoreactive proteins in extracts from the vagal trunk. Immunohistochemistry showed the presence of leptin receptors and the leptin‐induced transcription factor STAT3 in the cytoplasm of nodose ganglion cells. In cervical vagal segments, levels of leptin receptor protein displayed physiological regulation, with decreased amounts after feeding and increased levels after food restriction. In addition, leptin receptor and STAT3 immunoreactivities were detected in neurons of the nucleus of tractus solitarius (NTS) and the dorsal motor nucleus of the vagus nerve (DMNX) by immunofluorescence histochemistry. Furthermore, direct double‐labelling demonstrated colocalization of Ob‐Rb and STAT3 immunoreactivities in cholinergic vagal efferent cell bodies of the DMNX. It is speculated that vagal leptin receptors, apart from being activated by adipocyte‐derived leptin, may also be influenced by leptin produced by the stomach. This may explain the synergistic action of leptin and CCK on neuronal activity in the NTS and on food intake.
Chronic psychosocial stress differentially affects apoptosis in hippocampal subregions and cortex of the adult tree shrew
Authors: Lucassen Paul J. , Vollmann‐Honsdorf Gesa K. , Gleisberg Mira , Czéh Boldizsár , De Kloet E. Ron , Fuchs Eberhard
Published: March 2003

We studied the effect of chronic psychosocial stress on cell death and volume changes in the tree shrew hippocampus. In situ end labelling (ISEL) identified low frequent but convincing apoptosis in many hippocampal subregions. Also in entorhinal cortex, apoptosis was found, generally at higher frequencies. After 28 days of chronic stress, apoptosis was significantly reduced in the CA1 stratum radiatum, whereas an increase was observed in the hilus (P < 0.04). With all subregions taken together, the hippocampus showed a decrease, whereas in the cortex, an increase in apoptosis was found after stress (P < 0.04). In a parallel and similar chronic stress study, post mortem morphometry of the same brain regions was performed, revealing mild decreases (7.6%) in entire hippocampal volume. We conclude that (i) low frequent apoptosis occurs throughout the adult tree shrew brain, and (ii) 28 days of chronic stress differentially affects its occurrence in distinct hippocampal subregions and entorhinal cortex. As previous stereological investigations failed to detect any loss in the principal neuronal layers, psychosocial stress, therefore, must affect other (structural) parameters like dendritic tree, interneurons, neurogenesis, or glia.
Ca2+‐dependent regulation of synaptic δ2 glutamate receptor density in cultured rat Purkinje neurons
Authors: Hirai Hirokazu
Published: March 2003

The δ2 glutamate receptor (δ2 receptor), which is expressed abundantly at parallel fibre–Purkinje neuron synapses, has an important role in synaptogenesis and synaptic plasticity in the cerebellum. The present study examined the molecular mechanisms regulating synaptic δ2 receptor density. Immunocytochemistry, using two antibodies against the intracellular C‐terminal and extracellular N‐terminal regions of the δ2 receptor, indicated the reversible redistribution of postsynaptic δ2 receptors in response to either glutamatergic stimulation or enhancement of synaptic activity. The effect of glutamatergic stimulation was completely inhibited by either coapplication of the glutamate receptor antagonist or the removal of extracellular Ca2+ using EGTA and mimicked by selective activation of voltage‐gated Ca2+ channels (VGCCs) with KCl, suggesting the significant role of Ca2+ influx in δ2 receptor redistribution. Biochemical examination indicated that a large amount of δ2 receptor protein was internalized following glutamatergic stimulation. These results suggest that the number of synaptic δ2 receptors is controlled by endocytosis in a synaptic activity‐ and intracellular Ca2+‐dependent way, through which synaptogenesis and synaptic plasticity in Purkinje cells might be modulated.
Activation of muscarinic receptors induces protein synthesis‐dependent long‐lasting depression in the perirhinal cortex
Authors: Massey P. V. , Bhabra G. , Cho K. , Brown M. W. , Bashir Z. I.
Published: March 2003

