bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential increases in chromogranins, but not synapsin I, in cortical neurons following spreading depression: implications for functional roles and transmitter peptide release
Authors: Shen Pei‐Juan , Gundlach Andrew L.
Published: March 2003

Experimental damage of cerebral cortex induces a slow‐moving depolarization and subsequent depression of activity called cortical spreading depression (CSD) which is associated with various ionic, metabolic and genomic changes. Chromogranins are a family of water‐soluble acidic proteins with a widespread distribution in secretory, large dense‐core vesicles of neurons. We have earlier reported that secretogranin II (SgII) mRNA is increased in cerebral cortex hours after a unilateral craniotomy which would have induced CSD. To investigate further the regulation of chromogranin systems and the nature of genomic and biochemical changes produced by CSD, this study examined the temporal changes in chromogranin A (CgA), chromogranin B (CgB) and SgII mRNAs and CgB and SgII immunoreactivity (IR) in cerebral cortex and hippocampus following unilateral

KCl‐induced CSD. For comparison, the levels of mRNA for synapsin I, a protein present in small synaptic vesicles was also examined. Rats were killed at various times after 10 min or 2 h of CSD and levels of chromogranins mRNAs were determined by semiquantitative in situ hybridization histochemistry, while changes in corresponding peptide products were detected by immunohistochemistry. CSD increased both SgII and CgB mRNA levels in ipsilateral cortex – levels of SgII mRNA were significantly (P < 0.01) increased at 1–6 h after CSD (165–225% of levels in contralateral cortex), but were not significantly above control values at later time points. Increased expression of CgB mRNA was delayed and prolonged compared with SgII and was significantly (P < 0.05) increased between 3 and 24 h (120–145%) after CSD, peaked at 2 days (180%), and was still elevated at 1 week (130%) compared with contralateral cortex. No alteration in CgA mRNA was observed in the ipsilateral cortex of the same animals across the entire time‐course except for an increase in piriform cortex at 1–2 days. In contrast, levels of synapsin I mRNA in affected cortex were identical to those in contralateral cortex and cortex in sham‐operated rats, at all times after CSD. Levels of chromogranin (SN‐IR and PE‐11‐IR) were also increased in ipsilateral cortex following CSD. A strong increase in SN‐IR in neuronal cell bodies and fibres was observed at 12 h and a moderate increase in PE‐11‐IR was observed 24–72 h after CSD. These results demonstrate that chromogranin transcripts and gene products are differentially regulated by neuronal depolarization/depression occurring during CSD and suggest that these chromogranin proteins may have differing functional roles in peptide transmitter release and distinct effects on neuronal function in rat brain.
Increase in syntaxin 1B and glutamate release in mossy fibre terminals following induction of LTP in the dentate gyrus: a candidate molecular mechanism underlying transsynaptic plasticity
Authors: Helme‐Guizon Alix , Davis Sabrina , Israel Maurice , Lesbats Bernard , Mallet Jacques , Laroche Serge , Hicks Andrew
Published: March 2003

A growing body of evidence suggests that modulation of certain proteins of the exocytotic machinery is, in part, involved in the biochemical changes that underlie long‐term synaptic plasticity. We have previously shown that the induction of long‐term potentiation (LTP) at perforant path to dentate granule cell synapses in the rat hippocampus induces changes in the mRNA levels of syntaxin 1B and synapsin I, known to be involved in neurotransmitter release. Immunohistochemical staining suggested that concomitant changes in these proteins occurred at mossy fibre synapses, downstream of those synapses at which LTP was induced, leading us to postulate that such a mechanism might underlie a form of transsynaptic plasticity. Here we have used a specific mossy‐fibre synaptosome preparation to quantify levels of proteins and measure, using a chemiluminescent glutamate assay, depolarization‐induced glutamate release from these synaptosomes after induction of LTP in the dentate gyrus in vivo. We show that 5 h after the induction of LTP, there is an increase in the protein levels of syntaxin 1B and, although to a lesser extent, the synapsins I and II, associated with an increase in depolarization‐induced release of glutamate within these terminals. Increases in both the protein levels and glutamate release were not observed when dentate gyrus LTP was blocked by an NMDA receptor antagonist. From these results we propose a molecular mechanism for the propagation of synaptic plasticity through hippocampal circuits.
Piracetam facilitates long‐term memory for a passive avoidance task in chicks through a mechanism that requires a brain corticosteroid action
Authors: Loscertales María , Rose Steven P. R. , Daisley Jonathan N. , Sandi Carmen
Published: March 2003

We investigated the effects of piracetam, a nootropic, on learning and memory formation for a passive avoidance task in day‐old chicks. To test for the possible cognitive‐enhancing properties of piracetam, a weak learning version of this task – whereby chicks maintain a memory to avoid pecking at a bead coated in a diluted aversant for up to 10 h – was used. Post‐training (5, 30 or 60 min), but not pretraining, injections of piracetam (10 or 50 mg/kg, i.p.) increased recall for the task when the chicks were tested 24 h later. Because previous studies showed that long‐term memory for the passive avoidance task is dependent upon a brain corticosteroid action, and because the efficacy of piracetam‐like compounds is also modulated by corticosteroids, we tested whether the facilitating effect of piracetam was dependent upon a corticosteroid action through specific brain receptors (mineralocorticoid receptor and glucocorticoid receptor). First, increased plasma levels of corticosterone were found 5 min after piracetam injection. In addition, intracerebral administration of antagonists for each receptor type (RU28318, for mineralocorticoid receptors, and RU38486 for glucocorticoid receptors; i.c.) given before the nootropic inhibited the facilitative effect of piracetam on memory consolidation. These results give further support to a modulatory action of piracetam on the mechanisms involved in long‐term memory formation through a neural action that, in this learning model, requires the activation of the two types of intracellular corticosteroid receptors.
Promoter analysis of the neuronal nicotinic acetylcholine receptor α4 gene: methylation and expression of the transgene
Authors: Watanabe Hajime , Zoli Michele , Changeux Jean‐Pierre
Published: March 2003

