Neurotransmitter regulation of cellular activation and neuropeptide gene expression in the paraventricular nucleus of the hypothalamus

Abstract

Norepinephrine (NE), glutamate (Glu), and GABA have been identified as important neurotransmitters governing neuroendocrine mechanisms represented in the paraventricular nucleus of the hypothalamus (PVH). Microinjection studies were used to compare the efficacy of these transmitter mechanisms in stimulating PVH output neurons. Local administration of NE provoked an increase in plasma corticosterone levels and Fos induction in the both the parvocellular and magnocellular divisions of the nucleus. This treatment also stimulated a robust increase in corticotropin-releasing factor (CRF) heteronuclear (hn) RNA in the parvocellular PVH and a more subtle, although reliable, increase in arginine vasopressin (AVP) hnRNA in this same compartment. Local administration of the GABA(A) receptor antagonist bicuculline methiodide (BMI) resulted in increased plasma corticosterone and, in contrast to NE treatment, Fos induction limited primarily to the parvocellular PVH. BMI elicited marked increases in both CRH and AVP hnRNAs within the parvocellular division of the nucleus. Over a wide range of concentrations, Glu failed to produce reliable increases in corticosterone secretion and induced only weak activational responses limited primarily to non-neurosecretory regions of the PVH. Local Glu administration did, however, provoke Fos induction in identified GABAergic neurons immediately adjoining the PVH, suggesting that the muted response to Glu may be a consequence of concurrent activation of local inhibitory interneurons. These results support a differential involvement of adrenergic, glutamatergic and GABAergic mechanisms in regulating neurosecretory populations of the PVH and suggest that involvement of local circuit neurons must be carefully considered in the interpretation of microinjection studies.

Fig. 1.

The extent and placement of microinjections into the PVH. A, Dark-field photomicrograph showing the location and extent of a microinjection of 200 nl of [³H]NE (asterisk) into the region just dorsal to the PVH (arrow). B, Enlarged view of injection site showing that the gradient of diffused injectate spreads to involve the PVH. C, Bright-field photomicrographs of immunoperoxidase preparations (for Fos-IR) to show representative microinjection cannula placements in rats injected with saline (C), glutamate (C′) or norepinephrine (C"). Note that the placements shown inC and C" are centered at the level of the mid-PVH, although the one illustrated in C′ is situated more rostrally. Rats whose cannula tips were found to lay within a ventricle or >1 mm from the dorsal and/or lateral margin of the PVH were excluded from analysis. Induced Fos-IR is observed as a black reaction product. Magnifications: A, 8.5×;B, 70×; C, 25×. cc, Corpus callosum; Ctx, cerebral cortex;fx, fornix; HF, hippocampal formation;ic, internal capsule; och, optic chiasm;ot, optic tract; SCh, suprachiasmatic nucleus.

Fig. 2.

Plasma corticosterone levels are significantly increased above control levels by microinjections of norepinephrine or bicuculline, but not glutamate, into the PVH. A, Mean ± SEM plasma corticosterone (CORT) concentrations at various time points after PVH microinjection of saline (Sal), 25 nmol of NE, 100 pmol of BMI, or 1 nmol of Glu. n = 4 per group.B, Compared with saline-injected controls, a significant elevation in peak plasma corticosterone is observed at 30 min after NE or BMI injection (p < 0.05 by one-way ANOVA). Plasma corticosterone was not significantly increased after Glu microinjection.

Fig. 3.

Distinct patterns of cellular activation evoked by microinjection of bicuculline or norepinephrine. Bright-field photomicrographs of sections through the PVH on the sides ipsilateral (injected; left) and contralateral (noninjected;right) to microinjections of saline (top), 25 nmol of NE (middle), or 100 pmol of BMI (bottom) at the level of the mid-PVH. Saline-treated animals displayed very low-level Fos expression whose pattern and strength was indistinguishable from that seen in nonmanipulated rats. NE provoked a robust and lateralized Fos induction, highly focussed in the PVH, that encompassed all major functional compartments of the magnocellular and parvocellular divisions of the nucleus. BMI, in contrast, gave rise to Fos induction with the PVH that was highly confined to the parvocellular division of the nucleus and equally prominent in immediately adjoining areas, outside the PVH, proper. Magnification: 60×. mp, Medial parvocellular part (PVH); pm, posterior magnocellular part (PVH); fx, fornix. Asterisk indicates injection site.

Fig. 4.

