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A newly developed synaptosomal model was used to evaluate the in vivo effects of the GABA-elevating drugs aminooxyacetic acid (AOAA, 30 mg/kg i.p.) and valproic acid (VPA, 200 mg/kg i.p.) on GABA levels in nerve endings of 11 brain regions in rats as a function of time after administration. The data obtained were compared with the magnitude and time course of the effects of both drugs in rats on body temperature, pain response and against seizures induced by electroshock, pentylenetetrazol and 3-mercaptopropionic acid. Following AOAA, maximum increases in synaptosomal GABA levels of brain regions were observed 6 hr after administration. At this time, GABA was significantly elevated up to 300% over control values in synaptosomal fractions from all 11 regions. However, the hypothermic and antinociceptive effects of the drug as well as its anticonvulsant action against electroshock and pentylenetetrazol induced seizures were maximal 1 hr after injection and had vanished after 6 hr, i.e. at the time of maximum GABA increases in synaptosomes. The only pharmacological effect of AOAA which paralleled the time course of the synaptosomal GABA elevation was the attenuation of seizures induced by 3-mercaptopropionic acid. Following VPA, the effect on synaptosomal GABA levels was much more rapid in onset and significant increases were already determined 5 to 30 min after administration. Significant increases of up to 80% over control values were found in synaptosomal fractions from olfactory bulb, frontal cortex, hippocampus, hypothalamus, tectum, substantia nigra and cerebellum. In contrast to AOAA, the time course of the synaptosomal GABA increases, at least in some regions, was similar to the time course of VPA's antinociceptice effects and its anticonvulsant effects in the three seizure models studied. The data may suggest that AOAA and VPA increase different pools of GABA within nerve terminals, only one of which is involved in GABA-mediated neurotransmission.

Luteinizing hormone-releasing hormone immunoreactivity was studied in the olfactory system of the rat in combination with acetylcholinesterase histochemistry. Neuronal perikarya containing luteinizing hormone-releasing hormone lie in the medial septal nucleus, the vertical limb of the diagonal band of Broca, the olfactory tubercule and the ganglionated plexus of the terminal nerve. Labelled fibres spread in the superficial layers of the main and accessory olfactory bulbs, some encompassing the strongly acetylcholinesterase-positive atypical glomeruli. Others are observed on the medial side of the bulb, running along the terminal nerve bundles and ganglia. These fibres join the vomeronasal nerve branches and proceed distally towards the nasal cavity. In the septal submucosa, immunoreactive fibres are partly associated with the terminal nerve network. Conspicuous endings filled with luteinizing hormone-releasing hormone are observed on blood vessels of the olfactory mucosa. Such well-differentiated terminals might be the neurosecretory afferents of a new neurohemal area. Immunoreactive terminals are also observed around the excretory ducts of the anterior medial glands. We have failed to observe any labelled fibres in the olfactory and vomeronasal epithelia. The results of the present study are discussed with respect to possible functional interpretations. It is suggested that significant amounts of luteinizing hormone-releasing hormone could be released in the submucosal capillaries in spite of the scarcity of immunoreactive fibres. Similar afferents could also modulate the secretory activity of some nasal glands. Synaptic events involving the neuropeptide might occur in the olfactory bulb, particularly in atypical glomerular areas previously characterized by their high acetylcholinesterase content. Finally, no anatomical support for a chemosensory function of fibres containing luteinizing hormone-releasing hormone has been brought out by our work.

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.

The distribution of axons and axon varicosities containing GABA was studied in the superior cervical ganglion of rat by light and electron microscopic immunohistochemistry. Two different polyclonal antibodies were used, which had been made against GABA conjugated by glutardialdehyde to bovine serum albumin. GABA-like immunoreactivity occurred in many axons within the cervical sympathetic trunk and in axons and axon varicosities around the principal nerve cells in the superior cervical ganglion. GABA-positive axons were intermingled with non-stained axons, except for a small group of fibers in the trunk where the staining was absent. The rostral part of the ganglion and some scattered patches were more densely innervated by GABA-positive axons than the middle and caudal parts. Within dense areas, some of the large ganglion cells were abundantly surrounded by GABA-positive nerve fibers, while the vicinity of others was devoid of any immunoreactive axon terminals. None of the principal ganglion cells contained GABA-like immunoreactivity, although a class of small cells scattered within the ganglion was stained. Transection of the cervical sympathetic trunk for 11 days caused the disappearance of GABA-like positivity from most of the fibers, and only very little GABA-like staining was revealed in some small cells, which resembled satellite cells. Ultrastructurally, the GABA-positive nerve fibers were unmyelinated. However, their terminal branches and varicosities accumulated around the perikarya and dendrites of certain principal ganglion cells were partly wrapped in glial processes. The present results provide evidence that the superior cervical ganglion of adult rat receives a significant number of GABA-positive axons from the cervical sympathetic trunk and that these axons provide an innervation which is heterogeneously distributed within the superior cervical ganglion and on ganglionic cells. The source and function of the GABA-positive axons remain to be elucidated.

