Non-neuronal Elements Research Articles

IL-1beta in the trigeminal subnucleus caudalis contributes to extra-territorial allodynia/hyperalgesia following a trigeminal nerve injury

It has been reported that the whisker pad (WP) area, which is innervated by the second branch of the trigeminal nerve, shows allodynia/hyperalgesia following transection of the mental nerve (MN: the third branch of the trigeminal nerve). However, the mechanisms of this extra-territorial pain induction still remain unclear. Glia and cytokines are known to facilitate perception of noxious input, raising a possibility that these non-neuronal elements are involved in the induction and spread of allodynia/hyperalgesia at non-injured skin territory. One day after MN transection, tactile allodynia/hyperalgesia developed on the ipsilateral WP area, which is in the non-injured skin territory. The tactile allodynia/hyperalgesia lasted for more than 56 days. In response to MN transection, astrocytes and microglia appeared to be in an activated state, and interleukin (IL)-1beta was up-regulated in astrocytes in the trigeminal subnucleus caudalis (Vc). Allodynia/hyperalgesia at WP area induced by MN transection was attenuated dose-dependently by IL-1 receptor antagonist IL-1ra (i.t., 0.05, 0.5, and 5 pg/rat). Fos-like immunoreactive (Fos-Li) neurons were observed in the Vc after non-noxious mechanical stimulation of the WP area in the rats with MN transection. Administration of IL-1ra also attenuated the number of Fos-Li neurons dose-dependently. Administration of a noncompetitive antagonist of NMDA receptors MK-801 (i.t., 5 μg/rat) reversed allodynia/hyperalgesia. IL-1 receptor type I (IL-1RI) was localized in Fos- and phospho NR1-immunoreactive neurons. These results suggest that IL-1beta in the Vc plays an important role in the development of extra-territorial tactile allodynia/hyperalgesia after MN transection.

NMDA R1 receptor distribution in the cyprid of Balanus amphitrite (= Amphibalanus amphitrite) (Cirripedia, Crustacea)

The ontogenetic cycle of the barnacle Balanus amphitrite (= Amphibalanus amphitrite) (Darwin, 1854) includes a cyprid that binds submerged surfaces, metamorphosing into a sessile adult. γ-Aminobutyric acid (GABA) and GABA receptors have recently been located in its cyprid with a similar pattern to other crustaceans. Since NMDA R1 ionotropic glutamatergic receptors have been identified in crustacean neuromuscular junctions, we have investigated their presence in the B. amphitrite cyprid. The presence of NMDA R1 receptors might indicate a role for glutamate in neuromuscular control in B. amphitrite cyprids, therefore we studied the presence and distribution of the NMDA R1 by immunohistochemistry. Its distribution was observed in the peripheral nervous system and in non-neuronal elements. Actually, NMDA R1 immunoreactivity was detected in thoracic appendages, at the level of neuromuscular junctions, thus suggesting an involvement in motor control functions, as already demonstrated in other crustaceans. Immunoreactivity was also detected in ommatidia cells of the eye, in antennules, and in epidermal cells. The distribution pattern comparable to that of GABAergic molecules could indicate an interrelated agonistic/antagonistic role for these two systems, which could be considered as potential targets of combined antifouling strategies.

Abnormal distribution of sarcoglycan subcomplex in colonic smooth muscle cells of aganglionic bowel

Hirschsprung's disease (HD) is a development disorder of the enteric nervous system in which the altered innervation explains the inability of the aganglionic segment to relax. Impairment of cytoskeleton in SMC of aganglionic bowel has been shown. Sarcoglycan subcomplex (SG) may support the development and maintenance of muscle cells. We examined the SG subunit expression in colonic aganglionic and ganglionic specimens obtained from patients with HD. Full-thickness bowel specimens were obtained from six patients with HD. Six normal colon specimens were used as controls. Immunofluorescent analysis and reverse transcriptase polymerase chain reaction evaluation were performed for alpha-, beta-, gamma-, delta- and epsilon-SG. In control colon, the indirect immunofluorescence showed a strong staining pattern of beta- gamma- delta- and epsilon-SG while a weak positivity of alpha-SG was recorded. In aganglionic bowel, immunofluorescence intensity values documented a significant lack of epsilon-SG while an enhanced alpha-SG, coupled to a loss of epsilon-SG, was recorded in ganglionic bowel in HD-affected patients. Our observations underscore the assumption that non-neuronal elements of the colon might play a key role in the pathogenesis of HD and loss of epsilon-SG might critically alter the cytoskeleton in the aganglionic bowel segment. Up-regulation of alpha-SG is probably an acquired phenomenon to reinforce the sarcolemma and to perform a forceful contraction in dilated ganglionic HD-affected colon, related to chronic pseudo-obstruction, contributing to the intestinal dysmotility that persists in 20% of patients after resection of the aganglionic bowel.

