bluuebunny/crossref_metadata_2025 huggingface dataset:
The expression of vesicular glutamate transporters VGLUT1 and VGLUT2 in neurochemically defined axonal populations in the rat spinal cord with emphasis on the dorsal horn
Authors: Todd A. J. , Hughes D. I. , Polgár E. , Nagy G. G. , Mackie M. , Ottersen O. P. , Maxwell D. J.
Published: March 2003

Two vesicular glutamate transporters, VGLUT1 and VGLUT2, have recently been identified, and it has been reported that they are expressed by largely nonoverlapping populations of glutamatergic neurons in the brain. We have used immunocytochemistry with antibodies against both transporters, together with markers for various populations of spinal neurons, in an attempt to identify glutamatergic interneurons in the dorsal horn of the mid‐lumbar spinal cord of the rat. The great majority (94–100%) of nonprimary axonal boutons that contained somatostatin, substance P or neurotensin, as well as 85% of those that contained enkephalin, were VGLUT2‐immunoreactive, which suggests that most dorsal horn neurons that synthesize these peptides are glutamatergic. In support of this, we found that most somatostatin‐ and enkephalin‐containing boutons (including somatostatin‐immunoreactive boutons that lacked calcitonin gene‐related peptide and were therefore probably derived from local interneurons) formed synapses at which AMPA receptors were present.

We also investigated VGLUT expression in central terminals of primary afferents. Myelinated afferents were identified with cholera toxin B subunit; most of those in lamina I were VGLUT2‐immunoreactive, whereas all those in deeper laminae were VGLUT1‐immunoreactive, and some (in laminae III–VI) appeared to contain both transporters. However, peptidergic primary afferents that contained substance P or somatostatin (most of which are unmyelinated), as well as nonpeptidergic C fibres (identified with Bandeiraea simplicifolia isolectin B4) showed low levels of VGLUT2‐immunoreactivity, or were not immunoreactive with either VGLUT antibody. As all primary afferents are thought to be glutamatergic, this raises the possibility that unmyelinated afferents, most of which are nociceptors, express a different vesicular glutamate transporter.
The mouse MPTP model: gene expression changes in dopaminergic neurons
Authors: Kühn Kati , Wellen Jennifer , Link Nina , Maskri Lyutha , Lübbert Hermann , Stichel Christine C.
Published: March 2003

Parkinson's disease (PD) is a common neurodegenerative disorder, characterized by the progressive loss of dopaminergic neurons in the substantia nigra. Although valuable animal models have been developed, our knowledge of the aetiology and pathogenic factors implicated in PD is still insufficient to develop causal therapeutic strategies aimed at halting its progression. The neurotoxicity induced by 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) is one of the most valuable models for analysing pathological aspects of PD. In this paper we studied the gene expression patterns underlying the pathogenesis of MPTP‐induced neurodegeneration. We treated young and old C57BL/6 mice with different schedules of MPTP to induce degenerative processes that vary in intensity and time‐course. During the first week after intoxication we used nonradioactive in situ‐hybridization to investigate the expression patterns of genes associated with (i) dopamine metabolism and signalling; (ii) familial forms of PD; (iii) protein folding and (iv) energy metabolism. MPTP injections induced different severities of neuronal injury depending on the age of the animals and the schedule of administration as well as a significant degeneration in the striatum. In situ hybridization showed that MPTP intoxication initiated a number of gene expression changes that (i) were restricted to the neurons of the substantia nigra pars compacta; (ii) were correlated in intensity and number of changes with the age of the animals and the severity of histopathological disturbances; (iii) displayed in each a significant down‐regulation by the end of one week after the last MPTP injection, but (iv) varied within one MPTP regimen in expression levels during the observation period. The subacute injection of MPTP into one‐year‐old mice induced the most severe changes in gene expression. All genes investigated were affected. However, α‐synuclein was the only gene that was exclusively up‐regulated in MPTP‐treated animals displaying cell death.
Cannabinoid withdrawal syndrome is reduced in double mu and delta opioid receptor knockout mice
Authors: Castañé Anna , Robledo Patricia , Matifas Audrey , Kieffer Brigitte L. , Maldonado Rafael
Published: March 2003

Several studies have shown a functional relationship between the endogenous cannabinoid and opioid systems. However, acute effects of Δ9‐tetrahydrocannabinol (THC) and physical dependence were not modified in knockout mice with single deletion of mu (MOR), delta (DOR) or kappa (KOR) opioid receptors. To further investigate the neurobiological basis of cannabinoid dependence, we have evaluated acute pharmacological responses, rewarding effects, tolerance and dependence to THC in double MOR/DOR knockout mice. Antinociception and hypolocomotion induced by acute THC administration remained unaffected, whereas the hypothermic effect was slightly attenuated in these double knockout mice. During chronic THC treatment, knockout mice developed slower tolerance to the hypothermic effect, but the development of tolerance to antinociceptive and hypolocomotor effects was unchanged. The rewarding properties of THC, measured in the conditioned place preference paradigm, were reduced in knockout mice. Interestingly, the somatic manifestations of THC withdrawal were also significantly attenuated in mutant mice, suggesting that a cooperative action of MOR and DOR is required for the entire expression of THC dependence.
Behavioural learning‐induced increase in spontaneous GABAA‐dependent synaptic activity in rat striatal cholinergic interneurons
Authors: Bonsi Paola , Florio Tiziana , Capozzo Annamaria , Pisani Antonio , Calabresi Paolo , Siracusano Alberto , Scarnati Eugenio
Published: March 2003

