bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of a Large‐conductance Ca2+‐Dependent K+ Channel by Stimulation of Glutamate Phosphoinositide‐coupled Receptors in Cultured Cerebellar Granule Cells
Authors: Fagni L. , Bossu J. L. , Bockaert J.
Published: April 2006

Trans‐1‐amino‐cyclopentyl‐1,3‐dicarboxylic acid (trans‐ACPD), a specific agonist of the glutamate phosphoinositide‐coupled receptor (Qp receptor), increased the amplitude of the outward K+ current recorded in the whole‐cell configuration of the patch‐clamp technique in mouse cultured cerebellar granule cells. This effect was abolished by buffering internal Ca2+ with BAPTA [1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid]. Activation of a large‐conductance K+ channel was observed when trans‐ACPD or quisqualic acid (QA), another Qp receptor agonist, was applied outside the cell‐attached patch pipettes. No activation was observed with alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA), a specific agonist of ionotropic non‐N‐methyl‐d‐aspartate (non‐NMDA) receptors. The effects of trans‐ACPD or QA were potentiated in the presence of external Ca2+. The channel was also directly activated by both micromolar concentrations of internal Ca2+ and membrane depolarization. Its unitary conductance was 100–115 pS under asymmetrical K+ and 195–235 pS under high symmetrical K+ conditions. In the absence of agonist, the channel was blocked by 1 mM external tetraethylammonium. This is the first description of a large conductance Ca2+‐activated K+ channel in cultured cerebellar granule cells. It possesses properties similar to those of the so‐called ‘big K+ channel’ described in other preparations. Our cell‐attached experiments demonstrated an indirect coupling between Qp receptors and this channel. The most likely hypothesis is that the second messenger system inositol 1,4,5‐triphosphate (IP3)‐Ca2+ was involved in the coupling process. This hypothesis was further strengthened by our whole‐cell experiments. On the basis of the voltage‐ and Ca2+‐sensitivities of the studied channel, we estimated an increase of 350 to 570 nM in internal Ca2+ concentration when Qp receptors were stimulated by 100 μM trans‐ACPD. Under physiological conditions, stimulation of Qp receptors by the endogenous neurotransmitter should lead to similar K+ channel activation and therefore would tend to reduce the efficacy of ionotropic glutamate synaptic receptor stimulation responsible for cell excitation.
Single Channel Recording from Glial Cells on the Untreated Surface of the Frog Optic Nerve
Authors: Marrero Hector , Orkand Paula M. , Kettenmann Helmut , Orkand Richard K.
Published: April 2006

The patch clamp technique has been used to record single channel currents from the untreated surface of the intact frog optic nerve after the meninges and basal lamina have been mechanically removed. Cells filled via dialysis with Lucifer yellow (LY) from the patch pipette had a typical astrocyte morphology and were dye‐coupled to adjacent astrocytes. This is consistent with the electron‐microscopic observation that all the cells on the surface of this nerve are astrocytes. Two types of ion channels were studied in detached patches. One, identified as a K+ channel, had a conductance of 88±4 (S.E.) n= 9 pS and an equilibrium potential of −59±8 mV in physiological K+ solutions. The steady‐state open probability was not significantly altered by changing the membrane potential. A second channel had a large conductance of 300–1200 pS, a reversal potential of ∼ 0 mV in symmetrical and non‐symmetrical solutions, and was open only in the voltage range of ±20 mV. These are the characteristics of a large anionic channel described in other preparations including cultured astrocytes.
High‐Resolution Light and Electron Microscopic Immunocytochemistry of Colocalized GABA and Calbindin D‐28k in Somata and Double Bouquet Cell Axons of Monkey Somatosensory Cortex
Authors: DeFelipe J. , Jones E. G.
Published: April 2006

A simple method for high‐resolution immunocytochemical colocalization of different antigens in semithin sections 1 ‐ 3 μm thick was used to study the colocalization of the calcium binding protein calbindin D‐28k (calbindin) with γ‐aminobutyric acid (GABA) in double bouquet cells of monkey (Macaca fuscata) somatosensory cortex. Double bouquet cells were first visualized in vibratome sections by pre‐embedding immunocytochemical staining for calbindin. Sections containing calbindin‐immunoreactive somata and double bouquet cell axons were then osmicated, embedded in Araldite, resectioned at 1–3μm and stained for GABA by postembedding immunocytochemistry after elution of the bound anti‐calbindin antibodies. Other semithin sections adjacent to those eluted and still containing calbindin immunoreactive somata and processes were resectioned at 60–70 nm for electron microscopy and stained immunocytochemically for GABA by the postembedding immunogold procedure. Calbindin‐positive cells are most numerous in layer II and upper layer III, where they outnumber those in all other layers combined. In layers II and upper III, ‐30% of the stained cells are pyramidal and do not colocalize GABA. Only approximately two‐thirds of the calbindin‐stained nonpyramidal cells in these layers colocalize GABA, but among these virtually all the calbindin‐positive double bouquet cells and their axons are GABA‐immunoreactive. In deeper layers all calbindin‐positive cells are nonpyramidal and all colocalize GABA. At the electron microscopic level, however, significant numbers of calbindin‐positive axon terminals making symmetrical synapses are not GABA‐immunoreactive. These results suggest the calbindin cells of monkey somatosensory cortex are a heterogeneous population that includes GABAergic and non‐GABAergic cell types.
Accelerated Differentiation in Response to Retinoic Acid After Retrovirally Mediated Gene Transfer of GAP‐43 into Mouse Neuroblastoma Cells
Authors: Morton A. Jennifer , Buss Timothy N.
Published: April 2006

