bluuebunny/crossref_metadata_2025 huggingface dataset:
Lithium exposure enhances survival of NT2N cells (hNT neurons) in the hemiparkinsonian rat
Authors: Willing Alison E. , Zigova Tanja , Milliken Melissa , Poulos Steve , Saporta Samuel , McGrogan Michael , Snable Gary , Sanberg Paul R.
Published: March 2003

Lithium (Li +) treatment of NTera2/D1 (or hNT Neurons) in culture increases tyrosine hydroxylase (TH) expression in this cell‐line [Zigova et al., (1999) Exp. Neurol., 157, 251–258]. It is not known if these Li + treated cells maintain TH expression once transplanted into the striatum of the hemiparkinsonian rats. hNT neurons were either treated with 1 mm LiCl or left untreated and then transplanted into the striatum of Sprague‐Dawley rats. Some cells were exposed to the lithium for 24 h in culture while others were exposed only briefly (2–3 h) just prior to transplantation. We also examined whether Li + treatment of the animal after transplantation (0.24% w/w lithium carbonate in chow) was effective in increasing neuronal survival. One week after transplantation, the animals were perfused with 4% paraformaldehyde and immunocytochemistry was performed on 30 µm sections through the transplant. Human nuclear matrix antigen immunostaining demonstrated that there was significantly better survival of cells in the group treated briefly with lithium compared to all other groups. Brief exposure to lithium resulted in a greater expression of TH in situ as well. Neuron specific enolase immunohistochemistry showed that there was extensive fibre outgrowth in all groups. These results suggest that brief Li + exposure may enhance survival to over 60% and increase TH expression of hNT Neurons transplanted in the hemiparkinsonian rat nearly three‐fold.
Aggresome‐related biogenesis of Lewy bodies
Authors: McNaught Kevin St P. , Shashidharan P. , Perl Daniel P. , Jenner Peter , Olanow C. Warren
Published: March 2003

Neurodegenerative disorders such as Parkinson's disease (PD) and ‘dementia with Lewy bodies’ (DLB) are characterized pathologically by selective neuronal death and the appearance of intracytoplasmic protein aggregates (Lewy bodies). The process by which these inclusions are formed and their role in the neurodegenerative process remain elusive. In this study, we demonstrate a close relationship between Lewy bodies and aggresomes, which are cytoplasmic inclusions formed at the centrosome as a cytoprotective response to sequester and degrade excess levels of potentially toxic abnormal proteins within cells. We show that the centrosome/aggresome‐related proteins γ‐tubulin and pericentrin display an aggresome‐like distribution in Lewy bodies in PD and DLB. Lewy bodies also sequester the ubiquitin‐activating enzyme (E1), the proteasome activators PA700 and PA28, and HSP70, all of which are recruited to aggresomes for enhanced proteolysis. Using novel antibodies that are specific and highly sensitive to ubiquitin–protein conjugates, we revealed the presence of numerous discrete ubiquitinated protein aggregates in neuronal soma and processes in PD and DLB. These aggregates appear to be being transported from peripheral sites to the centrosome where they are sequestered to form Lewy bodies in neurons. Finally, we have shown that inhibition of proteasomal function or generation of misfolded proteins cause the formation of aggresome/Lewy body‐like inclusions and cytotoxicity in dopaminergic neurons in culture. These observations suggest that Lewy body formation may be an aggresome‐related event in response to increasing levels of abnormal proteins in neurons. This phenomenon is consistent with growing evidence that altered protein handling underlies the etiopathogenesis of PD and related disorders.
The effect of bilateral deafness on excitatory and inhibitory synaptic strength in the inferior colliculus
Authors: Vale Carmen , Sanes Dan H.
Published: March 2003

The consequences of deafness on the central auditory nervous system have been examined at many levels, from molecular to functional. However, there has never been a direct and selective measurement of excitatory synaptic function following total hearing loss. In the present study, gerbils were deafened at postnatal day 9, an age at which there is no deafferentation‐induced cell death of ventral cochlear nucleus neurons. One to five days after bilateral cochlear ablation, the amplitude of evoked excitatory postsynaptic currents (EPSC) was measured with whole‐cell voltage‐clamp recordings in an inferior colliculus (IC) brain slice preparation in response to electrical stimulation of the ipsilateral lateral lemniscus (LL) or the commissure of the inferior colliculus (CIC). Deafness resulted in larger LL‐ and CIC‐evoked EPSC amplitudes and durations. This result was observed at a depolarized holding potential. In addition, deafness caused a decrease in excitatory neurotransmitter release at the LL pathway, as assessed with a paired‐pulse stimulation protocol. In contrast to its effect on excitatory synapses, bilateral cochlear ablation reduced inhibitory synaptic strength in IC neurons. The effects included a postsynaptic decrease in IPSC conductance, a 25‐mV depolarization in the IPSC equilibrium potential and a decrease of neurotransmitter release. Thus normal innervation differentially affects excitatory and inhibitory synaptic strength in IC neurons, and these changes may contribute to alterations in auditory coding properties following sensory deprivation.
Selective ablation of olfactory receptor neurons without functional impairment of vomeronasal receptor neurons in OMP‐ntr transgenic mice
Authors: Ma Dan , Allen Nicholas D. , Van Bergen Yfke C. H. , Jones Camilla M. E. , Baum Michael J. , Keverne E. Barry , Brennan Peter A.
Published: March 2003

