Neurons expressing individual enzymes of dopamine synthesis in the mediobasal hypothalamus of adult rats: functional significance and topographic interrelations.

Besides dopaminergic (DA-ergic) neurons having all enzymes of DA synthesis, tyrosine hydroxylase (TH) and aromatic l-amino acid decarboxylase (AADC), "monoenzymatic" neurons expressing only one of them were found in the brain, mostly in the mediobasal hypothalamus (MBH). The aim of this study was to test our hypothesis that DA is synthesized by monoenzymatic neurons, i.e. l-3,4-dihydroxyphenylalanine (l-DOPA), which produced in the monoenzymatic TH neurons is transported in the monoenzymatic AADC neurons for DA synthesis. Incubation of MBH in Krebs-Ringer solution with l-leucine, a competitive inhibitor of l-DOPA uptake, was used to prevent a hypothetical l-DOPA capture into AADC-containing neurons. Incubation of the substantia nigra containing DA-ergic neurons under the same conditions served as the control. According to our data, the l-leucine administration provoked a decrease of DA concentration in MBH and in the incubation medium but not in the substantia nigra and respective incubation medium, showing a decrease of cooperative synthesis of DA in MBH. This conclusion was supported by an observation of higher concentration of l-DOPA in the incubation medium under perfusion of MBH with Krebs-Ringer solution containing tolcapone, an inhibitor of catechol-O-methyltransferase, and l-leucine than under perfusion with the same solution, but without l-leucine. Functional interaction between monoenzymatic TH and AADC neurons was indirectly confirmed by finding in electron microscopy their close relations in MBH. Besides monoenzymatic AADC neurons, any AADC-possessing neurons, catecholaminergic and serotoninergic, apparently, could participate in DA synthesis together with monoenzymatic TH neurons. This idea was confirmed by the observation of close topographic relations between monoenzymatic TH neurons and those containing both enzymes, i.e. DA-ergic, noradrenergic or adrenergic. Thus, monoenzymatic neurons possessing TH or AADC and being in close topographic relations can synthesize DA in cooperation.

Caudal neurosecretory system of the zebrafish: ultrastructural organization and immunocytochemical detection of urotensins.

The caudal neurosecretory system is described here for the first time in the zebrafish, one of the most important models used to study biological processes. Light- and electron-microscopical approaches have been employed to describe the structural organization of Dahlgren cells and the urophysis, together with the immunohistochemical localization of urotensin I and II (UI and UII) peptides. Two latero-ventral bands of neuronal perikarya in the caudal spinal cord project axons to the urophysis. The largest secretory neurons (approximately 20 microm) are located rostrally. UII-immunoreactive perikarya are much more numerous than those immunoreactive for UI. A few neurons are immunopositive for both peptides. Axons contain 75-nm to 180-nm dense-core vesicles comprising two populations distributed in two axonal types (A and B). Large dense vesicles predominate in type A axons and smaller ones in type B. Immunogold double-labelling has revealed that some fibres contain both UI and UII, sometimes even within the same neurosecretory granule. UII is apparently the major peptide present and predominates in type A axons, with UI predominating in type B. A surprising finding, not previously reported in other fish, is the presence of dense-core vesicles, similar to those in neurons, in astrocytes including their end-feet around capillaries. Secretory type vesicles are also evident in ependymocytes and cerebrospinal-fluid-contacting neurons in the terminal spinal cord. Thus, in addition to the urophysis, this region may possess further secretory systems whose products and associated targets remain to be established. These results provide the basis for further experimental, genetic and developmental studies of the urophysial system in the zebrafish.

Expression of tyrosine hydroxylase and vasopressin in magnocellular neurons of salt-loaded aged rats.