Cholinergic striatal interneurons play a crucial role in cognitive aspects of context‐dependent motor behaviours. They are considered to correspond to the tonically active neurons (TANs) of the primate striatum, which phasically decrease their discharge at the presentation of reward‐related sensory stimuli. The origin of this response is still poorly understood. Therefore, in the present paper, we have investigated whether synaptic changes establish in cholinergic interneurons from young rats that have learned a rewarded, externally cued sensorimotor task. Corticostriatal slices were prepared from both control and trained rats. No significant change in intrinsic membrane properties and evoked synaptic activity was observed in cholinergic interneurons, nor the responsiveness to exogenously applied dopaminergic and glutamatergic agonists was modified. Conversely, an increased occurrence of spontaneous bicuculline‐sensitive depolarizing postsynaptic potentials (sDPSP) was recorded. The frequency of the GABAA‐mediated sDPSP was increased in comparison to not‐conditioned rats. Overall, these results suggest that after learning a rewarded sensorimotor paradigm an increased GABA influence develops on cholinergic interneurons. The origin of this effect might be searched in collaterals of GABAergic output spiny neurons as well as in GABAergic striatal interneurons impinging onto cholinergic interneurons. This intrastriatal mechanism might be involved in the phasic suppression of discharge of TANs at the presentation of reward‐related sensory stimuli.
Neuronal activation in female budgerigars is localized and related to male song complexity
Authors: Eda‐Fujiwara Hiroko , Satoh Ryohei , Bolhuis Johan J. , Kimura Takeji
Published: March 2003

Females of several songbird species have been shown to respond preferentially to a more complex song. The male budgerigar (Melopsittacus undulatus) sings complex songs consisting of discrete components, known as syllables. We exposed female budgerigars to either standard male song, complex song, or simple song, the iteration of only one syllable (either frequency‐modulated or unmodulated). Using immunocytochemistry, we analysed the expression of the protein product of the immediate early gene ZENK in a number of forebrain regions. The level of Zenk protein expression caused by song stimuli varied among each of the brain regions. Expression was highest in the caudomedial neostriatum (NCM), lower in the caudomedial hyperstriatum ventrale (CMHV), and lowest in the hippocampus. There was a significant effect of song complexity on the number of Zenk‐immunoreactive cells in the NCM, but not in the hippocampus. Zenk protein expression correlated significantly and positively with the number of different syllables to which the females were exposed in the NCM and to a lesser extent in the CMHV, but not in the hippocampus. For the NCM this correlation was also significant within the group exposed to natural song. These results suggest that the NCM is involved in the perception of song complexity in female budgerigars.
Phasic activation of substantia nigra and the ventral tegmental area by chemical stimulation of the superior colliculus: an electrophysiological investigation in the rat
Authors: Coizet Véronique , Comoli Eliane , Westby G. W. Max , Redgrave Peter
Published: March 2003

The source of short‐latency visual input to midbrain dopaminergic (DA) neurons is not currently known; however, the superior colliculus (SC) is a subcortical visual structure which has response latencies consistently shorter than those recorded for DA neurons in substantia nigra and the ventral tegmental area. To test whether the SC represents a plausible route by which visual information may gain short latency access to the ventral midbrain, the present study examined whether experimental stimulation of the SC can influence the activity of midbrain DA neurons. In urethane‐anaesthetized rats, 63 pairs of extracellular recordings were obtained from neurons in the SC and ipsilateral ventral midbrain, before and after local disinhibitory injections of the GABA antagonist bicuculline (20–40 ng/200–400 nL saline) into the SC. Neurons recorded from substantia nigra and the ventral tegmental area were classified as putative DA (25/63, 39.7%) or putative non‐DA (38/63, 60.3%). In nearly half the cases (27/63, 42.8%), chemical stimulation of the SC evoked a corresponding increase in neural activity in the ventral midbrain. This excitatory effect did not distinguish between DA and non‐DA neurons. In 6/63 cases (9.5%), SC activation elicited a reliable suppression of activity, while the remaining 30/63 cases (47.6%) were unaffected. In almost a third of cases (16/57, 28.1%) intense phasic activation of the SC was associated with correlated phasic activation of neurons in substantia nigra and the ventral tegmental area. These data suggest that the SC is in a position to play an important role in discriminating the appropriate stimulus qualities required to activate DA cells at short latency.
Nicotinic activation of reticulospinal cells involved in the control of swimming in lampreys
Authors: Le Ray Didier , Brocard Frédéric , Bourcier‐Lucas Céline , Auclair François , Lafaille Philippe , Dubuc Réjean
Published: March 2003

In lampreys as in other vertebrates, brainstem centres play a key role in the initiation and control of locomotion. One such centre, the mesencephalic locomotor region (MLR), was identified physiologically at the mesopontine border. Descending inputs from the MLR are relayed by reticulospinal neurons in the pons and medulla, but the mechanisms by which this is carried out remain unknown. Because previous studies in higher vertebrates and lampreys described cholinergic cells within the MLR region, we investigated the putative role of cholinergic agonists in the MLR‐controlled locomotion. The local application of either acetylcholine or nicotine exerted a direct dose‐dependent excitation on reticulospinal neurons as well as induced active or fictive locomotion. It also accelerated ongoing fictive locomotion. Choline acetyltransferase‐immunoreactive cells were found in the region identified as the MLR of lampreys and nicotinic antagonists depressed, whereas physostigmine enhanced the compound EPSP evoked in reticulospinal neurons by electrical stimulation of this region. In addition, cholinergic inputs from the MLR to reticulospinal neurons were found to be monosynaptic. When the brainstem was perfused with d‐tubocurarine, the induction of swimming by MLR stimulation was depressed, but not prevented, in a semi‐intact preparation. Altogether, the results support the hypothesis that cholinergic inputs from the MLR to reticulospinal cells play a substantial role in the initiation and the control of locomotion.
Relationship between two types of vesicular glutamate transporters and neurokinin‐1 receptor‐immunoreactive neurons in the pre‐Bötzinger complex of rats: light and electron microscopic studies
Authors: Liu Ying‐Ying , Wong‐Riley Margaret T. T. , Liu Jin‐Ping , Jia Yi , Liu Hui‐Ling , Fujiyama Fumino , Ju Gong
Published: March 2003

