bluuebunny/crossref_metadata_2025 huggingface dataset:
Heat shock proteins in retinal neurogenesis: identification of the PM1 antigen as the chick Hsc70 and its expression in comparison to that of other chaperones
Authors: Morales Aixa V. , Hadjiargyrou Michael , Díaz Begoña , Hernández‐Sánchez Catalina , De Pablo Flora , De La Rosa Enrique J.
Published: March 2003

While the role of heat shock proteins under experimental stress conditions is clearly characterized, their expression in unstressed cells and tissues and their functions in normal cell physiology, besides their chaperone action, remain largely undetermined. We report here the identification in chicken of the antigen recognized by the monoclonal antibody PM1 [Hernández‐Sánchez et al. (1994) Eur. J. Neurosci.6,1801–1810) as the non‐inducible chaperone heat‐shock cognate 70 (Hsc70). Its identity was determined by partial peptide sequencing, immuno‐crossreactivity & two‐dimensional gel‐electrophoresis. In addition, we examined its expression during chick embryo retinal neurogenesis. The early widespread Hsc70 immunostaining corresponding to most, if not all, of the neuroepithelial cells becomes restricted to a subpopulation of these cells in the peripheral retina as development proceeds. On the other hand, retinal ganglion cells, differentiating in the opposite central‐to‐peripheral gradient, retained Hsc70 immunostaining. Other molecular chaperones, the heat‐shock proteins Hsp40, Hsp60 & Hsp90, did not seem to compensate the loss of Hsc70. They also showed decreasing immunostaining patterns as neurogenesis proceeds, although distinctive from that of Hsc70, whereas Hsp70 was not detected in the embryonic retina. This precise cellular & developmental regulation of Hsc70, a generally considered constitutive molecular chaperone, in unstressed embryos, together with the expression of other chaperones, provides new tools & a further insight on neural precursor heterogeneity & suggests possible specific cellular roles of chaperone function during vertebrate neurogenesis.
Systemic harmaline blocks associative and motor learning by the actions of the inferior olive
Authors: Welsh John P.
Published: March 2003

Is there a role for the inferior olive in learning? Novel paradigms of conditioning involving tongue protrusion were developed using the rat to test whether: (a) the indole alkaloid harmaline blocks associative learning via actions within the inferior olive, and (b) the inferior olive is required for associative and motor learning. Harmaline blocked associative learning as measured by the absence of conditioned responses to a tone over six daily sessions of conditioning and the absence of retention without harmaline. Harmaline's effect on associative learning was completely blocked by prior removal of the inferior olive with 3‐acetylpyridine. Rats whose inferior olives were chronically lesioned showed normal associative learning, normal associative memory, and could learn to modify tongue protrusion via a motor learning paradigm involving response shaping. Removal of the inferior olive degraded the performance of the licking motor system by increasing the latency of conditioned tongue protrusions and by increasing the temporal variability of rhythmic licking elicited by intraoral water. The experiments raise doubt as to whether the inferior olive encodes memory in the cerebellum but demonstrate that the inferior olive is essential for the temporal precision of movement. The results indicate that harmaline's antilearning action is produced by its ability to exaggerate the normal propensity of olivary neurons to fire rhythmically, a process that must be constrained under physiological conditions for normal learning to occur. It is concluded that there may be an important role for the rhythmic activity of inferior olivary neurons in the temporal processes that underlie both motor control and learning.
The murine Y1 receptor 5′ upstream sequence directs cell‐specific and developmentally regulated LacZ expression in transgenic mice CNS
Authors: Oberto Alessandra , Tolosano Emanuela , Brusa Rossella , Altruda Fiorella , Panzica Giancarlo , Eva Carola
Published: March 2003

The Y1 receptor for neuropeptide Y (NPY) is highly expressed in mammalian CNS where it mediates the activation of several neurobiological functions. We have previously demonstrated that a 1.3‐kb fragment upstream of the transcription initiation sites of the murine Y1 receptor gene is able to direct specific expression of reporter genes in neuronal cell cultures. In the present study transgenic mice harbouring this putative promoter region linked to the LacZ reporter gene were generated and analysed for temporal and spatial distribution. Ten transgenic lines expressed β‐galactosidase in the CNS but not in other organs such as heart, liver and kidney. Histochemical analysis of brain from adult transgenic mice showed specific expression of the transgene in specific brain regions with little variation. Four transgenic lines showed characteristic patterns of β‐galactosidase activity in the brain that are consistent with the expression of the endogenous gene. Prominent LacZ activity was present in several telencephalic and diencephalic structures, including deeper layers of cerebral cortex, amygdaloid complex, hippocampus, preoptic area, several thalamic and hypothalamic nuclei and habenula. The ontogeny analysis indicates that the LacZ expression agrees with the temporal expression pattern of rat Y1 receptor mRNA. These data demonstrate that the 1.3‐kb upstream region of the murine Y1 receptor gene contains the cis acting elements required for establishing a CNS‐restricted and developmental stage‐specific pattern of expression in vivo. Moreover they provide further information on the distribution of this NPY subtype receptor in mammalian brain.
Endogenous activation of metabotropic glutamate receptors contributes to burst frequency regulation in the lamprey locomotor network
Authors: Krieger P. , Grillner S. , Manira A. El
Published: March 2003

The effect of metabotropic glutamate receptor (mGluR) agonists and antagonists on the spinal cord network underlying locomotion in the lamprey has been analysed. The specific group I mGluR agonist (R,S)‐3,5‐dihydroxyphenylglycine (DHPG) and the broad‐spectrum mGluR agonist (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (1S,3R‐ACPD) both increased the burst frequency of N‐methyl‐d‐aspartic acid (NMDA)‐induced fictive locomotion and depolarized grey matter neurons. The burst frequency increase induced by the mGluR agonists was counteracted by the mGluR antagonists (+)‐alpha‐methyl‐4‐carboxyphenylglycine

