Interaction of thiocolchicoside with [3H]strychnine binding sites in rat spinal cord and brainstem

Radioreceptor binding assays and receptor autoradiography were used to investigate the activity of thiocolchicoside on strychnine-sensitive binding sites in rat brain and spinal cord using [3H]strychnine as a ligand. Thiocolchicoside displaced the binding of [3H]strychnine with an affinity similar to that of unlabeled glycine, and showed a Hill coefficient and proportionality parameter (P) less than unity. The activity of thiocolchicoside toward [3H]strychnine binding sites was confirmed in autoradiographic studies. The results suggest that thiocolchicoside behaves as an allosteric compound acting on the strychnine-sensitive glycine receptor in rat brainstem and spinal cord, and that this may provide a possible mechanism for the myorelaxant activity of this colchicoside derivative, the first clinically useful drug acting on this receptor.     

Comparative autoradiographic distribution of central omega (benzodiazepine) modulatory site subtypes with high, intermediate and low affinity for zolpidem and alpidem

Pharmacological characterization of [3H]benzodiazepine binding to membrane preparations of adult rat hippocampus and neonatal rat brain have demonstrated, in addition to the omega 1 and omega 2 populations of central omega benzodiazepine binding sites associated with GABAA receptors, the existence of binding sites with microM affinity for the imidazopyridines zolpidem and alpidem. In the present study we have investigated their comparative autoradiographic distribution using [3H]flumazenil as a ligand. In the neonatal rat CNS, the imidazopyridine derivatives zolpidem and alpidem were found to discriminate two [3H]flumazenil binding site populations with an IC50 value ratio of more than 200-fold. In the different regions investigated (spinal cord, striatum, neocortex and inferior colliculus) the low affinity component had IC50 values of 20-40 microM (zolpidem) and 5-15 microM (alpidem) and accounted for ca. 50% of the total binding site population. In the adult rat, these imidazopyridine derivatives displayed a greater displacing potency in the cerebellum (IC50 = 6 and 36 nM, respectively) than in the hippocampus (IC50 = 37 and 403 nM, respectively). In the cerebellum, [3H]flumazenil binding was fully displaced by 1 microM of either compound and Hill coefficients of displacement curves were close to unity. In the hippocampus, 25% of [3H]flumazenil binding were resistant to 3 microM zolpidem or 1 microM alpidem, but were displaced by 100 microM of either compound. CL 218,872 also displayed a greater displacing potency in the cerebellum (IC50 = 83 nM) than in the hippocampus (IC50 = 711 nM) but [3H]flumazenil binding in the hippocampus was fully displaced by 10 microM of this compound. In adult rat hippocampus, zolpidem and alpidem were found to discriminate between three central omega site subtypes which display high (IC50 = 31 and 6.1 nM, for these imidazopyridine derivatives. In contrast, CL 218,872 discriminated between omega 1 and omega 2 sites but not between two omega 2 receptor subpopulations. omega 1 sites were mainly localized in layer IV of the sensorimotor cortex, cerebellum, substantia nigra, olfactory bulb and inferior colliculus. omega 2I sites were present in the cortical mantle (with higher levels in the cingulate and olfactory than in the sensorimotor cortex) and in subcortical (hippocampus, hypothalamus and nucleus accumbens) limbic structures. In the hippocampus, hypothalamus, spinal cord and nucleus accumbens, omega 2L sites accounted for more than 25% of the specific [3H]flumazenil binding; the density of these sites was minor in the cortex and in most pyramidal and extrapyramidal system structures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Colocalization of cholinergic, adrenergic and peptidergic receptors on astrocytes

By means of combined immunohistochemical and auto-radiographic techniques we have studied the colocalization of cholinergic, adrenergic and peptidergic binding sites on astrocytes in explant cultures of rat spinal cord, brain stem and cerebellum. Many astrocytes which were immunostained by the monoclonal muscarinic receptor antibody M 35 were also intensely labelled by 3H-noradrenaline, the beta-adrenergic antagonist 3H-dihydroalprenolol and the peptides 125I-angiotensin II, 125I-neuropeptide Y and 3H-bradykinin. Electrophysiological studies demonstrating that muscarine, the adrenergic agonists noradrenaline and isoprenaline as well as angiotensin II, neuropeptide Y and bradykinin affect the membrane potential of the same astrocytes provide further evidence for the coexistence of cholinergic, adrenergic and peptidergic receptors on astrocytes.     