This study used transgenic mice, in which expression of a bacterial nitroreductase (ntr) gene was linked to the expression of olfactory marker protein (OMP). The nitroreductase enzyme is thus expressed in mature chemosensory neurons of these OMP‐ntr transgenic mice, and converts the pro‐drug CB1954 to a cytotoxic form, specifically killing these neurons. Systemic injections of the pro‐drug led to the ablation of receptor neurons in both the main olfactory and vomeronasal epithelia. Due to the anatomical separation of the epithelia, however, when the pro‐drug was administered by intranasal infusion only the receptors of the main olfactory epithelium were destroyed. This procedure resulted in a profound deficit in olfactory investigation and discrimination in a habituation–dishabituation test, whereas the pregnancy blocking effect of male pheromones, which is mediated via the vomeronasal system was unaffected. OMP‐ntr mice receiving intranasal infusion of pro‐drug had not recovered any significant main olfactory function at 24 days following treatment. This novel technique could potentially be applied to selectively ablate olfactory receptor neurons expressing a particular olfactory receptor by linking its expression to that of the nitroreductase enzyme.
The murine neurokinin NK1 receptor gene contributes to the adult hypoxic facilitation of ventilation
Authors: Ptak Krzysztof , Burnet Henri , Blanchi Bruno , Sieweke Michael , De Felipe Carmen , Hunt Stephen P. , Monteau Roger , Hilaire Gérard
Published: March 2003

Substance P and neurokinin‐1 receptors (NK1) modulate the respiratory activity and are expressed early during development. We tested the hypothesis that NK1 receptors are involved in prenatal development of the respiratory network by comparing the resting respiratory activity and the respiratory response to hypoxia of control mice and mutant mice lacking the NK1 receptor (NK1−/−). In vitro and in vivo experiments were conducted on neonatal, young and adult mice from wild‐type and NK1−/− strains. In the wild strain, immunohistological, pharmacological and electrophysiological studies showed that NK1 receptors were expressed within medullary respiratory areas prior to birth and that their activation at birth modulated central respiratory activity and the membrane properties of phrenic motoneurons. Both the membrane properties of phrenic motoneurons and the respiratory activity generated in vitro by brainstem‐spinal cord preparation from NK1−/− neonate mice were similar to that from the wild strain. In addition, in vivo ventilation recordings by plethysmography did not reveal interstrain differences in resting breathing parameters. The facilitation of ventilation by short‐lasting hypoxia was similar in wild and NK1−/− neonates but was significantly weaker in adult NK1−/− mice. Results demonstrate that NK1 receptors do appear to be necessary for a normal respiratory response to short‐lasting hypoxia in the adult. However, NK1 receptors are not obligatory for the prenatal development of the respiratory network, for the production of the rhythm, or for the regulation of breathing by short‐lasting hypoxia in neonates.
Extracellular acid increases the open probability of transduction channels in spider mechanoreceptors
Authors: Höger Ulli , French Andrew S.
Published: March 2003

Ion channels of the epithelial sodium channel, degenerin and acid‐sensitive channel (ENaC/DEG/ASIC) family share a number of structural and functional homologies. Several members of this group have been linked to mechanoreception and nociception, but there is no direct evidence that these molecules cause the transduction of mechanical stimuli in any mechanoreceptor. The receptor channels of a spider mechanoreceptor, the VS‐3 slit‐sense organ of Cupiennius salei, show several similarities to ENaC/DEG/ASIC channels, including Na+ selectivity and amiloride blockade. We recorded the receptor current under voltage clamp in VS‐3 neurons at different extracellular pH values. Acid pH partially blocked the delayed rectifier K+ current and increased the receptor current in these cells. Noise analysis of the receptor current showed that low pH increased the open probability of the receptor channels. Therefore, acid sensitivity is a further similarity between these mechanoreceptor channels and the ENaC/DEG/ASIC family.
Saturation and self‐inhibition of rat hippocampal GABAA receptors at high GABA concentrations
Authors: Mercik Katarzyna , Zarnowska Ewa D. , Mandat Maria , Mozrzymas Jerzy W.
Published: March 2003

Current responses to ultrafast γ‐aminobutyric acid (GABA) applications were recorded from excised patches in rat hippocampal neurons to study the gating properties of GABAA receptors at GABA concentrations close to saturating ones and higher. The amplitude of currents saturated at approximately 1 mm, while the onset rate of responses reached saturation at 4–6 mm GABA. At high GABA concentrations (> 10 mm), the amplitude of current responses was reduced in a dose‐dependent manner with a half‐blocking GABA concentration of approximately 50 mm. The peak reduction at high GABA doses was accompanied by a tendency to increase the steady‐state to peak ratio. At concentrations higher than 30 mm, this effect took the form of a rebound current, i.e. during the prolonged GABA applications, the current firstly declined due to desensitization onset and then, instead of decreasing towards a steady‐state value, clearly increased. Both the self‐inhibition of GABAA receptors by high GABA doses and rebound were clearly voltage dependent, being larger at positive holding potentials. The fast desensitization component accelerated with depolarization at all saturating [GABA] tested. The rebound phenomenon indicates that the self‐block of GABAA receptors is state dependent, and suggests that the sojourn in the desensitized conformation provides a ‘rescue’ from the block. We propose that high GABA concentrations inhibit the receptors by direct occlusion of the channel pore having no effect on the receptor gating.
Compartment‐specific changes in striatal neuronal activity during expression of amphetamine sensitization are the result of drug hypersensitivity
Authors: Vanderschuren Louk J. M. J. , Schoffelmeer Anton N. M. , Van Leeuwen Sarah D. C. , Hof Liesbeth , Jonker Allert J. , Voorn Pieter
Published: March 2003

