bluuebunny/crossref_metadata_2025 huggingface dataset:
Opioid Inhibition of Ca2+ Channel Subtypes in Bovine Chromaffin Cells: Selectivity of Action and Voltage‐dependence
Authors: Albillos Almudena , Carbone Emilio , Gandia Luis , Garcia Antonio G. , Polio Antonella
Published: April 2006

Bovine chromaffin cells possess a mixture of high‐voltage‐activated Ca2+ channel subtypes: L‐type, dihydropyridine‐sensitive channels, and N‐, P‐ and Q‐types, ω‐conotoxin MVIIC‐sensitive channels. In these cells, we studied the reversible, naloxone‐antagonized inhibition of Ba2+ currents by the opioid agonist met‐enkephalin (IC50= 272 nM). This inhibition could be resolved into a voltage‐dependent and a voltage‐independent component. The first was revealed by its slow Ba2+ current activation kinetics at 0 mV and by the current facilitation induced by short prepulses to +90 mV. The second was estimated as the residual inhibition persisting after the facilitation protocol. The two inhibitory components varied markedly from cell to cell and each contributed to about half of the total inhibition. Replacement of internal GTP by GDP‐β‐S or cell pretreatment with pertussis toxin completely abolished the voltage‐dependent inhibition by opioids, partially preserving the voltage‐independent component. The opioid‐induced inhibition was not selective for any Ca2+ channel subtype, being not prevented after the addition of specific Ca2+ channel antagonists. However, when separately analyzing the contribution of each channel type to the voltage‐dependent and voltage‐independent modulation, a clear‐cut distinction could be achieved. The voltage‐independent inhibition was effective on all Ca2+ channel subtypes but predominantly on L‐type Ca2+ channels. The voltage‐dependent process was abolished by ω‐conotoxin‐MVIIC, but unaffected by nifedipine, and was thus sharply restricted to non‐L‐type channels (N‐, P‐ and Q‐types). Our data suggest a functionally distinct opioid receptor‐mediated modulation of L‐ and non‐L‐type channels, i.e. of the two channel classes sharing major control of catecholamine secretion from bovine chromaffin cells.
A Glutamate‐sensitizing Activity in Conditioned Media Derived from Rat Cerebellar Granule Cells
Authors: Ciotti Maria Teresa , Giannetti Stefano , Mercanti Delio , Calissano Pietro
Published: April 2006

When cerebellar granule cells that had been cultured in vitro for 8 days were subjected to a cytotoxic glutamate pulse (100 μM, 30 min incubation), the response varied according to cell density and the volume of medium in which cells were grown. Thus, lowering the cell density by a factor of 4 compared with usual conditions (2.6 × 105 cells/cm2) or increasing the volume by an identical 4‐fold factor reduced cell death from 90‐95% to 20‐30%. Addition of a conditioned medium derived from high‐density to low‐density cultures or to high‐volume cultures markedly increased the sensitivity of the cells to glutamate. This glutamate‐sensitizing activity, which accelerated by several days the onset of the response of cerebellar cultures to glutamate, was inhibited by actinomycin D and was not detectable in conditioned medium derived from confluent cultures of cerebellar astroglia, or from cell lines such as PC12, GT1‐7, 3T3 and CHP 100. Glutamate‐sensitizing activity was not mimicked by triiodo‐L‐thyronine, insulin‐like growth factor‐I (IGF‐I), truncated IGF‐I, GPE [a tripeptide (gly‐pro‐glu) derived from IGF‐I], brain‐derived neurotrophic factor (BDNF), basic fibroblast growth factor or tumour necrosis factor‐α. However, IGF‐I added to cultures of granule cells plated at high density and grown in basal medium Eagle's without serum or any other constituent of chemically defined media was capable of supporting production of glutamate‐sensitizing activity to an extent similar to that shown by whole fetal calf serum. Under the same conditions triodoi‐L‐thyronine and BDNF did not support the production of glutamate‐sensitizing activity. Glutamate‐sensitizing activity was not mimicked by glutamate, NMDA, glycine or lactate, and was not inhibited by glucose, haemoglobin or N‐Ω‐nitro‐L‐arginine methyl ester. At variance with the response of granule cells, the response to glutamate of GABAergic cells present in the same culture was not affected by cell density or by glutamate‐sensitizing activity.
Characteristic Localization of gpl30 (the Signal‐transducing Receptor Component Used in Common for IL‐6/IL‐11/CNTF/LIF/OSM) in the Rat Brain
Authors: Watanabe D. , Yoshimura R. , Khalil M. , Yoshida K. , Kishimoto T. , Taga T. , Kiyama H.
Published: April 2006

The localization of gp130, the signal transducing receptor component used in common for interleukin (IL)‐6, IL‐11, ciliary neurotrophic factor (CNTF), LIF and OSM, in the rat brain was demonstrated by immunohistochemistry using an antibody specific to gpl30. The gp130 immunoreactivity was observed in both glial and neuronal cells. Two distinct neuronal staining patterns were observed. The first showed neuropil staining, observed mainly in telencephalic structures including the hippocampus, cerebral cortex and caudate‐putamen. The second pattern was observed on the cytoplasmic membrane of neuronal somata and was found primarily in the lower brainstem, in the large neurons of the reticular formation, and in spinal and cranial motor neurons. Electron‐microscopic analysis revealed that both types of gpl30 immunoreactivity were primarily associated with the cytoplasmic membrane and were not localized exactly at synaptic sites. Further, gpl30 immunoreactivity was also observed in the oligodendrocytes and subependymal zone. These widespread but characteristic patterns of gp130 immunoreactivity overlap well with those of IL‐6 receptor and CNTF alpha chains, suggesting a role of cytokines and growth factors such as IL‐6 and CNTF via gp130 in certain specific regions of the brain.
Developmentally Regulated Neurite Outgrowth Response from Dorsal Root Ganglion Neurons to Heparin‐binding Growth‐associated Molecule (HB‐GAM) and the Expression of HB‐GAM in the Targets of the Developing Dorsal Root Ganglion Neurites
Authors: Nolo Riitta , Kaksonen Marko , Rauvala Heikki
Published: April 2006

