European Journal of Neuroscience, Vol. 10, pp. 128–145, 1998 © European Neuroscience Association Characterization of the multisynaptic neuronal control of the rat pineal gland using viral transneuronal tracing P. J. Larsen, L. W. Enquist 1 and J. P. Card 2 Department of Anatomy, University of Copenhagen, Copenhagen, Denmark 1 Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA 2 Departments of Neuroscience and Psychiatry, University of Pittsburgh, Pittsburgh, PA 15260, USA Keywords: hypothalamic paraventricular nucleus, parasympathetic innervation, pseudorabies virus, suprachiasmatic nucleus, sympathetic innervation Abstract Knowledge of the polysynaptic pathway conveying photic information to the pineal gland is based upon studies employing lesions, knife cuts and classical tracers. In the present investigation we used viral transneuronal tracing to re-examine the organization of this circuitry. This was accomplished by injecting a neurotropic alpha herpesvirus (pseudorabies virus) into the gland and localizing viral antigen in infected neurones at various postinoculation intervals. This approach is based upon the demonstrated ability of this virus to invade axon terminals, replicate in neurones and pass retrogradely through a multisynaptic circuit. Immunohistochemical localization of viral antigen revealed the progressive appearance of infected neurones in the superior cervical ganglion (SCG), intermediolateral nucleus of the upper thoracic spinal cord (IML), parvicellular subdivisions of the hypothalamic paraventricular nucleus (PVN), and the suprachiasmatic nucleus (SCN). Other infected cell groups known to project to the IML also became infected. Infection of the PVN reproducibly involved neurones in the dorsal, medial and lateral parvicellular subdivisions and preceded the appearance of infected neurones in the SCN and other regions of hypothalamus. Topographic analysis of virus infected neurones within the SCN revealed differential infection of SCN subdivisions that suggested topography in the projection of the SCN to the PVN. Removal of the SCG eliminated infection within the aforementioned circuitry and revealed a parasympathetic innervation from the sphenopalatine ganglion. The data provide further detail on the cellular identity and synaptology of neural circuitry controlling the rhythmic secretion of melatonin by the rat pineal gland. Introduction Melatonin, the hormonal secretory product of the pineal gland, is released into the circulation in a rhythmic fashion with highest levels occurring during the dark phase of the photoperiod (cf. Bartness & Goldman, 1989 for review). In photoperiodic species the sensitivity of the rhythmic release of this hormone to light provides a mechanism for measuring day length that is critical for the synchronized expression of a number of physiological processes (Hastings, 1991). In 1662 Descartes proposed a pathway linking the eye and pineal gland (Descartes, 1662). Today, we know that photoperiodic information transduced in the retina is conveyed to the suprachiasmatic nuclei (Moore & Lenn, 1972; Hendrickson et al., 1972) and that the rhythmic output of this circadian oscillator is relayed to the pineal gland via a multisynaptic circuit involving both central and peripheral pathways (Moore, 1978; Moore & Klein, 1974; Meier & Rietveld, 1989; Hastings, 1991). The major neural input to the pineal is from the superior cervical ganglion (Kappers, 1965; Luebke & Wright, 1992). An innervation from parasympathetic ganglia and central pinealopetal fibres entering the gland via the pineal stalk has also been demonstrated in some species (Møller & Korf, 1983; Shiotani et al., 1986; Møller, 1992). Correspondence: P. J. Larsen, Department of Anatomy, Section B, The Panum Institute, Blegdamsvej 3, 2200 Copenhagen N, Denmark. E-mail: P.Larsen@mai.ku.dk Received 28 April 1997, revised 9 July 1997, accepted 18 July 1997 Although the physiological importance of this latter innervation is uncertain, it is well established that the nocturnal rise in melatonin secretion is dependent upon the sympathetic innervation (cf. Illnerova, 1991 for review). In rodents, the sympathetic projection to the pineal constitutes the final link in a multisynaptic pathway believed to originate in the SCN and involve the PVN (Fig. 1; cf. Moore, 1996 for a recent review). Evidence supporting this conclusion is found in studies demonstrating loss of photoperiodic regulation of melatonin secretion after lesions of the PVN or its descending projections (Klein & Moore, 1979; Klein et al., 1983a; Hastings & Herbert, 1986). These data have clarified the areas of CNS that influence the sympathetic outflow to the pineal, but the precise cell groups in the SCN and PVN involved in this projection pathway have not been defined. In this study we used a neurotropic swine alpha herpesvirus (pseudorabies virus, PRV) for transneuronal analysis of this circuitry. Substantial evidence supports the conclusion that the patterns of infection produced by this attenuated virus result from transynaptic passage of virions rather than lytic release into the extracellular space (cf. Strick & Card, 1992; Card & Enquist, 1994; Loewy, 1995;