bluuebunny/crossref_metadata_2025 huggingface dataset:
The effect of microglia on embryonic dopaminergic neuronal survival in vitro: diffusible signals from neurons and glia change microglia from neurotoxic to neuroprotective
Authors: Zietlow R. , Dunnett S. B. , Fawcett J. W.
Published: March 2003

When embryonic dopaminergic neurons are transplanted into the adult brain, ≈ 95% die within a few days. To assess whether microglia activated during transplantation might be responsible for this rapid death, we examined the effect of microglia on rat embryonic dopaminergic neurons in vitro. Conditioned medium from 7‐day‐old microglia was found to decrease the number of dopamine neurons surviving in primary culture, but activation of the microglia with N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) or Zymosan A did not increase the toxicity of the conditioned medium. We next tested the effect of coculturing microglia and dopaminergic neurons by placing microglia in semipermeable well inserts over the neuronal cultures. The presence of microglia now increased dopaminergic neuronal survival, microglial activation again having no effect. To increase yet further the possible interactions between microglia and neurons, the mesencephalic cells and microglia were mixed together and placed as a tissue in three‐dimensional culture, and here again the presence of microglia increased dopaminergic neuronal survival with no effect of activation. Contact of microglia with the mesencephalic cells therefore converted them from being toxic to dopaminergic neurons to promoting their survival. The change in microglial effect from toxic to protective was caused by soluble molecules secreted by cells in the neuronal cultures, as conditioned medium derived from microglia–neuronal cocultures also had a dopaminergic neuron survival effect, indicating that microglia in cocultures behave differently from microglia removed from neuronal and glial influence. Microglia cocultured with either neurons or astrocytes downregulated inducible nitric oxide synthase (iNOS), indicating a decrease in the production of nitric oxide and possibly other toxic molecules. These findings indicate that in their natural environment, microglia are likely to be beneficial for the survival of embryonic dopaminergic grafts.
Cytoskeleton mediates inhibition of the fast Na+ current in respiratory brainstem neurons during hypoxia
Authors: Mironov S. L. , Richter D. W.
Published: March 2003

Whole‐cell Na+ currents (INa) were recorded in inspiratory neurons in a medullary slice preparation from neonatal mouse that contains the functional respiratory network. Hypoxia and metabolic poisoning with KCN rapidly inhibited INa by reducing the number of Na+ channels available for opening during depolarization. Application of agents specific for G‐proteins, protein kinase C and A, intracellular Ca2+ and pH did not prevent the hypoxic inhibition of INa. The effects of hypo‐osmolarity and hypoxia were additive, whereas hyperosmolarity partially prevented a subsequent hypoxic inhibition of INa. Cytochalasin B and colchicine decreased, and taxol or phalloidin increased INa and reduced its hypoxic inhibition. We conclude that cytoskeleton rearrangements during hypoxia are responsible for suppression of a fast INa in brainstem respiratory neurons, which could be mediated by the uncoupling of channel inactivation gates from cytoskeletal elements.
The role of p75NTR in modulating neurotrophin survival effects in developing motoneurons
Authors: Wiese Stefan , Metzger Friedrich , Holtmann Bettina , Sendtner Michael
Published: March 2003

Neurotrophins exert their biological functions on neuronal cells through two types of receptors, the trk tyrosine kinases and the low‐affinity neurotrophin receptor (p75NTR), which can bind all neurotrophins with similar affinity. The p75NTR is highly expressed in developing motoneurons and in adult motoneurons after axotomy, suggestive of a physiological role in mediating neurotrophin responses under such conditions. In order to characterize this specific function of p75NTR, we have tested the effects of nerve growth factor (NGF) on embryonic motoneurons from control and p75NTR‐deficient mice. NGF antagonizes brain‐derived neurotrophic factor (BDNF)‐ and neurotrophin‐3 (NT‐3)‐mediated survival in control but not p75NTR‐deficient motoneurons. Survival of cultured motoneurons in the presence of 0.5 ng/mL of either ciliary neurotrophic factor (CNTF) or glial‐derived neurotrophic factor (GDNF) was not reduced by 20 ng/mL NGF. Dose–response investigations revealed that five times higher concentrations of BDNF are required for half‐maximal survival of p75NTR‐deficient motoneurons in comparison to motoneurons from wild‐type controls. After facial nerve lesion in newborn wild‐type mice, local administration of NGF reduced survival of corresponding motoneurons to less than 2% compared to the unlesioned control side. In p75NTR‐deficient mice, the same treatment did not enhance facial motoneuron death on the lesioned side. In the facial nucleus of 1‐week‐old p75NTR–/– mice, a significant reduction of motoneurons was observed at the unlesioned side in comparison to p75NTR +/+ mice. The observation that motoneuron cell numbers are reduced in the facial nucleus of newborn p75NTR‐deficient mice suggests that p75NTR might not function as a physiological cell death receptor in developing motoneurons.
Thalamic projections of the fusiform gyrus in man
Authors: Clarke Stephanie , Riahi‐Arya Sahba , Tardif Eric , Eskenasy Anne‐Claude Cottier , Probst Alphonse
Published: March 2003