Heparin‐binding growth‐associated molecule (HB‐GAM) is a highly conserved cell surface‐ and extracellular matrix‐associated protein that enhances neurite outgrowth in brain neurons in vitro. To study the possible response of peripheral neurons, we cultured chicken dorsal root ganglion neurons from different developmental stages from embryonic day 4.5 (E4.5; St 25) to E9 (St 35) on recombinant HB‐GAM. We discovered that the neurite outgrowth response to HB‐GAM is maximal at E5.5‐6.5 (St 28‐30). In order to correlate this in vitro phenomenon with in vivo phenomena, immunohistochemical staining and in situ hybridization were performed on cryosections. The protein expression of HB‐GAM peaked at E6 (St 29) and was most extensive on the dorsal spinal cord and dorsal roots. Using Dil labelling, we confirmed that at the time when sensory afferents travel longitudinally in the bundle of His of the spinal cord, HB‐GAM protein expression there is at its peak. Though HB‐GAM is a secreted protein, at the RNA level the timing of HB‐GAM appearance and existence in the spinal cord and sensory ganglia is in accordance with its protein expression. Our results demonstrate that peripheral neurons are responsive to substrate‐bound HB‐GAM in a developmentally regulated manner, and that the expression of both HB‐GAM mRNA and protein in vivo is spatially and temporally matched to this in vitro phenomenon. HB‐GAM is therefore a putative cue for the growth of sensory afferents to and within the dorsal spinal cord.
M‐cadherin Distribution in the Mouse Adult Neuromuscular System Suggests a Role in Muscle Innervation
Authors: Cifuentes‐Diaz Carmen , Goudou Danièle , Padilla Françoise , Facchinetti Patricia , Nicolet Marc , Mège René‐Marc , Rieger François
Published: April 2006

M‐cadherin belongs to the Ca2+‐dependent cadherin family of cell adhesion molecules and was first isolated from a mouse muscle cell line cDNA library. It is specifically expressed in muscle tissue during development and is supposed to play an important role in secondary myogenesis. In the present study the expression of M‐cadherin mRNA and protein and its localization were investigated in adult mouse skeletal muscle and peripheral nerve. The mRNA was abundant in embryonic legs from embryonic day (E)14 to E18. It remained expressed in new‐born and adult muscles. In the adult muscle M‐cadherin immunoreactivity was only detected at the neuromuscular junction, associated with perijunctional mononucleated cells and on intramuscular nerves. Peripheral nerves were also M‐cadherin‐positive. The molecule was found at the surface of myelinated nerve fibres where it was concentrated at the node of Ranvier. When a nerve was crushed and allowed to regenerate, M‐cadherin was over‐expressed at the site of nerve injury and in the distal stump. M‐cadherin was also up‐regulated on the sarcolemma of denervated muscle fibres. Taken together, these observations point toward a much wider tissue distribution of M‐cadherin than previously thought. M‐cadherin might be involved not only in specific steps of myogenesis but also in some aspects of synaptogenesis, axon/Schwann cell interactions and node of Ranvier structural maintenance.
Long‐term Survival of Retina Optic Nerve Section in Adult Ganglion Cells Following bcl‐2 Transgenic Mice
Authors: Cenni Maria Cristina , Bonfanti Lidia , Martinou Jean‐Claude , Ratto Gian Michele , Strettoi Enrica , Maffei Lam berto
Published: April 2006

The bcl‐2 gene codes for a protein that acts as a powerful inhibitor of active cell death. Since the transection of the optic nerve in adult mammalians starts a massive process of degeneration in retinal ganglion cells, we investigated whether the overexpression of bcl‐2 in adult transgenic mice can protect the axotomited ganglion cells. We performed intracranial optic nerve transection on both wild type and transgenic adult mice, and we tested cell survival 2 or 3.5 months after axotomy. The percentage of surviving ganglion cells after optic nerve section was computed by combining the counts of the optic nerve fibres in intact nerves with the cell density measures of the ganglion cell layer of axotomized retinae. From these data we found that in transgenic mice˜65% of ganglion cells survived 3.5 months after axotomy. In contrast, 2 months after surgery, <10% of ganglion cells were left in wild type retinae. We have also examined the morphology and fine structure of the proximal stump of the sectioned optic nerves by light and electron microscopy. In the transgenic mice a very large number of axons survived after surgery and they still exhibited fairly normal morphology and ultrastructure. On the other hand the wild type transected nerves had only a few visible axons that displayed clear signs of degeneration. We conclude that the overexpression of Bcl‐2 protein in central neurons is a very effective strategy to ensure long‐term survival in axotomized cells.
The Basomedial and Basolateral Amygdaloid Nuclei Contribute to the Induction of Long‐term Potentiation in the Dentate Gyrus In Vivo
Authors: Ikegaya Yuji , Saito Hiroshi , Abe Kazuho
Published: April 2006

