Association of dopaminergic fibers with corticotropin releasing hormone (CRH)-synthesizing neurons in the paraventricular nucleus of the rat hypothalamus

Summary Catecholamines are known to exert a central influence on the hypothalamo-hypophyseal-adrenal neuroendocrine system. The selective dopaminergic innervation of the hypothalamic paraventricular nucleus (PVN) and putative relationships between dopaminergic fibers and corticotropin releasing hormone (CRH)-synthesizing neurons were studied in the male rat by means of immunocytochemistry following the elimination of noradrenergic and adrenergic inputs to the hypothalamus. A 3.0-mm-wide coronal cut was placed unilaterally in the brain at the rostral level of the mesencephalon. All neuronal structures from the cortex to the ventral surface of the brainstem, including the ascending catecholaminergic fiber bundles were transected. This surgical intervention resulted in the accumulation of dopamine--hydroxylase (DBH)-immunoreactivity in axons proximal to the cut, and an almost complete disappearance of DBH activity in those located distal to the lesion. Two weeks following the operation, DBH immunoreactivity was significantly diminished in the PVN located on the side of lesion, while tyrosine hydroxylase (TH)-immunoreactivity was present in a substantial number of fibers in the same nucleus. Both DBH- and TH-immunoreactive axons were preserved in the contralateral PVN. Simultaneous immunocytochemical localization of either DBH- or TH-IR fibers and corticotropin releasing hormone-synthesizing neurons in the hypothalami from brainstem-lesioned, colchicine treated animals revealed that the distribution of catecholaminergic fibers and CRH neurons is homologous within the PVN of the intact side. Only a few scattered DBH-immunoreactive axons were detected among CRH-producing neurons in the PVN on the side of the lesion. In contrast, many tyrosine hydroxylase containing neurons and neuronal processes were observed on the lesioned side and the TH-IR fibers established juxtapositions with CRH-synthesizing neurons.These morphological data demonstrate that following the surgical ablation of noradrenergic and adrenergic afferents to the PVN, a substantial number of tyrosine hydroxylase-IR fibers remained in the nucleus and they were associated with corticotropin releasing hormone synthesizing neurons. Therefore, it is hypothesized that the paraventricular nucleus receives a selective dopaminergic innervation and these dopaminergic axons might influence the function of the pituitary and adrenal glands via the hypothalamic CRH system.Supported by grants from the National Science Foundation (NSF INT 8703030), the Hungarian Academy of Sciences (OTKA 104), the National Institutes of Health (NS 19266) and the National Foundation of Technical Development (OKKFT Tt 286/1986)     

Electron microscopic analysis of tyrosine hydroxylase,dopamine-β-hydroxylase and phenylethanolamine-N-methyltransferase immunoreactive innervation of the hypothalamic paraventricular nucleus in the rat

Summary The catecholaminergic innervation of the hypothalamic paraventricular nucleus (PVN) of the rat was studred by preembedding immunocytochemical methods utilizing specific antibodies which were generated against catecholamine synthesizing enzymes. Phenylethanolamine-N-methyltransferase (PNMT)-immunoreactive terminals contained 80–120 nm dense core granules and 30–50 nm clear synaptic vesicles. The labeled boutons terminated on cell bodies and dendrites of both parvo- and magnocellular neurons of PVN via asymmetric synapses. The parvocellular subnuclei received a more intense adrenergic innervation than did the magnocellular regions of the nucleus. Dopamine--hydroxylase (DBH)-immunopositive axons were most numerous in the periventricular zone and the medial paryocellular subnucleus of PVN. Labeled terminal boutens contained 70–100 nm dense granules and clusters of spherical, electron lucent vesicles. Dendrites, perikarya and spinous structures of paraventricular neurons were observed to be the postsynaptic targets of DBH axon terminals. These asymmetric synapses frequently exhibited subsynaptic dense bodies. Paraventricular neurons did not demonstrate either PNMT or DBH immunoreactivity. The fibers present within the nucleus which contained these enzymes are considered to represent extrinsic afferent connections to neurons of the PVN.Tyrosine hydroxylase (TH)-immunoreactivity was found both in neurons and neuronal processes within the PVN In TH-cells, the immunolabel was associated with rough endoplasmic reticulum, free ribosomes and 70–120 nm dense granules. Occasionally, nematosome-like bodies and cilia were observed in the TH-perikarya. Unlabeled axons established en passant and bouton terminaux type synapses with these TH-immunopositive cells. TH-immunoreactive axons terminated on cell bodies as well as somatic and dendritic spines of paraventricular parvocellular neurons. TH-containing axons were observed to deeply invaginate into both dendrites and perikarya of magnocellular neurons.These observations provide ultrastructural evidence for the participation of central catecholaminergic neuronal systems in the regulation of the different neuronal and neuroendocrine functions which have been related to hypothalamic paraventricular neurons.Supported by NIH Grant NS 19266 to W.K. Paull     