((+)‐MCPG), cyclopropan[b]chromen‐1a‐carboxylic acid ethylester (CPCCOEt) and (RS)‐1‐aminoindan‐1,5‐dicarboxylic acid (AIDA). Application of CPCCOEt alone reduced the locomotor burst frequency, indicating that mGluRs are endogenously activated during fictive locomotion. The mGluR antagonist CPCCOEt had no effect on NMDA‐, or (S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoazolepropionic acid (AMPA)‐induced depolarizations. The mGluR agonists 1S,3R‐ACPD and DHPG increased the amplitude of NMDA‐induced depolarizations, a mechanism which could account for the increase in burst frequency. The group III mGluR agonist L‐2‐amino‐4‐phosphonobutyric acid reduced intraspinal synaptic transmission and burst frequency.
NMDA and AMPA receptors on neocortical neurons are differentially distributed
Authors: Dodt H. ‐U. , Frick A. , Kampe K. , Zieglgänsberger W.
Published: March 2003

The distribution of glutamate receptor subtypes on the surface of neurons is highly relevant for synaptic activation and signal processing in the neocortex. As a novel approach we have used infra‐red videomicroscopy in combination with photostimulation or microiontophoresis in brain slices of rat neocortex to map the distribution of N‐methyl‐d‐aspartate (NMDA) and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptors on pyramidal neurons of layer V. Both modes of application revealed a spatially distinct distribution of glutamate receptor subtypes: the soma and the proximal dendrite of neurons are highly sensitive to NMDA, whereas the more distal parts of the dendrite are more sensitive to AMPA. An implication is that NMDA receptors near the soma might regulate the amplification of synaptic signals resulting from AMPA receptor activation on remote dendritic sites.
Delivery of a constitutively active form of the heat shock factor using a virus vector protects neuronal cells from thermal or ischaemic stress but not from apoptosis
Authors: Wagstaff M. J. D. , Smith J. , Collaco‐Moraes Y. , De Belleroche J. S. , Voellmy R. , Coffin R. S. , Latchman D. S.
Published: March 2003

The heat shock proteins (HSPs) are induced by stressful stimuli and have a protective effect. Different HSPs protect with different efficiencies against different stresses indicating that optimal protection would be obtained with a non‐stressful agent which induced a range of HSPs. We have prepared a herpesvirus vector expressing a constitutively active mutant form of heat shock factor 1 (HSF1) which, unlike the wild‐type form of this transcription factor, does not require stress for its activation. Upon infection of neuronal cells, this virus induced a more restricted range of HSPs than in non‐neuronal cells. Infection with the virus protected neuronal cells against subsequent thermal or ischaemic stress in accordance with its ability to induce HSP70 expression but did not protect them against apoptotic stimuli. The mechanisms of these effects and their significance for the use of HSF to manipulate HSP gene expression is discussed.
Pharmacological stimuli decreasing nucleus accumbens dopamine can act as positive reinforcers but have a low addictive potential
Authors: Marinelli Michela , Barrot Michel , Simon Hervé , Oberlander Claude , Dekeyne Anne , Moal Michel Le , Piazza Pier Vincenzo
Published: March 2003

Opioid peptides, through μ and δ receptors, play an important part in reward. In contrast, the role of κ receptors is more controversial. We examined the possible positive reinforcing effects of a selective κ agonist, RU 51599, by studying intravenous self‐administration in the rat. The effect of RU 51599 on dopamine release in the nucleus accumbens was also studied, as opioids and dopamine seem to interact in the mediation of reward. The behavioural and dopaminergic effects of RU 51599 were compared with those of the μ agonist heroin. Rats self‐administered both RU 51599 (6.5, 20 and 60 μg/inj) and heroin (30 μg/inj) at low ratio requirement. When the ratio requirement, i.e. the number of responses necessary to receive one drug infusion, was increased, self‐administration of RU 51599 rapidly extinguished, whereas self‐administration of heroin was maintained. Intravenous infusion of RU 51599 (100, 200 and 400 μg) dose‐dependently decreased (25, 30 and 40%, respectively) extracellular concentrations of dopamine, as measured by means of microdialysis in freely moving rats. In contrast, heroin increased accumbens dopamine (130% over baseline). These results indicate that κ receptors, similarly to μ ones, can mediate positive reinforcing effects of opioid peptides. However, the strength of the reinforcement is very low for κ receptors. This suggests that changes in accumbens dopamine do not correlate with the capacity of a stimulus to induce reward or aversion. In contrast, a parallel seems to exist between an increase in accumbens dopamine and the drive to reach or obtain a positive reinforcer.
Dual effects of botulinum neurotoxin A on the secretory stages of chromaffin cells
Authors: Gil Anabel , Viniegra Salvador , Gutiérrez Luis M.
Published: March 2003