Repeated exposure to drugs of abuse induces behavioural sensitization, i.e. a persistent hypersensitivity to the psychomotor stimulant effects of these drugs. This may be the result of increased responsiveness, to drugs, of mesostriatal dopamine systems and their projections, but it has also been suggested that acute and sensitized behavioural responses to psychostimulant drugs involve activation of distinct neuronal circuits. In order to distinguish between these possibilities, we studied amphetamine‐induced c‐fos immunoreactivity in subregions of rat striatum (patch and matrix compartments of caudate–putamen and nucleus accumbens core and shell) in drug‐naive rats, as well as during long‐term expression of amphetamine sensitization. We found that, in sensitized animals, amphetamine (1.0 mg/kg) evoked an increase in the ratio of c‐fos‐immunopositive cells in striatal patch and matrix compartments, suggesting a preferential involvement of striatal patches in the sensitized response to amphetamine. In drug‐naive rats, amphetamine (0.5–5.0 mg/kg) dose‐dependently increased c‐fos expression in all striatal subregions. Remarkably, the highest dose of amphetamine also evoked an increase in patch : matrix ratio of c‐fos immunoreactivity. In nucleus accumbens core and shell of amphetamine‐ and saline‐pretreated animals, amphetamine (1.0 mg/kg) evoked comparable increases in c‐fos expression. These data indicate that distinct striatal compartments display a differential sensitivity to amphetamine in both drug‐naive and amphetamine‐sensitized animals. In addition, they suggest that the shift in amphetamine‐induced c‐fos expression from striatal matrix to patches in sensitized animals is the consequence of a change in the sensitivity to amphetamine, rather than a long‐term circuitry reorganization that is exclusive to the sensitized state.
Calbindin neurons in the hamster suprachiasmatic nucleus do not exhibit a circadian variation in spontaneous firing rate
Authors: Jobst Erin E. , Allen Charles N.
Published: March 2003

The role of the mammalian suprachiasmatic nuclei (SCN) in generating circadian rhythms in behaviours and other physiological processes is well established. A prominent feature of SCN neurons is the circadian oscillation in action potential firing frequency, with a peak near midday. A subset of calbindin‐immunoreactive (CB+) neurons form a compact subnucleus (CBsn) in the hamster SCN. Restoration of rhythmicity using fetal SCN grafts in SCN‐lesioned hamsters is critically dependent upon the presence of CB+ neurons within the transplanted grafts [LeSauter & Silver (1999) J. Neurosci., 5574–5585]. The aim of the current study was to determine whether CB+ neurons within the CBsn of the hamster SCN fire action potentials in a circadian pattern as part of their output signal. Using patch‐clamp recording, we demonstrated that CB+ neurons in the CBsn do not express a circadian rhythm in spontaneous firing frequency under diurnal conditions in vitro. Furthermore, the percentage of silent CB– cells varies with zeitgeber time, whereas the percentage of silent CB+ cells does not. Immunohistochemical analysis revealed that the CBsn is a nonhomogeneous nucleus, containing many more CB– than CB+ cells. Our results reveal that CB+ neurons within the CBsn represent a functionally distinct neuronal subpopulation in which rhythmic action potential output may not be necessary for the restoration of behavioural circadian rhythmicity.
Specific contributions of the basal forebrain corticopetal cholinergic system to electroencephalographic activity and sleep/waking behaviour
Authors: Berntson G. G. , Shafi R. , Sarter M.
Published: March 2003

The present study examined the role of the basal forebrain corticopetal cholinergic projection in the regulation of cortical electroencephalographic activity across sleep/wake states in rats. Selective lesions of this projection were effected by local intraparenchymal infusions of the immunotoxin 192 IgG‐saporin. Lesions spared the septo‐hippocampal cholinergic system, as well as p75‐receptor‐bearing noncholinergic neurons in the suprachiasmatic nucleus. Relative to sham‐lesioned control animals, rats with lesions of basal forebrain cholinergic neurons displayed a significant reduction in high frequency EEG activity, characterized especially by a reduction in gamma EEG power. Lesions did not significantly alter the overall proportion of sleeping and waking states as defined behaviourally, but the attenuation of high frequency EEG activity was apparent across all stages, including REM‐like periods. Results are consistent with the view that the basal forebrain corticopetal cholinergic system exerts a general activational effect on the cortical mantle. Although this system may not be essential for sleep/wake stage‐switching, it does impact on the cortical states associated with those stages.
Increased BDNF and trk‐B mRNA expression in cortical and limbic regions following formation of a social recognition memory
Authors: Broad Kevin D. , Mimmack Mike L. , Keverne E Barry , Kendrick Keith M.
Published: March 2003