Induction of peripheral-type benzodiazepine receptors in mouse brain following thioacetamide-induced acute liver failure

To investigate the possible role of peripheral-type benzodiazepine receptors (PBR) in hepatic encephalopathy, we examined expression of PBR in mouse brain following thioacetamide (TAA)-induced acute liver failure. Treatment of mice with TAA resulted in an increase in the number of binding sites of the PBR ligand [3H] Ro5-4864 to brain homogenates, with no significant change in affinity of the ligand. The order of potency of different ligands to compete against [3H] Ro5-4864 binding in the brain of TAA-treated mice was Ro5-4864 > PK11195 > diazepam > protoporphyrin IX, findings similar to those in the control. Northern blot analysis revealed an increase in PBR/isoquinoline binding protein (PBR/IBP) mRNA in mouse brain following TAA treatment, in a time- and dose-dependent manner. These results indicate that the increased number of PBR in the brains of TAA-treated mice relates to the induction of PBR/IBP expression and suggest that the induction of PBR in brain may contribute to pathogenesis of hepatic encephalopathy.     

Differential distribution of [3H]sumatriptan binding sites (5-HT1B, 5-HT1D and 5-HT1F receptors) in human brain: focus on brainstem and spinal cord

We report on the autoradiographic distribution of 5-HT1B, 5-HT1D and 5-HT1F receptor subtypes in human brain, focusing on the brainstem and cervical spinal cord. We have used [3H]sumatriptan as a radioligand in the presence of suitable concentrations of 5-CT (5-carboxamidotryptamine) to define 5-HT1F receptors, and ketanserin, to discriminate between 5-HT1B and 5-HT1D receptors. In the brainstem the highest concentrations of [3H]sumatriptan binding sites were seen in substantia nigra. The spinal trigeminal nucleus, substantia gelatinosa of the spinal cord, nucleus of the tractus solitarius and periaqueductal grey, also showed significant levels of [3H]sumatriptan binding sites. In the brainstem and spinal cord the total population of 5-CT-insensitive receptors, corresponding to 5-HT1F receptors, ranged from 9.8% in the periaqueductal grey to 53.4% in the substantia gelatinosa. This population represented 67.0% of binding in layer V of the frontal cortex. The decrease in [3H]sumatriptan binding in the presence of 200 nM ketanserin, indicative of the presence of 5-HT1D receptors, was very limited throughout the human brain, only reaching 20% of total specific binding over the periaqueductal grey. The proportion of [3H]sumatriptan binding sites displaced by 5-CT and insensitive to ketanserin, corresponding to 5-HT1B receptors, was, in general, the most abundant, ranging from 43.8% in substantia gelatinosa to 69.9% in the periaqueductal grey. Significant levels of 5-HT1B and 5-HT1D receptors found in migraine control pain areas suggest their involvement in antinociceptive mechanisms.     