Neuronal nicotinic acetylcholine receptor (nAChR) subunit genes compose a family of genes. The major isoform of nAChR in the brain is made up of the α4 and β2 subunits and possesses a high affinity for nicotine. To investigate the mechanisms of the regulation of the nAChR α4 gene expression in mouse, its genomic DNA was cloned and characterized. The transcription initiation site was mapped by primer extension and RNase protection experiments and localized at about 254 bp upstream of the translation initiation site. The 5′ flanking region of this gene did not have typical TATA box but GC‐rich sequences were found around the initiation site. Methylation analysis of this region revealed that genomic DNAs from liver and muscle are partially methylated, whereas little methylation was observed in genomic DNA from brain. To characterize the cis‐acting elements driving cell‐specific expression of the α4 subunit gene, we produced lines of transgenic mice which carry a series of fragments of the α4 gene fused with bacterial lacZ as a reporter gene. An 11.5‐kb DNA fragment containing 9 kb of the region upstream of the transcription initiation site and the first intron was found to confer an expression pattern which coincides rather well with the endogenous gene expression pattern at early embryonic stages, suggesting that the elements necessary for the onset of α4 gene expression are located in this region. A DNA fragment containing the 1.8‐kb upstream sequence and the first intron drove expression of lacZ in a limited subset of α4 expressing cells, whereas the 1.8‐kb upstream sequence alone did not elicit any significant expression. These results show that both upstream and intronic sequences are important for cell‐specific expression of the nAChR α4 gene.
Regional cerebral blood flow changes in human brain related to ipsilateral and contralateral complex hand movements – a PET study
Authors: Kawashima Ryuta , Matsumura Michikazu , Sadato Norihiro , Naito Eichi , Waki Atsuo , Nakamura Satoshi , Matsunami Kenichi , Fukuda Hiroshi , Yonekura Yoshiharu
Published: March 2003

The purpose of this study was to investigate the cortical motor areas activated in relation to unilateral complex hand movements of either hand, and the motor area related to motor skill learning. Regional cerebral blood flow (rCBF) was measured in eight right‐handed healthy male volunteers using positron emission tomography during a two‐ball‐rotation task using the right hand, the same task using the left hand and two control tasks. In the two‐ball‐rotation tasks, subjects were required to rotate the same two iron balls either with the right or left hand. In the control task, they were required to hold two balls in each hand without movement. The primary motor area, premotor area and cerebellum were activated bilaterally with each unilateral hand movement. In contrast, the supplementary motor area proper was activated only by contralateral hand movements. In addition, we found a positive correlation between the rCBF to the premotor area and the degree of improvement in skill during motor task training. The results indicate that complex hand movements are organized bilaterally in the primary motor areas, premotor areas and cerebellum, that functional asymmetry in the motor cortices is not evident during complex finger movements, and that the premotor area may play an important role in motor skill learning.
Importance of neurokinin‐1 receptors in the nucleus tractus solitarii of mice for the integration of cardiac vagal inputs
Authors: Paton Julian F. R.
Published: March 2003

Unmyelinated vagal afferents from the heart terminate within the nucleus tractus solitarii (NTS) located in the dorsomedial medulla. The neurotransmitter and postsynaptic receptors mediating information from cardiac vagal receptors to the NTS are unknown. This study determined the effects of neurokinin‐1 (NK1) receptor blockade on: (i) the reflex response evoked following aortic root injection of either veratridine (1–3 μg/kg) or bradykinin (80–300 ng/kg) to stimulate cardiac receptors in in vivo anaesthetized mice; and (ii) the evoked synaptic response of cardioreceptive NTS neurons following both intraleft‐ventricular injection of veratridine or bradykinin, and electrical stimulation of the ipsilateral vagus nerve in an arterially perfused working heart‐brainstem preparation of mouse. Administration of CP‐99,994 (0.75–1.5 mg/kg i.v.), a specific NK1 antagonist, attenuated significantly the evoked reflex bradycardia and depressor response following cardiac receptor (n = 6), but not pulmonary chemoreflex stimulation in vivo. From extracellular recordings of cardioreceptive NTS neurons, CP‐99,994 reduced reversibly the total number of evoked spikes, peak firing frequency and response duration evoked by intraventricular injections of veratridine (n = 5) or bradykinin (n = 5). The number of evoked action potentials following electrical stimulation of the vagus nerve was also reduced. In five whole cell recordings of NTS neurons, both the evoked depolarization following cardiac receptor stimulation, and the peak amplitude and duration of vagus nerve‐evoked EPSPs were reduced by CP‐99 994; synaptic inputs from both peripheral chemoreceptors or pulmonary C‐fibres were unaffected. These data support a selective involvement of NK1 receptors in the transmission of cardiac vagal afferent inputs to NTS neurons integrating cardiorespiratory information.
Neuronal survival and calcium influx induced by basic fibroblast growth factor in chick ciliary ganglion neurons
Authors: Distasi Carla , Torre Marina , Antoniotti Susanna , Munaron Luca , Lovisolo Davide
Published: March 2003

