Neuropeptide FF (NPFF) control of magnocellular neurosecretory cells of the rat hypothalamic paraventricular nucleus (PVN)

Neuropeptide FF (NPFF) is an octapeptide belonging to an extended family of RF amide peptides that have been implicated in a wide variety of physiological functions in the brain. NPFF and its receptors are abundantly expressed in the rat brain and spinal cord including the hypothalamic paraventricular nucleus (PVN), an autonomic nucleus critical for the secretion of neurohormones and the regulation of sympathetic outflow. In this study, we sought to examine the effects of NPFF on GABAergic inhibitory synaptic input to magnocellular neurosecretory cells (MNCs) of the PVN, which secrete the neurohormones, vasopressin and oxytocin from their terminals in the neurohypophysis. Whole cell patch clamp recordings under voltage clamp conditions were performed from PVN MNCs in the brain slice. Bicuculline-sensitive inhibitory postsynaptic currents (IPSCs) were isolated in the presence of glutamate receptor blockers. In tetrodotoxin, NPFF (5 microM) caused an increase in frequency, but not amplitude of miniature inhibitory postsynaptic currents (mIPSCs) in MNCs indicating a presynaptic locus of action for this peptide. Intracerebroventricular application of NPFF resulted in an activation of GABAergic neurons located adjacent to the PVN as revealed by immunohistochemistry for Fos protein and in situ hybridization for glutamic acid decarboxylase (GAD67) mRNA. Based on these observations we conclude that NPFF facilitates inhibitory input to MNCs of the PVN via GABAergic interneurons located in immediate vicinity of the nucleus. These findings provide a cellular and anatomic basis for the NPFF-induced inhibition of vasopressin release has been reported consequent to hypovolemia and hyperosmolar stimulation.     

Circadian control of the daily plasma glucose rhythm: an interplay of GABA and glutamate

The mammalian biological clock, located in the hypothalamic suprachiasmatic nuclei (SCN), imposes its temporal structure on the organism via neural and endocrine outputs. To further investigate SCN control of the autonomic nervous system we focused in the present study on the daily rhythm in plasma glucose concentrations. The hypothalamic paraventricular nucleus (PVN) is an important target area of biological clock output and harbors the pre-autonomic neurons that control peripheral sympathetic and parasympathetic activity. Using local administration of GABA and glutamate receptor (ant)agonists in the PVN at different times of the light/dark-cycle we investigated whether daily changes in the activity of autonomic nervous system contribute to the control of plasma glucose and plasma insulin concentrations. Activation of neuronal activity in the PVN of non-feeding animals, either by administering a glutamatergic agonist or a GABAergic antagonist, induced hyperglycemia. The effect of the GABA-antagonist was time dependent, causing increased plasma glucose concentrations only when administered during the light period. The absence of a hyperglycemic effect of the GABA-antagonist in SCN-ablated animals provided further evidence for a daily change in GABAergic input from the SCN to the PVN. On the other hand, feeding-induced plasma glucose and insulin responses were suppressed by inhibition of PVN neuronal activity only during the dark period. These results indicate that the pre-autonomic neurons in the PVN are controlled by an interplay of inhibitory and excitatory inputs. Liver-dedicated sympathetic pre-autonomic neurons (responsible for hepatic glucose production) and pancreas-dedicated pre-autonomic parasympathetic neurons (responsible for insulin release) are controlled by inhibitory GABAergic contacts that are mainly active during the light period. Both sympathetic and parasympathetic pre-autonomic PVN neurons also receive excitatory inputs, either from the biological clock (sympathetic pre-autonomic neurons) or from non-clock areas (para-sympathetic pre-autonomic neurons), but the timing information is mainly provided by the GABAergic outputs of the biological clock.     