Truncation of the C‐terminal domain of the synaptosomal associated protein of 25 kDa (SNAP‐25) by botulinum neurotoxin A (BoNT A) has been shown to block neuroendocrine cell secretion. It is unclear, however, if toxin mechanism involved the affection of a single or more events of the exocytotic cascade. BoNT A induced changes in both the degree of inhibition and the kinetics of catecholamine secretion from populations of cultured bovine chromaffin cells. Ca2+‐dependent secretion from digitonin‐permeabilized cells showed partial inhibition associated with the alteration of a slow secretory phase at different toxin concentrations. In contrast, in intact cells stimulated by depolarization, cell treatment with low concentrations (1 nm) of the toxin affected the late phase of secretion, whereas 100 nm BoNT A‐poisoned cells showed an alteration even of fast components. The high degree of inhibition associated with fast secretory component alteration was dependent on Ca2+ entry through the Ca2+ channels, as it was absent from cells permeated with the A23187 Ca2+ ionophore. Vesicle pools implicated in the effect of BoNT A on the secretory response from single cells were identified using amperometry. These studies supported the macroscopic view by showing that secretion from BoNT A‐treated permeabilized cells presented specific inhibition of late vesicle fusions. Intact cells showed alterations in the late vesicle pool (t = 39 s) recruited during prolonged or repetitive KCl depolarizations using 1 nm BoNT A‐treated cells as well as in an intermediate kinetic pool (t = 18 s) at higher toxin concentrations (100 nm). The faster resolved component (t = 8 s) or the membrane fusion event itself were not affected. Our results demonstrate that removal of the last nine C‐terminal amino acids of SNAP‐25 by BoNT A has a specific effect on two different and distal secretory stages in chromaffin cells.
Intracellular calcium stores modulate miniature GABA‐mediated synaptic currents in neonatal rat hippocampal neurons
Authors: SaviĆ Nataša , Sciancalepore Marina
Published: March 2003

The whole‐cell configuration of the patch clamp technique was used to record miniature γ‐aminobutyric acidA (GABAA) receptor‐mediated currents (in tetrodotoxin, 1 μm and kynurenic acid 1 mm) from CA3 pyramidal cells in thin hippocampal slices obtained from postnatal (P) day (P6–9) old rats. Switching from a Ca2+‐containing to a nominally Ca2+‐free medium (in which Ca2+ was substituted with Mg2+, in the presence or in the absence of 100 μm EGTA) did not change significantly the frequency or amplitude of miniature events. Superfusion of thapsigargin induced a concentration‐dependent increase in frequency but not in amplitude of tetrodotoxin‐resistant currents that lasted for the entire period of drug application. Mean frequency ratio (thapsigargin 10 μm over control) was 1.8 ± 0.5, (n = 9). In nominally Ca2+‐free solutions thapsigargin was ineffective. When bath applied, caffeine (10 mm), reversibly reduced the amplitude of miniature postsynaptic currents whereas, if applied by brief pressure pulses, it produced an increase in frequency but not in amplitude of spontaneous GABAergic currents. Superfusion of caffeine (10 mm) reversibly reduced the amplitude of the current induced by GABA (100 μm) indicating a clear postsynaptic effect on GABAA receptor. Superfusion of ryanodine (30 μm), in the majority of the cells (n = 7) did not significantly modify the amplitude or frequency of miniature events. In two of nine cells it induced a transient increase in frequency of miniature postsynaptic currents. These results indicate that in neonatal hippocampal neurons, mobilization of calcium from caffeine–ryanodine‐sensitive stores facilitates GABA release.
Psychotomimetic‐induction of tissue plasminogen activator mRNA in corticostriatal neurons in rat brain
Authors: Hashimoto Takanori , Kajii Yasushi , Nishikawa Toru
Published: March 2003

We have studied in the rat the effects of acute subcutaneous injection of psychotomimetics including methamphetamine (MAP), cocaine and phencyclidine (PCP) on the expression of a brain plasticity‐related molecule, tissue plasminogen activator (tPA) mRNA, using non‐radioactive in situ hybridization histochemistry. In addition to the constitutive expression of tPA mRNA in cerebellar Purkinje cells, ventricular ependymal cells and meningeal blood vessel‐associated cells, MAP (1–4 mg/kg), cocaine (30 mg/kg) and PCP (1.25–5 mg/kg) caused a transient and dose‐dependent induction of the transcript with its peak at 3 h postinjection in a group of neurons of the medial and insular prefrontal cortices, and the piriform cortex. Another indirect dopamine agonist nomifensine (20–40 mg/kg) mimicked the tPA mRNA induction in the prefrontal cortical areas. Moreover, MAP induction of tPA mRNA was markedly inhibited by pretreatment with a D1 (R(+)‐SCH23390: R(+)‐7‐chloro‐8‐hydroxy‐3‐methyl‐1‐phenyl‐2,3,4,5‐tetra‐hydro‐1H‐3‐benzazepine hydrochloride) or a D2 (haloperidol) dopamine receptor‐preferring antagonist. Intramedial striatum, but not intrathalamic, application of a fluorescent tracer, fluorogold, retrogradely labelled the cortical cells expressing tPA mRNA. The present results suggest that acute injections of the above psychotomimetic drugs may induce tPA mRNA in a group of the prefrontal cortical neurons that project to the medial striatum. This tPA mRNA expression may be due to the activation of the dopamine neurotransmission. Because it is well documented that single or repeated administration of methamphetamine, cocaine and PCP produces enduring changes in responses to these drugs in humans and experimental animals (e.g. behavioural sensitization), the psychotomimetic‐induction of tPA mRNA could be implicated in an initial step in the plastic rearrangements in the neuronal circuits underlying long‐lasting changes in behavioural expression.
Novel pluripotential neural progenitor lines exhibiting rapid controlled differentiation to neurotransmitter receptor‐expressing neurons and glia
Authors: Jung Marion , Krämer Eva‐Maria , Müller Thomas , Antonicek Horst , Trotter Jacqueline
Published: March 2003