There is strong evidence that decrements in neuronal activation in perirhinal cortex when a novel stimulus is repeated provide a neural substrate of visual recognition memory. There is also strong evidence that muscarinic acetylcholine (ACh) receptors are involved in learning and memory. However, the mechanisms underlying neuronal decrements in the perirhinal cortex and the basis of ACh involvement in learning and memory are not understood. In an in vitro preparation of rat perirhinal cortex we now demonstrate that activation of ACh receptors by carbachol (CCh) produces long‐lasting depression (LLD) of synaptic transmission that is dependent on muscarinic M1 receptor activation. Crucially, the induction of this form of LLD requires neither N‐methyl‐d‐aspartate receptor activation nor synaptic stimulation. CCh‐induced LLD was not blocked by the protein kinase C inhibitors staurosporine or BIM, or by the protein phosphatase inhibitor okadaic acid. However, each of cyclopiazonic acid (an agent that depletes intracellular calcium stores) and anisomycin (an inhibitor of protein synthesis) significantly reduced the magnitude of CCh‐induced LLD. These mechanisms triggered by muscarinic receptor activation could play a role in the induction and/or expression of certain forms of activity‐dependent long‐term depression in perirhinal cortex. An understanding of CCh‐induced LLD may thus provide clues to the mechanisms underlying lasting neuronal decrements that occur in the perirhinal cortex and hence for neural substrates of visual recognition memory.
Fluorescent tracer in pilocarpine‐treated rats shows widespread aberrant hippocampal neuronal connectivity
Authors: Lehmann T.‐N. , Gabriel S. , Eilers A. , Njunting M. , Kovacs R. , Schulze K. , Lanksch W. R. , Heinemann U.
Published: March 2003

Neuronal fibres of the hippocampal formation of normal and chronic epileptic rats were investigated by fluorescent tracing methods using the pilocarpine model of limbic epilepsy. Two months after onset of spontaneous limbic seizures, hippocampal slices were prepared and maintained in vitro for 10 h. Small crystals of fluorescent dye [fluorescein (fluoro‐emerald®) and tetramethylrhodamine (fluoro‐ruby®)] were applied to different hippocampal regions. The main findings were: (i) in control rats there was no supragranular labelling when the mossy fibre tract was stained in stratum radiatum of area CA3. However, in epileptic rats a fibre network in the inner molecular layer of the dentate gyrus was retrogradely labelled; (ii) a retrograde innervation of area CA3 by CA1 pyramidal cells was disclosed by labelling remote CA1 neurons after dye injection into the stratum radiatum of area CA3 in chronic epileptic rats; (iii) labelling of CA1 neurons apart from the injection site within area CA1 was observed in epileptic rats but not in control animals; and (iv), a subicular‐hippocampal projection was present in pilocarpine‐treated rats when the tracer was injected just below the stratum pyramidale of area CA1. The findings show that fibre rearrangement in distinct regions of the epileptic hippocampal formation can occur as an aftermath of pilocarpine‐induced status epilepticus.
An essential role for the H218/AGR16/Edg‐5/LPB2 sphingosine 1‐phosphate receptor in neuronal excitability
Authors: MacLennan A. John , Carney Paul R. , Zhu Wei Jian , Chaves Alicia H. , Garcia Jairo , Grimes Jeremy R. , Anderson Kevin J. , Roper Steven N. , Lee Nancy
Published: March 2003

A wealth of indirect data suggest that the H218/AGR16/Edg‐5/LPB2 sphingosine 1‐phosphate (S1P) receptor plays important roles in development. In vitro, it activates several forms of development‐related signal transduction and regulates cellular proliferation, differentiation and survival. It is expressed during embryogenesis, and mutation of an H218‐like gene in zebrafish leads to profound defects in embryonic development. Nevertheless, the in vivo functions served by H218 signalling have not been directly investigated. We report here that mice in which the H218 gene has been disrupted are unexpectedly born with no apparent anatomical or physiological defects. In addition, no abnormalities were observed in general neurological development, peripheral axon growth or brain structure. However, between 3 and 7 weeks of age, H218–/– mice have seizures which are spontaneous, sporadic and occasionally lethal. Electroencephalographic abnormalities were identified both during and between the seizures. At a cellular level, whole‐cell patch‐clamp recordings revealed that the loss of H218 leads to a large increase in the excitability of neocortical pyramidal neurons. Therefore, H218 plays an essential, unanticipated and functionally important role in the proper development and/or mediation of neuronal excitability.
Spatiotemporal characterization of rhythmic activity in rat spinal cord slice cultures
Authors: Tscherter Anne , Heuschkel Marc O. , Renaud Philippe , Streit Jürg
Published: March 2003