Autonomic and neuroendocrine actions of adrenomedullin in the brain: mechanisms for homeostasis

In addition to its role as a potent vasodilator, adrenomedullin (ADM) affects an animal's physiological status through its effects in the brain. We have shown that circulating ADM activates neurons, including nitric oxide (NO)-producing neurons, in autonomic centers of the brain such as the hypothalamic paraventricular nucleus (PVN). Systemic ADM gains access to the brain through the area postrema (AP), a brainstem circumventricular organ, and the PVN is a major target of these ADM-sensitive AP neurons. Neurons expressing the preproADM (ppADM) gene are distributed throughout the brain, with high levels in autonomic centers. Lipopolysaccharide (LPS, immune stress), restraint (psychological stress), and 24 h dehydration all down-regulate ppADM gene expression in different subsets of autonomic centers. Receptor-activity-modifying protein (RAMP) 2 and RAMP3, ADM receptor subunits, are expressed in autonomic centers including the PVN and supraoptic nucleus. Intracerebroventricular injections of ADM increase arterial pressure, heart rate, tyrosine hydroxylase mRNA levels in the locus coeruleus, plasma levels of ACTH, and NO production in the hypothalamus. ADM excites putative GABAergic and cholinergic neurons in dissociated cells from a basal forebrain integrative center, the diagonal band of Broca. These results demonstrate that the signalling components necessary for ADM to influence physiological systems are present in the brain and that ADM is an important transmitter of brain autonomic pathways which are involved in regulating homeostatic balance.     

Neuronal organization of the avian paraventricular nucleus: intrinsic, afferent, and efferent connections

The heterogeneous paraventricular nucleus (PVN) of birds offers favorable conditions for the analysis of intrinsic, afferent, and efferent connections of neuroendocrine systems. Paraventricular neurons are successfully impregnated with the Golgi-technique. The findings indicate a direct influence of the cerebrospinal fluid (CSF) on the magnocellular neurons that, via their axon terminals in the neural lobe of the pituitary, are also exposed to the hemal milieu. The magnocellular neurons are intermingled with parvocellular elements which may represent local interneurons. A group of parvocellular nerve cells is identified as CSF-contacting neurons. This type of cell forms a basic morphologic component of the avian neuroendocrine apparatus. Immunocytochemical and ultrastructural studies further support the concept of neuronal interactions between parvocellular and magnocellular elements. Moreover, these findings speak in favor of the existence of recurrent collaterals of the magnocellular neurons. Nerve cells giving rise to afferent connections to the PVN are located in the limbic system and autonomic areas of the upper and lower brainstem. Further afferents may originate from the subfornical organ, the organon vasculosum laminae terminalis, the ventral tegmentum, and the area postrema. Via efferent projections, the PVN is connected to the nucleus accumbens, lateral septum, several hypothalamic nuclei, the neural lobe of the pituitary, the organon vasculosum laminae terminalis, the subfornical organ, the pineal organ, the area postrema, the lateral habenular complex, and various autonomic areas of the reticular formation in the upper and lower brainstem and the spinal cord. In conclusion, the PVN may be regarded as an integral component of the neuroendocrine apparatus reciprocally coupled to the limbic system, several circumventricular organs, and various autonomic centers of the brain.     

Adrenomedullin influences magnocellular and parvocellular neurons of paraventricular nucleus via separate mechanisms

We previously reported that adrenomedullin (AM) decreases blood pressure following microinjection into the paraventricular nucleus of the hypothalamus (PVN) of the rat. With the use of whole cell recordings in rat hypothalamic slice preparations, we characterized the effects of AM on electrophysiologically identified PVN neurons and described the membrane events underlying such actions. AM hyperpolarized magnocellular (type I) neurons in a dose-dependent manner, a response associated with an increase in the frequency and amplitude of inhibitory postsynaptic potentials. Blockade of action potentials with tetrodotoxin (TTX) abolished AM effects on membrane potential and synaptic activity in magnocellular neurons, suggesting direct actions on inhibitory interneurons. Furthermore, blockade of inhibitory synaptic transmission with the GABA(A) receptor antagonist bicuculline methiodide also abolished AM effects on membrane potential in magnocellular neurons. In contrast, parvocellular (type II) neurons depolarized following AM receptor activation. AM effects on parvocellular neurons were dose dependent and were maintained in the presence of TTX, indicating direct effects on this population of neurons. Voltage-clamp recordings from parvocellular neurons showed AM enhances a nonselective cationic conductance, suggesting a potential mechanism through which AM influences membrane potential. These observations show clear population-specific actions of AM on separate identified groups of PVN neurons. Such effects on magnocellular neurons likely contribute to the hypotensive actions of this peptide in PVN. Although the effects on parvocellular neurons may also contribute to such cardiovascular effects of AM, it is more likely that actions on this population of PVN neurons underlie the previously demonstrated activational effects of AM on the hypothalamic-pituitary-adrenal axis.     