Crosstalk between Spinal Astrocytes and Neurons in Nerve Injury-Induced Neuropathic Pain

Emerging research implicates the participation of spinal dorsal horn (SDH) neurons and astrocytes in nerve injury-induced neuropathic pain. However, the crosstalk between spinal astrocytes and neurons in neuropathic pain is not clear. Using a lumbar 5 (L5) spinal nerve ligation (SNL) pain model, we testified our hypothesis that SDH neurons and astrocytes reciprocally regulate each other to maintain the persistent neuropathic pain states. Glial fibrillary acidic protein (GFAP) was used as the astrocytic specific marker and Fos, protein of the protooncogene c-fos, was used as a marker for activated neurons. SNL induced a significant mechanical allodynia as well as activated SDH neurons indicated by the Fos expression at the early phase and activated astrocytes with the increased expression of GFAP during the late phase of pain, respectively. Intrathecal administration of c-fos antisense oligodeoxynucleotides (ASO) or astroglial toxin L-α-aminoadipate (L-AA) reversed the mechanical allodynia, respectively. Immunofluorescent histochemistry revealed that intrathecal administration of c-fos ASO significantly suppressed activation of not only neurons but also astrocytes induced by SNL. Meanwhile, L-AA shortened the duration of neuronal activation by SNL. Our data offers evidence that neuronal and astrocytic activations are closely related with the maintenance of neuropathic pain through a reciprocal “crosstalk”. The current study suggests that neuronal and non-neuronal elements should be taken integrally into consideration for nociceptive transmission, and that the intervention of such interaction may offer some novel pain therapeutic strategies.

Modulation of hemorheological parameters by the erythrocyte redox thiol status

There is growing knowledge about the association between hemorheological blood disorders and compromised microcirculation in erythrocyte abnormalities. Effects of the non-neuronal cholinergic elements, especially acetylcholinesterase, on the erythrocyte hemorheological parameters were characterized in the past. However, alterations of these parameters have not been studied under the influence of the cellular redox thiol status. Aliquots of venous blood from ten healthy male subjects were incubated in vitro with increasing concentrations of a thiol reducer agent (dithiothreitol 1, 10, 50 microM final concentrations) in the presence and absence of acetylcholinesterase substrate (acetylcholine) or inhibitor (velnacrine maleate). The following parameters were determined in all blood samples aliquots: erythrocyte aggregation, erythrocyte deformability and lipid membrane fluidity. Blood smears were performed. Dithiothreitol induces no significant changes on the hemorheological behaviour of human red cells. Upon intracellular thiol stimulation, the presence of AChE effectors (either acetylcholine or velnacrine) decreases erythrocyte aggregation and elongation indexes. The addition of DTT to blood samples aliquots, contributing to the redox thiol status, is not directly involved in the modulation of erythrocyte rheological properties. However, upon acetylcholinesterase modulation by its substrate or inhibitor, changes on the hemorheological parameters are triggered by DTT. Associated pharmacological interest is considerable to address the hemorheology-hemostasis-microcirculation triad disorders.

Long-term exposure of the rabbit eye to silicone oil causes optic nerve atrophy

A silicone oil endotamponade following vitrectomy has for decades been a standard method in human ophthalmology with a view to restoring a detached retina. However, severe functional deficiences may remain after treatment. In adult rabbits, the injection of silicone oil into the eye-ball following vitrectomy resulted in a decrease of 89% in the number of myelinated optic nerve fibres after a survival time of 1 year (418,313 ± 29,703 versus 45,620 ± 23,905). Concomitantly, the cross-sectional area of the optic nerve was also reduced significantly (0.853 ± 0.159 mm 2 versus 0.355 ± 0.107 mm 2). The number of non-neuronal elements of the optic nerve remained virtually unchanged immediately behind the eye-ball and in the middle part of the nerve, whereas it increased significantly close to the optic chiasm (3040 ± 433 versus 3888 ± 403). Thus, destruction of the myelinated optic nerve fibres is likely to be responsible for the functional deficiencies observed after silicone oil implantation.