Previous retrograde degeneration studies have shown that human extrastriate visual cortex receives projections from the pulvinar, but their precise topographical organization remained unknown. We report on the distribution of thalamic projections originating in the fusiform gyrus, as studied with the Nauta method for anterogradely degenerating axons, in a case of right fusiform gyrus infarction. Ipsilaterally to the lesion, high density of afferents was found in the inferior pulvinar nucleus and a low density in the medial pulvinar nucleus as well as in the postero‐inferior part of the reticular nucleus; no degenerating fibres were found in the lateral geniculate body. Degenerating axons were completely absent in the contralateral thalamus. Thus, there is a precise topographic relationship between parts of the extrastriate cortex and the pulvinar, suggesting segregated thalamocortical pathways for different parts of the extrastriate cortex. As in nonhuman primates, the human inferior temporal cortex has no direct output to the lateral geniculate body.
Astrocytic regulation of the recovery of extracellular potassium after seizures in vivo
Authors: Xiong Zhi‐Qi , Stringer Janet L.
Published: March 2003

Glial cells are believed to play a major role in the regulation of the extracellular potassium concentration ([K+]o), particularly when the [K+]o is increased. Using ion‐selective electrodes, we compared the [K+]o changes in the dentate gyrus of urethane‐anaesthetized adult rats in the presence of reactive astrocytes and after reduction of glial function. The regulation of [K+]o in the dentate gyrus was determined by measuring the ceiling level of [K+]o and the half‐time of recovery of [K+]o during and after seizures produced by 20 Hz trains of stimulation to the angular bundle. Reactive astrocytes were induced by repeated seizures and their presence was confirmed by a qualitative increase in glial fibrillary acidic protein (GFAP) and vimentin immunoreactivity. To inhibit glial function, fluorocitrate (FC), a reversible metabolic inhibitor, or α‐aminoadipate (α‐AA), an irreversible toxin, was injected into the dentate gyrus region, and the regulation of [K+]o was monitored for 8 h or 2 days later, respectively. After α‐aminoadipate, loss of astrocytes in the dentate gyrus was demonstrated by loss of staining for GFAP. In the presence of reactive astrocytes there was no significant change in the peak [K+]o during seizures or the half‐time of recovery of [K+]o after seizures compared to control animals. α‐Aminoadipate significantly slowed the rate of recovery of [K+]o, but did not change the ceiling [K+]o. Fluorocitrate reversibly decreased the ceiling [K+]o, but also slowed the rate of recovery of [K+]o. Overall our results suggest that normal glial function is required for the recovery of elevated [K+]o after seizures in vivo.
Biochemical and pharmacological evidence of a functional role of AMPA receptors in motor neuron dysfunction in mnd mice
Authors: Mennini T. , Cagnotto A. , Carvelli L. , Comoletti D. , Manzoni C. , Muzio V. , Rizzi M. , Vezzani A.
Published: March 2003

We studied ionotropic glutamate receptor subtypes and the effect of chronic treatment with NBQX [6‐nitro‐7‐sulphamoyl‐benzo(F)quinoxaline‐2,3‐dione], a selective (rs)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid (AMPA) receptor antagonist, in the spinal cord of mnd mice. NBQX (8 mg/kg daily i.p. for 3 weeks starting from 24 weeks old) significantly improved the behavioural scores (hind leg extension reflex, cage rung grasping and gait) in mnd mice, measured after the last drug injection, and increased the number of mice with ‘normal’ gait (from 50% to 90%, P < 0.05).

Receptor binding autoradiography of the competitive N‐methyl‐ d‐aspartate (NMDA) antagonist, [3H]CGP 39653, of [3H]AMPA and [3H]kainic acid in spinal cord sections, measured after 1 week of drug washout, were not significantly different in control and mnd mice, and were not modified by NBQX.

GluR2/3 immunoreactivity, assessed using Western blotting, was significantly enhanced (by 59%, P < 0.01) in the spinal cord but not in the brain of 28‐week‐old mnd mice compared to age‐matched control mice. NBQX treatment increased GluR2/3 immunoreactivity in the spinal cord of control mice and mnd mice by 327 ± 74% (P < 0.01) and 212 ± 52% (P < 0.01), respectively.

The changes in GluR2/3 subunits may involve adaptive mechanisms of the receptor and play some role in the protective effect of NBQX. These findings suggest that selective antagonism of ionotropic non‐NMDA receptors may be of value in the treatment of motor neuron disease.
Satellite‐cell‐derived nerve growth factor and neurotrophin‐3 are involved in noradrenergic sprouting in the dorsal root ganglia following peripheral nerve injury in the rat
Authors: Zhou Xin‐Fu , Deng Yan‐Shen , Chie Edward , Xue Qing , Zhong Jin‐Hua , McLachlan Elspeth M. , Rush Robert A. , Xian Cory J.
Published: March 2003

Injury to a peripheral nerve induces in the dorsal root ganglia (DRG) sprouting of sympathetic and peptidergic terminals around large‐diameter sensory neurons that project in the damaged nerve. This pathological change may be implicated in the chronic pain syndromes seen in some patients with peripheral nerve injury. The mechanisms underlying the sprouting are not known. Using in situ hybridization and immunohistochemical techniques, we have now found that nerve growth factor (NGF) and neurotrophin‐3 (NT3) synthesis is upregulated in satellite cells surrounding neurons in lesioned DRG as early as 48 h after nerve injury. This response lasts for at least 2 months. Quantitative analysis showed that the levels of mRNAs for NT3 and NGF increased in ipsilateral but not contralateral DRG after nerve injury. Noradrenergic sprouting around the axotomized neurons was associated with p75‐immunoreactive satellite cells. Further, antibodies specific to NGF or NT3, delivered by an osmotic mini‐pump to the DRG via the lesioned L5 spinal nerve, significantly reduced noradrenergic sprouting. These results implicate satellite cell‐derived neurotrophins in the induction of sympathetic sprouting following peripheral nerve injury.
Neutralization of endogenous NGF prevents the sensitization of nociceptors supplying inflamed skin
Authors: Koltzenburg Martin , Bennett David L. H. , Shelton David L. , McMahon Stephen B.
Published: March 2003

