bluuebunny/crossref_metadata_2025 huggingface dataset:
CaMKII‐dependent phosphorylation of NR2A and NR2B is decreased in animals characterized by hippocampal damage and impaired LTP
Authors: Caputi A. , Gardoni F. , Cimino M. , Pastorino L. , Cattabeni F. , Di Luca M.
Published: March 2003

The calcium‐calmodulin‐dependent protein kinase II (CaMKII) subserves activity‐dependent plasticity in central neurons. To examine in vivo the implication of CaMKII activity in synaptic plasticity, we used an animal model characterized by developmentally induced targeted neuronal ablation within the cortex and the hippocampus, and showing, at presynaptic level, molecular alterations leading to facilitation of glutamate release in hippocampal synapses (methylazoxymethanol‐treated rats, MAM‐rats). We report here that at the postsynaptic side, the activity of CaMKII is markedly decreased in MAM‐rats when compared to controls, although the concentration of the enzyme in Post Synaptic Density (PSD) is not altered. This effect is confined to PSD‐associated CaMKII, as enzyme activity tested in the soluble fraction is unchanged in MAM‐rats. In addition, the decreased activity is not due to inhibition by autophosphorylation in specific sites within the calmodulin‐binding domain, as preincubation with purified phosphatases 1 and 2A failed to restore CaMKII activity in PSD of MAM‐rats. The CaMKII‐dependent phosphorylation of NR2A/B subunits of NMDA receptor is lower in MAM‐rats when compared to controls (51.77 ± 7.39% of controls level), as revealed in back‐phosphorylation experiments. In addition, a treatment able to restore long‐term potentiation (LTP) in hippocampal slices from MAM‐rats, e.g. exposure to d‐serine, is able to restore CaMKII activity to the control value.
Differential effects of adenosine receptor subtypes on release and reuptake of hippocampal serotonin
Authors: Okada Motohiro , Kawata Yuko , Murakami Takuya , Wada Kazumaru , Mizuno Kazuhisa , Kondo Tuyoshi , Kaneko Sunao
Published: March 2003

To clarify the effects of adenosine receptor subtypes (A1, A2 and A3) on hippocampal serotonin (5‐HT) release and 5‐HT reuptake activity, hippocampal extracellular 5‐HT levels were determined in vivo by microdialysis in freely moving rats. Selective 5‐HT reuptake inhibitor (SSRI) fluoxetine and DU24565 increased extracellular 5‐HT levels. Adenosine and A1 receptor agonist, 2‐chloro‐N6‐cyclopentyl‐adenosine (CCPA), decreased extracellular 5‐HT levels, whereas non‐selective antagonist, caffeine, and A1 antagonist, 8‐cyclopentyl‐1,3‐dimethylxanthine (CPT) increased them. When 5‐HT reuptake activity was inhibited by DU24565 and fluoxetine, the effects of CPT and CCPA on 5‐HT level were enhanced. A2A receptor agonist, CGS21680, A2 receptor agonist, PD125944, A2 receptor antagonist, 3,7‐dimethyl‐1‐propargylxanthine (DMPX), and A3 receptor agonist, N6–2‐(4‐aminophenyl)ethyladenosine (APNEA) did not affect 5‐HT levels; however, when A1 receptor was blocked by CPT, 5‐HT levels were increased by adenosine, CGS21680 and PD125944, and decreased by DMPX and APNEA. Under conditions of A1 receptor blockade, pretreatment with DU24565 or fluoxetine, enhanced the stimulatory effects of CGS21680 and PD125944 as well as inhibitory effects of DMPX on 5‐HT level, whereas the inhibitory effect of APNEA was abolished. These results indicate that the stimulatory effects of A2 receptor and inhibitory effects of A3 receptor on hippocampal extracellular 5‐HT levels are masked or abolished by the inhibitory effects of A1 receptor. In addition, hippocampal serotonergic transmission might be modulated by hippocampal presynaptic adenosine receptor subtypes, and hippocampal 5‐HT reuptake activity might be modulated by hippocampal A3 receptor.
TrkA activation in the rat visual cortex by antirat trkA IgG prevents the effect of monocular deprivation
Authors: Pizzorusso Tommaso , Berardi Nicoletta , Rossi Francesco M. , Viegi Alessandro , Venstrom Kristine , Reichardt Louis F. , Maffei Lamberto
Published: March 2003

It has been recently shown that intraventricular injections of nerve growth factor (NGF) prevent the effects of monocular deprivation in the rat. We have tested the localization and the molecular nature of the NGF receptor(s) responsible for this effect by activating cortical trkA receptors in monocularly deprived rats by cortical infusion of a specific agonist of NGF on trkA, the bivalent antirat trkA IgG (RTA‐IgG). TrkA protein was detected by immunoblot in the rat visual cortex during the critical period. Rats were monocularly deprived for 1 week (P21–28) and RTA‐IgG or control rabbit IgG were delivered by osmotic minipumps. The effects of monocular deprivation on the ocular dominance of visual cortical neurons were assessed by extracellular single cell recordings. We found that the shift towards the ipsilateral, non‐deprived eye was largely prevented by RTA‐IgG. Infusion of RTA‐IgG combined with antibody that blocks p75NTR (REX), slightly reduced RTA‐IgG effectiveness in preventing monocular deprivation effects. These results suggest that NGF action in visual cortical plasticity is mediated by cortical TrkA receptors with p75NTR exerting a facilitatory role.
Chloride‐dependent transport of NH4+ into bee retinal glial cells
Authors: Marcaggi Païkan , Thwaites David T. , Deitmer Joachim W. , Coles Jonathan A.
Published: March 2003