The ultrastructural morphology and afferent sources of terminals containing substance P-like immunoreactivity were examined in the rat parabrachial region. In the first portion of the study, a polyclonal antiserum to substance P was localized in the ventrolateral parabrachial region using the peroxidase-antiperoxidase labeling technique combined with electron microscopy. The antiserum was tested for cross-reaction with substance P, physalaemin, substance K and neuromedins B, C and K. Cross-reactivity was most intense with substance P. However, substance K, neuromedin K and physalaemin also exhibited limited cross-reactions with the antiserum. In the ventrolateral parabrachial region of untreated adult animals, substance P-like immunoreactivity was localized in axon terminals containing numerous small (40-60 nm) clear vesicle and 1-3 large (90-120 nm) dense-core vesicles. At least 54% of the labeled terminals formed asymmetric synapses with unlabeled dendrites; and at least 30% of the recipient dendrites received more than one labeled axon terminal. In addition, the labeled terminals were associated less frequently with other unlabeled soma, axon terminals and blood vessels. In the second part of the study, we examined whether or not perikarya in various extrinsic regions contributed to the substance P-like immunoreactivity in axon terminals in the parabrachial region. Wheat-germ agglutinin conjugated horseradish peroxidase was injected unilaterally into the parabrachial region of adult rats two days prior to being killed and one day prior to intraventricular injection of colchicine (100 micrograms in 7.5 microliter saline) which enhanced the detection of immunoreactivity in perikarya. Sections were first processed by a tetramethylbenzidine reaction stabilized with cobalt-diaminobenzidine for demonstration of the transported peroxidase then were immunocytochemically labeled for substance P. Perikarya containing both the black granular retrograde labeling and brown peroxidase-immunoreactivity were found in the nuclei of the solitary tracts, the caudal ventrolateral reticular formation, the lateral dorsal tegmental nucleus and the paraventricular, dorsomedial and lateral hypothalamic nuclei. The projections were largely, but not exclusively, from perikarya located on the same side as the parabrachial injection. We conclude that substance P, or a closely related tachykinin, is a putative transmitter or modulator within a number of pathways to the parabrachial region and that these afferents act primarily through axodendritic synapses with intrinsic neurons.

Enkephalins have been detected in the cranial motor nuclei and may play a role in the motor system. The afferent source of leucine-enkephalin-like immunoreactive fibers in the facial nucleus was investigated using experimental immunohistochemistry. These leucine-enkephalin-containing fibers were markedly reduced on the operated side after the destruction of the ventrolateral part of the caudal medullary reticular formation, where a number of leucine-enkephalin-positive cell bodies were observed. This fact strongly suggests that these leucine-enkephalin-positive cells project ipsilaterally to the facial nucleus and so could have some effects on facial motoneurons.

In the present study the dopaminergic innervation of the prefrontal cortex was studied by means of a recently developed anti-dopamine serum. This method can demonstrate endogenous dopamine in a specific way, and offers the opportunity to study the distribution of dopaminergic fibres in the cortex in detail in counterstained sections. Furthermore, dopaminergic nerve endings can be visualized at the electron microscopic level. Light microscopic observations demonstrated that the highest density of dopaminergic fibres in the frontal cortex is found in the prefrontal cortex and the infralimbic cortex. Within the prefrontal cortex, a good correlation is found between regional differences in distribution of dopaminergic fibres and the cytoarchitectonic parcellation of this part of the cortex. Outside the prefrontal cortex dopaminergic fibres were observed in adjacent frontal areas, the cortex surrounding the entire rhinal sulcus and the retrosplenial cortex. Electron microscopic observations demonstrated dopaminergic terminals through all cortical layers. The majority of dopaminergic terminals in the prefrontal cortex from synaptic contacts with dendritic processes. The synaptic profiles were usually symmetric and were characterized by the presence of many clear vesicles and an occasional dense-core vesicle.

