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The peripheral benzodiazepine receptor (PBR), a benzodiazepine but not gamma-aminobutyric acid-binding mitochondrial membrane protein, has roles in steroid production, energy metabolism, cell survival and growth. PBR expression in the nervous system has been reported in non-neuronal glial and immune cells. We now show expression of both PBR mRNA and protein, and the appearance of binding of a synthetic ligand, [(3)H]PK11195, in dorsal root ganglion (DRG) neurons following injury to the sciatic nerve. In naïve animals, PBR mRNA, protein expression and ligand binding are undetectable in the DRG. Three days after sciatic nerve transection, however, PBR mRNA begins to be expressed in injured neurons, and 4 weeks after the injury, expression and ligand binding are present in 35% of L4 DRG neurons. PBR ligand binding also appears after injury in the superficial dorsal horn of the spinal cord. The PBR expression in the DRG is restricted to small and medium-sized neurons and returns to naïve levels if the injured peripheral axons are allowed to regrow and reinnervate targets. No non-neuronal PBR expression is detected, unlike its putative endogenous ligand the diazepam binding inhibitor (DBI), which is expressed only in non-neuronal cells, including the satellite cells that surround DRG neurons. DBI expression does not change with sciatic nerve transection. PBR acting on small-calibre neurons could play a role in the adaptive survival and growth responses of these cells to injury of their axons.

In the cerebral cortex of mammals, gamma-aminobutyric acid (GABA)ergic neurons represent 15-25% of all neurons, depending on the species and area being examined. Because converging evidence suggests that activity may play an important role in the neuritic maturation and synaptic function of GABAergic neurons, it is feasible that activity plays a role in the regulation of the proportion of GABAergic neurons. Here we provide direct evidence that early in cortical development activity blockade may deplete the network of a subpopulation of GABA immunoreactive neurons characterized by their small size and late generation in vitro. In a period of time coinciding with the emergence of synchronous network activity, the survival and morphological differentiation of GABAergic neurons was influenced by long-term blockade of synaptic activity. While GABA(A) receptor antagonists had a minor promoting effect on interneuronal survival during the second week in vitro, antagonists of ionotropic glutamate receptors strongly impaired survival and differentiation of immature GABAergic interneurons. Interneuronal loss was more severe when N-methyl-D-aspartate receptors were blocked than after blockade of alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA)/kainate receptors. The decrease in the density of GABAergic neurons was irreversible, but could be prevented by the simultaneous addition of brain-derived neurotrophic factor (BDNF). These results suggest that there is a narrow time window during neocortical development when glutamatergic activity, and specially NMDA receptor stimulation, is crucial to assure survival and maturation of a subpopulation of late developing GABAergic interneurons.

A synRas mouse model was used expressing constitutively activated Ha-Ras (Val12 mutation) in neurons to investigate the role of Ras-MAPkinase signalling for neuronal connectivity in adult brain. Expression of the transgene in the cortex of these mice starts after neuronal differentiation is completed and allows to directly investigate the effects of enhanced Ras activity in differentiated neurons. Activation of Ha-Ras induced an increase in soma size which was sensitive to MEK inhibitor in postnatal organotypic cultures. Adult cortical pyramidal neurons showed complex structural rearrangements associated with an increased size and ramification of dendritic arborization. Dendritic spine density was elevated and correlated with a twofold increase in number of synapses. In acute brain slices of the somatosensory and of the visual cortex, extracellular field potentials were recorded from layer II/III neurons. The input-output relation of synaptically evoked field potentials revealed a significantly higher basal excitability of the transgenic mice cortex compared to wild-type animals. In whole cell patch clamp preparations, the frequency of AMPA receptor-mediated spontaneous excitatory postsynaptic currents was increased while the ratio between NMDA and AMPA-receptor mediated signal amplitude was unchanged. A pronounced depression of paired pulse facilitation indicated that Ras contributes to changes at the presynaptic site. Furthermore, synRas mice showed an increased synaptic long-term potentiation, which was sensitive to blockers of NMDA-receptors and of MEK. We conclude that neuronal Ras is a common switch of plasticity in adult mammalian brain sculpturing neuronal architecture and synaptic connectivity in concert with tuning synaptic efficacy.

Cellular repair of neuronal circuitry affected by neurodegenerative disease or injury may be approached in the adult neocortex via transplantation of neural precursors ("neural stem cells") or via molecular manipulation and recruitment of new neurons from endogenous precursors in situ. A major challenge for potential future approaches to neuronal replacement will be to specifically direct and control progressive differentiation, axonal projection and connectivity of neural precursors along a specific neuronal lineage. This goal will require a progressively more detailed understanding of the molecular controls over morphologic differentiation of specific neuronal lineages, including neurite outgrowth and elongation, in order to accurately permit and direct proper neuronal integration and connectivity. Here, we investigate controls over the morphologic differentiation of a specific prototypical lineage of cortical neurons: callosal projection neurons (CPN). We highly enriched CPN to an essentially pure population, and cultured them at three distinct stages of development from embryonic and postnatal mouse cortex by retrograde fluorescence labelling, followed by fluorescence-activated cell sorting. We find that specific peptide growth factors exert direct stage-specific positive and negative effects over the morphologic differentiation and process outgrowth of CPN. These effects are distinct from the effects of these growth factors on CPN survival [Catapano et al. (2001)J. Neurosci., 21, 8863-8872]. These data may be critical for the future goal of directing lineage-specific neuronal differentiation of transplanted or endogenous precursors/"stem cells" toward cellular repair of complex cortical circuitry.

Barrington's nucleus impacts on bladder and distal colon function and relays pelvic visceral information to the forebrain. This study investigated processing of information from the bladder and the distal colon by Barrington's nucleus in the rat. The responses of individual Barrington's nucleus neurons to bladder and/or colon distention were characterized using extracellular recording and the recorded neurons were identified using juxtacellular labelling. Most neurons within Barrington's nucleus (79%) were activated by bladder distention, consistent with its role as a pontine micturition centre. Although no neurons were selectively responsive to colon distention, the majority of bladder-responsive neurons (73%) were also activated by colon distention. In a second study, Barrington's nucleus neurons were characterized with respect to their response to colon distention and their immunoreactivity for the stress-related neuropeptide corticotropin-releasing factor (CRF). Of 30 labelled neurons in the central part of Barrington's nucleus, 53% were activated by colon distention and 63% of these were CRF-ir. This is the first report demonstrating that Barrington's nucleus neurons are responsive to colon distention. The results provide evidence for convergence of information from the bladder and the colon onto individual Barrington's nucleus neurons. Taken with evidence that many Barrington's nucleus neurons are synaptically linked to the bladder and colon, the present study suggests a role for these neurons in coordinating peripheral parasympathetic and central responses to both viscera and implicate CRF as a neurotransmitter in this function. Dysfunctions in this circuit may underlie the coexistence of colon and bladder symptoms observed in functional bowel disorders.

The roles of protein kinase C and the MAP-kinase extracellular receptor kinase in structural changes associated with long-term potentiation of network activity were examined in cultured hippocampal neurons. A brief exposure to a conditioning medium that favours activation of the N-methyl-d-aspartate receptor caused a rapid and specific increase in staining of neurons for the phosphorylated form of extracellular receptor kinase as well as of cyclic AMP response element binding protein. Exposure of the cultures to the conditioning medium was followed by a protein synthesis-dependent formation of novel dendritic spines. An extracellular receptor kinase antagonist PD98059 blocked the phosphorylated form of extracellular receptor kinase response and the formation of novel spines. A selective protein kinase C agonist, phorbol 12-myristate 13-acetate, caused activation of extracellular receptor kinase and formation of novel spines. The protein kinase C antagonist GF109203x blocked the phosphorylated form of extracellular receptor kinase response and the subsequent spine formation by phorbol 12-myristate 13-acetate. Both the conditioning medium and phorbol 12-myristate 13-acetate caused a delayed increase in mean amplitude of miniature excitatory postsynaptic currents recorded in the hippocampal neurons. These results indicate that activation of extracellular receptor kinase mediates the effect of a conditioning protocol on the formation of dendritic spines. The formation of novel spines was associated with long-term increase in network activity and functional synaptic connectivity among the cultured neurons.