Although substantial evidence exists for the involvement of growth‐associated protein‐43 (GAP‐43) in neuronal development and regeneration, the precise role of this protein in neurite outgrowth is currently debated. To investigate the role of GAP‐43 in the initiation of neurite outgrowth, we transfected a full‐length cDNA coding for GAP‐43 into a mouse neuroblastoma cell line (Neuro‐2a) which can be differentiated to a neuronal phenotype using retinoic acid (RA). We show that the consequent overexpression of GAP‐43 results in a change in the basic morphology of these cells, but is not in itself sufficient to induce the extension of neurites. However, overexpression of GAP‐43 results in a marked acceleration of neurite formation in response to RA. We propose that while GAP‐43 does not trigger the initiation of neurite extension, its expression is rate‐limiting for neurite outgrowth in response to differentiation agents such as RA.
Long‐term Potentiation in the Striatum is Unmasked by Removing the Voltage‐dependent Magnesium Block of NMDA Receptor Channels
Authors: Calabresi P. , Pisani A. , Mercuri N. B. , Bernardi G.
Published: April 2006

We have studied the effects of tetanic stimulation of the corticostriatal pathway on the amplitude of striatal excitatory synaptic potentials. Recordings were obtained from a corticostriatal slice preparation by utilizing both extracellular and intracellular techniques. Under the control condition (1.2 mM external Mg2+), excitatory postsynaptic potentials (EPSPs) evoked by cortical stimulation were reversibly blocked by 10 μM 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), an antagonist of dl‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA) ionotropic glutamate receptors, while they were not affected by 30–50 μM 2‐amino‐5‐phosphonovalerate (APV), an antagonist of N‐methyl‐d‐aspartate (NMDA) glutamate receptors. In the presence of 1.2 mM external Mg2+, tetanic activation of cortical inputs produced long‐term depression (LTD) of both extracellularly and intracellularly recorded synaptic potentials. When Mg2+ was removed from the external medium, EPSP amplitude and duration increased. In Mg2+‐free medium, cortically evoked EPSPs revealed an APV‐sensitive component; in this condition tetanic stimulation produced long‐term potentiation (LTP) of synaptic transmission. Incubation of the slices in 30–50 μM APV blocked striatal LTP, while it did not affect LTD. In Mg2+‐free medium, incubation of the slices in 10 μM CNQX did not block the expression of striatal LTP. Intrinsic membrane properties (membrane potential, input resistance and firing pattern) of striatal neurons were altered neither by tetanic stimuli inducing LTD and LTP, nor by removal of Mg2+ from the external medium. These findings show that repetitive activation of cortical inputs can induce long‐term changes of synaptic transmission in the striatum. Under control conditions NMDA receptor channels are inactivated by the voltage‐dependent Mg2+ block and repetitive cortical stimulation induces LTD which does not require activation of NMDA channels. Removal of external Mg2+ deinactivates these channels and reveals a component of the EPSP which is potentiated by repetitive activation. Since the striatum has been involved in memory and in the storage of motor skills, LTD and LTP of synaptic transmission in this structure may provide the cellular substrate for motor learning and underlie the physiopathology of some movement disorders.
The Effect of Kainic Acid Lesions of the Cerebellar Cortex on the Conditioned Nictitating Membrane Response in the Rabbit
Authors: Hardiman M. J. , Yeo C. H.
Published: April 2006

In previous studies we have shown that aspiration lesions centred on lobule HVI in the cerebellar cortex of rabbits produce a profound loss of conditioned nictitating membrane (NM) responses. Because aspiration lesions of the cerebellar cortex cause retrograde degeneration in precerebellar nuclei we tested in rabbits whether excitotoxic lesions of the cerebellar cortex that spare these precerebellar nuclei also cause a loss of conditioned NM responses. Following discrete injections of kainic acid into HVI and rostral regions of the adjacent folia of crus I and crus II, we observed an immediate loss of conditioned NM responses. Following extensive retraining several subjects showed a gradual recovery of conditioned responses. But subjects with the most complete lesions never recovered more than a few conditioned responses. Kainic acid lesions did not change ipsilateral unconditioned reflex responses to a range of stimulus intensities. The kainic acid injections caused obvious degeneration of Purkinje and granule cells but not of the precerebellar nuclei. We conclude that HVI and parts of crus I and crus II are essential for normal retention of conditioned NM responses.
Endothelin‐evoked Release of Arachidonic Acid from Mouse Astrocytes in Primary Culture
Authors: Tencé Martine , Cordier Jocelyne , Glowinski Jacques , Prémont Joël
Published: April 2006