Basic fibroblast growth factor (bFGF/FGF2) exhibits widespread biological activities in the nervous system. However, little is known about the cascade of intracellular events that links the activation of its tyrosine kinase receptors to these effects. Here we report that, in ciliary ganglion neurons from chick embryo, this trophic factor significantly enhanced neuronal survival. The percentage of surviving neurons was reduced when intracellular calcium was chelated by adding a membrane‐permeable BAPTA ester to the culture medium, while antagonists of L‐ and N‐type voltage‐dependent calcium channels were ineffective. The ionic signals in response to bFGF stimulation have been studied using cytofluorimetric and patch‐clamp techniques. In single‐cell Fura‐2 measurements, bFGF elicited a long lasting rise of the cytosolic calcium concentration that was dependent on [Ca2+]o. In whole‐cell experiments, we observed a reversible depolarization of the membrane resting potential and an inward cationic current. Single channel experiments, performed in the cell‐attached configuration, provide evidence for the activation of two families of Ca2+‐permeable cationic channels. Moreover, inositol 1,4,5‐trisphosphate opens channels with similar properties, suggesting that this cytosolic messenger can be responsible for the calcium influx induced by bFGF.
Postnatal expression of H1‐receptor mRNA in the rat brain: correlation to l‐histidine decarboxylase expression and local upregulation in limbic seizures
Authors: Lintunen Minnamaija , Sallmen Tina , Karlstedt Kaj , Fukui Hiroyuki , Eriksson Krister S. , Panula Pertti
Published: March 2003

Histamine is implicated in the regulation of brain functions through three distinct receptors. Endogenous histamine in the brain is derived from mast cells and neurons, but the importance of these two pools during early postnatal development is still unknown. The expression of histamine H1‐receptor in the rat brain was examined using in situ hybridization during postnatal development and in adults. For comparison, the expression of l‐histidine decarboxylase (HDC) in the two pools was revealed. H1‐receptor was evenly expressed throughout the brain on the first postnatal days, but resembled the adult, uneven pattern already on postnatal day 5 (P5). HDC was expressed in both mast cells and tuberomammillary neurons from birth until P5, after which the mast cell expression was no more detectable. In adult rat brain, high or moderate levels of H1‐receptor expression were found in the hippocampus, zona incerta, medial amygdaloid nucleus and reticular thalamic nucleus. In most areas of the adult brain the expression of H1‐receptor mRNA correlates well with binding data and histaminergic innervation. A notable exception is the hypothalamus, with high fibre density but moderate or low H1‐receptor expression. Systemic kainic acid administration induced increased expression of H1‐receptor mRNA in the caudate‐putamen and dentate gyrus, whereas no change was seen in the hippocampal subfields CA1–CA3 or in the entorhinal cortex 6 h after kainic acid injections. This significant increase supports the concept that histaminergic transmission, through H1‐receptor, is involved in the regulation of seizure activity in the brain.
A peptide activator of endogenous tyrosine kinase enhances synaptic currents mediated by NMDA receptors
Authors: Lancaster B. , Rogers M. V.
Published: March 2003

N‐methyl‐d‐aspartic acid (NMDA) receptor currents in cultured cells or expression systems are increased by the addition of purified tyrosine kinases. However, there is no direct demonstration of this effect at NMDA receptors in intact synapses of rat brain slices. Transmitters which might be used to activate tyrosine kinases in situ are unlikely to have a sufficiently selective action to allow a clear interpretation of their effects. Therefore, we used a phosphotyrosine‐containing decapeptide which can be included in recording electrodes to activate postsynaptic src‐family tyrosine kinases. This peptide enhanced NMDA responses in dissociated hippocampal CA1 neurons. These effects were not reproduced by a non‐phosphorylated peptide or a scrambled‐sequence phosphopeptide. The enhancement of NMDA responses was blocked by a tyrosine kinase inhibitor. In brain slices the phosphopeptide, but not control peptide, increased NMDA receptor‐mediated synaptic current indicating that endogenous tyrosine kinase can upregulate the response of NMDA receptors at glutamatergic synapses in the hippocampus.
The voltage‐dependent conductances of rat neocortical layer I neurons
Authors: Budde Thomas , White John A.
Published: March 2003