Rat spinal networks generate a spontaneous rhythmic output directed to motoneurons under conditions of increased excitation or of disinhibition. It is not known whether these differently induced rhythms are produced by a common rhythm generator. To investigate the generation and the propagation of rhythmic activity in spinal networks, recordings need to be made from many neurons simultaneously. Therefore extracellular multisite recording was performed in slice cultures of embryonic rat spinal cords grown on multielectrode arrays. In these organotypic cultures most of the spontaneous neural activity was nearly synchronized. Waves of activity spread from a source to most of the network within 35–85 ms and died out after a further 30–400 ms. Such activity waves induced the contraction of cocultured muscle fibres. Several activity waves could be grouped into aperiodic bursts. Disinhibition with bicuculline and strychnine or increased excitability with high K+ or low Mg2+ solutions could induce periodic bursting with bursts consisting of one or several activity waves. Whilst the duration and period of activity waves were similar for all protocols, the duration and period of bursts were longer during disinhibition than during increased excitation. The sources of bursting activity were mainly situated ventrally on both sides of the central fissure. The pathways of network recruitment from one source were variable between bursts, but they showed on average no systematic differences between the protocols. These spatiotemporal similarities under conditions of increased excitation and of disinhibition suggest a common spinal network for both types of rhythmic activity.
The generation of rhythmic activity in dissociated cultures of rat spinal cord
Authors: Streit Jürg , Tscherter Anne , Heuschkel Marc O. , Renaud Philippe
Published: March 2003

Locomotion in vertebrates is controlled by central pattern generators in the spinal cord. The roles of specific network architecture and neuronal properties in rhythm generation by such spinal networks are not fully understood. We have used multisite recording from dissociated cultures of embryonic rat spinal cord grown on multielectrode arrays to investigate the patterns of spontaneous activity in randomised spinal networks. We were able to induce similar patterns of rhythmic activity in dissociated cultures as in slice cultures, although not with the same reliability and not always with the same protocols. The most reliable rhythmic activity was induced when a partial disinhibition of the network was combined with an increase in neuronal excitability, suggesting that both recurrent synaptic excitation and neuronal excitability contribute to rhythmogenesis. During rhythmic activity, bursts started at several sites and propagated in variable ways. However, the predominant propagation patterns were independent of the protocol used to induce rhythmic activity. When synaptic transmission was blocked by CNQX, APV, strychnine and bicuculline, asynchronous low‐rate activity persisted at ≈ 50% of the electrodes and ≈ 70% of the sites of burst initiation. Following the bursts, the activity in the interval was transiently suppressed below the level of intrinsic activity. The degree of suppression was proportional to the amount of activity in the preceding burst. From these findings we conclude that rhythmic activity in spinal cultures is controlled by the interplay of intrinsic neuronal activity and recurrent excitation in neuronal networks without the need for a specific architecture.
Discharge threshold is enhanced for several seconds after a single interictal spike in a model of focal epileptogenesis
Authors: De Curtis Marco , Librizzi Laura , Biella Gerardo
Published: March 2003

Interictal spikes (ISs) are typically observed between seizures in focal epilepsies. Whether ISs are causally involved or represent protective elements in the transition toward an ictal discharge is an open question. Previous studies suggested that inhibition or disfacilitation occurs during the period elapsing between two ISs induced by local application of either bicuculline or penicillin in the piriform cortex of the in vitro isolated guinea pig brain preparation. We further investigated this issue by studying responses to afferent stimulation during the interspike period (6.3 ± 2.5 s; mean ± SD). Properly set stimulation intensity of the lateral olfactory tract resets ISs exclusively (and not before) 4–10 s (5.6 ± 2.0 s; mean ± SD) after a preceding spontaneous spike. This finding demonstrates the existence of a period of enhanced threshold to stimulus‐evoked activation that coincides with the interspike interval in the absence of stimulation. Current source density analysis of depth laminar profiles demonstrated that both stimulus‐evoked and spontaneous ISs were generated by the activation of an identical cortical circuit. Our study suggests that interictal spiking could play a protective role or at least provide an effective restraint against the onset of a focal ictal discharge.
Patterns of colocalization of GABA, glutamate and glycine immunoreactivities in terminals that synapse on dendrites of noradrenergic neurons in rat locus coeruleus
Authors: Somogyi J. , Llewellyn‐Smith I. J.
Published: March 2003