Interrelations between monoaminergic afferents and corticotropin-releasing factor-immunoreactive neurons in the rat central amygdaloid nucleus: ultrastructural evidence for dopaminergic control of amygdaloid stress systems

Ample evidence implicates corticotropin-releasing factor (CRF)-producing neurons of the central amygdaloid nucleus (CeA) in vegetative, endocrine, and behavioral responses to stress and anxiety in laboratory rats. Monoaminergic systems are involved in modulating these responses. In the present paper, interrelations between CRF-immunoreactive (ir) neurons, and noradrenergic, serotonergic, and dopaminergic afferents were studied using single and double immunolabeling for light and electron microscopy in the rat CeA. Dopaminergic axons formed dense plexus in the CeA overlapping with the localization of CRF-ir neurons, and their terminals formed frequent associations with CRF-ir somata. Contacts of serotonergic axons on CRF-ir neurons were few, and contacts of noradrenergic axons were the exception. Ultrastructurally, symmetric synapses of dopaminergic terminals on CRF-ir somata and dendrites were found. More than 83% of CRF-ir somata were contacted in single ultrathin sections. About half of these possessed two or more contacts. Of non-ir somata, 37% were contacted by dopaminergic terminals, and only 13% of these had two or more contacts. Correlative in situ hybridization indicated that CeA CRF-ir neurons may express receptor subtype dopamine receptor subtype 2. In conclusion, dopaminergic afferents appear to specifically target CeA CRF neurons. They are thus in a position to exert significant influence on the rat amygdaloid CRF stress system.     

Electron microscopic analysis of tyrosine hydroxylase, dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase immunoreactive innervation of the hypothalamic paraventricular nucleus in the rat

The catecholaminergic innervation of the hypothalamic paraventricular nucleus (PVN) of the rat was studied by preembedding immunocytochemical methods utilizing specific antibodies which were generated against catecholamine synthesizing enzymes. Phenylethanolamine-N-methyltransferase (PNMT)-immunoreactive terminals contained 80-120 nm dense core granules and 30-50 nm clear synaptic vesicles. The labeled boutons terminated on cell bodies and dendrites of both parvo- and magnocellular neurons of PVN via asymmetric synapses. The parvocellular subnuclei received a more intense adrenergic innervation than did the magnocellular regions of the nucleus. Dopamine-beta-hydroxylase (DBH)-immunopositive axons were most numerous in the periventricular zone and the medial parvocellular subnucleus of PVN. Labeled terminal boutons contained 70-100 nm dense granules and clusters of spherical, electron lucent vesicles. Dendrites, perikarya and spinous structures of paraventricular neurons were observed to be the postsynaptic targets of DBH axon terminals. These asymmetric synapses frequently exhibited subsynaptic dense bodies. Paraventricular neurons did not demonstrate either PNMT or DBH immunoreactivity. The fibers present within the nucleus which contained these enzymes are considered to represent extrinsic afferent connections to neurons of the PVN. Tyrosine hydroxylase (TH)-immunoreactivity was found both in neurons and neuronal processes within the PVN. In TH-cells, the immunolabel was associated with rough endoplasmic reticulum, free ribosomes and 70-120 nm dense granules. Occasionally, nematosome-like bodies and cilia were observed in the TH-perikarya. Unlabeled axons established en passant and bouton terminaux type synapses with these TH-immunopositive cells. TH-immunoreactive axons terminated on cell bodies as well as somatic and dendritic spines of paraventricular parvocellular neurons. TH-containing axons were observed to deeply invaginate into both dendrites and perikarya of magnocellular neurons. These observations provide ultrastructural evidence for the participation of central catecholaminergic neuronal systems in the regulation of the different neuronal and neuroendocrine functions which have been related to hypothalamic paraventricular neurons.     