Brain‐derived neurotrophic factor (BDNF) and its receptor, tyrosine receptor kinase (trk‐B), play important roles in neural plasticity, long‐term potentiation and memory formation. Sheep form a selective recognition memory for their lambs within 2 h of birth. Initially, this memory is exclusively based on olfactory cues; however, as it consolidates over a 12‐h recognition period it extends to incorporate visual cues. We investigated whether changes in BDNF and trk‐B mRNA expression occurred in both olfactory and visual processing systems at 4.5 h postpartum, 2–3 h after the behavioural manifestations of an olfactory recognition memory were found. Animals that formed a recognition memory showed increased BDNF mRNA expression in the inferior part of the temporal cortex, subfield CA1 of the hippocampus, the diagonal band, basolateral amygdala and the anterior cingulate, medial frontal, entorhinal and pyriform cortices. No increases were observed in either the olfactory bulbs or the dentate gyrus. Expression of trk‐B mRNA was significantly increased only in the medial temporal, entorhinal and pyriform cortices. These findings demonstrate that by 2–3 h following the initial formation of olfactory recognition memory there are BDNF/trk‐B‐mediated plasticity changes in brain areas involved in the consolidation of olfactory memory (the pyriform and entorhinal cortices). However, similar changes also occur in areas of the brain involved in visual memory, face and object recognition (the temporal cortex, entorhinal cortex, hippocampal subfield CA1 and basolateral amygdala), and in areas of the brain with integrative and attentional functions (the medial frontal and anterior cingulate cortices and diagonal band). This suggests that reorganization of neural circuits underlying the visual recognition of lambs or the integration of olfactory/visual information is occurring even at this time even though accurate behavioural recognition at this stage can only be made using olfactory cues.
Spatial reconfiguration of charge transfer effectiveness in active bistable dendritic arborizations
Authors: Korogod S. M. , Kulagina I. B. , Kukushka V. I. , Gogan P. , Tyc‐Dumont . S.
Published: March 2003

The aim of this work was to explore the electrical spatial profile of the dendritic arborization during membrane potential oscillations of a bistable motoneuron. Computational simulations provided the spatial counterparts of the temporal dynamics of bistability and allowed simultaneous depiction the electrical states of any sites in the arborization. We assumed that the dendritic membrane had homogeneously distributed specific electrical properties and was equipped with a cocktail of passive extrasynaptic and NMDA synaptic conductances. The electrical conditions for evoking bistability in a single isopotential compartment and in a whole dendritic arborization were computed and showed differences, revealing a crucial effect of dendritic geometry. Snapshots of the whole arborization during bistability revealed the spatial distribution of the density of the transmembrane current generated at the synapses and the effectiveness of the current transfer from any dendritic site to the soma. These functional maps changed dynamically according to the phase of the oscillatory cycle. In the low depolarization state, the current density was low in the proximal dendrites and higher in the distal parts of the arborization while the transfer effectiveness varied in a narrow range with small differences between proximal and distal dendritic segments. When the neuron switched to high depolarization state, the current density was high in the proximal dendrites and low in the distal branches while a large domain of the dendritic field became electrically disconnected beyond 200 µm from the soma with a null transfer efficiency. These spatial reconfigurations affected dynamically the size and shape of the functional dendritic field and were strongly geometry‐dependent.
BDNF up‐regulates evoked GABAergic transmission in developing hippocampus by potentiating presynaptic N‐ and P/Q‐type Ca2+ channels signalling
Authors: Baldelli P. , Novara M. , Carabelli V. , Hernández‐Guijo J. M. , Carbone E.
Published: March 2003

Chronic application of brain‐derived neurotrophic factor (BDNF) induces new selective synthesis of non‐L‐type Ca2+ channels (N, P/Q, R) at the soma of cultured hippocampal neurons. As N‐ and P/Q‐channels support neurotransmitter release in the hippocampus, this suggests that BDNF‐treatment may enhance synaptic transmission by increasing the expression of presynaptic Ca2+ channels as well. To address this issue we studied the long‐term effects of BDNF on miniature and stimulus‐evoked GABAergic transmission in rat embryo hippocampal neurons. We found that BDNF increased the frequency of miniature currents (mIPSCs) by ≈40%, with little effects on their amplitude. BDNF nearly doubled the size of evoked postsynaptic currents (eIPSCs) with a marked increase of paired‐pulse depression, which is indicative of a major increase in presynaptic activity. The potentiation of eIPSCs was more relevant during the first two weeks in culture, when GABAergic transmission is depolarizing. BDNF action was mediated by TrkB‐receptors and had no effects on: (i) the amplitude and dose–response of GABA‐evoked IPSCs and (ii) the number of GABAA receptor clusters and the total functioning synapses, suggesting that the neurotrophin unlikely acted postsynaptically. In line with this, BDNF affected the contribution of voltage‐gated Ca2+ channels mediating evoked GABAergic transmission. BDNF drastically increased the fraction of evoked IPSCs supported by N‐ and P/Q‐channels while it decreased the contribution associated with R‐ and L‐types. This selective action resembles the previously observed up‐regulatory effects of BDNF on somatic Ca2+ currents in developing hippocampus, suggesting that potentiation of presynaptic N‐ and P/Q‐channel signalling belongs to a manifold mechanism by which BDNF increases the efficiency of stimulus‐evoked GABAergic transmission.
Forced swim stress activates rat hippocampal serotonergic neurotransmission involving a corticotropin‐releasing hormone receptor‐dependent mechanism
Authors: Linthorst Astrid C. E. , Peñalva Rosana G. , Flachskamm Cornelia , Holsboer Florian , Reul Johannes M. H. M.
Published: March 2003

