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. 2008 Jan 16;28(3):566-75.
doi: 10.1523/JNEUROSCI.3976-07.2008.

Visualizing cold spots: TRPM8-expressing sensory neurons and their projections

Ajay Dhaka  1 , Taryn J Earley, James Watson, Ardem Patapoutian
Affiliations

Visualizing cold spots: TRPM8-expressing sensory neurons and their projections

Ajay Dhaka et al. J Neurosci. .

Abstract

Environmental stimuli such as temperature and pressure are sensed by dorsal root ganglion (DRG) neurons. DRG neurons are heterogeneous, but molecular markers that identify unique functional subpopulations are mainly lacking. ThermoTRPs are members of the transient receptor potential family of ion channels and are gated by shifts in temperature. TRPM8 is activated by cooling, and TRPM8-deficient mice have severe deficits in cool thermosensation. The anatomical and functional properties of TRPM8-expressing fibers have not been not comprehensively investigated. We use mice engineered to express the farnesylated enhanced green fluorescent protein (EGFPf) from the TRPM8 locus (TRPM8(EGFPf)) to explore this issue. Virtually all EGFPf-positive cultured DRG neurons from hemizygous mice (TRPM8(EGFPf/+)) responded to cold and menthol. In contrast, EGFPf-positive DRGs from homozygous mice (TRPM8(EGFPf/EGFPf)) had drastically reduced cold responses and no menthol responses. In vivo, EGFPf-positive neurons marked a unique population of DRG neurons, a majority of which do not coexpress nociceptive markers. The fraction of DRG neurons expressing EGFPf was not altered under an inflammatory condition, although an increase in TRPV1-coexpressing neurons was observed. TRPM8(EGFPf) neurons project to the superficial layer I of the spinal cord, making distinct contacts when compared with peptidergic projections. At the periphery, TRPM8(EGFPf) projections mark unique endings in the most superficial layers of epidermis, including bush/cluster endings of the mystacial pads. We show that TRPM8 expression functionally associates with cold sensitivity in cultured DRGs, and provide the first glimpses of the unique anatomical architecture of cold fibers in vivo.

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Figures

Figure 1.
Figure 1.
EGFPf neurons from TRPM8EGFPf/+ mice are activated by cold and menthol. Dorsal root ganglion cells from TRPM8EGFPf/+ (A) and TRPM8EGFPf/EGFPf (B) mice were challenged with cold, followed by menthol (250 μm) and then capsaicin (Caps; 1 μm). Individual traces from all EGFPf-positive neurons are presented.
Figure 2.
Figure 2.
TRPM8EGFPf marks a unique population of DRG cell bodies. A–S, DRG neurons from TRPM8EGFPf/EGFPf mice were stained with antibodies against GFP and sensory neuron markers (red). TRPM8EGFPf neurons were visualized by endogenous EGFPf (green). Arrowheads indicate examples of double-labeled neurons. Scale bar (in S) is the same for all panels.
Figure 3.
Figure 3.
TRPV1 expression is upregulated in TRPM8EGFPf neurons after CFA injection. DRG neurons from a TRPM8EGFPf/+ mouse injected with CFA (A–C) and uninjected TRPM8EGFPf/+ control (D–F) were stained with antibodies against TRPV1 (red). TRPM8EGFPf neurons were visualized by endogenous EGFPf (green). Arrowheads indicate examples of double-labeled neurons. Scale bar (in C) is the same for all panels.
Figure 4.
Figure 4.
TRPM8EGFPf neurons terminate in lamina I of the spinal cord dorsal horn. A–R, Confocal images of the lumbar region of adult spinal cord from TRPM8EGFPf/EGFPf mice were stained with antibodies against GFP or sensory neuron markers (red). TRPM8EGFPf neurons were visualized by endogenous EGFPf (green). The boxed regions in I and O were reimaged at 60× with 2× digital zoom and shown in J–L and P–R, respectively. SP, Substance P. Scale bar (in C) is the same for A–I, M–O. Scale bar (in L) is the same for J–L, P–R.
Figure 5.
Figure 5.
TRPM8EGFPf fibers colocalize with spinal cord neurons present in lamina I of the spinal cord dorsal horn. Confocal images of the lumbar region of adult spinal cord from TRPM8EGFPf/EGFPf mice. A–O, were stained with spinal cord neuron markers (red). TRPM8EGFPf neurons were visualized by endogenous EGFPf (green). The boxed regions in F and L were reimaged at 60× with 2× digital zoom and shown in G–I and M–L, respectively. Scale bar (in C) is the same for A–F, J–L. Scale bar (in I) is the same for G–I, M–O.
Figure 6.
Figure 6.
TRPM8EGFPf epidermal free nerve endings project to multiple termination zones in glabrous skin. A–F, Confocal images of CGRP (red) and TRPM8EGFPf (green) epidermal free nerve endings in the glabrous skin from TRPM8EGFPf/EGFPf mice. ^ marks the stratum spinosum; * marks the stratum granulosum; arrows mark intertwined fibers. G–I, Blood vessel is innervated with CGRP (red) fibers and has no innervation from TRPM8EGFPf fibers. # symbol marks blood vessel. Scale bar (in C) is the same for all panels.
Figure 7.
Figure 7.
TRPM8EGFPf epidermal free nerve endings mark unique classes of sensory nerve endings in the mystacial pad. A–L, Confocal images of CGRP (red) and TRPM8EGFPf (green) epidermal free nerve endings in the mystacial pad from TRPM8EGFPf/+ mice. ^ marks example of radiating free nerve endings; * marks examples of bush/cluster free nerve endings; # marks example of pencillate/horizontal free nerve endings, and arrow marks example of TRPM8EGFPf fibers near sebaceous glands. Scale bar (in C) is the same for all panels.
Figure 8.
Figure 8.
TRPM8EGFPf epidermal free nerve endings innervate tissue around but not into taste buds of the tongue. A–I, Confocal images of CGRP (red) and TRPM8EGFPf (green) epidermal free nerve endings in fungiform papillae (A–F) and epidermal tissue (G–I) of the tongue. J–L, Confocal images of gustducin (red)-positive taste buds and TRPM8EGFPf (green) epidermal free nerve endings in fungiform papillae. Scale bar (in C) is the same for all panels.
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