Tyrosine hydroxylase (TH) is expressed in catecholaminergic neurons. However, under certain conditions it is also ectopically expressed in magnocellular neurons of the hypothalamus. To test the hypothesis that this expression of TH is related to the cellular activation of these neurons and/or to the vasopressin (VP) expression, we studied the expression of both TH and VP in control and salt-loaded aged rats. Our results demonstrate that aged rats show a marked TH expression in VP cells which is further increased by osmotic stimulation in the absence of increase in VP synthesis in the supraoptic nucleus. The presence of TH-immunopositive dendritic swellings in the ventral part of this nucleus reveals the high state of plasticity of these neurons. Furthermore, the lack of several actors of catecholamine biosynthesis in these neurons suggests a different role for TH. This study further demonstrates an ectopic expression of TH in hypothalamic neurons of aged rats and a TH expression linked to the activation of VP neurons but unrelated to VP synthesis.

[The synaptology of 2 types of neurons in the sympathetic ganglia of the frog]. / Sinaptologiia dvukh tipov neironov simpaticheskikh gangliev liagushki.

This work is based on the selective section of the B or C preganglionic fibres innervating respectively the B or C neurons of the last two abdominal ganglia of the sympathetic chain in the frog. A quantitative study of three morphological features of their synapses was performed for each of the two neuronal types. Significant differences were observed in the mean size of the preganglionic ending sections; on the contrary there were no differences in the mean length of the active zones. The subsynaptic apparatus, which is fairly frequent in this material, is not restricted to one neuronal type, although much more frequent in the synapses of B neurons. The modifications of the innervation of ganglionic neurons after selective section of one type of preganglionic fibres were followed measuring the synaptic density, using a synaptic index and an index of simple contacts. It appeared that, after selective degeneration of preganglionic C fibres, their regeneration being prevented, the preganglionic B fibres were able to from functional synapses on C neurons with a short latency, normal values of synaptic density being reached within 2 months. On the contrary, after degeneration of the preganglionic B fibres, the preganglionic C fibres reinnervated B neurons with a limited efficiency, in such a way that the synaptic density remained after several months much lower than normal and that electrophysiological recordings of synaptic transmission were limited to 44% of the impaled neurons. Possible interpretations of these data are discussed.

Reinnervation of frog sympathetic ganglia after selective denervation of B or C neurons.

Selective transection of the B or C preganglionic nerve fibres respectively innervating the B and C sympathetic neurons was carried out on the last two ganglia of the sympathetic chain of the frog Rana esculenta. At different times thereafter, the cross-reinnervation of one type of denervated neuron by nerve endings sprouting within the ganglia from intact fibres innervating the other type was investigated by both the quantitative morphology of the synaptic contacts and related structures and electrophysiological recordings of ganglionic transmission. As there are no fine ultrastructural criteria for distinguishing B from C neurons, the overall density of synapse, simple contact, and 'vacated' postsynaptic differentiation profiles was measured in the two cases of selective section and compared with the values for normal ganglia, therefore permitting the progress of cross-reinnervation with time for each type of neuron to be followed. At ten days after section of the C preganglionic fibres, immunocytochemistry showed that there were no anti-LH-RH-like peptide containing fibres within the ganglia. The B myelinated preganglionic fibres were able to reinnervate the denervated C neurons, with return to normal values of synaptic density and fully efficient transmission at two months in all tested C neurons. However, the latency of orthodromic action potentials was close to that of normally innervated B neurons. In contrast, the C non-myelinated preganglionic fibres reinnervated the denervated B neurons with limited efficiency, the synaptic density being two-thirds the normal value after five months, while subthreshold excitatory postsynaptic potentials or action potentials were only recorded in 44% of the tested B neurons. The latency of these orthodromic responses was close to that of normally innervated C neurons. It is postulated that the poor cross-reinnervation of B neurons could be due to insufficient sprouting of C fibres and/or lack of 'affinity' between C fibres and B neurons. In addition, these experiments demonstrated that the subsynaptic apparatus, fairly characteristic of frog ganglionic synapses, is present in both types of sympathetic neurons, although predominantly in B neurons.

Synaptic organization of amphibian sympathetic ganglia.