Serotonin is important for adequate coping with stress. Aberrant serotonin function is implicated in the aetiology of major depression and anxiety disorders. Dysregulation of the hypothalamic–pituitary–adrenocortical axis, involving elevated corticotropin‐releasing hormone (CRH) activity, also plays a role in these stress‐related illnesses. Here we studied the effects of stress on hippocampal serotonin and the role of the CRH system using in vivo microdialysis. First, rats were subjected to a forced swim stress, resulting in a dramatic increase in hippocampal serotonin (1500% of baseline), which was associated with the occurrence of diving behaviour. The diving‐associated increase in serotonin depended on activation of CRH receptors, as it was antagonized by intracerebroventricular pretreatment with D‐Phe‐CRH12−41. Secondly, the effects of intracerebroventricular administration of CRH and urocortin (0.03–1.0 µg) were studied. Both CRH and urocortin caused a dose‐dependent rise in hippocampal serotonin (maximally 350% of baseline) and 5‐hydroxyindoleacetic acid levels, suggesting the involvement of CRH receptor type 1. Because the effects of urocortin were prolonged, CRH receptor type 2 could play a role in a later phase of the neurotransmitter response. Experiments using adrenalectomized rats showed that CRH‐induced serotonin changes were adrenally independent. These data suggest that the raphe‐hippocampal serotonin system is able to mount, CRH receptor‐dependent, responses to specific stressful situations that surpass the usually observed maximal increases of about 300% of baseline during stress and enhanced vigilance.
Vasopressin and oxytocin reverse adenosine‐induced pituicyte stellation via calcium‐dependent activation of Cdc42
Authors: Rosso Lia , Peteri‐Brunbäck Brigitta , Vouret‐Craviari Valérie , Deroanne Christophe , Van Obberghen‐Schilling Ellen , Mienville Jean‐Marc
Published: March 2003

In view of the potential impact of pituicyte morphology on neurohypophysial hormone secretion, we have studied the mechanisms involved in the shape changes induced by vasopressin (AVP) and oxytocin (OXT) in cultured rat pituicytes. Pituicytes induced to become stellate in the presence of 10 µm adenosine revert to their nonstellate shape ∼20 min after application of AVP or OXT. The IC50 for this effect is 0.1 nm for AVP and 36 nm for OXT. Both agonists induce Ca2+ signals in pituicytes, comprised of a transient peak and a plateau phase that is dependent on the presence of extracellular Ca2+. The EC50 values of AVP for the transient and sustained responses are 4.5 and 0.1 nm, respectively; corresponding values for OXT are 180 and 107 nm. We determined pharmacologically that these hormone‐induced Ca2+ signals are mediated by the V1a subtype of vasopressin receptors, similar to what we previously observed for hormone‐induced reversal of stellation. Removal of extracellular Ca2+ or chelation of intracellular Ca2+ partially prevented AVP from reversing stellation, suggesting a role for Ca2+ in this event. We previously established that adenosine‐induced stellation of pituicytes occurs via RhoA inhibition. However, pharmacological experiments and pull‐down assays presented here show that AVP‐induced reversal of stellation does not involve RhoA activation. Rather, AVP was found to induce a time‐dependent activation of Cdc42, another small GTPase involved in cytoskeletal plasticity. Activation of Cdc42 by AVP is sensitive to intra‐ and extracellular Ca2+ depletion, similar to AVP‐induced reversal of stellation. Furthermore, AVP‐induced reversal of stellation is blocked by expression of an NWASP fragment known to inhibit endogenous Cdc42.
Characterization of the expression of PDZ‐RhoGEF, LARG and Gα12/Gα13 proteins in the murine nervous system
Authors: Kuner R. , Swiercz J. M. , Zywietz A. , Tappe A. , Offermanns S.
Published: March 2003

Small GTPases of the Rho‐family, like Rho, Rac and Cdc42, are involved in neuronal morphogenesis by regulating growth cone morphology or dendritic spine formation. G‐proteins of the G12‐family, G12 and G13, couple G‐protein‐coupled receptors (GPCRs) to the activation of RhoA. Recently, two novel Rho‐specific guanine nucleotide exchange factors (RhoGEFs), PDZ‐RhoGEF and LARG, have been identified to interact with the activated α‐subunits of G12/G13 and are thus believed to mediate GPCR‐induced Rho activation. Although studies in neuronal cell lines have shown that G12/G13 and PDZ‐RhoGEF mediate GPCR‐induced neurite retraction, the role, as well as the expression of this signalling pathway, in intact brain has not been adequately studied. In the present study, we have characterized systematically the expression of Gα12, Gα13, PDZ‐RhoGEF and LARG in various murine tissues as well as their subcellular localization in the central and peripheral nervous systems. By performing immunohistochemistry, using polyclonal antibodies raised against the above proteins, we observed that Gα12, Gα13 and their RhoGEF‐effectors are distributed widely in the mammalian nervous system. Moreover, these proteins localize to distinct morphological compartments within neurons. While LARG and Gα12 were mainly found in somata of the neurons, PDZ‐RhoGEF and Gα13 were predominantly localized in the neuropil of central neurons. Interestingly, PDZ‐RhoGEF is a neural‐specific protein, whereas LARG is nearly ubiqoutous. Our data provide evidence that the G12/13–RhoGEF‐mediated pathway is present throughout the adult brain and may be involved in regulation of neuronal morphogenesis and function via GPCRs.
Expression of functional NR1/NR2B‐type NMDA receptors in neuronally differentiated SK‐N‐SH human cell line
Authors: Pizzi Marina , Boroni Flora , Bianchetti Andrea , Moraitis Costantinos , Sarnico Ilenia , Benarese Marina , Goffi Francesca , Valerio Alessandra , Spano PierFranco
Published: March 2003