Innervation of somatostatin synthesizing neurons by adrenergic,phenylethanolamine-N-methyltransferase (PNMT)-immunoreactive axons in the anterior periventricular nucleus of the rat hypothalamus

Summary The adrenergic innervation of somatostatin synthesizing neurons located in the anterior region of the rat hypothalamic periventricular nucleus was studied by means of a light and electron microscopic immunocytochemical double labelling technique. This region which is the source of hypophysiotrophic somatostatin immunoreactive (IR) neurons also receives a dense plexus of adrenergic axons as determined by immunocytochemistry of phenylethanolamine-N-methyltransferase (PNMT), the marker enzyme for the central adrenergic system. The simultaneous detection of PNMT and somatostatin antigens in hypothalamic sections of colchicine pretreated animals revealed a congruency in the distribution of the labelled elements and also close juxtaposition of PNMT-IR axons to somatostatin producing neurons. At the ultrastructural level, axo-somatic and axo-dendritic synaptic connections were found between PNMT-containing axons and somatostatin expressing neurons. These morphological findings support the view that the central adrenergic system might influence the production and secretion of growth hormone in the pituitary gland by a direct monosynaptic interaction with somatostatin synthesizing neurons.     

Dopaminergic neurons in the brain and dopaminergic innervation of the albumen gland in mated and virgin helisoma duryi (mollusca: pulmonata)

Background

Dopamine was shown to stimulate the perivitelline fluid secretion by the albumen gland. Even though the albumen gland has been shown to contain catecholaminergic fibers and its innervation has been studied, the type of catecholamines, distribution of fibers and the precise source of this neural innervation has not yet been deduced. This study was designed to address these issues and examine the correlation between dopamine concentration and the sexual status of snails.

Results

Dopaminergic neurons were found in all ganglia except the pleural and right parietal, and their axons in all ganglia and major nerves of the brain. In the albumen gland dopaminergic axons formed a nerve tract in the central region, and a uniform net in other areas. Neuronal cell bodies were present in the vicinity of the axons. Dopamine was a major catecholamine in the brain and the albumen gland. No significant difference in dopamine quantity was found when the brain and the albumen gland of randomly mating, virgin and first time mated snails were compared.

Conclusions

Our results represent the first detailed studies regarding the catecholamine innervation and quantitation of neurotransmitters in the albumen gland. In this study we localized catecholaminergic neurons and axons in the albumen gland and the brain, identified these neurons and axons as dopaminergic, reported monoamines present in the albumen gland and the brain, and compared the dopamine content in the brain and the albumen gland of randomly mating, virgin and first time mated snails.     

Innervation of somatostatin synthesizing neurons by adrenergic, phenylethanolamine-N-methyltransferase (PNMT)-immunoreactive axons in the anterior periventricular nucleus of the rat hypothalamus

The adrenergic innervation of somatostatin synthesizing neurons located in the anterior region of the rat hypothalamic periventricular nucleus was studied by means of a light and electron microscopic immunocytochemical double labelling technique. This region which is the source of hypophysiotrophic somatostatin immunoreactive (IR) neurons also receives a dense plexus of adrenergic axons as determined by immunocytochemistry of phenylethanolamine-N-methyltransferase (PNMT), the marker enzyme for the central adrenergic system. The simultaneous detection of PNMT and somatostatin antigens in hypothalamic sections of colchicine pretreated animals revealed a congruency in the distribution of the labelled elements and also close juxtaposition of PNMT-IR axons to somatostatin producing neurons. At the ultrastructural level, axo-somatic and axo-dendritic synaptic connections were found between PNMT-containing axons and somatostatin expressing neurons. These morphological findings support the view that the central adrenergic system might influence the production and secretion of growth hormone in the pituitary gland by a direct monosynaptic interaction with somatostatin synthesizing neurons.     

