Characterization of two neuronal subclasses through constellation pharmacology

Abstract

Different types of neurons diverge in function because they express their own unique set or constellation of signaling molecules, including receptors and ion channels that work in concert. We describe an approach to identify functionally divergent neurons within a large, heterogeneous neuronal population while simultaneously investigating specific isoforms of signaling molecules expressed in each. In this study we characterized two subclasses of menthol-sensitive neurons from cultures of dissociated mouse dorsal-root ganglia. Although these neurons represent a small fraction of the dorsal-root ganglia neuronal population, we were able to identify them and investigate the cell-specific constellations of ion channels and receptors functionally expressed in each subclass, using a panel of selective pharmacological tools. Differences were found in the functional expression of ATP receptors, TRPA1 channels, voltage-gated calcium-, potassium-, and sodium channels, and responses to physiologically relevant cold temperatures. Furthermore, the cell-specific responses to various stimuli could be altered through pharmacological interventions targeted to the cell-specific constellation of ion channels expressed in each menthol-sensitive subclass. In fact, the normal responses to cold temperature could be reversed in the two neuronal subclasses by the coapplication of the appropriate combination of pharmacological agents. This result suggests that the functionally integrated constellation of signaling molecules in a particular type of cell is a more appropriate target for effective pharmacological intervention than a single signaling molecule. This shift from molecular to cellular targets has important implications for basic research and drug discovery. We refer to this paradigm as "constellation pharmacology."

Fig. 1.

Antagonists of the K_V1 family of voltage-gated K channels substantially amplified the response to menthol in M+A+ neurons but not in M+A− neurons. The experimental methods and figure format are described in Materials and Methods. Each trace represents the response of a different neuron. In a typical experimental trial, the responses of >100 individual neurons were monitored simultaneously. Selected traces are shown. The aforementioned facts are the same for all subsequent figures. The abbreviations used in all figures are defined in Table 1. (A) Two traces from different M+A+ neurons that responded to menthol. Their responses to menthol were amplified reversibly by α-Dtx. After application of α-Dtx, the average menthol-elicited response as a percentage of control peak height was 275 ± 29% for M+A+ neurons (n = 5 trials, 51 cells total). (B) Two traces from different M+A− neurons that responded to menthol. Their responses to menthol were not amplified by α-Dtx. After application of α-Dtx, the average menthol-elicited response as a percentage of control peak height was 99 ± 3% for M+A− neurons (n = 5 trials, 16 cells total). (C) (Upper) Menthol responses in this M+A+ neuron were amplified reversibly by both κM-RIIIJ and α-Dtx. With this experimental protocol, the average menthol-elicited response as a percentage of control peak height was 292 ± 21% after application of κM-RIIIJ and 352 ± 14% after application of α-Dtx for M+A+ neurons (n = 3 trials). (Lower) Menthol responses in this M+A−neuron were not amplified significantly by either κM-RIIIJ or α-Dtx. The average menthol-elicited response as a percentage of control peak height was 108 ± 8% after application of κM-RIIIJ and 110 ± 10% after application of α-Dtx for M+A− neurons (n = 3 trials).

Fig. 2.

A Ca_V1 antagonist blocked the response to high [K⁺]_o in M+A− neurons nearly completely, whereas a combination of Ca_V1 and Ca_V2.1 antagonists was required to achieve a similar degree of block in M+A+ neurons. Menthol was applied at 500 μM working concentration, in contrast to Fig. 1, where 250 μM menthol was used. Application of 500 μM menthol typically produced more robust [Ca²⁺]_i signals in M+A+ neurons than 250 μM (Fig. 1), but the [Ca²⁺]_i signals elicited by 500 μM menthol were excessively large in some M+A− neurons. Here, “K” indicates 30 mM KCl. This concentration was used to activate as many voltage-gated Ca channels as possible. Many neurons could not recover from higher concentrations of KCl. (A) Nicardipine inhibited the KCl-elicited calcium signals in M+A− neurons nearly completely, but it blocked KCl-elicited calcium signals in M+A+ neurons only modestly: The average KCl-elicited response as a percentage of control peak height was 14 ± 4% for M+A− neurons (n = 4 trials, 11 cells total) and 79 ± 2% for M+A+ neurons (n = 4 trials, 39 cells total). (B) GVIA did not augment the block by nicardipine in M+A+ neurons. After application of nicardipine and GVIA. The average KCl-elicited response as a percentage of control peak height was 19 ± 5% for M+A− neurons (n = 4 trials, 9 cells total) and 82 ± 3% for M+A+ neurons (n = 4 trials, 40 cells total). (C) The combination of nicardipine and MVIIC substantially inhibited the KCl-elicited calcium signal in both M+A− and M+A+ neurons. After application of the mixture, the average KCl-elicited response as a percentage of control peak height was 9 ± 2% for M+A− neurons (n = 5 trials, 18 cells total) and 24 ± 2% for M+A+ neurons (n = 5 trials, 57 cells total).

