Synaptic interaction of serotonergic axons and corticotropin releasing factor (CRF) synthesizing neurons in the hypothalamic paraventricular nucleus of the rat

Summary The morphological interrelationship between the central serotonergic and hypothalamic corticotropin-releasing factor (CRF) synthesizing systems was studied in the hypothalamic paraventricular nucleus (PVN) of colchicine pretreated male rats. The simultaneous immunocytochemical localization of the transmitter and peptide employed the peroxidase-antiperoxidase complex (PAP) technique using the silver-gold intensified (SGI) and non-intensified forms of the oxidized 3,3-diaminobenzidine (DAB) chromogen.The paraventricular nucleus received a moderate serotonergic innervation as compared with other diencephalic structures. The distribution and arborization of serotonergic axons were more prominent in the parvocellular subnuclei than in the magnocellular units of the nucleus. Serotonin containing axons formed terminal bouton and en passant type synapses with dendrites and somata of parvocellular neurons. The immunocytochemical double labelling technique revealed the overlapping of serotonergic axons and CRF-immunoreactive neurons. Vibratome (40 m) and semithin (1 m) sections indicated that the interneuronal communication may take place on both dendrites and cell bodies of CRF-immunoreactive neurons. Ultrastructural analysis demonstrated that serotonin-containing terminals formed axo-dendritic and axo-somatic synapses with CRF-immunoreactive neurons. These findings indicate that the central serotonergic neuronal system can influence the function of the pituitary-adrenal endocrine axis via a direct action upon the hypophysiotrophic CRF synthesizing neurons.Supported by NIH Grant NS19266     

Adrenergic innervation of corticotropin releasing factor (CRF)-synthesizing neurons in the hypothalamic paraventricular nucleus of the rat. A combined light and electron microscopic immunocytochemical study

Corticotropin releasing factor (CRF), a neuropeptide synthesized in the parvocellular subnuclei of the hypothalamic paraventricular nucleus (PVN), takes part in the regulation of different stress evoked responses of the organism. In order to elucidate the role of the central adrenergic system in the regulation of these CRF-synthesizing neurons, a novel ultrastructural immunocytochemical dual localization technique was utilized. Phenylethanolamine-N-methyltransferase (PNMT), a specific enzyme marker for the central adrenaline system, and CRF-immunoreactive elements were simultaneously visualized in hypothalamic sections. PNMT-immunoreactive axon terminals established synaptic connections with somata, dendrites and spinous structures of CRF-producing neurons. This morphological finding indicates that the central adrenergic system directly influences CRF-synthesizing neurons in the PVN and provides basis for a more definitive pharmacological manipulation of this system.     

Adrenergic innervation of corticotropin releasing factor (CRF) — synthesizing neurons in the hypothalamic paraventricular nucleus of the rat

Summary Corticotropin releasing factor (CRF), a neuropeptide synthesized in the parvocellular subnuclei of the hypothalamic paraventricular nucleus (PVN), takes part in the regulation of different stress evoked responses of the organism. In order to elucidate the role of the central adrenergic system in the regulation of these CRF-synthesizing neurons, a novel ultrastructural immunocytochemical dual localization technique was utilized. Phenylethanolamine-N-methyltransferase (PNMT), a specific enzyme marker for the central adrenaline system, and CRF-immunoreactive elements were simultaneously visualized in hypothalamic sections. PNMT-immunoreactive axon terminals established synaptic connections with somata, dendrites and spinous structures of CRF-producing neurons. This morphological finding indicates that the central adrenergic system directly influences CRF-synthesizing neurons in the PVN and provides basis for a more definitive pharmacological manipulation of this system.Supported by NIH grant NS19266     

Evidence for local corticotropin releasing factor (CRF)-immunoreactive neuronal circuits in the paraventricular nucleus of the rat hypothalamus. An electron microscopic immunohistochemical analysis