The innervation of the renal cortex in the dog

Summary Two cytochemical techniques were used at the ultrastructural level to study the distribution of specific axon types to different intrarenal structures in the dog. Using the chromaffin reaction to distinguish catecholaminergic fibres from other axon populations, it was found that the renal cortex of the dog is supplied only by catecholaminergic nerves. Immunostaining for tyrosine hydroxylase (TH) labelled all of the intracortical nerves, and 20% to 25% of these profiles also contained dopa decarboxylase (DDC)-immunoreactivity, indicating they were dopaminergic rather than noradrenergic. Both DDC-positive and DDC-negative axons were seen in close association (80 nm) with blood vessels and juxtaglomerular cells as well as tubular epithelial cells. The distribution of TH- and DDC-immunoreactive nerves in the renal cortex is compatible with existing functional evidence indicating that both dopaminergic and noradrenergic nerves are involved in the regulation of renal blood flow, tubular reabsorption and renin release.     

Two patterns of dopa decarboxylase immunoreactivity in sympathetic axons supplying rat renal cortex.

Three ultrastructural cytochemical methods have been used to classify the innervation of the rat renal cortex. Every axon seen contained chromaffin-reactive, dense core vesicles and stained for tyrosine hydroxylase, indicating that they were all catecholaminergic. About 10% of the axons associated with smooth muscle and juxtaglomerular cells of the arteriolar vessels also contained dopa decarboxylase, but this enzyme was not present in any of the peritubular axons. Our results are compatible with the possibility that, in the rat, the juxtaglomerular blood vessels, but not the renal tubules, are supplied by dopaminergic as well as by noradrenergic nerves.     

Hypophysiotrophic thyrotropin releasing hormone (TRH) synthesizing neurons

Summary The neuropeptide thyrotropin releasing hormone (TRH) is capable of influencing both neuronal mechanisms in the brain and the activity of the pituitary-thyroid endocrine axis. By the use of immunocytochemical techniques, first the ultrastructural features of TRH-immunoreactive (IR) perikarya and neuronal processes were studied, and then the relationship between TRH-IR neuronal elements and dopamine--hydroxylase (DBH) or phenylethanolamine-N-methyltransferase (PNMT)-IR catecholaminergic axons was analyzed in the parvocellular subnuclei of the hypothalamic paraventricular nucleus (PVN). In control animals, only TRH-IR axons were detected and some of them seemed to follow the contour of immunonegative neurons. Colchicine treatment resulted in the appearance of TRH-IR material in parvocellular neurons of the PVN. At the ultrastructural level, immunolabel was associated with rough endoplasmic reticulum, free ribosomes and neurosecretory granules. Non-labelled axons formed synaptic specializations with both dendrites and perikarya of the TRH-synthesizing neurons. TRH-IR axons located in the parvo-cellular units of the PVN exhibited numerous intensely labelled dense-core and fewer small electron lucent vesicles. These axons were frequently observed to terminate on parvocellular neurons, forming both bouton- and en passant-type connections. The simultaneous light microscopic localization of DBH or PNMT-IR axons and TRH-synthesizing neurons demonstrated that catecholaminergic fibers established contacts with the dendrites and cell bodies of TRH-IR neurons. Ultrastructural analysis revealed the formation of asymmetric axo-somatic and axo-dendritic synaptic specializations between PNMT-immunopositive, adrenergic axons and TRH-IR neurons in the periventricular and medial parvocellular subnuclei of the PVN.These morphological data indicate that the hypophysiotrophic, thyrotropin releasing hormone synthesizing neurons of the PVN are directly influenced by the central epinephrine system and that TRH may act as a neurotransmitter or neuromodulator upon other paraventricular neurons.Supported by NIH research grants NS19266 and DK34540     