The present study demonstrates that human SK‐N‐SH neuroblastoma cells, differentiated by retinoic acid (RA), express functional NMDA receptors and become vulnerable to glutamate toxicity. During exposure to RA, SK‐N‐SH cells switched from non‐neuronal to neuronal phenotype by showing antigenic changes typical of postmitotic neurons together with markers specific for cholinergic cells. Neuronally differentiated cells displayed positive immunoreactivity to the vesicular acetylcholine transporter and active acetylcholine release in response to depolarizing stimuli. The differentiation correlated with the expression of NMDA receptors. RT‐PCR and immunoblotting analysis identified NMDA receptor subunits NR1 and NR2B, in RA‐differentiated cultures. The NR1 protein immunolocalized to the neuronal cell population and assembled with the NR2B subunit to form functional N‐methyl‐d‐aspartate (NMDA) receptors. Glutamate or NMDA application, concentration‐dependently increased the intracellular Ca2+ levels and acetylcholine release in differentiated cultures, but not in undifferentiated SK‐N‐SH cells. Moreover, differentiated cultures became vulnerable to NMDA receptor‐mediated excitotoxicity. The glutamate effects were enhanced by glycine application and were prevented by the NMDA receptor blocker MK 801, as well as by the NR2B selective antagonist ifenprodil. These data suggest that SK‐N‐SH cells differentiated by brief treatment with RA may represent an unlimited source of neuron‐like cells suitable for studying molecular events associated with activation of human NR1/NR2B receptors.
Lateral hypothalamic lesions impair flavour‐nutrient and flavour‐toxin trace learning in rats
Authors: Touzani Khalid , Sclafani Anthony
Published: March 2003

Food‐restricted rats with ibotenic acid lesions of the lateral hypothalamus (LHx) learned to prefer a flavour paired with concurrent intragastric (i.g.) infusions of maltodextrin, although their preference was weaker than that displayed by sham controls. Unlike controls, the LHx rats failed to acquire a flavour preference when the i.g. maltodextrin infusion was delayed by 15 min. The same rats learned to avoid flavours paired with i.g. lactose or lithium chloride over short delays (15–30 min), but were impaired, relative to controls, at a long conditioned–unconditioned stimuli delay (2 h). These data indicate that the LH is critical for the formation of flavour‐postingestive consequence learning over a delay, particularly with nutrient reinforcement. Lateral hypothalamus lesions might specifically impair the processing of nutrient‐generated unconditioned stimuli and, more generally, could interfere with the maintenance of flavour memories.
Morphological alterations in the amygdala and hippocampus of mice during ageing
Authors: Von Bohlen und Halbach Oliver , Unsicker Klaus
Published: March 2003

Declines in memory function and behavioural dysfunction accompany normal ageing in mammals. However, the cellular and morphological basis of this decline remains largely unknown. It was assumed for a long time that cell losses in the hippocampus accompany ageing. However, recent stereological studies have questioned this finding. In addition, the effect of ageing is largely unknown in another key structure of the memory system, the amygdala. In the present study, we have estimated neuronal density and total neuronal numbers as well as density of fragments of degenerated axons in different hippocampal subfields and amygdaloid nuclei. Comparisons were made among aged (21–26 months old) mice and normal adult littermates (8 months old). No significant volume loss occurs in the hippocampus of aged mice. Small but insignificant reductions in total neuronal numbers were found in the hippocampus and in the amygdaloid nuclei. In contrast to the mild effects of ageing upon neuronal numbers, fragments of degenerated axons were increased in both hippocampus and amygdala of aged mice. These data suggest that ageing does not induce prominent cell loss in the hippocampus or amygdala, but leads to degeneration of axons that innervate these forebrain structures. Thus, mechanisms underlying age‐related dysfunction depend on parameters other than neuronal numbers, at least in the hippocampal formation and the amygdala.
mGluR1 in cerebellar Purkinje cells is required for normal association of temporally contiguous stimuli in classical conditioning
Authors: Kishimoto Yasushi , Fujimichi Ryoko , Araishi Kenji , Kawahara Shigenori , Kano Masanobu , Aiba Atsu , Kirino Yutaka
Published: March 2003

In metabotropic glutamate receptor‐subtype 1 (mGluR1)‐null (mGluR1–/–) mice, cerebellar long‐term depression (LTD) and several forms of memory are impaired. However, because mGluR1 is expressed in various brain regions in wild‐type mice, it has been difficult to identify which type of memory depends on mGluR1 expressed in a given brain region. Furthermore, severe ataxia in mGluR1–/– mice complicated interpretation of the data from non‐cerebellum‐dependent tasks. We have generated mGluR1‐rescue mice, which express mGluR1 only in Purkinje cells (PCs) of their cerebellum, by introducing the mGluR1α transgene into mGluR1–/– mice under the control of a PC‐specific promoter. The mGluR1‐rescue mouse has normal LTD and displays no apparent ataxia. Therefore, this mouse is the first animal model in which effects of mGluR1 deficiency outside PCs can be studied without cerebellar dysfunction. We used three eyeblink conditioning paradigms with different temporal specificities between conditioned stimulus (CS) and unconditioned stimulus (US). Delay conditioning, in which CS and US coterminate, was impaired in mGluR1–/– mice but normal in mGluR1‐rescue mice. However, both strains of mice displayed severe impairment in trace conditionings, in which a stimulus‐free interval of 250 or 500 ms intervened between CS and US. We also examined social transmission of food‐preference and novel‐object‐recognition memory tests. In these tasks, mGluR1‐rescue mice showed normal short‐term but impaired long‐term memory. We conclude that mGluR1 in PCs is indispensable for normal learning of association of temporally contiguous stimuli in associative conditioning. In contrast, mGluR1 in other cell types is required for associating discontiguous stimuli and long‐term memory formation in nonspatial hippocampus‐dependent learning.
Thalamocortical oscillations in a genetic model of absence seizures
Authors: D'Arcangelo Giovanna , D'Antuono Margherita , Biagini Giuseppe , Warren Richard , Tancredi Virginia , Avoli Massimo
Published: March 2003