Fig. 3.

Veratridine-elicited responses were blocked by TTX in both M+A− and M+A+ neurons. However, in the presence of both TTX and veratridine, KCl-elicited responses were amplified in M+A+ neurons but were blocked in M+A− neurons. The vertical dashed lines are for clarity only, indicating when veratridine was present in the well. (A) Both M+A− and M+A+ neurons responded immediately to application of veratridine (black bar) with an elevated and sustained increase in [Ca²⁺]_i. Here, “K” indicates 20 mM KCl. (B) The immediate responses to veratridine shown between the vertical dashed lines in A were blocked by TTX (open horizontal bars) in both M+A− and M+A+ neurons. However, in the presence of both TTX and veratridine, KCl-elicited responses were amplified in M+A+ neurons (Upper) but were partially blocked in M+A− neurons (Lower). After application of veratridine and TTX, the average KCl-elicited response as a percentage of control peak height was 271 ± 16% for M+A+ cells (n = 6 trials, 56 cells total) and 81 ± 10% for M+A− cells (n = 6 trials, 15 cells total). Here, “K” indicates 25 mM KCl at minute 1 and then 15 mM KCl at subsequent time points (15 mM KCl was used because it made the amplification observed in the upper trace of B more evident, while the block observed in the lower trace of B remained clear).

Fig. 4.

M+A− neurons responded to innocuous cool temperatures and noxious cold temperatures, but M+A+ neurons responded only to noxious cold temperatures. (A and B) Traces from neurons in the culture that did not respond to menthol or cold temperatures with an increase in [Ca²⁺]_i. (A) One neuron responded only to application of KCl. (B) The other neuron responded to KCl and ATP. In these two neurons, the slight downward inflection in the baseline observed when 4 °C bath solution was applied to the well suggests a decrease rather than an increase in [Ca²⁺]_i. (C) A trace from an M+A+ neuron that responded to noxious cold temperature (4 °C, 1-min application) but not to innocuous cool temperature (17 °C). The response to 4 °C reached its maximum relatively slowly over the time course of the application. (D) A trace from an M+A− neuron that responded to cool temperature (17 °C, 15-s application) and noxious cold temperature (4 °C). The M+A− neuron reached a maximum response to 4 °C quickly, and the calcium signal plateaued for the duration of the application.

Fig. 5.

The combination of nicardipine and κM-RIIIJ blocked responses to cold in M+A− neurons but amplified responses to cold in M+A+ neurons. Note that each application of 4 °C bath solution was for 30 s, designated by small black bars. Coapplication of nicardipine and κM-RIIIJ is designated by open horizontal bars. (A) Two traces from different M+A+ neurons. Their responses to 4 °C bath solution were amplified by a combination of κM-RIIIJ and nicardipine. After application of κM-RIIIJ and nicardipine, the average cold-elicited response as a percentage of control peak height was 294 ± 52% for M+A+ neurons (n = 5 trials, 81 cells total). (B) Two traces from different M+A− neurons. Their responses to the 4 °C bath solution were partially blocked by a combination of κM-RIIIJ and nicardipine. After application of κM-RIIIJ and nicardipine, the average cold-elicited response as a percentage of control peak height was 46 ± 4% for M+A− neurons (n = 5 trials, 19 cells total).