Evidence suggests that nerve growth factor (NGF) is an important mediator in inflammatory pain states: NGF levels increase in inflamed tissue, and neutralization of endogenous NGF prevents the hyperalgesia which normally develops during inflammation of the skin. Here we asked whether NGF contributes to sensitization of primary afferent nociceptors, which are an important component of pain and hyperalgesia in inflamed tissue. An in vitro skin nerve preparation of the rat was used to directly record the receptive properties of thin myelinated (Aδ) and unmyelinated (C) nociceptors innervating normal hairy skin, carrageenan‐inflamed skin and carrageenan‐inflamed skin where endogenous NGF had been neutralized by application of a trkA‐IgG (tyrosine kinase Aimmunoglobulin G) fusion molecule. Following carrageenan inflammation, there was a marked increase in the proportion of nociceptors which displayed ongoing activity (50% of nociceptors developed spontaneous activity compared to 4% of nociceptors innervating normal uninflamed skin), and this was reflected in a significant increase in the average ongoing discharge activity. Spontaneously active fibres were sensitized to heat and displayed a more than twofold increase in their discharge to a standard noxious heat stimulus. Furthermore, the number of nociceptors responding to the algesic mediator bradykinin increased significantly from 28% to 58%. By contrast, the mechanical threshold of nociceptive afferents did not change during inflammation. When the NGF‐neutralizing molecule trkA‐IgG was coadministered with carrageenan at the onset of the inflammation, primary afferent nociceptors did not sensitize and displayed essentially normal response properties, although the inflammation as evidenced by tissue oedema developed normally. We therefore conclude that NGF is a crucial component for the sensitization of primary afferent nociceptors associated with tissue inflammation.
Regrowth of axons into the distal spinal cord through a Schwann‐cell‐seeded mini‐channel implanted into hemisected adult rat spinal cord
Authors: Xu Xiao Ming , Zhang Shu‐Xin , Li Huaying , Aebischer Patrick , Bunge Mary Bartlett
Published: March 2003

Schwann cells (SCs) have been shown to be a key element in promoting axonal regeneration after being grafted into the central nervous system (CNS). In the present study, SC‐supported axonal regrowth was tested in an adult rat spinal cord implantation model. This model is characterized by a right spinal cord hemisection at the eighth thoracic segment, implantation of a SC‐containing mini‐channel and restoration of cerebrospinal fluid circulation by suturing the dura. We demonstrate that a tissue cable containing grafted SCs formed an effective bridge between the two stumps of the hemicord 1 month after transplantation. Approximately 10 000 myelinated and unmyelinated axons (1 : 9) per cable were found at its midpoint. In addition to propriospinal axons and axons of peripheral nervous system (PNS) origin, axons from as many as 19 brainstem regions also grew into the graft without additional treatments. Most significantly, some regenerating axons in the SC grafts were able to penetrate through the distal graft–host interface to re‐enter the host environment, as demonstrated by anterograde axonal labelling. These axons coursed toward, and then entered the grey matter where terminal bouton‐like structures were observed. In channels containing no SCs, limited axonal growth was seen within the graft and no axons penetrated the distal interface. These findings further support the notion that SCs are strong promotors of axonal regeneration and that the mini‐channel model may be appropriate for further investigation of axonal re‐entry, synaptic reconnection and functional recovery following spinal cord injury.
IPSPs elicited in CA1 pyramidal cells by putative basket cells in slices of adult rat hippocampus
Authors: Ali Afia B. , Bannister A. Peter , Thomson Alex M.
Published: March 2003

CA1 basket cells are identifiable by an axonal arbour largely confined to, and spanning, the entire depth of stratum pyramidale where they innervate pyramidal somata and proximal dendrites. Basket cells display a range of electrophysiological properties and the inhibitory postsynaptic potentials (IPSPs) they elicit in pyramidal cells vary widely in duration. To determine whether these parameters are correlated, we used paired intracellular recordings, with biocytin filling, in pyramidal cells of adult hippocampal slices, and studied γ‐aminobutyric acid (GABAA) IPSPs (n = 43) elicited by putative basket cells (n = 35) with axons largely confined to stratum pyramidale in simultaneously recorded pyramidal cells. Fast‐spiking interneurons elicited relatively brief IPSPs, while IPSPs elicited by burst‐firing cells were amongst the slowest. Regular spiking interneurons elicited fast and slow GABAA IPSPs, but any one interneuron elicited IPSPs with remarkably similar durations in two to four pyramidal targets. However, with different types of target for a single putative basket cell, IPSPs elicited in postsynaptic interneurons were briefer than in pyramidal cells. Vertical oriens cells with somata in stratum oriens and a narrow, sparse axonal arbour in stratum pyramidale in transverse hippocampal slices, elicited IPSPs whose rise times and half widths clustered around intermediate values. Durations of IPSPs in pyramidal cells thus correlate, to a degree, with the physiological properties of presynaptic basket cells. The seven‐fold range of durations observed (10–70 ms half widths) may underlie contributions made by different basket cells to hippocampal rhythms of different frequencies.
Hippocampal plasticity in Alzheimer's disease: changes in highly polysialylated NCAM immunoreactivity in the hippocampal formation
Authors: Mikkonen Mia , Soininen Hilkka , Tapiola Tero , Alafuzoff Irina , Miettinen Riitta
Published: March 2003