Amino acid transmitters play a key role in regulating the activity of noradrenergic neurons in the locus coeruleus. We investigated the anatomical substrate for this regulation by quantifying immunoreactivity for GABA, glutamate and glycine in terminals that contacted the dendrites of tyrosine hydroxylase‐immunoreactive principal neurons in rat locus coeruleus. Pre‐embedding peroxidase immunocytochemistry was used to detect tyrosine hydroxylase‐immunoreactivity in Vibratome sections of tissue perfused with 2.5% glutaraldehyde. GABA, glutamate and glycine were localized with postembedding immunogold labelling. Gold particle densities over terminals were measured in three semiserial ultrathin sections, each reacted for a different amino acid. More than 90% (range among rats, 89%−95%) of the terminals analyzed (n = 288) were immunoreactive for at least one amino acid. A high proportion (39%−49%) were positive for two or three amino acids. About two‐thirds (60%−69%) of the boutons contained GABA, of which more than half (51%−55%) also contained glycine. More than one‐third (36%−38%) of the terminals were positive for glycine. Terminals immunoreactive for glycine alone were rare (0%−2%). About one‐third of the terminals showed glutamate‐immunoreactivity (32%−37%). GABA and/or glycine occurred in one‐fifth to one‐third of these. These results show that amino acid‐immunoreactivity is present in almost all of the terminals that synapse on tyrosine hydroxylase‐positive dendrites in locus coeruleus. Glutamate provides a major excitatory input. The almost complete colocalization of glycine with GABA suggests that the inhibitory input to locus coeruleus is predominantly GABAergic with a contribution from glycine in about half of the GABAergic boutons.
Human brain activity in the control of fine static precision grip forces: an fMRI study
Authors: Kuhtz‐Buschbeck Johann P. , Ehrsson H. Henrik , Forssberg Hans
Published: March 2003

Dexterous manipulation of delicate objects requires exquisite control of fingertip forces. We have used functional magnetic resonance imaging to identify brain regions involved in the skilful scaling of these forces when normal human subjects (n = 8) held with precision grip a small object (weight 200 g) in the dominant right hand. In one condition, they used their normal, automatically scaled grip force. The object was held gently in a second condition; the isometric grip force was maintained just above the critical level at which the object would have slipped. In a third condition, the force was increased to hold the object with a more firm grip. The supplementary and cingulate motor areas were significantly more active during the gentle force condition than during either of the other conditions in all subjects, despite weaker contractions of the hand muscles. In addition, the left primary sensorimotor cortex, the ventral premotor cortex and the left posterior parietal cortex were more strongly activated during gentle than during normal grasping. These novel results suggest that these regions are specifically involved in dexterous scaling of fingertip forces during object manipulation.
Projections from the nociceptive area of the central nucleus of the amygdala to the forebrain: a PHA‐L study in the rat
Authors: Bourgeais Laurence , Gauriau Caroline , Bernard Jean‐François
Published: March 2003

The lateral capsular division (CeLC) of the central nucleus (Ce) of the amygdala, in the rat, has been shown to be the main terminal area of a spino(trigemino)‐parabrachio‐amygdaloid nociceptive pathway [Bernard & Besson (1990)J. Neurophysiol.63,473–490; Bernardet al. (1992)J. Neurophysiol.68,551–569; Bernardet al. (1993)J. Comp. Neurol.329,201–229]. The projections to the forebrain from the CeLC and adjacent regions were studied in the rat by using microinjections ofPhaseolus vulgarisleucoagglutinin (PHA‐L) restricted in subdivisions of the Ce and the basolateral amygdaloid nucleus anterior (BLA). Our data showed that the entire CeLC projects primarily and extensively to the substantia innominata dorsalis (SId). The terminal labelling is especially dense in the caudal aspect of the SId. The other projections of the CeLC in the forebrain were dramatically less dense. They terminate in the bed nucleus of the stria terminalis (BST) and the posterior hypothalamus (pLH). No (or only scarce) other projections were found in the remaining forebrain areas. The Ce lateral division (CeL) and the Ce medial division (CeM), adjacent to the CeLC, also project to the SId with slightly lower density labelling. However, contrary to the case of the CeLC, both the CeL and the CeM extensively project to the ventrolateral subnucleus of the BST (BSTvl) with a few additional terminals found in other regions of the lateral BST. Only the CeM projects densely to both the interstitial nucleus of the posterior limb of the anterior commissure and the caudal most portion of the pLH. The projections of the BLA are totally different from those of the Ce as they terminate in the dorsal striatum, the accumbens nucleus, the olfactory tubercle, the nucleus of olfactory tract and the rostral pole of the cingulate/frontal cortex. This study demonstrates that the major output of the nociceptive spino(trigemino)‐parabrachio‐CeLC pathway is to the SId. It is suggested that the CeLC–SId pathway could have an important role in anxiety, aversion and genesis of fear in response to noxious stimuli.
Exogenous NT‐3 and NGF differentially modulate PACAP expression in adult sensory neurons, suggesting distinct roles in injury and inflammation
Authors: Jongsma H. , Danielsen N. , Johnston J.M. , Gratto K.A. , Karchewski L.A. , Verge V.M.K.
Published: March 2003