Mammalian astrocytes convert glutamate to glutamine and bee retinal glial cells convert pyruvate to alanine. To maintain such amination reactions these glial cells may take up NH4+/NH3. We have studied the entry of NH4+/NH3 into bundles of glial cells isolated from bee retina by using the fluorescent dye BCECF to measure pH. Ammonium caused intracellular pH to decrease by a saturable process: the rate of change of pH was maximal for an ammonium concentration of about 5 mm. This acidifying response to ammonium was abolished by the loop diuretic bumetanide (100 μm) and by removal of extracellular Cl–. These results strongly suggest that ammonium enters the cell by cotransport of NH4+ with Cl–. Removal of extracellular Na+ did not abolish the NH4+‐induced acidification. The NH4+‐induced pH change was unaffected when nearly all K+ conductance was blocked with 5 mm Ba2+ showing that NH4+ did not enter through Ba2+‐sensitive ion channels. Application of 2 mm NH4+ led to a large increase in total intracellular proton concentration estimated to exceed 13.5 mEq/L. As the cell membrane appeared to be permeable to NH3, we suggest that when NH4+ entered the cells, NH3 left, so that protons were shuttled into the cell. This shuttle, which was strongly dependent on internal and external pH, was quantitatively modelled. In retinal slices, 2 mm NH4+ alkalinized the extracellular space: this alkalinization was reduced in the absence of bath Cl–. We conclude that NH4+ enters the glial cells in bee retina on a cotransporter with functional similarities to the NH4+(K+)‐Cl– cotransporter described in kidney cells.
Pontine and medullary control of the respiratory activity in the trigeminal and facial nerves of the newborn mouse: an in vitro study
Authors: Jacquin Thierry Didier , Sadoc Gérard , Borday Véronique , Champagnat Jean
Published: March 2003

In vitro, the respiratory activity in rodents is characterized by: (i) the rapidly peaking, slowly decrementing pattern of spontaneous and rhythmic active phases recorded from the motor rootlets, and (ii) the specific location of their rhythmic generator in the ventrolateral medulla. The aim of the present study was to assess whether the trigeminal and facial motor rootlets still exhibit respiratory activity in the absence of peripheral and higher cerebral structures, and to compare the onset of their active phases with that of other respiratory rootlets, using the in vitro isolated brainstem–spinal cord preparation of the newborn mouse and rat. Spontaneous rhythmic activity was recorded from the trigeminal and facial rootlets. It was regular and synchronized bilaterally and ipsilaterally with the hypoglossal or cervical C1–C6 rootlets. Brainstem transection experiments demonstrated that for both the trigeminal and facial rootlets, the spontaneous rhythmic activity originates from the medulla, in a region consistent with the pre‐Bötzinger complex and the rostral ventrolateral medulla. The pattern of the respiratory motor activity recorded from the trigeminal and facial rootlets was identical to the pattern recorded from the hypoglossal and cervical C1–C6 rootlets with rapidly peaking, slowly decrementing characteristics. The duration of the ascending part and the total duration of their active phases were similar. The onset of the active phases of the phrenic rootlets was delayed compared with that of the trigeminal, facial and hypoglossal rootlets. However, no difference in the onsets of the active phases of the cranial rootlets could be observed. Removal of the rostral pons suppressed the delay in onset of the active phases of the phrenic rootlets. Our findings show that: (i) rhythmic activities of the trigeminal and facial rootlets are preserved in absence of control by peripheral or high cerebral structures; (ii) the pattern and the location of the rhythmic generator for these activities are of the respiratory type; and (iii) the rostral pons is responsible for a delay in the onset of the active phases of the phrenic rootlets compared with that of the trigeminal, facial and hypoglossal rootlets.
Cerebellar granule‐cell‐specific GABAA receptors attenuate benzodiazepine‐induced ataxia: evidence from α6‐subunit‐deficient mice
Authors: Korpi Esa R. , Koikkalainen Paula , Vekovischeva Olga Y. , Mäkelä Riikka , Kleinz Raymonde , Uusi‐Oukari Mikko , Wisden William
Published: March 2003

Benzodiazepine‐ and alcohol‐induced ataxias in rodents have been proposed to be affected by the γ‐aminobutyric acid type A (GABAA) receptor α6 subunit, which contributes to receptors specifically expressed in cerebellar granule cells. We have studied an α6 –/– mouse line for motor performance and drug sensitivity. These mice, as a result of a specific genetic lesion, carry a precise impairment at their Golgi‐granule cell synapses. On motor performance tests (rotarod, horizontal wire, pole descending, staircase and swimming tests) there were no robust baseline differences in motor function or motor learning between α6 –/– and α6 +/+ mice. On the rotarod test, however, the mutant mice were significantly more impaired by diazepam (5–20 mg/kg, i.p.), when compared with α6 +/+ control and background C57BL/6J and 129/SvJ mouse lines. Ethanol (2.0–2.5 g/kg, i.p.) produced similar impairment in the α6 –/– and α6 +/+ mice. Diazepam‐induced ataxia in α6 –/– mice could be reversed by the benzodiazepine site antagonist flumazenil, indicating the involvement of the remaining α1β2/3γ2 GABAA receptors of the granule cells. The level of activity in this synapse is crucial in regulating the execution of motor tasks. We conclude that GABAA receptor α6 subunit‐dependent actions in the cerebellar cortex can be compensated by other receptor subtypes; but if not for the α6 subunit, patients on benzodiazepine medication would suffer considerably from ataxic side‐effects.
Differential projection patterns of superior and inferior collicular neurons onto posterior paralaminar nuclei of the thalamus surrounding the medial geniculate body in the rat
Authors: Linke Rüdiger
Published: March 2003

The thalamic nuclei at the medial border of the medial geniculate body (i.e. the suprageniculate nucleus, the medial division of the medial geniculate nucleus, the posterior intralaminar nucleus and the peripeduncular nucleus) which relay sensory information to the amygdala are thought to receive convergent input from multiple sites. In order to delineate the organization of these multimodal thalamic nuclei, the locations of superior and inferior collicular neurons projecting to these nuclei were studied by means of retrograde transport methods.