Whole cell patch‐clamp techniques were used to study voltage‐dependent sodium (Na+), calcium (Ca2+), and potassium (K+) conductances in acutely isolated neurons from cortical layer I of adult rats. Layer I cells were identified by means of γ‐aminobutyric acid (GABA) immunocytochemistry. Positive stainings for the Ca2+‐binding protein calretinin in a subset of cells, indicated the presence of Cajal–Retzius (C‐R) cells. All investigated cells displayed a rather homogeneous profile of voltage‐dependent membrane currents. A fast Na+ current activated at about −45 mV, was half‐maximal steady‐state inactivated at −66.6 mV, and recovery from inactivation followed a two‐exponential process (τ1 = 8.4 ms and τ2 = 858.8 ms). Na+ currents declined rapidly with two voltage‐dependent time constants, reaching baseline current after some tens of milliseconds. In a subset of cells (< 50%) a constant current level of < 65 pA remained at the end of a 90 ms step. A transient outward current (Ifast) activated ≈–40 mV, declined rapidly with a voltage‐insensitive time constant (τ≈ 350 ms) and was relatively insensitive to tetraethylammonium (TEA, 20 mm). Ifast was separated into two components based on their sensitivity to 4‐aminopyridine (4‐AP): one was blocked by low concentrations (40 μm) and a second by high concentrations (6 mm). After elimination of Ifast by a conditioning prepulse (50 ms to −50 mV), a slow K+ current (IKV) could be studied in isolation. IKV was only moderately affected by 4‐AP (6 mm), while TEA (20 mm) blocked most (> 80%) of the current. IKV activated at about −40 mV, declined monoexponentially in a voltage‐dependent manner (τ≈ 850 ms at −30 mV), and revealed an incomplete steady‐state inactivation. In addition to Ifast and IKV, indications of a Ca2+‐dependent outward current component were found. When Na+ currents, Ifast, and IKV were blocked by tetrodotoxin (TTX, 1 μm), 4‐AP (6 mm) and TEA (20 mm) an inward current carried by Ca2+ was found. Ca2+ currents activated at depolarized potentials at about −30 mV, were completely blocked by 50 μm cadmium (Cd2+), were sensitive to verapamil (≈ 40% block by 10 μm), and were not affected by nickel (50 μm). During current clamp recordings, isolated layer I neurons displayed fast spiking behaviour with short action potentials (≈ 2 ms, measured at half maximal amplitude) of relative small amplitude (≈ 83 mV, measured from the action potential threshold).
Modulation of big K+ channel activity by ryanodine receptors and L‐type Ca2+ channels in neurons
Authors: Chavis Pascale , Ango Fabrice , Michel Jean‐Marie , Bockaert Joel , Fagni Laurent
Published: March 2003

As metabotropic glutamate receptor type 1 (mGluR1) is known to couple L‐type Ca2+ channels and ryanodine receptors (RyR, Chavis et al. 1996) in cerebellar granule cells, we examined if such a coupling could activate a Ca2+‐sensitive K+ channel, the big K+ (BK) channel, in cultured cerebellar granule cells. We observed that (±)‐1‐amino‐cyclopentane‐trans‐1,3‐dicarboxylic acid (t‐ACPD) and quisqualate (QA) stimulated the activity of BK channels. On the other hand, (2S, 3S, 4S)‐α‐carboxycyclopropyl‐glycine (L‐CCG‐I) and l‐(+)‐2‐amino‐4‐phosphonobutyrate (L‐AP4) had no effect on BK channels, indicating a specific activation by group I mGluRs. Group I mGluRs stimulation of the basal BK channel activity was mimicked by caffeine and both effects were blocked by ryanodine and nifedipine. Interestingly, carbachol stimulated BK channel activity but through a pertussis toxin (PTX)‐sensitive pathway that was independent of L‐type Ca2+ channel activity. Our report indicates that unlike the muscarinic receptors, group I mGluRs activate BK channels by mobilizing an additional pathway involving RyR and L‐type Ca2+ channels.
Expression and presence of septal neurokinin‐2 receptors controlling hippocampal acetylcholine release during sensory stimulation in rat
Authors: Steinberg R. , Marco N. , Voutsinos B. , Bensaid M. , Rodier D. , Souilhac J. , Alonso R. , Oury‐Donat F. , Fur G. Le , Soubrie P.
Published: March 2003

We examined the expression and presence of NK2 receptors in the septal area of rat brain, and investigated their functional role in the regulation of the septohippocampal cholinergic system. Using reverse transcription‐polymerase chain reaction (RT‐PCR) analysis, we showed the presence of NK2 receptor mRNA expression in the septal area, and detected septal NK2 binding sites by using a fluorescent‐tagged neurokinin A (NKA) derivative. In vivo microdialysis was employed to explore the functional role of NK2 receptors in the release of hippocampal acetylcholine evoked by tactile stimulation in freely moving rats. Two sessions of stroking of the neck and back of the rat for 30 min, at 90 min intervals, produced a marked and reproducible increase in hippocampal acetylcholine release. This effect was dose‐dependently prevented by intraperitoneal administration of the two selective non‐peptide tachykinin NK2 receptor antagonists SR144190 (0.03–0.3 mg/kg, i.p.) and SR48968 (0.3 and 1 mg/kg, i.p.), but not by the inactive enantiomer of SR48968 (SR48965, 1 mg/kg) nor by the two non‐peptide NK1 receptor antagonists SR140333 (3 mg/kg, i.p.) and GR205171 (1 mg/kg, i.p.). Furthermore, the intraseptal application of SR144190 (10–8m) reduced the sensory response. Finally, intraseptal perfusion of neurokinin A (0.01–10 μm) in anaesthetized rats produced a concentration‐dependent increase in hippocampal acetylcholine release. The response to neurokinin A (0.1 μm) was prevented by SR144190 (0.03–0.3 mg/kg, i.p.) and SR48968 (0.3–1 mg/kg, i.p.). In conclusion, this study provides direct evidence for the role of endogenous NKA/substance P, through the activation of NK2 receptors, in regulating the septohippocampal cholinergic function.
PET study of human voluntary saccadic eye movements in darkness: effect of task repetition on the activation pattern
Authors: Dejardin S. , Dubois S. , Bodart J. M. , Schiltz C. , Delinte A. , Michel C. , Roucoux A. , Crommelinck M.
Published: March 2003

Using H215O 3D Positron Emission Tomography (PET), regional cerebral blood flow (rCBF) was measured in six human subjects under two different conditions: at rest and while performing self‐paced horizontal saccadic eye movements in darkness. These two conditions were repeated four times each.

 First, the comparison between the four saccadic and four resting conditions was investigated in a group and a single subject analysis. Saccades elicited bilateral rCBF increases in the medial part of the superior frontal gyrus (supplementary eye field), precentral gyrus (frontal eye field), superior parietal lobule, anterior medial part of the occipital lobe involving striate and extrastriate cortex (lingual gyrus and cuneus), and in the right inferior parietal lobule. At the subcortical level, activations were found in the left putamen. These results mainly replicate previous PET findings on saccadic control.