In striatal astrocytes, receptors for the vasoactive peptide endothelin (ET) are associated with several intracellular signalling pathways: ET‐1 increases the breakdown of phosphoinositides, induces a sustained influx of Ca2+ and inhibits the isoproterenol‐induced formation of cAMP (Marin et al., J. Neurochem., 56, 1270–1275, 1991). In the present study, it will be shown that ET‐1 and ET‐3 markedly stimulate the release of arachidonic acid (AA) from cultured astrocytes from the mouse striatum (EC50= 3 and 7 nM for ET‐1 and ET‐3, respectively), mesencephalon and cerebral cortex. The ET‐1‐evoked release of AA probably resulted from the activation of a phospholipase A2, since it required extracellular Ca2+ and was prevented by mepacrine but not by RHC 80267, an inhibitor of diacylglycerol lipase. The ET‐1‐induced release of AA was shown to be partially mediated by a guanine nucleotide‐binding protein sensitive to pertussis toxin but not to cholera toxin. A cAMP‐dependent process is not involved since the ET‐1‐evoked release of AA was not affected when cells were incubated with either isoproterenol or 8‐bromo‐cAMP. The ET‐1‐evoked release of AA could be mimicked by the co‐application of a calcium ionophore and a protein kinase C activator. However, staurosporine, a potent inhibitor of protein kinase C, which blocked the release of AA induced by the combined application of ionomycin and phorbol 12‐myristate 12‐acetate (PMA), was without effect on the ET‐1‐evoked response, indicating that protein kinase C is not directly involved in the ET‐1‐induced release of AA. Furthermore, the responses induced by ET‐1 and by PMA were found to be additive. These results suggest that (1) ET‐1 receptors are coupled to the release of AA by a mechanism independent of both protein kinase C activation and the adenylate cyclase pathway, possibly via the activation of phospholipase A2, (2) different mechanisms (or different phospholipase A2 subtypes) are involved in the control of AA release in astrocytes.
Simulation of the Effect of Synapses: the Significance of the Dendritic Diameter in Impulse Propagation
Authors: Wolf E. , Birinyi A. , Székely G.
Published: April 2006

The effectiveness of synapses at various sites of the dendritic tree was studied using a segmental cable model with a program developed by Hines (Int. J. Biomed. Comput., 24, 55–68, 1989). The model rendered possible a high‐fidelity simulation of the dendritic geometry of a frog motoneuron described in the accompanying paper (Birinyi et al., Eur. J. Neurosci., 1003–1012, 1992). The model was used in the passive membrane mode and the synaptic activity was simulated with current injections into large and small diameter dendrites at proximal and distal locations. Synaptic efficiency was defined by the charge transfer ratio expressed as the proportion of the injected current which appeared at the soma. The charge transfer ratio was determined with uniform and non‐uniform distribution of specific membrane resistance over the soma–dendrite surface while the diameter of selected dendrite segments changed. The best charge transfer ratio was found with the largest dendrite membrane resistance, and the maximum efficiency of synaptic activity appeared at the original size of the dendrite segment stimulated. The amount of current that flowed in the proximal and distal directions from the segment stimulated depended on the diameter of that segment. The increase in diameter of proximal dendrites increased synaptic efficiency on distal dendrites, whereas the reverse caused a decline in synaptic efficiency on proximal dendrites. In addition to the diameter of dendrites, the arborization pattern also played a significant role in this mechanism. It is concluded that the cellulipetal increase in dendrite diameter greatly increases synaptic efficiency.
Altered Topography in the Geniculo‐cortical Projection of the Golden Hamster Following Neonatal Monocular Enucleation
Authors: Trevelyan A. J. , Thompson I. D.
Published: April 2006

The consequence of neonatal eye removal on the adult organization of the geniculo‐cortical pathway was studied anatomically in hamsters. Separate discrete injections of rhodamine‐ and green‐fluorescent latex microspheres were made into the primary visual cortex of adult hamsters. The distribution of labelling in the dorsal lateral geniculate nucleus (dLGN) of normal animals was compared with that seen in animals monocularly enucleated at birth. In the normal animals, as expected, the projection has a precise topographic order. This is also true of the projection contralateral to the remaining eye in the enucleated animals. However, on the side ipsilateral to the remaining eye, the visual cortex appears to receive two convergent projections from the deafferented dLGN, one mirroring the other. A single injection made in very lateral cortex labels cells in two discrete regions of the dLGN. As the injection is made progressively more medial, the two patches of labelled cells converge. Eventually, the two patches are no longer discrete so that injections into central area 17 produce just one, extended patch of labelling. These results suggest that the altered retinal input to the dLGN may affect the subsequent development of ordered geniculo‐cortical projections.
Cells Expressing mRNA for Neurotrophins and their Receptors During Embryonic Rat Development
Authors: Ernfors Patrik , Merlio Jean‐Phillipe , Persson Håkan
Published: April 2006

In situ hybridization analysis of cells expressing messenger RNAs (mRNAs) for the neurotrophins nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 (NT‐3) and their high‐affinity receptors (trk, trkB and trkC) in the rat embryo revealed a complex but specific expression pattern for each of these mRNAs. For all mRNAs a developmentally regulated expression was seen in many different tissues. BDNF and NT‐3 mRNAs were expressed in the sensory epithelia of the cochlea and vestibule macula of the sacculus and utricle, and both trkB and trkC mRNA were expressed in the spiral and vestibule ganglia innervating these sensory structures. NGF and NT‐3 mRNA were found in the iris, innervated by the sympathetic neurons of the superior cervical ganglion and sensory neurons from the trigeminal ganglion, which expressed both trk and trkC mRNAs. Both NGF and NT‐3 mRNAs were also expressed in other target fields of the trigeminal ganglion, the epithelium of the whisker follicles (NT‐3 mRNA) and in the epithelium of the nose, tongue and jaw. NT‐3 mRNA was found in the cerebellar external granule layer and trkC mRNA in the Purkinje layer of the cerebellar primordia. These sites of synthesis are consistent with a target‐derived neurotrophic interaction for NGF, BDNF and NT‐3. However, in some cases mRNAs for both the neurotrophins and their high‐affinity receptors were detected in the same tissue, including the dorsal root, geniculate, superior, jugular, petrose and nodose ganglia, as well as in the hippocampus, frontal cortical plate and pineal recess, implying a local mode of action. Combined, these data suggest a broad function for the neurotrophins and their receptors in supporting neural innervation during embryonic development. The results also identify several novel neuronal systems that are likely to depend on the neurotrophins in vivo.
Widespread Distribution of Synaptophysin, a Synaptic Vesicle Glycoprotein, in Growing Neurites and Growth Cones
Authors: Phelan Pauline , Gordon‐Weeks Phillip R.
Published: April 2006

