Some claustro-cortical connections in the cat and baboon as studied by retrograde horseradish peroxidase transport.

1. The mammalian Claustrum (Cl) is a convergent multisensory structure of unknown function, and disputed ontogenetic origin. Its cortical projections, hitherto unknown, have been studied in cat and baboon by means of the horseradish peroxidase (HRP) technique. HRP was injected into the gyrus proreus (frontal eye field) of cats, and separately into the frontal eye fields, visual areas, and motor-premotor areas of the baboon cortex. 2. Differential retrograde transport to the Cl was demonstrated, such that in the cat the ipsilateral dorsal Cl was shown to be the principal origin of claustroproreate projections. In the baboon, the whole Cl projects onto area 8, while only the posteroventral part of the nucleus sends efferents to the visual cortex. The projection to the motor and premotor areas is present, but does not seem to be "essential." 3. Discussion of the physiological literature, together with anatomical evidence of reciprocal cortico-claustral projections to closely similar regions of the Cl lead to the suggestion that the Cl is concerned with the integration of messages subserving visually-directed movements. Some other functional implications are also discussed.

Monoamine innervation of the oculomotor nucleus in the rat. A radioautographic study.

The serotonin and noradrenaline innervations of the rat oculomotor nucleus were examined by high resolution radioautography after in vivo labeling with tritiated 5-hydroxytryptamine and dopamine, respectively. Noradrenaline as well as serotonin endings (axonal varicosities) pervaded the entire nucleus, but the latter were at least six times more numerous (1.3 X 10(6) per mm3 of tissue) and were often found in the immediate vicinity of neuronal somata and proximal dendrites. The axon terminals of both types were of similar size and exhibited some large dense-cored vesicles in association with aggregated small and clear vesicles. The dense-cored vesicles were, however, more frequent and the content in clear vesicles more pleomorphic in serotonin than noradrenaline endings. In single thin sections, the proportion of noradrenaline and serotonin profiles exhibiting a synaptic junction was relatively small (15%). These were either symmetrical or asymmetrical when made on dendritic branches but invariably symmetrical on spines. In addition, a significant number of serotonin terminals were seen in close apposition or synaptic contact with neuronal perikarya and large dendrites, allowing for a direct, "proximal" action of serotonin. Moreover, many such terminals appeared to be coupled with unlabeled endings of another category, characterized by dispersed, uniformly round and clear synaptic vesicles, providing an alternate route for a proximal effect of serotonin in the oculomotor nucleus. In line with previous investigations on other motor nuclei, these data support the likelihood of a close involvement of both noradrenaline and serotonin in the control of motoneuronal activity.

Up-regulation of substance P binding sites in the vagus nerve projection area of the cat brainstem after nodosectomy. A quantitative autoradiographic study.

Substance P (SP) regulates visceral functions in the nucleus of the solitary tract (NST) area. High affinity SP binding sites labelled with [3H]SP or [125I]SP show a heterogeneous distribution in the cat medulla with high densities in the rostral and dorso-caudal parts of both the median subnucleus of NST and the dorsal motor nucleus (DMN). We previously observed a significant loss of SP immunoreactivity in the vagal area of the cat after an ipsilateral nodosectomy. It was thus important to study the correlated plasticity of SP binding in the context of the regulation of receptor function. Whichever labelled ligand was used, a unilateral nodose excision was followed by an ipsilateral increase in SP binding in the NST (200%) and the DMN (300%) after 30 days of survival. This increase was region-specific and did not match exactly the decrease in SP immunoreactivity following nodosectomy. This SP receptor density up-regulation could be due to long-term deprivation of SP afferent fibres in the NST and partly in the DMN. In the latter the increase of SP receptors occurred in both the cytoplasm of large neurons and the neuropile and did not affect the glia. The up-regulation phenomenon seems to be specific for SP receptors in the cat (at least in the DMN) and may constitute a reactive mechanism against the injury of axotomy of DMN neurons.

Substance P-immunoreactivity in the dorsal medial region of the medulla in the cat: effects of nodosectomy.