Modulation of neuropeptide FF receptors by guanine nucleotides and cations in membranes of rat brain and spinal cord

Using a radioligand binding assay, we examined ionic modulation and G protein coupling of neuropeptide FF (NPFF) receptors in membranes of rat brain and spinal cord. We found that NaCl (but not KCl or LiCl) and MgCl2 increased specific 125I-YLFQPQRFamide (125I-Y8Fa) binding to NPFF receptors in both tissues in a dose-dependent manner, with optimal conditions being 60 mM NaCl and 1 mM MgCl2. Guanine nucleotides dose-dependently inhibited specific 125I-Y8Fa binding to rat brain and spinal cord membranes with maximal effects of 64 +/- 6 and 71 +/- 2%, respectively. The order of potency was nonhydrolyzable GTP analogues > GTP > or = GDP > GMP, ATP. The guanine nucleotide inhibition was observed in the absence and presence of NaCl and MgCl2. The mechanism of inhibition in spinal cord membranes appeared to be a reduction in the number of NPFF receptors; in one experiment, control KD and Bmax values were 0.068 nM and 7.2 fmol/mg of protein, respectively, and with 0.1 microM guanylylimidodiphosphate the respective values were 0.081 nM and 4.9 fmol/mg, a 32% reduction in receptor number. Similar results were obtained with guanosine 5'-O-(3-thiotriphosphate). Our data suggest that 125I-Y8Fa binding sites in rat CNS are G protein-coupled NPFF receptors regulated by GTP and cations.     

L-364,718 and L-365,260, two CCK antagonists, have no affinity for central benzodiazepine binding sites in chickens

It has recently been demonstrated that L-365,260, a CCK-B antagonist in mammals, causes an increase in food intake in chickens. In contrast, L-364, 718, a CCK-A antagonist in mammals, shows this effect only at very high dose levels. It has been shown that L-365,260 has very low affinity for chicken CCK receptors. Thus, the mechanism of action of L-365,260 remains unknown. As L-365,260 is a benzodiazepine derivative, one may hypothesize that it would be acting on benzodiazepine binding sites. The aims of this work were to establish the existence of benzodiazepine binding sites in the chicken brain, and to check the possibility that L-365,260 was acting on these receptors, determining the affinity of L-364,718 and L-365,260 for them. We have found specific binding for tritiated flunitrazepam (a benzodiazepine agonist) ([3H]-flunitrazepam) in chicken brain membranes. A single binding site was detected with a Kd of 3.58 +/- 0.97 nM and a Bmax of 451.6 +/- 23.3 fmol/mg protein L-365,260 and L-364,718 exhibited very low affinity for these binding sites (Ki = 1.17 x 10(-6) +/- 0.16 x 10(-6) M and Ki > 10(-5) M, respectively). Thus, these results demonstrate that the increase in food intake caused by L-365,260 in the chicken is not due to a direct action on benzodiazepine receptors. Other possible explanations for its effect are discussed.     

M2, M3 and M4, but not M1, muscarinic receptor subtypes are present in rat spinal cord

Muscarinic receptors in the spinal cord have been shown to mediate antinociception and alter blood pressure. Currently, there is much interest in identifying which muscarinic receptor subtypes regulate these functions. Toward that end, this study aimed to identify and localize the muscarinic receptor subtypes present in spinal cord using in vitro receptor autoradiography with [3H]-pirenzepine and [3H]-N-methylscopolamine. The results showed that M2 binding sites were distributed throughout the dorsal and ventral horns, whereas M3 binding sites were localized to laminae I to III of the dorsal horn. Only background levels of M1 binding sites were detected. Saturation binding assays using [3H]-pirenzepine in spinal cord homogenates confirmed the absence of M1 receptors. Competition membrane receptor assays using [3H]-N-methylscopolamine and the unlabeled antagonists pirenzepine, 11-2[(-[(diethylamino)methyl]-1-piperidinyl)-acetyl]-5, 11-dihydro 6H-pyrido(2, 3-b)(1, 4) benzodiazepine-one, methoctramine, and methoctramine in combination with atropine corroborated the autoradiographic findings and also revealed the presence of M4 binding sites. The finding that M2 and M3 binding sites were localized to the superficial laminae of the dorsal horn where nociceptive A delta and C fibers terminate suggests the possibility that either or both of these muscarinic receptor subtypes modulate antinociception. The present demonstration of M4 binding sites in spinal cord is consistent with the possibility that M2 and/or M4 receptors are involved in the regulation of blood pressure at the spinal level.     