Recent behavioural studies have provided evidence that the amygdala modulates hippocampal‐dependent memory. To test the possibility that the amygdala modulates hippocampal synaptic plasticity, we investigated the effects of surgical lesions of the amygdaloid nuclei on the induction of long‐term potentiation (LTP) in the dentate gyrus of anaesthetized rats. Previously we reported that LTP in the dentate gyrus was attenuated by lesion of the basolateral amygdala, but was not affected by lesion of the central amygdala. In the present study, dentate gyrus LTP was significantly attenuated by basomedial amygdala lesion but not by medial amygdala lesion. These results suggest that, among the amygdaloid nuclei, the basomedial and basolateral nuclei are involved in the modulation of hippocampal plasticity. The roles of the basomedial and basolateral amygdala were further supported by experiments examining the effects of electrical stimulation of these nuclei. High‐frequency stimulation of the basomedial amygdala alone did not induce dentate gyrus LTP, but when applied at the same time as tetanic stimulation of the perforant path increased the magnitude of the dentate gyrus LTP. Similarly, high‐frequency stimulation of the basolateral amygdala enhanced LTP induced by tetanic stimulation of the perforant path. Furthermore, facilitation of dentate gyrus LTP by basomedial or basolateral amygdala stimulation was observed even in rats lesioned in either amygdala, suggesting that neurons in the basomedial and basolateral amygdala can modulate dentate gyrus LTP independently. Activity‐dependent facilitation of hippocampal plasticity by the basomedial and basolateral amygdala may underlie memory processing associated with emotion.
The Glutamate Transport Inhibitor L‐trans‐pyrrolidine‐2,4‐dicarboxylate Indirectly Evokes NMDA Receptor Mediated Neurotoxicity in Rat Cortical Cultures
Authors: Blitzblau Rachel , Gupta Shalini , Djali Sina , Robinson Michael B. , Rosenberg Paul A.
Published: April 2006

Because of the well‐documented importance of glutamate uptake in protecting neurons against glutamate toxicity, we were interested in testing the effects of L‐trans‐pyrrolidine‐2,4‐ dicarboxylate (PDC) on rat cortical cultures. This compound is a substrate for glutamate transporters and is a potent glutamate transport inhibitor that does not interact significantly with glutamate receptors. Using a 30 min exposure, and assessing neuronal survival after 20‐24 h, PDC was neurotoxic in conventional astrocyte‐rich cortical cultures, with an EC50 in these cultures of 320 ± 157 μM. In astrocyte‐poor cultures, an EC50 for PDC of 50 ± 5 μM was determined. The neurotoxicity of PDC in both astrocyte‐rich and astrocyte‐poor cultures was blocked by the NMDA antagonist MK‐801, but not by the non‐NMDA receptor antagonist 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX). We tested the possibility that the neurotoxicity of PDC might be due to release of excitatory amino acids using several approaches. After pre‐loading cells with the non‐metabolizable analogue of glutamate, [3H]‐D‐aspartate, first we demonstrated that PDC caused significant efflux of [3H]‐D‐aspartate. This effect of PDC was dependent upon extracellular sodium. In contrast with glutamate neurotoxicity, PDC neurotoxicity was inhibited by removal of extracellular sodium. In the presence of 1 mM PDC, sodium caused neurotoxicity with an EC50 of 18 ± 7.6 mM. Tetrodotoxin had no effect on either PDC neurotoxicity or on PDC‐evoked [3H]‐D‐aspartate release. PDC‐evoked release of [3H]‐D‐aspartate was demonstrable in astrocyte cultures with no neurons present. PDC also evoked release of endogenous glutamate. Finally, the neurotoxicity of PDC was blocked by coincubation with glutamate‐pyruvate transaminase plus pyruvate to degrade extracellular glutamate. These results demonstrate the neurotoxicity of PDC, and suggest that the mechanism of this toxicity is the glutamate transporter‐dependent accumulation of glutamate in the extracellular space.
Plasticity of Metabolic Whisker Maps in Somatosensory Brainstem and Thalamus of Mice with Neonatal Lesions of Whisker Follicles
Authors: Melzer Peter , Smith Carolyn B.
Published: April 2006

We employed the autoradiographic deoxyglucose method to study metabolic whisker maps of the adult mouse somatosensory brainstem and thalamus after the neonatal removal of left whisker follicles C1, C2 and C3. Left whiskers B1–3 and D1–3 were deflected to metabolically activate the somatosensory pathway. Unoperated mice that were stimulated in the same fashion served as controls. Whisker stimulation resulted in an ipsilateral increase in metabolic activity in the three trigeminal brainstem structures in which the whiskers are represented topologically by segments of high cytochrome oxidase activity, i.e. subnucleus caudalis, subnucleus interpolaris and nucleus principalis. In the two subnuclei of mice with lesions and of controls, there was an increase in metabolic activity of the representations of the deflected whiskers, whereas the metabolic activity of representations A1–3 and E1–3 was low. Apart from these similarities, the metabolic activation of the representations originally representing whiskers C1–3 was remarkably greater in mice with lesions than in controls. This increase reached statistical significance in subnucleus caudalis and approached statistical significance in subnucleus interpolaris. In nucleus principalis the deprived territory was only partially activated and the degree of metabolic activation was less than in the subnuclei. In the thalamic ventrobasal complex of mice with lesions metabolic activity was unpatterned whereas two areas of metabolic activation were distinct in controls. Hence, the removal of whisker follicles in newborn mice resulted in the suppression of localized metabolic responses to whisker stimulation in the thalamus, whereas in the brainstem stimulus‐related activity was prominent and the deprived territory became responsive to the stimulation of whisker follicles adjacent to the lesion. Apparently, the modification of the whisker representation at the first synapse of the pathway induces a diminution of localized responsivity in the thalamus.
Neurotrophins and their Receptors in the Adult Hypo‐ and Hyperthyroid Rat after Kainic Acid Injection: an In Situ Hybridization Study
Authors: Calzà L. , Giardino L. , Ceccatelli S. , Hökfelt T.
Published: April 2006