The adrenergic innervation of the renal artery and vein of the rat

Summary The adrenergic innervation of the extrarenal blood vessels of the rat left kidney was investigated by fluorescence histochemistry and by electron microscopy. The trunk of the renal artery proximal to the aorta is elastic and appears to be very sparsely innervated. In contrast, near the kidney the renal artery—which divides into 3 to 4 large branches of the muscular type possesses a dense adrenergic innervation. The adrenergic terminal axons are situated in the adventitia close to the external elastic lamella, but only rarely in close contact with smooth muscle cells. In most instances several terminal axons are grouped and enclosed by a Schwann cell, single axons being rare. All terminal axons are able to take up and to store 5-hydroxydopamine which strongly suggests that they are adrenergic. The innervation of the renal vein is more sparse than that of the muscular arteries but somewhat denser than that of the elastic artery. In addition, close to the origin of the renal artery the presence of small intensively fluorescent (SIF) cells as well as of some adrenergic ganglion cells is noted. The latter are situated in the adrenergic nonterminal axon bundles, which run parallel to the blood vessels.It is concluded that the uneven adrenergic innervation along the artery as well as individual variations in the branching of the artery are the main causes of the unusually high individual variations of the NA content of this organ such as used in pharmacological experiments.     

Catecholamine-synthesizing enzymes in the rat stomach

Local production of catecholamines in the stomach of the rat was studied by immunohistochemical demonstration of tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH) and phenylethanolamine-N-methyltransferase (PNMT), the enzymes catalyzing the formation of dopamine, noradrenaline and adrenaline, respectively. A rich innervation of TH- and DBH-immunoreactive nerve fibers was seen in the muscular layers and the myenteric plexus, in the submucosa and in the walls of submucosal blood vessels and in the lamina propria at the base of the epithelial layer. In addition, TH-, but not DBH-immunoreactive nerve fiber networks surrounding ganglion cells in the myenteric plexus were frequently observed, indicating dopaminergic preganglionic innervation of the myenteric plexus. In the oxyntic epithelium, single TH- and DBH-immunoreactive fibers extended in the strands of lamina propria as far as the middle portion of the gastric glands. A small population of single angulate cells in the oxyntic epithelium showed TH-, but not DBH-immunoreactivity. No specific PNMT immunoreactivity was observed.     

Studies on the acetylcholinesterase (ache)-positive and -negative autonomic axons supplying smooth muscle in the normal and 6-hydroxydopamine (6-OHDA)treated rat iris

Summary The adrenergic and cholinergic innervation to the rat iris has been studied at a light and electron microscopic level. Catecholamine fluorescence histochemistry showed adrenergic nerves to be present in both the dilatator and the constrictor pupillae regions. At a fine structural level the terminal innervation of the iris was studied and criteria for the differentiation between presumptive adrenergic and presumptive cholinergic axon terminals were examined. To aid this examination presumptive adrenergic axons were either labelled with the false adrenergic transmitter, 5-hydroxydopamine, or chemical sympathectomy performed using 6-hydroxydopamine. The value of using acetylcholinesterase staining as a marker for cholinergic nerve terminals was also studied.Results showed a mixed adrenergic/cholinergic innervation to the dilatator pupillae. In the constrictor pupillae an exclusively cholinergic innervation was found although adrenergic and cholinergic nerves were found supplying the blood vessels and at the dilatator-constrictor interface. These findings are discussed with regard to innervation-function relationships in the iris.     

Catecholamines and catecholamine-synthesizing enzymes in guinea-pig sensory ganglia

Summary Cranial and spinal sensory ganglia of the guinea-pig were investigated by means of histochemistry and biochemistry for the presence of catecholamines and catecholamine-synthesizing enzymes. Sensory neurons exhibiting immunoreactivity to the rate-limiting enzyme of catecholamine synthesis, tyrosine nydroxylase (TH), were detected by immunohistochemistry in lumbo-sacral dorsal root ganglia, the nodose ganglion and the petrosal/jugular ganglion complex. The carotid body was identified as a target of TH-like-immunoreactive (TH-LI) neurons by the use of combined retrograde tracing and immunohistochemistry. Double-labelling immunofluorescence revealed that most TH-LI neurons also contained somatostatin-LI, but TH-LI did not coexist with either calcitonin gene-related peptide- or substance P-LI. TH-LI neurons did not react with antibodies to other enzymes involved in catecholamine synthesis, i.e., aromatic amino acid decarboxylase (AADC), dopamine--hydroxylase (DH), and phenylethanolamine-N-methyltransferase (PNMT). Petrosal neurons as well as their endings in the carotid body lacked dopamine- and L-DOPA-LI. Sensory neurons did not display glyoxylic acid-induced catecholamine fluorescence. Ganglia containing TH-LI neurons were kept in short-term organ culture after crushing their roots and the exiting nerve in order to enrich intra-axonal transmitter content at the ganglionic side of the crush. However, even under these conditions, catecholamine fluorescence was not detected in axons projecting peripherally or centrally from the ganglia. Sympathetic noradrenergic nerves entered the ganglia and terminated within them. Accordingly, biochemical analyses of guinea-pig sensory ganglia revealed noradrenaline but no dopamine. In conclusion, catecholamines within guinea-pig sensory ganglia are confined to sympathetic nerves, which fulfill presently unknown functions. The TH-LI neurons themselves, however, lack any additional sign of catecholamine synthesis, and the presence of enzymatically active TH within these neurons is questionable.     

