Structural Organization Of The Connections Between Neurons Of The Paraventricular And Lateral Hypothalamic Regions In The Adult Male Rat

The hypothalamus is a major integrator of homeostatic signals in the brain; within the hypothalamus, there are sub-regions with specific roles in regulating energy balance. For example, the lateral hypothalamic area (LHA) has been classically associated with feeding behaviors, and contains two major orexigenic cell populations expressing the neuropeptides melanin concentrating hormone (MCH) or hypocretin 1/orexin A (H/O). Similarly, the paraventricular nucleus of the hypothalamus (PVH) integrates information to mediate neuroendocrine and autonomic responses related to ingestive behaviors. The PVH has also been implicated directly in the control of feeding. However, the precise structural and functional interactions between these two hypothalamic regions have not been fully established. In the present study, neuroanatomical tract tracing techniques were employed to target a cocktail of anterograde and retrograde tracers, Phaseolus vulgaris- leucoagglutinin (PHA-L) and cholera toxin subunit B (CtB), respectively; or only the retrograde tracer Fluorogold (FG), into the PVH. The resulting pattern of labeled neurons and fibers within the forebrain was then visualized, analyzed and mapped; with special attention to the LHA. Multi-fluorescence immunohistochemistry and confocal/wide-field imaging were used to identify tracer-filled cell bodies expressing the calcium-binding protein, calbindin (CaBn), H/O, or MCH; or a combination of these markers. The distribution of these neuronal populations was mapped to a canonical rat brain atlas (Swanson\u27s Brain Maps: Structure of the Rat Brain, 3rd edition, 2004). Retrogradely filled cells were found in the LHA mainly on the side ipsilateral to the PVH injection site. The majority of these cells were in the suprafornical, juxtadorsomedial, and the juxtaventromedial (dorsal and ventral zones) sub-regions. In addition, the dorsomedial, ventromedial, and arcuate hypothalamic nuclei; and posterior hypothalamus also contained abundant retrogradely labeled neurons. While only some of the retrogradely filled neurons were H/O- immunoreactive (-ir) or MCH-ir, many more co-expressed CaBn; of these, a small percentage was also H/O-ir. The results just described corroborate several published investigations where anterogradely transported tracers were used to identify PVH afferents, and identify for the first time (a) novel CaBn-expressing neuronal population(s) sending axonal projections to the PVH; these neurons are distributed widely throughout the LHA and other hypothalamic regions. Projections from these areas might be involved in the modulation of ingestive, neuroendocrine, and pre-autonomic functions, and may have a role in orchestrating reproductive behaviors as well. Previous work demonstrates that CaBn functions intracellularly within certain hypothalamic neurons to modulate firing rates through its direct Ca2+-buffering actions. Thus, in addition to providing the first high-resolution maps for this novel group of neurons, this work suggests that these neurons are likely to have unique firing patterns that might confer specific and perhaps unique functional properties to those neurons modulating PVH function. In addition, a PHA-L/CtB injection site confined exclusively to the periventricular part of the PVH (PVHpv) and anterior part of the periventricular hypothalamus (PVa), a region that harbors a somatostatin-expressing neuronal population, enabled characterization of the afferents and efferents for this PVH sub-region for the first time at high resolution. Projections to the PVHpv/PVa were found to originate mainly from several hypothalamic nuclei. First, confirming the anterograde analysis of ventral premammillary (PMv) projections provided by Canteras et al., 1992 (J. Comp. Neurol. 324:195-212), robustly labeled, retrogradely filled neurons were found within the PMv. Second, several additional hypothalamic regions also displayed retrogradely labeled neurons, including the arcuate nucleus, the dorsomedial and ventromedial nuclei of the hypothalamus, and the LHA. Back-filled neurons were also found in the ventral part of the orbitofrontal cortex. The axonal projections of the PVHpv/PVa, in turn, target mainly different hypothalamic regions; most remarkable are the projections to the median eminence. Efferents to hindbrain regions include dense projections to the nucleus of the solitary tract. Collectively, the data suggest that PVHpv/PVa neurons exert a complex control of growth and reproductive function through the release of key signals