Thyroid hormone plays a key role in trophic events during development of the central nervous system. In spite of neurological and psychiatric symptoms associated with adult hypothyroidism, the role of thyroid hormone in mature brain function is less clear. In this paper we investigated the effect of thyroid status on kainic acid‐induced up‐regulation of mRNAs for members of the nerve growth factor family and related receptors in adult male rats by means of in situ hybridization. We found that in hypothyroid rats there is a dramatic attenuation of the kainic acid‐induced up‐regulation of mRNA levels for nerve growth factor, brain‐derived neurotrophic factor and tyrosine kinase trkB in euthyroid rats. A trend to reduced c‐fos mRNA up‐regulation, which did not reach significance, was also found, whereas the increase in c‐jun mRNA after kainic acid was similar in eu‐, hypo‐ and hyperthyroid rats. These data indicate a severe impairment of the regulation of neurotrophin synthesis after excitotoxin administration in the hippocampus during adult hypothyroidism. Possible roles of thyroid hormone in molecular, biochemical and metabolic mechanisms of this defect are discussed.
Cholinergic Responses in Developing Outer Hair Cells of the Rat Cochlea
Authors: Dulon Didier , Lenoir Marc
Published: April 2006

Acetylcholine‐evoked currents were investigated using the conventional whole‐cell patch‐clamp recording technique in developing outer hair cells (OHCs). The cells were isolated from the rat cochlea at different stages of postnatal development ranging from day 4 (P4) to P30. Acetylcholine‐evoked currents could be recorded at P6 and P8. At this developmental stage, the majority of OHCs displayed inward nicotinic‐like currents near the resting membrane potential. These cholinergic currents zeroed near 0 mV, as expected for a non‐selective cation current, and could be reversibly blocked by d‐tubocurarine. At P12 and adult stage, the cholinergic response of OHCs switched to an outward current reversing near EK and displaying a bell shape peaking between ‐40 and ‐30 mV. This change in polarity of the acetylcholine response during postnatal development might be explained by progressive functional coupling between acetylcholine ionotropic receptors permeable to Ca2+ and nearby Ca2+‐activated K+ channels at the synaptic pole of OHCs.
Evidence for Reactive Astrocytes in Rat Vestibular and Cochlear Nuclei Following Unilateral Inner Ear Lesion
Authors: de Waele C. , Torres A. Campos , Josset P. , Vidal P. P.
Published: April 2006

We investigated whether unilateral removal of the labyrinthine and cochlear receptors induces a macroglial reaction in rat vestibular and cochlear nuclei using vimentin and glial fibrillary acidic protein (GFAP) immunochemical markers. Antibody binding was visualized using the avidin‐biotin method and 3,3′‐diaminobenzidine as the peroxidase substrate. In addition, double‐labelling experiments were performed using specific secondary fluorescent antibodies. Potentially degenerating axon terminals were also studied using a silver impregnation method. In normal adult rats, vimentin was found only in ependymal cells, tanicytes around the fourth ventricle, endothelial cells in the blood vessels and Bergmann glia in the molecular layer of the cerebellum. In lesioned rats, all deafferented vestibular and ventral cochlear nuclei showed strong vimentin immunoreactivity. Furthermore, double‐labelling experiments demonstrated that these vimentin‐positive cells were also GFAP‐positive. The reaction became evident on the second day after the lesion, was intense for 3–8 days and then declined until day 21. No vimentin immunoreactivity could be detected at the level of the ipsilateral dorsal cochlear nucleus. Therefore, unilateral inner ear lesion induced an astroglial reaction within the deafferented vestibular and cochlear nuclei. The decrease in the resting discharge of the primary vestibular afferents and/or in the deafferented central vestibular neurons may induce the glial reaction in the vestibular complex, whereas both degeneration and silence of the cochlear nerve and central cochlear neurons are most probably responsible for the cochlear vimentin‐immunoreactive staining. The role of the reactive astrocytes in the vestibular compensation process remains to be determined.
The Competitive Transport Inhibitor L‐trans‐pyrrolidine‐2,4‐dicarboxylate Triggers Excitotoxicity in Rat Cortical Neuron‐Astrocyte Co‐cultures via Glutamate Release rather than Uptake Inhibition
Authors: Volterra Andrea , Bezzi Paola , Rizzini Barbara Lodi , Trotti Davide , Ullensvang Kyrre , Danbolt Niels C. , Racagni Giorgio
Published: April 2006

We studied the early and late effects of L‐trans‐pyrrolidine‐2,4‐dicarboxylate (PDC), a competitive inhibitor of glutamate uptake with low affinity for glutamate receptors, in co‐cultures of rat cortical neurons and glia expressing spontaneous excitatory amino acid (EAA) neurotransmission. At 100 or 200 μM, PDC induced different patterns of electrical changes: 100 μM prolonged tetrodotoxin‐sensitive excitation triggered by synaptic glutamate release; 200 μM produced sustained, tetrodotoxin‐insensitive and EAA‐mediated neuronal depolarization, overwhelming synaptic activity. At 200 μM, but not at 100 μM, PDC caused rapid elevation of the glutamate concentration ([Glu]0) in the culture medium, resulting in NMDA receptor‐mediated excitotoxic death of neurons 24 h later. The increase in [Glu]0 was largely insensitive to tetrodotoxin, independent of extracellular Ca2+, and present also in astrocyte‐pure cultures. By the use of glutamate transporters functionally reconstituted in liposomes, we showed directly that PDC activates carrier‐mediated release of glutamate via heteroexchange. Glutamate release and delayed neurotoxicity in our cultures were suppressed if PDC was applied in a Na+‐free medium containing Li+. However, replacement of Na+ with choline instead of Li+ did not result in an identical effect, suggesting that Li+ does not act simply as an external Na+ substitute. In conclusion, our data indicate that alteration of glutamate transport by PDC has excitotoxic consequences and that active release of glutamate rather than just uptake inhibition is responsible for the generation of neuronal injury.
The Influence of Nitric Oxide on Perigeniculate GABAergic Cell Activity in the Anaesthetized Cat
Authors: Rivadulla C. , Rodriguez R. , Martinez‐Conde S. , Acuña C. , Cudeiro J.
Published: April 2006