To explore the role of brain-derived neurotrophic factor for survival and generation of striatal neurons after stroke, recombinant adeno-associated viral vectors carrying brain-derived neurotrophic factor or green fluorescent protein genes were injected into right rat substantia nigra 4-5 weeks prior to 30 min ipsilateral of middle cerebral artery occlusion. The brain-derived neurotrophic factor-recombinant adeno-associated viral transduction markedly increased the production of brain-derived neurotrophic factor protein by nigral cells. Brain-derived neurotrophic factor was transported anterogradely to the striatum and released in biologically active form, as revealed by the hypertrophic response of striatal neuropeptide Y-positive interneurons. Animals transduced with brain-derived neurotrophic factor-recombinant adeno-associated virus also exhibited abnormalities in body posture and movements, including tilted body to the right, choreiform movements of left forelimb and head, and spontaneous, so-called 'barrel' rotation along their long axis. The continuous delivery of brain-derived neurotrophic factor had no effect on the survival of striatal projection neurons after stroke, but exaggerated the loss of cholinergic, and parvalbumin- and neuropeptide Y-positive, gamma-aminobutyric acid-ergic interneurons. The high brain-derived neurotrophic factor levels in the animals subjected to stroke also gave rise to an increased number of striatal cells expressing doublecortin, a marker for migrating neuroblasts, and cells double-labelled with the mitotic marker, 5-bromo-2'-deoxyuridine-5'monophosphate, and early neuronal (Hu) or striatal neuronal (Meis2) markers. Our findings indicate that long-term anterograde delivery of high levels of brain-derived neurotrophic factor increases the vulnerability of striatal interneurons to stroke-induced damage. Concomitantly, brain-derived neurotrophic factor potentiates the stroke-induced neurogenic response, at least at early stages.

In view of the potential impact of pituicyte morphology on neurohypophysial hormone secretion, we have studied the mechanisms involved in the shape changes induced by vasopressin (AVP) and oxytocin (OXT) in cultured rat pituicytes. Pituicytes induced to become stellate in the presence of 10 micro m adenosine revert to their nonstellate shape approximately 20 min after application of AVP or OXT. The IC50 for this effect is 0.1 nm for AVP and 36 nm for OXT. Both agonists induce Ca2+ signals in pituicytes, comprised of a transient peak and a plateau phase that is dependent on the presence of extracellular Ca2+. The EC50 values of AVP for the transient and sustained responses are 4.5 and 0.1 nm, respectively; corresponding values for OXT are 180 and 107 nm. We determined pharmacologically that these hormone-induced Ca2+ signals are mediated by the V1a subtype of vasopressin receptors, similar to what we previously observed for hormone-induced reversal of stellation. Removal of extracellular Ca2+ or chelation of intracellular Ca2+ partially prevented AVP from reversing stellation, suggesting a role for Ca2+ in this event. We previously established that adenosine-induced stellation of pituicytes occurs via RhoA inhibition. However, pharmacological experiments and pull-down assays presented here show that AVP-induced reversal of stellation does not involve RhoA activation. Rather, AVP was found to induce a time-dependent activation of Cdc42, another small GTPase involved in cytoskeletal plasticity. Activation of Cdc42 by AVP is sensitive to intra- and extracellular Ca2+ depletion, similar to AVP-induced reversal of stellation. Furthermore, AVP-induced reversal of stellation is blocked by expression of an NWASP fragment known to inhibit endogenous Cdc42.

The aim of this work was to explore the electrical spatial profile of the dendritic arborization during membrane potential oscillations of a bistable motoneuron. Computational simulations provided the spatial counterparts of the temporal dynamics of bistability and allowed simultaneous depiction the electrical states of any sites in the arborization. We assumed that the dendritic membrane had homogeneously distributed specific electrical properties and was equipped with a cocktail of passive extrasynaptic and NMDA synaptic conductances. The electrical conditions for evoking bistability in a single isopotential compartment and in a whole dendritic arborization were computed and showed differences, revealing a crucial effect of dendritic geometry. Snapshots of the whole arborization during bistability revealed the spatial distribution of the density of the transmembrane current generated at the synapses and the effectiveness of the current transfer from any dendritic site to the soma. These functional maps changed dynamically according to the phase of the oscillatory cycle. In the low depolarization state, the current density was low in the proximal dendrites and higher in the distal parts of the arborization while the transfer effectiveness varied in a narrow range with small differences between proximal and distal dendritic segments. When the neuron switched to high depolarization state, the current density was high in the proximal dendrites and low in the distal branches while a large domain of the dendritic field became electrically disconnected beyond 200 micro m from the soma with a null transfer efficiency. These spatial reconfigurations affected dynamically the size and shape of the functional dendritic field and were strongly geometry-dependent.

Recent immunocytochemical stainings on cat visual cortex, visually stimulated for 1 h, showed a strong induction of Fos expression in cortical neurons. We initiated immunocytochemical double staining experiments with different cytochemical markers to investigate the neurochemical and morphological character of these activated neurons showing Fos induction after sensory stimulation. Double staining with Fos and glutamic acid decarboxylase (GAD) demonstrated the presence of Fos in the nuclei of GABAergic neurons of the primary visual cortex. To further subdivide this Fos/GABAergic cell population we investigated whether Fos colocalized with parvalbumin, calbindin or calretinin. Colocalization of Fos with these calcium-binding proteins delineated distinct neuronal subclasses of Fos-immunoreactive neurons in supra- and infragranular layers of cat area 17. Quantitative analysis of the proportion of immunoreactive local circuit neurons revealed that 35% of the GABAergic neurons showed Fos induction in supragranular layers, whereas in infragranular layers a mere 10% of the GABAergic cells revealed Fos expression. Fos coexisted in about 24% of the calbindin-immunopositive cells within supra- and infragranular layers, but only a minority of the parvalbumin and the calretinin neuronal subgroups were immunopositive for Fos in the corresponding layers of area 17. These findings suggest that visual stimulation induces Fos expression in distinct subsets of inhibitory neurons in cat primary visual cortex.

High threshold noxious heat-activated currents and vanilloid receptor-like protein-1 expression were studied in rat cultured primary sensory neurons to find out the molecule(s) responsible for high threshold noxious heat-sensitivity. The average temperature threshold and amplitude of high threshold noxious heat-activated currents were 51.6 +/- 0.13 degrees C and -2.0 +/- 0.1nA (at a holding potential of -60 mV), respectively. The current-voltage relationship of high threshold noxious heat-activated currents was linear at positive membrane potentials, while it showed a weak inward rectification at negative membrane potentials. The average reversal potential measured in control intracellular and extracellular solutions was 4.5 +/- 0.9 mV (n = 6). Ionic substitutions revealed that the high threshold noxious heat-activated current is a nonselective cationic current with calculated ionic permeabilities of Cs+ : Na+ : Ca2+ (1 : 1.3 : 4.5). Consecutive stimuli reduced the heat threshold from 52.2 +/- 1 to 48.4 +/- 1.4 degrees C and then to 44 +/- 0.7 degrees C (n = 3). High threshold noxious heat-activated currents could dose-dependently and reversibly be reduced by ruthenium red (100 nm-10 micro m) but not by capsazepine (10 micro m). The average longest diameter of high threshold noxious heat-sensitive neurons was 31.48 +/- 0.5 micro m (A = approximately 778 micro m2; n = 77). Twenty-three percent of the total neuronal population expressed vanilloid receptor-like protein-1. The average area of the vanilloid receptor-like protein-1-immunopositive cells was 1,696 +/- 65.3 micro m2 (d = approximately 46 micro m). Vanilloid receptor-like protein-1-expressing neurons did not express the vanilloid receptor 1. Comparison of our data with results obtained in vanilloid receptor-like protein-1-expressing non-neuronal cells and previous immunohistochemical findings suggests that high threshold noxious heat-activated currents are produced by vanilloid receptor-like protein-1 and that high threshold heat-sensitive dorsal root ganglion neurons are the perikarya of type I noxious heat-sensitive fibers.