 Small injections of the tracer Miniruby were made into single paralaminar thalamic nuclei. Injections of Miniruby into the suprageniculate nucleus labelled predominantly neurons in the stratum opticum of the superior colliculus, whereas injections into the medial division of the medial geniculate body, the posterior intralaminar nucleus and the peripeduncular nucleus labelled predominantly neurons in the deep layers of the superior colliculus. These injections also labelled neurons in the inferior colliculus. The majority of retrogradely labelled neurons were found in the external nucleus of the inferior colliculus and here predominantly in layer 2. Injections focused onto the medial division of the medial geniculate body additionally labelled magnocellular neurons in layer 3 of the external nucleus and a few neurons in the central nucleus. More ventrally located injections, focused onto the posterior intralaminar and peripeduncular nucleus, almost exclusively labelled neurons in layer 1 of the external nucleus and the dorsal part of the dorsal nucleus. After injections into the suprageniculate nucleus, only neurons in layer 2 were found. Neurons in the central nucleus of the inferior colliculus were only found after injections that involved the medial division of the medial geniculate body.

 The present results suggest that, despite a considerable degree of convergence in this thalamic region, each of these thalamic nuclei receives a unique pattern of projections from the superior and inferior colliculi. It appears that the thalamic nuclei may be concerned mainly, but not exclusively, with a single sensory modality, and give rise to parallel multimodal and unimodal pathways to the amygdala.
GABAC receptor ρ subunits are heterogeneously expressed in the human CNS and form homo‐ and heterooligomers with distinct physical properties
Authors: Enz Ralf , Cutting Garry R.
Published: March 2003

In the central nervous system, receptors for γ‐aminobutyric acid (GABA) are responsible for inhibitory neurotransmission. Anatomical and electrophysiological studies indicate that GABAC receptors are composed of ρ subunits. While the ρ1 subunit of various species forms homooligomeric receptors with GABAC‐like properties, molecular cloning has identified additional ρ subunits whose functional role is unclear. By RT‐PCR, we demonstrated that ρ1 expression is primarily restricted to the retina, whereas the ρ2 subunit was present in all brain regions tested. Transfection of HEK‐293 cells with ρ2 cDNA resulted in GABA‐gated whole‐cell currents that differed from those mediated by the ρ1 subunit in two respects: maximal amplitude (ρ1:ρ2 ≈ 4:1) and inactivation time course (ρ1:ρ2 ≈ 2:1). Cotransfection of ρ1 and ρ2 cDNA in a 1:1 ratio generated whole‐cell currents with large amplitudes characteristic of ρ1 but more rapid inactivation typical for ρ2. This observation suggested formation of heterooligomeric GABAC receptors with distinct features. Therefore, we tested the assembly of ρ1 and ρ2 subunits by cotransfecting ρ2 cDNA together with a chimeric ρ1β1 subunit, known to interfere with ρ1 assembly in a dominant‐negative fashion. Reduction of ρ2 generated currents correlated with the ratio of chimeric to ρ2 cDNA. Secondly, we determined that the picrotoxinin sensitivity of cells transfected with various ratios of ρ1 and ρ2 cDNA differed from that expected of a pure mixture of homooligomeric receptors. The latter two observations support the idea that ρ1 and ρ2 subunits form heterooligomeric GABAC receptors in mammalian cells. Together, our results indicate that the presence of both ρ subunits enables the formation of heterooligomeric receptors with physical properties distinct from homooligomers, thus increasing the diversity of GABAC receptors in the CNS.
Characteristics of slow and fast phases of the optocollic reflex (OCR) in head free pigeons (Columba livia): influence of flight behaviour
Authors: Gioanni H. , Sansonetti A.
Published: March 2003

The effect of behavioural context on the properties of slow and fast phases of the horizontal optocollic reflex (OCR) were investigated in head free pigeons for two situations, i.e.: (i) animals were hung in a harness (‘resting condition’); (ii) animals were additionally submitted to a frontal airflow that provoked a flight posture (‘flying condition’) [Bilo & Bilo (1983) J. Comp. Physiol., 153, 111]. A ‘transient flight’ was also provoked in the ‘resting condition’ by tapping the breastbone region. Stimuli consisted either of velocity steps (30–300 °/s) or of an increasing velocity stimulus (0–300 °/s).

 The amplitude of nystagmic beats and the OCR gain increased in the ‘flying condition’ and during ‘transient flight’ as compared to the ‘resting condition’. The OCR working range was considerably extended toward high velocities by the flying behaviour. In the ‘resting condition’, spontaneous head oscillations generally triggered a high‐gain OCR, close to that obtained in the ‘flying condition’. One‐third of the animals showed a higher gain in response to an increasing velocity stimulus than with step stimuli, in the high velocity range.

 The linear relation between amplitude and peak velocity of OCR fast phases was independent of the stimulation velocity in the ‘resting condition’, whereas the amplitude and peak velocity increased with the stimulation velocity in the ‘flying condition’. In this condition, the fast phase velocity was correlated with the slow phase velocity, but not with the retinal slip velocity.