Association of dopaminergic fibers with corticotropin releasing hormone (CRH)-synthesizing neurons in the paraventricular nucleus of the rat hypothalamus

Catecholamines are known to exert a central influence on the hypothalamo-hypophyseal-adrenal neuroendocrine system. The selective dopaminergic innervation of the hypothalamic paraventricular nucleus (PVN) and putative relationships between dopaminergic fibers and corticotropin releasing hormone (CRH)-synthesizing neurons were studied in the male rat by means of immunocytochemistry following the elimination of noradrenergic and adrenergic inputs to the hypothalamus. A 3.0-mm-wide coronal cut was placed unilaterally in the brain at the rostral level of the mesencephalon. All neuronal structures from the cortex to the ventral surface of the brainstem, including the ascending catecholaminergic fiber bundles were transected. This surgical intervention resulted in the accumulation of dopamine-beta-hydroxylase (DBH)-immunoreactivity in axons proximal to the cut, and an almost complete disappearance of DBH activity in those located distal to the lesion. Two weeks following the operation, DBH immunoreactivity was significantly diminished in the PVN located on the side of lesion, while tyrosine hydroxylase (TH)-immunoreactivity was present in a substantial number of fibers in the same nucleus. Both DBH- and TH-immunoreactive axons were preserved in the contralateral PVN. Simultaneous immunocytochemical localization of either DBH- or TH-IR fibers and corticotropin releasing hormone-synthesizing neurons in the hypothalami from brainstem-lesioned, colchicine treated animals revealed that the distribution of catecholaminergic fibers and CRH neurons is homologous within the PVN of the intact side. Only a few scattered DBH-immunoreactive axons were detected among CRH-producing neurons in the PVN on the side of the lesion. In contrast, many tyrosine hydroxylase containing neurons and neuronal processes were observed on the lesioned side and the TH-IR fibers established juxtapositions with CRH-synthesizing neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Noradrenergic innervation of the heart in untreated and daunomycin-pretreated animals

Summary The innervation of guinea-pig and rat cardiac tissue consists of thin noradrenergic axons running parallel to the myocardial fibres and producing well-developed fluorescent networks which are denser in guinea-pig tissue. Transverse sections of arterial vessels exhibit a marked polar-like disposition of adrenergic nerves, which mainly appear to be concentrated in two opposite areas. Tissue sections from daunomycin-treated animals fluoresced a striking organge-red. This drug-specific fluorescence emanated from all parts of the ventricular tissue, with particular intensity in the nuclei. Neither the density nor the shape of the adrenergic nerves were affected by treatment with the drug. Despite penetration and storage of the antibiotic into the ventricular tissue, cardiotoxicity was not found in the present study.     