It has been suggested that altered retrograde neurotrophin support contributes to the phenotypic switch observed in BDNF expression in injured sensory neurons. Thus, modulatory influences of NGF and NT-3 on BDNF expression in injured adult rat DRG neurons were examined using in situ hybridization and immunohistochemical approaches. Quantitative analysis reveals a biphasic response to sciatic nerve injury, whereby in the first day following injury, BDNF expression is up-regulated in approximately 83% of injured neurons including all small neurons, and a larger pool of trkB expressing neurons than in intact. By 1 week and up to 3 weeks later expression is still seen in approximately 66% of injured neurons, but the characteristic phenotypic switch in the subpopulations expressing BDNF occurs, whereby expression in the trkA population is reduced and expression in trkB- and in trkC-positive neurons is elevated. NGF infusion results in elevated levels of BDNF expression in both intact and injured trkA-positive neurons, accompanied by reduced trkB expression. NT-3 acts in an opposite fashion effecting a down-regulation in BDNF expression in intact neurons and preventing/reducing the injury-associated increases in BDNF expression in both trkC- and nontrkC-expressing subpopulations of injured neurons. These effects suggest NGF can regulate BDNF expression in trkA-expressing neurons regardless of the axonal state and that elevated levels of BDNF may contribute to the down-regulation in trkB expression associated with these states. Furthermore, the findings demonstrate that NT-3 can act in an antagonistic fashion to NGF in the regulation of BDNF expression in intact neurons, and mitigate BDNF's expression in injured neurons.

The pre-Bötzinger complex (preBötC) in the ventrolateral medulla is thought to be the kernel for respiratory rhythm generation. Neurons in the preBötC contain intense neurokinin-1 receptor (NK1R) immunoreactivity. Some of these neurons in the adult preBötC are presumed to be the pre-inspiratory interneurons that are essential for generating respiratory rhythm in the neonate. Chloride-mediated synaptic inhibition is critical for rhythmogenesis in the adult. The present study used immunofluorescence histochemistry and immunogold-silver staining to determine the inhibitory synaptic relationship between glutamic acid decarboxylase (GAD)- or glycine transporter 2 (GlyT2)-immunoreactive (ir) boutons and NK1R-ir neurons in the preBötC of adult rats. Under the confocal microscope, we found that GAD- and GlyT2-ir boutons were in close apposition to NK1R-ir somas and dendrites in the preBötC. Under the electron microscope, GAD- and GlyT2-ir terminals were in close apposition to NK1R-ir somas and dendrites. Symmetric synapses were identified between GAD- or GlyT2-ir terminals and NK1R-ir neurons. A total of 51.6% GAD-ir and 38.2% GlyT2-ir terminals were found to contact or make synapses with NK1R-ir profiles, respectively. GAD- and GlyT2-ir terminals synapsed not only upon NK1R-ir neurons but also upon NK1R immuno-negative neurons. NK1R-ir neurons received both symmetric (presumed inhibitory) and asymmetric (presumed excitatory) synapses. Thus, the present findings provide the morphological basis for inhibitory inputs to NK1R-ir neurons in the preBötC, consistent with the suggestion that chloride-mediated synaptic inhibition may contribute importantly to rhythm generation by controlling the membrane potential trajectory and resetting rhythmic bursting of the kernel neurons in the adult.

During postnatal development of CA1 pyramidal neurons, GABAergic synapses are excitatory and established prior to glutamatergic synapses. As interneurons are generated before pyramidal cells, we have tested the hypothesis that the GABAergic interneuronal network is operative before glutamate pyramidal neurons and provides the initial patterns of activity. We patch-clamp recorded interneurons in foetal (69 neurons) and neonatal P0 (162 neurons) hippocampal slices and performed a morphofunctional analysis of biocytin-filled neurons. At P0, three types of interneurons were found: (i) non-innervated "silent" interneurons (5%) with no spontaneous or evoked synaptic currents; (ii) G interneurons (17%) with GABA(A) synapses only; and (iii) GG interneurons with GABA and glutamatergic synapses (78%). Relying on the neuronal capacitance, cell body size and arborization of dendrites and axons, the three types of interneurons correspond to three stages of development with non-innervated neurons and interneurons with GABA(A) and glutamatergic synapses being, respectively, the least and the most developed. Recordings from both pyramidal neurons and interneurons in foetuses (E18-20) revealed that the majority of interneurons (65%) had functional synapses whereas nearly 90% of pyramidal neurons were quiescent. Therefore, interneurons follow the same GABA-glutamate sequence of synapse formation but earlier than the principal cells. Interneurons are the source and the target of the first synapses formed in the hippocampus and are thus in a position to modulate the development of the hippocampus in the foetal stage.

Dopamine (DA) has a crucial role in the modulation of striatal neuron activity. Along with projection cells, striatal interneurons receive dense dopaminergic innervation from midbrain neurons, thus, also suggesting that these intrinsic cells represent a synaptic target for DA action in the striatum. In the present study, we investigated the effects of DA on low-threshold spike (LTS) interneurons of the rat striatum, by means of in vitro whole-cell patch-clamp electrophysiological recordings. Dopamine depolarized LTS cells, a pharmacological effect prevented by D1- but not D2-like DA receptor antagonists. The membrane depolarization produced by DA was sufficient to trigger action potential discharge in the recorded cells and was insensitive to tetrodotoxin and glutamate receptor antagonists. In addition, this pharmacological effect was mimicked by D1- but not D2-like DA receptor agonists, implying the selective involvement of D1-like receptors in this action.

Systemic administration of nitroglycerin, a nitric oxide donor, triggers in migraine patients a delayed attack of unknown mechanism. After puberty migraine is more prevalent in women. Attacks can be triggered by abrupt falls in plasma estrogen levels, which accounts in part for sexual dimorphism, but lacks an established neurobiological explanation. We studied the effect of nitroglycerin on the innervated area of calcitonin gene-related peptide (CGRP) and serotonin-immunoreactive afferents to the superficial laminae of the spinal portion of trigeminal nucleus caudalis, and its modulation by estrogen. In male rats, nitroglycerin produced after 4 h a significant decrease of the area innervated by CGRP-immunoreactive afferents and an increase of that covered by serotonin-immunoreactive fibres. These effects were not observed in the superficial laminae of thoracic dorsal horns. The effect of nitroglycerin was similar in ovariectomized females. In estradiol-treated ovariectomized females the area in the spinal portion of trigeminal nucleus caudalis laminae I-II covered by CGRP-immunoreactive fibres was lower and that of serotonin-immunoreactive fibres was higher than in males and for both transmitters not significantly changed after nitroglycerin. The bouton size of CGRP profiles was smaller in estradiol-treated ovariectomized females, whereas after nitroglycerin it decreased significantly but only in males and ovariectomized females. Nitroglycerin, i.e. nitric oxide, is thus able to differentially influence afferent fibres in the superficial laminae of rat spinal trigeminal nucleus caudalis. Estradiol modulates the basal expression of these transmitters and blocks the nitroglycerin effect. These data may contribute to understanding the mechanisms by which estrogens influence migraine severity and the triggering of attacks by nitric oxide.

Utrophin, the autosomal homologue of dystrophin, the Duchenne muscular dystrophy gene product, is a cytoskeletal protein found in many tissues. In muscle fibers, the level and localization of utrophin depend on their state of differentiation and innervation. Transgenic overexpression of utrophin prevents degeneration of dystrophin-deficient muscle fibers. In brain, in addition to its enrichment in blood vessels, utrophin is associated primarily with the plasma membrane of large sensory and motor brainstem neurons, suggesting a contribution to their structural stability. Here, we examined the role of utrophin for long-term survival of dentate granule cells, which become markedly hypertrophic in a mouse model of temporal lobe epilepsy. This morphogenetic change is induced several weeks after a unilateral intrahippocampal injection of kainic acid (KA), while mice experience chronic focal seizures. Using in situ hybridization and immunohistochemistry, we show that dispersion and hypertrophy of granule cells in KA-treated wildtype mice are accompanied by a strong and long-lasting expression of utrophin in somata and proximal dendrites. Utrophin knockout mice had a normal hippocampal cytoarchitecture but were more sensitive to KA-induced excitotoxicity, as shown by increased mortality and faster progression of the lesion. At 6 weeks post-KA, the numerical density of granule cells and thickness of the granule cell layer were significantly reduced ipsilaterally in mutant mice, indicating a profound reduction in total cell number in the absence of utrophin. These findings suggest that utrophin contributes to protect CNS neurons against pathological insults, in particular, stimuli leading to massive neuronal hypertrophy.