Synaptophysin, a 38‐kD glycoprotein, is one of the most abundant of the integral membrane proteins of small synaptic vesicles. The protein is widely distributed at synapses throughout the nervous system, where it is believed to be involved in the exocytosis of stored neurotransmitter. We show here that synaptophysin is also widely expressed in growing neurites and growth cones both in vitro and in vivo. In dissociated rat cerebral cortical cultures anti‐synaptophysin antiserum (G‐95) stains growth cones punctately as soon as they emerge from the cell body. In early cultures all neurites are immunoreactive. Later, synaptophysin is redistributed to become concentrated in axonal varicosities. In developing rat embryos, synaptophysin is expressed in the growing axons of, for instance, the spinal commissural interneurons and the parallel fibres of the cerebellar granule cells long before these neurons have established synaptic connections. These observations suggest that synaptic vesicle proteins like synaptophysin are functionally important in neuronal development.
Noradrenergic, but not Adrenergic Chromaffin Cells in the Adrenal Gland Express Neuromodulin (GAP‐43)
Authors: Grant Nancy J. , König Fabienne , Deloulme Jean‐Christophe , Aunis Dominique , Langley Keith
Published: April 2006

Neuroendocrine chromaffin cells of the adrenal gland express certain molecular markers either transiently during development or permanently. In the present study, the expression of neuromodulin (GAP‐43), a neuronal protein often associated with neurite outgrowth, was examined in adult adrenals. Neuromodulin was detected by Western blot analysis in extracts of both rat adrenals and cultured bovine chromaffin cells, and was localized in situ in a subpopulation of chromaffin cells, as well as in nerve fibres and Schwann cells. The use of anti‐tyrosine hydroxylase or anti‐phenylethanolamine N‐methyltransferase antibodies in combination with anti‐neuromodulin antibodies in double immunofluorescent labelling of cryostat sections of rat glands demonstrated that neuromodulin is expressed by noradrenergic, and not by adrenergic chromaffin cells. The results provide further evidence that neuromodulin is not limited to neurons; it is also expressed in a subpopulation of neuroendocrine chromaffin cells. Neuromodulin may play a role in the development of the adrenal medulla or in the specific regulation of noradrenalin secretion from chromaffin cells.
Sodium and Calcium Currents in Glial Cells of the Mouse Corpus Callosum Slice
Authors: Berger Thomas , Schnitzer Jutta , Orkand Paula M. , Kettenmann Helmut
Published: April 2006

We studied Na+ and Ca2+ currents in glial cells during the development of the corpus callosum in situ. Glioblasts and oligodendrocytes from frontal brain slices of postnatal day (P) 3 to P18 mice were identified based on morphological and ultrastructural features after characterization of the currents with the patch‐clamp technique. Slices from P3‐P8 mice contained predominantly glioblasts with immature morphological features. These cells showed Na+ and Ca2+ currents, but the population with these currents decreased between P3 and P8. Na+ currents were blocked in Na+‐free bathing solution and in the presence of tetrodotoxin, Ca2+ currents were only observed when a high concentration of extracellular Ba2+ was present. The cells from the corpus callosum of P10 – P18 mice predominantly had morphological features of oligodendrocytes. In these cells, which in some cases were shown to form myelin, neither Na+ nor Ca2+ currents were detected. To compare these in situ results with those from the electrophysiologically and immunocytochemically well‐characterized cultured glial cells, we determined the expression pattern of stage‐specific antigens in the corpus callosum in situ. The first O4 antigen‐positive glial precursors were observed at P1, the earliest stage examined. The oligodendrocytic antigens O7 and O10 appeared at P6 and P14, respectively, and prominent labelling with the corresponding markers was seen at P12 and P18, respectively. Despite the existence of numerous mature, O10‐positive oligodendrocytes at P18, which expressed Ca2+ channels in vitro, we failed to detect Ca2+ currents in situ at this stage.
Heterogeneous Expression Patterns of Mammalian Potassium Channel Genes in Developing and Adult Rat Brain
Authors: Kues Wilfried A. , Wunder Frank
Published: April 2006

Voltage‐gated K+ channels in the mammalian brain are functionally heterogeneous. Mechanisms which may underlie heterogeneity are the expression of multiple K+ channel subunit genes, alternative splicing and the formation of heteromultimers from different subunits. To examine the molecular basis of regional and cell‐specific K+ channel expression in rat brain in situ hybridization techniques were used. The transcript distribution patterns of 11 cloned mammalian K+ channel genes encoding both slow‐ and fast‐inactivating K+ channels from four different gene families were examined at different stages of development. The results show that each subunit‐specific messenger RNA (mRNA) is independently expressed and is characterized by an individual expression pattern. In the hippocampal formation transcripts of RCK2, RCK3, RCK4, RCK5, Raw3 and rat Shal genes are heterogeneously expressed and regulated during postnatal development. RCK1, Raw1, Raw2 and DRK1 mRNAs, on the other hand, are present in the hippocampus throughout postnatal life. The expression patterns of the 11 genes partially overlap, suggesting the formation of different heteromultimeric K+ channel complexes.
Light and Electron Microscopic Localization of the Neutral Metalloendopeptidase EC 3.4.24.16 in the Mesencephalon of the Rat
Authors: Woulfe John , Checler Frederic , Beaudet Alain
Published: April 2006