The interrelationships of corticotropin-releasing factor (CRF) immunoreactive neuronal cell bodies and processes have been examined in the paraventricular nucleus (PVN) of adrenalectomized-dexamethasone treated rats. Antisera generated against ovine CRF (oCRF) were used in the peroxidase-anti-peroxidase-complex (PAP)-immunocytochemical method at both the light and electron microscopic levels. In this experimental model, a great number of CRF-immunoreactive neurons were detected in the parvocellular subdivisions of the PVN and a few scattered labelled parvocellular neurons were also observed within the magnocellular subunits. Characteristic features of immunolabeled perikarya included hypertrophied rough endoplasmic reticulum with dilated endoplasmic cisternae, well developed Golgi complexes and increased numbers of neurosecretory granules. These features are interpreted to indicate accelerated hormone synthesis as a result of adrenalectomy. Afferent fibers communicated with dendrites and somata of CRF-immunoreactive neurons via both symmetrical and asymmetrical synapses. Some neurons exhibited somatic appendages and these structures were also observed to receive synaptic terminals. Within both the PVN and its adjacent neuropil, CRF-immunoreactive axons demonstrated varicosites which contained accumulations of densecore vesicles. CRF-containing axons were observed to branch into axon collaterals. These axons or axon collaterals established axo-somatic synapses on CRF-producing neurons in the parvocellular regions of the PVN, while in the magnocellular area of the nucleus they were found in juxtaposition with unlabeled magnocellular neuronal cell bodies or in synaptic contact with their dendrites. The presence of CRF-immunoreactive material in presynaptic structures suggests that the neurohormone may participate in mechanisms of synaptic transfer. These ultrastructural data indicate that the function of the paraventricular CRF-synthesizing neurons is adrenal steroid hormone dependent. They also provide morphological evidence for the existence of a neuronal ultrashort feed-back mechanism within the PVN for the regulation of CRF production and possibly that of other peptide hormones contained within this complex.     

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.     

Evidence for local corticotropin releasing factor (CRF)-immunoreactive neuronal circuits in the paraventricular nucleus of the rat hypothalamus

Summary The interrelationships of corticotropin-releasing factor (CRF) immunoreactive neuronal cell bodies and processes have been examined in the paraventricular nucleus (PVN) of adrenalectomized-dexamethesone treated rats. Antisera generated against ovine CRF (oCRF) were used in the peroxidase-anti-peroxidase-complex (PAP)-immunocytochemical method at both the light and electron microscopic levels. In this experimental model, a great number of CRF-immunoreactive neurons were detected in the parvocellular subdivisions of the PVN and a few scattered labelled parvocellular neurons were also observed within the magnocellular subunits. Characteristic features of immunolabeled perikarya included hypertrophied rough endoplasmic reticulum with dilated endoplasmic cisternae, well developed Golgi complexes and increased numbers of neurosecretory granules. These features are interpreted to indicate accelerated hormone synthesis as a result of adrenalectomy. Afferent fibers communicated with dendrites and somata of CRF-immunoreactive neurons via both symmetrical and asymmetrical synapses. Some neurons exhibited somatic appendages and these structures were also observed to receive synaptic terminals. Within both the PVN and its adjacent neuropil, CRF-immunoreactive axons demonstrated varicosites which contained accumulations of densecore vesicles. CRF-containing axons were observed to branch into axon collaterals. These axons or axon collaterals established axo-somatic synapses on CRF-producing neurons in the parvocellular regions of the PVN, while in the magnocellular area of the nucleus they were found in juxtaposition with unlabeled magnocellular neuronal cell bodies or in synaptic contact with their dendrites. The presence of CRF-immunoreactive material in presynaptic structures suggests that the neurohormone may participate in mechanisms of synaptic transfer.These ultrastructural data indicate that the function of the paraventricular CRF-synthesizing neurons is adrenal steroid hormone dependent. They also provide morphological evidence for the existence of a neuronal ultrashort feedback mechanism within the PVN for the regulation of CRF production and possibly that of other peptide hormones contained within this complex.Supported by NIH grant NS 19266 to WKP     

Neuropeptide-Y and ACTH-immunoreactive innervation of corticotropin releasing factor (CRF)-synthesizing neurons in the hypothalamus of the rat. An immunocytochemical analysis at the light and electron microscopic levels

Corticotropin releasing factor (CRF), synthesized in neurons of the hypothalamic paraventricular nucleus (PVN), is one of the main regulators of the pituitary-adrenal cortex endocrine axis. In order to elucidate the possible involvement of the central neuropeptide-Y (NPY)- and adrenocorticotroph hormone (ACTH)-immunoreactive (IR) systems in the innervation of hypophysiotrophic CRF-synthesizing neurons, immunocytochemical double labelling studies were conducted in the hypothalamus of the rat to localize CRF-synthesizing neurons, as well as neuronal fibers exhibiting NPY and ACTH-immunoreactivity, respectively. The parvocellular subnuclei of the PVN received an intense NPY- and ACTH-IR innervation. At the light microscopic level, these peptidergic axons were associated with the dendrites and perikarya of CRF-IR neurons. Ultrastructural analysis revealed that NPY- and ACTH-IR axons established synaptic specializations with parvocellular neurons expressing CRF-immunoreactivity. These findings indicate that both neuropeptide-Y and adrenocorticotroph hormone containing neuronal systems of the brain are capable of influencing adrenal function via synaptic interactions with hypophysiotrophic CRF-synthesizing neurons. The data also support the concept that NPY and ACTH might be utilized as neuromodulators within the PVN.     