Gamma-aminobutyric acid and related molecules in the sea fan Eunicella cavolini (Cnidaria: Octocorallia): a biochemical and immunohistochemical approach

The aim of this study has been the biochemical demonstration of the presence of gamma-aminobutyric acid (GABA) in the Mediterranean sea fan Eunicella cavolini by means of high-performance liquid chromatography, and the description of the distribution pattern of GABA and its related molecules, glutamic acid decarboxylase (GAD), vesicular GABA transporter (VGAT) and one of the GABA receptors (GABA(B) R) by immunohistochemical methods. The interrelationships of GABA, GAD and GABA receptor immunoreactivity have been established by using double-immunohistochemical methods and confocal microscopy. The immunodetection of monoclonal and/or polyclonal antibodies has revealed GABA immunoreactivity throughout the polyp tissue, both in neuronal and non-neuronal elements. GAD immunoreactivity has been mostly localized in the neuronal compartment, contacting epithelial and muscular elements. GABA(B) R immunoreactivity appears particularly intense in the nematocytes and in the oocyte envelope; its presence in GAD-immunoreactive neurons in the tentacles suggests an autocrine type of regulation. Western blot analysis has confirmed that a GABA(B) R, with a molecular weight of 142 kDa, similar to that of rat brain, is present in E. cavolini polyp tissue. The identification of the sites of the synthesis, vesicular transport, storage and reception of GABA strongly suggests the presence of an almost complete set of GABA-related molecules for the functioning of the GABAergic system in this simple nervous system. The distribution of these different immunoreactivities has allowed us to hypothesize GABA involvement in nematocyst discharge, in body wall and enteric muscular contraction, in neuronal integration and in male gametocyte differentiation.

Encoding of the cough reflex

Coughing can be both voluntarily induced and involuntarily initiated by activation of vagal afferent nerves innervating the airways and lungs. Centrally, cough is regulated at the level of the brainstem through integration of vagal afferent nerve input by relay neurones in the nucleus tractus solitarius (nTS). Projections to and from the nTS add further complexity to cough regulation, as do the profound influences of psychological and social factors known to regulate cough. Peripherally, both neuronal and non-neuronal elements in the airways regulate the excitability of the vagal afferent nerve terminals regulating cough. These multiple levels of integration and encoding of the cough reflex may render this defensive respiratory response highly susceptible to modulation both by disease processes and through therapeutic intervention.

Localization of the Na +-coupled neutral amino acid transporter 2 in the cerebral cortex

We studied the distribution and cellular localization of Na +-coupled neutral amino acid transporter 2, a member of the system A family of amino acid transporters, in the rat and human cerebral cortex using immunocytochemical methods. Na +-coupled neutral amino acid transporter 2-positive neurons were pyramidal and non-pyramidal, and Na +-coupled neutral amino acid transporter 2/GABA double-labeling studies revealed that Na +-coupled neutral amino acid transporter 2 was highly expressed by GABAergic neurons. Double-labeling studies with the synaptophysin indicated that rare axon terminals express Na +-coupled neutral amino acid transporter 2. Na +-coupled neutral amino acid transporter 2-immunoreactivity was also found in astrocytes, leptomeninges, ependymal cells and choroid plexus. Electron microscopy showed robust Na +-coupled neutral amino acid transporter 2-immunoreactivity in the somato-dendritic compartment of neurons and in glial processes, but, as in the case of double-labeling studies, failed to reveal Na +-coupled neutral amino acid transporter 2-immunoreactivity in terminals. To rule out the possibility that the absence of Na +-coupled neutral amino acid transporter 1-[Melone M, Quagliano F, Barbaresi P, Varoqui H, Erickson JD, Conti F (2004) Localization of the glutamine transporter SNAT1 in rat cerebral cortex and neighboring structures, with a note on its localization in human cortex. Cereb Cortex 14:562–574] and Na +-coupled neutral amino acid transporter 2-positive terminals was due to insufficient antigen detection, we evaluated Na +-coupled neutral amino acid transporter 1/synaptophysin and Na +-coupled neutral amino acid transporter 2/synaptophysin coexpression using non-standard immunocytochemical procedures and found that Na +-coupled neutral amino acid transporter 1 and Na +-coupled neutral amino acid transporter 2+ terminals were rare in all conditions. These findings indicate that Na +-coupled neutral amino acid transporter 1 and Na +-coupled neutral amino acid transporter 2 are virtually absent in cortical terminals, and suggest that they do not contribute significantly to replenishing the Glu and GABA transmitter pools through the glutamate–glutamine cycle. The strong expression of Na +-coupled neutral amino acid transporter 2 in the somato-dendritic compartment and in non-neuronal elements that are integral parts of the blood–brain and brain–cerebrospinal fluid barrier suggests that Na +-coupled neutral amino acid transporter 2 plays a role in regulating the levels of Gln and other amino acids in the metabolic compartment of cortical neurons.