 Second, the interaction between the experimental conditions and their repetition was examined. When activations throughout repetition of the same saccadic task are compared, the supplementary eye fields show a progressive increase of activation. On the contrary, the activation in the cerebellum, left superior parietal lobule and left occipital cortex progressively decreases during the scanning session. Given the existence of such an interaction, the pattern of activations must be interpreted as a function of task repetition. This may be a factor explaining some apparent mismatch between different studies.
A population of cholinergic neurons is present in the macaque monkey thalamus
Authors: Rico Beatriz , Cavada Carmen
Published: March 2003

Three new cholinergic markers were employed to study the cholinergic innervation in the thalamus of adult macaque monkeys. They were: two antibodies against choline acetyltransferase (ChAT), one polyclonal and one monoclonal; and a polyclonal antibody against the vesicular transporter of acetylcholine (VAChT), a powerful new marker that colocalizes with ChAT. This approach led to an unexpected finding: the three antibodies positively immunostained a population of neurons in the paracentral nucleus. The immunostained cells are confined to the dorsal region of this nucleus along its rostrocaudal extent. Measurement of the somatic areas of the immunostained neurons indicated that they correspond to a population of large neurons thought to be projection neurons. Because dorsal paracentral neurons are known to project to the dorsal striatum and specific cortical areas involved in visual and visuomotor mechanisms, these structures might be modulated by cholinergic thalamic neurons.
Amelioration of spatial navigation and short‐term memory deficits by grafts of foetal basal forebrain tissue placed into the hippocampus and cortex of rats with selective cholinergic lesions
Authors: Leanza G. , Martìnez‐Serrano A. , Björklund A.
Published: March 2003

Impairments in learning and memory, induced by surgical or excitotoxic lesions of the septo‐hippocampal or basalo‐cortical pathways, can be ameliorated by grafts of cholinergic‐rich foetal basal forebrain tissue into the hippocampus and/or neocortex. However, the effects of such grafts have been only partial, which may be due to the non‐specific nature of the lesioning procedures used in these studies, known to destroy both cholinergic and non‐cholinergic neuronal projections. In the present study, we have explored the effects of cholinergic‐rich grafts in rats subjected to selective cholinergic lesions, induced by intraventricular injections of the immunotoxin 192 IgG‐saporin. This lesion, which selectively destroyed 85–95% of the cholinergic neurons in both the septal‐diagonal band and nucleus basalis, produced a long‐lasting, substantial impairment in both the acquisition of spatial reference memory in the Morris water maze task and delay‐dependent short‐term memory performance, as seen in a delayed matching‐to‐position test. Foetal cholinergic grafts (but not control grafts of cerebellar tissue) implanted at multiple sites into both the hippocampus and fronto‐parietal neocortex, bilaterally, completely reversed the acquisition deficit in place navigation in the water maze, to an extent that greatly exceeded that previously seen in animals with non‐selective lesions. Most notably, however, the impairment in short‐term memory was only partially and inconsistently affected, and only at the longest delay times. The morphological analysis, performed at about 7 months after transplantation, showed that the grafts had re‐established a close to normal cholinergic innervation in the initially denervated cortical and hippocampal territories. It is proposed that the differential effects of cholinergic‐rich transplants on different aspects of cognitive performance may define intrinsic limitations to the functional capacity of the ectopically placed grafts, which may be due to incomplete integration of the grafted cholinergic neurons into functional regulatory circuitries normally available to the basal forebrain cholinergic system.
Serotonin inhibits synaptic glutamate currents in rat nucleus accumbens neurons via presynaptic 5‐HT1B receptors
Authors: Muramatsu Masatoshi , Lapiz Maria Danet S. , Tanaka Eiichiro , Grenhoff Johan
Published: March 2003

Neurons in the nucleus accumbens septi in brain slices from adult male rats were studied with patch clamp recording in the whole‐cell conformation. Cells filled with Lucifer Yellow were identified as medium spiny neurons. Electrical stimulation close to the recorded cell evoked excitatory and inhibitory synaptic currents. In the presence of picrotoxin or bicuculline, stimulation at a holding potential of −90 mV evoked an inward excitatory current that was blocked by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX, 10 μm), identifying it as an excitatory postsynaptic current (EPSC) mediated by glutamate acting at AMPA/kainate receptors. Serotonin (5‐hydroxytryptamine, 5‐HT; 3–100 μm in the bath) decreased the EPSC in about 90% of the cells. The action of 5‐HT was mimicked by N‐(3‐trifluoromethylphenyl)‐piperazine HCl (TFMPP), but not by (±)‐8‐hydroxy‐dipropylaminotetralin (8‐OH‐DPAT) or (±)‐2,5‐dimethoxy‐4‐iodoamphetamine HCl (DOI). The 5‐HT effect was antagonized by pindolol or cyanopindolol, but not by spiperone, ketanserin or tropisetron. Taken together, these results indicate that 5‐HT acts at 5‐HT1B receptors. The effect of 5‐HT was potentiated by cocaine (0.3–3 μm) or the selective serotonin reuptake inhibitor citalopram. Miniature synaptic currents recorded in the presence of tetrodotoxin were inhibited by CNQX, identifying them as spontaneous miniature EPSCs. 5‐HT reduced the frequency of these miniature EPSCs without affecting their amplitude, which indicates a presynaptic site of action. This presynaptic inhibition by 5‐HT might be involved in the behavioural effects of cocaine.
Increase of preprotachykinin mRNA and substance P immunoreactivity in spared dorsal root ganglion neurons following partial sciatic nerve injury
Authors: Ma Weiya , Bisby Mark A.
Published: March 2003

