Conjugated quantum dots allow for the co-localization of endogenous proteins in submicroscopic structures of the rat cerebellum.

GABA and glutamate are known as the principal inhibitory and excitatory neurotransmitters in the vertebrate central nervous system, respectively. However, recent electro-physiological and immunogold literature data indicate that GABA may undergo also an excitatory action on presynaptic varicosities of parallel fibers (PFs) in the molecular layer of the rat cerebellum. PFs are axonal extensions, with a cross section of about 0.1 microm, of the glutamatergic granule cells. Such an unexpected excitatory action of GABA indicates clearly the presence of GABA receptors in the PFs of granule cells. We show in this study that quantum dots may be used specifically and efficiently to label two endogenous synaptic proteins, namely R-GABA(A)-alpha1 receptors (GABA(A) Rs) and glutamate transporters (VGLUT1) in order to target their localization in very small structures such as the presynaptic varicosities of the PFs.

Analysis of temporal and spatial patterns of rat vestibular hair cell differentiation by tritiated thymidine radioautography.

The location in time and space of the terminal mitoses of type I and type II sensory hair cells (HCI and HCII) of the developing crista ampullaris in rat lateral semicircular canal and macula utriculi was determined by radioautographs of specimens exposed to tritiated thymidine from the 13th to the 20th day of gestation. Qualitative analysis and statistical treatment of the percentages of labeled HCI and HCII show that the terminal mitoses occur first in the macula utriculi with a maximum percentage of the 14th day of gestation, for the HCI, and on the 15th day of gestation, for the HCII. In the lateral crista, the maximum percentage of labeled HCI occurs on the 17th and 18th day of gestation and on the 19th day of gestation for the HCII. A spatial distribution of this labeling activity is also described: the older cells are located at the top of the crista and at the level of the striola of the macula utriculi while the younger cells are found at the bottom of the crista and on the sides of the utricle. A study of the vestibular receptors in the fetuses shows that synaptic contacts already exist on the 18th day of gestation in the macula utriculi at the level of the striola and on the 19th day at the top of the crista; the cells situated on the periphery are still immature. The first hair cells to undergo their terminal mitoses are, therefore, connected first. These results also suggest that the two types of cells are genetically programmed and that the HCI start functioning first during the development of the labyrinth.

Catecholaminergic and GABAergic anatomical relationship in the rat substantia nigra, locus coeruleus, and hypothalamic median eminence: immunocytochemical visualization of biosynthetic enzymes on serial semithin plastic-embedded sections.

The visualization of protein antigens has been performed on semithin sections embedded in Araldite. After partial removal of the resin and a light proteolytic treatment of the tissue we were able to localize several biosynthetic enzymes: tyrosine hydroxylase (TH), phenylethanolamine N-methyltransferase (PNMT), and glutamic acid decarboxylase (GAD), which are, respectively, markers of catecholaminergic, adrenergic, gamma-aminobutyric acid (GABA)ergic systems. This technique afforded a high resolution of light microscopy details and immunostaining of TH, GAD, and PNMT on serial sections enabled us to compare with great precision GABAergic and adrenergic innervations in the rat locus coeruleus. In addition, it allows us to study the possible relationship between these terminals and the noradrenergic neurons. We also compared the general pattern of distribution of TH- and GAD-positive endings in the hypothalamic median eminence. The preliminary results obtained with this technique revealed some interesting facts previously unseen when preparations with lower histological resolution were used.

Anatomical distribution and characteristics of [(3)H]muscimol high affinity binding sites in the dorsolateral pontine tegmentum of the rat brain.

The pharmacological characterization and fine anatomical distribution of [(3)H]muscimol high affinity binding sites in the dorsolateral pontine tegmentum (region of locus coeruleus) of the rat was established using biochemical and autoradiographic techniques. On crude synaptosomal membranes obtained from microsamples of locus coeruleus, as well as on frozen sections of the dorsolateral pontine tegmentum, [(3)H]muscimol binding was saturable, displaced by 80-90% with 10(?5) M GABA or 5.10(?4) M (+) bicuculline, but unchanged in the presence of 10(?4) M nipecotic acid. Analysis of the saturation curve suggested the existence of one population of high affinity binding sites with K(d) = 10.5 nM, B(max) = 2.15 pmol/mg of crude synaptosomal membrane proteins, and K(d) = 14.3 nM, B(max) = 0.36 pmol/mg proteins on frozen tissue sections. Autoradiographic data on (3)H ultrofilms evidenced low densities of sites in this region compared to cerebellum or cortex as stated by previous authors. The highest densities of sites were observed in nuclei locus coeruleus and cuneiform, and in central grey. Dorsal and ventral parabrachial, ventral tegmental, motor trigeminal and superior vestibular nuclei exhibited slightly lower densities of sites, and brachium conjunctivum and medial longitudinal fasciculus presented no binding sites. In addition, light microscopy autoradiographic observations revealed [(3)H]muscimol binding sites between the structures previously quoted. In the locus coeruleus, [(3)H]muscimol binding sites were not strictly superimposed with cell bodies. These data are discussed in relation with the distribution of GABAergic terminals in the various areas of the dorso-lateral pontine tegmentum.