Cannabinoids: Between Neuroprotection and Neurotoxicity

Cannabinoids, such as the delta9-tetrahydrocannabinol (THC), present in the cannabis plant, as well as anandamide and 2-arachidonoyl glycerol, produced by the mammalian body, have been shown to protect the brain from various insults and to improve several neurodegenerative diseases. The current review summarizes the evidence for cannabinoid neuroprotection in vivo, and refers to recent in vitro studies, which help elucidate possible molecular mechanisms underlying this protective effect. Some of these mechanisms involve the activation of CB1 and CB2 cannabinoid receptors, while others are not dependent on them. In some cases, protection is due to a direct effect of the cannabinoids on neuronal cells, while in others, it results from their effects on non-neuronal elements within the brain. In many experimental set-ups, cannabinoid neurotoxicity, particularly by THC, resides side by side with neuroprotection. The current review attempts to shed light on this dual activity, and to dissociate between the two contradictory effects.

Characterization of non-neuronal elements within fibronectin mats implanted into the damaged adult rat spinal cord

Previous studies have shown that mats made from fibronectin (FN) integrate well into spinal cord lesion sites and support extensive axonal growth. Using immunohistochemistry, we have investigated the non-neuronal factors that contribute to these properties. Extensive vascularization was observed in FN mats by 1 week along with heavy macrophage infiltration by 3 days post-implantation. By 1 week post-implantation, laminin tubules had formed and were associated with axons and p75 immunoreactive Schwann cells. By 4 weeks post-implantation, most axons were associated with Schwann cell derived myelin. Few oligodendrocytes were present within the mat, even with an increase in the number of oligodendrocyte precursors around the implant site by 7 days post-implantation. Astrocyte proliferation also occurred in the intact tissue, with a prominent glial scar forming around the implant within 4 weeks. However, by 2 months post-implantation astrocytes were present in the FN implant site and were intermingled with the axons. Axonal ingrowth and integration of the FN mats is probably due to the ability of FN mats to support and organize infiltration of Schwann cells and deposition of laminin. At later time points, myelinated axons remain in the implant site, even after other elements (e.g. macrophages and laminin) have disappeared. Both of these properties are likely to be important in the design of biomaterial bridges for CNS regeneration.

Plasticity of nonneuronal brain tissue: roles in developmental disorders.

Neuronal and nonneuronal plasticity are both affected by environmental and experiential factors. Remodeling of existing neurons induced by such factors has been observed throughout the brain, and includes alterations in dendritic field dimensions, synaptogenesis, and synaptic morphology. The brain loci affected by these plastic neuronal changes are dependent on the type of experience and learning. Increased neurogenesis in the hippocampal dentate gyrus is a well-documented response to environmental complexity ("enrichment") and learning. Exposure to challenging experiences and learning opportunities also alters existing glial cells (i.e., astrocytes and oligodendrocytes), and up-regulates gliogenesis, in the cerebral cortex and cerebellum. Such glial plasticity often parallels neuronal remodeling in both time and place, and this enhanced morphological synergism may be important for optimizing the functional interaction between glial cells and neurons. Aberrant structural plasticity of nonneuronal elements is a contributing factor, as is aberrant neuron plasticity, to neurological and developmental disorders such as epilepsy, autism, and mental retardation (i.e., fragile X syndrome). Some of these nonneuronal pathologies include abnormal cerebral and cerebellar white matter and myelin-related proteins in autism; abnormal myelin basic protein in fragile X syndrome (FXS); and abnormal astrocytes in autism, FXS, and epilepsy. A number of recent studies demonstrate the possibility of using environmental and experiential intervention to reduce or ameliorate some of the neuronal and nonneuronal abnormalities, as well as behavioral deficits, present in these neurological and developmental disorders.