Galanin-receptor ligand M40 peptide distinguishes between putative galanin-receptor subtypes

The galanin-receptor ligand M40 [galanin-(1-12)-Pro3-(Ala-Leu)2-Ala amide] binds with high affinity to [mono[125I]iodo-Tyr26]galanin-binding sites in hippocampal, hypothalamic, and spinal cord membranes and in membranes from Rin m5F rat insulinoma cells (IC50 = 3-15 nM). Receptor autoradiographic studies show that M40 (1 microM) displaces [mono[125I]iodo-Tyr26]galanin from binding sites in the hippocampus, hypothalamus, and spinal cord. In the brain, M40 acts as a potent galanin-receptor antagonist: M40, in doses comparable to that of galanin, antagonizes the stimulatory effects of galanin on feeding, and it blocks the galaninergic inhibition of the scopolamine-induced acetylcholine release in the ventral hippocampus in vivo. In contrast, M40 completely fails to antagonize both the galanin-mediated inhibition of the glucose-induced insulin release in isolated mouse pancreatic islets and the inhibitory effects of galanin on the forskolin-stimulated accumulation of 3',5'-cAMP in Rin m5F cells; instead M40 is a weak agonist at the galanin receptors in these two systems. M40 acts as a weak antagonist of galanin in the spinal flexor reflex model. These results suggest that at least two subtypes of the galanin receptor may exist. Hypothalamic and hippocampal galanin receptors represent a putative central galanin-receptor subtype (GL-1-receptor) that is blocked by M40. The pancreatic galanin receptor may represent another subtype (GL-2-receptor) that recognizes M40, but as a weak agonist. The galanin receptors in the spinal cord occupy an intermediate position between these two putative subtypes.     

Functional interactions between spinal cord grafts suggest asymmetries dictated by graft maturity

Fetal spinal cord tissue grafts have been advocated as a possible repair strategy for spinal cord injury. In the present study, we used intraocular spinal cord grafts to model the interactions which may occur between fetal and adult spinal cord after making such a graft and to study to which extent functional connections can be expected to occur between the host and graft tissue. We first grafted fetal spinal cord to the anterior chamber of the eye where it was allowed to mature. A second piece of fetal spinal cord was then sequentially grafted in contact with the first graft. Electrophysiological recordings made from the older graft while electrically stimulating the younger graft provided evidence for an excitatory innervation from the younger spinal cord graft to the mature spinal cord which appeared to be glutamatergic. However, we only rarely found excitatory inputs from the first, mature spinal cord graft to the younger graft. Fiber connections between the two spinal cord grafts were verified by retrograde tracing and neurofilament immunohistochemistry. In no case was a trophic influence on graft volume observed between spinal cord grafts regardless of whether the transplantations were performed sequentially or at the same time. Even the introduction of a second graft to immature spinal cord tissue was ineffective. In contrast, we found a marked trophic, neuron-rescuing effect of spinal cord grafts upon cografts of fetal dorsal root ganglia. This latter observation is consistent with the hypothesis that spinal cord tissue can exert a trophic effect on developing sensory ganglia and demonstrates that many sensory neurons can survive in the presence of a central target and in the absence of the appropriate peripheral target. These intraocular experiments predict that fetal spinal cord grafted to the injured adult spinal cord may develop effective excitatory inputs with the host, while host-to-graft inputs may develop to a considerably smaller extent. Our results also suggest that the adult spinal cord does not exert marked trophic effects on growth of fetal spinal cord, while it does exert a trophic influence on central projections of dorsal root ganglia.     