The immortalization of progenitor cells from embryonic murine hippocampus using oncogene‐carrying retroviral vectors is described. Use of a vector encoding the oncogene v‐myc results in lines of nestin‐positive progenitor cells. Limited differentiation ensues if the cells are cultured in the presence of dibutyryl cyclic adenosine monophosphate. In contrast, use of a vector in which the extracellular portion of the epidermal growth factor (EGF) receptor is fused to the neu tyrosine kinase generates lines of pluripotential nestin‐positive progenitor cells, which differentiate upon withdrawal of EGF into neurons and glia. Differentiated neurons expressing action potentials and neurotransmitter receptors make up a high proportion of the cells. These cell lines are useful tools to investigate the characteristics of differentiating neurons and glia, as well as to screen neuroactive drugs. This work has been reported in preliminary form as an abstract (1996 Society for Neuroscience Abstract, #606.20, p. 1537).
Attenuation of 6‐OHDA‐induced neurotoxicity in glutathione peroxidase transgenic mice
Authors: Bensadoun J. C. , Mirochnitchenko O. , Inouye M. , Aebischer P. , Zurn A. D.
Published: March 2003

Normal cellular metabolism produces oxidants which are neutralized within cells by antioxidant enzymes and other antioxidants. An imbalance between oxidants and antioxidants has been postulated to lead to the degeneration of specific populations of neurons in neurodegenerative diseases, e.g. Parkinson's disease. The present study investigates whether overexpression of glutathione peroxidase, the enzyme which metabolizes hydrogen peroxide to water, can prevent or slow down neuronal injury in an animal model of Parkinson's disease. Transgenic mice overexpressing the human glutathione peroxidase gene under the control of the mouse hydroxymethylglutaryl‐coenzyme A promoter and genetically matched control mice were injected intracerebroventricularly with the dopaminergic neurotoxin 6‐hydroxydopamine. Seven days after injection, the number of tyrosine hydroxylase‐positive nigral dopaminergic neurons was decreased by 52.4% and 20.5% in 6‐hydroxydopamine‐injected control and glutathione peroxidase transgenic mice, respectively. Similarly, 3 days after injection of the neurotoxin, striatal dopamine was decreased by 71.2% and 56.5%, respectively. Overexpression of glutathione peroxidase therefore partially protects dopaminergic neurons against 6‐hydroxydopamine‐induced toxicity.
Differential time course of neuronal and glial apoptosis in neonatal rat dorsal root ganglia after sciatic nerve axotomy
Authors: Whiteside G. , Doyle C. A. , Hunt S. P. , Munglani R.
Published: March 2003

Sensory neurons in neonatal rat lumbar dorsal root ganglia die after sciatic nerve axotomy, and previous studies have estimated the total cell loss to be 40–95%. We have used the terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick end labelling (TUNEL) technique, combined with immunohistochemistry, to investigate the contribution of apoptosis to the cell loss that occurs after unilaterally transecting the sciatic nerve of new‐born rats.

  TUNEL‐positive cells were detected 1 day post‐lesion, and their number peaked 3 days after the injury. Combining TUNEL labelling with immunohistochemistry, for neuron‐specific neurofilament 150 kDa, or glial‐specific S‐100β, enabled us to identify dying neurons and dying glia. One day after axotomy, most of the TUNEL‐positive cells (58%) were neurons, whereas 3 days post‐injury, only a small number of dying cells (6%) were neuronal. This lower incidence was due to a decrease in neuronal death and an increase in glial death. The glia in the dorsal root ganglia therefore die subsequent to the neurons. The apoptotic nature of the cell death was confirmed by electron microscopy, with fine structural features of apoptotic cell death, e.g. chromatin compaction and membrane blebbing, being observed in both glia and neurons.

  Our results confirm that extensive apoptosis occurs in the neonatal lumbar dorsal root ganglia after sciatic nerve section, and show that neurons and glial cells die with different time‐courses. The results suggest a neuron‐glia trophic interdependence in the dorsal root ganglia.
Regional and differential expression of gelatinases in rat brain after systemic kainic acid or bicuculline administration
Authors: Zhang J. Wenjun , Deb Suman , Gottschall Paul E.
Published: March 2003

Indirect evidence from in vitro studies implicates a functional role for matrix metalloproteinases (MMPs) in the central nervous system (CNS), including induction of neuronal migration during development and enhancement of neurite extension. Few reports have documented the expression of these enzymes in the brain, especially after injury in vivo. The objective of this study was to determine whether MMPs are expressed in various regional areas of rat brain after administration of the neurotoxin, kainic acid. Limbic motor seizures and neuronal degeneration were induced in Sprague–Dawley rats by systemic administration of kainate (10 mg/kg). Rats were subsequently divided into convulsive and non‐convulsive groups, after observing their behaviour in response to the drug. Animals were killed 6, 12, 24, 72 and 168 h (7 days) after injection of kainate. Gelatinases were extracted from various brain regions and assayed by gelatin‐substrate zymography. Levels of glial fibrillary acidic protein (GFAP) in corresponding regions were measured by ELISA. In the absence of treatment, MMP‐2 and MMP‐9 activities were expressed differentially in various brain regions with the highest levels in the hippocampus and the lowest in the cerebellum. In areas from convulsive rats, MMP‐9 activity was markedly elevated at 6 h, and reached a maximum at 12 h after injection of kainate (8.1‐fold hippocampus, 7.7‐fold diencephalon, 7.2‐fold striatum, 5.7‐fold frontal cortex, 5.5‐fold cerebellum, 2.6‐fold midbrain). MMP‐2 activity was induced more than two‐fold in the hippocampus, diencephalon and striatum, to a lesser extent in the frontal cortex and midbrain, and was unchanged in the cerebellum, 72 h after injection. Neither MMP activity was altered in any brain region derived from non‐convulsive rats. Treatment with the GABAA antagonist, bicuculline, resulted in increased levels of MMP‐9, 12 h after drug administration, but no change in levels of MMP‐2 up to 3 days following treatment. GFAP levels were induced 3 days after kainic acid injection in brain regions where MMP‐2 was elevated. Nissl staining displayed the classical, regional neurodegeneration in kainate‐treated animals that exhibited seizures. No obvious degeneration was detected in kainate‐treated, non‐convulsive rats or bicuculline‐treated animals. These data demonstrate that MMP‐9 and MMP‐2 are differentially expressed with respect to time after kainic acid injection, and suggest that they are regulated by convulsion and/or neurodegenerative‐associated mechanisms, respectively. Although similar in catalytic activity, MMP‐9 and MMP‐2 may play different roles in response to kainic acid‐induced seizure and neuronal degeneration.
European Neuroscience Association Pre‐ and post‐synaptic muscarinic receptors in thin slices of rat adrenal gland
Authors: Barbara Jean‐Gaël , Lemos Virginia Soares , Takeda Kenneth
Published: March 2003