The distribution of substance P in the vagal system of the cat was studied by immunohistochemistry. Substance P-immunoreactive cell bodies and fibres were observed in the nodose ganglion. Numerous substance P-immunoreactive terminals and fibres were localized in their bulbar projection area, i.e. throughout the caudo-rostral extent of the nucleus of the solitary tract. Four subnuclei, among the nine forming the nucleus of the solitary tract, were strongly labelled: interstitial, gelatinosus, dorsal and commissural. The dorsal motor nucleus of the vagus nerve also exhibited numerous substance P-immunoreactive terminals, sometimes closely apposed on the somata of preganglionic neurons. To determine the substance P component of the vagal afferent system a nodose ganglion was removed on one side. The ablation triggered ipsilaterally a large decrease of substance P immunoreactivity in the four subnuclei strongly labelled on normal cats. These results suggest the involvement of substance P-containing vagal fibres in integrative processes of the central regulation of cardiovascular, digestive and respiratory systems, viscerotopically organized throughout these four subnuclei. The nodose ablation also resulted in a decrease of substance P immunoreactivity in the ipsilateral dorsal motor nucleus of the vagus nerve, suggesting monosynaptic vago-vagal interactions.

Transient increase in [3H]Ro 15-4513 specific binding in the superficial gray layer of the rat superior colliculus induced by visual deafferentation.

The imidazodiazepine compound [3H]Ro 15-4513, a partial inverse agonist of benzodiazepine receptors of the central type, binds with high affinity (order of 10(-8) M) to a single population of benzodiazepine binding sites in the mammalian central nervous system. A quantitative autoradiographic study was carried out to determine the effects of one eye removal on [3H]Ro 15-4513 specific binding to rat brain sections in the superficial gray layer or stratum griseum superficiale (SGS) of the superior colliculus. Retinal afferent degeneration due to right eye removal, performed 3 and 7 days before sacrifice, led to a significant and symmetrical increase in the [3H]Ro 15-4513 specific binding in both right and left SGS by enhancing the binding affinity of the radioligand. This transient phenomenon disappeared when a longer survival period of 45 days was allowed to elapse. Conversely, unilateral lesion of the primary visual areas had no apparent effects on the specific binding of the radioligand. The absence of any loss of binding sites after either type of lesion suggests that the benzodiazepine receptors are probably not situated on the optic nerve axon terminals, nor on the cortical axon terminals originating from primary visual areas. In the SGS, as in other rat brain structures, benzodiazepine receptors of the central type are functionally coupled with GABAA receptors and form 'GABAA receptors/benzodiazepine receptors/chloride channel' complexes. The involvement of the local GABAergic system in the postlesion plasticity of benzodiazepine receptors was studied by testing the effects of exogenously applied GABA on [3H]Ro 15-4513 specific binding.(ABSTRACT TRUNCATED AT 250 WORDS)

High-affinity serotonin binding sites: autoradiographic evidence for their location on retinal afferents in the rat superior colliculus.

High affinity 5-HT binding sites (5-HT1) were labeled in vitro on mounted rat brain slices using [3H]5-HT as a radioligand. In the first stage of experimentation, the bound radioactivity was measured on slices by liquid scintillation count in order to define the biochemical characteristics of the binding. Saturation curves were drawn, as well as association and elution curves for a 2 nM radioligand concentration. The mean affinity constant of the specific binding (Kd) was found to be 2.9 nM. In the second stage, the experimental parameters giving optimum binding were applied to the frozen slices prepared exactly as for the biochemical approach in order to investigate the effects of degeneration of retinal axon terminals on the distribution of 5-HT1 sites in the visual upper layers of the superior colliculus. The optical densities directly measured from tritium-sensitive film clearly indicate that the ablation of one eye causes a progressive reduction in the binding in the contralateral, largely deafferented, stratum griseum superficiale (SGS); with a 24-day survival period, the reduction was about 35-40%. In the homologous region of the ipsilateral colliculus, the binding decreased by about 10-15%. It is concluded that at least two populations of 5-HT1 binding sites coexist in the visual collicular layers, one of which is probably located on the axon terminals of retinal afferents. The present results confirm a previous hypothesis based on iontophoretic data, according to which this monoamine is involved in retino-collicular transmission. As far as the retinofugal terminal binding sites are concerned, 5-HT seems to exert a presynaptic control on visual inputs.