Preproinsulin I and II mRNAs and insulin electron microscopic immunoreaction are present within the rat fetal nervous system

Insulin-like substance has been found within the nervous system. In the rat, preproinsulin II mRNA was shown within the brain and preproinsulin I mRNA within the retina. The present study demonstrates the presence of preproinsulin mRNAs within the 15, 17 and 19 day gestational age fetal rat brain, spinal cord and dorsal root ganglia (DRG), employing RNA template-specific polymerase chain reaction (RS-PCR), semi-nested PCR and RNase protection assay. Preproinsulin I mRNA was present in the 17 and 19 day gestational age brain, spinal cord and DRG, and only in the brain of the 15 day gestational age brain. Preproinsulin II mRNA was present in all the gestational ages studied in the brain, spinal cord and DRG. The RS-PCR and the semi-nested PCR demonstrated products that co-migrated with the pancreatic control. The semi-nested products were characterized as preproinsulin I and II by restriction enzyme digestion and sequence. RNase protection assay using specific cRNA for preproinsulin I and II showed a band that co-migrated with pancreatic preproinsulin I and II mRNAs, and confirmed the PCR results. In addition, insulin receptor mRNA was detected by RS-PCR. Ultrastructural studies showed insulin immunoreaction within the endoplasmic reticulum, Golgi apparatus, cytoplasm, axon, dendrites, and in relation to the synapses. Thus, we demonstrated the presence of preproinsulin I and II mRNA, insulin receptor mRNA and insulin immunoreaction within the rat fetal central and peripheral nervous system.     

Involvement of peripheral-type benzodiazepine receptors in the intracellular transport of heme and porphyrins

To investigate the involvement of peripheral-type benzodiazepine receptors (PBR) in heme metabolism, we examined the interaction of [55Fe]heme with PBR. Transfection of the cloned mouse PBR-isoquinoline carboxamide-binding protein (PBR/IBP) cDNA into monkey kidney Cos-1 cells resulted in a 2.5-fold increase in [55Fe]hemin binding sites, concomitant with the increase in [3H]PK11195 binding sites, as compared with those seen in antisense PBR/IBP cDNA-transfected cells. The binding of hemin to the transfected receptors exhibited a relatively high affinity with a Kd of 12 nM, and was inhibited by several benzodiazepine ligands, including PK11195, Ro 5-4864, diazepam and protoporphyrin IX. When mouse liver mitochondria were incubated with [55Fe]hemin, the binding to PBR had a Kd of 15 +/- 1.8 nM. The Bmax of [55Fe]hemin binding to the mitochondria was 6.88 +/- 0.76 pmol/mg of protein, a value consistent with that of [3H]PK11195 binding, with a lower affinity. Coproporphyrinogen III, a precursor porphyrin produced in the cytosol, is translocated into mitochondria, then is converted to protoporphyrinogen IX; this conversion decreased in the presence of benzodiazepine ligands. To examine whether this decrease was related to a decrease in the binding of coproporphyrinogen to the mitochondria, the effects of benzodiazepines on the binding of coproporphyrinogen were examined. As the binding was dose-dependently inhibited by PK11195, Ro 5-4864, and diazepam, porphyrins are likely to be endogenous ligands for PBR. We propose that PBR play a role in the intracellular transport of porphyrins and heme.     

Postnatal development of multiple opioid receptors in the spinal cord and development of spinal morphine analgesia