The effects of activation of muscarinic receptors on chromaffin cells and splanchnic nerve terminals were studied in a rat adrenal slice preparation. In chromaffin cells, muscarine induced a transient hyperpolarization followed by a depolarization associated with cell spiking. The hyperpolarization was blocked by charybdotoxin (1 μm) and tetraethylammonium chloride (TEA, 1 mm), but was not affected by 200 μm Cd2+ or removal of external Ca2+, consistent with activation of BK channels. This would follow internal Ca2+ mobilization, as shown by Ca2+ imaging with fura‐2 on isolated chromaffin cells in culture. Under voltage‐clamp, outward BK currents were insensitive to MT3 toxin, a specific muscarinic m4 receptor antagonist. In contrast, muscarine‐induced depolarization was due to a m4 receptor‐mediated inward current blocked by MT3 toxin. This current was permeable to cations and was associated with Ca2+ entry and subsequently, Ca2+‐induced Ca2+ release. Finally, both muscarine (25 μm) and oxotremorine (10 μm) decreased the amplitude and frequency of KCl‐evoked excitatory postsynaptic currents, without affecting quantal size, consistent with a presynaptic inhibitory effect. Taken together, our data suggest that activation of m4 and probably m3 muscarinic receptors results in a strong, long‐lasting excitation of chromaffin cells, as well as an uncoupling of synaptic inputs onto these cells.
Molecular cloning and characterization of an invertebrate homologue of a neuropeptide Y receptor
Authors: Tensen Cornelis P. , Cox Kingsley J. A. , Burke Julian F. , Leurs Rob , Van Der Schors Roel C. , Geraerts Wijnand P. M. , Vreugdenhil Erno , Van Heerikhuizen Harm
Published: March 2003

Neuropeptide Y is an abundant and physiologically important peptide in vertebrates having effects on food intake, sexual behaviour, blood pressure and circadian rhythms. Neuropeptide Y homologues have been found in invertebrates, where they are very likely to play similar, important roles. Although five neuropeptide Y‐receptor subtypes have been identified in mammals, none has been reported from invertebrates. Here we describe the cloning of a neuropeptide Y‐receptor from the brain of the snail Lymnaea stagnalis. The identity of the receptor was deduced by expressing the neuropeptide Y‐receptor‐encoding cDNA in Chinese Hamster Ovary cells, which were subsequently challenged with size‐fractionated Lymnaea brain extracts. An active peptide, selected on the basis of its ability to induce changes in cAMP levels, was purified to homogeneity, analysed by mass spectrometry and amino acid sequence determination, and turned out to be a Lymnaea homologue of neuropeptide Y.
Involvement of GABAB receptors in convulsant‐induced epileptiform activity in rat neocortex in vitro
Authors: Sutor Bernd , Luhmann Heiko J.
Published: March 2003

The role of γ‐aminobutyric acid B (GABAB) receptors in the generation and maintenance of bicuculline‐induced epileptiform activity in rat neocortical slices was studied using electrophysiological methods. A block of GABAB receptors in the presence of functional GABAA receptor‐mediated inhibition was not sufficient to induce epileptiform activity. In the presence of the GABAA receptor antagonist bicuculline (10 μm) and at suprathreshold stimulation, the GABAB receptor antagonist CGP 35348 (10–300 μm) significantly potentiated epileptiform activity. With stimulation at threshold intensity, low concentrations of CGP 35348 (10–30 μm) potentiated bicuculline‐induced activity, whereas higher concentrations (100–300 μm) invariably led to a reversible suppression of stimulus‐evoked epileptiform discharges. CGP 35348 also enhanced picrotoxin‐induced epileptiform activity, but at higher concentrations it was considerably less effective in suppressing such epileptiform discharges. The GABA uptake inhibitor nipecotic acid partially mimicked the actions of CGP 35348: with stimulation at threshold intensity, it reversibly suppressed bicuculline‐induced epileptiform field potentials, but it did not influence epileptiform activity induced by picrotoxin. We conclude that a postsynaptic blockade of GABAB receptors induces an amplification of epileptiform activity in neocortical slices disinhibited by GABAA receptor antagonists. An additional blockade of presynaptic GABAB receptors, especially under conditions of weak stimulation of the neurons, reduces the inhibitory auto‐feedback control of GABA release, leading to a displacement of competitive antagonists from the postsynaptic GABAA receptor and hence, to a suppression of epileptiform activity induced by competitive GABAA receptor antagonists.
Corticotrophin‐releasing factor produces a long‐lasting enhancement of synaptic efficacy in the hippocampus
Authors: Wang H. L. , Wayner M. J. , Chai C. Y. , Lee E. H. Y.
Published: March 2003