Complete sciatic nerve injury reduces substance P (SP) expression in primary sensory neurons of the L4 and L5 dorsal root ganglia (DRG), due to loss of target‐derived nerve growth factor (NGF). Partial nerve injury spares a proportion of DRG neurons, whose axons lie in the partially degenerating nerve, and are exposed to elevated NGF levels from Schwann and other endoneurial cells involved in Wallerian degeneration. To test the hypothesis that SP is elevated in spared DRG neurons following partial nerve injury, we compared the effects of complete sciatic nerve transection (CSNT) with those of two types of partial injury, partial sciatic nerve transection (PSNT) and chronic constriction injury (CCI). As expected, a CSNT profoundly decreased SP expression at 4 and 14 days postinjury, but after PSNT and CCI the levels of preprotachykinin (PPT) mRNA, assessed by in situ hybridization, and the SP immunoreactivity (SP‐IR) of the L4 and L5 DRGs did not decrease, nor did dorsal horn SP‐IR decrease. Using retrograde labelling with fluorogold to identify spared DRG neurons, we found that the proportion of these neurons expressing SP‐IR 14 days after injury was much higher than in neurons of normal DRGs. Further, the highest levels of SP‐IR in individual neurons were detected in ipsilateral L4 and L5 DRG neurons after PSNT and CCI. We conclude that partial sciatic nerve injury elevates SP levels in spared DRG neurons. This phenomenon might be involved in the development of neuropathic pain, which commonly follows partial nerve injury.
Action sequencing is impaired in D1A‐deficient mutant mice
Authors: Cromwell Howard C. , Berridge Kent C. , Drago John , Levine Michael S.
Published: March 2003

The role of dopamine in the production of behaviour is multifarious in that it can influence different aspects of movement (e.g. movement initiation, sensorimotor integration, and movement sequencing). A characteristic of the dopamine system which seems to be critical for the expression of this diverse influence is its varied receptor population. Previous studies have shown that specific receptor subtype activation leads to specific behavioural responses or alterations of selective aspects of movement. It is known that one of the important influences of dopamine includes sequential co‐ordination of ‘syntactic’ patterns of grooming movements because moderate loss of the dopaminergic nigrostriatal projections specifically disrupts these patterns without affecting grooming actions in a general fashion (Berridge, K.C. Psychobiology, 15, 336 1989). The specific receptors of the dopamine family which play a key part in this co‐ordination of movement sequences is not known. In the present study, we examined the serial order of particular syntactic sequences or chains of grooming actions in mice lacking D1A receptors to explore the relationship between this receptor subtype and movement sequencing. Mutant mice had shorter grooming bouts and a disruption of the organization of sequential patterns compared with wild‐type littermate controls. Sequential disruption was reflected in the failure of D1A mutants to follow the syntactic pattern of grooming to completion. This sequential disruption deficit appeared to be specific, as mutant mice initiated more syntactic chains than wild‐type controls even though they were less likely to complete them. These results support the hypothesis that D1A receptor activation plays a part in the sequencing of natural action. This conclusion has important implications for the understanding of the functional heterogeneity of dopamine receptor subtypes and of the aetiology of symptoms observed in patients with basal ganglia disease.
Cytokine‐induced changes in the ability of astrocytes to support migration of oligodendrocyte precursors and axon growth
Authors: Fok‐Seang Juin , DiProspero Nicholas A. , Meiners Sally , Muir Elizabeth , Fawcett James W.
Published: March 2003

Repair of demyelination in the CNS requires that oligodendrocyte precursors (OPs) migrate, divide and then myelinate. Repair of axon damage requires axonal regeneration. Limited remyelination and axon regeneration occurs soon after injury, but usually ceases in a few days. In vivo and in vitro experiments have shown that astrocytic environments are not very permissive for migration of OPs or for axonal re‐growth. Yet remyelination and axon sprouting early after injury occurs in association with astrocytes, while later astrocytes can exclude remyelination and prevent axon regeneration. A large and changing cast of cytokines are released following CNS injury, so we investigated whether some of these alone or in combination can affect the ability of astrocytes to support migration of OPs and neuritic outgrowth. Interleukin (IL) 1α, tumour necrosis factor α, transforming growth factor (TGF) β, basic fibroblast growth factor (bFGF), platelet‐derived growth factor and epidermal growth factor alone exerted little or no effect on migration of OPs on astrocytes, whereas interferon (IFN) γ was inhibitory. The combination of IL‐1α + bFGF was found to be pro‐migratory, and this effect could be neutralized by TGFβ. We also examined neuritic outgrowth from dorsal root ganglion explants in three‐dimensional astrocyte cultures treated with cytokines and found that IL‐1α + bFGF greatly increased axon outgrowth and that this effect could be blocked by TGFβ and IFNγ. All these effects were absent or much smaller when OP migration or axon growth was tested on laminin, so the main effect of the cytokines was via astrocytes. The cytokine effects did not correlate with expression on astrocytes of laminin, fibronectin, tenascin, chondroitin sulphate proteoglycan, N‐cadherin, polysialyated NCAM (PSA‐NCAM), tissue plasminogen activator (tPA) or urokinase (uPA).
Mechanisms of spontaneous cytosolic Ca2+ transients in differentiated human neuronal cells
Authors: Gao Zhi‐yong , Chen Meng , Collins Heather W. , Matschinsky Franz M. , Lee Virginia M. ‐Y. , Wolf Bryan A.
Published: March 2003