Expression of pituitary adenylate cyclase‐activating polypeptide in sensory neurons varies with injury or inflammation. The neurotrophins NGF and NT‐3 are profound regulators of neuronal peptidergic phenotype in intact and injured sensory neurons. This study examined their potential for modulation of PACAP expression in adult rat with intact and injured L4–L6 spinal nerves with or without immediate or delayed intrathecal infusion of NT‐3 or NGF. Results indicate that in L5 DRG, few trkC neurons express high levels of PACAP mRNA in the intact state, but many do following injury. The elevated expression in injured neurons is mitigated by NT‐3 infusion, suggesting a role for NT‐3 in returning the ‘injured phenotype’ back towards an ‘intact phenotype’. NGF dramatically up‐regulated PACAP expression in trkA‐positive neurons in both intact and injured DRGs, implicating NGF as a positive regulator of PACAP expression in nociceptive neurons. Surprisingly, NT‐3 modulates PACAP expression in an antagonistic fashion to NGF in intact neurons, an effect most evident in the trkA neurons not expressing trkC. Both NT‐3 and NGF infusion results in decreased detection of PACAP protein in the region of the gracile nuclei, where central axons of the peripherally axotomized large sensory fibers terminate. NGF infusion also greatly increased the amount of PACAP protein detected in the portion of the dorsal horn innervated by small–medium size DRG neurons, while both neurotrophins appear able to prevent the decrease in PACAP expression observed in these afferents with injury. These results provide the first insights into the potential molecules implicated in the complex regulation of PACAP expression in sensory neurons.
Multiple pertussis toxin‐sensitive G‐proteins can couple receptors to GIRK channels in rat sympathetic neurons when expressed heterologously, but only native Gi‐proteins do soin situ
Authors: Fernández‐Fernández José M. , Abogadie Fe C. , Milligan Graeme , Delmas Patrick , Brown David A.
Published: March 2003

Although many G‐protein‐coupled neurotransmitter receptors are potentially capable of modulating both voltage‐dependent Ca2+channels (ICa) and G‐protein‐gated K+channels (IGIRK), there is a substantial degree of selectivity in the coupling to one or other of these channels in neurons. Thus, in rat superior cervical ganglion (SCG) neurons, M2muscarinic acetylcholine receptors (mAChRs) selectively activateIGIRKwhereas M4mAChRs selectively inhibitICa. One source of selectivity might be that the two receptors couple preferentially to different G‐proteins. Using antisense depletion methods, we found that M2mAChR‐induced activation ofIGIRKis mediated by Giwhereas M4mAChR‐induced inhibition ofICais mediated by GoA. Experiments with the βγ‐sequestering peptides α‐transducin and βARK1C‐terindicate that, although both effects are mediated by G‐protein βγ subunits, the endogenous subunits involved inIGIRKinhibition differ from those involved inICainhibition. However, this pathway divergence does not result from any fundamental selectivity in receptor–G‐protein–channel coupling because bothIGIRKandICamodulation can be rescued by heterologously expressed Gior Goproteins after the endogenously coupled α‐subunits have been inactivated withPertussistoxin (PTX). We suggest instead that the divergence in the pathways activated by the endogenous mAChRs results from a differential topographical arrangement of receptor, G‐protein and ion channel.
Astrocytes down‐regulate neuronal β‐amyloid precursor protein expression and modify its processing in an apolipoprotein E isoform‐specific manner
Authors: Vincent Bruno , Smith Jonathan D.
Published: March 2003

Alzheimer's disease is the most frequent neurodegenerative disorder in the aged population and is characterized by the deposition of the 40/42‐residue amyloid β protein (Aβ), a proteolytic fragment of the β‐amyloid precursor protein (APP). A common apolipoprotein E (apoE) polymorphism is associated with an increased risk of developing the disease. In order to assess the putative relationship between apoE and amyloidogenesis in the CNS, we prepared primary cortical neurons overexpressing humanized APP695 bearing the Swedish mutation (hAPP695sw) and we analysed APP expression and processing after: (i) coculture with primary astrocytes from wild‐type, apoE‐deficient (E0) mice, or mice overexpressing human apoE2, E3, or E4; (ii) treatment with conditioned media from apoE0, E2, E3 or E4 astrocytes; and (iii) treatment with human recombinant ApoE or human apoE purified from conditioned media of stably transfected RAW264 cells (E2, E3 and E4). Interestingly, a strong decrease in APP expression was observed only when neurons were cocultured with astrocytes (and independently of the apoE genotype considered), suggesting that cell–cell contact is required. Moreover, apoE4‐secreting astrocytes, but not recombinant or purified apoE4, significantly increased Aβ production and decrease sAPPα secretion only when cultured in direct contact with neurons, whereas apoE2 astrocytes had a protective effect. We conclude that astrocytes: (i) strongly regulate neuronal APP expression in primary neurons, and (ii) promote the amyloidogenic pathway in an apoE4‐dependent manner. Thus, apoE and astrocytic factor(s) may modulate the pathogenesis of Alzheimer's disease.
Effect of microiontophoretic application of dopamine on subthalamic nucleus neuronal activity in normal rats and in rats with unilateral lesion of the nigrostriatal pathway
Authors: Ni Zhongge , Gao Dongming , Bouali‐Benazzouz Rabia , Benabid Alim‐Louis , Benazzouz Abdelhamid
Published: March 2003

