Mark Zylka, PhD
Assistant Professor

Education:

BS, Virginia Tech 1994
PhD, Harvard University 1999
Postdoc, Caltech 2000-2005

Molecules and Mechanisms for Pain

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.
Figure 1.

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.

Figure 2. Nerve endings in glabrous skin.			Figure 3. Dorsal spinal cord. CGRP (red) and Mrgprd (green) axons. PKCγ interneurons (blue).

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)

Positions Available : (Contact Dr. Zylka by email for more information)

Graduate Students – Rotations and permanent positions available. Students from any graduate program can be readily accommodated.

Postdoctoral – Molecular / Genetics / Neurobiology.  Creation and analysis of knock-in mouse lines, tissue culture, mouse behavioral experiments.  Background in neuroscience preferred.  Please email Dr. Zylka your CV.

Selected Recent Publications:

Dussor G, Zylka MJ, Anderson DJ, McCleskey EW. (2008). Cutaneous sensory neurons expressing the Mrgprd receptor sense extracellular ATP and are putative nociceptors. J. Neurophysiol. 99:1581-9.

Liu Y, Yang FC, Okuda T, Dong X, Zylka MJ, Chen CL, Anderson DJ, Kuner R, Ma Q. (2008) Mechanisms of compartmentalized expression of Mrg class G protein-coupled sensory receptors. J. Neurosci. 28: 125-32.

Campagnola L, Wang H, Zylka MJ. (2008) Fiber-coupled light-emitting diode for localized photostimulation of neurons expressing channelrhodopsin-2.  J Neurosci Methods. 169:27-33.

Liu Q, Vrontou S, Rice FL, Zylka MJ, Dong XD, Anderson DJ. (2007) Molecular genetic visualization of a rare subset of unmyelinated sensory neurons that may detect gentle touch. Nature Neurosci. 10:946-8

Zylka MJ. (2005) Nonpeptidergic circuits feel your pain. Neuron 47:771-772.

Zylka M, Rice FL, Anderson, DJ. (2005) Topographically distinct epidermal nociceptive circuits revealed by axonal tracers targeted to Mrgprd. Neuron 45:17-25.

Zylka MJ , Dong X, Southwell AL, Anderson DJ. (2003) Atypical expansion in mice of the sensory neuron-specific Mrg G protein-coupled receptor family. Proc. Natl. Acad. Sci . USA 100:10043-10048.

Han S-K, Dong X, Hwang J-I, Zylka MJ, Anderson DJ, Simon MI. (2002) Orphan G protein-coupled receptors MrgA1 and MrgC11 are distinctively activated by RF-amide-related peptides through the G a q/11 pathway. Proc. Natl. Acad. Sci . USA 99:14740-14745.

Dong X, Han S-K, Zylka MJ, Simon MI, Anderson DJ. (2001) A diverse family of GPCRs expressed in specific subsets of nociceptive sensory neurons. Cell 106:619-632.

Kume K*, Zylka MJ*, Sriram S*, Shearman LP*, Weaver DR, Jin XJ, Maywood ES, Hastings MH, Reppert SM. (1999) mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop. Cell 98:193-205.

Jin X*, Shearman LP*, Weaver DR, Zylka MJ, De Vries GJ, Reppert SM. (1999) A molecular mechanism regulating rhythmic output from the suprachiasmatic circadian clock. Cell 96:57-68.

Zylka MJ, Reppert SM. (1999) Discovery of a putative heme -binding protein family (SOUL/ HBP ) by two-tissue suppressive subtractive hybridization and database searches. Molecular Brain Research 74:175-181.

Zylka MJ, Shearman LP, Levine JD, Jin X, Weaver DR, Reppert SM. (1998) Molecular analysis of mammalian Timeless. Neuron 21:1115-1122.

Zylka MJ*, Shearman LP*, Weaver DR, Reppert SM. (1998) Three period homologs in mammals: Differential light responses in the suprachiasmatic circadian clock and oscillating transcripts outside of brain. Neuron 20:1103-1110.

Shearman LP*, Zylka MJ*, Weaver DR, Kolakowski LF Jr., Reppert SM. (1997) Two period homologs: Circadian expression and photic regulation in the suprachiasmatic nuclei. Neuron 19:1261-1269.

* = Authors contributed equally to the paper