We have tested the effect of iontophoretic application of the nitric oxide synthase inhibitor l‐nitroarginine on the activity of a population of 53 perigeniculate (PGN) cells, recorded extracellularly in the anaesthetized paralysed cat. In all cells tested with visual stimulation during l‐nitroarginine application (n= 15), the visually elicited responses were markedly reduced, on average by 63 ± 15%, and there was a reduction in spontaneous activity too. This effect was blocked by co‐application of the substrate for nitric oxide synthase, l‐arginine, but not by the inactive d‐isoform, although application of l‐arginine alone was without effect. Pressure application of the nitric oxide donor S‐nitroso‐N‐acetylpenicillamine (SNAP) elevated both visual responses and spontaneous discharge, an effect also seen with a second nitric oxide donor, sodium nitroprusside (n= 12). The nitric oxide synthase inhibitor l‐nitroarginine was applied to a sub‐population of seven cells and it selectively decreased NMDA mediated excitation (reduction 80 % 14%) with little or no effect on the excitation mediated by α‐amino‐3‐hydroxy‐5‐5‐methyl‐4‐isoxazole‐propionic acid (AMPA) or quisqualate (effects not statistically significant), and it had no effect (n= 7) on excitation mediated by the metabotropic agonist (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (ACPD). Furthermore, application of SNAP also increased the magnitude of excitatory responses mediated by NMDA receptors. On a different population of seven cells, application of the new NO donor diethylamine‐nitric oxide (DEA‐NO) enhanced the actions Of NMDA without an effect on responses to AMPA. These effects are qualitatively and quantitatively similar to those we have previously described for X and Y type cells in the dorsal lateral geniculate nucleus (dLGN), despite the known opposite effects of acetylcholine (ACh) application in the dLGN and PGN (ACh is co‐localized with nitric oxide synthase at both sites). We propose that within the PGN nitric oxide acts to enhance transmission utilizing NMDA receptors selectively (thereby interacting with the globally inhibiting effect of ACh at this site) to enhance visual responses, reducing or removing the non‐specific inhibitory drive from PGN to dLGN seen in the spindling activity of slow‐wave sleep. These effects will act in concert with the facilitatory actions of both ACh and nitric oxide within the dLGN proper, and will thereby enhance the faithful transmission of visual information from retina to cortex.
Structural Analysis of the Murine Cell Adhesion Molecule L1 by Electron Microscopy and Computer‐assisted Modelling
Authors: Drescherl Bernd , Spies Eberhard , Schachner Melitta , Probstmeier Rainer
Published: April 2006

In the present study we have analysed the morphology of two fragments with apparent molecular weights of 180 and 140 kDa (L1‐180 and L1‐140) derived from the extracellular region of the murine neural cell adhesion molecule L1. The fragment L1‐180 consists of almost the entire extracellular part of the molecule, and is built up of six immunoglobulin‐like and five fibronectin type III‐like domains. Fragment L1‐140 lacks one‐half of the third, the fourth and the fifth fibronectin type III‐like domains. By electron microscopic analysis of rotary‐shadowed molecules, L1‐140 and L1‐180 revealed fibrillar structures 31‐43 nm long and 7‐12 nm wide with one pronounced globular terminal domain. As determined by complex formation with an L1 antibody, this terminal part of the molecule is formed by the fibronectin type III‐like domains. The individual structures showed variation and complexity, and four distinct aspects were identified. These different forms probably represent two‐dimensional projections of the same three‐dimensional helical structure. Computer‐assisted modelling of the L1 molecule, i.e. the protein backbone, showed no strong intramolecular interaction between the different fibronectin type III‐ or Ig‐like domains, suggesting that the formation of the globular part of the molecule is probably achieved by protein‐carbohydrate and/or carbohydrate‐carbohydrate rather than protein‐protein interactions. In addition, our model proposes that interactions occur within the interfaces between the different domains. The highly conserved amino acid residues in these regions point to the necessity of maintaining the orientation between the different domains.
Regulation of VIP and other Neuropeptides by c‐Jun in Sensory Neurons: Implications for the Neuropeptide Response to Axotomy
Authors: Mulderry Peter K. , Dobson Stephen P.
Published: April 2006

Peripheral axotomy of adult rat sensory neurons causes induction of the transcription factor c‐Jun and increased expression of the neuropeptides vasoactive intestinal polypeptide (VIP), galanin and neuropeptide Y. To determine whether VIP induction is dependent on transcriptional regulation by c‐Jun, we exploited the fact that c‐Jun and VIP are also induced in cultured sensory neurons. We blocked c‐Jun synthesis by microinjecting antisense oligonucleotides and found that VIP expression, determined by quantitative immunofluorescence, was specifically reduced. Blockade of c‐Jun expression also resulted in reduced neuropeptide Y expression but left galanin, substance P and calcitonin gene‐related peptide unaffected. Since in vitro electrophoretic mobility shift assays showed that a nominal cyclic AMP responsive element (CRE) associated with the rat VIP gene could bind c‐Jun‐containing transcription factor complexes, we next investigated whether VIP expression in sensory neurons might depend on transcription factor binding to the CRE. When a DNA plasmid containing multiple copies of the CRE was injected into newly cultured sensory neurons to sequester transcription factors binding the endogenous CRE, there was a selective reduction in VIP expression. VIP induction in sensory neurons therefore probably results from transcriptional activation by c‐Jun acting in combination with other factor(s), possibly acting through the CRE. These results show that c‐Jun can regulate transcription of other genes affected by axotomy and imply that it could be a key regulator of the neuronal axotomy response.
Developmental Expression and Self‐regulation of Ca2+ Entry via AMPA/KA Receptors in the Embryonic Chick Retina
Authors: Allcorn Suzette , Catsicas Marina , Mobbs Peter
Published: April 2006