The highly polysialylated neural cell adhesion molecule (PSA‐NCAM) is one of the most promising molecules that contributes to plasticity in the central nervous system. We evaluated PSA‐NCAM immunoreactivity in the hippocampal formation of Alzheimer's disease (AD) patients. We found significant increases over control levels in the optical density ratios of PSA‐NCAM immunoreactivity in the outer molecular layer/granule cell layer (ODoml/grl) and in the inner molecular layer/granule cell layer (ODiml/grl) in the dentate gyrus of AD patients. The optical density of the granule cell layer in the dentate gyrus did not differ significantly between AD patients and control subjects. However, the number of PSA‐NCAM‐immunopositive infragranule cells was higher in the AD group compared with control subjects. The major finding in the CA1, subiculum and entorhinal cortex of AD patients was the disorganization of PSA‐NCAM‐immunoreactive fibres. These results indicate that neuronal remodelling occurs, especially in the dentate gyrus of patients with AD.
Investigation by ion channel domain transplantation of rat glutamate receptor subunits, orphan receptors and a putative NMDA receptor subunit
Authors: Villmann Carmen , Strutz Nathalie , Morth Tanja , Hollmann Michael
Published: March 2003

Among the 18 ionotropic glutamate receptor subunits identified in the mammalian central nervous system, five (delta1, delta2, GluR7, chi2 and NR3A, formerly called NMDAR‐L or chi1) reportedly fail to form functional ion channels in heterologous expression systems. Four of these subunits, delta1, delta2, chi2 and NR3A, have not even been shown to bind glutamatergic ligands, relegating them to the status of ‘orphan’ receptors. We used a domain transplantation approach to investigate potential functional properties of the putative ion channel domains of four of these subunits. By exchanging ion pore domains between functional glutamate receptors (GluR1, GluR6 and NMDAR1) with known pore properties we first tested the feasibility of the domain swapping method. We demonstrate that ion channel domains can be transplanted between all three functional subfamilies of ionotropic glutamate receptors. Furthermore, exchange of ion pore domains allows identification of those channel properties determined exclusively by the ion pore. We then show that transplanting the pore domain of GluR7 into either GluR1 or GluR6 generates perfectly functional ligand‐gated ion channels that allow characterization of electrophysiological and pharmacological properties of the GluR7 pore domain. In contrast, delta1, delta2 and NR3A do not produce functional receptors when their pore domains are transplanted into either the AMPA receptor, GluR1, the kainate receptor, GluR6, or the NMDA receptor, NMDAR1. We speculate that the orphan receptors delta1 and delta2, and the NMDA receptor‐like subunit NR3A may serve some modulatory function, rather than contributing to the formation of ion channels.
Release properties and functional integration of noradrenergic‐rich tissue grafted to the denervated spinal cord of the adult rat
Authors: Leanza G. , Cataudella T. , Dimauro R. , Monaco S. , Stanzani S.
Published: March 2003

Noradrenaline‐ (NA‐) containing grafts of central (embryonic locus coeruleus, LC) or peripheral (juvenile adrenal medullary, AM, autologous superior cervical ganglionic, SCG) tissue were implanted unilaterally into rat lumbar spinal cord previously depleted of its NA content by 6‐hydroxydopamine (6‐OHDA) intraventricularly. A microdialysis probe was implanted in the spinal cord 3–4 months after transplantation, and extracellular levels of noradrenaline were monitored in freely moving animals during basal conditions and following administration of pharmacological or behavioural stimuli. Age‐matched normal and lesioned animals both served as controls. Morphometric analyses were carried out on horizontal spinal sections processed for dopamine‐β‐hydroxylase (DBH) immunocitochemistry, in order to assess lesion‐ or graft‐induced changes in the density of spinal noradrenergic innervation, relative to the normal patterns. In lesioned animals, the entire spinal cord was virtually devoid of DBH‐positive fibers, resulting in a dramatic 88% reduction in baseline NA, compared with that in controls, which did not change in response to the various stimuli. LC and SCG grafts reinstated ≈ 80% and 50% of normal innervation density, respectively, but they differed strikingly in their release ability. Thus, LC grafts restored baseline NA levels up to 60% of those in controls, and responded with significantly increased NA release to KCl‐induced depolarization, neuronal uptake blockade and handling. In contrast, very low NA levels and only poor and inconsistent responses to the various stimuli were observed in the SCG‐grafted animals. In AM‐grafted animals, spinal extracellular NA levels were restored up to 45% of those in controls, probably as a result of nonsynaptic, endocrine‐like release, as grafted AM cells retained the chromaffine phenotype, showed no detectable fibre outgrowth and did not respond to any of the pharmacological or behavioural challenges. Thus, both a regulated, impulse‐dependent, and a diffuse, paracrine‐like, NA outflow may play roles in the recovery of lesion‐induced sensory and/or motor impairments previously reported with these types of grafts following transplantation into the severed spinal cord.
μ‐ and δ‐opioid receptor agonists inhibit DARPP‐32 phosphorylation in distinct populations of striatal projection neurons
Authors: Lindskog Maria , Svenningsson Per , Fredholm Bertil , Greengard Paul , Fisone Gilberto
Published: March 2003