The topographic and cellular distribution of the neurotensin‐hydrolysing neutral metalloendopeptidase 24.16 (EC 3.4.24.16) was examined by light and electron microscopic immunohistochemistry in adult rat mesencephalon. Light microscopic immunoradioautography revealed a ubiquitous distribution of the enzyme throughout the midbrain with a relative enrichment of grey matter areas including the substantia nigra, ventral tegmental area, interfascicular nucleus, interpeduncular nucleus, rostral and caudal linear raphe nuclei, central grey and superficial grey of the superior colliculus. Peroxidase‐antiperoxidase immunocytochemistry revealed two distinct cellular patterns of immunostaining: (1) weakly labelled neuronal perikarya more or less uniformly distributed throughout the grey matter, and (2) intensely immunoreactive glial cells heterogeneously distributed across the mesencephalon. Areas exhibiting dense concentrations of endopeptidase 24.16‐containing glial cells corresponded to those displaying enhanced immunoreactivity in immunoradioautographs, suggesting that a major proportion of brain endopeptidase 24.16 is associated with glia. Electron microscopic examination of the substantia nigra and ventral tegmental area confirmed the association of the enzyme with a subpopulation of neurons and allowed identification of labelled glial cells as protoplasmic astrocytes. In neurons, endopeptidase 24.16 immunoreactivity was distributed heterogeneously within the cytoplasm of perikarya, dendrites and axons. Reaction product was also characteristically associated with restricted zones of the plasma membrane and underlying neuroplasm. In astrocytes, endopeptidase 24.16 immunostaining was densely and uniformly distributed throughout the cytoplasm of cell bodies and processes. Many of these processes were in direct contact with endopeptidase 24.16‐immunopositive neuronal elements. The present results demonstrate that within the midbrain, endopeptidase 24.16 is both intracytoplasmic and membrane‐associated in neurons and predominantly intracytoplasmic in glia. The presence of a large number of immunostained elements within areas of the midbrain known to display high levels of neurotensin and/or neurotensin receptors, together with the demonstrated catabolic activity of the enzyme on neurotensin in vitro, is consistent with a role of endopeptidase 24.16 in the functional inactivation of endogenous neurotensin in this region of the brain.
Effects of P‐Aminophenyl Dichloroarsine on Reduced High‐affinity [3H]Nicotine Binding Sites from Chick Brain: A Covalent, Yet Reversible, Agent for Neuronal Nicotinic Receptors
Authors: Pike Ashley , Loring Ralph H.
Published: April 2006

Neuronal nicotinic acetylcholine receptor (nAChR) α‐subunits contain a conserved disulphide that is essential for function. Here, we have examined the effects of sulphydryl redox reagents on [3H]nicotine binding to chick brain nAChR immunoisolated with the monoclonal antibody mAb35. The disulphide reducing agent, dithiothreitol (DTT), inhibited [3H]nicotine binding [50% inhibitory concentration (IC50) = 146 μM] but this effect was reversed (93±1.5%) by subsequent reoxidation with 1 mM dithio‐bis(nitrobenzoic acid) (DTNB). The trivalent arsenical, p‐aminophenyl dichloroarsine (APA), which reacts with pairs of spatially close sulphydryls, was a potent inhibitor of reoxidation by DTNB (IC50= 35 nM). However, application of the ‘anti‐arsenical’, 2,3‐dimercaptopropane sulphonic acid (DMPS), restored agonist binding after APA treatment (50% effective concentration = 120 μM). Paradoxically, DMPS was also found to be a potent oxidizing agent of these receptors. Affinity alkylation of reduced nAChRs with bromoacetylcholine (BAC; 100 μM) irreversibly blocked nicotine binding (>90%). We propose (but have not proven) that APA interacts with the cysteines homologous to Cys192–193 in Torpedo AChRs, since APA pretreatment of reduced neuronal receptors protected against irreversible BAC alkylation, as shown by subsequent reversal of DMPS (2 mM; 20 min). This study illustrates the potent and reversible nature of the arsenical's covalent interaction with an isolated nAChR and suggests that modified arsenicals could be useful nAChR probes.
MAP2 Isoforms in Developing Cat Cerebral Cortex and Corpus Callosum
Authors: Riederer Beat M. , Innocenti Giorgio M.
Published: April 2006

The microtubule‐associated protein MAP2 was studied in the developing cat visual cortex and corpus callosum. Biochemically, no MAP2a was detectable in either structure during the first postnatal month; adult cortex revealed small amounts of MAP2a. MAP2b was abundant in cortical tissue during the first postnatal month and decreased in concentration towards adulthood; it was barely detectable in corpus callosum at all ages. MAP2c was present in cortex and corpus callosum at birth; in cortex it consisted of three proteins of similar molecular weights between 65 and 70 kD. The two larger, phosphorylated forms disappeared after postnatal day 28, the smaller form after day 39. In corpus callosum, MAP2c changed from a phosphorylated to an unphosphorylated variant during the first postnatal month and then disappeared. Immunocytochemical experiments revealed MAP2 in cell bodies and dendrites of neurons in all cortical layers, from birth onwards. In corpus callosum, in the first month after birth, a little MAP2, possibly MAP2c, was detectable in axons. The present data indicate that MAP2 isoforms differ in their cellular distribution, temporal appearance and structural association, and that their composition undergoes profound changes during the period of axonal stabilization and dendritic maturation.
Bulbectomy Enhances Neurogenesis and Cell Turnover of Primary Olfactory Neurons But Does Not Abolish Carnosine Expression
Authors: Biffo Stefano , Pognetto Marco Sassoè , di Cantogno Ludovica Verdun , Perroteau Isabelle , Fasolo Aldo
Published: April 2006