[Postnatal maturation of vestibular nuclei in the cat: histological study by the Golgi-Cox method (author's transl)]. / Maturation postnatale des noyaux vestibulaires chez le chat: étude histologique par la méthode de Golgi-Cox

Postnatal maturation of vestibular nuclei in the cat was studied from both the qualitative and quantitative points of view, following impregnation of the sections by the Golgi-Cox method. Three principal data arise from our results. (1) The different vestibular neurones undergo significant growth during the first two weeks of extra-uterine life: during this time, the progressive disappearance of dendritic growth cones and filopodia, an increase in area and volume of the perikarya as well as that of dendritic fields are remarkable; the smallest neurones completing their maturation before the largest. (2) Average sized neurones of the superior vestibular nucleus show a significant increase in the number of dendritic spines between birth and the age of 3 days. (3) On the 12th day, a decrease in the number of dendritic spines is noted. This decrease is accompagnied by a modification in spine density along the dendrites.

Identification of catecholamine cell bodies in the pons and pons-mesencephalon junction of the cat brain, using tyrosine hydroxylase and dopamine-beta-hydroxylase immunohistochemistry.

Using a double labeling immunohistochemical method with antibodies to tyrosine hydroxylase and dopamine-beta-hydroxylase, we reexamined the topographical distribution of dopaminergic (DA) and noradrenergic (NA) cells in the pons-mesencephalon junction of the cat. Besides DA cells of SN and its extensions, DA cells were also observed more caudally in nucleus raphe linearis intermedius and dorsalis, decussation of brachium conjunctivum, fasciculus longitudinalis medialis and periaqueductal gray. In nucleus locus coeruleus, cells exhibited variable levels of DBH, but dopaminergic cells were not evidenced in this structure.

Drug metabolizing enzyme activities and superoxide formation in primary and immortalized rat brain endothelial cells.

The activities of several enzymes involved in drug metabolism, NADPH-cytochrome P450 reductase, cytochrome P450 isoforms CYP1A and CYP2B, and uridine diphosphate glucuronosyltransferase (UGT) have been measured in primary cultures of rat cerebrovascular endothelial cells and in the immortalized rat brain endothelial cell line RBE4. These drug metabolizing activities were similar in the microsomes prepared from both cell types, even after 20 passages for RBE4 cells. These results were confirmed by Western immunoblotting analysis, using polyclonal antibodies raised against rat liver enzymes. The superoxide production observed during NADPH-cytochrome P450 reductase-dependent monoelectronic reduction of four xenobiotics, menadione, anthraquinone, nitrofurazone and diquat, was also investigated in these cultured cells at confluence. The rates of radical production were concentration-dependent. The superoxide formation induced by quinone metabolism was comparable in both cell cultures, and high amounts of superoxide radicals were produced even after 20 passages of RBE4 cells. On the other hand, nitrofurazone and diquat metabolism produced weak amounts of superoxide radicals in both cell types. Taken together, these results suggest that RBE4 cell line seems to constitute a valuable in vitro model for studies on the activity of some enzymatic systems involved in drug metabolism at the blood-brain barrier and the functional consequences of their activity.

Effects of Ginkgo biloba extract (EGb 761) on the guinea pig vestibular system.

Previous studies have demonstrated that the administration of Ginkgo biloba extract (EGb 761) improves the compensation of the vestibular syndrome induced by transection of the VIIIth nerve. To investigate the mechanisms at play, the vestibular nuclei of alert guinea pigs were perfused with EGb 761. This perfusion always induced a stereotyped reversible postural syndrome that was the mirror image of the syndrome provoked by the unilateral lesion of the otolithical receptors. This result supports the hypothesis that EGb 761 has a direct excitatory effect on the lateral vestibular nuclei (LVN) neurons. In a second step, we quantified the horizontal vestibuloocular reflex (HVOR) of the normal guinea pig following IP injection of EGb 761. In normal guinea pig, IP administration of EGb 761 led to a reversible, dose-dependent decrease of the HVOR gain without affecting the phase of the reflex. These data help to explain the therapeutic effects of EGb 761 during vestibular syndromes and strongly suggest an impact at the neuronal level.