Our previous study demonstrated GABAergic and glycinergic synapses onto neurokinin‐1 receptor (NK1R)‐immunoreactive (ir) neurons in the pre‐Bötzinger complex (pre‐BötC), the hypothesized kernel of normal respiratory rhythmogenesis. In the present study, we aimed to identify glutamatergic synapses onto NK1R‐ir pre‐BötC neurons, as excitatory synaptic transmission is a prerequisite to normal respiratory rhythmogenesis. Two types of vesicular glutamate transporters (VGLUT), VGLUT1 and VGLUT2, have been recently implicated in glutamate‐mediated transmission. The present study used immunofluorescence and immunogold‐silver staining to determine the relationship between the transporters and NK1R‐ir neurons in the pre‐BötC of adult rats. Under the confocal laser‐scanning microscope, VGLUT2‐ir boutons were found to be widely distributed in the pre‐BötC, some of which were in close apposition to NK1R‐ir somas and dendrites. VGLUT1‐ir boutons were relatively rare and only a few were found to be in close apposition to NK1R‐ir somas and dendrites. Electron microscopic observation revealed that approximately 41% of VGLUT2‐ir terminals were in close apposition to, or made asymmetric synapses with NK1R‐ir somas and dendrites in the pre‐BötC. On the other hand, 50.5% of NK1R‐ir dendrites were closely apposed to, or synapsed with VGLUT2‐ir terminals. Occasionally, VGLUT1‐ir terminals were found in close apposition to NK1R‐ir somas or dendrites, but we were unable to identify synapses between them. The present findings provide the morphological basis for excitatory synaptic inputs onto NK1R‐ir neurons in the pre‐BötC. VGLUT2 may be involved in a dominant excitatory synaptic pathway for normal respiratory rhythmogenesis.
Developmental plasticity of mouse visual acuity
Authors: Prusky Glen T. , Douglas Robert M.
Published: March 2003

Monocular deprivation in mice between postnatal days 19 and 32 has been reported to significantly shift ocular dominance within the binocular region of primary visual cortex; however, it is not known whether visual deprivation in mice during this physiologically defined critical period also results in amblyopia, as it does in other mammals. We addressed this uncertainty by psychophysically assessing in adulthood (postnatal day 70 or older) the grating acuity of normal and monocularly deprived mice, using the Visual Water Task. The visual acuity of mice tested with their nondeprived eyes was equivalent to that of normal mice (≈ 0.5 cycles/degree); however, acuity measured with eyes monocularly deprived of vision transiently between postnatal days 19 and 32 was reduced by over 30% (≈ 0.31 cycles/degree). Identical binocular deprivation produced a significant, but smaller, decrease in acuity (≈ 0.38 cycles/degree). The effects of monocular and binocular deprivation were long lasting and occurred only if visual deprivation occurred between postnatal days 19 and 32. These data indicate that the deleterious effects of early visual deprivation on visual acuity in mice are similar to those reported in other mammals, and together with electrophysiological evidence of ocular dominance plasticity, suggest that the mechanisms of mouse visual plasticity are fundamentally the same as that in other mammals. Therefore, the mouse is probably a good model for investigating the basic cellular and molecular mechanisms underlying visual developmental plasticity and amblyopia.
Lesions of basolateral amygdala impair extinction of CS motivational value, but not of explicit conditioned responses, in Pavlovian appetitive second‐order conditioning
Authors: Lindgren John L. , Gallagher Michela , Holland Peter C.
Published: March 2003

The basolateral amygdala (BLA) is important for the modification of the motivational significance of events through associative learning. In previous work, we found that BLA was critical for the acquisition of conditioned reinforcement value to a visual conditioned stimulus (CS) paired with food. Unlike normal rats, rats with neurotoxic lesions of the BLA failed to acquire Pavlovian second‐order conditioning to an auditory stimulus paired with the first‐order visual CS in the absence of food. In this experiment, we examined the role of BLA in the extinction of the previously acquired conditioned reinforcement value of a Pavlovian CS. Rats received first‐order visual CS‐food pairings prior to either BLA‐ or sham‐lesions. Subsequent CS‐alone extinction training reduced the ability of the visual CS to reinforce second‐order conditioning of an auditory stimulus in the sham‐lesioned rats, but not in the BLA‐lesioned rats. Despite this persistence of the conditioned reinforcement value of the visual first‐order CS in the BLA‐lesioned rats, no effects of the lesions were observed on extinction of the explicit behavioural conditioned responses elicited by that CS.
Ciproxifan, a histamine H3‐receptor antagonist/inverse agonist, modulates the effects of methamphetamine on neuropeptide mRNA expression in rat striatum
Authors: Pillot Catherine , Héron Anne , Schwartz Jean‐Charles , Arrang Jean‐Michel
Published: March 2003