Primary olfactory neurons located in the olfactory neuroepithelium project to the ipsilateral olfactory bulb and undergo a continuous process of neurogenesis and differentiation. We describe, in the adult rat, the kinetics of proliferation, differentiation and survival of primary olfactory neurons either in the presence or absence of their target, the olfactory bulb. The experimental design included unilateral bulbectomy, coupled with a single bromodeoxyuridine pulse 35 days after surgery. The rate of proliferation and survival of olfactory neurons was then examined by immunohistochemistry for bromodeoxyuridine, and the differentiation status by in situ hybridization for calmodulin messenger RNA in immature and mature olfactory neurons and immunohistochemistry for the dipeptide carnosine in mature olfactory neurons. We show that primary olfactory neurons can synthesize carnosine in the absence of the olfactory bulb. However, the number of carnosine‐immunopositive neurons in the absence of their target is dramatically reduced to less than one‐fourth, whereas the number of olfactory neurons expressing calmodulin messenger RNA is only slightly reduced. The numeric reduction of camosine‐positive neurons in the target‐deprived neuroepithelium is correlated with a dramatic reduction in the survival rate of olfactory neurons, since newly generated olfactory neurons are completely lost 35 days after the bromodeoxyuridine pulse. In contrast, in the normal olfactory neuroepithelium almost one‐third of newly generated olfactory neurons survive 35 days after the bromodeoxyuridine pulse. On the whole, these data indicate that most of the primary olfactory neurons have a short lifespan but that once they have connected with the olfactory bulb they may persist longer, and suggest that throughout adulthood olfactory neurons are overproduced, differentiate independently from their target, and then undergo a process of target‐induced neuronal selection.
Activity in the Precentral Motor Areas After Presentation of Targets for Delayed Reaching Movements Varies with the Initial Arm Position
Authors: Bauswein Erhard , Fromm Christoph
Published: April 2006

We studied single unit activity in the primary motor and premotor cortex during preparation for unrestrained arm movements that the monkey made to a right or left visual target. ‘Set‐related’ activity occurring during an instructed delay period could not only vary with the target location, but also with the initial arm posture which was changed via a motor‐driven forearm support between two positions. Modulation with arm displacement of some cells was contingent upon prior instruction, i.e. it occurred only if a target for the upcoming movement was presented, and for the majority of neurons it was different from the effects caused by the same passive arm shift when being applied before the visual instruction cue. Furthermore, responses to presentation of the same target were found to depend on the starting position of the hand. These findings suggest that both kinaesthetic and visual information interact in motor cortical cells in an appropriate manner for elaborating the movement trajectory.
Development of Light‐activated Pupilloconstriction in Rats as Mediated by Normal and Transplanted Retinae
Authors: Radel J. D. , Das S. , Lund R. D.
Published: April 2006

The relationship between the development of the pupilloconstriction response to changes in light levels and retinal maturation was studied in normal rats and rats that had received intracranial retinal transplants at birth. A pupillary response to light was first observed between postnatal days 7 and 9 in normal rats, and was typically of small amplitude and sluggish. By the time the eyelids first open, 2 weeks after birth, the pupillary response had improved to near adult levels. The inception of the pupillary response correlates with the first appearance of conventional synaptic contacts in the inner and outer plexiform layers of the retina, while improved responses correlate with maturation of photoreceptor outer segments and formation of synaptic ribbons in the inner plexiform layer. When embryonic retinae were transplanted to intracranial locations in newborn hosts and the transplants later illuminated as the host matured, the onset of a pupillary response to transplant illumination was delayed in proportion to the developmental disparity between the transplant and the host. The pattern of anatomical development in transplanted retinae was also similar, but delayed in time, compared to normal retinae. This indicates that the limiting factors for expression of light‐activated pupilloconstriction exist within the retina, rather than being intrinsic to the central nuclei or to the output pathway subserving the response.
Left Unilateral Inferior Pedunculotomy Prevents Neuronal Death During Postnatal Development of the Remaining Left Inferior Olivary Complex in the Rat
Authors: Armengol J. A. , López‐Román A.
Published: April 2006

Neuronal death in the inferior olivary complex (IOC) was studied in control and unilaterally pedunculotomized newborn rats, from postnatal day 1 (P1) to P30, in order to test whether the approximately two‐fold increase in available specific targets (i.e. Purkinje cells) that is theoretically provided by sectioning one inferior cerebellar peduncle to the developing climbing fibres of the remaining IOC could prevent the loss of inferior olivary neurons taking place during the first 2 weeks of postnatal life in the rat. Numerical estimation of the number of inferior olivary neurons in control and experimental rats showed that (i) in pedunculotomized rats, the number of inferior olivary neurons of the remaining inferior olivary complex was always greater than that encountered in control rats, (ii) the consistent decrease in the number of inferior olivary neurons observed in control animals between P2 and P8 was absent in cell counts of the pedunculotomized rats, and (iii) the increase in olivary cell number following the phase of cell decrease was also absent in pedunculotomized rats. It is concluded that the increase of available Purkinje cells during early postnatal development of the olivocerebellar projection prevents neuronal death in the remaining inferior olivary complex following pedunculotomy.
Differential Effects of a Distant Noxious Stimulus on Hindlimb Nociceptive Withdrawal Reflexes in the Rat
Authors: Kalliomäki J. , Schouenborg J. , Dickenson A. H.
Published: April 2006