In the striatum, DARPP‐32 (dopamine‐ and cAMP‐regulated phosphoprotein of 32 kDa) is highly expressed by virtually all projection medium‐sized spiny neurons. cAMP‐dependent phosphorylation of DARPP‐32 is stimulated via activation of dopamine D1 receptors in striatonigral neurons, and via activation of adenosine A2A receptors in striatopallidal neurons. In this study, we have examined the contribution of μ‐, δ‐ and κ‐opioid receptors to the regulation of DARPP‐32 phosphorylation, in rat striatal slices. The results show that, at low concentrations (100 pm–1 nm), the μ‐opioid agonist, Tyr‐D‐Ala‐Gly‐N‐Me‐Phe‐glycinol (DAMGO), inhibits the increase in DARPP‐32 phosphorylation induced by activation of D1, but not by activation of A2A receptors. Conversely, the δ‐receptor agonist, Tyr‐D‐Pen‐Gly‐Phe‐D‐Pen (DPDPE), inhibits DARPP‐32 phosphorylation induced by activation of A2A, but not by activation of D1 receptors. The κ‐receptor agonist, U50488, does not affect DARPP‐32 phosphorylation induced by either D1 or A2A agonists. Thus, μ‐opioid receptors interact with dopamine D1 receptors on striatonigral neurons, whereas δ‐opioid receptors interact with adenosine A2A receptors on striatopallidal neurons. These results suggest that regulation of DARPP‐32 phosphorylation is involved in mediating some of the effects exerted by enkephalin on striatal medium‐sized spiny neurons.
Observation and execution of movement: similarities demonstrated by quantified electroencephalography
Authors: Cochin Stéphanie , Barthelemy Catherine , Roux Sylvie , Martineau Joëlle
Published: March 2003

Quantified electroencephalography (qEEG) was used to compare cerebral electrical variations while human subjects (10 males and 10 females) were observing and executing finger movements and while they were resting. Video recording enabled elimination of subjects performing involuntary movements. EEGs were recorded from 14 sites in seven frequency bands: theta 1, theta 2, alpha 1, alpha, beta 1, beta 2 and beta 3. Analyses were performed on logarithmically transformed absolute spectral power values. Both observation and execution of finger movements involved a decrease in spectral power compared with resting. This decrease was significant only for the alpha 1 frequency band (7.5–10.5 Hz) and it involved nine of the 14 electrode locations (F7, F8, F4, T6, T5, C3, C4, P3 and P4). This indicates that the motor cortex and the frontal cortex are specifically activated by both observation and execution of finger movements. These results provide evidence that observation and execution of movement share the same cortical network.
Amyloid precursor protein of Alzheimer's disease: evidence for a stable, full‐length, trans‐membrane pool in primary neuronal cultures
Authors: Storey Elsdon , Katz Melissa , Brickman Yardenah , Beyreuther Konrad , Masters Colin. L.
Published: March 2003

We and colleagues have shown that the amyloid protein precursor of Alzheimer's disease (APP) is distributed along the surface of neurites of fixed but nonpermeabilized neurons in primary culture in a segmental pattern, which shows colocalization with some markers of adhesion patches. This is in contrast to the diffuse pattern of immunoreactivity seen after permeabilization. We have also recently demonstrated that the APP in these surface patches is likely to be integral to the membrane rather than secreted and re‐adsorbed, based on alkali stripping experiments and on soluble APP adsorption experiments. Total cellular APP has previously been shown to have a short half‐life of ≈ 30–60 min. We confirm this in neurons in primary culture in pulse‐chase experiments using short labelling times. Additionally, we provide evidence that a separate, stable pool of neuronal APP can be demonstrated in pulse‐chase experiments using long labelling times. Experiments involving inhibition of protein synthesis suggest that this corresponds with the surface, segmental pool. Metabolic labelling followed by surface biotinylation and two‐stage precipitation demonstrates that the surface APP is trans‐membrane and full‐length (not carboxyl‐terminal truncated), and confirms that the surface APP belongs to the stable pool. This two‐stage procedure is necessary as the surface APP appears to be present in low copy number, and is difficult to detect by direct labelling. This information is consistent with a role for APP in stable cell‐matrix or cell–cell interactions.
Autoradiographic studies of 5‐HT1A‐receptor‐stimulated [35S]GTPγS‐binding responses in the human and monkey brain
Authors: Dupuis Delphine S. , Pauwels Petrus J. , Radu Diana , Hall Håkan
Published: March 2003

G‐protein activation mediated by serotonin 5‐HT1A receptors in human and monkey brain was investigated by using quantitative autoradiography of agonist‐stimulated [35S]GTPγS binding to whole‐hemisphere brain sections. [35S]GTPγS binding was stimulated by the mixed 5‐HT1A/1B/1D agonist L 694247 (10 μm) in human brain regions enriched in 5‐HT1A binding sites [e.g. hippocampus (132–137%), superficial layers of the neocortex (37–61%), and cingulate and entorhinal cortex (34 and 32%, respectively)]. L 694247 caused virtually no stimulation in regions with 5‐HT1B/1D receptors, such as substantia nigra, caudate nucleus and putamen. Similar results were obtained with monkey brain sections. The L 694247‐mediated [35S]GTPγS‐binding responses in human and monkey brain sections were antagonized by the selective, silent 5‐HT1A antagonist WAY 100635 (10 μm). The 5‐HT1B inverse agonist SB 224289 (10 μm) did not affect the [35S]GTPγS‐binding response of L 694247. The distribution pattern of the [35S]GTPγS‐binding response and the antagonist profile suggest the L 694247‐induced response in human and monkey brain is mediated by 5‐HT1A receptors. A weak stimulation of [35S]GTPγS binding was also observed in human hippocampus with either 10 μm 8‐OH‐DPAT (25 ± 4%) or naratriptan (42 ± 2%) compared with that obtained with L 694247. In conclusion, G‐protein activation by 5‐HT1A receptors can be measured in human and monkey brain sections. L 694247 appears to possess higher efficacy at 5‐HT1A receptors compared with 8‐OH‐DPAT and naratriptan.
Amino‐terminal region of secreted form of amyloid precursor protein stimulates proliferation of neural stem cells
Authors: Ohsawa Ikuroh , Takamura Chizuko , Morimoto Takako , Ishiguro Mariko , Kohsaka Shinichi
Published: March 2003