Rostrally placed norepinephrine injections give rise to bilateral activation of both the PVH and the supraoptic nucleus. Bright-field photomicrographs of sections through the PVH (top) and the supraoptic nucleus (middle) on the sides ipsilateral (injected; left) and contralateral (noninjected; right) to a microinjection of 25 nmol of NE centered at the level of the rostral PVH. In contrast to the highly lateralized effects seen after mid-PVH injections (see Fig. 3), more rostrally placed injections gave rise to a bilateral pattern of activation in the PVH, which included the supraoptic nucleus, also bilaterally. This distinctive pattern of activation could be attributable to concurrent involvement of a cell group that lay within the sphere of the injection site, exhibits NE sensitivity, and projects bilaterally to both the PVH and supraoptic nucleus. These attributes are displayed by the median preoptic nucleus (bottom), which exhibited robust Fos induction in response to NE administration at rostral, but not mid-PVH, levels. Magnification: 60×. ac, Anterior commissure;MePO, median preoptic nucleus; SO, supraoptic nucleus.

Fig. 5.

Local glutamate injection produces a modest activational response in the PVH. Top, Bright-field photomicrographs showing Fos-IR expression in and around the PVH on the side ipsilateral (injected; left) and contralateral (noninjected; right) to a microinjection of 1 nmol of Glu. Only a modest activational response, localized principally to the autonomic-related dorsal and ventral medial parts of the parvocellular division of the nucleus, was observed. Note also the substantial induction of Fos-IR in regions immediately adjoining the PVH.Bottom, In contrast to the PVH, microinjection of 1 nmol of Glu into the hippocampal formation produces a marked increase in Fos-IR on the injected side of the brain, particularly in the granule cell layer of the dentate gyrus. Asterisk marks tip of injection cannula. Magnification: 60×. mp, Medial parvocellular part (PVH); pm, posterior magnocellular part (PVH); fx, fornix.

Fig. 6.

Glutamate microinjection activates perinuclear GABAergic neurons. Bright-field photomicrographs showing Fos-IR expression (brown nuclear product) combined with hybridization histochemical localization of GAD-67 mRNA (black cytoplasmic grains) in the anterior hypothalamic area (AHA; top) and perinuclear zone of the PVH (peri-PVH;bottom) of control (saline-injected;left) and glutamate-injected (right) rats. Under control conditions, a majority of the relatively small number of cells displaying Fos-IR are colabeled for GAD-67 mRNA. Local glutamate administration substantially increases the number of Fos-immunoreactive neurons in these regions (but not in the PVH itself); most of the glutamate-sensitive perinuclear neurons also display the marker for the GABAergic phenotype. Arrowsdenote examples of double-labeled cells. Magnification: 230×.

Fig. 7.

Both NE and BMI microinjection induce CRF gene transcription in the parvocellular division of the PVH.Left, Dark-field photomicrographs depicting CRF hnRNA expression in the parvocellular division of the PVH after microinjection of saline (Sal), 100 pmol of BMI, and 25 nmol of NE. Animals were killed 20 min after microinjection. Both NE and BMI microinjection produce a robust increase in hnRNA encoding for CRH peptide in the parvocellular division of the PVH. Magnification: 45×. Right, Mean ± SEM relative levels of CRF hnRNA in the parvocellular PVH of rats killed 20 min after saline, NE, or BMI injection. n = 4–6 per group. *p < 0.05 by one-way ANOVA.

Fig. 8.

AVP gene transcription in the parvocellular division of the PVH in response to microinjection of BMI or NE.Left, Dark-field photomicrographs showing AVP hnRNA expression in the parvocellular division of the PVH after microinjection of saline (Sal), 100 pmol of BMI, and 25 nmol of NE. Animals were killed 90 min after microinjection. Whereas robust expression of the primary AVP transcripts in the magnocellular division of the PVH is evident under each condition, AVP hnRNA expression in the parvocellular PVH is induced in response to BMI injection, with a substantially more muted response seen after NE administration. Magnification: 45×. Right, Mean ± SEM relative levels of AVP hnRNA after PVH microinjection of saline, NE, and BMI, as assessed by population densitometry (open bars) and cell counts (filled bars).n = 4–6 per group. *p < 0.05 by one-way ANOVA. Only the BMI effect on AVP hnRNA was significant based on densitometric analysis, although both drugs elicited reliable increases in the number of neurons exhibiting positive hybridization signal in the parvocellular division of the PVH.