Immunoreactive substance P is not part of the retinohypothalamic tract in the rat

The retinohypothalamic tract (RHT) is a monosynaptic retinofugal pathway mediating information concerning the light/dark cycle from the retina to the brain's biological clock located in the suprachiasmatic nucleus (SCN). Light information, which daily adjusts (entrains) the rhythms of behaviour and physiology generated by the SCN, is mediated by two neurotransmitters, viz. glutamate and pituitary adenylate cyclase activating polypeptide (PACAP), co-stored in the RHT. Substance P (SP) modulates photic- and glutamate-induced phase shifts but data on its possible presence in the RHT are conflicting. By labelling the RHT projection in the SCN with the anterograde tracer cholera toxin subunit B (ChB) and antibodies against PACAP, we have shown that SP immunoreaction is absent from the PACAP/ChB-labelled nerve fibres in the SCN, indicating that the SP-immunoreactive nerve fibres are not part of the RHT but may originate from SP-immunoreactive cell bodies located within the SCN. In the retina, SP immunoreactivity occurs in amacrine cells in the inner nuclear cell layer, in a few displaced amacrine cells in the ganglion cell layer and in a dense plexus of SP-immunoreactive nerve terminals of the inner plexiform layer. Double immunostaining has revealed that SP-immunoreactive cells and fibres in the retina are not identical with the PACAP-immunoreactive ganglion cells that constitute the RHT. These findings together with the demonstration that bilateral eye enucleation does not decrease the number of SP-immunoreactive nerve fibres in the SCN indicate that SP is not a neurotransmitter in the RHT but could be an intrinsic neurotransmitter of the SCN modulating photic input to the clock.