Touch sensation is initiated by diverse mechanosensory neurons that innervate distinct skin structures; however, little is known about how touch receptors are patterned during mammalian skin development. During the course of my PhD training, I analyzed embryonic and neonatal development of mouse touch domes, which contain Merkel cell-neurite complexes that encode pressure and object features. I found that developing touch domes share three key features with canonical sensory placodes: discrete patches of specialized epithelial, co-clustered mesenchymal cells capable of engaging in molecular crosstalk with the epithelium, and selective recruitment of sensory neurons. During embryogenesis, molecularly distinct patches of epithelial Merkel cells and keratinocytes clustered with a previously unsuspected population of BMP4-expressing dermal fibroblasts in nascent touch domes. Concurrently, two populations of sensory neurons preferentially targeted touch domes compared with other skin regions. Surprisingly, only one neuronal population persisted in mature touch domes. Overexpression of Noggin, a BMP antagonist, in epidermis at embryonic age 14.5 resulted in fewer touch domes, a loss of Merkel cells, and decreased innervation density in skin areas where touch domes are typically found. Thus, touch domes bear hallmarks of placode-derived sensory epithelia that require BMP signaling for proper specification.

The peripheral nervous system (PNS) is composed of a diverse array of neurons that mediate sensation. This includes sensory circuits that encode external stimuli, as well as circuits that provide information flow from our internal organs. My PhD training has focused on addressing two questions: 1) what molecular mechanisms underlie this functional diversity, and 2) can we engineer non-invasive therapeutics to modulate PNS activity? To study the molecular mechanisms of sensory function, I employed the Merkel-cell neurite complex as a model system. Merkel cells are mechanosensory epidermal cells that have long been proposed to activate neuronal afferents through chemical synaptic transmission. RNA sequencing of adult mouse Merkel cells demonstrated that they express presynaptic molecules and biosynthetic machinery for adrenergic transmission. Moreover, live-cell imaging showed that Merkel cells mediate activity- and VMAT- dependent release of fluorescent catecholamine neurotransmitter analogues. Touch-evoked firing in Merkel-cell afferents was inhibited either by silencing of SNARE-mediated vesicle fusion from Merkel cells or by neuronal deletion of b2-adrenergic receptors. Next, to develop non-invasive technologies for peripheral nerve modulation, I employed targeted focused ultrasound (FUS) stimulation and electrophysiology to record activity of individual mechanosensory neurons. Parameter space exploration showed that stimulating neuronal receptive fields with high-intensity, millisecond FUS sonication reliably and repeatedly evoked action potentials in peripheral neurons. FUS elicited action potentials with latencies comparable to electrical stimulation, demonstrating both speed and reliability of the technique. Lastly, I show that peripheral neurons can be both excited by FUS stimulation targeted to either skin receptive fields or peripheral nerve trunks, a key finding that increases the therapeutic range of FUS-based peripheral neuromodulation.

Signals for each of the five tastes have previously been shown to be processed by distinct labeled lines from taste receptor cells (TRCs) on the tongue to the ganglion neurons that innervate them. Furthermore, different tastes have been shown to be represented by distinct neurons in the taste cortex. We recorded calcium activity using fiber photometry from genetically defined populations in the mouse rostral nucleus of the solitary tract (rNST), the first brain station receiving taste signals from the tongue. We found that Somatostatin- (Sst) expressing cells respond exclusively to bitter chemicals while Calretinin- (Calb2) expressing cells respond exclusively to sweet chemicals. Immunostaining and viral strategies demonstrated that Sst and Calb2 mark distinct neuronal populations in the rNST. We then showed that optogenetic activation of Sst and Calb2 cells elicits prototypical bitter and sweet behaviors, respectively and demonstrate that ablation of these cells strongly impairs aversion to bitter tastants and attraction to sweet tastants, respectively. These findings reveal how taste information is propagated into the brain.

The nuclei of the midline thalamus are an important part of the brain’s limbic system. Previous work has described the presence of dopamine D2 receptors in the midline thalamus in humans, non-human primates, and rodents. A similar body of literature has also demonstrated dopaminergic innervation of the midline thalamus across these species. However, little is known regarding a) the source of dopaminergic innervation to the midline thalamus in rodents and b) the function of D2R in the midline thalamus in any species. I begin this thesis with a review of the literature examining the anatomy, electrophysiological properties, and role in behavior of the paraventricular nucleus of the thalamus (PVT), a region where D2R mRNA and protein is expressed. I next describe a series of three sets of experiments aimed toward examining the anatomical, electrophysiological, and behavioral role of D2R in the PVT in mice. In the first set of experiments, I used anatomical methods to show that D2R are particularly enriched in neurons of the PVT. I focused on D2R-expressing PVT neurons specifically and show their afferent and efferent projections throughout the brain. In addition, I describe a set of experiments aimed to establish a dopaminergic innervation to the PVT. In the second set of experiments, I used electrophysiological methods to study D2R-expressing PVT neurons. Here, I establish that tonic firing in D2R-expressing thalamic relay neurons in the PVT is inhibited by quinpirole, a D2R/D3R agonist, and increased by sulpiride, a D2R/D3R antagonist. In the third set of experiments, I assessed the behavioral function of D2R in PVT neurons since this has never been studied in any species. I directly manipulated PVT D2R in two directions: a) by overexpressing D2R, and b) by downregulating D2R. Here I show PVT D2R plays a role in both cocaine locomotor sensitization as well as contextual fear expression. Our findings demonstrate for the first time the role of D2R in the PVT and add to literature suggesting that the PVT is an important component of the neural circuitry underlying fear behavior and drug reward. I conclude this thesis with a discussion of the findings described in the three sets of experiments as well as a proposal for future experiments.

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