The postnatal ontogeny of mu, delta and kappa opioid receptor binding sites in the spinal cord of rat pups at various postnatal days was determined using in vitro autoradiographical methods. The functional effect of spinal morphine was also assessed using in vivo electrophysiological methods in rats at P14, P21 and adults (P56). Both mu and kappa opioid receptor binding-sites are present from P0 and spread relatively diffusely throughout the spinal cord. Overall binding peaks at P7 and subsequently decreases to adult levels with the mu opioid receptor binding sites regressing to become denser in the superficial dorsal horn. delta Opioid receptor binding was first seen at P7, and no distinction between superficial and deeper laminae was seen. In the adult, the relative proportions of the opiate receptors in the superficial dorsal horn are 63%, 22% and 15%, for mu, delta and kappa receptor binding sites, respectively. C-fibre evoked dorsal horn neuronal responses recorded from anaesthetized rat pups were highly sensitive to spinal morphine at P21, (EC50 0.005 microgram), compared to the adult (EC50 0.9 microgram). However, the EC50 (0.2 microgram) at P14 was greater than at P21 despite the fact that mu receptor binding was greater at P14. Opioid receptor binding is developmentally regulated and undergoes substantial postnatal reorganization. However, the number of mu receptor binding sites appears not to be the only determinant of functional sensitivity to spinal morphine. Other factors, such as coupling of the receptors are likely to be important.     

Dynorphin A(1-17) mediates midazolam antagonism of morphine antinociception in mice

Studies have shown that midazolam acts in the brain to antagonize the antinociception produced by morphine. The purpose of this study was to determine if spinal dynorphin A(1-17) (Dyn) was involved in the antagonistic effects of midazolam. A number of drugs when administered intracerebroventricularly (ICV) to mice release Dyn in the spinal cord to antagonize morphine-induced antinociception. In the present study using the mouse tail-flick test, midazolam administered ICV produced a dose related reduction of the antinociception induced by morphine given intrathecally (IT). The antagonistic action of midazolam against morphine-induced antinociception involved the release of Dyn in the spinal cord, as evidenced by the following results. 1) Administration of naloxone, nor-binaltorphimine and dynorphin antiserum, IT, eliminated the antagonistic effect of midazolam, given ICV, against morphine. Treatment with these opioid antagonists and dynorphin antiserum is known to inhibit the action of spinally released Dyn. 2) Production of desensitization to the effect of spinal Dyn by pretreating with morphine, 10 mg/kg subcutaneously 3 h before the tail-flick test, abolished the antagonistic action of midazolam given ICV. A 3-h pretreatment with midazolam, ICV, also produced desensitization to the antianalgesic action of Dyn given IT. 3) Elimination of the Dyn component of action of midazolam by administration of naloxone, nor-binaltorphimine and dynorphin antiserum, IT, uncovered slight antinociceptive activity of midazolam, given ICV. Coadministration of flumazenil (a benzodiazepine antagonist), bicuculline (a GABA antagonist) and picrotoxin (a chloride ion channel blocker) inhibited the midazolam effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for the existence of the beta-endorphin-sensitive "epsilon-opioid receptor" in the brain: the mechanisms of epsilon-mediated antinociception

Recently, mu-, delta- and kappa-opioid receptors have been cloned and relatively well-characterized. In addition to three major opioid receptor types, more extensive studies have suggested the possible existence of other opioid receptor types that can be classified as non-mu, non-delta and non-kappa. Based upon anatomical and binding studies in the brain, the sensitive site for an endogenous opioid peptide, beta-endorphin, has been postulated to account for the unique characteristics of the opioid receptor defined as a putative epsilon-opioid receptor. Many epsilon-opioid receptors are functionally coupled to G-proteins. The functional epsilon-opioid receptors in the brain are stimulated by bremazocine and etorphine as well as beta-endorphin, but not by selective mu-, delta- or kappa-opioid receptor agonists. Epsilon-opioid receptor agonists injected into the brain produce profound antinociception. The brain sites most sensitive to epsilon-agonist-induced antinociception are located in the caudal medial medulla such as the nucleus raphe obscures, nucleus raphe pallidus and the adjacent midline reticular formation. The stimulation of epsilon-opioid receptors in the brain facilitates the descending enkephalinergic pathway, which probably originates from the brainstem terminating at the spinal cord. The endogenous opioid Met-enkephalin, released in the spinal cord by activation of supraspinal epsilon-opioid receptors, stimulates spinal delta2-opioid receptors for the production of antinociception. It is noteworthy that the epsilon-opioid receptor-mediated pain control system is different from that of other opioid systems. Although there appears to be no epsilon-selective ligand currently available, these findings provide strong evidence for the existence of the putative epsilon-opioid receptor and its unique function in the brain.     