Neurons in the hippocampal formation of the rat that project to the medial nucleus accumbens were identified following the retrograde transport of a conjugate of horseradish peroxidase with wheat germ agglutinin. The great majority of such projecting neurons were located in the ventral subiculum and were pyramidal in shape; the pyramidal nature of 25 such retrogradely labelled neurons was established by Golgi impregnation. In material processed to reveal both retrogradely labelled cells and cholecystokinin-immunoreactivity, no immunoreactive projecting neurons were found. However, 48 identified projecting neurons, probably pyramidal, were found to receive input from cholecystokinin-immunoreactive boutons that formed symmetrical synaptic contacts with the soma or proximal dendrites. It is suggested that one function of cholecystokinin-immunoreactive neurons in the hippocampal formation might be to influence the output of the pyramidal neurons that project to the nucleus accumbens. Since this pathway is one of the main links between the limbic system and the basal ganglia, it is conceivable that changes in the cholecystokinin levels in the hippocampus, as found in schizophrenia, might influence behaviour through the pathway connecting the hippocampus with the nucleus accumbens.

Serotonin and gamma-aminobutyric acid (GABA) neurons in the nucleus raphe dorsalis were identified by immunocytochemistry using antibodies to 5-hydroxytryptamine or GABA. The pattern of the 5-hydroxytryptamine and GABA immunostaining presented similar features: 5-hydroxytryptamine or GABA immunoreactive somata were fusiform or ovoid (15-20 micron) and positive dendritic profiles were found either without any connection with other nerve elements or in contact with one or several terminals. In addition, some 5-hydroxytryptamine nerve endings were apposed to 5-hydroxytryptamine immunoreactive cell bodies or dendrites; also some GABA-immunopositive terminals were in contact with GABA-immunopositive nerve cell bodies. On the other hand, GABA and 5-hydroxytryptamine patterns may be differentiated in several respects: the 5-hydroxytryptamine-reactive nerve cell bodies were more numerous than the GABA ones. Some small, round (8-10 micron) nerve cell bodies were reactive with GABA antiserum, but no neurons of this type were reactive with a 5-hydroxytryptamine antiserum; finally, GABA nerve terminals were more numerous than 5-hydroxytryptamine ones. In order to understand the relationship between GABA and 5-hydroxytryptamine neurons, radioautographic and immunocytochemical procedures were combined: 5-hydroxytryptamine and GABA immunocytochemistry was combined with radioautography of [3H]GABA and [3H]5-hydroxytryptamine uptake, respectively. Some nerve cell bodies, dendrites or terminals, which were 5-hydroxytryptamine-immunopositive, were also capable of accumulating [3H]GABA and, conversely, some GABA-immunopositive elements were capable of accumulating [3H]5-hydroxytryptamine. Moreover, several nerve elements were reactive with both glutamate decarboxylase and 5-hydroxytryptamine antisera. These data confirm in electron microscopy previous studies suggesting the coexistence of both GABA and 5-hydroxytryptamine in the same neurons. The presence of uptake mechanisms for GABA and 5-hydroxytryptamine may indicate the action of both neurotransmitters in the same neuron. On the other hand, the [3H]GABA-labelled nerve endings in contact with 5-hydroxytryptamine-positive dendrites or nerve cell bodies indicate the possibility of a GABAergic control of the activity of some 5-hydroxytryptamine neurons; this corroborates biochemical and electrophysiological studies whereby a trans-synaptic control of the 5-hydroxytryptamine neurons by GABA may be envisaged.

The morphology and distribution of serotonin-containing axon terminals in the rat hypoglossal nucleus (XII) was investigated immunocytochemically at the electron microscopic level. Serotonin-positive profiles were found throughout all regions of XII and included unmyelinated axons, varicosities and axon terminals. Most labeled profiles (68.1%) were nonsynaptic unmyelinated axons and varicosities, while synaptic profiles, ending on dendrites and somata, were seen less frequently (28.7%). The majority of labeled axon terminals (76.9%) ended on small-to-medium-sized dendrites. Most axodendritic terminals contained small, round agranular vesicles (20-55 microns), several large (60-100 microns) dense core vesicles, and were associated with a pronounced asymmetric postsynaptic specialization. By contrast, labeled axosomatic terminals were seen less often than those ending on dendrites (23.0%). Axosomatic terminals typically contained small, round, agranular and large dense core vesicles and were associated with a symmetric or no postsynaptic specialization. These results provide the structural substrates for elucidating the functional role of serotonin in tongue control.

