Voltage-gated calcium ion (CaV) channels play important roles in sensory transduction and are important targets of non-opioid analgesics. CaV3.2 channels are the dominant T-type current in DRG and exhibit interesting properties, such as activation near the resting membrane potential and slow kinetics of closing. These properties allow them to control neuronal excitability, lower activation thresholds, and prolong Ca2+ entry to boost calcium signaling. CaV3.2-knockout mice have impaired nociception and CaV3.2 channel expression is increased in a cell specific manner in several chronic pain models. Although it is clear that CaV3.2 channels are critical for the development of hyperalgesia, the biological function of CaV3.2-expressing neurons is still debated. In this work, I seek to define CaV3.2-expressing sensory neurons using functional anatomy and pharmacology and elucidate the role of CaV3.2 channels in hyperalgesia. In order to accomplish this, I have expressed the light-activated ion channel channelrhodopsin-2 (ChR2) specifically in neurons that express CaV3.2 channels using transgenic knock-in mice. This allows for the identification and control of CaV3.2-expressing neurons in vitro and in vivo. I propose a strategy to answer two questions: 1) What anatomical and functional characteristics define CaV3.2-expressing inputs to spinal dorsal horn? 2) How do CaV3.2- containing and CaV3.2-lacking neurons contribute to hyperalgesia? To examine the neuronal subpopulations that express CaV3.2 channels, as well as the mechanism by which they contribute to chronic pain, I will use a combination of behavior and slice physiology. In awake behaving mice, nociceptor activation elicits a rapid paw withdrawal. Using acute spinal cord slices, I will examine the functional distribution of CaV3.2- expressing afferent fibers into the spinal cord and test their pharmacological sensitivity to opiate and non-opiate analgesics. I hypothesize that CaV3.2 expressing sensory neurons represent a heterogeneous group of high-threshold and low-threshold sensory neurons and co-express ?-opioid receptors, but not -opoid receptors. To induce hyperalgesia, a feature of many chronic pain syndromes, I will use prolonged activation of ChR2-expressing sensory neurons. Preliminary data suggests that prolonged stimulation of CaV3.2-expressing sensory neurons is sufficient to generate mechanical hyperalgesia. In this proposal, I propose experiments testing for the contribution of CaV3.2-expressing and CaV3.2-lacking sensory neurons to mechanical hyperalgesia and allodynia, as well as thermal hyperalgesia. Using acute spinal cord slices, I will examine the mechanism of these behavioral effects by recording fEPSPs in acute spinal cord sections. I will test for increased contribution o CaV3.2 channels to neurotransmission using the CaV3 inhibitor mibefradil. This study will be the first to examine the entire population of CaV3.2-expressing sensory neurons in vivo and in vitro and will expand our understanding of the peripheral mechanism underlying chronic pain.
Chronic pain affects millions of individuals world-wide and current therapies are ineffective, addictive, or come with severe side effects. The lack of sufficient therapies is largely due to our incomplete understanding of the cellular and molecular mechanisms involved in developing and maintaining a chronic pain state. In this proposal, I examine the contribution of a genetically-identified population of sensory neurons to hypersensitivity in whole mice and in acute spinal cord sections in order to aid in the understanding chronic pain syndromes.