We have studied Ca2+ homeostasis in a unique model of human neurons, the NT2N cell, which differentiates from a human teratocarcinoma cell line, NTera2/C1.D1 by retinoic acid treatment. When perifused with Krebs–HEPES buffer containing 2.5 mm CaCl2, fura‐2 loaded NT2N cells produced spontaneous cytosolic Ca2+ oscillations, or Ca2+ transients. These cytosolic Ca2+ transients were not blocked by antagonists of glutamate (6‐cyano‐7‐nitroquinoxaline‐2,3‐dione and d(–)‐2‐amino‐5‐phosphonopentanoic acid) or muscarinic (atropine) receptors. Omission of extracellular Ca2+ completely abolished Ca2+ oscillations and decreased the average Ca2+ level from 106 ± 14 nm to 59 ± 8 nm. Addition of the L‐type Ca2+ channel blocker nifedipine (1 or 10 μm) or of the N‐type inhibitor ω‐conotoxin GVIA (5 μm) significantly, although incompletely, suppressed Ca2+ oscillations, while ω‐conotoxin MVIIC (5 μm), a selective antagonist of P‐ and Q‐channels, had no effect. Ni2+, at 100 μm, a concentration selective for T‐type channels, did not inhibit Ca2+ transients. Non‐specific blockage of Ca2+ channels by higher concentrations of Ni2+ (2–5 mm) or Co2+ (1 mm) abolished Ca2+ oscillations completely. The endoplasmic reticulum Ca2+‐ATPase inhibitor, thapsigargin (1 μm), slightly decreased Ca2+ oscillation frequency, and induced a small transitory increase in the average cytosolic Ca2+ concentration. The mRNAs of L‐ (α1D subunit) and N‐type (α1B subunit) Ca2+ channel were present in NT2N cells, while that of a T‐type Ca2+ channel (α1‐subunit) was not present in the NT2N cells as shown by reverse transcription–polymerase chain reaction. In conclusion, NT2N neuronal cells generate cytosolic Ca2+ oscillations mainly by influx of extracellular Ca2+ through multiple channels, which include L‐ and N‐type channels, and do not require activation of glutamate or muscarinic receptors.
Immunorecognition, ultrastructure and phosphorylation status of astrocytic gap junctions and connexin43 in rat brain after cerebral focal ischaemia
Authors: Li W. E. I. , Ochalski P. A. Y. , Hertzberg E. L. , Nagy J. I.
Published: March 2003

Gap junctions between astrocytes support a functional syncytium that is thought to play an important role in neural homeostasis. In order to investigate regulation of this syncytium and of connexin43 (Cx43), a principal astrocytic gap junction protein, we determined the sequelae of gap junction and Cx43 disposition in a rat cerebral focal ischaemia model with various ischaemia/reperfusion times using sequence‐specific anti‐Cx43 antibodies (designated 13‐8300, 18A, 16A and 71‐0700) that exhibit differential recognition of Cx43, perhaps reflecting functional aspects of gap junctions. Antibody 13‐8300 specifically detects only an unphosphorylated form of Cx43 in both Western blots and tissue sections. In hypothalamus after brief (15 min) ischaemic injury, Cx43 at intact gap junctions undergoes dephosphorylation, accompanied by reduced epitope recognition by antibodies 16A and 71‐0700. Tissue examined 24 h after reperfusion showed that these effects were reversible. Astrocytic gap junction internalization occurring 1 h after ischaemia was accompanied by decreased immunodetection with 13‐8300. At this time, gap junctions were absent in the ischaemic core, coinciding with a loss of Cx43 recognition with 18A and 13‐8300, but elevated labelling of internalized Cx43 with 16A and 71‐0700. Unphosphorylated Cx43 persisted at intact gap junctions confined to a thin corridor at the ischaemic penumbra which contained presumptive apoptotic cell profiles. Similar results were obtained in ischaemic striatum and cerebral cortex, though with a delayed time course that depended on the severity of the ischaemic insult. These results demonstrate that astrocytic Cx43 epitope masking, dephosphorylation and cellular redistribution occur after ischaemic brain injury, proceed as a temporally and spatially ordered sequence of events and culminate in differential patterns of Cx43 modification and sequestration at the lesion centre and periphery. These observations suggest an attempt by astrocytes in the vicinity of injury to remodel the junctional syncytium according to altered tissue homeostatic requirements.
Learning‐specific, time‐dependent increases in hippocampal Ca2+/calmodulin‐dependent protein kinase II activity and AMPA GluR1 subunit immunoreactivity
Authors: Cammarota Martín , Bernabeu Ramón , Levi de Stein Miguelina , Izquierdo Iván , Medina Jorge H.
Published: March 2003

Ca2+/calmodulin‐dependent protein kinase II (CAMK II) and one of its target, α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA), glutamate receptors have been shown to participate in both long‐term potentiation (LTP) in the hippocampus, and in spatial, as well as in a variety, of learning paradigms. Recently, we were able to demonstrate that the intrahippocampal infusion of a specific inhibitor of CAMK II (KN62) provoked full retrograde amnesia of an inhibitory avoidance learning in rats when given immediately, but not 120 or 240 min, after training. Furthermore, this task is accompanied by a rapid, selective and reversible increase in hippocampal [3H] AMPA receptor binding. Here we report the effect of this aversively motivated learning task on CAMK II activity, and AMPA GluR1 subunit phosphorylation and immunoreactivity in the hippocampus. One trial inhibitory avoidance training is associated with a learning‐specific, time‐dependent increase (25–78%) in both total and Ca2+‐independent activities of CAMK II in the hippocampus of rats killed immediately (0 min), but not 120 min, after training. In addition, immunoblotting experiments showed an increment in the amount of the α‐subunit of CAMK II at 0, 30 and 120 min after training. An increase in the in vitro phosphorylation of α‐ and β‐subunits of CAMK II was also observed in hippocampal synaptosomal membranes (SPM) of trained rats killed immediately and 30 min post‐training.

