Hypothalamic and brainstem sources of pituitary adenylate cyclase-activating polypeptide nerve fibers innervating the hypothalamic paraventricular nucleus in the rat

Abstract

The hypothalamic paraventricular nucleus (PVN) coordinates major neuroendocrine and behavioral mechanisms, particularly responses to homeostatic challenges. Parvocellular and magnocellular PVN neurons are richly innervated by pituitary adenylate cyclase-activating polypeptide (PACAP) axons. Our recent functional observations have also suggested that PACAP may be an excitatory neuropeptide at the level of the PVN. Nevertheless, the exact localization of PACAP-producing neurons that project to the PVN is not understood. The present study examined the specific contribution of various brain areas sending PACAP innervation to the rat PVN by using iontophoretic microinjections of the retrograde neuroanatomical tracer cholera toxin B subunit (CTb). Retrograde transport was evaluated from hypothalamic and brainstem sections by using multiple labeling immunofluorescence for CTb and PACAP. PACAP-containing cell groups were found to be retrogradely labeled from the PVN in the median preoptic nucleus; preoptic and lateral hypothalamic areas; arcuate, dorsomedial, ventromedial, and supramammillary nuclei; ventrolateral midbrain periaqueductal gray; rostral and midlevel ventrolateral medulla, including the C1 catecholamine cell group; nucleus of the solitary tract; and dorsal motor nucleus of vagus. Minor PACAP projections with scattered double-labeled neurons originated from the parabrachial nucleus, pericoeruleus area, and caudal regions of the nucleus of the solitary tract and ventrolateral medulla. These observations indicate a multisite origin of PACAP innervation to the PVN and provide a strong chemical neuroanatomical foundation for interaction between PACAP and its potential target neurons in the PVN, such as parvocellular CRH neurons, controlling physiologic responses to stressful challenges and other neuroendocrine or preautonomic PVN neurons.

Fig. 1

CTb injection sites. A–C: Photomicrographs showing a typical CTb injection site in the PVN (No. 520301) in three coronal levels rostrocaudally, labeled with the immunoperoxidase method with diaminobenzidine as chromogen. D: Summary drawing of successful PVN injection sites centered at midlevel of the PVN (Nos. 450702, 452702, 511002, 520301). Scale bar = 200 μm.

Fig. 2

Fluorescent images from two distinct cases (Nos. 511002, 520301) showing the localization of CTb (red fluorescence) injection sites with CRH, oxytocin, or vasopressin (blue fluorescence) neurons as markers of PVN neurons. A1–A3: A CTb injection site using CRH as marker of PVN; A1: CTb injection site; A2: CRH neurons; A3: superimposition of CTb and CRH immunolabeling showing the localization of the injection in the PVN. B1–B3: A CTb injection site using oxytocin as marker of the PVN; B1: CTb injection site; B2: oxytocin neurons in the PVN; B3: superimposition of CTb and oxytocin immunolabeling showing the localization of the injection. C1–C3: A CTb injection site using vasopressin as marker of the PVN; C1: CTb injection site; C2: vasopressin neurons; C3: superimposition of CTb and vasopressin immunolabeling showing the localization of the injection. Scale bar = 200 μm.

Fig. 3

Fluorescent images showing examples of CTb or PACAP or PACAP and CTb double-labeled neurons in selected forebrain and midbrain areas. PACAP neurons show green fluorescence, CTb labeling shows red fluorescence, and double-labeled neurons are indicated by the presence of a composite yellow/orange color (arrows). A: Medial preoptic nucleus. B: Anterior tuberal lateral hypothalamic area. C: Arcuate nucleus. D: Dorsomedial nucleus. E: Supramammillary nucleus. F: Ventrolateral periaqueductal gray. Scale bar = 100 μm.

Fig. 4

Schematic maps showing the distribution of the Ctb, PACAP, or Ctb-PACAP double-labeled neurons in representative levels of coronal rat hypothalamic sections ipsilateral to the injection site. A: Preoptic region. B: Caudal hypothalamus (–3.5 mm from Bregma). Symbols: ×, PACAP only; open circles, Ctb only; solid circles, PACAP/Ctb double-labeled neurons. Scale bar = 200 μm.

Fig. 5

Fluorescent images showing examples of Ctb or PACAP or PACAP and Ctb double-labeled neurons in selected pontine and medullary areas. PACAP neurons show green fluorescence, Ctb labeling shows red, and double-labeled neurons appear yellow/orange (arrows). A: Parabrachial nucleus. B: Nucleus of the solitary tract/dorsal motor nucleus of vagus. C. Ventrolateral medulla. D: Ventral medulla, gigantocellular nucleus. Scale bar = 100 μm.

Fig. 6

Schematic maps of the distribution of CTb, PACAP, or CTb/PACAP double-labeled neurons in representative levels of the medulla ipsilateral to the injection site. A: Rostral medulla (Bregma –12.0 mm). B: Midcaudal medulla (Bregma –13.3 mm). Symbols: ×, PACAP only; open circles, CTb only; solid circles, PACAP/CTb double-labeled neurons. Each symbol represents a single neuron. Scale bar = 400 μm.

Fig. 7

Fluorescent images showing examples of PACAP-, CTb-, and PNMT-labeled neurons in the rostral ventrolateral medulla. A: PACAP. B: CTb. C: PNMT immunolabeling. PACAP neurons show green fluorescence, CTb-labeled neurons show red, and PNMT-labeled neurons are indicated by blue fluorescence. Neurons simultaneously labeled with PACAP, CTb, and PNMT antibodies (triple-labeled neurons) are marked with arrows. Scale bar = 100 μm.