We have previously demonstrated that intra‐hippocampal injection of corticotrophin‐releasing factor improved memory retention of an inhibitory avoidance learning in rats; while the electrophysiological effects corticotrophin‐releasing factor produces on hippocampal neurons are largely uncharacterized. In the present study, we found that corticotrophin‐releasing factor injected into the dentate gyrus of hippocampus produced a dose‐dependent and long‐lasting enhancement in synaptic efficacy of these neurons, as measured by an increase in the amplitude and slope of population excitatory postsynaptic potentials, as well as the amplitude of population spike. The onset of corticotrophin‐releasing factor‐induced potentiation was slow. It was observed approximately 40–60 min after corticotrophin‐releasing factor administration and lasted for more than 5 h. This effect of corticotrophin‐releasing factor was blocked by pretreatment with the cyclase‐adenosine‐3,5‐monophosphate (cAMP) inhibitor Rp‐adenosine‐3,5‐cyclic monophosphothiolate triethylamine (Rp‐cAMPS) and partially blocked by the N‐methyl‐D‐aspartate receptor antagonist MK‐801. Further, pretreatment with corticotrophin‐releasing factor receptor antagonist dose‐dependently diminished tetanization‐induced long‐term potentiation, and corticotrophin‐releasing factor and tetanic stimuli had an additive effect on hippocampal neuron excitation. Moreover, direct injection of corticotrophin‐releasing factor increased cAMP level in the dentate gyrus. These results together suggest that corticotrophin‐releasing factor‐induced potentiation simulates the late phase of tetanization‐induced long‐term potentiation and cAMP seems to be the messenger mediating this effect. Moreover, corticotrophin‐releasing factor‐induced potentiation and long‐term potentiation may share some similar mechanisms, and corticotrophin‐releasing factor is probably involved in the neural circuits underlying long‐term potentiation. Thus, corticotrophin‐releasing factor may play an important role in modulating synaptic plasticity in the hippocampus.
Effect of GABA on the processing of interaural time differences in nucleus laminaris neurons in the chick
Authors: Brückner Susanne , Hyson Richard L.
Published: March 2003

Neurons in the avian nucleus laminaris (NL) are the first to receive binaural information and are presumed to play a role in encoding interaural time differences (ITD). NL not only receives excitatory projections from the ipsi‐and contralateral nucleus magnocellularis, but also receives inhibitory (GABAergic) input. This study investigates how GABA (γ‐aminobutyric acid) influences ITD coding in NL.

  Intracellular responses of chick NL neurons were studied in a brain slice preparation. Both excitatory inputs to NL were electrically activated and the delay between trains of bilateral stimuli (simulated‐interaural time difference [s‐ITD]) was varied. The resulting s‐ITD functions were recorded in the presence of 0–75 μm GABA.

  The discharge rate of NL neurons varied with s‐ITD. Cells responded maximally using s‐ITDs at which the peak of the ipsi‐ and contralateral excitatory postsynaptic potentials occurred simultaneously (favourable s‐ITD). At unfavourable s‐ITDs, the discharge rates usually fell below unilateral levels.

  GABA had contrary effects on the s‐ITD functions depending on the drug concentration. A low GABA dose enhanced excitability at favourable s‐ITD, but not at unfavourable s‐ITDs. In contrast, higher GABA concentrations diminished excitability. Moderate GABA concentrations had no consistent effect.

  These results suggest that the GABAergic input to NL will either increase or decrease the excitability of the NL neuron depending on the degree to which this GABAergic input is activated. A gain control hypothesis is presented in which the GABAergic input makes ITD processing in NL independent of the stimulus intensity by adjusting the excitability of NL neurons.
P2X3 is expressed by DRG neurons that terminate in inner lamina II
Authors: Vulchanova L. , Riedl M. S. , Shuster S. J. , Stone L. S. , Hargreaves K. M. , Buell G. , Surprenant A. , North R. A. , Elde R.
Published: March 2003

The P2X3 receptor subunit, a member of the P2X family of ATP‐gated ion channels, is almost exclusively localized in sensory neurons. In the present study, we sought to gain insight into the role of P2X3 and P2X3‐containing neurons in sensory transmission, using immunohistochemical approaches. In rat dorsal root ganglia (DRG), P2X3‐immunoreactivity (‐ir) was observed in small‐ and medium‐sized neurons. Approximately 40% of DRG neuronal profiles in normal rats contained P2X3‐ir. In rats that had received neonatal capsaicin treatment, the number of P2X3‐positive neurons was decreased by approximately 70%. Analysis of the colocalization of P2X3‐ir with cytochemical markers of DRG neurons indicated that approximately 94% of the P2X3‐positive neuronal profiles were labelled by isolectin B4 from Bandeiraea simplicifolia, while only 3% contained substance P‐ir, and 7% contained somatostatin‐ir. In dorsal horn of rat spinal cord, P2X3‐ir was observed in the inner portion of lamina II and was reduced subsequent to dorsal rhizotomy, as well as subsequent to neonatal capsaicin treatment. Finally, P2X3‐ir accumulated proximal to the site of sciatic nerve ligation, and was seen in nerve fibres in skin and corneal epithelium. In summary, our results suggest that P2X3 is expressed by a functionally heterogeneous population of BSI‐B4‐binding sensory neurons, and is transported into both central and peripheral processes of these neurons.
AMPA receptor subunits are differentially expressed in parvalbumin‐ and calretinin‐positive neurons of the rat hippocampus
Authors: Catania Maria Vincenza , Bellomo Maria , Giuffrida Raffaella , Giuffrida Rosario , Stella Anna Maria Giuffrida , Albanese Vincenzo
Published: March 2003