Excessive activation of glutamate receptors in the late embryonic and adult retina leads to excitotoxic cell death through an increase in intracellular calcium concentration. Here we use the cobalt‐staining technique of Pruss et al. to investigate the developmental expression of Ca2+‐permeable α‐amino‐3–hydroxy‐5–methyl‐isoxazole‐4‐propionic acid/kainate (AMPNKA) receptors in the embryonic chick retina, and the effects of AMPNKA receptor activation on cell survival and AMPNKA receptor expression. Ca2+‐permeable AMPNKA receptors are present in the retina as early as embryonic day 6 (E6). While sustained activation of these receptors with KA led to massive cell death in explant and dissociated cultures of the chick retina late in development, continuous application of high doses of KA from early times was not excitotoxic. Cell survival in KA is correlated with both a reduction in cobalt staining and the KA‐evoked membrane current, and thus with a reduction in the Ca2+entry into cells via AMPNKA receptors. The effects of KA could be blocked by the non‐N‐methyl‐d‐aspartic acid (NMDA) receptor antagonist 6‐cyano‐7‐nitro‐quinoxaline‐2,3–dione (CNQX), but not by the NMDA receptor antagonist D‐2‐amino‐5‐phosphonovalerate (AP5) nor the L‐type Ca2+ channel blockers diltiazem and nifedipine. The action of AP5 was mimicked by exposure to glutamate but not by the metabotropic receptor agonist 1S,3R‐1–aminocyclopentane‐1,3–dicarboxylic acid. Thus exposure of retinal neurons to glutamate early in development may protect them from its excitotoxic actions later on.
Biphasic Response of Spinal GABAergic Neurons after a Lumbar Rhizotomy in the Adult Rat
Authors: Dumoulin Andréa , Alonso Gérard , Privat Alain , Feldblum Sophie
Published: April 2006

The expression of γ‐aminobutyric acid (GABA) and of the isoforms of the enzyme involved in its synthesis, glutamic acid decarboxylase (GAD), is modified in several rat brain structures in different injury models. The aim of the present work was to determine whether such plasticity of the GABAergic system also occurred in the deafferented adult rat spinal cord, a model where a major reorganization of neural circuits takes place. GABAergic expression following unilateral dorsal rhizotomy was studied by means of non‐radioactive in situ hybridization to detect GADs67 mRNA and by immunohistochemistry to detect GAD67 protein and GABA. Three days following rhizotomy the number of GAD67 mRNA‐expressing neurons was decreased in the superficial layers of the deafferented horn, while GABA immunostaining of axonal fibres located in this region was highly increased. Seven days after lesion, on the other hand, many GAD67 mRNA‐expressing neurons were bilaterally detected in deep dorsal and ventral layers, this expression being correlated with the increased detection of GADs67 immunostained somata and with the reduction of GABA immunostaining of axons. GABA immunostaining was frequently found to be associated with reactive astrocytes that exhibited intense immunostaining for glial fibrillary acidic protein (GFAP) but remained GADs67 negative. These results indicate that degeneration of afferent terminals induces a biphasic response of GABAergic spinal neurons located in the dorsal horn and show that many spinal neurons located in deeper regions re‐express GAD67, suggesting a possible participation of the local GABAergic system in the reorganization of disturbed spinal networks.
Lesion Study of the Distribution of Serotonin 5‐HT4 Receptors in Rat Basal Ganglia and Hippocampus
Authors: Compan Valérie , Daszuta Annie , Salin Pascal , Sebben Michèle , Bockaert Joël , Dumuis Aline
Published: April 2006

The regional distribution of 5‐hydroxytryptamine (5‐HT4) receptors labelled with [3H]GR113808 was examined in rat basal ganglia and hippocampus after specific lesions. Lesion of serotonin neurons induced by injections of 5,7‐dihydroxytryptamine into the dorsal and medial raphe nuclei resulted in increased 5‐HT4 receptor binding in most regions examined, compared with controls. More precisely, there was a 78% increase in the rostral but no change in the caudal part of caudate‐putamen, and 83% and 54% increases in the shell and core of the nucleus accumbens respectively. In the substantia nigra, the increase in 5‐HT4 binding was larger (72%) than that in the globus pallidus (32%). In the hippocampus, 63%, 30% and 28% increases were measured in CA2, CA1 and CA3 respectively. Following lesion of dopamine neurons by intranigral injection of 6‐hydroxydopamine, increased 5‐HT4 receptor binding was observed in the caudal (59%), but not the rostral part of caudate‐putamen, as well as in the globus pallidus (93%). Since no decreases in 5‐HT4 receptor density were detected after the dopamine lesion, it was concluded that these receptors are not expressed in dopamine neurons. Kainic acid lesions of the caudate‐putamen were associated with dramatic local decreases in 5‐HT4 receptor binding on the injected side (‐89%), which suggested that striatal neurons express 5‐HT4 receptors. Corresponding decreases of 72 and 20% in receptor density were detected in globus pallidus and substantia nigra, consistent with a presumed localization of 5‐HT4 receptors on striatal GABA neurons projecting to these regions. In the substantia nigra, the decrease in [3H]GR113808 binding was localized to the pars lateralis, indicating that striatal neurons belonging to the cortico‐striato‐nigrotectal pathway, and containing GABA and dynorphin, express 5‐HT4 receptors.
Induction of Stat3, a Signal Transducer and Transcription Factor, in Reactive Microglia following Transient Focal Cerebral Ischaemia
Authors: Planas Anna M. , Soriano Marc A. , Berruezo Meritxell , Justicia Carles , Estrada Anna , Pitarch Silvia , Ferrer Isidre
Published: April 2006

