Elsevier

Neuroscience

Volume 119, Issue 4, 16 July 2003, Pages 1071-1083
Neuroscience

Cold transduction in rat trigeminal ganglia neurons in vitro

https://doi.org/10.1016/S0306-4522(03)00225-2Get rights and content

Abstract

Three sub-populations of sensory neurons may be distinguished based on responses to a decrease in temperature: one has a relatively low threshold for activation (cool fibers), a second has a high threshold for activation (cold nociceptors), and the third is unresponsive to a decrease in temperature. Results from several recent studies suggest that the ability to detect a decrease in temperature reflects an intrinsic property(ies) of sensory neurons and therefore may be characterized via the study of the sensory neuron cell body in vitro. However, while three unique ionic mechanisms of cold transduction have recently been identified (i.e. activation of the transient receptor potential channel M8 [TRPM8] or an epithelial Na+ channel [ENaC] or inhibition of two pore K+ channel [TREK-1]), the possibility that these “mechanisms” may be differentially distributed among sensory neurons in a manner consistent with predictions based on in vivo observations has not been investigated. To investigate this possibility, we have characterized the influence of cooling on isolated trigeminal ganglion (TG) neurons from adult rats in vitro with Ca2+ microfluorimetry in combination with a series of pharmacological interventions. We report that neurons responded to a decrease in temperature from approximately 34 °C to approximately 12 °C in one of two ways: 1) with a low threshold (30.1±0.6 °C) for activation demonstrating an increase in fluorescence with a minimal decrease in bath temperature (12.3%); 2) with a high threshold for activation (21.5±0.6 °C), demonstrating an increase in fluorescence only after a substantial decrease in bath temperature (13.3%); 74.4% did not respond to a decrease in temperature with an increase [Ca2+]i. These responses also were distinguishable on the basis of their rate of activation and degree of desensitization in response to prolonged application of a cold stimulus: low threshold responses were associated with a rapid (τ=12.0±5.7 s) increase in [Ca2+]i and a time constant of desensitization of 85.8±20.7 s while high threshold responses were associated with a slow (τ=38.1±8.2 s) increase in [Ca2+]i and demonstrated little desensitization over 4 min of stimulation. We refer to low threshold and high threshold cold responsive TG neurons as LTcool and HTcool neurons, respectively. LTcool and HTcool neurons were distributed among two distinct subpopulations of TG neurons distinguishable on the basis of cell body size and isolectin B4 staining. Both ENaC and TRPM8 appear to contribute to cold transduction, but neither is sufficient to account for all aspects of cold transduction in either population of TG neurons. Furthermore, inhibition of Ba2+ and/or Gd3+ sensitive two-pore K+ channels (i.e. TREK-1 and TRAAK) was insufficient to account for cold transduction in HTcool or LTcool neurons. Our results suggest that cold transduction in sensory neurons is a complex process involving the activation and inhibition of several different ion channels. In addition, there appear to be both similarities and differences between mechanisms underlying cold transduction in LTcool and HTcool neurons. Identification of specific mechanisms underlying cold transduction in LTcool and HTcool neurons may enable the development of novel therapeutic interventions for the treatment of pathological conditions such as cold allodynia.

Section snippets

Cell culture

Male Sprague–Dawley rats weighing 225±25 g, purchased from Harlan Sprague–Dawley (Indianapolis, IN, USA) were housed in a vivarium under a 12-h light/dark cycle and were fed standard rat diet and water ad libitum. All procedures concerning the care and use of animals in this study were approved by the University of Maryland Institutional Animal Care and Use Committee and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. We

Cold responsive tg neurons: response properties

TG neurons in short-term (<24 h) culture responded to a decrease in temperature from 34 to 12 °C (at approximately 1 °C/s) in one of three ways (Fig. 1A): 1) with a increase in fluorescence that occurred soon after initiation of the temperature ramp (i.e. a low threshold response); 2) with an increase in fluorescence that did not develop until well after initiation of the temperature ramp (i.e. a high threshold response); and 3) no change in fluorescence in response to a decrease in bath

Discussion

We have employed fura-2 microfluorimetry on dissociated rat TG neurons in order to test the hypothesis that there are two populations of neurons responsive to a decrease in temperature. In addition, we have determined the extent to which putative cold transducers underlie the responses observed in TG neurons. Consistent with our hypothesis, we observed two populations of TG neurons responsive to a decrease in temperature. These populations were distinguishable on the basis of their unique

Note added in proof

Another cold responsive member of the transient receptor potential (TRP) family of ion channels was recently identified. This channel, called AnkTM1 is menthol insensitive, has a high threshold for activation and is co-expressed with TRPV1 (the capsaicin receptor) (Story et al., 2003). These properties make this channel a likely candidate for an underlying mechanism of transduction in HTcool neurons.

Acknowledgements

The authors would like to thank Dr. Lei Zhang for technical assistance with experimental procedures and Drs. Daniel Weinreich and Michael Caterina and Michele Nealen for helpful discussions during preparation of the manuscript. Support for this work was obtained from the National Institutes of Health with grants RO3DA13274 (M.S.G.) and T32DE073093 (P.D.T.).

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