The synaptic organization of the amphibian sympathetic ganglia was studied, especially in the last two abdominal paravertebral ganglia of the frog. These ganglia appear to form a monosynaptic relay, not containing interneurons. They consist of two systems working in parallel: the principal neurons, by far the most numerous, and a small number of chromaffin (i.e., SIF) cells, usually arranged in clusters. Each principal neuron is innervated by a preganglionic branch forming a set of cholinergic synapses which exhibit classical ultrastructure. The only peculiarity is the presence of a subsynaptic apparatus in a variable percentage of synaptic complexes. Electrophysiological studies have demonstrated that synaptic transmission is due to ACh release and involves several postsynaptic potentials. Moreover, the principal neurons are of two types, B and C, whose preganglionic axons and their own axons have different conduction velocities. C neurons tend to be small in diameter, and B neurons are larger, but the size distribution of the two populations overlaps. More recently, it was demonstrated that these two neuronal systems have different immunocytochemical features. The C preganglionic fibers contain an LHRH-like peptide, which is responsible for late synaptic events. The B preganglionic fibers contain CGRP, whose role has not yet been established. The principal neurons all contain adrenaline, but neuropeptide Y is also present in C neurons and could be a second transmitter at peripheral junctions. SP-containing fibers also pass through the ganglia, but give rise to intraganglionic synapses only rarely, except in the celiac plexus. Galanin can coexist with neuropeptide Y in certain C neurons. Numerous principal neurons are immunoreactive for VIP. Chromaffin cells contain noradrenaline and metenkephalin, and some contain SP or LHRH; they are endocrine cells controlled by preganglionic fibers and can have a modulatory effect on principal neurons endowed with appropriate receptors. The accessibility of frog abdominal ganglia and the anatomical separation of B and C preganglionic fiber pathways provide interesting systems in which to carry out experimentation on the stability and specificity of synaptic contacts. After postganglionic axotomy, the majority of synapses disappear by disruption of synaptic contacts. There is a certain discrepancy between the recovery of synaptic transmission and the reappearance of morphologically identifiable synapses, suggesting that a certain amount of transmission is possible at contacts devoid of synaptic complexes. The selective deafferentation of B or C neurons showed that the subsynaptic apparati are mainly found at B neuron synapses. The course of reinnervation following selective deafferentation reveals the existence of different specificities at B and C synapses: C neurons are easily reinnervated by B preganglionic fibers, whereas C fibers appear fairly ineffective at reinnervating B neurons, even after a long interval. Attempts were made to reinnervate ganglionic neurons with somatic motor nerve fibers. Reinnervation was achieved only rarely, and it is concluded that the ganglionic synapses in the frog have a higher specificity and lower plasticity than in mammals.

Reinnervation of frog sympathetic ganglia with somatic nerve fibres.

The formation of synapses in the last two ganglia of the sympathetic chain of the frog Rana esculenta was investigated after anastomosing the 6th spinal nerve to the denervated ganglia in order to evaluate the reinnervation of deafferented sympathetic neurons with somatic cholinergic axons. The same ganglia were examined both electrophysiologically and morphologically from 25 to 280 days after the operation. In response to electrical stimulations of the anastomosed spinal nerve, synaptic transmission was analysed with intracellular microelectrodes placed into B or C sympathetic neurons. Synaptic density was quantified using electron microscopy by a synaptic index defined as the ratio of the number of synapses encountered to the number of perykarya examined. After ganglionic deafferentation, post-synaptic membrane differentiations persisted without any pre-synaptic element and an index of the 'vacated' post-synaptic differentiations was calculated. Although somatic axons were growing into all ganglia studied, no sign of neuronal reinnervation was detected in ganglia of 8 of the 31 frogs (26%) taken from 29 to 210 days after the anastomosis. Moreover, in 18 out of 31 frogs (58%) analysed at different times after the operation, the ganglia were reinnervated with regenerating preganglionic axons in spite of care taken to avoid it. However, even after 3 months, certain neurons of these ganglia were not reinnervated and the synaptic index approximated the value of normal ganglia only in the 8th ganglion. In addition, post-synaptic membrane differentiations could still persist and coexist with normal synapses. It was only beyond three months after the anastomosis that the ganglia of 5 of the 31 frogs (16%) were reinnervated with regenerating somatic axons. Reinnervated B and C neurons were polyinnervated. But in 3 out of these 5 frogs the ganglia were also reinnervated with preganglionic axons and several B and C neurons received a double reinnervation. The synaptic indices were far from the value of normal ganglia except for the 8th ganglion of one frog reinnervated by both types of axons and the indices of vacated differentiations were close to that of ganglia with no reinnervated neurons. Contrary to mammals, frog somatic axons are, therefore, relatively ineffective at reinnervating sympathetic neurons, probably because in amphibian ganglia, synapses between the pre- and post-synaptic elements require higher specificity.