 Thus, both the slow and fast phases of the OCR are dependent on the behavioural context.
Impaired glucocorticoid receptor function evolves in aberrant physiological responses to bacterial endotoxin
Authors: Linthorst Astrid C. E. , Karanth Sharada , Barden Nicholas , Holsboer Florian , Reul Johannes M. H. M.
Published: March 2003

The consequences of glucocorticoid receptor (GR) dysfunction for neuroimmunoendocrine responses to an inflammatory challenge were studied in transgenic mice expressing antisense RNA directed against the GR [GR‐impaired (GR‐i) mice]. Mice were implanted intraperitoneally with a biotelemetry transmitter to monitor body temperature and locomotion. GR‐i mice showed decreased locomotion and body temperature during the dark phase of the diurnal cycle. Intraperitoneal administration of saline caused a rapid increase in body temperature in control mice, which was terminated within 90 min. In GR‐i mice, however, body temperature remained elevated for about 6 h. Intraperitoneal injection of endotoxin (10 μg/mouse) produced a biphasic fever in control mice. However, in endotoxin‐injected GR‐i mice, body temperature was not significantly different from their saline‐injected controls during the first 6 h. Body temperature then increased and remained elevated during the night period. Both strains showed hypolocomotion after endotoxin. In a second experiment, mice were injected intraperitoneally with saline or endotoxin and killed after 1, 3, 6 or 24 h. In GR‐i mice, endotoxin caused an augmented rise in plasma ACTH, but not in corticosterone levels. The endotoxin‐induced increase in serum levels of interleukin‐1β and interleukin‐6 was not different between the strains. However, whereas in control mice tumour necrosis factor‐α levels were below detection at the time points studied, substantial levels of this cytokine were found in the serum of GR‐i mice 1 h after endotoxin administration.

 It may be concluded that life‐long impairment of GR evolves in aberrant physiological and humoral responses to an acute inflammatory challenge. These findings expand our understanding about the neuroendocrine and physiological disturbances associated with stress‐related disorders.
Characterization of cultured dorsal root ganglion neuron P2X receptors
Authors: Grubb B. D. , Evans R. J.
Published: March 2003

P2X receptors for adenosine 5′‐triphosphate (ATP) comprise a family of ligand‐gated cation channels with distinct characteristics which are dependent on the receptor subunits (P2X1–7) expressed, and the homomeric or heteromeric assembly of protein subunits in individual cells. We describe the properties of P2X receptors expressed by cultured adult rat dorsal root ganglion cells on the basis of the time course of responses to ATP, α,β‐methylene adenosine 5′‐triphosphate (α,β‐meATP) and 2‐methyl‐thioadenosine 5′‐triphosphate (2‐meSATP), and using the antagonists 2′,3′‐O‐(2,4,6‐trinitrophenyl) ATP (TNP‐ATP), a novel and highly selective purinoceptor antagonist, suramin and iso‐pyridocalphosphate‐6‐azophenyl‐2′,5′ disulphonic acid (PPADS). ATP (10 μm) evoked inward currents in ≈ 95% of neurons tested and > 80% responded with a fast transient inward current that rapidly inactivated during the continued presence of ATP. Of the remaining neurons, ≈ 4% showed a sustained response and ≈ 10% showed a combination of transient and sustained components. Rapid application of ATP, α,β‐meATP and 2meSATP demonstrated these to be full agonists of the rapidly inactivating P2X receptor (pA50 values = 5.83, 5.86 and 5.55, respectively), whilst uridine triphosphate (UTP) and 1‐β,γ‐methyleneadenosine 5′‐triphosphate (1‐β,γ‐meATP) were ineffective as agonists. These rapidly inactivating responses could be inhibited by TNP‐ATP, suramin and PPADS (pIC50 = 9.5, 6.5, 6.4, respectively). Using inactivation protocols, we demonstrate the presence of homomeric P2X3‐like receptors and non‐inactivating P2X receptors, which indicates that individual subsets of adult dorsal root ganglion neurons have distinct P2X receptor phenotypes, and that individual DRG neurons may express multiple P2X receptor subtypes.
Modulation of neurotransmitter release by dihydropyridine‐sensitive calcium channels involves tyrosine phosphorylation
Authors: Evans Gareth J. O. , Pocock Jennifer M.
Published: March 2003

Cultured rat cerebellar granule cells depolarized by high KCl, display a large component of Ca2+ influx through L‐type voltage‐dependent Ca2+ channels as defined by a sensitivity to 1 μm nifedipine. This Ca2+ influx is not coupled to neurotransmitter exocytosis but has implications for neuronal development. KCl stimulation in the absence of external Ca2+ followed by the re‐addition of Ca2+ allows the coupling of a class of L‐type Ca2+ channels to neurotransmitter exocytosis as assessed by loading of glutamatergic pools with [3H]‐d‐aspartate. KCl stimulation in the absence of external Ca2+ (‘predepolarization’) enhances tyrosine phosphorylation of several cellular proteins, and inhibitors of tyrosine kinases block both phosphorylation and the neurotransmitter release coupled to the L‐type Ca2+ channel. More specifically, an inhibitor of src family tyrosine kinases, PP1, blocks the effects of predepolarization suggesting a role for a src family kinase in the process. Furthermore, L‐type Ca2+ channel recruitment and modulation of release could be activated with the tyrosine phosphatase inhibitor sodium orthovanadate. The phosphoproteins enhanced by predepolarization, which include the cytoskeletal proteins focal adhesion kinase (FAK) and vinculin, are also highly phosphorylated early on in culture when neurite outgrowth occurs. As the neurons develop a network of neurites, both tyrosine phosphorylation and L‐type Ca2+ channel activity decrease. These results show a novel mechanism for the recruitment of L‐type Ca2+ channels and their coupling to neurotransmitter release which involves tyrosine phosphorylation. This phenomenon has a role in cerebellar granule cell development.
Subcellular distribution of survival motor neuron (SMN) protein: possible involvement in nucleocytoplasmic and dendritic transport
Authors: Béchade Catherine , Rostaing Philippe , Cisterni Claire , Kalisch Raffaël , Bella Vincenzo La , Pettmann Brigitte , Triller Antoine
Published: March 2003

