A functional characterisation of retrorubral field dopaminergic neurons

Characterising the function of the brain in health is critical if the changes that occur during disease states are to be fully understood. This is particularly true of the midbrain dopaminergic system, which is compromised in disorders such as Parkinson’s disease and drug addiction. Much research concerning the midbrain dopaminergic system focuses on the two largest cell groups, namely those situated in the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA). Caudal of the SNc and VTA is the third, relatively understudied group of dopaminergic neurons in the retrorubral field (RRF). The overarching goal of my thesis work was to characterise the function of RRF dopaminergic neurons in the mouse, in terms of their molecular makeup, connectivity and electrophysiological activity.
Molecular markers expressed by RRF dopaminergic neurons shared features of both the SNc and VTA. Like the SNc, some neurons in the RRF express Sox6 but lack Otx2; like the VTA, some neurons in the RRF express calbindin-1 and calretinin. Moreover, I determined that while many RRF dopaminergic neurons express calbindin-1 and few express aldehyde dehydrogenase 1a1, the opposite is true for the caudal SNc. Thus, this combination of molecular markers is useful for delineating the border between the rostral RRF and caudal SNc. Intermingled amongst dopaminergic neurons in the RRF were many putative GABAergic and other non-dopaminergic neurons, indicating a diversity of cell types within this region.
Cell type-selective anterograde and retrograde tracing of RRF dopaminergic neurons revealed their brain-wide connectivity. Examination of axonal projections uncovered a dense innervation of the ventrolateral striatum (VLS) and interstitial nucleus of the posterior limb of the anterior commissure (IPAC), in addition to a sparse innervation of the dorsolateral striatum (DLS) and olfactory tubercle. Although the projections of RRF dopaminergic neurons share features of both the nigrostriatal and mesolimbic pathways, the prominence of projections in ventral forebrain structures suggests that RRF neurons contribute more to the mesolimbic pathway. I observed that neurons providing monosynaptic inputs to RRF dopaminergic neurons are located in many brain regions. The densest clusters of input neurons were located in the bed nucleus of the stria terminalis (BNST), IPAC, and central amygdala. Overall, the results of these tracing experiments indicate that while the axonal projections of RRF dopaminergic neurons terminate in the striatum, their most prominent inputs arise from the extended amygdala. In the case of the IPAC, there appears to be prominent reciprocal connectivity with RRF dopaminergic neurons.
Electrophysiological in vivo recording experiments demonstrated that the spontaneous firing properties of identified RRF dopaminergic neurons are broadly similar to their counterparts in SNc and VTA, specifically a slow firing rate (< 10 spikes/s) and relatively long action potential duration (> 1 ms). Foot shocks applied to the hindpaw induced a brief pause (∼220 ms) in the firing of both identified RRF and SNc dopaminergic neurons, followed by rebound activation. Together, the results from these electrophysiological recordings demonstrate that the firing properties of dopaminergic neurons in the RRF are typical of midbrain dopaminergic neurons and that they are equipped to represent aversive stimuli with a pause in firing activity.
Next, I used an optogenetic approach to mimic the brief pause in firing observed in response to foot shock. Anaesthetised in vivo electrophysiological validation experiments demonstrated that ArchT-transduced dopaminergic neurons reliably pause their firing in response to brief (250 ms), physiologically-relevant yellow laser pulses. In freely-moving mice, ‘positive control’ experiments in which VTA dopaminergic neurons were briefly (250 ms) paused did not induce place aversion in either a bias conditioned place preference test or real-time place preference test.
Collectively, the work in this thesis provides important new information on the substrates for RRF dopaminergic neuron function. I have shown that the functional properties of these neurons are partly overlapping with respect to neighbouring SNc and VTA dopaminergic neurons, in terms of molecular makeup, connectivity and electrophysiological activity. I conclude that RRF dopaminergic neurons are equipped to fulfil distinct and specialised roles in the brain as compared to their midbrain counterparts.

Keywords: Midbrain dopamine
Retrorubral field
RRF
Advisor: Magill, P
Walton, M
Dodson, P