The subthalamic nucleus (STN) receives dopamine inputs from the substantia nigra but their implication in the pathophysiology of parkinsonism is still debated. Extracellular microrecordings were used to study the effect of microiontophoretic injection of dopamine and the D1 receptor agonist SKF 38393 on the activity of STN neurons in normal and 6‐hydroxydopamine‐lesioned rats under urethane anaesthesia. Dopamine and SKF induced an increase in the firing rate of the majority of STN neurons in both normal and 6‐OHDA rats. In rats with 6‐OHDA lesions, the percentage of firing rate increase did not differ from that of controls. When GABA, glutamate and dopamine were all applied to the same individual STN neurons, GABA induced an inhibitory effect and glutamate and dopamine caused an excitatory effect in both groups. This excitatory response was suppressed by the application of GABA. Systemic administration of apomorphine provoked a decrease in the firing rate of STN neurons in rats with 6‐OHDA lesions. These results show that dopamine exerts an excitatory influence on STN neurons, suggesting that the inhibitory effect induced by the systemic injection of apomorphine is due to the GABAergic inputs from the globus pallidus as predicted by the current model of basal ganglia organization. In addition, we show that dopamine, GABA and glutamate can act on the same STN neuron and that GABA can reverse the excitatory effect of dopamine and glutamate, suggesting the predominant influence of GABAergic inputs to the subthalamic nucleus.
Early behavioural changes in scrapie‐affected mice and the influence of dapsone
Authors: Guenther Katja , Deacon Robert M. J. , Perry V. Hugh , Rawlins J. Nicholas P.
Published: March 2003

Behavioural testing can reveal effects in scrapie‐infected mice long before overt clinical signs appear (Betmouni et al., 1999, Psychobiology, 27, 63–71). These effects may be partly attributable to an early, atypical inflammatory response in the brain (Betmouni et al., 1996, Neuroscience, 74, 1–5). The present study replicated and extended these findings, and examined the effect of chronic treatment with dapsone. This anti‐inflammatory compound has been reported to delay disease onset in a rat model of Creutzfeldt–Jakob disease (Manuelidis et al., 1998, Lancet, 352, 456). Although the doses used in the present study were higher than those of Manuelidis et al. (1998), no attenuation of the disease was seen in either behavioural or subsequent histological tests. Burrowing, i.e. displacing food pellets from a tube in the home cage, decreased from around week 12 in scrapie‐infected mice, as did consumption of palatable glucose solution. Concurrently, ambulation in an open field increased, as did rearing at around week 17. Spontaneous alternation was impaired around this time. Around 18 weeks, motor performance on an inverted screen, horizontal bar, rotating rod and static rods decreased. Nest construction was impaired at 20 weeks. Overt clinical signs (reduction in mobility, hunched posture, poor coat condition, bladder enlargement) only occurred after week 20, when the mice were prepared for histology. The ME7 scrapie‐infected mice thus showed a characteristic complex of neurological and behavioural changes during the course of the disease that were not ameliorated by dapsone. These changes appeared well before clinical signs were prominent.
A common inhibitory binding site for zinc and odorants at the voltage‐gated K+ channel of rat olfactory receptor neurons
Authors: Seebungkert Benchamaporn , Lynch Joseph W.
Published: March 2003

This study compared the effects of zinc and odorants on the voltage‐gated K+ channel of rat olfactory neurons. Zinc reduced current magnitude, depolarized the voltage activation curve and slowed activation kinetics without affecting inactivation or deactivation kinetics. Zinc inhibition was potentiated by the NO compound, S‐nitroso‐cysteine. The pH‐ and diethylpyrocarbonate‐dependence of zinc inhibition suggested that zinc acted by binding to histidine residues. Cysteine residues were eliminated as contributing to the zinc‐binding site. The odorants, acetophenone and amyl acetate, also reduced current magnitude, depolarized the voltage activation curve and selectively slowed activation kinetics. Furthermore, the diethylpyrocarbonate‐ and pH‐dependence of odorant inhibition implied that the odorants also bind to histidine residues. Zinc inhibitory potency was dramatically diminished in the presence of odorants, implying competition for a common binding site. These observations indicate that the odorants and zinc share a common inhibitory binding site on the external surface of the voltage‐gated K+ channel.
Interleukin‐6 (IL6) and cellular response to facial nerve injury: effects on lymphocyte recruitment, early microglial activation and axonal outgrowth in IL6‐deficient mice
Authors: Galiano Matthias , Liu Zhi Qiang , Kalla Roger , Bohatschek Marion , Koppius Andrea , Gschwendtner Andreas , Xu ShengLi , Werner Alexander , Kloss Christian U.A. , Jones Leonard L. , Bluethmann Horst , Raivich Gennadij
Published: March 2003