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-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     

Neuropeptide-Y and ACTH-immunoreactive innervation of corticotropin releasing factor (CRF)-synthesizing neurons in the hypothalamus of the rat

Summary Corticotropin releasing factor (CRF), synthesized in neurons of the hypothalamic paraventricular nucleus (PVN), is one of the main regulators of the pituitaryadrenal cortex endocrine axis. In order to elucidate the possible involvement of the central neuropeptide-Y (NPY)-and adrenocorticotroph hormone (ACTH)-immunoreactive (IR) systems in the innervation of hypophysiotrophic CRF-synthesizing neurons, immunocytochemical double labelling studies were conducted in the hypothalamus of the rat to localize CRF-synthesizing neurons, as well as neuronal fibers exhibiting NPY and ACTH-immunoreactivity, respectively.The parvocellular subnuclei of the PVN received an intense NPY-and ACTH-IR innervation. At the light microscopic level, these peptidergic axons were associated with the dendrites and perikarya of CRF-IR neurons. Ultrastructural analysis revealed that NPY- and ACTH-IR axons established synaptic specializations with parvocellular neurons expressing CRF-immunoreactivity. These findings indicate that both neuropeptide-Y and adrenocorticotroph hormone containing neuronal systems of the brain are capable of influencing adrenal function via synaptic interactions with hypophysiotrophic CRF-synthesizing neurons. The data also support the concept that NPY and ACTH might be ntilized as neuromodulators within the PVN.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Synaptic communication between somatostatinergic axons and growth hormone-releasing factor (GRF) synthesizing neurons in the arcuate nucleus of the rat

Growth hormone (GH) production of the anterior pituitary gland is controlled by inhibiting and releasing hormones that are synthesized in the diencephalon. In order to elucidate the possible interrelationships between somatostatin and growth hormone-releasing factor (GRF) synthesizing neurons at the hypothalamic level, immunocytochemical double labelling studies were performed on sections containing the arcuate nucleus (ARC) of the rat. Somatostatin producing neurons were located in the dorsomedial part of the ARC, while somatostatin immunoreactive (IR) axons were found in the ventro-lateral part of the nucleus, an area containing GRF-synthesizing cells. The use of the dual antigen localization technique revealed the approach and juxtaposition of somatostatin containing axons to dendrites and cell bodies of GRF-synthesizing neurons. At the light microscopic level, several somatostatinergic axon varicosities were clustered around single GRF-synthesizing cells. Ultrastructural analysis of the ventro-lateral part of the ARC showed that (i), somatostatinergic axons established synaptic connections (ii), GRF-producing neurons received axons terminals on their somata and dendrites and (iii), somatostatin-IR axons formed asymmetric synaptic specializations with both dendrites and somata of GRF-synthesizing neurons. These morphological findings indicate that the hormone production and release of hypophysiotrophic GRF-IR neurons can be influenced by the central somatostatin system via direct synaptic mechanisms. The data support the concept, that the interaction of inhibiting and releasing hormones, which determines responses of the pituitary target cells, may take place also at the hypothalamic level.     

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     

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.     

Synaptic communication between somatostatinergic axons and growth hormone-releasing factor (GRF) synthesizing neurons in the arcuate nucleus of the rat

Summary Growth hormone (GH) production of the anterior pituitary gland is controlled by inhibiting and releasing hormones that are synthesized in the diencephalon. In order to elucidate the possible interrelationships between somatostatin and growth hormone-releasing factor (GRF) synthesizing neurons at the hypothalamic level, immunocytochemical double labelling studies were performed on sections containing the arcuate nucleus (ARC) of the rat. Somatostatin producing neurons were located in the dorsomedial part of the ARC, while somatostatin immunoreactive (IR) axons were found in the ventro-lateral part of the nucleus, an area containing GRF-synthesizing cells. The use of the dual antigen localization technique revealed the approach and juxtaposition of somatostatin containing axons to dendrites and cell bodies of GRF-synthesizing neurons. At the light microscopic level, several somatostatinergic axon varicosities were clustered around single GRF-synthesizing cells. Ultrastructural analysis of the ventrolateral part of the ARC showed that (i), somatostatinergic axons established synaptic connections (ii), GRF-producing neurons received axons terminals on their somata and dendrites and (iii), somatostatin-IR axons formed asymmetric synaptic specializations with both dendrites and somata of GRF-synthesizing neurons.These morphological findings indicate that the hormone preduction and release of hypophysiotrophic GRF-IR neurons can be influenced by the central somatostatin system via direct synaptic mechanisms. The data support the concent that the interaction of inhibiting and releasing hormones, which determines responses of the pituitary target cells, may take place also at the hypothalamic level.Supported by grants from the National Institute of Health (NIH NS 19266), the National Science Foundation (INT 8703030, 8602688), the Hungarian Academy of Sciences (OTKA 104) and the National Foundation of Technical Development (OKKFT Tt 286/1986)     