We have explored the effect of histamine H3‐receptor ligands on the regulation of neuropeptide mRNA expression in the striatum by using in situ hybridization performed with proenkephalin, prodynorphin, substance P and proneurotensin riboprobes. Acute administration of ciproxifan, an H3‐receptor antagonist/inverse agonist, or (R)‐α‐methylhistamine, an H3‐receptor agonist, did not modify the striatal expression of the neuropeptides by itself. However, ciproxifan strongly and differentially modulated the effect of a single administration of 3 mg/kg methamphetamine on neuropeptide mRNA expression. This modulation was suppressed by the administration of (R)‐α‐methylhistamine and occurred in both the caudate–putamen and nucleus accumbens. Ciproxifan strongly potentiated the decrease of proenkephalin mRNA expression induced by methamphetamine. In contrast, it suppressed the increase in prodynorphin and substance P mRNA expression induced by methamphetamine. Methamphetamine alone or with ciproxifan did not modify proneurotensin mRNA expression. These neurochemical findings indicate that ciproxifan differentially regulates the effect of methamphetamine on the neuropeptides contained in striatonigral and striatopallidal neurons. They suggest that endogenous histamine and dopamine cooperate to modulate the activity of striatal projection neurons and strengthen the interest of H3‐receptors as new targets for the treatment of psychotic disorders and drug abuse.
Subpial neuronal migration in the medulla oblongata of Pax‐6‐deficient rats
Authors: Horie Masao , Sango Kazunori , Takeuchi Kosei , Honma Shizuka , Osumi Noriko , Kawamura Koki , Kawano Hitoshi
Published: March 2003

In rat fetuses, neurons generated in the lower rhombic lip on embryonic day 14 (E14) specifically express the transcription factor Pax‐6 and migrate circumferentially in the subpial region along axons which are immunoreactive for TAG‐1, a neural cell adhesion molecule. These neurons ultimately settle from E17 in the contralateral medulla oblongata to form the precerebellar nuclei, the lateral reticular and external cuneate nuclei. We have examined this migratory process in rat small eye (rSey2) which has a mutation in the Pax‐6 gene. Both the migration and settlement of neurons labelled with 5‐bromo‐2′‐deoxyuridine (BrdU) at E14 delayed in the mutant. Furthermore, in the ventrolateral part of the E16 mutant medulla oblongata, cells both expressing Pax‐6 mRNA and prelabelled with BrdU at E14 were ectopically localized in the deep zone of the medullary parenchyma in addition to the normal subpial location. These ectopic neurons remained in close contact with ectopic TAG‐1‐immunoreactive axons. These results indicate that Pax‐6 plays a role in the migration of medullary precerebellar neurons, although neurons generated in the lower rhombic lip can nevertheless migrate and settle to form the external cuneate nucleus in the absence of Pax‐6.
The mouse VPAC2 receptor confers suprachiasmatic nuclei cellular rhythmicity and responsiveness to vasoactive intestinal polypeptide in vitro
Authors: Cutler David J. , Haraura Mai , Reed Helen E. , Shen Sanbing , Sheward W. John , Morrison Christine F. , Marston Hugh M. , Harmar Anthony J. , Piggins Hugh D.
Published: March 2003

Expression of coherent and rhythmic circadian (≈ 24 h) variation of behaviour, metabolism and other physiological processes in mammals is governed by a dominant biological clock located in the hypothalamic suprachiasmatic nuclei (SCN). Photic entrainment of the SCN circadian clock is mediated, in part, by vasoactive intestinal polypeptide (VIP) acting through the VPAC2 receptor. Here we used mice lacking the VPAC2 receptor (Vipr2−/−) to examine the contribution of this receptor to the electrophysiological actions of VIP on SCN neurons, and to the generation of SCN electrical firing rate rhythms SCN in vitro. Compared with wild‐type controls, fewer SCN cells from Vipr2−/− mice responded to VIP and the VPAC2 receptor‐selective agonist Ro 25‐1553. By contrast, similar proportions of Vipr2−/− and wild‐type SCN cells responded to gastrin‐releasing peptide, arginine vasopressin or N‐methyl‐d‐aspartate. Moreover, VIP‐evoked responses from control SCN neurons were attenuated by the selective VPAC2 receptor antagonist PG 99‐465. In firing rate rhythm experiments, the midday peak in activity observed in control SCN cells was lost in Vipr2−/− mice. The loss of electrical activity rhythm in Vipr2−/− mice was mimicked in control SCN slices by chronic treatment with PG 99‐465. These results demonstrate that the VPAC2 receptor is necessary for the major part of the electrophysiological actions of VIP on SCN cells in vitro, and is of fundamental importance for the rhythmic and coherent expression of circadian rhythms governed by the SCN clock. These findings suggest a novel role of VPAC2 receptor signalling, and of cell‐to‐cell communication in general, in the maintenance of core clock function in mammals, impacting on the cellular physiology of SCN neurons.
PACAP and NGF regulate common and distinct traits of the sympathoadrenal lineage: effects on electrical properties, gene markers and transcription factors in differentiating PC12 cells
Authors: Grumolato Luca , Louiset Estelle , Alexandre David , Aït‐Ali Djida , Turquier Valérie , Fournier Alain , Fasolo Aldo , Vaudry Hubert , Anouar Youssef
Published: March 2003