Extrinsic adrenergic innervation of the extrahepatic biliary duct system in guinea-pigs,cats and rhesus monkeys

Summary The localization of catecholamines has been investigated in the extrahepatic biliary duct system of cats, guinea-pigs and rhesus monkeys. In fluorimetric determinations noradrenaline was found to be the main primary catecholamine present in the biliary tract of rhesus monkeys. There exist regional differences in the noradrenaline content: Fairly low amounts were detected in the lower fundus of the gall-bladder (0.28 g/g). Increasing concentrations were measured in the corpus vesicae felleae (0.35 g/g), reaching a maximum level in the collum vesicae (0.49 g/g) and the ductus cysticus (0.50 g/g). The noradrenaline content of the choledochus and the choledocho-duodenal junction including Oddi's sphincter was much lower: 0,27 and 0,25 g/g respectively. The noradrenaline level in the small intestine of the rhesus monkey amounted to less than half the concentration found in the biliary ducts. Neither dopamine nor adrenaline have been detected. Fluorescence microscopical analysis reveals the presence of adrenergic nerves in the bile ducts which correspond to the measured noradrenaline concentrations: All parts of the biliary duct system in the different species investigated contain an elaborate perivascular adventitial plexus and adrenergic fibres confined to adventitial non-adrenergic ganglia. In guinea-pigs adrenergically innervated ganglia extend into the smooth muscle layer. The smooth muscle layer of the gall-bladder and the terminal choledochus in cats and rhesus monkeys is penetrated by a wide-meshed adrenergic ground plexus. This plexus was absent in guinea-pigs. The smooth musculature of the sphincter Oddi lacks a specialized adrenergic nerve supply in all species investigated. Finally, bound to the arterial vascular bed inside the propria in all parts of the biliary tract from all species investigated a prominent perivascular plexus is present. It is concluded that the smooth musculature of the gall-bladder and the terminal choledochus (the sphincter region excluded) in cats and monkeys receives 1. a direct sympathetic noradrenergic inhibitory innervation and 2. an indirect sympathetic noradrenergic inhibitory innervation which acts on intrinsic excitatory neurons and is present in all species investigated. The functional significance of the direct and indirect inhibitory innervation to the smooth musculature of the gall-bladder is discussed in detail.Dedicated to Professor Bengt Falck.Supported by the Deutsche Forschungsgemeinschaft and Joachim-Jungius-Gesellschaft zur Förderung der Wissenschaften, Hamburg.     

Innervation of regenerated spindles in muscle grafts of the rat

Summary Features of the nerve supply and the encapsulated fibers of muscle spindles were assessed in grafted and normal extensor digitorum longus (EDL) muscles of rats by analysis of serial 10-m frozen transverse sections stained for enzymes which delineated motor and sensory endings, oxidative capacity and muscle fiber type.The number of fibers was significantly more variable, and branched fibers were more frequently observed in regenerated spindles than in control spindles. Forty-eight percent of regenerated spindles received sensory innervation. Spindles reinnervated by afferents had a larger periaxial space than did spindles which were not reinnervated by afferents. Regenerated fibers innervated by afferents had small cross-sectional areas, equatorial regions with myofi-brils restricted to the periphery of fibers, unpredictable patterns of nonuniform and nonreversible staining along the length of the fiber for myofibrillar adenosine triphosphatase (mATPase) after acid and alkaline preincubation. In contrast, regenerated fibers devoid of sensory innervation resembled extrafusal fibers in that they usually exhibited myofibrils throughout the length of the fiber, no central aggregations of myonuclei, uniform staining for mATPase and a reversal of staining for mATPase after preincubation in an acid or alkaline medium. Approximately thirty percent of encapsulated fibers devoid of sensory innervation stained analogous to a type I extrafusal fiber, a pattern of staining never observed in intrafusal fibers of normal spindles. Groups of encapsulated fibers all exhibiting this pattern of staining reflect that either these fibers may have been innervated by collaterals of skeletomotor axons that originally innervated type I extrafusal fibers or that fibers innervated by only fusimotor neurons express patterns of staining for mATPase similar to extrafusal fibers in the absence of sensory innervation. Sensory innervation may also influence the reestablishment, of multiple sites of motor endings on regenerated intrafusal fibers. Those regenerated fibers innervated by afferents had more motor endings than did regenerated fibers devoid of sensory innervation.Differences in size, morphology, and patterns of staining for mATPase and numbers of motor endings between fibers innervated by afferents and fibers devoid of sensory innervation reflect that afferents can influence the differentiation of muscle cells and the reestablishment of motor innervation other than during the late prenatal/early postnatal period when muscle spindles form and differentiate in rats.     