Recent studies indicate that the nociceptive withdrawal reflexes to individual muscles are evoked by separate reflex pathways. The present study examines whether nociceptive withdrawal reflexes to different muscles are subject to differential supraspinal control in rats. A distant noxious stimulus was used to activate a bulbospinal system which selectively inhibits ‘multireceptive’ neurons (i.e. neurons receiving excitatory tactile and nociceptive inputs) in the dorsal horn of the spinal cord. Withdrawal reflexes, recorded with electromyographic techniques in single hindlimb muscles, were evoked by standardized noxious pinch. Thirty‐seven rats, anaesthetized with halothane and nitrous oxide, were used. Whereas withdrawal reflexes to the extensor digitorum longus and brevis, tibialis anterior and biceps posterior muscles were strongly inhibited, reflexes to interossei muscles were potentiated during noxious pinch of the nose. Reflexes to peronei muscles were not significantly changed. The effects on the reflexes usually had an onset latency of <0.5 s and outlasted the conditioning stimulation by up to 2 s. The monosynaptic la reflex to the deep peroneal nerve, innervating dorsiflexors of the digits and ankle, was not significantly changed during noxious pinch of the nose. Hence, the inhibitory effects on the hindlimb withdrawal reflexes induced by the conditioning stimulation were presumably exerted on reflex interneurons. It is concluded that nociceptive withdrawal reflexes to different hindlimb muscles are differentially controlled by descending pathways activated by a distant noxious stimulus. The results support our previous conclusion that there are separate nociceptive withdrawal reflex pathways to different hindlimb muscles.
Blockade of Glutamatergic Neurotransmission in the Suprachiasmatic Nucleus Prevents Cellular and Behavioural Responses of the Circadian System to Light
Authors: Vindlacheruvu R. R. , Ebling F. J. P. , Maywood E. S. , Hastings M. H.
Published: April 2006

The aim of this study was to test the role of glutamatergic neurotransmission in photic entrainment of the circadian oscillator of the suprachiasmatic nuclei (SCN) in the Syrian hamster. The response of the oscillator to a brief pulse of light was assessed using two independent indices, the phase shift of the free‐running activity rhythm, and the photically induced expression of the immediate‐early gene c‐fos within neurons of the SCN. The behavioural and the cellular responses to light were compared in animals which received intra‐cerebroventricular (icv) infusions into the region of the SCN of either a vehicle solution or a solution of γd‐glutamyl‐glycine (DGG), a competitive antagonist at both N‐methyl‐d‐aspartate (NMDA) and non‐NMDA types of glutamate receptor. Infusions of vehicle or DGG (200 nmol) were given 10 min before presentation of a 15‐min light pulse at either circadian time (CT) 14 or CT20 (onset of activity defined as CT12). As anticipated, animals treated with vehicle and light at CT14 exhibited phase delays in the activity rhythm, whereas animals treated at CT20 exhibited phase advances. Central infusion of DGG prior to a light pulse at CT14 blocked the phase‐delaying effect of light, and DGG delivered before a light pulse at CT20 markedly attenuated the phase‐advancing effect of light. In a separate group of animals, the expression of the immediate‐early gene c‐fos was assessed by immunocytochemical staining for its protein product Fos. Exposure of vehicle‐infused animals to light at CT14 caused extensive expression of c‐fos throughout the retinorecipient region of the SCN. However, when the light pulse was preceded by icv fusion of DGG at a dose which would block the phase‐shifting response to light, the total number of neurons immunopositive for Fos was significantly reduced (∼50%) and the expression was confined to a restricted area of the dorsolateral SCN. The precise correlation between the effects of glutamatergic blockade upon both the behavioural and the cellular responses of the circadian system to light demonstrates that effective glutamatergic neurotransmission within or adjacent to the SCN is a necessary component of the mechanism which mediates photic entrainment of the circadian clock. The results further demonstrate a pharmacological and anatomical compartmentalization of the retinorecipient zone of the SCN, consistent with the view that retinal afferents to the ventral region employ glutamate as a transmitter, whereas more dorsal input may be dependent upon non‐glutamatergic (DGG‐insensitive) pathways.
Different Types of Potassium Outward Current in Relay Neurons Acutely Isolated from the Rat Lateral Geniculate Nucleus
Authors: Budde Thomas , Mager Ralph , Pape Hans‐Christian
Published: April 2006

Different classes of potassium (K+) outward current activated by depolarization were characterized in relay neurons acutely isolated from the rat lateral geniculate nucleus (LGN), using the whole‐cell version of the patch‐clamp technique. A fast‐transient current (IA), activated at around –70 mV, declined rapidly with a voltage‐dependent time constant (τ= 6 ms at + 45 mV), was 50% steady‐state inactivated at –70 mV, and rapidly recovered from inactivation with a monoexponential time course (τ= 21 ms). IA was blocked by 4‐aminopyridine (4‐AP, 2–8 mM) and was relatively insensitive to tetraethylammonium (TEA, 2–10 mM). After elimination of IA by a conditioning prepulse (30 ms to –50 mV), a slow‐transient K+ current could be studied in isolation, and was separated into three components, IKm, IKs and a calcium (Ca2+)‐dependent current, IK[Ca]. The slow‐transient current was not consistently affected by 4‐AP (up to 8 mM), while TEA (2–10 mM) predominantly blocked IKs and IK[Ca]. The component IKm persisted in a solution containing TEA and 4‐AP, activated at around –55 mV, declined monoexponentially during maintained depolarization (τ= 98 ms at +45 mV), was 50% inactivated at –39 mV, and recovered with τ= 128 ms from inactivation. IKs activated at a similar threshold, but declined much slower with τ= 2662 ms at +45 mV. Steady‐state inactivation of IKs was half‐maximal at –49 mV, and recovery from inactivation occurred relatively fast with τ= 116 ms. From these data and additional current‐clamp recordings it is concluded that the K+ currents, due to their wide range of kinetics and dependence on membrane voltage or internal Ca2+ concentration, are capable of cooperatively controlling the firing threshold and of shaping the different states of electrophysiological behaviour in LGN relay cells.
A Quantitative Description of Low‐ and High‐threshold Ca2+ Spikes in Rat Sensory Neurons: A Perforated‐patch Study
Authors: Wanke Enzo , Becchetti Andrea , Biella Gerardo , Del Bo Roberto , Ferroni Arnaldo
Published: April 2006