Spinal muscular atrophy (SMA) is among the most common recessive autosomal diseases and is characterized by the loss of spinal motor neurons. A gene termed ‘Survival of Motor Neurons' (SMN) has been identified as the SMA‐determining gene. Recent work indicates the involvement of the SMN protein and its associated protein SIP1 in spliceosomal snRNP biogenesis. However, the function of SMN remains unknown. Here, we have studied the subcellular localization of SMN in the rat spinal cord and more generally in the central nervous system (CNS), by light fluorescence and electron microscopy. SMN immunoreactivity (IR) was found in the different regions of the spinal cord but also in various regions of the CNS such as the brainstem, cerebellum, thalamus, cortex and hippocampus. In most neurons, we observed a speckled labelling of the cytoplasm and a discontinuous staining of the nuclear envelope. For some neurons (e.g. brainstem nuclei, dentate gyrus, cortex: layer V) and, in particular in motoneurons, SMN‐IR was also present as prominent nuclear dot‐like‐structures. In these nuclear dots, SMN colocalized with SIP1 and with fibrillarin, a marker of coiled bodies. Ultrastructural studies in the anterior horn of the spinal cord confirmed the presence of SMN in the coiled bodies and also revealed the protein at the external side of nuclear pores complexes, in association with polyribosomes, and in dendrites, associated with microtubules. These localizations suggest that, in addition to its involvement in the spliceosome biogenesis, the SMN protein could also play a part in nucleocytoplasmic and dendritic transport.
Different cortical organization of visceral and somatic sensation in humans
Authors: Schnitzler Alfons , Volkmann Jens , Enck Paul , Frieling Thomas , Witte Otto W. , Freund Hans‐Joachim
Published: March 2003

Sensory stimuli from the visceral domain exhibit perceptual characteristics different from stimuli applied to the body surface. Compared with somatosensation there is not much known about the cortical projection and functional organization of visceral sensation in humans. In this study, we determined the cortical areas activated by non‐painful electrical stimulation of visceral afferents in the distal oesophagus, and somatosensory afferents in the median nerve and the lip in seven healthy volunteers using whole‐head magnetoencephalography. Stimulation of somatosensory afferents elicited short‐latency responses (≈ 20–60 ms) in the primary somatosensory cortex (SI) contralateral (median nerve) or bilateral (lip) to the stimulated side, and long‐latency responses (≈ 60–160 ms) bilaterally in the second somatosensory cortex (SII). In contrast, stimulation of visceral oesophageal afferents did not evoke discernible responses in SI but well reproducible bilateral SII responses (≈ 70–190 ms) in close vicinity to long‐latency SII responses following median nerve and lip stimuli. Psychophysically, temporal discrimination of successive stimuli became worse with increasing stimulus repetition rates (0.25 Hz, 0.5 Hz, 1 Hz, 2 Hz) only for visceral oesophageal, but not for somatosensory median nerve stimuli. Correspondingly, amplitudes of the first cortical response to oesophageal stimulation emerging in the SII cortex declined with increasing stimulus repetition rates whereas the earliest cortical response elicited by median nerve stimuli (20 ms SI response) remained unaffected by the stimulus frequency. Our results indicate that visceral afferents from the oesophagus primarily project to the SII cortex and, unlike somatosensory afferents, lack a significant SI representation. We propose that this cortical projection pattern forms the neurophysiological basis of the low temporal and spatial resolution of conscious visceral sensation.
Myelin does not influence the choice behaviour of entorhinal axons but strongly inhibits their outgrowth length in vitro
Authors: Savaskan Nicolai E. , Plaschke Martina , Ninnemann Olaf , Spillmann Adrian A. , Schwab Martin E. , Nitsch Robert , Skutella Thomas
Published: March 2003

Myelin is crucial for the stabilization of the entorhinohippocampal projection during late development and is a non‐permissive substrate for regrowing axons after lesion in the adult brain. We used two in vitro assays to analyse the impact of myelin on rat entorhinohippocampal projection neurons. A stripe assay was used to study the impact of myelin on the choice behaviour of axons from the entorhinal cortex (EC). Given a choice between alternating hippocampal membrane lanes from developmental stages ranging from early postnatal to adult, EC axons preferred to extend on early postnatal hippocampal membranes. Neither the neutralization of myelin‐associated factors by a specific antibody (IN‐1) nor the separation of myelin from membranes interfered with the axons' choice behaviour. The entorhinal axons showed no preference in the membrane combination of adult and myelin‐free adult hippocampal membranes. These stripe assay experiments demonstrate that support for EC axon choice in the developing hippocampus is maturation‐dependent and is not influenced by myelin. The application of IN‐1 in the outgrowth assay and the separation of myelin from membranes, enhanced elongation of outgrowing entorhinal axons on adult hippocampal membranes, whereas a control antibody did not. This shows that myelin‐associated factors have a strong inhibitory effect on the outgrowth length of entorhinal axons. In conclusion, we suggest that axonal elongation in the entorhinohippocampal system during development is strongly influenced by myelin‐associated growth inhibition factors and that specific target finding of entorhinal axons is regulated by a different mechanism.
Local inhibition of hippocampal nitric oxide synthase does not impair place learning in the Morris water escape task in rats
Authors: Blokland Arjan , De Vente Jan , Prickaerts Jos , Honig Wiel , Van Ittersum Marjanne Markerink‐ , Steinbusch Harry
Published: March 2003