Effects of axotomy, deafferentation, and reinnervation on sympathetic ganglionic synapses: a comparative study.

The main physiological and morphological features of the synapses in the superior cervical ganglia of mammals and the last two abdominal ganglia of the frog sympathetic chain are summarized. The effects of axotomy on structure and function of ganglionic synapses are then reviewed, as well as various changes in neuronal metabolism in mammals and in the frog, in which the parallel between electrophysiological and morphological data leads to the conclusion that a certain amount of synaptic transmission occurs at "simple contacts." The effects of deafferentation on synaptic transmission and ultrastructure in the mammalian ganglia are reviewed: most synapses disappear, but a number of postsynaptic thickenings remain unchanged. Moreover, intrinsic synapses persist after total deafferentation and their number is strongly increased if axotomy is added to deafferentation. In the frog ganglia, the physiological and morphological evolution of synaptic areas is comparable to that of mammals, but no intrinsic synapses are observed. The reinnervation of deafferented sympathetic ganglia by foreign nerves, motor or sensory, is reported in mammals, with different degrees of efficiency. In the frog, the reinnervation of sympathetic ganglia with somatic motor nerve fibers is obtained in only 20% of the operated animals. The possible reasons for the high specificity of ganglionic connections in the frog are discussed.

Subarachnoid midazolam: histologic study in rats and report of its effect on chronic pain in humans.

Subarachnoid administration via a catheter of a water-soluble benzodiazepine, midazolam, was tested in the control of cancer pain. First, the lack of its toxicity during constant subarachnoid administration (50 micrograms per day) was assessed in the rat. After 15 days of treatment, a histologic examination of the spinal cord revealed the same amount of fibrosis, infiltration, and deformation in the control group (n = 14), which had received only saline, as in the test group (n = 18), treated with subarachnoid midazolam. Therefore, the histologic changes observed in the spinal cord probably are related to the presence of the catheter. After these results, a mixture of 2 mg midazolam and a variable dose of subarachnoid morphine was injected in two patients presenting chronic neoplastic pain resistant to high doses of morphine. In these two cases, the addition of midazolam appeared to be effective in controlling intractable neoplastic pain.

Effects of axotomy on synaptic transmission and structure in frog sympathetic ganglia.