Lesions of the mammalian visual cortex cause the retrograde degeneration of the thalamic neurons projecting to the damaged cortex. The proto-oncogene bcl-2 is known to inhibit neuronal apoptosis induced by a variety of noxious stimuli and preserve the functional integrity of the injured cells. Here we have tested whether the overexpression of bcl-2 via adeno-associated virus (AAV) vectors is able to protect the neurons in the lateral geniculate nucleus after visual cortex ablation in adult rats. Recombinant AAV vectors encoding Bcl-2 (AAV-Bcl-2) or green fluorescent protein (AAV-GFP) as a control were stereotaxically injected into the geniculate. Three weeks after vector injection, the ipsilateral visual cortex was removed by aspiration, and cell survival was assessed 2 weeks later. We found that 20% of the geniculate neurons were transduced by the Bcl-2 vector. These cells were completely protected from death following cortical ablation. Delivery of AAV-GFP transduced an identical number of geniculate neurons but had no effect on cell survival after lesion. The total number of surviving geniculate neurons was found to be significantly higher in animals injected with AAV-Bcl-2 than in rats injected with AAV-GFP or in control lesioned rats. These data indicate that Bcl-2 gene therapy with AAV vectors represents an effective treatment to promote neuronal survival after central nervous system insults.

Whole-cell recording techniques were used to characterize ionic membrane currents and odourant responses in honeybee olfactory receptor neurons (ORNs) in primary cell culture. ORNs of workerbee (female) and drone (male) were isolated at an early stage of development before sensory axons connect to their target in the antennal lobe. The results collectively indicate that honeybee ORNs have electrical properties similar, but not necessarily identical to, those currently envisaged for ORNs of other species. Under voltage clamp at least four ionic currents could be distinguished. Inward currents were made of a fast transient, tetrodotoxin-sensitive sodium current. In some ORNs a cadmium-sensitive calcium current was detected. ORNs showed heterogeneity in their outward currents: either outward currents were made of a delayed rectifier type potassium current, which was partially blocked by tetraethyl ammonium or quinidine, or were composed of a delayed rectifier type and a transient calcium-dependent potassium current, which was cadmium-sensitive and abolished by removal of external calcium. The proportion of each of the two outward currents, however, was different within the ORNs of the two sexes suggesting a gender-specific functional heterogeneity. ORNs showed heterogeneity in action potential firing properties: depolarizing current steps elicited either one action potential or, as in most of the cells, it led to repetitive spiking. Action potentials were tetrodotoxin-sensitive suggesting they are carried by sodium. Odourant stimulation with different mixtures and pure substances evoked depolarizing receptor potentials with superimposed action potentials when spike threshold was reached. In summary, honeybee ORNs are remarkably mature at early stages in their development.

The time-dependent decline in the ability of motoneurons to regenerate their axons after axotomy is one of the principle contributing factors to poor functional recovery after peripheral nerve injury. A decline in neurotrophic support may be partially responsible for this effect. The up-regulation of BDNF after injury, both in denervated Schwann cells and in axotomized motoneurons, suggests its importance in motor axonal regeneration. In adult female Sprague-Dawley rats, we counted the number of freshly injured or chronically axotomized tibial motoneurons that had regenerated their axons 1 month after surgical suture to a freshly denervated common peroneal distal nerve stump. Motor axonal regeneration was evaluated by applying fluorescent retrograde neurotracers to the common peroneal nerve 20 mm distal to the injury site and counting the number of fluorescently labelled motoneurons in the T11-L1 region of the spinal cord. We report that low doses of BDNF (0.5-2 microg/day for 28 days) had no detectable effect on axonal regeneration after immediate nerve repair, but promoted axonal regeneration of motoneurons whose regenerative capacity was reduced by chronic axotomy 2 months prior to nerve resuture, completely reversing the negative effects of delayed nerve repair. In contrast, high doses of BDNF (12-20 microg/day for 28 days) significantly inhibited motor axonal regeneration, after both immediate nerve repair and nerve repair after chronic axotomy. The inhibitory actions of high dose BDNF could be reversed by functional blockade of p75 receptors, thus implicating these receptors as mediators of the inhibitory effects of high dose exogenous BDNF.

The effects of water on cortical excitability, measured using magnetoencephalographic recordings, were investigated in a sample of 19 healthy volunteers in a double-blind, placebo experiment comparing water with saline solution. Spontaneous magnetoencephalogram as well as auditory-evoked magnetic fields were recorded before and after the drinking of 750 mL water (9 subjects) or saline solution (10 subjects) and during and after hyperventilation following the drinking conditions. Hyperventilation was used to enhance the hypothesized synchronizing effect of water on spontaneous magnetoencephalographic activity. In addition, the magnetic fields were measured during a dichotic listening task under attended and unattended conditions. The prediction, that intake of water, because of induced cell swelling, will increase neuronal excitability and lead to an increased synchronization of the spontaneous magnetoencephalogram during hyperventilation was confirmed. Hyperventilation induced an increase of spectral power in all frequency bands particularly theta and delta power after water drinking. Furthermore, there was an increase of magnetic mismatch negativity (MMNm) amplitude in attended conditions and a simultaneous decrease in unattended conditions after water drinking. N1m (magnetic N1 wave) revealed significant changes during experimental conditions: increase after drinking and decrease after hyperventilation in both groups. MMNm for attended conditions showed a high positive correlation with osmolality changes (difference in the mol solute per kg water before and after drinking); N1m and PNm (magnetic processing negativity) as well as MMNm for unattended conditions showed significant correlations with subjective ratings of thirst and mood state.

Infrared videomicroscopy and differential interference contrast optics were used to identify medium- and large-sized neurons in striatal slices from young rats. Whole-cell patch-clamp recordings were obtained to compare membrane currents evoked by application of N-methyl-d-aspartate (NMDA) and kainate. Inward currents and current densities induced by NMDA were significantly smaller in large- than in medium-sized striatal neurons. The negative slope conductance for NMDA currents was greater in medium- than in large-sized neurons and more depolarization was required to remove the Mg2+ blockade. In contrast, currents induced by kainate were significantly greater in large-sized neurons whilst current densities were approximately equal in both cell types. Spontaneous excitatory postsynaptic currents occurred frequently in medium-sized neurons but were relatively infrequent in large-sized neurons. Excitatory postsynaptic currents evoked by electrical stimulation were smaller in large- than in medium-sized neurons. A final set of experiments assessed a functional consequence of the differential sensitivity of medium- and large-sized neurons to NMDA. Cell swelling was used to examine changes in somatic area in both neuronal types after prolonged application of NMDA or kainate. NMDA produced a time-dependent increase in somatic area in medium-sized neurons whilst it produced only minimal changes in large interneurons. In contrast, application of kainate produced significant swelling in both medium- and large-sized cells. We hypothesize that reduced sensitivity to NMDA may be due to variations in receptor subunit composition and/or the relative density of receptors in the two cell types. These findings help define the conditions that put neurons at risk for excitotoxic damage in neurological disorders.

Ethanol effects on the central nervous system have been well investigated and described in recent years; modulations, by ethanol, of several ligand-gated and voltage-gated ion channels have been found. In this paper, we describe a shortening of action potential duration (APD) by ethanol in approximately equal to 40% of small diameter neurons in rat dorsal root ganglia (DRG). In these neurons, designated as group A neurons, we observed an ethanol-induced increase in whole-cell outward-current. As iberiotoxin, a specific blocker of large-conductance calcium-activated K+ channels (BK(Ca) channels), blocks the effects of ethanol, we investigated the interaction between these channels and ethanol in outside-out patches. Open probability of BK(Ca) channels was increased 2-6 x depending on the concentration (40-80 mM approximately equal to 2-4 per thousand v/v) of ethanol. Functional consequences were a prolongation of the refractory period, which was reversible after addition of iberiotoxin, and reduced firing frequency during ethanol application. In contrast, another type of neuron (group B) showed a prolonged APD during application of ethanol which was irreversible in most cases. In 90% of cases, neurons of group A showed a positive staining for isolectin B4 (I-B4), a marker for nociceptive neurons. We suggest that the activation of BK(Ca) channels induced by clinically relevant concentrations of ethanol, the resulting modulations of APD and refractory period of DRG neurons, might contribute to clinically well-known ethanol-induced analgesia and paresthesia.