Coexistence of CRF peptide and oxytocin mRNA in the paraventricular nucleus

Several studies have reported coexistences of peptides in parvocellular neurons of the paraventricular nucleus (PVN). However, the coexistence of peptides in the magnocellular PVN is less clear. Controversy exists in particular about the coexistence of corticotropin-releasing factor (CRF) and oxytocin (OX). Although these peptides are present in distinct areas of the PVN, some overlap may exist. This study investigated a potential coexistence of OX and CRF in magno- and parvocellular PVN. The data demonstrate with clarity that neurons containing both the mRNA for OX and the peptide CRF are present in subpopulations of magnocellular and parvocellular neurons of the PVN.     

Fos,RVLM-projecting neurons,and spinally projecting neurons in the PVN following hypertonic saline infusion

Hypertonic saline (HTS; 1.7 M) infused intravenously into conscious rats increases the production of Fos, a marker of cell activation, in the hypothalamic paraventricular nucleus (PVN). The parvocellular PVN contains subpopulations of neurons. However, which subpopulations are activated by HTS is unknown. We determined whether PVN neurons that innervate the rostral ventrolateral medulla (RVLM) or the spinal cord (important autonomic sites) expressed Fos following HTS. Experiments were performed 24-96 h after chronic implantation of an intravenous cannula. HTS significantly increased the number of Fos-positive cells. In the parvocellular PVN, the maximum number of Fos-positive cells occurred rostral of the anterior-posterior level at which the number of neurons that projected to the medulla or spinal cord peaked. Compared with controls, HTS did not significantly increase the number of double-labeled neurons. These findings demonstrate that an elevation in plasma osmolality activates PVN neurons but not the subgroups of PVN neurons with projections to the RVLM or to the spinal cord.     

Neurocircuitry of stress integration: anatomical pathways regulating the hypothalamo-pituitary-adrenocortical axis of the rat

The hypothalamo-pituitary-adrenocortical (HPA) axis is recruitedby the organism in response to real or perceived threats tohomeostasis ("stress"). Regulation of this neuroendocrine systemis accomplished by modulation of secretory tone in hypophysiotrophicneurons of the medial parvocellular paraventricular nucleus.Excitation of these neurons is mediated by several sources:direct (and perhaps indirect) inputs from brainstem neuronsregulating autonomic tone/arousal; circumventricular organsmonitoring blood and CSF constituents; and local-circuit neuronswithin the hypothalamus and basal forebrain. The latter arepredominantly GABAergic; notably, these areas are targets fordescending GABAergic input from limbic structures, and may promotePVN secretory activity via disinhibition. Neurosecretory paraventricularnucleus neurons are inhibited by glucocorticoid–dependentand –independent mechanisms. Glucocorticoid negative feedbackappears to act both locally and in extrahypothalamic loci, andis likely integrated in a region- and stressor-specific manner.Inhibitory input to the medial parvocellular paraventricularnucleus emanate predominantly from the bed nucleus of the striaterminalis and hypothalamus, and are likely regulated by neuroendocrinehomeostats. Descending limbic inhibitory information appearsto act through excitation of these inhibitory inputs. Overall,integration of stressful information is a multi-faceted processintegrating prior experience and real or anticipated homeostaticdisruption into appropriate activation and deactivation of thehypothalamo-pituitary-adrenocortical axis.     

Anatomical evidence for interaction of ACTH1-39 immunostained fibers and hypothalamic paraventricular neurons that project to the dorsal vagal complex

Anatomical evidence is presented for an interaction of ACTH1-39 immunostained fibers and a specific population of hypothalamic paraventricular (PVN) neurons; these neurons project to the dorsal vagal complex (DVC) of brainstem medulla. Bilateral injection of 10% HRP-WGA into DVC is incorporated into nerve terminals and transported retrogradely to cell bodies in the parvocellular subdivision of PVN, as revealed by standard HRP-WGA histochemistry or antibody to wheatgerm agglutinin followed by immunocytochemical techniques. Labeled cells are localized predominantly in the ventral portion of the caudal medial parvocellular subdivision and ventrolaterally in the posterior subnucleus of PVN. Few labeled cells are seen in the anterior parvocellular PVN, rostrally in the medial parvocellular component and in the dorsal cap. HRP-WGA cells are rarely observed in the magnocellular divisions of PVN. Dual-staining immunocytochemical-retrograde tracing techniques in the same tissue section demonstrate ACTH1-39 fibers in intimate anatomical proximity to parvocellular PVN neurons that project to DVC. It is suggested that this interaction may partially account for the known cardiovascular effects of opiocortins and supports the role of the paraventricular nucleus in hypothalamic integration and modulation of cardiovascular control.     