Exacerbation of Motor Neuron Disease by Chronic Stimulation of Innate Immunity in a Mouse Model of Amyotrophic Lateral Sclerosis

Innate immunity is a specific and organized immunological program engaged by peripheral organs and the CNS to maintain homeostasis after stress and injury. In neurodegenerative disorders, its putative deregulation, featured by inflammation and activation of glial cells resulting from inherited mutations or viral/bacterial infections, likely contributes to neuronal death. However, it remains unclear to what extent environmental factors and innate immunity cooperate to modulate the interactions between the neuronal and non-neuronal elements in the perturbed CNS. In the present study, we addressed the effects of acute and chronic administration of lipopolysaccharide (LPS), a Gram-negative bacterial wall component, in a genetic model of neurodegeneration. Transgenic mice expressing a mutant form of the superoxide dismutase 1 (SOD1(G37R)) linked to familial amyotrophic lateral sclerosis were challenged intraperitoneally with a single nontoxic or repeated injections of LPS (1 mg/kg). At different ages, SOD1(G37R) mice responded normally to acute endotoxemia. Remarkably, only a chronic challenge with LPS in presymptomatic 6-month-old SOD1(G37R) mice exacerbated disease progression by 3 weeks and motor axon degeneration. Closely associated with the severity of disease is the stronger and restricted upregulation of the receptor of innate immunity Toll-like receptor 2 and proinflammatory cytokines in degenerating regions of the ventral spinal cord and efferent fiber tracts of the brain from the LPS-treated SOD1(G37R) mice. This robust immune response was not accompanied by the establishment of acquired immunity. Our results provide solid evidence that environmental factors and innate immunity can cooperate to influence the course of disease of an inherited neuropathology.

Altered synaptic and electrical properties of lumbar motoneurons in the neurological glial mutant taiep rat.

Maturation and differentiation of electrical properties of neurons and synaptic transmission are modulated by neuronal interaction. In vitro experiments have shown that these processes also seem to be regulated by signals from non-neuronal elements such as glial cells. It is not known, however, whether glial alterations in intact neural networks may also affect the maturation of electrical properties and synaptic transmission during development. We used the taiep rat, a neurological mutant with a progressive demyelination and astrogliosis, as an experimental model to study the postnatal development of motoneurons in an altered glial environment. Using the patch-clamp technique, we made intracellular recording from motoneurons of Rexed's lamina IX in spinal cord slices of neonatal rats (postnatal day P4-P10). The electrical properties of normal motoneurons changed significantly with age, showing decreasing input resistance (R(in)) and increasing membrane capacity (C(m)). The rheobase increased with age, accompanied by an increase of the amplitude and a decrease of the duration of action potentials (APs). In contrast, mutant neurons showed no age-dependent changes of R(in), C(m), or AP characteristics. After blocking inhibitory transmission, intralaminar bipolar stimulation elicited, in both control and taiep motoneurons, fast glutamatergic excitatory postsynaptic potentials (EPSPs). Two types of taiep motoneurons were identified according to the temporal patterns of synaptic responses; (1). taiep(SYN) neurons, which showed no significant differences to control motoneurons, and (2). taiep(ASYN) neurons, in which the initial EPSP was followed by a variable number of delayed, asynchronous EPSP responses (for up to 300 ms). All these electrophysiological findings suggest that the mutation in taiep rats interfered with the development of the electrical properties of neurons and with the maturation of synaptic transmission, probably due to alterations in the neuron-glia interactions.

Expression pattern of the ether-a-gogo-related (ERG) K+ channel-encoding genes ERG1, ERG2, and ERG3 in the adult rat central nervous system.