To determine the possible role of pituitary adenylate cyclase‐activating polypeptide (PACAP) in the development of the sympathoadrenal cell lineage, we have examined the effects of this neurotrophic peptide, in comparison to nerve growth factor (NGF), on the morphology, electrophysiological properties, expression of neuronal and neuroendocrine marker genes, and activity of transcription factors during differentiation of sympathoadrenal‐derived cells, using the rat pheochromocytoma PC12 cell model. Both PACAP and NGF elicited rapid neurite outgrowth, which was accompanied by induction of cell excitability and the development of both sodium and calcium currents. Concurrently, PACAP and NGF increased the expression of a marker of synaptic vesicles. By contrast, PACAP, but not NGF, regulated the expression of different constituents of neuroendocrine large dense core vesicles in PC12 cells. Furthermore, PACAP and NGF differentially regulated the expression of mammalian achaete‐scute homologue and paired homeobox 2b genes, transcription factors instrumental for sympathoadrenal development. To compare downstream effectors activated by PACAP and NGF, we studied the effects of these factors on the binding activity of consensus 12‐O‐tetradecanoylphorbol‐13‐acetate‐ and cAMP‐responsive elements to nuclear extracts of differentiating PC12 cells. We found that both PACAP and NGF markedly increase the binding activity of these cis‐regulatory sequences and that PACAP preferentially recruits activator protein‐1‐like transcription factors to these elements. Taken together, these results show that PACAP and NGF exert common as well as different effects on neuronal and neuroendocrine traits in differentiating PC12 cells, strongly suggesting that these two trophic factors could play complementary roles in the development of the sympathoadrenal cell lineage.
Developmental and circadian changes in Ca2+ mobilization mediated by GABAA and NMDA receptors in the suprachiasmatic nucleus
Authors: Ikeda Masayuki , Yoshioka Tohru , Allen Charles N.
Published: March 2003

The hypothalamic suprachiasmatic nucleus (SCN) develops as the circadian pacemaker during postnatal life. Although both GABAA and NMDA receptors are expressed in the majority of SCN neurons, postnatal development of their functions has not been analysed. Thus, we studied the receptor‐mediated Ca2+ responses in mouse hypothalamic slices prepared on postnatal days (P) 6–16. The NMDA‐induced Ca2+ flux was prominent in the SCN and maximal Ca2+ responses in Mg2+‐free conditions had no day–night variations in P14–16 mice. At P6–7, extracellular Mg2+ reduced the NMDA‐induced Ca2+ flux irrespective of the circadian time whereas, after P9–10, Mg2+ produced a larger reduction at night than during the daytime. Muscimol also significantly increased Ca2+ in the developing SCN. Voltage‐sensitive Ca2+ channel blockers inhibited the muscimol‐induced Ca2+ increase whereas tetrodotoxin had no effect, suggesting that stimulation of postsynaptic GABAA receptors depolarizes SCN neurons to increase Ca2+. Macroscopic imaging analysis demonstrated a developmental reduction in the muscimol‐induced Ca2+ increase preferentially in the nighttime group older than P9–10. The day–night variation in the magnitude of the Ca2+ response was due to two cell populations, one of which exhibited an increase and the other a decrease in Ca2+ in response to muscimol. Because the critical developmental stages for exhibiting day–night variations in the receptor‐mediated Ca2+ responses overlapped the maturation of firing rhythms in SCN neurons, the Ca2+ signalling may be necessary for or regulated by the mature circadian clock.
Modulation of ACh release by presynaptic muscarinic autoreceptors in the neuromuscular junction of the newborn and adult rat
Authors: Santafé Manel M. , Salon Isabel , Garcia Neus , Lanuza M. Angel , Uchitel Osvaldo D. , Tomàs Josep
Published: March 2003

We studied the presynaptic muscarinic autoreceptor subtypes controlling ACh release and their relationship with voltage‐dependent calcium channels in the neuromuscular synapses of theLevator auris longusmuscle from adult (30–40 days) and newborn (3–6 and 15 days postnatal) rats. Using intracellular recording, we studied how several muscarinic antagonists affected the evoked endplate potentials. In some experiments we previously incubated the muscle with calcium channel blockers (nitrendipine, ω‐conotoxin‐GVIA and ω‐Agatoxin‐IVA) before determining the muscarinic response. In the adult, the M1 receptor‐selective antagonist pirenzepine (10 µm) reduced evoked neurotransmission (≈ 47%). The M2 receptor‐selective antagonist methoctramine (1 µm) increased the evoked release (≈ 67%). Both M1‐ and M2‐mediated mechanisms depend on calcium influx via P/Q‐type synaptic channels. We found nothing to indicate the presence of M3 (4‐DAMP‐sensitive) or M4 (tropicamide‐sensitive) receptors in the muscles of adult or newborn rats. In the 3–6‐day newborn rats, pirenzepine reduced the evoked release (≈ 30%) by a mechanism independent of L‐, N‐ and P/Q‐type calcium channels, and the M2 antagonist methoctramine (1 µm) unexpectedly decreased the evoked release (≈ 40%). This methoctramine effect was a P/Q‐type calcium‐channel‐dependent mechanism. However, upon maturation in the first two postnatal weeks, the M2 pathway shifted to perform the calcium‐dependent release‐inhibitory activity found in the adult. We show that the way in which M1 and M2 muscarinic receptors modulate neurotransmission can differ between the developing and adult rat neuromuscular synapse.
Glutamate transporters in the guinea‐pig cochlea: partial mRNA sequences, cellular expression and functional implications
Authors: Rebillard G. , Ruel J. , Nouvian R. , Saleh H. , Pujol R. , Dehnes Y. , Raymond J. , Puel J. L. , Devau G.
Published: March 2003