Nerve injury triggers numerous changes in the injured neurons and surrounding non‐neuronal cells. Of particular interest are molecular signals that play a role in the overall orchestration of this multifaceted cellular response. Here we investigated the function of interleukin‐6 (IL6), a multifunctional neurotrophin and cytokine rapidly expressed in the injured nervous system, using the facial axotomy model in IL6‐deficient mice and wild‐type controls. Transgenic deletion of IL6 caused a massive decrease in the recruitment of CD3‐positive T‐lymphocytes and early microglial activation during the first 4 days after injury in the axotomized facial nucleus. This was accompanied by a more moderate reduction in peripheral regeneration at day 4, lymphocyte recruitment (day 14) and enhanced perikaryal sprouting (day 14). Motoneuron cell death, phagocytosis by microglial cells and recruitment of granulocytes and macrophages into injured peripheral nerve were not affected. In summary, IL6 lead to a variety of effects on the cellular response to neural trauma. However, the particularly strong actions on lymphocytes and microglia suggest that this cytokine plays a central role in the initiation of immune surveillance in the injured central nervous system.
Subcellular localization of calcium‐permeable AMPA receptors in spinal motoneurons
Authors: Vandenberghe Wim , Bindokas Vytautas P. , Miller Richard J. , Robberecht Wim , Brorson James R.
Published: March 2003

Activation of Ca2+‐permeable α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA) receptors has been linked to potent effects on survival and dendritic outgrowth of spinal motoneurons. Ca2+ permeability of AMPA receptors is controlled by the GluR2 subunit. Whole‐cell electrophysiological studies have suggested that GluR2‐containing and GluR2‐lacking AMPA receptors may coexist in individual motoneurons. However, there has not been a direct demonstration of heterogeneity in AMPA receptor subunit composition in single motoneurons, nor of distinct subcellular distributions of GluR2‐containing and GluR2‐lacking receptors. In the present study, we have used confocal microscopy, immunocytochemistry and Ca2+ imaging to characterize the subcellular localization of AMPA receptors in cultured rat spinal motoneurons. Immunoreactivity for GluR2 and GluR4 was concentrated in clusters, the vast majority of which were found in dendrites at synapses. Double‐labelling for GluR2 and GluR4 revealed variability in relative expression of GluR2 and GluR4 between clusters within individual motoneurons; most AMPA receptor clusters were immunoreactive for both GluR2 and GluR4, but a significant minority of clusters were immunoreactive for GluR2 only or for GluR4 only. The majority of GluR2‐immunonegative AMPA receptor clusters was present in dendrites, but the relative proportion of GluR2‐immunonegative and GluR2‐immunopositive clusters was similar in dendrites and soma. Imaging of [Ca2+]i rises triggered by AMPA receptor activation confirmed Ca2+ influx in motoneuron dendrites. These findings strongly support a model in which GluR2‐containing and GluR2‐lacking AMPA receptors coexist in motoneurons, clustered at synapses, and mixed in a relative proportion that varies considerably between cell membrane microdomains.
Astrocytic factors protect neuronal integrity and reduce microglial activation in an in vitro model of N‐methyl‐d‐aspartate‐induced excitotoxic injury in organotypic hippocampal slice cultures
Authors: Hailer Nils P. , Wirjatijasa Florentina , Roser Nadine , Hischebeth Gunnar T. R. , Korf Horst‐Werner ,   Faramarz Dehghani
Published: March 2003