β‐Amyloid precursor protein (APP) has been reported to be expressed in the CNS from the early stages of development. However, the functional role of APP during early development remains unclear. In the present study, we found that the secreted form of APP (sAPP) significantly enhanced proliferation of neural stem cells. Cells were prepared from 13‐day embryonic rat neocortex, which was dissected with a Pasteur pipette to make cell clusters. After 12 h of cultivation in the medium without serum, cells around the centre of the cluster were still nestin‐positive proliferative cells, i.e. neural stem cells. To determine whether the proliferation of cells was regulated by sAPP, cultures were treated with recombinant sAPP695, the secreted form of human APP695 produced by yeast. Both DNA synthesis and expression of proliferating cell nuclear antigen markedly increased after 5 h of sAPP695 addition. The enhancement of DNA synthesis by sAPP695 stimulation was blocked by the 22C11 monoclonal antibody specific for the amino‐terminal region of sAPP. Then, we examined the effect of the amino‐terminal fragment of sAPP and the epitope peptide of 22C11 antibody, and found that both of them also promoted DNA synthesis, suggesting that the amino‐terminal region of sAPP is responsible for the biological activity. Our findings indicate the possibility that sAPP enhances proliferation of neural stem cells in vivo and plays an important role during the early CNS development.
Involvement of primary afferent C‐fibres in touch‐evoked pain (allodynia) induced by prostaglandin E2
Authors: Minami Toshiaki , Okuda‐Ashitaka Emiko , Hori Yuuichi , Sakuma Satoru , Sugimoto Tetsuo , Sakimura Kenji , Mishina Masayoshi , Ito Seiji
Published: March 2003

Nociceptive primary afferents have the capacity to induce a state of increased excitability in dorsal horn neurons of the spinal cord or central sensitization causing thermal hyperalgesia and touch‐evoked pain (allodynia). It is believed that primary afferent C‐fibres become hypersensitive and induce hyperalgesia and that low‐threshold Aβ‐fibres are responsible for induction of allodynia, the mechanisms of which remain elusive. We previously showed that intrathecal administration of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α) induce allodynia in conscious mice. Here we demonstrated that selective elimination of C‐fibres by neonatal capsaicin treatment resulted in the disappearance of allodynia induced by PGE2, but not that by PGF2α. PGE2‐induced allodynia was not observed in N‐methyl‐D‐aspartate (NMDA) receptor ε1 subunit knockout mice and was sensitive to morphine. In contrast, PGF2α‐induced allodynia was not observed in NMDA ε4 subunit knockout mice and was insensitive to morphine. Furthermore, while PGF2α showed a capsaicin‐insensitive feeble facilitatory action on evoked excitatory postsynaptic currents in dorsal horn neurons, PGE2 induced a long‐lasting facilitation of evoked excitatory postsynaptic currents in a capsaicin‐sensitive manner. Taken together, the present study demonstrates that there are two pathways for induction of allodynia and that capsaicin‐sensitive C‐fibres may participate in PGE2‐induced allodynia.
Morphine injected into the paraventricular nucleus of the hypothalamus prevents noncontact penile erections and impairs copulation: involvement of nitric oxide
Authors: Melis Maria Rosaria , Succu Salvatora , Spano Maria Sabrina , Argiolas Antonio
Published: March 2003

Male rats show four to six penile erection episodes when put in the presence of an inaccessible receptive female for 80 min. These noncontact erections occur concomitantly with an increase in nitric oxide production in the paraventricular nucleus of the hypothalamus. This is shown by the increases in the NO2– and NO3– concentrations in the paraventricular dialysate obtained from these males by in vivo microdialysis. The NO2– concentration increased from 0.75 ± 0.10 μm to 2.89 ± 0.39 μm and that of NO3– from 4.13 ± 0.58 μm to 9.5 ± 1.2 μm. Morphine (0.5, 1 and 5 μg), given unilaterally into the paraventricular nucleus 15 min before the introduction of the receptive female, prevented the NO2– and NO3– increases, and noncontact erections, dose‐dependently. In contrast, the κ opioid receptor agonist U‐69 593 (5 μg) was ineffective. The effects of morphine on NO2– and NO3–, and on noncontact erections, were prevented by the opiate receptor antagonist naloxone (10 μg) injected into the paraventricular nucleus 15 min before morphine. The NO2– and NO3– concentrations were also increased in the paraventricular dialysate of male rats during copulation, i.e. when in copula penile erections occurred. As found with noncontact erections, morphine, but not U‐69 593, injected into the paraventricular nucleus prevented the NO2– and NO3– increases and impaired copulatory behaviour, and naloxone prevented these responses when given before morphine. Although some diffusion of the opiate to surrounding brain areas cannot be completely ruled out, the present results suggest that morphine acts through μ receptors in the paraventricular nucleus to impair noncontact erections and copulation. These effects of morphine are apparently mediated by a prevention of the increased nitric oxide production that occurs in the paraventricular nucleus of the hypothalamus of male rats during sexual activity.
Presynaptic calcium‐channel currents in normal and csp mutant Drosophila peptidergic terminals
Authors: Morales Miguel , Ferrús Alberto , Martínez‐Padrón Manuel
Published: March 2003