Voltage-dependent K(+) channels play a pivotal role in controlling cellular excitability within the nervous system. The aim of the present study was to investigate the expression in the adult rat brain of the three ether-a-gogo-related gene (ERG) family members ERG1, ERG2, and ERG3, encoding for K(+) channel subunits. To this aim, the distribution of ERG transcripts was studied by means of reverse-transcription polymerase chain reaction (RT-PCR) and nonradioactive in situ hybridization histochemistry (NR-ISH). Furthermore, ERG1 subunit distribution was studied by immunohistochemical analysis. RT-PCR analysis revealed ERG1, ERG2, and ERG3 expression in the olfactory bulb, cerebral cortex, hippocampus, hypothalamus, and cerebellum. NR-ISH experiments detected transcripts encoded by all three ERG genes in the cerebral cortex and in all CA subfields and in the granular cell layer of the dentate gyrus of the hippocampus; strong ERG1 signals were also detected in scattered large elements throughout the oriens, pyramidal, and radiatum layers, and in the hilus of the dentate gyrus. In the thalamus, positively labeled neurons were detected in the reticular nucleus with ERG1 and ERG3 and in the anterodorsal nucleus with ERG2 riboprobes. Transcripts for ERG1 and, to a lesser degree, also for ERG3, were detected in the basal ganglia and in several brainstem nuclei. All three ERG genes appeared to be expressed in cerebellar Purkinje cells. Finally, ERG1 expression was also revealed in non-neuronal elements such as ependymal and subependymal cells along the ventricular walls and hippocampal astrocytes. These results suggest that the K(+) channel isoforms of the ERG family appear to be expressed in different central nervous system regions where they might differentially control the firing of neurons engaged in several networks.

Distribution and densitometry mapping of L1-CAM Immunoreactivity in the adult mouse brain – light microscopic observation

BackgroundThe importance of L1 expression in the matured brain is suggested by physiological and behavioral studies showing that L1 is related to hippocampal plasticity and fear conditioning. The distribution of L1 in mouse brain might provide a basis for understanding its role in the brain.ResultsWe examined the overall distribution of L1 in the adult mouse brain by immunohistochemistry using two polyclonal antibodies against different epitopes for L1. Immunoreactive L1 was widely but unevenly distributed from the olfactory bulb to the upper cervical cord. The accumulation of immunoreactive L1 was greatest in a non-neuronal element of the major fibre bundles, i.e. the lateral olfactory tract, olfactory and temporal limb of the anterior commissure, corpus callosum, stria terminalis, globus pallidus, fornix, mammillothalamic tract, solitary tract, and spinal tract of the trigeminal nerve. High to highest levels of non-neuronal and neuronal L1 were found in the grey matter; i.e. the piriform and entorhinal cortices, hypothalamus, reticular part of the substantia nigra, periaqueductal grey, trigeminal spinal nucleus etc. High to moderate density of neuronal L1 was found in the olfactory bulb, layer V of the cerebral cortex, amygdala, pontine grey, superior colliculi, cerebellar cortex, solitary tract nucleus etc. Only low to lowest levels of neuronal L1 were found in the hippocampus, grey matter in the caudate-putamen, thalamus, cerebellar nuclei etc.ConclusionL1 is widely and unevenly distributed in the matured mouse brain, where immunoreactivity was present not only in neuronal elements; axons, synapses and cell soma, but also in non-neuronal elements.

Selective expression of L-serine synthetic enzyme 3PGDH in schwann cells, perineuronal glia, and endoneurial fibroblasts along rat sciatic nerves and its upregulation after crush injury.

Non-essential amino acid L-serine functions as a highly potent, glia-derived neurotrophic factor, because it is a precursor for syntheses of proteins, other amino acids, membrane lipids, and nucleotides, and also because its biosynthetic enzyme 3-phosphoglycerate dehydrogenase (3PGDH) is preferentially expressed in particular glial cells within the brain. Here we pursued 3PGDH expression in peripheral nerves and its change after crush injury. In the pathway of rat sciatic nerves, 3PGDH was selectively expressed in non-neuronal elements: Schwann sheaths and endoneurial fibroblasts in sciatic nerves, satellite cells in dorsal root ganglia, and astrocytes and oligodendrocytes in the spinal ventral horn. In contrast, 3PGDH was immunonegative in axons, somata of spinal motoneurons and ganglion cells, and endoneurial macrophages. One week after crush injury, 3PGDH was upregulated in the distal segment of injured nerves, where 3PGDH was intensified in activated Schwann cells and fibroblasts. 3PGDH was still negative in activated macrophages, which were instead associated or surrounded by activated Schwann cells with intensified 3PGDH. These results suggest that in the peripheral nervous system, these non-neuronal cells synthesize and may supply L-serine to satisfy metabolic demands for maintenance and regeneration of peripheral nerves and for proliferation and activation of macrophages upon nerve injury.