Recent studies suggest a functional diversity of native α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionate‐type glutamate receptor channels (AMPARs). In several types of interneurons, AMPARs are characterized by higher Ca2+ permeability and faster kinetics than AMPARs in principal cells. We studied the expression profile of AMPAR subunits in the hippocampal parvalbumin (PV)‐ and calretinin (CR)‐positive cells, which represent different populations of non‐principal cells. To this end, non‐radioactive in situ hybridization with AMPAR subunit specific cRNAs was combined with immunocytochemistry for PV or CR. Double‐immunolabelling using antibodies against AMPAR subunits and PV or CR was also performed. PV‐containing neurons represent a fairly homogeneous population of cells expressing high levels of GluR‐A and GluR‐D mRNAs, moderate levels of GluR‐C and low levels of GluR‐B mRNAs in all the examined regions of hippocampus. The vast majority of CR‐containing cells have a much lower expression of GluR‐A, ‐C and ‐D mRNA than PV‐positive neurons, although similarly featuring low levels of GluR‐B mRNA. Only a subpopulation of CR‐containing cells, the spiny neurons of the dentate gyrus and CA3 region of the hippocampus were characterized by a strong expression of GluR‐A and ‐D subunit mRNAs. The differential pattern found for the AMPAR subunit mRNA expression was confirmed by immunocytochemistry at protein level. Despite the common feature of low GluR‐B subunit expression, PV‐ and CR‐containing interneurons differ with respect to the density and combination of their expressed AMPAR subunits. The different combination of subunits might subserve different properties of the AMPA channels featured by these cell types, with implications for the functioning of the hippocampal network.
Dopaminergic modulation of early signs of excitotoxicity in visualized rat neostriatal neurons
Authors: Cepeda Carlos , Colwell Christopher S. , Itri Jason N. , Gruen Ehud , Levine Michael S.
Published: March 2003

Cell swelling induced by activation of excitatory amino acid receptors is presumably the first step in a toxic cascade that may ultimately lead to cell death. Previously we showed that bath application of N‐methyl‐d‐aspartate (NMDA) or kainate (KA) produces swelling of neostriatal cells. The present experiments examined modulation of NMDA and KA‐induced cell swelling by dopamine (DA) and its receptor agonists. Nomarski optics and infra‐red videomicroscopy were utilized to visualize neostriatal medium‐sized neurons in thick slices from rat pups (12–18 postnatal days). Increase in somatic cross‐sectional area served as the indicator of swelling induced by bath application of glutamate receptor agonists. NMDA induced cell swelling in a dose‐dependent manner. Activation of DA receptors in the absence of NMDA did not produce swelling. DA and the D1 receptor agonist SKF 38393, increased the magnitude of swelling produced by NMDA. This effect was reduced in the presence of the D1 receptor antagonist, SCH 23390. In contrast, activation of D2 receptors by quinpirole decreased the magnitude of NMDA‐induced cell swelling. DA slightly attenuated cell swelling induced by activation of KA receptors. Quinpirole produced a significant concentration‐dependent reduction in KA‐induced swelling while SKF38393 increased KA‐induced swelling, but only at a low concentration of KA. Together, these results provide additional support for the hypothesis that the direction of DA modulation depends on the glutamate receptor subtype, as well as the DA receptor subtype activated. One possible consequence of these observations is that endogenous DA may be an important contributing factor in the mechanisms of cell death in Huntington's disease.
Spreading depression determines acute cellular damage in the hippocampal slice during oxygen/glucose deprivation
Authors: Obeidat Akef S. , Andrew R. David
Published: March 2003

During ischaemia neurons depolarize and release the neurotransmitter l‐glutamate, which accumulates extracellularly and binds to postsynaptic receptors. This initiates a sequence of events thought to culminate in immediate and delayed neuronal death. However, there is growing evidence that during ischaemia the development of spreading depression (SD) can be an important determinant of the degree and extent of ischaemic damage. In contrast, SD without metabolic compromise (as occurs in migraine aura) causes no discernible damage to brain tissue. SD is a profound depolarization of neurons and glia that propagates like a wave across brain tissue. Brain cell swelling, an early event of both the excitotoxic process and of SD, can be assessed by imaging associated intrinsic optical signals (IOSs). We demonstrate here that IOS imaging clearly demarcates the ignition site and migration of SD across the submerged hippocampal slice of the rat. If SD is induced by elevating [K+]o, the tissue fully recovers, but in slices that are metabolically compromised at 37.5 °C by oxygen/glucose deprivation (OGD) or by ouabain exposure, cellular damage develops only where SD has propagated. Specifically, the evoked CA1 field potential is permanently lost, the cell bodies of involved neurons swell and their dendritic regions increase in opacity. In contrast to OGD, bath application of l‐glutamate (6–10 mm) at 37.5 °C evokes a non‐propagating LT increase in CA1 that reverses without obvious cellular damage. Moreover, application of 2–20 mm glutamate or various glutamate agonists fail to evoke SD in the submerged hippocampal slice. We propose that SD and OGD together (but not alone) constitute a ‘one‐two punch’, causing acute neuronal death in the slice that is not replicated by elevated glutamate. These findings support the proposal that SD generation during stroke promotes and extends acute ischaemic damage.
The NO‐cGMP pathway in the rat locus coeruleus: electrophysiological, immunohistochemical and in situ hybridization studies
Authors: Xu Zhi‐Qing David , De Vente Jan , Steinbusch Harry , Grillner Sten , Hökfelt Tomas
Published: March 2003