Axotomy was carried out on frog sympathetic neurons of the two last lumbar chain ganglia. At different times thereafter, synaptic transmission was analysed electrophysiologically by intracellular microelectrodes and compared with synaptic density, measured by electron microscopy in the same ganglia. For this purpose, modifications in synaptic transmission were estimated first, by the numbers of B and C sympathetic neurons exhibiting subthreshold excitatory postsynaptic potentials in response to 10 Hz orthodromic stimulation of preganglionic fibres, and second, by the amplitude and number of excitatory postsynaptic potentials occurring over 5-10 s periods of 10 Hz stimulation. By distinguishing two types of morphological relationships between the pre- and postsynaptic elements, two contact indices were defined: a synaptic index (ratio of the number of synapses encountered to the number of perikarya explored) and a simple contact index corresponding to the same type of contacts, but without any membrane differentiation. Both the electrophysiological and morphological results showed that the first effects were detectable 4 days after axotomy, and that the main alterations in synaptic transmission and density occurred at 2 weeks. In addition, while in normal ganglia the excitatory postsynaptic potentials of B and C neurons reached the threshold for action potential generation in response to 10 Hz stimulation, about 29% of the axotomized neurons had subthreshold excitatory postsynaptic potentials 1 week after section. At 2 weeks, this proportion reached 65%, and the synaptic and simple contact indices, at 90% and 60% respectively, were significantly lower than the control ganglion indices. At longer times after axotomy, there was a discrepancy between the morphological and electrophysiological results: at 1 month, the synaptic index seemed to rise as the decline in the efficacy of synaptic transmission became more marked. The amplitude of the subthreshold excitatory postsynaptic potentials recorded in B neurons was 5.5 +/- 2.8 mV (mean +/- SD, n = 18); this value was significantly lower by about 50% than that measured 1 week after axotomy. In addition, the number of excitatory postsynaptic potentials in B neurons reached an average maximum of 83 +/- 29 for 100 stimuli applied at 10 Hz. Similar results were obtained for C neurons. Two months after axotomy, the physiological and morphological parameters of synaptic efficacy began to recover and return to normal values, but had not reached them by 4 months. These observations show that some synaptic transmission remains possible, even with a much reduced number of synaptic complexes. It is suggested that after axotomy, simple contacts also might be involved in synaptic transmission.

Ultrastructural changes in the shape of neurons and related structures in frog sympathetic ganglia after axotomy.

Changes in the shape of neuronal perikarya and other ganglionic structures were observed by electron microscopy in the frog sympathetic ganglia at different times after axotomy. Degenerating and hypertrophic profiles appeared to reflect a remodelling process affecting preganglionic fibres. The shape of neuronal perikarya was modified by the formation of infoldings occupied by preganglionic fibres and/or by that of short winding dendrites often bearing a synapse. The origin of these changes is discussed. In frog sympathetic ganglia, the period of recovery after axotomy was marked by specific reactions which affected neuronal shape and preganglionic fibres, and are not known to occur in the ganglia of mammals.

[Synaptic contacts between SIF cells immunoreactive to cholecystokinin (CCK-8) in inferior mesenteric ganglia in the guinea-pig]. / Contacts synaptiques entre cellules SIF immunoréactives à la cholécystokinine (CCK-8) dans le ganglion mésentérique inférieur du cobaye.

Cell bodies and processes of SIF cells were found to be CCK-like immunoreactive within the mesenteric ganglion of the guinea-pig. Immunoreactivity was contained in granular vesicles, the labeling being chiefly associated with granules, about 100 nm in diameter. Asymmetric synaptic contacts between CCK-like immunoreactive SIF cells have been identified and a possible functional implication regarding intraganglionic connections for these SIF cells is discussed.

[Ganglionic synapses and synaptic transmission after axotomy of sympathetic neurons in the frog]. / Synapses ganglionnaires et transmission synaptique après axotomie des neurones sympathiques chez la Grenouille.

In frog ganglia, efficacy of synaptic transmission was analyzed in parallel with the number of synapses at different times after axotomy of sympathetic neurons: the number of synapses was at their minimum at two weeks whereas depression of synaptic transmission was strongest at one month. The relationship between the presence of synaptic dense specializations and the existence of efficacious transmission is discussed.

[Morphofunctional characteristics of serotonin-like neurons in the hypothalamus of rats in ontogeny]. / Morfofunktsional'naia kharakteristika serotoninopodobnykh neironov v gipotalamuse krys v ontogeneze.