Recent studies have provided evidence that nitric oxide (NO) has a role in certain forms of memory formation. Spatial learning is one of the cognitive abilities that has been found to be impaired after systemic administration of an NO‐synthase inhibitor. As the hippocampus has a pivotal role in spatial orientation, the present study examined the role of hippocampal NO in spatial learning and reversal learning in a Morris task in adult rats. It was found that Nω‐nitro‐l‐arginine infusions into the dorsal hippocampus affected the manner in which the rats were searching the submerged platform during training, but did not affect the efficiency to find the spatial location of the escape platform. Hippocampal NO‐synthase inhibition did not affect the learning of a new platform position in the same water tank (i.e. reversal learning). Moreover, no treatment effects were observed in the probe trials (i.e. after acquisition and after reversal learning), indicating that the rats treated with Nω‐nitro‐l‐arginine had learned the spatial location of the platform. These findings were obtained under conditions where the NO synthesis in the dorsal hippocampus was completely inhibited. On the basis of the present data it was concluded that hippocampal NO is not critically involved in place learning in rats.
A new pyrrolyl‐quinoxalinedione series of non‐NMDA glutamate receptor antagonists: pharmacological characterization and comparison with NBQX and valproate in the kindling model of epilepsy
Authors: Löscher W. , Lehmann H. , Behl B. , Seemann D. , Teschendorf H. J. , Hofmann H. P. , Lubisch W. , Höger T. , Lemaire H. ‐G. , Groß G.
Published: March 2003

Antagonists at the ionotropic non‐NMDA [AMPA (amino‐methyl proprionic acid)/kainate] type of glutamate receptors have been suggested to possess several advantages compared to NMDA (N‐methyl‐d‐aspartate) receptor antagonists, particularly in terms of risk/benefit ratio, but the non‐NMDA receptor antagonists available so far have not fulfilled this promise. From a large series of pyrrolyl‐quinoxalinedione derivatives, we selected six new competitive non‐NMDA receptor antagonists. The basis of selection was high potency and selectivity for AMPA and/or kainate receptors, high in vivo potency after systemic administration, and an acceptable ratio between neuroprotective or anticonvulsant effects and adverse effects. Pharmacological characteristics of these novel compounds are described in this study with special emphasis on their effects in the kindling model of temporal lobe epilepsy, the most common type of epilepsy in humans. In most experiments, NBQX and the major antiepileptic drug valproate were used for comparison with the novel compounds. The novel non‐NMDA receptor antagonists markedly differed in their AMPA and kainate receptor affinities from NBQX. Thus, while NBQX essentially did not bind to kainate receptors at relevant concentrations, several of the novel compounds exhibited affinity to rat brain kainate receptors or recombinant kainate receptor subtypes in addition to AMPA receptors. One compound, LU 97175, bound to native high affinity kainate receptors and rat GluR5–GluR7 subunits, i.e. low affinity kainate binding sites, with much higher affinities than to AMPA receptors. All compounds potently blocked AMPA‐induced cell death in vitro and, except LU 97175, AMPA‐induced convulsions in vivo. In the kindling model, compounds with a high affinity for GluR7 (LU 97175) or compounds (LU 115455, LU 136541) which potently bind to AMPA receptors and low affinity kainate receptor subunits were potent anticonvulsants in the kindling model, whereas the AMPA receptor‐selective LU 112313 was the least selective compound in this model, indicating that non‐NMDA antagonists acting at both AMPA and kainate receptors are more effective in this model than AMPA receptor‐selective drugs. Three of the novel compounds, i.e. LU 97175, LU 115455 and LU 136541, exerted potent anticonvulsant effects without inducing motor impairment in the rotarod test. This combination of actions is thought to be a prerequisite for selective anticonvulsant drug action.
Intrastriatal grafting of a GDNF‐producing cell line protects striatonigral neurons from quinolinic acid excitotoxicity in vivo
Authors: Pérez‐Navarro E. , Arenas E. , Marco S. , Alberch J.
Published: March 2003

Glial cell line‐derived neurotrophic factor (GDNF) is a neurotrophic factor with a therapeutic potential in neurodegenerative disorders. GDNF is expressed in the adult striatum, but its signalling tyrosine kinase receptor, c‐ret, has not been detected in this structure by in situ hybridization. In the present work, we first examined c‐ret and GDNF receptor α1 (GFR‐α1) expression using an RNAse protection assay, and found that both receptors are expressed in the adult rat striatum. We then examined whether GDNF was able to regulate the phenotype and/or prevent the degeneration of striatal projection neurons in a well‐characterized model of excitotoxic damage. A fibroblast cell line, engineered to overexpress GDNF, was grafted in adult rats striatum 24 h before quinolinic acid (QUIN) injection. QUIN injection alone or in combination with the control cell line induced a loss of glutamic acid decarboxylase 67 (GAD)‐, preprotachykinin A (PPTA)‐, prodynorphin (DYN)‐ and preproenkephalin (PPE)‐positive neurons. GDNF selectively prevented: (i) the loss of a subpopulation of striatonigral neurons expressing GAD and PPTA; (ii) the atrophy of PPTA‐positive neurons; and (iii) the decrease in GAD, PPTA and DYN mRNA expression, after QUIN injection. Moreover, in unlesioned animals, GDNF increased the size of PPTA‐positive neurons and up‐regulated their mRNA levels. In contrast, GDNF showed no effect in intact or lesioned striatopallidal PPE‐positive neurons. Thus, our findings show that GDNF selectively regulates the phenotype and protects striatonigral neurons from QUIN‐induced excitotoxicity, suggesting that GDNF may be used for the treatment of striatonigral degenerative disorders, e.g. Huntington's disease and multiple system atrophy.
Increased cyclin D1 in vulnerable neurons in the hippocampus after ischaemia and epilepsy: a modulator of in vivo programmed cell death?
Authors: Timsit Serge , Rivera Santiago , Ouaghi Philippe , Guischard Frédérique , Tremblay Évelyne , Ben‐Ari Yezekiel , Khrestchatisky Michel
Published: March 2003

