Members of all vertebrate classes are known to use vocalizations in reproductive contexts. In fish and the tetrapod classes surveyed to date (amphibians, birds and mammals), experiments have established a role for the forebrain in the modulation of vocalizations and have shown the preoptic area (POA) and ventral telencephalon to function in vocal regulation. Hindbrain motor pattern generating circuitry which innervates the vocal musculature has also been outlined in all vertebrate classes. However, no investigations have addressed the issue of how the POA or other forebrain vocal centers which may integrate a variety of reproductive behaviors modulate hindbrain circuitry to effect a change in vocal output. We hypothesize that: (1) the preoptic area (POA) and regions of the ventral telencephalon influence the production of vocal behavior in midshipman, and (2) this forebrain regulation occurs, in part, via dopamine and arginine vasotocin (AVT) innervation of the vocal motor network. These hypotheses are strongly supported by the following data for midshipman and closely related species: 1) Vocal activity may be elicited by electrical stimulation of the POA and ventral telencephalon. 2) The hindbrain vocal region is bordered by a density of dopaminergic and AVT-immunoreactive (-ir) fibers and terminals. 3) The POA and ventral telencephalon contain a large number of dopaminergic cell bodies, while the POA contains all AVT-ir cell bodies present in the midshipman brain. Vocalizing teleost fish provide the only system in which hindbrain vocal activity has been monitored quantitatively using intracellular recording techniques. Importantly, the vocal activity of midshipman fish is isomorphically related to the neuronal firing in the hindbrain vocal pacemaker-motoneuron (PN-MN) circuit. Thus, forebrain modulation of vocal motor output may be monitored directly at the level of the PN-MN circuit and the output of the circuit may be classified as to which species-typical agonistic or courtship vocalization is being generated. The primary goal of the experiments described here is to determine how forebrain activity modulates a rhythmically-active, hindbrain vocal motor circuit at physiological, neurochemical, and anatomical levels. This will be performed in the plainfin midshipman (Porichthys notatus), a teleost fish with a well-characterized hindbrain vocal PN-MN circuit. Our primary goal will be realized through the achievement of four specific aims: Hence, we will address our primary goal by testing our hypotheses through the achievement of three specific aims: (1) Forebrain connectivity to the known vocal circuitry will be investigated by iontophoresis of biotin compounds into forebrain, midbrain and hindbrain sites which have been identified as part of the vocal motor network by neurophysiological assessment of vocal motor output from the hindbrain pattern generator. (2) Forebrain modulation of vocal motor circuitry will be delineated via iontophoretic injection of dopamine and AVT into vocal motor regions, coupled with neurophysiological assessment of vocal motor output from the hindbrain pattern generator. (3) The dopaminergic or AVT signature of vocal motor afferents will be characterized by combined retrograde tract-tracing and immunocytochemistry (ICC) using well characterized antibodies to dopamine and AVT.