Cellular localization of monoamines in the median eminence and the infundibular stem of some mammals

Summary A histochemical analysis of the monoamines which are strongly accumulated in the median eminence and the proximal part of infundibular stem of all species examined (mouse, rat, guinea pig, hamster, rabbit, and cat) was performed with the help of a highly specific and sensitive fluorescence method. Strong evidence was obtained for the view that the monoamines are localized in very high concentrations to the terminal parts of non-sympathetic nerve fibres, which — mainly at least — converge to the primary plexus of the hypophyseal portal system. The capillaries are densely and closely surrounded by the nerve fibres.Pharmacological experiments, involving the administration of reserpine, nialamide, m-tyrosine and -methyl-m-tyrosine, furnished good evidence for the view that primary catecholamines, probably mainly DA but also NA, are the predominant monoamines present. The experiments also revealed the existence of catecholamine-containing nerve cells in the arcuate nuclei and the ventral portion of the anterior periventricular nuclei. These nerve cells, situated in the regions where the tubero-infundibular tract arises, may be the cell bodies of adrenergic neurons to which the amine-containing nerve fibres at least partly belong.The findings indicate that primary catecholamines are released to the primary plexus of the hypophyseal portal system and thus transported to the anterior lobe. These amines may consequently act as neuro-humoral transmittors for the regulation of the activity of the anterior pituitary. — No direct adrenergic innervation of the cells in the pars tuberalis and anterior lobe was found. The portal vesstes in the pars tuberalis receive a very sparse adrenergic innervation and the vessels in the anterior lobe receive no or very few adrenergic nerves. Pars intermedia, on the other hand, may have a non-sympathetic adrenergic innervation.The Following Abbreviations are Used DA Dopamine - NA Noradrenaline - A Adrenaline - 5-HT 5-hydroxytryptamine For generous supplies of drugs we are indepted to Swedish Ciba, Stockholm (reserpine), and Swedish Pfizer, Stockholm (nialamide). The investigation was supported by research grants from the United States Public Health Service (NB 02854-03), the Swedish Medical Research Council, and the Therese and Johan Andersson Memorial Foundation. The excellent technical assistance of Miss M. Gustafsson is gratefully acknowledged.     

Hypophysiotrophic thyrotropin releasing hormone (TRH) synthesizing neurons. Ultrastructure, adrenergic innervation and putative transmitter action

The neuropeptide thyrotropin releasing hormone (TRH) is capable of influencing both neuronal mechanisms in the brain and the activity of the pituitary-thyroid endocrine axis. By the use of immunocytochemical techniques, first the ultrastructural features of TRH-immunoreactive (IR) perikarya and neuronal processes were studied, and then the relationship between TRH-IR neuronal elements and dopamine-beta-hydroxylase (DBH) or phenylethanolamine-N-methyltransferase (PNMT)-IR catecholaminergic axons was analyzed in the parvocellular subnuclei of the hypothalamic paraventricular nucleus (PVN). In control animals, only TRH-IR axons were detected and some of them seemed to follow the contour of immunonegative neurons. Colchicine treatment resulted in the appearance of TRH-IR material in parvocellular neurons of the PVN. At the ultrastructural level, immunolabel was associated with rough endoplasmic reticulum, free ribosomes and neurosecretory granules. Non-labelled axons formed synaptic specializations with both dendrites and perikarya of the TRH-synthesizing neurons. TRH-IR axons located in the parvocellular units of the PVN exhibited numerous intensely labelled dense-core and fewer small electron lucent vesicles. These axons were frequently observed to terminate on parvocellular neurons, forming both bouton- and en passant-type connections. The simultaneous light microscopic localization of DBH or PNMT-IR axons and TRH-synthesizing neurons demonstrated that catecholaminergic fibers established contacts with the dendrites and cell bodies of TRH-IR neurons. Ultrastructural analysis revealed the formation of asymmetric axo-somatic and axo-dendritic synaptic specializations between PNMT-immunopositive, adrenergic axons and TRH-IR neurons in the periventricular and medial parvocellular subnuclei of the PVN. These morphological data indicate that the hypophysiotrophic, thyrotropin releasing hormone synthesizing neurons of the PVN are directly influenced by the central epinephrine system and that TRH may act as a neurotransmitter or neuromodulator upon other paraventricular neurons.