Research reportSuprachiasmatic pacemaker organization analyzed by viral transynaptic transport
Introduction
The suprachiasmatic nucleus (SCN) of the hypothalamus is the principal circadian pacemaker in mammals responsible for the generation and regulation of rhythms in behavioral state, performance, hormonal secretion, and physiological function 24, 29, 31. The SCN is a paired structure lying dorsal to the optic chiasm and lateral to the third ventricle. Each nucleus contains two subdivisions: (a) a core receiving direct and indirect visual input, a dense projection from the midbrain raphe, and input from the hypothalamus and thalamus and (b) a shell receiving input from the basal forebrain, thalamus, and brainstem [34]. All SCN neurons appear to be circadian oscillators 26, 59and each nucleus is capable of generating independent circadian rhythms 17, 61. Although the two subdivisions can function independent of one another under certain circumstances, both subdivisions and both nuclei normally function as a single unit 43, 44, 61. The anatomical basis for this coupling is unknown. For example, it is not known whether there are local connections between the two subdivisions and commissural projections between the two SCNs which would support unitary function. Prior studies provide limited, fragmentary answers to this question, largely because the SCN is very small and not readily amenable to conventional anatomical analysis. To avoid this problem we injected two targets of the SCN, the subparaventricular zone (sPVZ) and the dorsomedial hypothalamic nucleus (DMH) 56, 57, 58with a retrograde transynaptic tracer, the swine herpesvirus (pseudorabies virus, PRV). By sacrificing animals at various survival timepoints after the retrograde transport of PRV to the SCN, the transynaptic passage of virus could be followed through local circuits within the SCN. This novel technique reveals, for the first time, a highly topographically organized intrinsic and commissural circuitry. Furthermore, small iontophoretic injections of a dilute solution of HRP conjugated to fluorescein made into different parts of the SCN labeled the homologous region on the contralateral side. These data support the view that the mammalian pacemaker is divided into two units which differ in their inputs, outputs, and local connections. The significance of these findings with respect to the flow of information through the SCN pacemaker is discussed.
Section snippets
Animals and surgery
Twenty albino rats of both sexes (Harlan Sprague–Dawley, 200–450 g) were handled in accordance with the NIH Guide for the Care and Use of Laboratory Animals using an experimental protocol approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. Animals were pair-housed in polypropylene cages and maintained in a 12:12 photoperiod with free access to tap water and Purina Rat Chow. Surgeries, perfusions, and the housing of PRV infected animals took place in a
Results
The centers of representative PRV injection sites in the DMH (Fig. 1) and sPVZ (Fig. 2) are shown at varying survival times. Injections in the DMH (n=10) label cells in that nucleus at 45 h survival (Fig. 1A). The labeling of DMH neurons at 45 h reflects uptake by terminals of recurrent collaterals of DMH neuron axons and, perhaps, some uptake by damaged elements. Two differing patterns of retrograde labeling are evident at 45–54 h (n=6) and 66–72 h (n=4) after virus injection into the DMH. At
Discussion
The present findings are the first demonstration that the intrinsic and commissural connections of the SCN are topographically organized in a manner consistent with previous divisions of the nucleus into a core and shell. Two primary lines of evidence support the conclusion that the SCN is comprised of two subdivisions. First, analysis of cytoarchitecture and peptide phenotype indicates that there is a dorsal division, or shell, with a large population of small, closely compacted
Acknowledgements
We thank Joan C. Speh and Nadine Suhan for excellent technical advice and help. Supported by NIH Grant NS-16304 (R.Y.M.), MHJ3574 (J.P.C.), and by the University of Pittsburgh MHCRC.
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