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.

Ultrastructural changes of synaptic glomeruli following intrathecal injection of distilled water to rats.

We attempted to identify synaptic glomeruli in the spinal substantia gelatinosa of rats by injecting distilled water into the subarachnoid space. The altered synaptic glomeruli were visualized when injecting 1.0 ml of distilled water, though the incidence of degenerative synaptic glomeruli was rare in the case of an injection of 0.5 ml or less. We assume that when the distilled water is injected, there is a sudden change in homeostatic balance in cerebrospinal fluid, following inhibition of nerve impulses in the dorsal root and the altered synaptic glomeruli. Ultrastructurally, the injection of distilled water led to a preferential degeneration of synaptic glomeruli derived from the C-fibers.

Ultrastructural evidence for a functional unit between nerve fibers and type II cerebral mast cells in the cerebral vascular wall.

By histofluorescence microscopic examinations of pial arteries from rats and rabbits, we have observed that the routes of adrenergic fibers were apparently organized along successive sites of granular autofluorescent cells present in the adventitia. Subsequent electron microscopic studies showed that these cells were often situated in close apposition (80 to 200 nm) to the adventitial nerve bundles. The granular cells and nerve varicosities were frequently enclosed within the same basement membrane, with a membrane-to-membrane distance as small as 20 nm. However, no clear membrane differentiation was seen. These granular cells were identified histochemically by staining with Sudan Black, Oil Red O, Toluidine Blue, Alcian Blue, together with ultrastructural and pharmacological methods (48/80 compound and carbachol intracarotid infusions). The cells, many of which contained large amounts of lipids, showed morphological ultrastructural and pharmacological similarities to peripheral mast cells. Nerve bundles contained two types of varicosities: some of them degenerated after superior cervical ganglionectomy and were thus of sympathetic origin, whereas the others contained small clear vesicles (probably cholinergic) and/or large dense-cored vesicles (probably peptidergic). As we have shown that cholinomimetics induce exocytosis of these granular cells, the close relationship between these cells and the nerve fibers may indicate a neurogenic control of the cerebrovascular mast cell secretion. As these cells contain potent vasoactive substances, this relationship may be of importance in the genesis of physiological or pathological cerebrovascular events which are, as yet, poorly understood.

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.

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.

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.

Development of the hypothalamic serotoninergic system during ontogenesis in rats. Immunocytochemical and radioautographic study.

The development of the hypothalamic serotoninergic system has been studied in rats from the 14th fetal till the 9th postnatal day. Serotoninergic elements were detected with immunocytochemistry using antiserum to serotonin (5-HT) and radioautography following the injections of [3H]5-HT into the cerebral ventricles. Immunocytochemistry failed to recognize hypothalamic 5-HT neurons either in fetuses or in neonatal rats. Conversely, radioautography demonstrated sparse radioactively labeled cells, undifferentiated in appearance, at the 16th and 18th fetal days. Moreover, at the 18th fetal day an accumulation of radioactively labeled typical neurons appeared in the suprachiasmatic region. By the 9th postnatal day, practically all radioactively labeled cells looking like highly differentiated neurons were concentrated in the dorsomedial nucleus. As to nerve fibers, a small number of 5-HT-immunoreactive axons arising from the raphe nucleus were first detected in the hypothalamus at the 16th fetal day. Two days later, both 5-HT-immunoreactive and radioactively labeled fibers became widely distributed through the hypothalamus with especially high concentrations in and around the optic chiasma. By the 9th postnatal day, the frequency of 5-HT fibers increased considerably both in the anterior and middle hypothalamus. Some 5-HT fibers were apposed to unlabeled neurons and others abutted on the capillaries either in the organum vasculosum of the lamina terminalis or in the median eminence. The present morphologic study suggests that during the perinatal period the hypothalamic 5-HT system becomes developed enough to be involved in the regulation of some neuroendocrine functions.

Immunocytochemical and radioautographic study of serotonin projections to cerebral ventricles of perinatal rats.

Anatomical relationships between serotoninergic (5-HT) fibers and cerebral ventricles were studied in rats from the 16th fetal day until the 9th postnatal day with immunocytochemistry and radioautography. In the last case, 5-HT neuronal elements were detected according to their specific uptake of intraventricularly injected [3H]5-HT. At the 16th fetal day, occasional 5-HT fibers first spread from the main place of their origin in the raphe nuclei to the dorsocaudal portion of the 3rd ventricle and aqueduct. Two days later, a more extensive network of 5-HT fibers appeared around the dorsal portion of the 3rd ventricle whereas fibers only rarely penetrated toward its ventral portion. By the 9th postnatal day, extensive networks of supraependymal fibers became noticeable in the lateral ventricles and in the dorsal portion of the 3rd ventricle. In addition, a number of 5-HT fibers surrounded the infundibular and preoptic recesses and sometimes penetrated to the ventricular cavity. The functional significance of hypothalamic and ventricular 5-HT as a modulator of either the growth and differentiation of the developing brain or of some specific neuroendocrine functions is discussed.

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.

[Immunocytochemical and autoradiographic research on the growth of serotoninergic fibers to the cerebral ventricles in the perinatal period in rats]. / Immunotsitokhimicheskoe i avtoradiograficheskoe issledovaniia prorastaniia serotoninergicheskikh volokon k zheludochkam mozga v perinatal'nom periode u krys.

Anatomical relationships between serotoninergic (5-HT) fibers and cerebral ventricles were studied in rats from the 16th fetal day until the 9th postnatal day with immunocytochemistry and radioautography. In the latter case, 5-HT neuronal elements were detected according to their specific uptake of intraventricularly injected 3H-5-HT. On the 16th fetal day, occasional 5-HT fibers first spread from the main place of their origin in the raphe nuclei to the dorsocaudal portion of the 3rd ventricle and aqueduct. Two days later, a more extensive network of 5-HT fibers appeared around the dorsal portion of the 3rd ventricle, whereas fibers only rarely penetrated fibers became noticeable in the lateral and 3rd ventricles. The functional significance of hypothalamic and ventricular 5-HT is discussed from the standpoint of its being either a modulator of growth and differentiation of the developing brain, or a factor involved in some specific neuroendocrine functions.

[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.

[Radioautographic study of the hypothalamic serotoninergic structures in the perinatal period of the rat]. / Radioavtograficheskoe issledovanie serotoninergicheskikh struktur gipotalamusa v perinatal'nom periode krysy.

The distribution of 3H-serotonin-binding structures in hypothalamus of 16 and 18 day old fetuses and of 9 day old rats was studied after intraventricular injection of 3H-serotonin. Rare 3H-serotonin-binding little differentiated cells were found predominantly in the intermediate zone of the 3rd ventricle in the retrochiasmatic area wall on the 16th and 18th days of prenatal development. In addition, an aggregate of heavily labeled neurones was observed in the suprachiasmatic area. At the same time 3H-serotonin-binding fibers first appeared, predominantly in the optic chiasma and perichiasmatic area. Radioactively labelled cells, which can be characterized by their morphology as differentiated neurones, were located in the dorsomedial nucleus on the 9th day of postnatal development. The number of serotonin-binding fibers markedly increased but the pattern of their distribution was, on the whole, similar to that in fetuses. The data obtained suggest that the main stages of structural organization of serotoninergic system of hypothalamus in rats are realized during perinatal period.

[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.

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.