Distribution of benzodiazepine receptors in the rat superior colliculus: a light and electron microscope quantitative autoradiographic study.

The distribution of benzodiazepine (Bdz) receptors of the central type was analysed in the superficial grey layer of the rat superior colliculus from light and electron microscope autoradiographs, using the highly specific partial reverse agonist [3H]Ro 15-4513, a radioligand which can be crosslinked to its binding sites by ultraviolet rays. Biochemical characteristics of the binding were first defined by liquid scintillation count on unfixed cryostat mesencephalic brain slices. Saturation curves (1.6-20 nM) and Scatchard plot indicated that the radioligand bound with a high affinity (Kd = 11 nM) to a single population of sites (Bmax = 650 fmol/mg dry tissue). A slight primary chemical fixation of the brain did not significantly modify the binding characteristics. The consolidation of the specific binding by ultraviolet light on prefixed brain slices was found to be optimal after a 45-min illumination period. The distribution of Bdz sites on light and electron microscope autoradiographs was then analysed by applying these binding conditions. Prefixed brain slices (50 micron thick, Vibratome) were incubated in the 15 nM radioligand in the absence (total binding) or in the presence (non-specific binding) of the non-radioactive antagonist Ro 15-1788 (10(-5) M). Quantitative light microscopic study of Epon-embedded semithin sections showed that 95% of the silver grains of the specific label were located on the neuropil to the detriment of the neuronal and glial cell compartments. In the electron microscopic study, the distribution of the specific binding sites was statistically analysed over a total of more than 10 identified single or junctional tissue compartments, using the 50% probability circle method (Williams, 1969). Apart from a slight labeling of varicose profiles, the specific labeling was found to be concentrated on two particular tissue compartments: the percentage of grains associated with contacts between varicosities and dendrites was 32%, and that associated with axodendritic synapses was 16% of the total specific labeling measured over all compartments combined. A low proportion (33%) of the labeled axodendritic interfaces was characterized by a synaptic differentiation. These results suggest that both synaptic and non-synaptic Bdz receptors are present in the rat superior colliculus, and may each modulate neuronal cell activity in a different way.

A new 5-hydroxy-indole derivative with preferential affinity for 5-HT1B binding sites.

The affinities of several 5-hydroxy-indole derivatives for serotonin-1 (5-HT1) binding site subtypes, labeled with 2 nM [3H]5-HT, were assessed by quantitative autoradiography on rat brain sections. The results obtained with known ligands, namely 5-hydroxytryptamine (5-HT), 5-methoxytryptamine (5-Me-OT), 5-methoxy-N,N- dimethyl-tryptamine (5-Me-ODMT), 5-hydroxy-N,N-dimethyl-tryptamine (bufotenine) and 8-hydroxy-2-[di-N-propylamino]tetralin (8-OH-DPAT) demonstrate the reliability and the advantages of this technique for pharmacological studies. Novel serotonin derivatives were synthesized by carboxymethylation of the hydroxyl group. One of those new ligands, serotonin-O-carboxy-methyl- glycyl-tyrosinamide (S-CM-GTNH2), inhibited 2 nM [3H]5-HT binding to the substantia nigra with an IC50 of 22.4 nM, a value which is 22 times lower than that found in the dentate gyrus and choroid plexus. This demonstrates the preferential affinity of S-CM-GTNH2 for 5-HT1B versus 5-HT1A and 5-HT1C binding sites. S-CM-GTNH2 contains a tyrosine residue, which may be useful for the synthesis of a radioactive iodinated molecule and for the preparation of 'long-lasting ligands' linked through peptide bonds with a protein. These derivatives could be of great interest for ultrastructural and behavioral studies relevant to 5-HT1B sites.

Subcellular localization of GABAA/benzodiazepine receptor-like immunoreactivity in the superficial gray layer of the rat superior colliculus.