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.     

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.     

Ultrastructural alterations of the paraventriculo-infundibular Corticotropin Releasing Factor (CRF)-immunoreactive neuronal system in long term adrenalectomized rats

The corticotropin releasing factor (CRF)-immunoreactive paraventriculo-infundibular neuronal system of long-term adrenalectomized and adrenalectomized-short term dexamethasone treated rats was analyzed at the ultrastructural level using the preembedding peroxidase anti-peroxidase complex (PAP)-immunohistological method. In both groups of animals, parvocellular neurons located in the medial and dorsal subnuclei of the paraventricular nucleus (PVN) showed CRF-like immunoreactivity. The perikarya contained hypertrophied rough endoplasmic reticulum (rER) with dilated cisternae, active Golgi-complexes and numerous neurosecretory granules. The majority of the neurosecretory granules measured 80–120 nm. Dendrites of CRF-immunoreactive neurons contained labeled vesicles, secretory granules, bundles of microtubules, a well-developed smooth endoplasmic reticulum (sER) complex and free ribosomes. Unlabeled terminal boutons of axons were observed to synapse on dendrites and somata of CRF-neurons. In addition, CRF perikarya were found in direct somato-somatic apposition with both CRF-immunopositive and immunonegative parvocellular cells. Retraction of glial processes and the existence of puncta adherentia between the cell membranes characterized these appositions. Varicose CRF axons within the median eminence contained hypertrophied sER, labeled vesicles and neurosecretory granules. The preterminal portions of the CRF-axons were dilated and possessed many labeled 80–120 nm diameter granules. CRF-terminals were greatly enlarged and established direct neurohemal contacts with the external limiting basal lamina of portal vessels without the interposition of tanycytic ependymal foot-processes. These tanycytes were not CRF immunopositive. CRF positive terminals contained clusters of microvesicles, labeled small vesicles and multivesicular bodies, but fewer granular elements than were observed within the preterminals. Many of the labeled organelles were attached to tubules of sER. Occasionally, CRF-axons were observed within the pericapillary space adjacent to portal vessels. The ultrastructural features of CRF-neurons, obtained from adrenalectomized and adrenalectomized plus short-term dexamethasone treated rats did not differ significantly from each other. The hormone content of the entire CRF-neuron was greater in the steroid treated group. Adrenocorticotrophic hormone (ACTH) synthesizing cells in the pars distalis of adrenalectomized-dexamethasone treated rats also showed increased numbers of immunopositive secretory granules (150–320 nm in diameter). These ultrastructural morphological results provide evidence that the function of the paraventriculo-infundibular CRF-system is adrenal steroid hormone dependent and suggest the participation of glial and ependymal elements in the regulation of the system in this hyperfunctional state. The observed membrane specializations are indicative of ephaptic interactions between CRF-neurons and may serve a synchronizing function in adrenalectomized animals.     

GABA-immunohistological observations, at the electron-microscopical level, of the neurons of isthmic nuclei in chicken, Gallus domesticus

Following a demonstration of Golgi-impregnated neurons and their terminal axon arborization in the optic tectum, the neurons of the nucleus parvocellularis and magnocellularis isthmi were studied by means of postembedded electron-microscopical (EM) γ-aminobutyric acid (GABA)-immunogold staining. In the parvocellular nucleus, none of the neuronal cell bodies or dendrites displayed GABA-like immunoreactivity in EM preparations stained by postembedded GABA-immunogold. However, numerous GABA-like immunoreactive and also unlabeled terminals established synapses with GABA-negative neurons. GABA-like immunoreactive terminals were usually found at the dendritic origin. Around the dendritic profiles, isolated synapses of both GABA-like immunoreactive and immunonegative terminals established glomerulus-like structures enclosed by glial processes. All giant and large neurons of the magnocellular nucleus of the isthmi displayed GABA-like immunoreactivity. Their cell surface was completely covered by GABA-like immunoreactive and unlabeled terminals that established synapses with the neurons. These neurons are thought to send axon collaterals to the parvocellular nucleus; their axons enter the tectum opticum. The morphological characteristics of neurons of both isthmic nuclei are like those of interneurons, because of their numerous axosomatic synapses with both asymmetrical and symmetrical features. These neurons are not located among their target neurons and exert their modulatory effect on optic transmission in the optic tectum at a distance.     