The effect of two nitric oxide (NO) donors, SIN‐1 and DEA/NO, as well as of the inactive SIN‐1 derivative molsidomin, was studied on locus coeruleus (LC) neurons in a slice preparation using intracellular recordings. In addition, the effect of the guanylate cyclase inhibitor ODQ was analysed. Furthermore, the effect of NO donors on cyclic guanosine monophosphate (GMP) levels in the LC was studied using the indirect immunofluorescence technique, and the expression of soluble guanylyl cyclase with in situ hybridization. In 36 of 66 LC neurons extracellular application of SIN‐1 and DEA/NO caused a hyperpolarization and a decrease in apparent input resistance. In almost 20% of neurons SIN‐1 increased the firing rate. No effect could be recorded with the brain‐inactive SIN‐1 derivative molsidomin. The membrane permeable cGMP analogue 8‐bromo‐cGMP imitated the action of SIN‐1. The hyperpolarizing effect of SIN‐1 and DEA/NO was attenuated by preincubation with the guanylyl cyclase inhibitor ODQ. The immunohistochemical analysis revealed lack of cGMP immunostaining in non‐stimulated slices, whereas SIN‐1 dramatically increased this staining in about 40% of the LC neurons, and these neurons were all tyrosine hydroxylase positive, that is noradrenergic. A large proportion of the LC neurons expressed soluble guanylyl cyclase mRNA. The present and previous results suggest that NO, released from a small number of non‐noradrenergic neurons in the LC, mainly has an inhibitory influence on many noradrenergic neurons, by upregulating cGMP levels via stimulation of soluble guanylyl cyclase. As nitric oxide synthase is present only in a small number of non‐noradrenergic neurons (Xu et al. 1994), a few neurons may influence a large population of noradrenergic LC neurons, which in turn may control activity in many regions of the central nervous system.
Select types of supporting cell in the inner ear express aquaporin‐4 water channel protein
Authors: Takumi Yutaka , Nagelhus Erlend Arnulf , Eidet Jo , Matsubara Atsushi , Usami Shin‐ichi , Shinkawa Hideichi , Nielsen Søren , Ottersen Ole Petter
Published: March 2003

Aquaporins (AQPs) confer a high water permeability on cell membranes and play important parts in secretory and absorptive epithelia in kidney and other organs. Here we investigate whether AQPs are expressed in the sensory epithelia of the inner ear, where a precise volume regulation is crucial. By use of specific antibodies it was found that the inner ear contains AQP1 and 4 while being devoid of detectable levels of AQP2, 3 or 5. Immunofluorescence and postembedding immunogold labelling revealed a strictly non‐epithelial distribution of AQP1, confirming previous data. In contrast, AQP4 protein and mRNA (visualized by in situ hybridization) were concentrated in select types of supporting cell, including Hensen's cells and inner sulcus cells. Immunogold particles signalling AQP4 were confined to the basolateral plasma membrane of Hensen's cells and to the basal plasma membrane of Claudius cells and inner sulcus cells. AQP4 was also found in supporting cells of the vestibular end organs, but was absent from transitional epithelial cells and dark cells. Strong labelling for AQP4 and AQP4‐mRNA was associated with the central part of the cochlear and vestibular nerves. Hair cells were consistently unlabelled. Our findings indicate that AQP4 may facilitate osmotically driven water fluxes in the sensory epithelia of the inner ear and thus contribute to the volume and ion homeostasis at these sites.
Matrix‐assisted laser desorption/ionization time of flight mass spectrometric analysis of the pattern of peptide expression in single neurons resulting from alternative mRNA splicing of the FMRFamide gene
Authors: Worster Belinda M. , Yeoman Mark S. , Benjamin Paul R
Published: March 2003

MALDI‐ToF MS (matrix‐assisted laser desorption/ionization time of flight mass spectrometry) has become a fast, reliable and sensitive technique for the identification of neuropeptides in biological tissues. Here, we applied this technique to identified neurons of the cardioregulatory network in the snail Lymnaea that express the FMRFamide gene. This enabled us to study the complex processing of the FMRFamide gene at the level of single identified neurons. In the CNS of Lymnaea, FMRFamide‐like and additional peptides are encoded by a common, multiexon gene. Alternate mRNA splicing of the FMRFamide gene leads to the production of two different mRNAs. Type 1 mRNA (exon II) encodes for the tetrapeptides (FLRF/FMRFamide), whereas Type 2 (exons III–V) encodes for the heptapeptides (SDPFLRFamide/GDPFLRFamide). Previous in situ hybridization and immunocytochemical studies indicated that these two transcripts are expressed in the CNS neurons of Lymnaea in a differential and mutually exclusive manner. Two single identified neurons of the cardiorespiratory network, the Ehe neuron and the visceral white interneuron (VWI), were known to express the FMRFamide gene (Ehe, type 1 mRNA; VWI, type 2 mRNA). MALDI‐ToF MS analysis of these neurons and other neurons expressing the FMRFamide gene confirmed the mutually exclusive expression of the distinct sets of peptides encoded on the two transcripts and revealed the pattern of post‐translational processing of both protein precursors. From the gene sequence it was predicted that 16 final peptide products from the two precursor proteins could possibly exist. We showed that most of these peptides were indeed present in the identified neurons (13) while others were not (three), suggesting that not all of the potential cleavage sites within the two precursors are utilized. In this way, the neuronal expression of the full range of the peptide products resulting from alternative mRNA splicing was revealed for the first time.