Stat3, a member of the family of cytoplasmic signal transducers and activators of transcription, was found in the rat brain in vivo under physiological conditions and was stimulated following transient focal cerebral ischaemia. A transient episode of middle cerebral artery occlusion induced a strong microglial response in the areas undergoing neural cell death from 4 days after middle cerebral artery occlusion. This was accompanied by increased expression of Stat3 in the ipsilateral cortex and striatum, as revealed by Western blotting of tissue extracts. immunohistochemistry showed strong induction of Stat3 in reactive microglial cells 4, 7 and 15 days after cerebral ischaemia. Stat3 was seen in the microglia cytoplasm, but in many microglial cells immunoreactivity was also distributed within the nucleus. These results suggest that Stat3 mediates signal transduction and activates transcription in reactive microglia in vivo following brain ischaemia.
The Nitric Oxide/Cyclic GMP Messenger System in Olfactory Pathways of the Locust Brain
Authors: Bicker Gerd , Schmachtenberg Oliver , De Vente Jan
Published: April 2006

Nitric oxide is generated by a Ca2+/calmodulin‐stimulated nitric oxide synthase and activates soluble guanylyl cyclase. Using NADPH diaphorase (NADPHd) staining as a marker for the enzyme nitric oxide synthase and an antiserum against cGMP, we investigated the cellular organization of nitric oxide donor and target cells in olfactory pathways of the brain of the locust (Schistocerca gregaria). A small subset of neuronal and glial cells expressed cGMP immunoreactivity after incubation of tissue in a nitric oxide donor. Nitric oxide‐induced increases in cGMP immunoreactivity were quantified in a tissue preparation of the antennal lobe and in primary mushroom body cell cultures. The mushroom body neuropil is a potential target of a transcellular nitric oxide/ cGMP messenger system since it is innervated by extrinsic NADPHd‐positive neurons. The mushroom body‐intrinsic Kenyon cells do not stain for NADPHd but can be induced to express cGMP immunoreactivity. The colocalization of NADPHd and cGMP immunoreactivity in a cluster of interneurons of the antennal lobe, the principal olfactory neuropil of the insect brain, suggests a role of the nitric oxide/cGMP system in olfactory sensory processing. Colocalization of NADPHd staining and cGMP immunoreactivity was also found in certain glial cells. The cellular organization of the nitric oxide/cGMP system in neurons and glia raises the possibility that nitric oxide acts not only as an intercellular but also as an intracellular messenger molecule in the insect brain.
Sodium Current Amplitude Increases Dramatically in Human Retinal Glial Cells during Diseases of the Eye
Authors: Francke M. , Pannicke T. , Biedermann B. , Faude F. , Reichelt W.
Published: April 2006

Müller cells, the main macroglial cells of the retina, express several types of voltage and ligand‐activated ion channels, including Na‐ channels. Using the whole‐cell voltage‐clamp technique, we studied the expression of Na‐ currents in acutely isolated, non‐cultivated human Muller cells from retinas of healthy organ donors and patients suffering from different eye diseases. In both types of retinas transient Na+ currents could be recorded from Muller cells. The tetrodotoxin‐resistant Na+ currents, which were not completely blocked even at a concentration of 10 $muM tetrodotoxin, had a mean current density of 3.0± 3.0 pA/pF (mean ± SD, n = 10) in Muller cells from donor retinas and of 12.2± 2 9.6 pA/pF (n= 74) in Muller cells from patient retinas. Only 33.3% of healthy but 88.4% of pathological Muller cells depicted such currents. The GNa+/GK+ ratio was very high in several Muller cells from patient retinas, such that action potential‐like activity could be generated after prehyperpolarizing current injection in some of these cells. Apparently, the Na’channels, due to their negative steady‐state inactivation curve (Vh= ‐84.5 mV), do not influence the lowered membrane potential of the pathological cells, since they are inactivated at these voltages. Currently, we do not have an explanation for the increase in amplitude and frequency of Na+ currents in human Muller cells under pathological conditions. However, the up‐regulation of Na channels may mirror a basic glial response to pathological conditions, since it has also been found previously in human hippocampal astrocytes from epileptic foci and in rat cortex stab wounds lined by an astrocytic scar.
Rat Brain: Distribution of Immunoreactivity of PE‐11, a Peptide Derived from Chromogranin B
Authors: Kroesen S. , Marksteiner J. , Leitner B. , Hogue‐Angeletti R. , Fischer‐Colbrie R. , Winkler H.
Published: April 2006

An antiserum was raised against the peptide PE‐11 whose sequence is present in the chromogranin B molecule. The antiserum reacts only with the free C‐terminal end of this peptide. PE‐11 immunoreactivity in brain was characterized by molecular size exclusion high performance liquid chromatography. Only the free peptide and a N‐terminally elongated peptide were detected, indicating that proteolytic processing of chromogranin B in brain is quite extensive. In immunohistochemistry PE‐11 immunoreactivity was found in varicosities, fibres and perikarya throughout the brain. Strong staining was detected in the shell sector of the nucleus accumbens, in the lateral septum, in subregions of the extended amygdala, in some areas of the hippocampus and of the hypothalamus, in the locus coeruleus, in the Purkinje cells of the cerebellum and in the dorsal horn of the spinal cord. Our results, which demonstrate significant processing of chromogranin B in brain and its widespread distribution, can be taken as an indication that chromogranin B represents a precursor of peptides with functional relevance for this organ.
Developmental Study of N‐Methyl‐d‐Aspartate‐induced Firing Activity and Whole‐cell Currents in Nucleus Tractus Solitarii Neurons
Authors: Vincent Anne , Jean André , Tell Fabien
Published: April 2006