In the cochlea, glutamate plays a major role in synaptic transmission between the inner hair cell and the primary auditory neurons. Extracellular glutamate concentration must be regulated to prevent excitotoxicity. This regulation is mediated by excitatory amino acid transporters, membrane proteins that remove glutamate from the synaptic cleft. In this study, we investigated the distribution and activity of three excitatory amino acid transporters subtypes in the guinea‐pig cochlea: glutamate aspartate transporter, glutamate transporter and excitatory amino acid carrier. A partial messenger ribonucleic acid sequence was determined for each of these transporters, by polymerase chain reaction with degenerate primers, using guinea‐pig brain complementary deoxyribonucleic acid as the template. Primers specific for each transporter were then designed and used to screen a dissected organ of Corti complementary deoxyribonucleic acid library. The cellular distribution of each transporter was examined by immunocytochemistry. We investigated the functional consequences of inhibiting glutamate uptake by recording cochlear potentials during intracochlear perfusion with either l‐trans‐pyrrolidine‐2,4‐dicarboxylic acid or dihydrokainate. At the end of the electrophysiological session, cochleas were processed for electron microscopy. Only the glutamate aspartate transporter messenger ribonucleic acid was detected in the organ of Corti. Consistently, glutamate aspartate transporter protein was detected in the inner hair cell‐supporting cells and in the ganglion of Corti satellite cells. Glutamate transporter and excitatory amino acid carrier were found in the afferent auditory neurons. Only intracochlear perfusions with l‐trans‐pyrrolidine‐2,4‐dicarboxylic acid resulted in a dose‐dependent decrease in the amplitude of the cochlear compound action potential, leaving cochlear microphonic potential unaffected. After l‐trans‐pyrrolidine‐2,4‐dicarboxylic acid perfusion, cochleas displayed a swelling of the afferent endings typical of excitotoxicity. [(–)1‐(4‐aminophenyl)‐4‐methyl‐7,8‐methylenedioxy‐4,5‐dihydro‐3‐methylcarbamyl‐2,3‐benzodiazepine], a selective α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid receptor antagonist protects the cochlea against l‐trans‐pyrrolidine‐2,4‐dicarboxylic acid effect.
Patterns of calcium‐binding proteins support parallel and hierarchical organization of human auditory areas
Authors: Chiry Oriana , Tardif Eric , Magistretti Pierre J. , Clarke Stephanie
Published: March 2003

The human primary auditory cortex (AI) is surrounded by several other auditory areas, which can be identified by cyto‐, myelo‐ and chemoarchitectonic criteria. We report here on the pattern of calcium‐binding protein immunoreactivity within these areas. The supratemporal regions of four normal human brains (eight hemispheres) were processed histologically, and serial sections were stained for parvalbumin, calretinin or calbindin. Each calcium‐binding protein yielded a specific pattern of labelling, which differed between auditory areas. In AI, defined as area TC [see C. von Economo and L. Horn (1930) Z. Ges. Neurol. Psychiatr.,130, 678–757], parvalbumin labelling was dark in layer IV; several parvalbumin‐positive multipolar neurons were distributed in layers III and IV. Calbindin yielded dark labelling in layers I–III and V; it revealed numerous multipolar and pyramidal neurons in layers II and III. Calretinin labelling was lighter than that of parvalbumin or calbindin in AI; calretinin‐positive bipolar and bitufted neurons were present in supragranular layers. In non‐primary auditory areas, the intensity of labelling tended to become progressively lighter while moving away from AI, with qualitative differences between the cytoarchitectonically defined areas. In analogy to non‐human primates, our results suggest differences in intrinsic organization between auditory areas that are compatible with parallel and hierarchical processing of auditory information.
Expression of the circadian clock gene Period 1 in neuroendocrine cells: an investigation using mice with a Per1::GFP transgene
Authors: Kriegsfeld Lance J. , Korets Ruslan , Silver Rae
Published: March 2003

The circadian clock located in the suprachiasmatic nuclei (SCN) of the hypothalamus regulates daily temporal organization in behaviour and neuroendocrine function. The molecular basis for circadian rhythm generation is an interacting transcriptional/translational feedback loop comprised of several ‘clock genes’ and their respective protein products. Clock genes are expressed not only in the SCN but also in numerous other locations throughout the brain, including regions rich in neuroendocrine cells. In order to investigate whether neuroendocrine cells function as autonomous oscillators, we used female transgenic mice in which an unstable, degradable jellyfish green fluorescent protein (GFP) gene is driven by a mouse Period 1 (Per1) gene promoter. Mice were injected (s.c.) with fluorogold (FG) in order to label neuroendocrine cells and brain sections were double‐labelled for either FG and Per1 mRNA (labelled by GFP immunostaining) or FG and PER1 protein using fluorescence immunocytochemistry. Mice were killed during either the day or night. Neuroendocrine cells contained Per1 mRNA and PER1 protein in several brain regions with the greatest proportion of double‐labelled cells occurring in the arcuate nucleus (Arc). The number of neuroendocrine cells labelled was not affected by the stage of the estrous cycle. Fewer FG‐labelled cells expressed Per1 message and protein during the night compared to the day. In the Arc, staining for tyrosine hydroxylase revealed that neuroendocrine cells expressing Per1 message and protein were dopaminergic. Together, these findings suggest that neuroendocrine cells contain the molecular machinery necessary to oscillate independently. It remains to be determined whether these cells actually function as autonomous oscillators or whether these rhythms are driven by signals from the SCN. These findings also indicate that the endocrine system represents an opportunity to study the interactions between central (SCN and neuroendocrine cells) and peripheral circadian (endocrine gland) oscillators.
Neurogenesis in explants from the walls of the lateral ventricle of adult bovine brain: role of endogenous IGF‐1 as a survival factor
Authors: Pérez‐Martín M. , Cifuentes M. , Grondona J. M. , J. Bermúdez‐Silva F. , Arrabal P. M. , Pérez‐Fígares J. M. , Jiménez A. J. , García‐Segura L. M. ,   P. Fernández‐Llebrez
Published: March 2003

