Final Report Summary - RPINBIRDS (Reproductive pathways in birds) In the periodic report, we noted that, of all the groups of neurons retrogradely labelled in the brainstem from injections in the sacral spinal cord, nucleus retroambigualis (RAm) was the most likely to target cloacal motor neurons, on the basis of comparative considerations. Therefore, during the second part of our studies, we targeted RAm for injection of anterograde tracers in a series of male and female quail. In most of these cases, we also injected mSC with a different fluorescent tracer so that we could distinguish the descending axons of RAm neurons from retrogradely labelled mSC motor neurons and their dendrites. In practice, because of the small size of RAm, its location at the very caudal end of the narrowing brainstem, and the surgical impossibility of using vertical penetrations for injection, we used angled penetrations and made electrophysiological recordings of respiratory-related activity that we knew from previous work was characteristic of at least some RAm neurons (Wild, J. M., Brain Research, 606: 119 - 124, 1993). In other words, we used the respiratory-related activity to guide us to the target, and here we made our injections of anterograde tracers. The results of several cases of these double injections in RAm and mSC were quite dramatic. The RAm injections covered all the nucleus, including, of course, those neurons projecting upon spinal motor neurons in the lower thoracic and upper lumbar regions that innervate expiratory muscles (Wild, 1993). For the first time, however, we discovered that RAm injections led to the labelling of axons that descended to sacral spinal levels. In transverse sections, these labelled axons were seen to be grouped together to occupy the medial part of the dorsolateral funiculus (where dorsally directed mSC dendrites were seen to reach), and then formed a tangled mass of axons and apparent terminations closely corresponding to the distribution of retrogradely labelled mSC motor neurons and their dendrites in the ventral horn and adjoining lateral funiculus. No major differences between males and females in this pattern of projections were noted. But despite the clear areal overlap of descending axons and mSCmotor neurons, much further work is required using confocal microscopy to indicate the possibility of actual contacts between the descending axons and mSC motor neurons. Nevertheless, the results of retrograde labelling from the sacral spinal cord and anterograde labelling of RAm axons in proximity to mSC motor neurons provides a strong suggestion of a functional relationship between RAm and at least one major group of motor neurons involved in reproductive behaviour.The nature of the neural tracers used in our studies usually ensures that both retrograde and anterograde transport result from the injections. This is certainly the case with the tracer we injected into RAm (cholera toxin B-chain, abb. CTB), which in addition to anterogradely labelling the descending projections to the spinal cord, retrogradely identified a host of neurons in the brainstem and forebrain. As expected from previous work (Wild et al., 1997), a nucleus called DM within the intercollicular complex (ICo) of the midbrain was strongly labelled; but the extent of this nucleus in the quail was totally unsuspected. Suffice it to say that nothing like it has ever been seen before in any avian species. We went on to show using electrical stimulation that DM drives crowing in male quail and other vocalisations in female quail. Using anterograde tracing, we also showed that DM projects specifically upon RAm in the caudal medulla, including very caudal regions where neurons were retrogradely labelled from sacral spinal cord injections in the same bird.We next sought to identify inputs to DM and other regions of ICo. Injections of tracers into ICo and centered on DM retrogradely labelled neurons in the hypothalamus, including some in the medial preoptic nucleus (POM), the nucleus that has been at the centre of research on avian reproductive behaviour in Dr Balthazart's and others' labs for many years. However, hypothalamic injections that included POM led to an interesting pattern of terminations in ICo. At caudal levels terminations clearly included neurons that were retrogradely labelled from RAm in the same bird. More rostrally in ICo terminations avoided the closely packed DM cell bodies, but densely surrounded them within ICo. It was noticed, however, that some processes of DM neurons extended dorsally into the surrounding ICo, possibly indicating that contact between the axons of POM neurons and DM neurons are made in this region, rather than on DM cell bodies themselves.Thus, we now have a reasonably complete pathway related to reproductive behaviour, from POM in the hypothalamus to cloacal motor neurons in the sacral spinal cord, via DM in the midbrain and RAm in the caudal medulla. It should be understood, however, that although our results constitute a 'first' with regard to the identification of reproductive pathways in birds, we regard them as preliminary, and note that a great deal of further work is required to corroborate and build on them with confidence. Nevertheless, the pathways so far identified in the quail have clear similarities to those defined in mammals for the control and modulation of reproductive behaviour, despite the very different peripheral mechanisms of reproduction in the two classes of animal. This may be reassuring from the evolutionary point of view, in that a broad plan of neural connectivity has apparently been conserved across classes, yet we remain ignorant of a host of important details concerning the control of reproductive behaviour in birds. Not least of these are the roles played by other nuclei and pathways, ranging from those involved in the control of ejaculation, on the one hand, to those involved in visual, auditory, tactile, or even olfactory control of reproductive behaviour, on the other.