Changes in [3H]flunitrazepam binding in the brain of rats made tolerant to and dependent upon pentobarbital

Changes in benzodiazepine binding sites labeled by [3H]flunitrazepan (FNZ) in twenty discrete brain regions of rats made tolerant to and dependent upon pentobarbital were examined. Animals were rendered tolerant by intracerebroventricular (i.c.v.) infusion with pentobarbital (300 micrograms/ 10 microliters/ hr for six days) through pre-implanted cannulae connected to osmotic mini-pumps. The pentobarbital dependence was assessed 24 hr after abrupt withdrawal from pentobarbital. In the tolerant rats, a significant increase in [3H]FNZ binding sites was found in layer IV of frontal cortex and the molecular layer of olfactory bulb. [3H]FNZ binding sites in the pentobarbital dependent rats were significantly increased in layers I-III and V-VI of frontal cortex, caudate-putamen, olfactory tubercle, globus pallidus and ventral pallidum, in addition to those observed in the tolerant group. There was, however, no significant difference in the hippocampus and several regions in the hindbrain in either pentobarbital-treated group. Taken together with characteristics of subtypes of benzodiazepine receptors and changes in GABA-benzodiazepine receptor complexes elucidated in our previous studies, these findings suggest that both types of benzodiazepine receptors are involved in the development of pentobarbital intoxication mediated by GABAA receptors.     

Segregation of optic axons based on central target: the medial optic tract in Rana pipiens

The electrophysiological integrity of the adult rat spinal cord was assessed at the lumbar, lower cervical and cortical levels after the animals sustained a severe contusion injury at the mid-thoracic level (T8) and received either carbon filament cultured with fetal spinal cord tissue implants, fetal tissue implants, or carbon filament implants alone. Somatosensory evoked potentials (SSEPs) and motor evoked potentials (MEPs) were recorded from all animal groups at the end of the 8-week survival period. The results of this study demonstrate that the spinal cord injured animals that received carbon filament cultured with fetal spinal cord tissue implants had the highest degree of electrophysiological recovery, indicating that this combination plays an important role in promoting recovery after injury.     

Glutamate transporter GLT-1 is transiently localized on growing axons of the mouse spinal cord before establishing astrocytic expression

The glutamate transporter GLT-1 is expressed in astrocytes of the mature brain and spinal cord. In the present study, we examined its expression in the developing mouse spinal cord. By in situ hybridization, 35S-labeled antisense oligonucleotide probes for GLT-1 mRNA consistently labeled the mantle zone/gray matter from embryonic day 11 through the adult stage. However, immunohistochemistry with a specific antibody visualized distinct regional and cellular localizations during the time between the fetal and postnatal stages. At fetal stages, GLT-1 immunoreactivity predominated in the marginal zone/white matter, observed as tiny puncta in cross-sections and as thin fibers in longitudinal sections. The GLT-1-immunopositive structures were also labeled for neuron-specific enolase, a glycolytic enzyme specific to postmitotic neurons and endocrine cells. By electron microscopy, GLT-1 immunoreactivity was detected in axons forming frequent enlargements and was focally localized on a small portion of the axolemma, particularly that facing adjacent axons. At early postnatal stages, GLT-1 disappeared from axons in white matter tracts and, instead, appeared in astrocytic processes surrounding various neuronal elements in the gray matter. Therefore, before switching to astrocytic expression, GLT-1 is transiently expressed in neurons and localized in differentiating axons. Together with our previous finding on the localization of glutamate transporter GLAST in radial glial fibers, GLT-1 and GLAST are thus localized during development on distinct directional cellular elements along which young neurons elongate their axons or move their cell bodies, respectively.