Action potentials generated by voltage‐dependent Ca2+ conductances were studied at 25°C with the perforated‐patch technique, in freshly dispersed adult rat sensory neurons perfused with Na‐free solutions containing tetraethylammonium. Brief depolarizing currents from membrane potentials negative to –75 mV always elicited long (>100 ms) plateau spikes which had different thresholds in different neurons: a low threshold around –60/–50 mV and a high‐threshold at –30/–20 mV. Stimulations from potentials positive to –55 mV, on the contrary, elicited spikes originating only in the high threshold region and sensitive to 25 μM Cd2+, designated high‐threshold spikes. In neurons which showed spikes with low threshold, addition of 25 μM Cd2+ disclosed a smaller and shorter regenerative response, the low‐threshold spike. Moreover, the classical ‘anode‐break’ stimulation from –50/–60 mV uncovered isolated low‐threshold spikes, indicating a time‐ and voltage‐dependent de‐inactivating process. From the properties of the low (LVA) and high (HVA) voltage‐activated Ca2+ currents, recorded under the same extracellular conditions, a Hodgkin–Huxley model was derived and used to reconstruct all the features of the recorded spikes. The model was also able to simulate experimental blocking of LVA channels by amiloride, modulation of HVA channels by baclofen and induced oscillatory firing. This agreement between the behaviour of recorded spikes and their mathematical description led us to conclude that the LVA and HVA Ca2+ currents underlie the low‐ and high‐threshold Ca2+ spikes, respectively. Furthermore, our data suggest that complex behaviour known to be typical of central nervous system neurons is also present in sensory peripheral neurons.
Increase in Specific Proteins and mRNAs Following Transient Anoxia‐Aglycaemia in Rat CA1 Hippocampal Slices
Authors: Charriaut‐Marlangue C. , Pollard H. , Kadri‐Hassani N. , Khrestchatisky M. , Moreau J. , Dessi F. , Kang K. I. , Ben‐Ari Y.
Published: April 2006

Incorporation of [35S]methionine into proteins and two‐dimensional gel autoradiograms was used to characterize early post‐anoxia–aglycaemia protein synthesis in the CA1 area of rat hippocampal slices maintained in vitro. We have compared the effects of 3–4 min and 5–10 min insults, since the former but not the latter produces a reversible block of synaptic transmission (see companion paper). An insult of between 3 min 30 s and 4 min induces a transient increase in the labelled proteins during the first hour of reoxygenation, as compared to control. The increase in protein synthesis is conspicuous for several proteins, including actin, α‐tubulin and heat‐shock proteins (hsp70c and hsp90), as determined by immunoblotting. In the case of α‐tubulin, we show with in situ hybridization and polymerase chain reaction procedures that the increase in protein synthesis is associated with a marked increase in the expression of the corresponding messenger RNAs. The results demonstrate that, in addition to regulatory proteins such as hsps, the synthesis of several polypeptides, including those associated with the cytoskeleton, is altered in anoxic damage.
Evidence Against a Role for Protein Kinase C in the Inhibition of the Calcium‐activated Potassium Current IAHP by Muscarinic Stimulants in Rat Hippocampal Neurons
Authors: Sim J. A. , Gerber U. , Knöpfel T. , Brown D. A.
Published: April 2006

The possible role of protein kinase C activation in the inhibitory action of cholinergic transmitters on the slow Ca‐dependent afterhyperpolarizing current (IAHP) in hippocampal CA3 pyramidal neurons was investigated using hippocampal slice cultures. IAHP was inhibited reversibly by methacholine (100–600 nM) and irreversibly by the protein kinase C activator, phorbol‐12,13‐dibutyrate (PDBu, 10 nM to 1 μM). The inhibitory action of PDBu was antagonized by prior (15–60 min) exposure to staurosporin (1 μM). In contrast, the inhibitory effect of methacholine on IAHP was not reduced after up to 3 h of exposure to this compound. In addition, methacholine produced a reversible inward current at the holding potential, which was augmented by staurosporin. However, prior exposure to PDBu reduced the effect of methacholine on IAHP and occluded the methacholine‐induced inward current. This effect of PDBu was also observed in the presence of staurosporin, suggesting that it might be exerted through a protein kinase C‐independent pathway. Noradrenalin (2–5 μM) and 8‐bromo cyclic adenosine 3′,5′monophosphate (8‐Br‐cAMP, 1 mM) also produced a reversible block of IAHP. Their action was antagonized by staurosporin, probably via its effect on protein kinase A. Thus the present experiments suggest that the action of muscarinic agonists on IAHP cannot be explained by an effect on protein kinase C, but support a role for protein kinase A in mediating the action of noradrenalin.