Knockout Otx1 mice present a microcephalic phenotype mainly due to reduced deep neocortical layers and spontaneous recurrent seizures. We investigated the excitable properties of layer V pyramidal neurons in neocortical slices prepared from Otx1-/- mice and age-matched controls. The qualitative firing properties of the neurons of Otx1-/- mice were identical to those found in wild-type controls, but the proportion of intrinsically bursting (IB) neurons was significantly smaller. This is in line with the lack of the Otx1 gene contribution to the generation and differentiation of neurons destined for the deep neocortical layers, in which IB neurons are located selectively in wild-type rodents. The pyramidal neurons recorded in Otx1-/- mice responded to near-threshold electrical stimulation of the underlying white matter, with aberrant polysynaptic excitatory potentials often leading to late action potential generation. When the strength of the stimulus was increased, the great majority of the Otx1-/- neurons (78%) responded with a prominent biphasic inhibitory postsynaptic potential that was significantly larger than that observed in the wild-type mice, and was often followed by complex postinhibitory depolarizing events. Both late excitatory postsynaptic potentials and postinhibitory excitation were selectively suppressed by NMDA receptor antagonists, but not by AMPA antagonists. We conclude that the cortical abnormalities of Otx1-/- neocortex due to a selective loss of large projecting neurons lead to a complex rearrangement of local circuitry, which is characterized by an excess of N-methyl-d-aspartate-mediated polysynaptic excitation that is counteracted by GABA-mediated inhibition in only a limited range of stimulus intensity. Prominent postsynaptic inhibitory potentials may also act as a further pro-epileptogenic event by synchronizing abnormal excitatory potentials.

The circadian pacemaker housed in the suprachiasmatic nucleus (SCN) synchronizes daily sleep-wake cycles, presumably by modulating the sleep-wake regulatory system, including ventrolateral preoptic area (VLPO) neurons. We used whole-cell patch-clamp recording to study the projections from the SCN to the VLPO in horizontal slices of rat hypothalamus. Single-pulse stimulation of the SCN region elicited postsynaptic currents (PSCs) in 20 of 66 neurons (30%) recorded within the VLPO region as verified by intracellular biocytin labelling. At a holding potential of -60 mV, the evoked PSCs had an amplitude of 17.6 +/- 3.2 pA (SEM) and a latency of 6.3 +/- 0.5 ms (n = 10). There was a trend for simple excitatory postsynaptic currents (EPSCs) to be evoked in the VLPO cluster, simple inhibitory postsynaptic currents (IPSCs) in the extended VLPO, and a combination of EPSCs and IPSCs in both regions. IPSCs were blocked reversibly by bicuculline (10 microm, n = 11). In both the presence and absence of bicuculline, EPSCs had fast and slow components that were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX; 10 microm; n = 7), and (+/-)3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP; 10 microm, n = 6), respectively. Reversal potentials for the evoked IPSCs and EPSCs were consistent with mediation via GABAA and ionotropic glutamate receptors, respectively. These results suggest that the SCN region provides both inhibitory and excitatory inputs to single VLPO neurons, which are mediated, respectively, by GABAA receptors and by both non-NMDA and NMDA glutamate receptors. These projections may play important roles in conveying circadian input to systems in the preoptic area that regulate sleep and waking.

Acute CNS lesions lead to neuronal injury and a parallel glial activation that is accompanied by the release of neurotoxic substances. The extent of the original neuronal damage can therefore be potentiated in a process called secondary damage. As astrocytes are known to secrete immunomodulatory and neuroprotective substances, we investigated whether astrocytic factors can attenuate the amount of neuronal injury as well as the degree of microglial activation in a model of excitotoxic neurodegeneration. Treatment of organotypic hippocampal slice cultures with N-methyl-D-aspartate (NMDA) resulted in a reproducible loss of viable granule cells, partial destruction of the regular hippocampal cytoarchitecture and a concomitant accumulation of amoeboid microglial cells at sites of neuronal damage. Astrocyte-conditioned media reduced the amount of NMDA-induced neuronal injury by 45.3%, diminished the degree of microglial activation and resulted in an improved preservation of the hippocampal cytoarchitecture. Transforming growth factor (TGF)-beta failed to act as a neuroprotectant and even enhanced the amount of neuronal injury by 52.5%. Direct effects of astrocytic factors on isolated microglial cells consisted of increased microglial ramification and down-regulated expression of intercellular adhesion molecule-1, whereas incubation with TGF-beta had no such effects. In summary, our findings show that hitherto unidentified astrocyte-derived factors that are probably not identical with TGF-beta can substantially enhance neuronal survival, either by eliciting direct neuroprotective effects or by modulating the microglial response to neuronal injury.

Changes in phenotype or connectivity of primary afferent neurons following peripheral nerve injury may contribute to the hyperalgesia and allodynia associated with neuropathic pain conditions. Although earlier studies using partial nerve injury models have focused on the role of damaged fibres in the generation of ectopic discharges and pain, it is now thought that remaining undamaged fibres may be equally important. We have examined the expression of the sensory neuron-specific cation channel Vanilloid Receptor 1 (VR1), an important transducer of noxious stimuli, in three models of nerve injury in the rat, using anatomical separation or fluorescent retrograde tracers to identify damaged or undamaged sensory neurons. After total or partial sciatic nerve transection, or spinal nerve ligation, VR1-immunoreactivity (IR) was significantly reduced in the somata of all damaged dorsal root ganglion (DRG) neuronal profiles, compared to controls. However, after partial transection or spinal nerve ligation, VR1 expression was greater in the undamaged DRG somata than in controls. Unexpectedly, after L5 spinal nerve ligation, VR1-IR of the A-fibre somata increased approximately 3-fold in the uninjured L4 DRG compared to controls; a much greater increase than seen in the somata with C-fibres. Furthermore, we found that VR1-IR persisted in the transected sciatic nerve proximal to the lesion, despite its down-regulation in the damaged neuronal somata. This persistence in the nerve proximal to the lesion after nerve section, together with increased VR1 in DRG neurons left undamaged after partial nerve injury, may be crucial to the development or maintenance of neuropathic pain.

Vasodilatation in the dura mater has been implicated in migraine pathogenesis. Anti-migraine triptan drugs block vasodilatation by binding to 5-HT1B/1D receptors localized on the peripheral sensory terminals and dural blood vessel smooth muscles. Previous studies suggest that calcitonin gene-related peptide (CGRP) released from Adelta-fibres plays a more important role than substance P (SP) released from C-fibres in inducing dural vasodilatation and that one of the antimigraine mechanisms of triptan drugs is inhibiting CGRP release. In the present study, the relationship between CGRP and 5-HT1B/1D receptors, and between CGRP and SP in the trigeminal ganglion neurons in rats was examined by double immunohistochemical staining. CGRP, 5-HT1B, 5-HT1D and SP-positive trigeminal ganglion neurons were all predominantly small and medium-sized. In the trigeminal ganglia, approximately 50% of CGRP-positive neurons were 5-HT1B positive. Similarly, approximately 55% of CGRP-positive neurons were 5-HT1D immunoreactive. Approximately 50% of CGRP-positive neurons were SP-positive, while 93% of SP-positive neurons were CGRP-positive, suggesting that nearly all SP-positive neurons also contain CGRP. The fibre types of the 5-HT1B- and 5-HT1D-positive neurons were further investigated with an antibody against the A-fibre marker 200-kDa neurofilaments (NF200). Approximately 46% of the 5-HT1B-positive and 43% of the 5-HT1D-positive trigeminal ganglion neurons were also NF200 positive, indicating that many A-fibre trigeminal neurons express 5-HT1B or 5-HT1D receptors. These results support the hypothesis that one important action of antimigraine drugs is the inhibition of CGRP release and that Adelta-fibres may play an important role in migraine pathogenesis.

Mossy fibres are the main afferent input to the granular layer of the cerebellar cortex. In this study, the spatial distribution of the mossy fibres' presynaptic enlargements - the so-called rosettes - were analysed on the single fibre level. Data obtained from the cerebella of cat and mice were compared to look for species differences, and the cerebella of the adult and young mice were also compared to look for developmental changes. The results show that there is a spatial anisotropy in all mossy fibres studied, with neighbouring rosettes being about three times further away from each other along the parasagittal axis and closer to each other in the mediolateral direction. Furthermore, these results suggest that this anisotropy is established at an early developmental stage. The anisotropic orientation of mossy fibres at the single fibre level supports the hypothesis of a timing mechanism in cerebellar function.