Previous studies have shown the existence of proliferating cells in explants from bovine (Bos Taurus) lateral ventricle walls that were maintained for several days in vitro in the absence of serum and growth factors. In this study we have characterized the nature of new cells and have assessed whether the insulin‐like growth factor‐1 (IGF‐1) receptor regulates their survival and/or proliferation. The explants were composed of the ependymal layer and attached subependymal cells. Ependymal cells in culture were labelled with glial markers (S‐100, vimentin, GFAP, BLBP, 3A7 and 3CB2) and did not incorporate bromodeoxiuridine when this molecule was added to the culture media. Most subependymal cells were immunoreactive for βIII‐tubulin, a neuronal marker, and did incorporate bromodeoxiuridine. Subependymal neurons displayed immunoreactivity for IGF‐1 and its receptor and expressed IGF‐1 mRNA, indicating that IGF‐1 is produced in the explants and may act on new neurons. Addition to the culture media of an IGF‐1 receptor antagonist, the peptide JB1, did not affect the incorporation of bromodeoxiuridine to proliferating subependymal cells. However, JB1 significantly increased the number of TUNEL positive cells in the subependymal zone, suggesting that IGF‐1 receptor is involved in the survival of subependymal neurons. In conclusion, these findings indicate that neurogenesis is maintained in explants from the lateral cerebral ventricle of adult bovine brains and that IGF‐1 is locally produced in the explants and may regulate the survival of the proliferating neurons.
Blockade of HMG‐CoA reductase activity causes changes in microtubule‐stabilizing protein tau via suppression of geranylgeranylpyrophosphate formation: implications for Alzheimer's disease
Authors: Meske V. , Albert F. , Richter D. , Schwarze J. , Ohm T. G.
Published: March 2003

Histopathologically, Alzheimer's disease is characterized by plaques and tangles that develop progressively over time. Experimental data described a statin‐induced decrease in β‐amyloid production, a major constituent of the plaques. Others reported data on statin‐mediated changes in neuronal survival and cytoskeleton, including the microtubule‐associated protein tau, a major constituent of the tangles. However, these latter reports remain contradictory. To clarify and extend our knowledge on the effect of statin on the cytoskeleton, we challenged rat primary neuron cultures by lovastatin and determined the metabolite that is critical for structural integrity and survival of neurons. During the blockade of 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase, the neuritic network was affected and eventually was completely destroyed. This process was not part of the execution phase of apoptosis and was marked by alterations in the microfilament and microtubule system. The distribution and phosphorylation of protein tau changed. Immunoblot analysis and indirect immunofluorescence revealed a transient increase in tau phosphorylation, which ceased during the execution of apoptosis. All of these effects could be linked to the lack of the geranylgeranylpyrophosphate intermediate. Inhibition of the geranylgeranylation of Rho family GTPases (geranylgeranyl‐transferase I) evoked similar changes in neurons. These data and our findings that statin treatment reduced the membrane‐bound fraction of RhoA‐GTPase in neurons suggest that reduced levels of functional small G proteins are responsible for the observed effects. Our data demonstrate that lovastatin concentrations able to suppress not only cholesterol but also geranylgeranylpyrophosphate formation may evoke phosphorylation of tau reminiscent of preclinical early stages of Alzheimer's disease and, when prolonged, apoptosis.
Regulation of tyrosine hydroxylase levels and activity and Fos expression during opioid withdrawal in the hypothalamic PVN and medulla oblongata catecholaminergic cell groups innervating the PVN
Authors: Benavides Marta , Laorden M. Luisa , García‐Borrón J. Carlos , Milanés M. Victoria
Published: March 2003

Morphine withdrawal increases the hypothalamic–pituitary–adrenocortical (HPA) axis activity, which is dependent on an hyperactivity of noradrenergic pathways innervating the hypothalamic paraventricular nucleus (PVN). However, the possible adaptive changes that can occur in these pathways during morphine dependence are not known. We studied the alterations in tyrosine hydroxylase (TH; the rate‐limiting enzyme in catecholamines biosynthesis) immunoreactivity levels and TH enzyme activity in the rat NTS‐A2/VLM‐A1 noradrenergic cell groups and in the PVN during morphine withdrawal. In the same paradigm, we measured Fos expression as a marker of neuronal activation. TH and Fos immunoreactivity was determined by quantitative Western blot analysis, combined with immunostaining for TH and Fos for immunohistochemical identification of active neurons during morphine withdrawal. Dependence on morphine was induced by a 7‐day s.c. implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by an injection of naloxone (5 mg/kg s.c.). Morphine withdrawal induced the expression of Fos in the PVN and NTS/VLM, which indicates an activation of neurons in these nuclei. TH immunoreactivity in the NTS/VLM was increased 90 min after morphine withdrawal, whereas there was a decrease in TH levels in the PVN at the same time point. Following withdrawal, Fos immunoreactivity was present in most of the TH‐positive neurons of the A2 and A1 neurons. TH activity was measured in the PVN, a projection area of noradrenergic neurons arising from NTS‐A2/VLM‐A1. Morphine withdrawal was associated with an increase in the enzyme activity at different time points after naloxone‐precipitated morphine withdrawal. The present results suggest that an increase in TH protein levels and TH enzyme activity might contribute to the enhanced noradrenergic activity in the PVN in response to morphine withdrawal.
Impact of high and low anxiety on cognitive performance in a modified hole board test in C57BL/6 and DBA/2 mice
Authors: Ohl Frauke , Roedel Angelika , Binder Elke , Holsboer Florian
Published: March 2003