The monoclonal antibody bd-17, which recognizes the beta 2 and beta 3-subunits of GABAA/benzodiazepine receptors, was used to determine the cellular and subcellular localization of receptor-like immunoreactivity in the superficial gray layer of the rat superior colliculus. In numerous dendrites, very strong immunostaining was present in the cytoplasm and on the postsynaptic dendritic membrane of synaptic junctions. The extrasynaptic portion of the dendritic membrane also very often showed [beta 2 + beta 3]-like immunoreactivity. However, due to methodological limitations, it could not be stated with certainty whether presynaptic beta 2- and beta 3-subunits of GABAA/benzodiazepine receptors actually occur in this mesencephalic visual structure. In conclusion, these results strongly suggest that synaptic and non-synaptic GABAA/benzodiazepine receptors are present in the superficial gray layer of the rat superior colliculus. These receptors may modulate neuronal cell activity in different ways, depending on their location.

The action of lithium carbonate on the sleep-waking cycle in the cat.

The therapeutic effect of lithium carbonate on manic-depressive psychosis is now universally recognized. In view of this positive action on the cyclic endogenous manifestations, it was interesting to investigate the possible effects of lithium on the sleep-waking cycle in the cat. Polygraphic recording from three adults cats was carried out during 24 h periods before, and on different days after, beginning treatment with lithium carbonate. With low doses (30 and 50 mg/kg/day), important morphological changes were observed 5 days later while a new balance of the sleep-waking stages occurred. The EEG of each stage was characterized by slowing and amplitude increase of the different frequencies. The rhythms which appeared in long runs during quiet wakefulness and paradoxical sleep (PS) in the somaethetic cortex (mu rhythm) and the visual areas (alpha rhythm) were increased, slowed and almost continuously present. From the quantitative point of view, the percentage of time of deep slow wave sleep (DSWS) was increased from 38% to 55%. Conversely, the waking and PS times were both reduced, the latter from 18% to 10%. In contrast with human data, the mean duration of PS episodes was unchanged. Furthermore, lithium induced a slight dissociation between EEG activity and waking behaviour. With toxic dose (90 mg/kg/day) all the above changes were again observed, but more conspicuously. During wakefulness and SWS, bursts of generalized paroxysmal events appeared, in frequent association with a myoclonic jerk. SP became atypical and its percentage of time was drastically reduced.

Electron microscopic study of GABA-immunoreactive neuronal processes in the superficial gray layer of the rat superior colliculus: their relationships with degenerating retinal nerve endings.

GABA-immunoreactive neuronal elements were detected in the stratum griseum superficiale or superficial gray layer of the rat superior colliculus in an electron microscopic study, using postembedding immunocytochemistry with protein A-gold as a marker. In addition to neuronal somata, two types of GABA-immunoreactive neuronal processes were observed. Numerous profiles of axon terminals (1 microns in diameter) with clear round or pleomorphic synaptic vesicles and mitochondria were found to establish mostly symmetrical synaptic contacts with GABA-immunonegative dendrites of various diameters. Some axosomatic synapses could also be observed. The gold particle density in this axon terminal compartment was between seven and 13 times the background level. The stratum griseum superficiale also included GABA-immunoreactive dendrites, some of which contained clear synaptic vesicles. These dendritic profiles always formed the presynaptic component of dendrodendritic synaptic contacts. The density of the gold particles in the dendritic compartment, taken as a whole, was between three and 13 times the background level. Furthermore, the relationship between the GABA-immunoreactive neuronal elements and degenerating retinal nerve endings identified in the left stratum griseum superficiale following enucleation of the right eye was investigated after a 7-day survival period. The profiles of degenerating retinal nerve endings (0.7 microns in diameter) were found to be devoid of any specific labelling. Most of the retinal boutons established axodendritic synapses of the asymmetrical type with an immunonegative dendrite, which was also contacted in some cases by a GABA-immunopositive axon terminal. Other retinal endings were presynaptic to GABA-immunopositive dendritic profiles with synaptic vesicles, some of which were found to contact in turn an unlabelled dendrite, thereby completing serial synaptic relationships. More rarely, retinal endings formed the presynaptic component of possible axoaxonic synapses with GABA-positive terminals presumed to be axonic in nature. It can be concluded that the retinal input to the superficial gray layer often converges with a GABAergic axonal input on a dendritic target, the neurotransmitter specificity of which is unknown. In other cases, retinal terminals synaptically contact GABA-immunolabelled conventional and presynaptic dendrites and probably also some axon terminals; this might provide an anatomical substrate for the control of GABA release from these GABAergic processes. These results indicate that transmitter GABA plays an important role in retinocollicular transmission.