The heat-transducing receptor VR1 cloned from rat sensory neurons can be activated by both noxious heat and capsaicin. As the response of sensory neurons to capsaicin is species dependent, it is conceivable that the responses to noxious heat and to capsaicin are transduced by distinct receptors across different species. Therefore, we investigated responses to noxious heat from a capsaicin-insensitive (chick) and a capsaicin-sensitive (rat) species. In chick, whole-cell patch-clamp experiments in isolated dorsal root ganglion neurons revealed two populations of neurons with different thresholds to noxious heat, activated at approximately 43 degrees C and approximately 53 degrees C. In cobalt uptake experiments, the proportion of neurons showing a heat-induced response increased with increasing heat stimuli. Application of capsaicin (1-10 microM) did not result in inward currents or cobalt uptake. Rat neurons yielded comparable results in heat experiments, but were capsaicin-sensitive. Although chick neurons are insensitive to capsaicin, the competitive capsaicin antagonist capsazepine (1-10 microM) was effective in blocking heat-induced responses, verified by patch-clamp and cobalt uptake methods. The noncompetitive capsaicin antagonist ruthenium red (10 microM) reduced to almost nil the proportion of heat-responsive neurons identified with the cobalt uptake method. These findings suggest that chick DRG neurons express a low-threshold heat-transducing receptor with a pharmacological profile distinct from the low-threshold heat receptor VR1 cloned from rat DRG neurons. The data support the idea that there might be heat receptor subtypes with differences in the capsaicin binding site.

Although it is widely believed that astrocytes lack excitability in adult tissue, primitive action potential-like responses have been elicited from holding potentials negative to -80 mV, in cultured and injury-induced gliotic rodent astrocytes and in human glia under pathological conditions such as glioblastomas and temporal lobe epilepsy. The present study was designed to investigate the properties of astrocytes (identified by immunoreactivity for glial fibrillary acidic protein) derived from multipotent human embryonic CNS stem cells and cultured for 12-25 days in differentiating conditions. We describe here for the first time that brief (1 ms) current pulses elicit spikes from a resting potential (VREST) of approximately -37 mV and, more interestingly, that spontaneous firing can be occasionally recorded in human astrocytes. A voltage-clamp study revealed that in these cells: (i) the half-inactivation of the tetrodotoxin (TTX)-sensitive Na+ channels is around VREST; (ii) the delayed rectifier K+ current is very small; (iii) the ever-present transient outward A-type K+ channels are paradoxically capable of inhibiting the action potentials elicited from a negative membrane potential (-55 to -60 mV); and (iv) inwardly rectifying currents are not present. The responses predicted from a simulation model are in agreement with the experiments. As suggested by recent studies, the decrease of Na+ channel expression and the changes of the electrophysiological properties during the postnatal maturation of the CNS seem to exclude the possibility that astrocytes may play an excitable role in adult tissue. Our data show that excitability and firing should be considered an intrinsic attribute of human astrocytes during CNS development. This is likely to have physiological importance because the role of astrocytes during development is different from the [K+]o-buffering role played in adult CNS, namely the glutamate release and/or the guiding of migrating neurons.

The molecular and cellular characteristics of the gonadotropin-releasing hormone (GnRH) neurons have been difficult to ascertain due to their scattered distribution within the basal forebrain. Using morphological criteria coupled with single cell RT-PCR postidentification, we have developed a method for investigating native GnRH neurons in the mouse brain and used it to examine the development of GABA(A) receptor signalling in this phenotype. Following the harvesting of the cytoplasmic contents of individual GnRH neurons, single cell multiplex RT-PCR experiments demonstrated that GABAA receptor alpha1-5, beta1-3 and gamma2 & 3 subunit transcripts were expressed by both neonatal (postnatal day 5) and juvenile (day 15-20) GnRH neurons in a heterogeneous manner. Following puberty, this profile was reduced to a predominant alpha1, alpha5, beta1, gamma2 subunit complement in rostral preoptic area GnRH neurons of the adult female. Whole-cell patch-clamp recordings revealed little difference between juvenile and adult GnRH neurons in their resting membrane potential and spontaneous firing rates. All GnRH neurons were found to be subjected to a tetrodotoxin-insensitive, tonic GABAergic barrage signalling through the GABA(A) receptor. However, marked heterogeneity in the sensitivity of individual juvenile GnRH neurons to GABA was revealed and, in parallel with the change in subunit mRNA profile, this was dramatically reduced in the reproductively competent adult GnRH neurons. These findings provide the first electrical and molecular characterization of the GnRH phenotype and demonstrate a novel pattern of late postnatal reorganization of native GABA(A) receptor gene expression and signalling in the GnRH neuronal population.

The modulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazol-propionate (AMPA) receptor-mediated currents by cyclothiazide was investigated in acutely isolated cells from rat striatum with whole-cell patch-clamp recording. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) was used to identify medium spiny and giant aspiny neurons and to determine their AMPA receptor subunit composition mostly in separate experiments. After pretreatment with cyclothiazide, kainate-induced AMPA responses were more strongly potentiated in medium spiny than in giant aspiny neurons; cyclothiazide induced a ninefold leftward shift in the kainate concentration-response curve for medium spiny neurons (not giant aspiny neurons). The EC50s for the cyclothiazide potentiation did not differ substantially between medium spiny neurons and giant aspiny neurons. The recovery of kainate-activated currents from modulation by cyclothiazide was slower for medium spiny neurons than for giant aspiny neurons. Medium spiny neurons expressed GluR-A, GluR-B and GluR-C, but not GluR-D subunits in both flip and flop splice variants. All giant aspiny neurons expressed GluR-A and GluR-D, exclusively in the flop form, half of them also expressed GluR-B and GluR-C. This is in keeping with slow and fast desensitization kinetics in medium spiny neurons and giant aspiny neurons, respectively, and differences in cyclothiazide modulation. The rate of cyclothiazide dissociation from the AMPA receptor, activated by glutamate, was approximately 90 times slower in medium spiny neurons than in giant aspiny neurons. In giant aspiny neurons (not medium spiny neurons) this rate was strongly dependent on the presence of an agonist; 1 mM glutamate increased it 30-fold. Thus, two major cell groups in the striatum display distinct AMPA receptor compositions carrying specific properties of glutamate responses. Excitatory transmission will thus be differentially affected by cyclothiazide-type compounds.

Using classical neuroanatomical retrograde tracing methods we investigated the retinal ganglion cells projecting to the nucleus of the optic tract and dorsal terminal nucleus of the accessory optic system (NOT-DTN) in macaque monkeys. Our main aim was to quantify the strength of the projection from the ipsilateral retina to the NOT-DTN. We therefore examined the number, distribution, and soma size of retinal ganglion cells involved in this projection. Electrophysiologically controlled small injections into the NOT-DTN revealed a clearly bilateral retinal projection originating mainly from the central retina but also involving peripheral retinal regions. Labelled cells were found nasally in the contralateral retina and temporally in the ipsilateral retina with some overlap in the fovea. The projection from the ipsilateral retina was 36-43% of that from the contralateral retina. On average, only 1-6% of the local population of ganglion cells projected to the NOT-DTN. Small soma size and large dendritic fields imply that in monkey rarely encountered, 'specialized' ganglion cells provide the direct retinal input to the accessory optic system (AOS). These results are discussed with respect to the symmetry of monocular horizontal optokinetic nystagmus (OKN) in primates.

The molecular mechanisms underlying dendritic differentiation in neurons are currently poorly understood. We used slice cultures from rat cerebellum of postnatal day 8 to investigate the effect of protein kinase C (PKC) activity on dendritic development of Purkinje cells. After 12 days in culture under control conditions, Purkinje cells had developed a typical dendritic tree consisting of a few long primary dendrites with shorter side branches. Following treatment with the PKC agonist, phorbol-12-myristate-13-acetate (PMA), the dendritic tree area was strongly reduced to 32% of control and primary dendrites were short with only a few side branches. Delayed addition of PMA after 6 days resulted in a retraction of existing dendrites, whereas discontinuation of PMA treatment after 6 days resulted in a recovery of the dendritic tree to almost control values. In the presence of the PKC inhibitor, 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl)maleimide (GF109203X), the dendritic tree area was increased to 158% of control with much more ramified branches after 12 days. The overall morphology of the cultures and the survival of Purkinje cells were unaffected by PKC modulators. Our data show that increased activity of PKC inhibits, and reduced activity of PKC promotes dendritic growth. This suggests that PKC activity is a critical regulator of dendritic growth and differentiation in cerebellar Purkinje cells.