Acute CNS lesions lead to neuronal injury and a parallel glial activation that is accompanied by the release of neurotoxic substances. The extent of the original neuronal damage can therefore be potentiated in a process called secondary damage. As astrocytes are known to secrete immunomodulatory and neuroprotective substances, we investigated whether astrocytic factors can attenuate the amount of neuronal injury as well as the degree of microglial activation in a model of excitotoxic neurodegeneration. Treatment of organotypic hippocampal slice cultures with N‐methyl‐d‐aspartate (NMDA) resulted in a reproducible loss of viable granule cells, partial destruction of the regular hippocampal cytoarchitecture and a concomitant accumulation of amoeboid microglial cells at sites of neuronal damage. Astrocyte‐conditioned media reduced the amount of NMDA‐induced neuronal injury by 45.3%, diminished the degree of microglial activation and resulted in an improved preservation of the hippocampal cytoarchitecture. Transforming growth factor (TGF)‐β failed to act as a neuroprotectant and even enhanced the amount of neuronal injury by 52.5%. Direct effects of astrocytic factors on isolated microglial cells consisted of increased microglial ramification and down‐regulated expression of intercellular adhesion molecule‐1, whereas incubation with TGF‐β had no such effects. In summary, our findings show that hitherto unidentified astrocyte‐derived factors that are probably not identical with TGF‐β can substantially enhance neuronal survival, either by eliciting direct neuroprotective effects or by modulating the microglial response to neuronal injury.
Developmental regulation and overexpression of the transcription factor AP‐2, a potential regulator of the timing of Schwann cell generation
Authors: Stewart Helen J. S. , Brennan Angela , Rahman Mary , Zoidl Georg , Mitchell Pamela J. , Jessen Kristján R. , Mirsky Rhona
Published: March 2003

There is now evidence from in vivo and in vitro studies that the rate of Schwann cell generation is regulated by the balance of two opposing signals, β neuregulins and endothelins. The β neuregulins promote the development of precursors to Schwann cells whereas endothelins retard it through an action on endothelin‐B receptors. The present work has shown additional controls of this transition, and implicates AP‐2 transcription factors, in particular AP‐2α, as negative regulators of Schwann cell generation. We found that both AP‐2α and AP‐2γ are present in early embryonic nerves, whereas AP‐2β was not. Isoform‐specific analysis of AP‐2α showed that isoform 3 was most abundant with isoforms 1 and 2 present in lesser amounts; isoform 4 was absent. Maximal AP‐2α and AP‐2γ mRNA expression occurred at embryonic day (E) 12/13 in the mouse and at E14/15 in the rat, which correlates with the presence of Schwann cell precursors in the nerve. In both rats and in mice, in vivo and in vitro, downregulation of AP‐2α mRNA and protein coincided with one of the main steps in Schwann cell development, the precursor–Schwann cell transition. Moreover, Schwann cell generation was delayed if this downregulation was prevented by enforced expression of AP‐2α in precursors. These studies suggest that AP‐2 is involved in the control of the timing of Schwann cell development.
Bradykinin‐induced nociceptor sensitization to heat is mediated by cyclooxygenase products in isolated rat skin
Authors: Pethö Gabor , Derow Alexandra , Reeh Peter W.
Published: March 2003

Bradykinin can excite C‐polymodal nociceptors and sensitize them to heat and it can also enhance prostaglandin synthesis, but it is unclear whether these effects are causally related. The role of cyclooxygenase products was investigated using two enantiomers of the cyclooxygenase inhibitor flurbiprofen of which S(+)‐ is more potent than R(–)‐flurbiprofen. Single‐unit activity was recorded from mechano‐heat‐sensitive, polymodal C‐fibers in the isolated rat skin‐saphenous nerve preparation. Bradykinin pretreatment (10 µm, 5 min) induced a 219 ± 26% increase in the number of spikes evoked by noxious heat stimulation and a drop in the heat threshold by 5.2 ± 0.6 °C in a fully reproducible manner. S(+)‐flurbiprofen (1 µm) abolished the bradykinin‐induced heat sensitization but did not alter the unconditioned heat response itself. Under R(–)‐flurbiprofen (1 µm) bradykinin still induced a significant heat sensitization which was reduced by 33 ± 21% (P = 0.11) of its previous extent; this effect may be due to the limited purity of the enantiomer preparation or to a cyclooxygenase‐independent action of flurbiprofen. The heat sensitization suppressed by S(+)‐flurbiprofen could be significantly restored (to 43 ± 12%) by addition of PGE2 plus PGI2 (10 µm both) to bradykinin. Neither S(+)‐ nor R(–)‐flurbiprofen had an influence on the magnitude of the excitatory effect of bradykinin. It is concluded that (i) cyclooxygenase products are the main mediators of nociceptor sensitization to heat following bradykinin treatment in the isolated rat skin; (ii) PGE2/I2 are essential but perhaps not the only relevant cyclooxygenase products involved and (iii) neither S(+)‐ nor R(–)‐flurbiprofen inhibit the unconditioned noxious heat response and the excitatory bradykinin response of the polymodal C‐nociceptors.