GABA innervation in adult rat oculomotor nucleus: a radioautographic and immunocytochemical study.

GABA innervation in the adult rat oculomotor nucleus (n.III) was investigated using two complementary approaches: radioautography after incubation of brain slices with tritiated GABA ([3H]GABA) along with local in vivo microinjections of the tracer, and GABA immunocytochemical procedures involving antibodies directed against a GABA-glutaraldehyde-protein conjugate. As determined by radioautography after in vitro or in vivo labelling, the [3H]GABA uptake sites in the n.III mainly involved axon terminals. These were distributed throughout the neuropil and were often closely apposed to unlabelled motoneuron somata. A small number of glial cells also showed preferential accumulation of the tracer. The GABA-immunostaining likewise involved axon terminals throughout the nucleus, but no glial cells were immunopositive. In the dorsal region of the structure, occasional GABA-immunostained internuclear neurons were observed among unstained motoneuron cell bodies. Electron microscopic examination of [3H]GABA-labelled or GABA-immunostained profiles in n.III revealed axon terminals of around 1 micron in diameter, always filled with small, round synaptic vesicles homogeneously distributed throughout the axoplasm. These boutons frequently contained mitochondria and one or more large granular vesicles. In single thin sections, 35% of [3H]GABA-labelled, and 19% of GABA-immunostained varicose profiles exhibited a synaptic differentiation, suggesting the existence of a predominantly if not entirely junctional innervation. These synapses mostly involved dendritic trunks or dendritic branches and were usually of the symmetrical type. A few, which were always symmetrical, were also observed on large somata of motoneurons. Some of the dendrites synaptically contacted by GABA-immunostained axon terminals were themselves GABA immunoreactive. These data substantiate the idea that GABA is involved in the control of motoneuron activity in n.III, and provide a structural basis for the inhibitory role of this transmitter in oculomotor function.

[Slow posterior rhythm: electroclinical study of 100 new cases]. / Le rythme lent postérieur: étude électroclinique de 100 nouveaux cas.

One hundred patients with slow rhythmical electro-encephalographic (EEG) activity in the posterior regions were classified according to their clinical symptomatology. Correlations were established between the occurrence of the slow posterior rhythm (SPR) and head injury, and psychological, autonomic or vascular disturbances. In contrast to most previous publications, the patients with head injury constituted only one-half of the series. Autonomic and psychological complaints were frequently encountered in this group. A second group of 11 patients had some type of vascular pathology. A third group of 39 patients had symptoms of anxiety and autonomic system disturbance. The importance of head injury as a factor responsible for SPR seems to have been overrated. Regardless of classification, psychological symptoms were found in 50% and autonomic dysfunction in 53% of all patients. It is apparent that the origin and significance of slow posterior rhythm have not yet been eludicated.

Radioautographic study of 3H-GABA uptake in the oculomotor nucleus of the cat.

The uptake of tritiated gamma-aminobutyric acid (3H-GABA) in the oculomotor nucleus of the cat was studied, using light and electron microscopic examination of radioautograms after intracerebral in vivo administration of the amino-acid. A glial uptake by oligodendrocytes was seen together with a neuronal uptake of the tracer in a certain type of axon terminals found in synaptic contact with both dendrites and soma, some of them exhibiting all the ultrastructural features of motoneurons. Previous neurochemical, electrophysiological and immunocytochemical studies indicate that GABA might well be the inhibitory neurotransmitter in the vestibuloocular reflex arc. The present results show that a morphological substrate exists for the presumed postsynaptic GABAergic inhibition of ocular motoneurons, at least in the oculomotor nucleus of the cat.

[Synthesis and pharmacological study of radioiodinated serotonin derivative specific of 5-HT1B and 5-HT1D binding sites of the central nervous system]. / Synthèse et étude pharmacologique d'un dérivé radioiodé de la sérotonine spécifique des sites de liaison 5-HT1B et 5-HT1D du système nerveux central.