An attempt has been made to reveal 5-HT immunopositive (IP) neurones in the hypothalamus of intact foetuses (18th day of gestation) and neonatal (9-day) rats under normal conditions and after their treatment with drugs involved into 5-HT metabolism or into regulation of its uptake by serotoninergic neurones. 5-HTIP cells were not observed in intact animals as well as after L-tryptophan treatment, whereas two large colonies of these neurones were found in the anterio-lateral hypothalamus and dorsomedial nucleus after subsequent injections of monoamine oxidase inhibitor, pargyline, and amino acid precursor of 5-HT synthesis, L-tryptophan. Significantly less intensive reaction was observed after injections of another precursor of 5-HT synthesis, 5-hydroxytryptophan, or pargyline only. Immunostaining evoked by pargyline or L-tryptophan can be prevented by preliminary injections of fluoxetine, a specific inhibitor of 5-HT uptake by serotoninergic neurones. These data suggest that the immunostaining of hypothalamic neurones is due to their capacity to take up specifically 5-HT from the environment rather than to its intraneuronal synthesis from L-tryptophan. However, 5-HT synthesis from 5-hydroxytryptophan in the same cells may also take place. The uptake of extracellular 5-HT by catecholaminergic neurones is absent, since nomifensine, a specific inhibitor of this uptake, does not affect immunostaining.

On the distribution and morpho-functional characteristics of 5-HT-immunoreactive cells in the hypothalamus of fetuses and neonatal rats.

This study attempted to visualize serotonin (5-HT)-immunoreactive (IR) neurons in the hypothalamus of intact fetuses (E18) and neonatal rats (P9) as well as after their pretreatment with some drugs interfering with the 5-HT metabolism and uptake in the serotoninergic neurons (L-tryptophan, pargyline, 5-hydroxytryptophan, fluoxetine). The 5-HT-IR cells were not observed in the hypothalamus of normal, untreated fetuses and neonatal rats. However, two large accumulations of 5-HT-IR neurons appeared in the anterolateral hypothalamus and in the dorsomedial nucleus after the subsequent injections of the monoamine oxidase inhibitor, pargyline, and the amino acid precursor of the 5-HT synthesis, L-tryptophan. A significantly less intensive reaction was observed after injections either of the second precursor of the 5-HT synthesis, 5-hydroxytryptophan instead of L-tryptophan, or pargyline only. Immunostaining, provoked by the pargyline and L-tryptophan pretreatment, was completely blocked by the injection of the specific 5-HT uptake inhibitor, fluoxetine. It means that the 5-HT immunostaining of the hypothalamic neurons may be accounted for by their capacity to take up specifically 5-HT from the environment rather than by its intraneuronal synthesis from L-tryptophan. Nevertheless, the 5-HT synthesis from 5-hydroxytryptophan in these cells cannot be excluded. The uptake of extracellular 5-HT into catecholaminergic neurons can be excluded as nomifensine, the specific inhibitor of the uptake to these neurons, did not modify the immunostaining.

Immunohistochemical identification of serotonin and noradrenalin containing structures within the nerve plexuses of rat ileum.

The distribution of 5-hydroxytryptamine (= serotonin = 5-HT) and noradrenalin (NA) in the enteric plexuses of the rat ileum was studied using immunocytochemical techniques. 5-HT-like immunoreactive fibers were observed only in the myenteric plexus, surrounding the ganglionic cells, which are all unreactive. NA-like immunoreactive fibers were present in all layers of the ileum: in the myenteric plexus, they were localized in the nodes, forming a network all round the neuronal perikarya; in the Meissner plexus, positive axons were arranged in a delicate network; submucosal blood vessels were often provided by NA-immunopositive nerve plexus. In the inner circular muscle layer the immunoreactive NA-positive fibers run within nerve bundles mainly parallel with the smooth muscle cells. The 5-HT immunoreactive material was depleted by treatment with reserpine; depletion of NA by 6-hydroxy-dopamine was also observed; on the contrary, no depletion of 5-HT by 5,7-dihydroxytryptamine was obtained. To confirm the validity of these results, specific antibodies to tyrosine hydroxylase (TH) and aromatic 1-aminoacid-decarboxylase (AADC), two enzymes involved in the synthesis of catecholamines, were used. In conclusion these experiments indicate that 5-HT is present, probably as a transmitter, in certain fibres of the rat myenteric plexus, distributed in a way similar to that of NA-containing fibers. However, at variance with NA fibers, 5-HT fibers are not present in other regions of the intestine wall.