We used field potential recordings in an in vitro thalamocortical slice preparation to compare the rhythmic oscillations generated by reciprocally connected networks of the thalamus and cerebral cortex obtained from epileptic (> 160 days old) WAG/Rij and age‐matched, nonepileptic control (NEC) rats. To increase neuronal excitability, and thus to elicit spontaneous field potential activity in vitro, we applied medium containing: (i) zero [Mg2+]; (ii) high [K+] (8.25 mm); or (iii) low concentrations of the K+ channel blocker 4‐aminopyridine (4AP, 0.5–1 µm). Of these procedures, only the last was effective in triggering oscillatory activity that depended on the type of tissue. Thus, during 4AP application: (i) sequences of fast (intraburst frequency 9.5–16.1 Hz) and slower (5–8.9 Hz) field potential oscillations (FPOs) were recorded in WAG/Rij slices (n = 23), but (ii) only fast FPOs were seen in NEC slices (n = 7). Slower FPOs in WAG/Rij slices reflected a larger degree of thalamocortical synchronization than fast FPOs, and disappeared after surgical separation of cortex and thalamus (n = 5); under these conditions fast FPOs continued to occur in thalamus only. In addition, fast and slower FPOs disappeared in all areas of the WAG/Rij slice during thalamic application of the excitatory amino acid receptor antagonist kynurenic acid (n = 3), while fast FPOs continued to occur in thalamus when kynurenic acid was applied to the cortex (n = 4). Bath application of the N‐methyl‐d‐aspartic acid (NMDA) receptor antagonist 3,3‐(2‐carboxypiperazine‐4‐yl)‐propyl‐1‐phosphonate (CPP) abolished slower FPOs in WAG/Rij cortex and thalamus (n = 6) without infuencing fast FPOs recorded in WAG/Rij (n = 6) or NEC slices (n = 4). Moreover, cortical application of CPP (n = 6) abated slower FPOs although they persisted following CPP application to the thalamus (n = 7). Our data demonstrate that highly synchronized, slower FPOs can occur during 4AP application in WAG/Rij but not in NEC slices. This activity, which may represent an in vitro hallmark of thalamocortical epileptogenicity, requires the function of reciprocally connected thalamic and cortical networks and depends on cortical NMDA receptor‐mediated mechanisms.
Intrastriatal sonic hedgehog injection increases Patched transcript levels in the adult rat subventricular zone
Authors: Charytoniuk D. , Traiffort E. , Hantraye P. , Hermel J. M. , Galdes A. , Ruat M.
Published: March 2003

The morphogen sonic hedgehog (Shh) is implicated in neural tissue patterning and the growth of brain structures during embryogenesis and postnatal development and is also present in the adult brain. Shh signals through interaction with the tumour suppressor Patched (Ptc). This receptor for Shh is associated with Smoothened (Smo), a protein with high homology to the G‐protein coupled receptors. However, little is known about the transduction mechanisms implicated in Shh signalling in the adult brain. The study described here shows that injection of aminoterminal myristoylated Shh (myrShhN) into the adult rat striatum robustly increases the levels of Ptc transcripts in selective brain areas including the subventricular zone (SVZ). The adult SVZ contains cell progenitors, which can proliferate and differentiate into new neurons and glia. In the myrShhN injected animals, proliferation and differentiation of these SVZ precursor cells were not affected as demonstrated by BrdU incorporation and immunohistochemistry performed with specific antibodies for nestin (uncommitted neural progenitors), PSA‐NCAM (migrating neuroblasts) or GFAP (astrocytes). Together with the presence of Smo expressing cells and amino‐terminal Shh (ShhN) protein in SVZ area of untreated animals, the data presented here supports the hypothesis that the Shh pathway may be activated in the adult brain, and that a niche for Shh signalling exists within the adult SVZ.
Locus coeruleus activation modulates firing rate and temporal organization of odour‐induced single‐cell responses in rat piriform cortex
Authors: Bouret Sebastien , Sara Susan J.
Published: March 2003