We investigated the interaction between behavioural dimensions and cognitive performance in the inbred mouse strains C57BL/6 and DBA/2, which have previously been found to differ in cognitive performance and emotionality. Because it has never been evaluated whether cognitive performance and emotional behaviour are interrelated in these strains, we analysed various behavioural dimensions and cognitive functions in parallel using the modified hole board test. We could show that naive BL6 and DBA mice distinctly differed in terms of anxiety‐related behaviour. Principal component analysis on the phenotyping data showed that anxiety‐related behaviour was described by identical parameters and was not correlated to locomotion in the two strains. During cognitive testing, DBA mice habituated faster and performed better than BL6 mice. Principal component analysis indicated a close correlation between anxiety‐related behaviour and cognitive performance in DBA mice, being associated with a highly successful cognitive performance. In BL6 mice, cognition was correlated to general exploration. This correlation turned out to be less successful in performing the modified hole board test. Our findings support the idea that high anxiety may interact with specific cognitive processing, thus offering a promising animal model for future preclinical research on the interaction of anxiety and cognition.
5‐HT1B receptor knockout mice show a compensatory reduction in 5‐HT2C receptor function
Authors: Clifton Peter G. , Lee Michelle D. , Somerville Elizabeth. M. , Kennett Guy A. , Dourish Colin T.
Published: March 2003

Although null mutant (‘knockout’) mice have provided valuable animal models to complement traditional approaches to psychopharmacology, such animals may also show complex adaptations to the induced mutation. Here we demonstrate that serotonin1B (5‐HT1B) receptor knockout (KO) mice show adaptations in serotonin2C (5‐HT2C) receptor‐mediated functions. They show smaller reductions in food intake and locomotor activity in response to administration of 5‐HT2C receptor agonists that are not accounted for by altered drug disposition. These effects are not mimicked by pretreatment of wildtype (WT) mice with a 5‐HT1B receptor antagonist showing that they result from a longer term adaptation to the loss of 5‐HT1B receptor function and not from a short‐term interaction between 5‐HT1B‐ and 5‐HT2C‐mediated functions. In addition, we show that 5‐HT1B receptor KO mice have a lowered hypothalamic c‐fos response to the administration of 5‐HT2C receptor agonists. These results demonstrate that compensatory adaptations to the constitutive loss of 5‐HT1B receptors may be an important determinant of the altered response of 5‐HT1B KO mice to a variety of pharmacological challenges.
Grasp observation influences speech production
Authors: Gentilucci Maurizio
Published: March 2003

Subjects pronounced either the syllable ‘BA’ or ‘GA’ while observing motor acts of hand grasp directed to objects of two sizes (experiment 1). Kinematics of lip aperture and amplitude spectrum of voice were influenced by the observation of the different grasp kinematics depending on the size of the target objects. Specifically, both lip aperture and voice peak amplitude were greater when the observed hand grasp was directed to the large object. Two control experiments ruled out that the different arm velocity when reaching objects varying in size (experiment 2), and overt visual analysis of the target‐object (experiment 3), affected lip movement and voice emission. Results provide behavioural evidence in favour of the hypothesis that the system involved in observation (and preparation) of grasp movements partially shares the cortical areas involved in speech production.
Increase of morphine withdrawal in mice lacking A2a receptors and no changes in CB1/A2a double knockout mice
Authors: Berrendero Fernando , Castañé Anna , Ledent Catherine , Parmentier Marc , Maldonado Rafael , Valverde Olga
Published: March 2003

CB1 cannabinoid and A2a adenosine receptors are highly expressed in the central nervous system where they modulate numerous physiological processes including emotional behaviour and the responses of several drugs of abuse. To investigate the contribution of these receptors in emotional‐like responses and opioid dependence we have generated CB1/A2a double deficient mice (\mathrm{CB}^{‐/‐}_{1}/\mathrm{A}^{‐/‐}_{2\mathrm{a}}). The spontaneous locomotor activity was reduced in double knockout as compared to wild‐type animals. Emotional‐like responses of \mathrm{CB}^{‐/‐}_{1}/\mathrm{A}^{‐/‐}_{2\mathrm{a}} mice were investigated using the elevated plus‐maze and the lit‐dark box. Mutant mice exhibited an increased level of anxiety in both behavioural models. The specific involvement of CB1 and A2a receptors in morphine dependence was evaluated by using A2a knockout mice and CB1/A2a double mutant mice. The severity of naloxone‐precipitated morphine withdrawal syndrome was significantly increased in the absence of A2a adenosine receptors whereas no modifications were observed in the double knockout mice. These results suggest that both receptors participate in the control of emotional behaviour and seem to play an opposite role in the expression of opioid physical dependence.