Identification and characterization of novel mammalian neuropeptide FF-like peptides that attenuate morphine-induced antinociception

The two mammalian neuropeptides NPFF and NPAF have been shown to have important roles in nociception, anxiety, learning and memory, and cardiovascular reflex. Two receptors (FF1 and FF2) have been molecularly identified for NPFF and NPAF. We have now characterized a novel gene designated NPVF that encodes two neuropeptides highly similar to NPFF. NPVF mRNA was detected specifically in a region between the dorsomedial and ventromedial hypothalamic nuclei. NPVF-derived peptides displayed higher affinity for FF1 than NPFF-derived peptides, but showed poor agonist activity for FF2. Following intracerebral ventricular administration, a NPVF-derived peptide blocked morphine-induced analgesia more potently than NPFF in both acute and inflammatory models of pain. In situ hybridization analysis revealed distinct expression patterns of FF1 and FF2 in the rat central nervous system. FF1 was broadly distributed, with the highest levels found in specific regions of the limbic system and the brainstem where NPVF-producing neurons were shown to project. FF2, in contrast, was mostly expressed in the spinal cord and some regions of the thalamus. These results indicate that the endogenous ligands for FF1 and FF2 are NPVF- and NPFF-derived peptides, respectively, and suggest that the NPVF/FF1 system may be an important part of endogenous anti-opioid mechanism.     

Heterogeneous distribution of basal cyclic guanosine monophosphate within distinct neuronal populations in the hypothalamic paraventricular nucleus

The supraoptic (SON) and the paraventricular (PVN) hypothalamic nuclei constitute major neuronal substrates underlying nitric oxide (NO) effects on autonomic and neuroendocrine control. Within these nuclei, constitutively produced NO restrains the firing activity of magnocellular neurosecretory and preautonomic neurons, actions thought to be mediated by a cGMP-dependent enhancement of GABAergic inhibitory transmission. In the present study, we expanded on this knowledge by performing a detailed anatomical characterization of constitutive NO-receptive, cGMP-producing neurons within the PVN. To this end, we combined tract-tracing techniques and immunohistochemistry to visualize cGMP immunoreactivity within functionally, neurochemically, and topographically discrete PVN neuronal populations in Wistar rats. Basal cGMP immunoreactivity was readily observed in the PVN, both in neuronal and vascular profiles. The incidence of cGMP immunoreactivity was significantly higher in magnocellular (69%) compared with preautonomic ( approximately 10%) neuronal populations (P < 0.01). No differences were observed between oxytocin (OT) and vasopressin (VP) magnocellular neurons. In preautonomic neurons, the incidence of cGMP was independent of their subnuclei distribution, innervated target (i.e., intermediolateral cell column, nucleus tractus solitarii, or rostral ventrolateral medulla) or their neurochemical phenotype (i.e., OT or VP). Finally, high levels of cGMP immunoreactivity were observed in GABAergic somata and terminals within the PVN of eGFP-GAD67 transgenic mice. Altogether, these data support a highly heterogeneous distribution of basal cGMP levels within the PVN and further support the notion that constitutive NO actions in the PVN involve intricate cell-cell interactions, as well as heterogeneous signaling modalities.     