Whole‐cell recordings of rat nucleus tractus solitarii (NTS) neurons were performed on a slice preparation. We investigated possible postnatal changes in firing activities and currents induced by N‐methyl‐d‐aspartate (NMDA) application. A total of 42 neurons were selected and fell into the following age groups: 0‐5 days (n= 15), 10‐15 days (n= 9) and 30‐60 days (adult, n= 18). During this period, input resistance and spike duration decreased by‐˜40%. At all ages, bath application of NMDA elicited a bursting firing activity when the membrane potential was held between ‐60 and ‐75 mV. However, in the youngest cells the rhythmic bursting activity was irregular and was characterized by a progressive firing inactivation during a burst. In a tetrodotoxin‐containing saline, NMDA‐induced oscillations of membrane potential were retained in all age groups. The membrane current‐voltage relationship of the NMDA‐induced inward current (INMDA) was characterized by a region of negative slope conductance which was similar in all age groups. Thus the voltage‐dependent block of INMDA is present in NTS neurons from birth, allowing NTS neurons to display membrane potential oscillations. However, postnatal maturation of repolarizing conductances, as suggested by changes in spike characteristics, could render the oscillatory activity more stable than at birth.
Characterization of a Distinctive Motif of the Low Molecular Weight Neurotrophin Receptor that Modulates NGF‐mediated Neurite Growth
Authors: Dostaler S. M. , Ross G. M. , Myers S. M. , Weaver D. F. , Ananthanarayanan V. , Riopelle R. J.
Published: April 2006

The cytoplasmic region of the common neurotrophin receptor (p75NGFR) (rat, human, chick) contains a putative membrane‐associating domain implicated in intracellular signalling. A peptide (R3) identical to this domain (p75NGFR 367–379) and various analogues of this peptide displayed circular dichroism spectra in aqueous and non‐polar environments identical to the amphiphilic tetradecapeptide mastoparan (MP) and were internalized by PC12 rat pheochromocytoma cells. The R3 peptide enhanced neurite growth in PC12 cells, embryo chick primary sensory neurons and fetal rat primary sensory neurons in vitro in the presence of sub‐saturating concentrations of NGF. Peptide analogues of R3 not faithful to the distance and angular relationships of ionic groups and the putative amphiphilic structure of p75NGFR 367–379 displayed reduced potency to enhance NGF‐mediated neurite growth. Exposure of NGF and the R3 peptide to a cell line displaying predominantly p75NGFR (PC12nnr5), had no influence on neurite growth. The R3 peptide had no effects on cell survival, cell binding or uptake of [125I]NGF, affinity cross‐linking of [125I]NGF to p75NGFR or trkA monomers and homodimers, or NGF‐mediated trkA monomer tyrosine phosphorylation. The studies implicate a role for a highly conserved motif of p75NGFR in the downstream modulation of NGF‐mediated neurite growth.
Cranial Motor Axons Respond Differently to the Floor Plate and Sensory Ganglia in Collagen Gel Co‐cultures
Authors: Tucker Anna , Lumsden Andrew , Guthrie Sarah
Published: April 2006

Within the developing chick hindbrain, motor neurons differentiate in columns on either side of the ventral midline floor plate. Along the rostrocaudal axis, populations of motor neurons are organized segmentally with the trigeminal (V) and facial (VII) nuclei occupying successive pairs of rhombomeres. To reach their targets, motor axons follow stereotyped pathways. Branchiomotor and visceral motor axons of the Vth and VIIth nerves first project in a dorsal (lateral) direction away from the floor plate and towards the nerve exit point located in the alar plate of the even‐numbered rhombomere of the pair. Having exited the hindbrain, axons grow in association with the cranial sensory ganglia before branchiomotor axons enter the branchial arches. We have investigated some of the factors that might guide cranial motor axons using a three‐dimensional collagen gel culture system. When explants of hindbrain basal plate containing trigeminal or facial motor neurons were co‐cultured with floor plate explants, axon outgrowth from the side facing the floor plate was inhibited in a manner consistent with chemorepulsion. When basal plate explants that contained an exit point were cultured alone, motor axons grew to the exit point and then stopped. When basal plate explants were co‐cultured with trigeminal ganglia, motor outgrowth was increased in comparison with that in control cultures, suggesting a trophic influence. The findings presented here indicate that motor pathways are elaborated due to a progression of signals to which the growth cones respond in sequence.
Involvement of γ and β Actin Isoforms in Mouse Neuroblastoma Differentiation
Authors: Ulloa Luis , Avila Jesus
Published: April 2006

Two actin isoforms, γ and β, are contained within neuroblastoma cells. However, the relative amount and distribution of both isoforms within the cells are differentially regulated during neurite extension. The proportion of γ actin isoform became about four times greater than that of β actin during neuroblastoma cell differentiation. Additionally, whereas β actin appears to be concentrated in the cell cortex, γ actin is also present throughout the cell body. Upon differentiation, neuroblastoma cells reorganize their actin cytoskeleton and γ actin is induced to polymerize whereas β actin polymers are partially disassembled. Moreover, both actin isoforms are differentially distributed within differentiated cells. Thus, γ actin polymers are located both in the soma and proximal regions of extended neurites, whereas β actin is enriched in the terminal tip of the neurites. Our results strongly suggest that both actin isoforms are involved in a different way in neuroblastoma cell differentiation.