Several observations suggest that delayed neuronal death in ischaemia, epilepsy and other brain disorders includes an apoptotic component, involving programmed cell death (PCD). PCD is hypothesized to result, in part, from aberrant control of the cell cycle. Because they are instrumental in mitosis, cyclins D are key markers to evaluate whether neurons indeed progress into the cell cycle in situations of pathology. Therefore, we investigated in rat brains, the expression of cyclins D in the delayed neuronal death that occurs following transient global ischaemia and kainate‐induced seizures. Following a four‐vessel occlusion insult, quantitative in situ hybridization revealed a highly significant and persistent 100% increase of cyclin D1 mRNA in the vulnerable pyramidal neurons of the CA1 hippocampal region. Ischaemia also induced a smaller and transient cyclin D1 mRNA increase in the resistant CA3 area and dentate gyrus. In contrast, the cyclin D2 and D3 mRNAs, expressed constitutively in the adult rat hippocampus, were not upregulated. Following kainate‐induced seizures, cyclin D1 mRNA was induced in the vulnerable CA3 region, and to a lesser extent, in non‐vulnerable regions. Cyclin D1 immunohistochemistry revealed increased protein levels in the cytoplasm and nucleus of neurons commited to die after ischaemia. Double labelling experiments indicate that cyclin D1 is also expressed in reactive astrocytes but not in microglial cells. Finally, we report that in neurons, cyclin D1 expression peaks before nuclear condensation and the appearance of DNA fragmentation. We propose that cyclin D1, when expressed at high levels in lesioned neurons, may act as a modulator of apoptosis.
Nitric oxide is required for expression of LTP that is induced by stimulation phase‐locked with theta rhythm
Authors: Hölscher Christian
Published: March 2003

Long‐term potentiation (LTP) can be induced by giving only one burst (five stimuli at 200 Hz) on the positive phase of sensory‐induced theta rhythm in awake or anaesthetized rats, a stimulation protocol that mimics naturally occurring neuronal activity. Nitric oxide has been discussed as an important neuronal messenger in the induction of LTP. However, experiments testing inhibitors of nitric oxide synthase (NOS) in vitro produced contradictory results. The non‐specific NOS inhibitor Nitro‐l‐arginine (L‐NARG) impaired LTP induced by high‐frequency stimulation (HFS) [from 155 ± 7% to 122 ± 8%), but completely blocked theta‐dependent LTP induction (161 ± 8% to 102 ± 5%). NOS inhibitors, e.g. 7‐nitro indazole (7‐NI) or 1‐(2‐trifluoromethylphenyl) imidazole (TRIM) that are more selective for neuronal NOS and affect blood pressure less also impaired HFS‐induced LTP (186 ± 11% to 135 ± 9% for TRIM) but completely blocked theta‐dependent LTP (154 ± 7 to 91 ± 8). l‐Arginine reversed the effects of the NOS inhibitors tested. Therefore, NO appears to be a modulator that is important for synaptic plasticity in this more physiological stimulation technique in vivo. NO is not released in slice preparations in sufficient quantities or at the right timing. Instead, the unphysiologically strong HFS protocol appears to induce an NO‐independent type of LTP in some cases.
Zn2+ entry produces oxidative neuronal necrosis in cortical cell cultures
Authors: Kim Eun Young , Koh Jae Young , Kim Yang Hee , Sohn Seonghyang , Joe Eunhye , Gwag Byoung Joo
Published: March 2003

Evidence has accumulated that Zn2+ plays a central role in neurodegenerative processes following brain injuries including ischaemia or epilepsy. In the present study, we examined patterns and possible mechanisms of Zn2+ neurotoxicity. Inclusion of 30–300 μm Zn2+ for 30 min caused neuronal necrosis apparent by cell body and mitochondrial swelling in cortical cell cultures. This Zn2+ neurotoxicity was not attenuated by antiapoptosis agents, inhibitors of protein synthesis or caspase. Blockade of glutamate receptors or nitric oxide synthase showed no beneficial effect against Zn2+ neurotoxicity. Interestingly, antioxidants, trolox or SKF38393, attenuated Zn2+‐induced neuronal necrosis. Pretreatment with insulin or brain‐derived neurotrophic factor increased the Zn2+‐induced free radical injury. Kainate or AMPA facilitated Zn2+ entry and potentiated Zn2+ neurotoxicity in a way sensitive to trolox. Reactive oxygen species and lipid peroxidation were generated in the early phase of Zn2+ neurotoxicity. These findings indicate that entry and accumulation of Zn2+ result in generation of toxic free radicals and then cause necrotic neuronal degeneration under certain pathological conditions in the brain.
Rapid desensitization converts prolonged glutamate release into a transient EPSC at ribbon synapses between retinal bipolar and amacrine cells
Authors: Maguire Greg
Published: March 2003

The mechanisms underlying the conversion of prolonged glutamate release from ribbon synapses in bipolar cells to sustained and transient excitatory postsynaptic responses in identified retinal amacrine cells were studied in tiger salamander (Ambystoma tigrinum) retina. A retina slice preparation with whole cell patch recording techniques under voltage‐ and current‐clamp conditions was used to assay the electrical properties of bipolar and amacrine cells. Amacrine cells were categorized into two basic forms: (i) transient amacrine cells that respond to a step of light with a burst of spikes only at the transitions of the step; and (ii) sustained amacrine cells that respond with continuous spiking during the entire light step. The two cell types each had a characteristic morphology: transient amacrine cells possessed wide dendritic fields (χ = 375 μm), while sustained cells had much more narrowly confined dendritic fields (χ = 85 μm). Whole cell voltage‐gated currents of the transient and sustained cell types were not significantly different. Both cell types had spikes that were sensitive to tetradotoxin (TTX, 0.3 μm) with voltage deflections of up to 100 mV. Light‐evoked excitatory synaptic currents relaxed rapidly in transient neurons (τ1/2 = 100 ms) and more slowly in sustained neurons (τ1/2 = 1.2 s). EPSCs in both cells reversed near 0 mV. Rapid application of glutamate or kainate elicited rapidly desensitizing ionic currents (τ1/2 = 85 ms) followed by a slowly desensitizing component. Cyclothiazide, a drug that eliminates rapid desensitization, lengthened the time course of the glutamate gated current from τ1/2 = 85 ms to about 3 s, and the relaxation kinetics of the glutamatergic EPSC from τ1/2 = 85 ms to about 1.0 s. These data suggest that a key determinant in forming transient versus sustained responses in amacrine cells of vertebrate retina is the differences in their excitatory, glutamatergic synaptic inputs, and that rapid desensitization of glutamate receptors plays a role in converting the presynaptic signal associated with sustained glutamate release into a postsynaptic, transient signal at the ribbon synapse.
Synaptic and intrinsic mechanisms shape synchronous oscillations in hippocampal neurons in culture
Authors: Bacci Alberto , Verderio Claudia , Pravettoni Elena , Matteoli Michela
Published: March 2003