Neuropeptide FF receptors exert contractile activity via inhibition of nitric oxide release in the mouse distal colon

Neuropeptide FF (NPFF) and NPVF, two closely NPFF related peptides, have different affinities for the two NPFF receptors (NPFF1 and NPFF2). To assess the peripheral effects of NPFF receptors in the gastrointestinal tract motility, NPFF and NPVF were tested in the mouse isolated distal colon. Both NPFF (1-15 microM) and NPVF (1-15 microM) dose-dependently caused significant colonic contractions. Pre-treatment with the putative NPFF antagonist, BIBP3226 (30 microM) abolished the contractile responses to the two neuropeptides (3 microM). They had no additional contractile activities in colonic preparations contracted by Nomega-nitro-L-arginine (30 microM). Moreover, the contractions of these two neuropeptides were weakened by L-arginine (2 mM). The responses to NPFF (5 microM) and NPVF (5 microM) were not modified by atropine or naloxone (1 microM). Furthermore, NPFF (1 microM) and NPVF (1 microM) did not influence the contractive responses to acetylcholine (0.1-10 microM), morphine (1 microM) or nociceptin (0.1 microM). These data suggest that NPFF and NPVF cause contractions of the mouse distal colon via their NPFF receptors and this effect is mediated by NO but not by cholinergic pathways, independently from opioid system. In addition, the isolated bioassay may be applied as a simple parameter to characterize the potential NPFF agonists and antagonists.     

Plasticity of pre- and postsynaptic GABAB receptor function in the paraventricular nucleus in spontaneously hypertensive rats

GABA(B) receptor function is upregulated in the paraventricular nucleus (PVN) of the hypothalamus in spontaneously hypertensive rats (SHR), but it is unclear whether this upregulation occurs pre- or postsynaptically. We therefore determined pre- and postsynaptic GABA(B) receptor function in retrogradely labeled spinally projecting PVN neurons using whole cell patch-clamp recording in brain slices in SHR and Wistar-Kyoto (WKY) rats. Bath application of the GABA(B) receptor agonist baclofen significantly decreased the spontaneous firing activity of labeled PVN neurons in both SHR and WKY rats. However, the magnitude of reduction in the firing rate was significantly greater in SHR than in WKY rats. Furthermore, baclofen produced larger membrane hyperpolarization and outward currents in labeled PVN neurons in SHR than in WKY rats. The baclofen-induced current was abolished by either including G protein inhibitor GDPbetaS in the pipette solution or bath application of the GABA(B) receptor antagonist in both SHR and WKY rats. Blocking N-methyl-d-aspartic acid receptors had no significant effect on baclofen-elicited outward currents in SHR. In addition, baclofen caused significantly greater inhibition of glutamatergic excitatory postsynaptic currents (EPSCs) in labeled PVN neurons in brain slices from SHR than WKY rats. By contrast, baclofen produced significantly less inhibition of GABAergic inhibitory postsynaptic currents (IPSCs) in labeled PVN neurons in SHR than in WKY rats. Although microinjection of the GABA(B) antagonist into the PVN increases sympathetic vasomotor tone in SHR, the GABA(B) antagonist did not affect EPSCs and IPSCs of the PVN neurons in vitro. These findings suggest that postsynaptic GABA(B) receptor function is upregulated in PVN presympathetic neurons in SHR. Whereas presynaptic GABA(B) receptor control of glutamatergic synaptic inputs is enhanced, presynaptic GABA(B) receptor control of GABAergic inputs in the PVN is attenuated in SHR. Changes in both pre- and postsynaptic GABA(B) receptors in the PVN may contribute to the control of sympathetic outflow in hypertension.     

Leptin Activates Oxytocin Neurons of the Hypothalamic Paraventricular Nucleus in Both Control and Diet-Induced Obese Rodents

The adipocyte-derived hormone leptin acts in the brain to reduce body weight and fat mass. Recent studies suggest that parvocellular oxytocin (OXT) neurons of the hypothalamic paraventricular nucleus (PVN) can mediate body weight reduction through inhibition of food intake and increased energy expenditure. However, the role of OXT neurons of the PVN as a primary target of leptin has not been investigated. Here, we studied the potential role of OXT neurons of the PVN in leptin-mediated effects on body weight regulation in fasted rats. We demonstrated that intracerebroventricular (ICV) leptin activates STAT3 phosphorylation in OXT neurons of the PVN, showed that this occurs in a subpopulation of OXT neurons that innervate the nucleus of the solitary tract (NTS), and provided further evidence suggesting a role of OXT to mediate leptin’s actions on body weight. In addition, our results indicated that OXT neurons are responsive to ICV leptin and mediate leptin effects on body weight in diet induced obese (DIO) rats, which are resistant to the anorectic effects of the hormone. Thus, we conclude that leptin targets a specific subpopulation of parvocellular OXT neurons of the PVN, and that this action may be important for leptin’s ability to reduce body weight in both control and obese rats.     