Rostrocaudal and lateromedial density distributions of superior colliculus neurons projecting in the predorsal bundle and to the spinal cord: a retrograde HRP study in the cat.

Efferent neurons of the cat superior colliculus (SC) which project in the predorsal bundle (PDB) and to the spinal cord (PDB neurons) form a major pathway by which the SC controls the changes of the direction of gaze in response to stimuli of visual and other modalities. Knowledge of rostrocaudal and lateromedial density distributions of different groups of PDB neurons within the SC is necessary to analyse their relationships with the topography of sensory and motor maps. Density gradients may also bear on the efficacy of connections originating from topographically different collicular regions. In the present study, large injections of HRP/WGA-HRP were made in the C1 segment of the spinal cord and in the pontobulbar tegmentum. Judged by several morphological criteria, axons of passage, including those not subjected to a direct mechanical damage, were participating in the uptake of tracers. Therefore, labeled SC neurons corresponded to the nearly total populations of contralaterally projecting tectospinal neurons (TSNs) and neurons projecting in the PDB, respectively. Subtraction of the TSN density map from that of the whole PDB population was used to infer the distribution of tectal neurons terminating in the rhombencephalic tegmentum (TRhN). This subtotal labeling method proved useful in resolving the contradictions between the earlier HRP studies on the TSN and TRhN topography. The following density distributions were obtained for different groups of PDB neurons: 1) The mean TSN density is more than two times higher in the lateral half of the SC, representing the lower visual field. In this region the density remains constant from rostral to caudal, i.e., from the representation of vertical meridian to large contralateral azimuths. In the medial half, the average density decreases from rostral to caudal. Consequently, TSNs do not show the caudalward increment predicted by the higher efficacy of caudal stimulation points in eliciting head movements. 2) The distribution of PDB neurons is symmetrical with respect to the representation of the horizontal meridian. It is close to homogeneous at all azimuths of the retinotopic map and within the zone limited by small (10-15 degrees) upward and downward elevations.(ABSTRACT TRUNCATED AT 400 WORDS)

The control of slow orienting eye movements by tectoreticulospinal neurons in the cat: behavior, discharge patterns and underlying connections.

The activity of tectoreticulospinal neurons (TRSN) during orienting gaze shifts was studied in alert, head-fixed cats by intra-axonal recordings. The scope of the study was to evaluate the role of this class of superior colliculus neurons in the generation of slow eye movements (drifts) which often follow main-sequence saccades and sometimes appear as an independent motor event of orienting. The parameters of such movements are described in the first part of the paper. The organization of underlying pathways in the lower brainstem has been studied by intra-axonal horseradish peroxidase (HRP) tracing. The mean amplitude of postsaccadic drifts (PSD) is 1.21 degrees (SD 0.63), but it can eventually reach 6-8 degrees. PSDs have mean velocity of 14.9 degrees/s (SD 4.28) and mean duration of 104.2 ms (SD 50.8). These two parameters are positively correlated with PSD amplitude. The presence of PSDs is usually associated with an increased neck muscle activity on the side toward which the eyes move. The durations of these two motor events show a reliable positive correlation. PSDs appear to occur when gaze error persists after a saccade and a correction is attempted by means of a slow eye movement and a head turn. The durations of TRSN bursts are, on average, longer than the sum of the lead time and the saccade duration. Bursts associated with combinations of saccades and PSD are significantly longer than those recorded in the absence of PSDs. The probability of occurrence of PSDs is higher when firing of TRSNs continues after saccade termination. Such prolonged discharges usually coincide with a combination of PSDs and phasic activation of the neck electromyogram. The mean firing rate of TRSNs during PSDs is 62% of that during saccade-related portions of the burst and declines to 45% after the end of PSDs. According to its timing and intensity, postsaccadic firing of TRSNs is appropriate as a signal underlying slow, corrective eye movements and later portions of phasic neck muscle contractions during orienting. Intra-axonal HRP labeling showed that visuomotor TRSNs of the X type (n = 3) terminate in the abducens nucleus, with 145-331 boutons terminaux and en passant. Average bouton densities in the nucleus are lower than in the periabducens reticular formation, but higher than in more rostral paramedian pontine reticular formation (PPRF) regions. Terminal fields in the PPRF match the locations of "eye-neck' reticulospinal neurons (RSNs) and exitatory burst neurons.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of glutamatergic receptors and GAD mRNA-containing neurons in the vestibular nuclei of normal and hemilabyrinthectomized rats.