The study of regulated vesicle exocytosis, which underlies neurotransmitter and neuropeptide release, has benefited from a convergence of several independent approaches. These include the use of genetically tractable organisms and model preparations that allow a direct characterization of presynaptic ionic currents. Aiming for a comprehensive analysis of release, we had already developed a Drosophila preparation in which electrophysiological recordings from peptidergic terminals are feasible. Here, we report on the characterization of the Ca2+‐channel currents present in these terminals. With Ba2+ as the charge carrier, the presynaptic membrane expresses a current type with high‐activation threshold and little inactivation. This current is blocked by verapamil and diltiazem at micromolar concentrations, it is relatively insensitive to nifedipine and completely resistant to non‐L‐type Ca2+‐channel antagonists. As a comparison, we also analysed the pharmacology of high‐threshold Ba+2 currents on muscle fibres. A high‐activation threshold Ca2+‐channel current is also present in muscle fibres, albeit with a distinct pharmacological profile. Thus, peptidergic terminals and muscle fibres exhibit different subtypes of voltage‐gated Ca2+ channels. The putative role of cysteine string protein (CSP) as a neuronal Ca2+‐channel modulator was tested by examining the peptidergic presynaptic current in csp null mutants. We show that CSP is expressed in peptidergic boutons and abolished in the mutant. Direct recordings, under conditions that inhibit calcium influx into glutamatergic terminals, show that Ca2+‐currents in peptidergic csp terminals are entirely normal. This result indicates that CSP is not a generic Ca2+‐channel modulator and it might perform different functions in fast versus slow forms of release.
In vitro electrophysiological properties of rat dorsal column nuclei neurons
Authors: Nuñez Angel , Buño Washington
Published: March 2003

The dorsal column nuclei include the gracile and cuneate nuclei, which receive somatosensory information from the periphery and project to the ventroposterior nucleus of the contralateral thalamus. The aim of this study was to determine the electrophysiological and morphological characteristics of the neurons of the dorsal column nuclei and to identify synaptic events evoked by electrical stimulation of the dorsal column, using an in vitro slice preparation. The results show two types of neurons, termed type I and II. A repolarizing sag distinguished type I cells during hyperpolarizing current injection, suggesting the activation of a Q‐current. Moreover, type I cells, but not type II cells, were capable of maintaining spontaneous rhythmic activity at 9–15 Hz. Both types of cells displayed a delay in their return to the resting membrane potential following hyperpolarizing current pulses, indicating the existence of an A‐current. Electrical stimuli applied to the dorsal column elicited brief EPSPs and IPSPs in both cell types. EPSPs were abolished by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, indicating that they were mediated through non‐NMDA receptors. IPSPs were blocked by picrotoxin, implying the activation of GABAA receptors. Intracellular staining with carboxyfluoresceine revealed that type I neurons had elongated somas and primary dendrites that extended radially. Type II cells were smaller and had round somas with few primary dendrites, most of them emerging from one pole of the soma. The axon of many type I neurons was stained and could be followed running ventrally and in rostral direction.
Intrinsic optical signal measurements reveal characteristic features during different forms of spontaneous neuronal hyperactivity associated with ECS shrinkage in vitro
Authors: Buchheim Katharina , Schuchmann Sebastian , Siegmund Herbert , Gabriel Hans Jürgen , Heinemann Uwe , Meierkord Hartmut
Published: March 2003

We induced three different forms of spontaneous synchronous hyperactivity in adult rat hippocampal–entorhinal cortex slices in order to investigate effects on the intrinsic optical signal and associated changes in the extracellular space (ECS) volume. Low‐Mg2+ artificial cerebrospinal fluid (ACSF) and the addition of 4‐aminopyridine induced synchronous hyperactivity resulting mainly from increased synaptic transmission, while low‐Ca2+ ACSF induced hyperactivity in the absence of evoked synaptic transmission. In the two models of enhanced synaptic transmission, spontaneous activity lead to an immediate increase of light transmission. In contrast, a decrease of light transmission took place during low‐Ca2+‐induced hyperactivity. All three forms of synchronous neuronal hyperactivity were associated with a shrinkage of the ECS volume, as revealed by the tetraethylammonium signal, measured with ion‐sensitive microelectrodes. This indicates that the change in the intrinsic optical signal is not simply related to a shrinkage in ECS volume. We conclude that different forms of spontaneous synchronous neuronal hyperactivity are associated with characteristic optical signals and that the direction of the change in intrinsic optical signal does not reflect ECS shrinkage alone.
Functional characteristics of skate connexin35, a member of the γ subfamily of connexins expressed in the vertebrate retina
Authors: White Thomas W. , Deans Michael R. , O'Brien John , Al‐Ubaidi Muayyad R. , Goodenough Daniel A. , Ripps Harris , Bruzzone Roberto
Published: March 2003