Anatomical evidence for interaction of ACTH1–39 immunostained fibers and hypothalamic paraventricular neurons that project to the dorsal vagal complex

Summary Anatomical evidence is presented for an interaction of ACTH^1–39 immunostained fibers and a specific population of hypothalamic paraventricular (PVN) neurons; these neurons project to the dorsal vagal complex (DVC) of brainstem medulla. Bilateral injection of 10% HRP-WGA into DVC is incorporated into nerve terminals and transported retrogradely to cell bodies in the parvocellular subdivision of PVN, as revealed by standard HRP-WGA histochemistry or antibody to wheatgerm agglutinin followed by immunocytochemical techniques. Labeled cells are localized predominantly in the ventral portion of the caudal medial parvocellular subdivision and ventrolaterally in the posterior subnucleus of PVN. Few labeled cells are seen in the anterior parvocellular PVN, rostrally in the medial parvocellular component and in the dorsal cap. HRP-WGA cells are rarely observed in the magnocellular divisions of PVN. Dual-staining immunocytochemical-retrograde tracing techniques in the same tissue section demonstrate ACTH^1–39 fibers in intimate anatomical proximity to parvocellular PVN neurons that project to DVC. It is suggested that this interaction may partially account for the known cardiovascular effects of opiocorins and supports the role of the paraventricular nucleus in hypothalamie integration and modulation of cardiovascular control.     

Angiotensin depolarizes parvocellular neurons in paraventricular nucleus through modulation of putative nonselective cationic and potassium conductances

Neurosecretory parvocellular neurons in the hypothalamic paraventricular nucleus (PVN) exercise considerable influence over the adenohypophysis and thus play a critical role in neuroendocrine regulation. ANG II has been demonstrated to act as a neurotransmitter in PVN, exerting significant impact on neuronal excitability and also influencing corticotrophin-releasing hormone secretion from the median eminence and, therefore, release of ACTH from the pituitary. We have used whole cell patch-clamp techniques in hypothalamic slices to examine the effects of ANG II on the excitability of neurosecretory parvocellular neurons. ANG II application resulted in a dose-dependent depolarization of neurosecretory neurons, a response that was maintained in tetrodotoxin (TTX), suggesting a direct mechanism of action. The depolarizing actions of this peptide were abolished by losartan, demonstrating these effects are AT(1) receptor mediated. Voltage-clamp analysis using slow voltage ramps revealed that ANG II activates a voltage-independent conductance with a reversal potential of -37.8 +/- 3.8 mV, suggesting ANG II effects on a nonselective cationic current. Further, a sustained potassium current characteristic of I(K) was significantly reduced (29.1 +/- 4.7%) by ANG II. These studies identify multiple postsynaptic modulatory sites through which ANG II can influence the excitability of neurosecretory parvocellular PVN neurons and, as a consequence of such actions, control hormonal secretion from the anterior pituitary.     

Orexin depolarizes rat hypothalamic paraventricular nucleus neurons

Orexins, also called hypocretins, are newly discovered hypothalamic peptides that are thought to be involved in various physiological functions. In spite of the fact that orexin receptors, especially orexin receptor 2, are abundant in the hypothalamic paraventricular nucleus (PVN), the effects of orexins on PVN neurons remain unknown. Using a whole cell patch-clamp recording technique, we investigated the effects of orexin-B on PVN neurons of rat brain slices. Bath application of orexin-B (0.01-1.0 microM) depolarized 80.8% of type 1 (n = 26) and 79.2% of type 2 neurons tested (n = 24) in the PVN in a concentration-dependent manner. The effects of orexin-B persisted in the presence of TTX (1 microM), indicating that these depolarizing effects were generated postsynaptically. Addition of Cd(2+) (1 mM) to artificial cerebrospinal fluid containing TTX (1 microM) significantly reduced the depolarizing effect in type 2 neurons. These results suggest that orexin-B has excitatory effects on the PVN neurons mediated via a depolarization of the membrane potential.     

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.     

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.