New dopaminergic terminal fields have been visualized in the rat cerebral neocortex, using two morphological methods based on distinct properties of the dopaminergic system: presence of the first synthetic enzyme, tyrosine hydroxylase, and high-affinity uptake of amines. Tyrosine hydroxylase was used as an immunocytochemical marker after destruction of the cortical noradrenergic system, induced either neonatally by 6-hydroxydopamine or later on by DSP4, and controlled by the absence of dopamine beta-hydroxylase immunoreactivity. The uptake and storage of exogenous amines in tissue sections, in the presence of selective high-affinity transport inhibitors, enabled the specific visualization of the dopaminergic system with fluorescence histochemistry. A dopaminergic innervation of low density was observed along a dorsal sagittal strip which extended from the genu of corpus callosum until about 2 mm behind the splenium and encompassed several distinct cytoarchitectonic areas in the sensorimotor and visual cortex (medial and lateral agranular field, area 18b), as well as in discrete zones of the retrosplenial granular 29c,b, and agranular 29d areas. The distribution of these dopaminergic fields suggested a columnar organization. Several characteristics of the dopaminergic innervation were similar to that of the superficial anterior cingulate cortex (area 24): the laminar distribution to the superficial I-III layers, the secondarily developed varicose aspect in catecholamine fluorescence histochemistry and the delayed postnatal ingrowth in contrast with the early prenatal dopaminergic input to the prefrontal cortex. These similarities suggested that the subpopulation of dopaminergic neurons which provides projections to the anterior cingulate cortex could also contribute to the motor and visual cortex and thus play a role in sensorimotor integration. The predictive value of these results in the ascent of the phylogenetic scale are further considered.

Immunocytochemical methods were used to examine the ultrastructural features and cellular interrelationships of GABA and serotonin afferent fibers to the rat red nucleus. GABAergic nerve endings were identified in two ways, either using a pre-embedding immunoperoxidase procedure with an antibody against glutamate decarboxylase, the GABA-synthesizing enzyme, or after post-embedding immunogold labelling with an anti-GABA antibody. With the latter approach, it was possible to simultaneously visualize the GABAergic and serotoninergic innervation of the red nucleus (magnocellular part) in electron microscope preparations. This procedure involved GABA labelling of ultrathin sections obtained from specimens previously immunostained for serotonin using the pre-embedding peroxidase-antiperoxidase technique. The doubly stained sections showed gold and peroxidase markers to be present in two distinct populations of axonal varicosities. Unlike the GABAergic nerve endings, which were found to be profusely distributed throughout the nucleus, the serotonin nerve endings were relatively scarce. They contacted dendrites of large-sized neurons usually endowed with several GABA-gold labelled terminals. Not uncommonly, direct appositions between serotonin and GABA-positive terminals were also encountered. These data provide morphological evidence that red nucleus outputs may be dually regulated by GABAergic and serotoninergic afferents, while suggesting that presynaptic GABA/serotonin interactions might also play a significant part in red nucleus functions.

Immunocytochemical and electron microscopic methods were used to examine neurons in regio superior of rat hippocampus displaying cholecystokinin octapeptide-like immunoreactivity. Cholecystokinin-immunoreactive synaptic terminals and somata are found in all layers of regio superior but are most numerous in stratum pyramidale. The vast majority of terminals form symmetric synaptic contacts onto the somata and proximal dendrites of hippocampal pyramidal cells and onto smaller dendrites which may also arise from pyramidal cells. A very small number of cholecystokinin-immunoreactive terminals form synapses that appear asymmetric and contact dendritic shafts or spines. The somata of some pyramidal cells receive symmetric synapses from cholecystokinin-immunoreactive terminals that are joined by cytoplasmic bridges to form parts of pericellular baskets. These and adjacent pyramidal cell somata are also contacted by terminals that are not immunoreactive for cholecystokinin. No cholecystokinin-positive terminals contacted the initial segments of pyramidal cell axons. Cholecystokinin-immunoreactive cells are found in all layers of regio superior. Their somata receive a few symmetric synapses, most of which are formed by terminals not immunoreactive for cholecystokinin. Their dendrites receive a greater number of both symmetric and asymmetric contacts, some of which are immunoreactive for cholecystokinin. We conclude the following: The localization of cholecystokinin immunoreactivity in synaptic terminals contacting the somata and dendrites of hippocampal pyramidal cells is consistent with the suggestion that cholecystokinin acts as a neurotransmitter at these sites and at sites in other parts of the cerebral cortex. Results from the present and previous studies suggest that cholecystokinin-like immunoreactivity may co-exist with gamma-aminobutyrate in some non-pyramidal neurons of regio superior. Cholecystokinin-immunoreactive terminals arise mainly from non-pyramidal cells intrinsic to the hippocampus, one class of which appears to be a type of basket cell.