 In addition, inhibitory avoidance is accompanied by a 20% increase in GluR1 phosphorylation and a 33% increase in GluR1 immunoreactivity 120 min after training. No significant changes were observed in shocked animals. Phosphorylation of hippocampal SPM from naive control animals in conditions suitable for CAMK II activation resulted in a large increase in the density of [3H] AMPA binding (+ 100%). Taken together, these findings confirm and extend previous data suggesting that CAMK II and AMPA glutamate receptors in the hippocampus participate in the early phase of memory formation of an inhibitory avoidance learning.
Action potential broadening induced by lithium may cause a presynaptic enhancement of excitatory synaptic transmission in neonatal rat hippocampus
Authors: Colino Asunción , García‐Seoane Jorge J. , Valentín Antonio
Published: March 2003

Lithium enhances excitatory synaptic transmission in CA1 pyramidal cells, but the mechanisms remain unclear. The present study demonstrates that lithium enhances the N‐methyl‐d‐aspartate (NMDA) and α‐amino‐3‐hydroxy‐5‐methyl‐isoxazole propionic acid (AMPA) receptor‐mediated components of the excitatory postsynaptic current (EPSC). Lithium decreased the magnitude of paired‐pulse facilitation and presented an inverse correlation between the lithium‐induced enhancement of synaptic transmission and initial paired‐pulse facilitation, which is consistent with a presynaptic mode of action. The enhancement of synaptic strength is likely to act, at least in part, by increasing the amplitude of the presynaptic Ca2+ transient. One mechanism which could account for this change of the presynaptic Ca2+ transient is an increase in the duration of the action potential. We investigated action potential in hippocampal pyramidal neurons and found that lithium (0.5–6 mm) increased the half‐amplitude duration and reduced the rate of repolarization, whereas the rate of depolarization remained similar. To find out whether the lithium synaptic effects might be explained by spike broadening, we investigated the field recording of the excitatory postsynaptic potential (EPSP) in hippocampal slices and found three lines of evidence. First, the prolongation of the presynaptic action potential with 4‐aminopyridine and tetraethylammonium blocked or reduced the synaptic effects of lithium. Second, the lithium‐induced synaptic enhancement was modulated when presynaptic Ca2+ influx was varied by changing the external Ca2+ concentration. Finally, both effects, the synaptic transmission increment and the action potential broadening, were independent of inositol depletion. These results suggest that lithium enhances synaptic transmission in the hippocampus via a presynaptic site of action: the mechanism underlying the potentiating effect may be attributable to an increased Ca2+ influx consequent to the broadening effect of lithium on the action potential.
Dihydrokainate‐sensitive neuronal glutamate transport is required for protection of rat cortical neurons in culture against synaptically released glutamate
Authors: Wang Guang Jian , Chung Hye Joo , Schnuer Jamie , Lea Elizabeth , Robinson Michael B. , Potthoff William K. , Aizenman Elias , Rosenberg Paul A.
Published: March 2003

Glutamate transport in nearly pure rat cortical neurons in culture (less than 0.2% astrocytes) is potently inhibited by dihydrokainate, l‐serine‐O‐sulphate, but not by l‐α‐amino‐adipate. This system allows for a test of the hypothesis that glutamate transport is important for protecting neurons against the toxicity of endogenous synaptically released glutamate. In support of this hypothesis, a 20–24 h exposure to 1 mm dihydrokainate reduced cell survival to only 14.8 ± 9.8% in neuronal cultures (P < 0.001;n = 3), although it had no effect on neuronal survival in astrocyte‐rich cultures (P > 0.05;n = 3). Dihydrokainate also significantly caused accumulation of glutamate in the extracellular medium of cortical neuronal cultures (6.6 ± 4.9 μm, compared to 1.2 ± 0.3 μm in control, n = 14, P < 0.01). The neurotoxicity of dihydrokainate was blocked by 10 μm MK‐801, 10 μm tetrodotoxin, and an enzyme system that degrades extracellular glutamate. The latter two also abolished the accumulation of glutamate in the extracellular medium. Dihydrokainate (1 mm) inhibited the 45calcium uptake stimulated by 30 μmN‐methyl‐d‐aspartate (NMDA), but not by higher concentrations consistent with a weak antagonist action of dihydrokainate at the NMDA receptor. Whole cell recordings showed that 1 mm dihydrokainate produced ≈ 25% inhibition of 30 μm NMDA‐induced current in cortical neurons. Dihydrokainate (1 mm) alone generated a small current (17% of the current produced by 30 μm NMDA) that was blocked by 30 μm 5,7‐dichlorokynurenate and only weakly by 10 μm 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX). These results suggest that the toxicity of dihydrokainate in neuronal cultures is due to its ability to block glutamate transport in these cultures, and that dihydrokainate‐sensitive neuronal glutamate transport may be important in protecting neurons against the toxicity of synaptically released glutamate.