Unlabelled: Microglial cells serve as pathologic sensors of the brain. They are highly abundant in all regions of the central nervous system (CNS) and are characterized by a ramified morphology within the normal tissue. In the present study, we have developed a procedure to study the membrane properties of identified, in situ microglia in acutely isolated brain slices from rat cortex, striatum and facial nucleus. Unlike the well characterized cultured microglial cells, ramified microglia of the slice are characterized by little, if any, voltage-gated membrane currents and a very low membrane potential. They are thus distinct from neurons, other glial cells and nonbrain macrophages. To study the consequences of microglial activation on the membrane channel pattern, we compared cells in the normal facial nucleus and at defined times after facial nerve axotomy. Within 12 h of axotomy, microglial cells expressed a prominent inward rectifier current and thus acquired the physiological properties of cultured microglia. Within 24 h of the lesion, the cells expressed an additional outward current, which is typical for lipopolysaccharide (LPS)-activated microglia in vitro. Seven days after the lesion, at a time of major regenerative processes in the facial nucleus, the physiological properties of microglial cells had reverted to those present prior to the pathological event., In Conclusion: (i) ramified microglial cells represent a physiologically unique population of cells in the brain; (ii) are distinct from their cultured counterparts; and (iii), undergo a defined pattern of physiological states in the course of pathologic events.

The first neuropile, or lamina, of the fly's optic lobe comprises a model set of identified neurons that are arrayed in cylindrical modules, called cartridges. The cartridge acquires adult form only in the second half of the fly's pupal life. All cells are by then correctly located within each of the lamina's cartridges (Drosophila, Musca), becoming invested by glial cells after 75% of pupal development (P + 75%). In adult cartridges, two lamina cells, L1 and L2, receive input from photoreceptor terminals R1-R6, at so-called tetrad synapses that form in the pupa when these cells' dendrites contact R1-R6. Single-section electron microscopy (EM, Drosophila) and serial-EM reconstructions of L1 and L2 (Musca) reveal relationships between the morphogenesis of L1/L2 dendrites and the formation of tetrads. Neurite outgrowth is initially (P + 55%) random and neurites are unbranched; many neurites invaginate surrounding terminals of R1-R6 but, later, embrace the outer surfaces of these. The maximum profusion of neurites at P + 74% coincides with peak numbers of nascent tetrads; neurites then branch vertically, in the lamina's depth. Later, neurites failing to reach R1-R6's outer surfaces regress. Down the length of their axons, L1 and L2's neurites initially form a random sequence, L1 partnering L1 as often as L2, etc., but beginning at P + 74%, L1 partners L2, and L2 partners L1, with progressive strictness. L1 has more neurites overall than L2. These observations are consistent with the following hypotheses: a neurite only survives if it contacts a presynaptic site; a synapse only survives if it progressively acquires the appropriate number and combination of postsynaptic neurites, culminating in a tetrad; an interaction exists between the neurites of L1 and L2, so that the growth of one respects the pattern of growth of the other.

Age-dependent changes in noradrenergic innervations of the hippocampal dentate gyrus (DG) and the frontal cortex (FC) have been studied in male F344 rats. The projections from the nucleus locus coeruleus (LC) to DG or FC with advancing age (from 7 to 27 months) in rats have been quantified by electrophysiological and immunohistochemical methods. In the electrophysiological study, we observed that the percentage of LC neurons activated antidromically by electrical stimulation (P-index) of DG or FC decreased with age. We found that the percentage of LC neurons showing multiple antidromic latencies (M-index), which suggests axonal branching of individual LC neurons, increased markedly between 15 and 17 months in DG or FC. In DG, the M-index increased steadily between 15 and 24 months. In contrast, the increased M-index in FC was maintained until 24 months. The increased M-index in both targets declined at 27 months. These results suggest that LC neurons give rise to axonal branching following the loss of projections to DG or FC with age. In the immunohistochemical study, the density of dopamine-beta-hydroxylase-positive axonal varicosities was measured in molecular, granule cell and polymorphic layers of DG. The density in the polymorphic layer significantly decreased in the earlier stage of ageing (7-19 months), whilst the density in the molecular and granule cell layers decreased in the later stage (27 months). These findings suggested that a layer-specific decline occurred with age in the noradrenergic axon terminals in DG.

The p75 low affinity neurotrophin receptor (p75) can induce apoptosis in various neuronal and glial cell types. Because p75 is expressed in the cholinergic neurons of the basal forebrain, p75 knockout mice may be expected to show an increased number of neurons in this region. Previous studies, however, have produced conflicting results, suggesting that genetic background and choice of control mice are critical. To try to clarify the conflicting results from previous reports, we undertook a further study of the basal forebrain in p75 knockout mice, paying particular attention to the use of genetically valid controls. The genetic backgrounds of p75 knockout and control mice used in this study were identical at 95% of loci. There was a small decrease in the number of cholinergic basal forebrain neurons in p75 knockout mice at four months of age compared with controls. This difference was no longer apparent at 15 months due to a reduction in numbers in control mice between the ages of 4 and 15 months. Cholinergic cell size in the basal forebrain was markedly increased in p75 knockout mice compared with controls. Spatial learning performance was consistently better in p75 knockout mice than in controls, and did not show any deterioration with age. The results indicate that p75 exerts a negative influence on the size of cholinergic forebrain neurons, but little effect on neuronal numbers. The markedly better spatial learning suggests that the function, as well as the size, of cholinergic neurons is negatively modulated by p75.

Brain derived neurotrophic factor (BDNF) is normally expressed by a small number of predominantly trkA-expressing dorsal root ganglion cells. Using immunocytochemistry and in situ hybridization, we have examined the effect of sciatic nerve section on the expression of BDNF in the adult rat. Following axotomy there was a long lasting (4-week) increase in BDNF mRNA and protein in large-diameter, trkB- and trkC-expressing dorsal root ganglion cells. By 2 days postaxotomy, expression of BDNF mRNA had increased from 2% of trkB cells to 50%, and from 18% of trkC cells to 56%. In contrast, BDNF expression in most trkA cells was unchanged, although was increased in the small population of medium- and large-sized trkA cells. Following axotomy, BDNF-immunoreactive terminals appeared in the central axonal projections of large-diameter cells, including the deep dorsal horn and gracile nucleus. Neuropeptide Y was also upregulated following axotomy and was coexpressed with BDNF in the cell bodies and central terminals of the large cells. Ultrastructural analysis in lamina IV of the spinal cord revealed that BDNF terminals in these central projections establish synaptic contacts. Immunoreactivity at 4 weeks was also observed in pericellular baskets that contained calcitonin gene-related peptide (CGRP) and surrounded trkA- and trkB-expressing cells in L4 and L5 lumbar ganglia. These baskets are likely to arise from local, highly immunoreactive, BDNF/CGRP/trkA-expressing cells. Our results identify several novel targets for BDNF and imply that it acts locally in both autocrine and paracrine modes, as well as centrally in a synaptic mode, to modulate the response of somatosensory pathways in nerve injury.

Repeated treatment with psychostimulant drugs produces changes in brain and behaviour that far outlast their initial neuropharmacological actions. The nature of persistent drug-induced neurobehavioural adaptations is of interest because they are thought to contribute to the development of dependence and addiction, and other forms of psychopathology, e.g. amphetamine psychosis. There are many reports that psychostimulants produce biochemical adaptations in brain monoamine systems, especially dopamine systems. The purpose of the present study was to determine if they might also alter the morphology of neurons in brain regions that receive monoaminergic innervation. Rats were given repeated injections of either amphetamine or cocaine, or, to control for general motor activity, allowed access to a running wheel. They were then left undisturbed for 24-25 days before their brains were processed for Golgi-Cox staining. Treatment with either amphetamine or cocaine (but not wheel running experience) increased the number of dendritic branches and the density of dendritic spines on medium spiny neurons in the shell of the nucleus accumbens, and on apical dendrites of layer V pyramidal cells in the prefrontal cortex. Cocaine also increased dendritic branching and spine density on the basilar dendrites of pyramidal cells. In addition, both drugs doubled the incidence of branched spines on medium spiny neurons. It is suggested that some of the persistent neurobehavioural consequences of repeated exposure to psychostimulant drugs may be due to their ability to reorganize patterns of synaptic connectivity in the nucleus accumbens and prefrontal cortex.

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 gamma-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.