Retinal neurons are coupled by electrical synapses that have been studied extensively in situ and in isolated cell pairs. Although many unique gating properties have been identified, the connexin composition of retinal gap junctions is not well defined. We have functionally characterized connexin35 (Cx35), a recently cloned connexin belonging to the γ subgroup expressed in the skate retina, and compared its biophysical properties with those obtained from electrically coupled retinal cells. Injection of Cx35 RNA into pairs of Xenopus oocytes induced intercellular conductances that were voltage‐gated at transjunctional potentials ≥ 60 mV, and that were also closed by intracellular acidification. In contrast, Cx35 was unable to functionally interact with rodent connexins from the α or β subfamilies. Voltage‐activated hemichannel currents were also observed in single oocytes expressing Cx35, and superfusing these oocytes with medium containing 100 μm quinine resulted in a 1.8‐fold increase in the magnitude of the outward currents, but did not change the threshold of voltage activation (membrane potential = +20 mV). Cx35 intercellular channels between paired oocytes were insensitive to quinine treatment. Both hemichannel activity and its modulation by quinine were seen previously in recordings from isolated skate horizontal cells. Voltage‐activated currents of Cx46 hemichannels were also enhanced 1.6‐fold following quinine treatment, whereas Cx43‐injected oocytes showed no hemichannel activity in the presence, or absence, of quinine. Although the cellular localization of Cx35 is unknown, the functional characteristics of Cx35 in Xenopus oocytes are consistent with the hemichannel and intercellular channel properties of skate horizontal cells.
Intramembranous structure of synaptic membranes with special reference to spinules in the rat sensorimotor cortex
Authors: Bozhilova‐Pastirova A. , Ovtscharoff W.
Published: March 2003

The intramembranous structure of the synaptic contact zone at presynaptic and postsynaptic membranes in the rat sensorimotor cortex was examined by means of the freeze‐etching technique. In axospinous synapses, the synaptic contact zone is characterized by perforated and nonperforated aggregates of intramembranous particles at the extracellular half or E‐face of the postsynaptic membrane. On some perforated synaptic contact zones, both synaptic membranes are marked by so called spinules. These invaginations of the postsynaptic membrane and the parallel presynaptic membrane into the axon terminal are situated at the particle free zones among the postsynaptic E‐face intramembranous particle aggregates or in close proximity to it. Intramembranous characteristics of the spinules at both freeze‐etched faces of presynaptic and postsynaptic membranes and their density of perforated axospinous synapses were analysed. The results are discussed in terms of plasticity at the synaptic contact zone of the axospinous synapses of the sensorimotor cortex in the rat.
Raf‐1 and B‐Raf proteins have similar regional distributions but differential subcellular localization in adult rat brain
Authors: Morice Cecile , Nothias Fatiha , König Sandra , Vernier Philippe , Baccarini Manuela , Vincent Jean‐Didier , Barnier Jean Vianney
Published: March 2003

The Raf kinases play an important and specific role in the activation of extracellular signal‐regulated kinases (ERK) cascade. Beside its role in the control of proliferation and differentiation, the ERK cascade has also been implicated in neuron‐specific functions. In order to gain clues on the function of Raf kinases in the adult central nervous system (CNS), we performed a comparative analysis of the distribution and subcellular localization of the different Raf kinases in rat brain with antibodies specific for the different Raf kinases. We show that B‐Raf and Raf‐1 proteins are present in most brain areas, whereas A‐Raf is not detected. Interestingly, the two Raf proteins have an approximately similar pattern of distribution with a rostro‐caudal decreasing gradient of expression. These two kinases are colocalized in neurons but they are differentially located in subcellular compartments. Raf‐1 is localized mainly in the cytosolic fraction around the nucleus, whereas B‐Raf is widely distributed in the cell bodies and in the neuritic processes. In addition, we demonstrated that numerous B‐Raf isoforms are present in the brain. These isoforms have a differential pattern of distribution, some of them being ubiquitously expressed whereas others are localized to specific brain areas. These isoforms also have a clear differential subcellular localization, specially in Triton‐insoluble fractions, but also in synaptosomal, membrane and cytosolic compartments. Altogether these results suggest that each Raf protein could have a distinct signalling regulatory function in the brain with regard to its subcellular localization.
Mint2/X11‐like colocalizes with the Alzheimer's disease amyloid precursor protein and is associated with neuritic plaques in Alzheimer's disease
Authors: McLoughlin Declan M. , Irving Nicholas G. , Brownlees Janet , Brion Jean‐Pierre , Leroy Karelle , Miller Christopher C. J.
Published: March 2003

Aberrant metabolism of the amyloid precursor protein (APP) is believed to be at least part of the pathogenic process in Alzheimer's disease. The carboxy‐terminus of APP has been shown to interact with the Mint/X11 family of phosphotyrosine binding (PTB) domain‐bearing proteins. It is via their PTB domains that the Mints/X11s bind to APP. Here we report the cloning of full‐length mouse Mint2 and demonstrate that in primary cortical neurons, Mint2 and APP share highly similar distributions. Mint2 also colocalizes with APP in transfected CHO cells. In Mint2/APP‐cotransfected cells, Mint2 reorganizes the subcellular distribution of APP and also increases the steady‐state levels of APP. Finally, we demonstrate that Mint2 is associated with the neuritic plaques found in Alzheimer's disease but not with neurofibrillary tangles. These results are consistent with a role for Mint2 in APP metabolism and trafficking, and suggest a possible role for the Mints/X11s in the pathogenesis of Alzheimer's disease.