Apoptosis following peripheral sensory deafferentation in the olfactory bulb of adult zebrafish.

Removal of the olfactory organ in adult zebrafish results in a significant decrease in volume of the ipsilateral olfactory bulb. The current study investigated the potential role of apoptosis in this phenomenon. It was hypothesized that cells in the adult olfactory bulb normally undergo minimal apoptosis and that apoptosis increases upon removal of sensory stimulation. By using both the terminal transferase-mediated deoxyuridine nick-end labeling method and bis-benzimide labeling, the current study showed that, in the normal adult olfactory bulb, cells exhibiting apoptotic profiles were scarce and were localized to the outer layers of the bulb. However, in deafferented animals, there was a significant increase in the number of apoptotic cells. The apoptotic response occurred in two phases and was confined to the rostral half of the bulb. The first phase of cell death peaked at 1 hour postsurgery. These apoptotic profiles appeared to be primarily nonneuronal in nature, in that they exhibited no immunohistochemical labeling to the neuron-specific protein Hu. The second phase of cell death peaked at 24 hours and declined to normal levels by 1 week. At the 24 hour time point, only a fraction of the apoptotic cells was neuronal in nature. Thus, apoptosis of nonneuronal and neuronal elements accounts for at least part of the deafferentation-induced volume decrease in the zebrafish olfactory bulb. This model of anterograde transneuronal degeneration will be useful in elucidating the afferent signals involved in survival and maintenance of mature brain neurons.

Localisation of the high-affinity choline transporter-1 in the rat skeletal motor unit.

The rate-limiting step in neuronal acetylcholine (ACh) synthesis is the uptake of choline via a high-affinity transporter. We have generated antisera against the recently identified transporter CHT1 to investigate its distribution in rat motor neurons and skeletal muscle and have used these antisera in combination with (1) antisera against the vesicular acetylcholine transporter (VAChT) to identify cholinergic synapses and (2) Alexa-488-labelled alpha-bungarotoxin to identify motor endplates. In the motor unit, immunohistochemistry and RT-PCR have demonstrated that CHT1 is restricted to motoneurons and absent from the non-neuronal ACh-synthesizing elements, e.g. skeletal muscle fibres. In addition, CHT1 is also present in parasympathetic neurons of the tongue, as evidenced by immunohistochemistry and RT-PCR. CHT1 immunoreativity is principally found at all segments (perikaryon, dendrites, axon) of the motoneuron but is enriched at neuro-neuronal and neuro-muscular synapses. This preferential localisation matches well with its anticipated pivotal role in synaptic transmitter recycling and synthesis.

Right thing at a wrong time? Adenosine A3 receptors and cerebroprotection in stroke.

The involvement of adenosine A3 receptors in normal and pathologic functions of the brain remains to be defined. Previous studies have shown that chronic preischemic administration of the agonist [N6-(3-iodobenzyl)-5'-N-methylcarboxoamidoadenosine or IB-MECA) results in a significant protection of neurons in selectively vulnerable brain regions and in an equally significant reduction of the subsequent mortality. Acute administration of the drug, on the other hand, resulted in a pronounced worsening of these parameters. We now report that the effect of administration of IB-MECA depends on the timing of treatment with respect to the onset of the focal insult, and provide the first data supporting speculation that treatment with adenosine A3 receptor agonists may decrease the infarct size following focal brain ischemia. Treatment with IB-MECA administered 20 min prior to transient middle cerebral ischemia (MCAOt = 30 min) resulted in a significant increase of the infarct size (p < 0.01), whereas administration 20 min after ischemia resulted in statistically significant decrease of the infarct volume. Postischemic treatment results in improved neuronal preservation, decreased intensity of reactive gliosis, and pronounced reduction of microglial infiltration. The data indicate that the effects of adenosine A3 receptor stimulation depend on the differential impact of these receptors on both neuronal and non-neuronal elements of the cerebral tissue, for example, astrocytes, microglia, and vasculature.