Ontogenesis of tyrosine hydroxylase-immunopositive structures in the rat hypothalamus. An atlas of neuronal cell bodies.

The development of the catecholaminergic system in the hypothalamus and in the septal region was studied in rats from the 12th fetal day until the 9th postnatal day. Catecholaminergic structures were visualized with pre-embedding immunocytochemistry using antiserum to tyrosine hydroxylase. An intensification of diaminobenzidine product with silver and gold was additionally applied to make the immunocytochemical technique more sensitive. In this paper only the data on the appearance and distribution of the tyrosine hydroxylase-immunopositive neurons (cell bodies) are presented, whereas the catecholaminergic innervation of the hypothalamus with the tyrosine hydroxylase-immunopositive fibers is the topic of an accompanying paper. Sparse tyrosine hydroxylase-immunopositive neurons were first observed in the anlage of the hypothalamus and septal region on the 13th fetal day. Their number increased progressively with age and by the 15th fetal day they already gave rise to a large dorsal accumulation. From the 18th fetal day on, tyrosine hydroxylase immunopositive neurons began to occupy their definitive positions, mainly concentrating within the hypothalamus: in the zona incerta, periventricular and arcuate nuclei. To a lesser extent, they were concentrated in the medial preoptic area, suprachiasmatic, supraoptic, paraventricular, dorsomedial, and anterior hypothalamic nuclei. The data on the distribution of the tyrosine hydroxylase-immunopositive neurons both in the hypothalamus and in the septal region during ontogenesis are summarized in the precise atlas. Primarily small bi- and unipolar catecholaminergic neurons first observed in the youngest fetuses undergo cytodifferentiation during ontogenesis, giving rise to at least two different populations localized ventrally, mainly in the arcuate nucleus, and dorsally, in the zona incerta. The neurons of the former population remain similar to those of the youngest fetuses, whereas the neurons of the latter increase significantly in size, forming several long, highly ramified processes.

Ontogenesis of tyrosine hydroxylase-immunopositive structures in the rat hypothalamus. Fiber pathways and terminal fields.

The innervation of the hypothalamus and septal region by catecholaminergic fibers was studied in rats from the 12th fetal day until the 9th postnatal day. Catecholaminergic fibers were visualized with preembedding immunocytochemistry using antibodies to tyrosine hydroxylase. An intensification of diaminobenzidine product with silver and gold was additionally applied to increase the sensitivity and resolution power of the routine immunocytochemical technique. It has been demonstrated that, from the 13th fetal day, the hypothalamus and the septal region receive catecholaminergic fibers either belonging to the hypothalamic neurons or coming with the medial forebrain bundle from the outside of the hypothalamus. As the development of the hypothalamus proceeds, these fibers form the extensive networks within some neurosecretory centers either containing (the zona incerta, periventricular nucleus, etc.) or almost lacking (suprachiasmatic and paraventricular nuclei) the catecholaminergic neurons. In the former case, they terminate on the processes or perikarya of catecholaminergic neurons, while in the latter case their varicosities surround the immunonegative presumptive neurons in a basket-like manner. Moreover, from the 18th fetal day catecholaminergic fibers penetrate between the ependymal cells towards the 3rd ventricle and the primary capillary plexus of the hypophysial portal circulation, apparently providing the release of catecholamines to the cerebrospinal fluid and portal blood, respectively. The data obtained in this study are considered as the morphological basis for the involvement of the hypothalamic catecholamines in neuroendocrine regulations during ontogenesis.