Vestibular compensation is an attractive model for investigations of cellular mechanisms underlying post-lesional plasticity in the adult central nervous system. Immediately after hemilabyrinthectomy, the spontaneous activity in the deafferented second-order vestibular neurons falls to zero, resulting in a strong asymmetry between the resting discharge of the vestibular complexes on the lesioned and intact sides. This asymmetry most probably causes the static and dynamic vestibular deficits observed in the acute stage. After approximately 50 h, the deafferented vestibular neurons recover a quasi-normal resting activity which is thought to be the key of the compensation of the static vestibular syndromes. However, the molecular mechanisms underlying this recovery are unknown. In this study, we investigate possible changes in the distribution of glutamatergic N-methyl-D-aspartate (NMDA) and glutamate metabotropic receptors and of glutamate decarboxylase 67k (GAD 67k) mRNAs in the deafferented vestibular neurons induced by the labyrinthine lesion. Specific radioactive oligonucleotides were used to probe sections of rat vestibular nuclei according to in situ hybridization methods. Animals were killed at different times (5 h, 3 days and 3 weeks) following the lesion. Signal was detected by means of film or emulsion autoradiography. In the normal animals, several brainstem regions including the medial, lateral, inferior and superior vestibular nuclei were densely labelled by the antisense oligonucleotide NMDAR1 probe. However, the vestibular nuclei were not labelled by the glutamate metabotropic oligonucleotide antisense probe (mGluR 1). The GAD 67k antisense oligonucleotide probe labelled numerous small- to medium-sized central vestibular neurons but not the larger cell bodies in the lateral vestibular nucleus. This agrees with previous studies. In the hemilabyrinthectomized rats, no asymmetry could be detected, at either the autoradiographic or cellular levels, between the two medial vestibular nuclei whatever the probe used and whatever the delay following the lesion. However, for the NMDAR1 probe, the mean density of silver grains in both the deafferented and intact medial vestibular neurons was 20% lower 5 h after the lesion. Three days and 3 weeks later, the intensity of labelling over all cells was the same as in the control group. Further studies are necessary to confirm the relatively weak modification of the NMDAR1 mRNAs expression and to exclude a change of GAD 65 and of other NMDA subunit mRNAs during the vestibular compensation process.

Evidence for neurogenic transmission inducing degenerative cartilage damage distant from local inflammation.

OBJECTIVE: To investigate involvement of the nervous system in ipsilateral and contralateral joint inflammation. METHODS: Freund's complete adjuvant (CFA; 1 mg or 1 microg) was injected unilaterally and the messages (a) from the hind paw to the ipsilateral and contralateral knees and (b) from one knee to the contralateral knee were analyzed. The degenerative impact of the local injury on distant cartilage was assessed using patellar proteoglycan synthesis as an indicator. Neurogenic mechanisms were blocked either by spinal cord compression or by injection of neurokinin 1 (NK-1) antagonist, or they were mimicked by intraarticular injection of substance P. The data were compared with those gathered in a model of systemic inflammation, characterized by fever and serum interleukin-6 production. RESULTS: After unilateral subcutaneous injection of CFA, proteoglycan anabolism decreased bilaterally. Spinal cord compression and administration of the NK-1 antagonist inhibited the response in the contralateral limb. Following 1 mg CFA subcutaneous injection, the ipsilateral response implicated both neurogenic and systemic mechanisms, whereas the nervous system alone was implicated after 1 microg subcutaneous CFA injection. The 1 microg CFA intraarticular injection induced a degenerative contralateral signal, which was abolished by spinal cord compression and by pretreatment with the NK-1 antagonist. Intraarticular injection of 1 microg CFA also induced an ipsilateral increase of anabolism, which was enhanced by spinal cord compression. Similar results were obtained after intraarticular injections of substance P. These effects were not reproduced with turpentine treatment, a systemic model, in which spinal cord compression had no effect. CONCLUSION: A unilateral inflammation can induce, by neurogenic mechanisms, distal bilateral degeneration of articular cartilage, implicating involvement of neuropeptides.