A combined light and electron microscopic immunocytochemical method for the simultaneous localization of multiple tissue antigens. Tyrosine hydroxylase immunoreactive innervation of corticotropin releasing factor synthesizing neurons in the paraventricular nucleus of the rat

In order to study the morphological interrelationships between immunocytochemically identified neuronal systems, a double labelling procedure - suitable for correlative light and electron microscopic observations - is introduced. The technique is based on the consecutive use of the silver-gold (SG) intensified and non-intensified forms of the oxidized 3,3'-diaminobenzidine (DAB) chromogen in the framework of the peroxidase-antiperoxidase complex (PAP) indirect immunocytochemical procedure. The first tissue antigen is detected by the SG intensified DAB chromogen, which has a black color and high electron density. The structures containing the second antigen are visualized by the non-intensified DAB-endproduct, which is less electron-dense than the silver-gold amplified form and is brown. The metallic shield that forms around the labeled antibody sequences associated with the first antigen prevents non-specific binding of immunoglobulins used for the detection of the second tissue antigen. The application of this method for the simultaneous detection of tyrosine hydroxylase (TH)- and corticotropin releasing factor (CRF)-immunoreactive structures revealed that black colored TH-immunopositive fibers contacted brown colored CRF-synthesizing neurons in the hypothalamic paraventricular nucleus. The juxtaposition of TH- and CRF-containing elements was apparent in both thick vibratome (40 micron) and semithin (1 micron) sections. At the ultrastructural level, TH-positive terminals - labeled by silver-gold grains - were observed to establish asymmetric synapses with both CRF- and TH-immunoreactive neurons. The former finding indicates a direct, TH-immunopositive, catecholaminergic influence upon the hypothalamic CRF system, while the latter demonstrates the existence of intrinsic connections between TH-positive elements.     

Ultrastructural localization of glucocorticoid receptor (GR) in hypothalamic paraventricular neurons synthesizing corticotropin releasing factor (CRF)

Summary Corticotropin releasing factor (CRF) synthesizing neurons, located in the hypothalamic paraventricular nucleus (PVN), are the main central regulators of the pituitary-adrenal cortex endocrine axis. The hormone production and release of CRF-synthesizing neurons is regulated by neuronal messages and feedback action(s) of glucocorticoids secreted by the adrenal gland. In order to characterize the latter mechanism, glucocorticoid receptor (GR)-immunoreactive (IR) sites were studied in hypothalamic paraventricular neurons of intact, long-term adrenalectomized, and adrenalectomized plus glucocorticoid treated animals, by means of ultrastructural immunocytochemical labelling. In intact animals, glucocorticoid receptor immunoreactivity was found predominantly in the nuclei of parvocellular neurons. Following adrenalectomy GR-immunoreactivity was localized in the cytoplasm of the cells, and there was a concomitant disappearance of the label from the nuclei. After corticosterone administration to adrenalectomized animals, GR-IR sites were again concentrated within the cell nuclei. Immunocytochemical double labelling studies performed on adrenalectomized plus corticosterone-replaced animals demonstrated glucocorticoid receptor-IR sites in the cell nuclei of parvocellular paraventricular neurons that expressed CRF-immunoreactivity in their cytoplasm.These ultrastructural data indicate that the intracellular location of glucocorticoid receptor is dependent on the availability of glucocorticoids by the neurons. The simultaneous expression of GR- and CRF-immunoreactivity in parvocellular paraventricular neurons supports the concept of a direct feedback action of glucocorticoids upon CRF-synthesizing neurons.Supported by NIH Research Grants NS19266 (W.K.P. and Zs.L.), NS20832 (M.C.B.) and a joint grant (INT-8703030) awarded by the National Science Foundation and the Hungarian Academy of Sciences (Zs.L. and W.K.P.). R.M.U. is a recipient of NIMH Pre-doctoral Fellowship and M.C.B. an NIH Research Carcer Development Award