Piriform cortex (PCx) is the primary cortical projection region for olfactory information and has bidirectional monosynaptic connections with olfactory bulb and association cortices. PCx neurons display a complex receptive field, responding to odours rather than their molecular components, suggesting that these neurons are involved in higher order olfactory processing. Neuromodulators, especially noradrenaline (NA), have important influences on sensory processing in other cortical regions and might be responsible for the plasticity observed in PCx during learning. The present study is the first attempt to examine in vivo the actions of NA on sensory responses in the PCx. Stimulation of the noradrenergic nucleus locus coeruleus (LC) was used to induce release of NA in the forebrain in urethane‐anaesthetized rats. Extracellular recording of single units was made simultaneously in anterior and posterior PCx. The responses to an odour stimulus were measured over 25 trials. Twenty‐five subsequent odour presentations were preceded by stimulation of the ipsilateral LC through a bipolar electrode, previously placed in the LC under electrophysiological control. This priming stimulation modified the activity of 77 of the 135 recorded neurons. For most cells, LC stimulation enhanced cortical responses to odour in terms of both spike count and temporal organization, with some differential effects in anterior and posterior regions. These results are the first to show enhancement of sensory responses in the olfactory cortex by LC activation. Spontaneous activation of LC neurons such as occurs during learning could serve to enhance olfactory perception and promote learning.
Environmental conditions influence hippocampus‐dependent behaviours and brain levels of amyloid precursor protein in rats
Authors: Teather Lisa A. , Magnusson Jane E. , Chow Christina M. , Wurtman Richard J.
Published: March 2003

Sprague‐Dawley rats were reared in enriched (EC; group housing, exposure to stimulating objects, frequent handling) or restricted (RC; individual housing, no exposure to stimulating objects, minimal handling) environments starting on day 23 of life. At six months of age, they underwent behavioural tests to assess ‘cognitive’ and ‘stimulus‐response’ memory, selective attention, and inflammatory pain processing. Alterations in synapses and cell survival may occur as a result of environment differences; therefore we assessed the brain levels of several proteins implicated in neurite outgrowth, synaptogenesis, and cell survival. Brains were dissected and analysed for amyloid precursor protein (APP) and other synaptic and cytoskeletal proteins using Western blotting. The performance of EC animals in a hidden platform water maze task, and in a test of selective attention (both of which are thought to involve the hippocampus) was superior to that of RC animals. In contrast, performance of RC animals on two stimulus‐response tasks, the visible platform water maze test and simple visual discrimination (both of which are thought to be hippocampal independent) was indistinguishable from that of EC animals. Male EC rats displayed a different behavioural response to formalin during the inflammatory phase of nociception – the phase affected by hippocampal processing; a similar trend was observed in females. Female but not male RC rats exhibited elevated plasma corticosterone levels; adrenal weights were unaffected by environmental conditions. Region‐specific increases in brain levels of APP, neurofilament‐70 (NF‐70), and platelet‐activating factor receptor (PAF‐R) were found in EC rats. These data suggest that enriched animals manifest enhanced functioning of certain hippocampus‐mediated behaviours when compared with that of their restricted counterparts; and that brain levels of various synaptic and structural proteins involved in neurite outgrowth, cell survival, and synaptogenesis, are affected by environmental factors.
Patterns of cation‐chloride cotransporter expression during embryonic rodent CNS development
Authors: Li Hong , Tornberg Janne , Kaila Kai , Airaksinen Matti S. , Rivera Claudio
Published: March 2003

Intracellular Cl– plays a key role in cellular volume regulation, cell cycle control and shaping the polarity of inhibitory postsynaptic responses mediated by anion‐permeable GABA and glycine receptors. In this study, we have investigated the expression patterns of members of the cation‐chloride cotransporters (CCCs), including the K‐Cl cotransporters KCC1‐4 and the Na‐K‐2 Cl cotranporter NKCC1 during rodent embryonic brain development. At the time of neurogenesis (embryonic days; E12.5–14.5), KCC1 was only detectable in the developing choroid plexus. KCC2 mRNA was detectable as early as E12.5 in the ventral part of the (cervical) spinal cord, and by E14.5, the expression had spread to TUJ1‐positive differentiating regions of the rhombencephalon, diencephalon and olfactory bulb, in parallel with neuronal maturation. KCC3 mRNA was scarce in the cortical plate at E14.5, and slightly up‐regulated at birth. In contrast, KCC4 mRNA was abundantly expressed in the ventricular zone and was down‐regulated perinatally. At E14.5, NKCC1 was highly expressed in the vimentin‐positive radial glia of the proliferative zone of the subcortical region. At later embryonic stages, during gliogenesis (E17‐P0), there was a shift in NKCC1 expression to the neuron specific Class III β‐tubulin (βIII) positive region of the cortical plate. These unique spatiotemporal expression patterns of distinct CCCs during embryonic development suggests that Cl– regulatory mechanisms are critically involved in the control of neuronal development.
Altering electrical connections in the nervous system of the pteropod mollusc Clione limacina by neuronal injections of gap junction mRNA
Authors: Kelmanson Ilya V. , Shagin Dmitry A. , Usman Natalia , Matz Mikhail V. , Lukyanov Sergey A. , Panchin Yury V.
Published: March 2003

Neurons can communicate with each other either via exchange of specific molecules at synapses or by direct electrical connections between the cytoplasm of either cell [for review see Bruzzone et al. (1996) Eur. J. Biochem., 238, 1–27]. Although electrical connections are abundant in many nervous systems, little is known about the mechanisms which govern the specificity of their formation. Recent cloning of the innexins – gap junction proteins responsible for electrical coupling in invertebrates (Phelan et al. (1998) Trends Genet., 14, 348–349], has made it possible to study the molecular mechanisms of patterning of the electrical connections between individual neurons in model systems. Here we demonstrate that intracellular injection of mRNA encoding the molluscan innexin Panx1 (Panchin et al. 2000 Curr. Biol., 10, R473‐R474) drastically alters the specificity of electrical coupling between identified neurons of the pteropod mollusc Clione limacina.