We describe here the synthesis of a new serotonin conjugate, S-CM-GTNH2, and its radioiodinated derivative. Quantitative autoradiographic studies on rat and guinea pig brain sections incubated with 2 nM [3H]5-HT showed a preferential affinity of S-CM-GTNH2 for 5-HT1B and 5-HT1D sites. Autoradiograms from brain sections incubated with 0.02 nM S-CM-G[125I]TNH2 showed a heterogeneous anatomical distribution of the labelling with high densities in regions rich in 5-HT1B or 5-HT1D binding sites, and with no labelling of those rich in 5-HT1A or 5-HT1C sites. The pharmacological profiles of the binding sites corresponded to those of 5-HT1B and 5-HT1D receptor subtypes. The radioligand S-CM-G[125I]TNH2 is a good probe for the study of these sites and will be used for their subcellular localization in electron microscopy.

Pharmacological characterization of serotonin-O-carboxymethyl-glycyl-tyrosinamide, a new selective indolic ligand for 5-hydroxytryptamine (5-HT)1B and 5-HT1D binding sites.

The affinity of a new serotonin (S) derivative, serotonin-O-carboxymethyl-glycyl-tyrosinamide (S-CM-GTNH2), for the various 5-hydroxytryptamine (5-HT)1 receptor subtypes was tested using quantitative autoradiography on rat and guinea pig brain sections. In the rat, S-CM-GTNH2 is 57 and 24 times more potent at 5-HT1B sites (IC50 = 28 nM) than at 5-HT1A (IC50 = 1600 nM) and 5-HT1C sites (IC50 = 670 nM), respectively. In the guinea pig, the affinity of S-CM-GTNH2 for 5-HT1D sites (IC50 = 67 nM) is 21 times higher than at 5-HT1A sites (IC50 = 1400 nM). S-CM-GTNH2 shows a low affinity (less than 10 microM) for 5-HT2 and 5-HT3 binding sites. This new ligand is therefore highly specific for 5-HT1B and 5-HT1D binding sites and can be used to further characterize the involvement of these subtypes in physiological studies focusing particularly on behavioral effects.

Biochemical and pharmacological characterization of serotonin-O-carboxymethylglycyl[125I]iodotyrosinamide, a new radioiodinated probe for 5-HT1B and 5-HT1D binding sites.

There is a lack of radioactive probes, particularly radioiodinated probes, for the direct labeling of serotonin-1B (5-HT1B) and serotonin-1D (5-HT1D) binding sites. Serotonin-O-carboxymethylglycyltyrosinamide (S-CM-GTNH2) was shown previously to be specific for these two subtypes; we, therefore, linked a 125I to its tyrosine residue. Biochemical and pharmacological properties of S-CM-G[125I]TNH2-binding sites were studied by quantitative autoradiography on rat and guinea pig brain sections. S-CM-G[125I]TNH2 binding is saturable and reversible with a KD value of 1.3 nM in the rat and 6.4 nM in the guinea pig. Binding is heterogeneous, paralleling the anatomical distribution of 5-HT1B sites in the rat and of 5-HT1D sites in the guinea pig. The binding of 0.02 nM S-CM-G[125I]TNH2 was inhibited by low concentrations of 5-HT, S-CM-GTNH2, CGS 12066 B, 5-methoxytryptamine, and tryptamine in both species. Propranolol inhibited the radioligand binding with a greater affinity in the rat than in the guinea pig. Conversely, 8-hydroxy-2-(di-n-propylamino)tetralin inhibited S-CM-G[125I]TNH2 binding with a greater affinity in the guinea pig than in the rat. Other competitors, specific for 5-HT1C, 5-HT2, 5-HT3, and adrenergic receptors, inhibited S-CM-G[125I]TNH2 binding in rat and guinea pig substantia nigra and in other labeled structures known to contain these receptors, but only at high concentrations. S-CM-G[125I]TNH2 is then a useful new probe for the direct study of 5-HT1B and 5-HT1D binding sites.