Anatomical and physiological investigation of pathways mediating the effects of electrical stimulation of the external auricle of the ear on autonomic nervous system activity in rats

The Auricular Branch of the Vagus Nerve (ABVN) is a sensory nerve that innervates select areas of the external auricular dermatome. Electrical stimulation of the auricular region innervated by the ABVN influences the autonomic nervous system, observed by changes in control of the heart in humans and animals. However, the pathways and mechanisms for these effects are unknown. This thesis investigated in rats the pathways mediating the effects of electrical stimulation of the external auricle, comparing an ABVN innervated site of the external ear (the tragus) to an area not reported to receive ABVN innervation, the earlobe. Injection of the neuronal tracer cholera toxin B chain (CTB) into the right tragus (n=4) and right earlobe (n=4) revealed a large degree of similarity in sensory afferent termination sites. Afferent terminals were predominantly labelled ipsilateral to the injection site, with the densest labelling within laminae III-IV of the dorsal horn of the upper cervical spinal cord. In the medulla oblongata, CTB labelled afferents were observed in the paratrigeminal nucleus, cuneate nucleus, and to a minor extent in the nucleus tractus solitarius. Efforts were made to identify the targets of labelled afferents using immunofluorescence for choline acetyltransferase, calbindin, parvalbumin, glutamate decarboxylase 67 and neurokinin receptor 1 expressing cells, but inputs to each cell type were rare. Physiological recordings of the responses to ear stimulation were made in an anaesthetic free Working Heart Brainstem Preparation (WHBP) of the rat. Autonomic profiles of WHBP rats were first examined. Recordings made from rats at night time, revealed more robust sympathetic activity in comparison to day time rats, thus subsequent experiments were conducted in rats at night time. Electrical stimulation (100 Hz, 2.5 mA) was delivered for 5 minutes into the auricular stimulation sites in the WHBP. Direct recording from the sympathetic chain revealed a central sympathoinhibition from both tragus and earlobe stimulation. Sectioning of upper cervical afferent nerve roots silenced the sympathoinhibitory effects of tragus stimulation. Considering the predominance of afferent labelling in the cervical spinal cord dorsal horn and that cervical afferent nerve section reduced the sympathoinhibition evoked by tragus stimulation, this suggests that the autonomic effects of auricular stimulation are conveyed through somatosensory afferents rather than the ABVN.