We have detected spontaneous, synchronous calcium oscillations, associated with variations in membrane potential, in hippocampal neurons maintained in primary culture. The oscillatory activity is synaptically driven, as it is blocked by tetrodotoxin, by the glutamate receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) and by toxins inhibiting neurotransmitter release from presynaptic nerve endings. Neuronal oscillations do not require for their expression the presence of a polyneuronal network and are not primarily influenced by the γ‐aminobutyric acid (GABAA) receptor antagonist picrotoxin, suggesting that they entirely rely on glutamatergic neurotransmission. Synaptic and intrinsic conductances shape the synchronized oscillations in hippocampal neurons. The concomitant activation of N‐methyl‐d‐aspartate (NMDA) receptors and voltage‐activated L‐type calcium channels allows calcium entering from the extracellular medium and sustaining the long depolarization, which shapes every single calcium wave.
Stimulation of phospholipase C by the cloned μ, δ and κ opioid receptors via chimeric Gαq mutants
Authors: Joshi Sushma , Lee Jonathan W. M. , Wong Yung H.
Published: March 2003

Opioid receptors (μ, δ and κ) are known to regulate diverse physiological functions and yet, at the molecular level, they are coupled to a seemingly identical set of G proteins. A recent study has discerned subtle differences between the opioid receptors in their ability to activate the pertussis toxin‐insensitive G16. Differences in microarchitecture might be magnified when these receptors are provided with ‘non‐native’ partners. Here, we examined whether the opioid receptors can interact productively with a set of chimeric Gαq subunits which are known to link many Gi‐coupled receptors to phosphoinositide‐specific phospholipase C (PI‐PLC). The qi5, qo5 and qz5 chimeras have the last five residues of Gαq replaced by those of Gαi, Gαo and Gαz, respectively. Except for μ‐receptor and qo5, each pair of opioid receptor and Gαq chimera allowed opioid agonists to stimulate PI‐PLC in transfected COS‐7 cells. The Gαq chimera‐mediated responses were ligand selective, agonist dose dependent and saturable. The most robust responses were obtained with κ‐receptor and qi5 or qz5, whereas the coupling of δ‐ and μ‐receptors to Gαq chimeras produced much weaker responses. Among the Gαq chimeras, qo5 was less efficiently coupled to the opioid receptors. As revealed by radioligand binding assays and immunoblot analysis, differences in the efficiency of coupling were not due to variations in the expression of receptors and Gαq chimeras. Differences in the magnitude of PI‐PLC responses are thus likely to represent structural incompatibility between opioid receptors and Gαq chimeras, suggesting that each opioid receptor possesses unique structural surfaces for the binding of G proteins.
Xenopus muscle‐specific kinase: molecular cloning and prominent expression in neural tissues during early embryonic development
Authors: Fu Amy K. Y. , Smith Frances D. , Zhou Hong , Chu Apple H. , Tsim Karl W. K. , Peng Benjamin H. , Ip Nancy Y.
Published: March 2003

A muscle‐specific receptor tyrosine kinase, designated MuSK, mediates agrin‐induced aggregation of acetylcholine receptors at the vertebrate neuromuscular junction. cDNAs encoding Xenopus MuSK were isolated from embryonic cDNA libraries. The full‐length MuSK cDNA encodes for a polypeptide of 948 amino acids and possesses the features unique to mammalian MuSK, including four Ig‐like domains, C6 box, transmembrane region and an intracellular tyrosine kinase domain. Interestingly, Xenopus MuSK also contains a kringle domain similar to that previously reported for Torpedo MuSK. The overall amino acid sequence identity of Xenopus MuSK with mammalian MuSK is ≈ 65%. Northern blot analysis demonstrated the presence of three MuSK transcripts (≈ 1 kb, ≈ 3 kb and ≈ 7 kb) which were differentially expressed during development. The expression of the ≈ 7 kb MuSK transcript remained as the predominant species in adult tissues, e.g. skeletal muscle, spleen and lung. Immunocytochemical analysis with a MuSK‐specific antibody revealed that Xenopus MuSK was colocalized with AChRs at neuromuscular junctions as well as in spontaneous acetylcholine receptor hot spots of cultured muscle cells. In situ hybridization revealed prominent expression of MuSK transcripts in neural tissues and myotomal muscle during the period of neurulation and synaptogenesis. The MuSK transcript detected at abundant levels in the central nervous system (CNS) was localized to the brain, spinal cord and eye vesicles during early embryonic development. In addition, the MuSK protein in the developing eye was found to be prominently expressed during embryonic stages of 32 and 35. These findings raise an intriguing possibility that, in addition to the known function in the formation of the neuromuscular junctions, MuSK may be involved in neural development.