Zylka Lab Research

Molecules and Mechanisms for Pain

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We recently found that nociceptive (pain-sensing) circuits in mammals are highly organized at molecular and neuroanatomical levels. In our laboratory, we are using molecular, genetic, electrophysiological and behavioral approaches to study these pain circuits in mice.  Our ultimate goal is to identify new analgesics so that debilitating chronic pain conditions can be more effectively treated.

Neural circuit-based approaches:

In mammals, pain signals are transmitted from the periphery to the CNS by two neuronal circuits; the so called peptidergic and non-peptidergic circuits (Fig. 1). Peptidergic neurons contain neuropeptides, like CGRP and Substance P, while non-peptidergic neurons bind the lectin IB₄.

We recently found a G protein-coupled receptor (GPCR or GPR) called Mrgprd (Mas-related GPR) which is expressed in a majority of all non- peptidergic neurons. Mrgprd is not expressed in CGRP⁺ neurons, nor is it expressed anywhere else in the brain or body.

To identify the tissues that Mrgprd-expressing neurons innervate, we engineered knock-in mice that express a membrane-tethered version of enhanced Green Fluorescent Protein (EGFPf) from the Mrgprd locus (MrgprdΔ^EGFPf). Surprisingly, we found that Mrgprd-expressing neurons only innervate the epidermis of the skin (Fig. 2). Joints and internal organs, were not innervated, suggesting pain signals are transmitted from these tissues by molecularly-distinct circuits.

In addition to molecular differences, Mrgprd-expressing axons and CGRP⁺ axons terminate within different zones of the epidermis (Fig. 2). Mrgprd-expressing axons (green) also terminate beneath the red-labeled CGRP lamina in the dorsal spinal cord (Fig. 3). Taken together, these findings suggest peptidergic and non-peptidergic neurons might have unique functions and connectivity. Although studied for over 20 years, it is still not known why mammals have peptidergic and non- peptidergic circuits, both of which respond to noxious stimuli. Do these two molecularly different circuits have redundant or non-redundant functions in nociception? In our laboratory, we are trying to answer this fundamental question using a variety of approaches. As an example, we are making and studying “circuit knockout” mice. These mice are specifically missing either peptidergic or non-peptidergic neurons. We are studying the consequences of these ablations using molecular, electrophysiological and behavioral methodologies. We are also using genetically-encoded transneuronal tract tracers to better understand how these circuits interface with pain-related regions of the brain.

Functional genomics approaches:

Nociceptive neurons are activated by diverse thermal, mechanical and chemical stimuli. What genes are required for processing these diverse stimuli? And, are there genes that mark functionally and anatomically distinct pain circuits? We use fluorescence activated cell sorting to purify live, EGFP-expressing neurons and then profile gene expression of genetically-defined neurons with Affymetrix GeneChip arrays. We then use molecular, bioinformatics and functional genomics approaches to study gene function. One of the many genes we are focused on is Mrgprd, a receptor that can be activated by beta-alanine.  Receptor activation might alter the excitability of sensory neurons and reduce pain signaling. 

Techniques used in our lab:

• Molecular biology and cell culture
• In situ hybridization and immunofluorescence staining
• Construction and characterization of knock-in and transgenic mice
• Mouse behavioral experiments
• Bioinformatics
• FACS of neurons
• Expression profiling with Affymetrix GeneChip arrays
• Calcium imaging
• Patch Clamp Electrophysiology (single cell and slice)

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