Combination of morphological and electrophysiological techniques provided data, suggesting existence in the young rat striatum of a peculiar class of neurons, the neurogliaform or dwarf neurons. Striatal neurons (n = 92), intracellularly recorded from rat brain slices, were filled (one in each slice) with the intracellular marker biocytin, to compare physiological and morphological properties in the same cell. Moreover, some neurons (n = 7) were filled with biocytin plus the fluorescent calcium indicator fura-2, identifying cells during electrophysiological recording. Electrophysiological recording showed that striatal neurons had different firing patterns, suggestive in most cases (n = 80) of spiny neuron class and in others (n = 12) of interneuron class. Fura-2 injection clearly identified the body of six medium-sized cells and of one distinctive tiny cell. This small cell, however, showed a resting membrane potential and spontaneous and evoked firing pattern characteristic of striatal interneurons. Moreover, the fura-2 injected in such small neuron also completely filled the cell body of a near large neuron; the fura-2 fluorescence changed synchronously in the two paired neurons after electrical stimulation of the impaled small one. Accordingly, the biocytin staining identified the morphology of the small recorded neuron as a neurogliaform-like cell apposed to a dendrite of an aspiny neuron, suggesting that the dye injected in one neuron had diffused to the other of a different type. Furthermore, such heterologous dye coupling unexpectedly involved seven pairs of cells detected with biocytin staining (7.6% of the recorded neurons), invariably represented by a medium or large neuron on one side, and on the other side by a small (5.44 +/- 0.15 x 9.14 +/- 0.7 microns, mean +/- SD; n = 7) neurogliaform cell, roundish in shape with few slender and short processes, usually apposed to a dendrite of the companion neurons (six out of seven). In the other cases, the biocytin staining revealed in each slice either the morphology of single spiny or aspiny neurons (80.4% of recorded neurons), or of two-three medium-sized spiny neurons detected near to each other, suggesting that dye coupling had occurred typically between similar neurons (11.9% of the recorded neurons). These data suggest that some neurogliaform cells in the striatum of young rat can be identified as dwarf interneurons, that may be dye-coupled with neurons of different classes.

Neurons of a distinct type in CA1 area stratum radiatum of the rat hippocampus have been found to express a direct cellular form of long-term potentiation (LTP, Maccaferri & McBain, 1996, J. Neurosci. 16, 5334), but their functional identity, i.e. whether interneuron or principal cell, remained unknown. Whole cell recording from hippocampal slices in vitro was combined with light and electron microscopy to answer this question. LTP was robustly induced by a pairing protocol and physiological properties were measured in radiatum giant cells (RGCs) using biocytin containing pipettes. Reconstruction of the cells' dendritic and axonal arbor revealed morphological properties similar to CA1 pyramidal cells with some characteristic differences. They typically had two large diameter apical dendrites, or when only one dendrite arose, it soon bifurcated. Apical dendrites formed a dendritic tuft in stratum lacunosum-moleculare and the dendrites, but not the somata, were densely covered with conventional spines. The axon arose from the basal pole of the soma, descended to stratum oriens and emitted several axon terminals bearing collaterals that travelled horizontally, remaining in stratum oriens. The main, myelinated axon trunks turned towards the fimbria. In the electron microscope axon terminals were found to form asymmetrical synapses on postsynaptic dendritic shafts and dendritic spines in stratum oriens. The dendrites received asymmetrical synapses, mostly on their spines. The axon initial segments also received several synapses, a feature never observed on interneurons. All the above characteristics support the conclusion that RGCs are excitatory principal neurons.

Following spinal cord injury, projection neurons are frequently axotomized and many of the cells subsequently die. One goal in spinal injury research is to preserve damaged neurons so that ultimately they are accessible to regeneration-promoting strategies. Here we ask if neurotrophin treatment can prevent atrophy and death of axotomized sensory projection neurons. In adult rats, a hemisection was made in the thoracic spinal cord and axotomized neurons were retrogradely labelled with Fluoro-Gold. Four distinct populations of cells were identified in the lumbar spinal cord, and both numbers and sizes of labelled cells were assessed at different time points postlesion. A progressive and significant degeneration was observed over time with severe atrophy apparent in all cell populations and significant cell loss evident by 4 weeks postlesion. This time point was used to assess neurotrophin effects. Hemisected rats were treated with either neurotrophin 3 (NT-3) or brain-derived neurotrophic factor (BDNF, 12 microg/day for each), or a vehicle solution, delivered continuously to the lesion site via an osmotic minipump. Treatment with NT-3, but not BDNF, completely reversed cell atrophy in three of the four cell populations and also induced a significant increase in the number of surviving cells. In situ hybridization experiments showed trkB and trkC mRNA to be expressed in the majority of ascending spinal projection neurons, suggesting that these cells should be responsive to both BDNF and NT-3. However, only NT-3 treatment was neuroprotective, indicating that BDNF may not have reached the cell bodies of injured neurons. These results demonstrate that NT-3 may be of benefit in preventing the secondary cell loss that occurs following spinal injury.

The growth of retinal axons was investigated in different regions of the optic chiasm in C57 pigmented mouse embryos aged embryonic day 13 (E13) to E15. Individual retinal axons and their growth cones were labelled anterogradely by DiI and imaged using a confocal imaging system. In aldehyde-fixed embryos, retinal growth cones display a simple form in the optic nerve and become more complex in morphology in the chiasm. The complex form is particularly prominent in those axons that turn to the ipsilateral tract in the premidline region of chiasm. Moreover, complex growth cones are also commonly found in axons in the postmidline chiasm, which are markedly different in morphology from those axons in the premidline region, suggesting that the postmidline chiasm contains a novel environment for the pathfinding of retinal axons. In another experiment, the dynamic growth of retinal axons is studied in a brain slice preparation of the living retinofugal pathway. Retinal axons show an intermittent growth across the premidline and postmidline chiasm. Extensive remodelling of growth cone form followed by a shift in growth direction is commonly seen during the pause periods, indicating that signals that guide axon growth across the chiasm are not restricted to the midline, but are laid down throughout the chiasm. Moreover, dramatic changes in axon trajectory are noted first at the premidline chiasm where the uncrossed axons segregate from the crossed axons, and second at the postmidline chiasm where specific sorting of retinal axons according to their position in the dorsal ventral retinal axis and their ages are known to take place. These results show that there are two distinct environments, separated by the midline in the chiasm, where axons show different responses to local guidance cues and develop the distinct fibre orders.

The clinically employed general anaesthetic halothane was shown to exert action on the peripheral nervous system by suppressing spinal reflexes, but it is still unclear which mechanisms underlie this action. The present study addressed the question whether blockade of tetrodotoxin-sensitive (TTXs) and -resistant (TTXr) Na+-channels in rat dorsal root ganglia (DRG) neurons by halothane could explain its peripheral effects. Two types of TTXr Na+-currents, fast and slow, with distinct activation and inactivation kinetics were found in small (< 25 micrometer) and medium sized (25-40 micrometer) DRG neurons. These currents were blocked by halothane with IC50 values of 5.4 and 7.4 mmol/L, respectively. Additionally, in a concentration-dependent manner halothane accelerated the inactivation kinetics of both currents and shifted the inactivation curves to more hyperpolarized potentials. Neither the activation curves of both TTXr Na+-currents were influenced by halothane nor a voltage-dependent block at test potentials of the currents was seen. In contrast to that of fast current, the time-to-peak for slow current was changed in the presence of halothane. The TTXs Na+-current which prevailed in large neurons (> 40 micrometer) was blocked by halothane with an IC50 of 12.1 mmol/L. Its inactivation curve was also shifted to more hyperpolarized potentials and the inactivation kinetics accelerated with increasing halothane concentration. Similarly to TTXr Na+-currents, the activation curve of TTXs Na+-current and its time-to-peak were not influenced by halothane. It is suggested that two types of TTXr Na+-currents can explain the heterogeneity in kinetic data for TTXr Na+-currents. Furthermore, the incomplete blockade of Na+-currents might underlie the incomplete reduction of spinal reflexes at clinically used concentrations of halothane.

Mice with a targeted mutation of the p75 low affinity neurotrophin receptor display smaller peripheral nerves and dorsal root ganglia. Here we show that transgenic mice have a significant elevation of thresholds to noxious mechanical and heat stimuli compared with p75+/+ control mice. Immunocytochemical analysis using antibodies against PGP 9.5 (a panaxonal marker) and calcitonin gene related peptide (CGRP, which labels peptidergic neurons) showed a reduction to 73% and 54%, respectively, of the epidermal innervation density. However, analysis of the cell size distribution of toluidine blue-stained dorsal root ganglia showed no selective loss of neurons of particular diameters. Moreover, the neurochemical profile of dorsal root ganglia cells as defined by trkA, CGRP, IB4 and RT97 immunostaining revealed no significant differences in comparison with p75+/+ animals. Staining of the dorsal horn of the spinal cord for CGRP and IB4 was also normal in p75-/- animals. Taking into account a previously reported loss of approximately 50% dorsal root ganglion neurons, we conclude that all types of sensory neurons are equally depleted in p75-/- mice and that the absence of p75 impedes the development of more than one neuronal subtype.