Role of thalamic phospholipase C[beta]4 mediated by metabotropic glutamate receptor type 1 in inflammatory pain

Abstract

Phospholipase C (PLC) beta4, one of the four isoforms of PLCbetas, is the sole isoform expressed in the mouse ventral posterolateral thalamic nucleus (VPL), a key station in pain processing. The mouse thalamus also has been shown to express a high level of metabotropic glutamate receptor type 1 (mGluR1), which stimulates PLCbetas through activation of Galphaq/11 protein. It is therefore expected that the thalamic mGluR1-PLCbeta4 cascade may play a functional role in nociceptive transmission. To test this hypothesis, we first studied behavioral responses to various nociceptive stimuli in PLCbeta4 knock-out mice. We performed the formalin test and found no difference in the pain behavior in the first phase of the formalin test, which is attributed to acute nociception, between PLCbeta4 knock-out and wild-type mice. Consistent with this result, acute pain responses in the hot plate and tail flick tests were also unaffected in the PLCbeta4 knock-out mice. However, the nociceptive behavior in the second phase of the formalin test, resulting from the tissue inflammation, was attenuated in PLCbeta4 knock-out mice. In the dorsal horn of the spinal cord where PLCbeta1 and PLCbeta4 mRNAs are expressed, no difference was found between the wild-type and knock-out mice in the number of Fos-like immunoreactive neurons, which represent neuronal activity in the second phase in the formalin test. Thus, it is unlikely that spinal PLCbeta4 is involved in the formalin-induced inflammatory pain. Next, we found that pretreatment with PLC inhibitors, mGluR1 antagonists, or both, by either intracerebroventricular or intrathalamic injection, attenuated the formalin-induced pain behavior in the second phase in wild-type mice. Furthermore, activation of mGluR1 at the VPL enhanced pain behavior in the second phase in the wild-type mice. In contrast, PLCbeta4 knock-out mice did not show such enhancement, indicating that mGluR1 is connected to PLCbeta4 in the VPL. Finally, in parallel with the behavioral results, we showed in an electrophysiological study that the time course of firing discharges in VPL corresponds well to that of pain behavior in the formalin test in both wild-type and PLCbeta4 knock-out mice. These findings indicate that the thalamic mGluR1-PLCbeta4 cascade is indispensable for the formalin-induced inflammatory pain by regulating the response of VPL neurons.

Figure 1.

Attenuation of the second phase of formalin-induced nociceptive behavior in PLC β4-/- knock-out mice. Time courses of pain behavior in the formalin test with wild-type mice (open squares; n = 10) and PLCβ4 knock-out mice (filled circles; n = 10) are shown. Each point represents the mean ± SEM of cumulative durations of paw licking and lifting every 5 min immediately after the formalin injection. ^**p < 0.01 compared with the wild-type mice.

Figure 2.

Acute pain responses to thermal stimulus in the tail flick test (A) and hot plate test (B). No significant differences in the latencies of withdrawal (mean ± SEM) from thermal stimuli were observed between wild-type mice (open bars) and knock-out mice (filled bars) in the tail flick test (n = 19) or the hot plate test (n = 19).

Figure 3.

PLCβ isoforms transcribed in the mouse lumbar cord (A-D) and forebrain (E-H). Note the reciprocal expression of PLCβ1 and PLCβ4 mRNAs in the thalamic VPL and VPM and layer IV of somatosensory cortex S1. PLCβ1 and PLCβ4 mRNAs are, in contrast, both expressed concomitantly in the spinal cord, including the ventral horn (VH) and dorsal horn (DH). Scale bars, 1 mm.

Figure 4.

Fos-LI neurons of the dorsal horn of the spinal cord after formalin injection in knock-out and wild-type mice. A, B, Fos-LI cells in the dorsal horn of the spinal cord ipsilateral to the formalin injection site in wild-type mice (A) and PLCβ4 knock-out mice (B). Scale bar, 50 μm. C, Numbers of Fos-LI neurons (mean ± SD) in laminae I and II, III and IV, and V and VI of the ipsilateral spinal cord of wild-type and knock-out mice injected with saline (Control: wild type, n = 9; knock-out, n = 6) or with formalin (Formalin treated: wild type, n = 9, knock-out, n = 6) into the hindpaw. **p < 0.01 compared with the wild-type and knock-out controls, respectively.

Figure 5.

Attenuation of the second phase of the formalin-induced nociceptive behavior in a dose-dependent manner after intracerebroventricular injection of U73122 and AIDA. Time courses after 10 min of pretreatment with 5.4 nmol of U73122 (n = 8; A), 100 nmol of AIDA (n = 8; B) and 100 nmol of MPEP (n = 5; B) compared with those after vehicle injection (n = 8) are shown. Each data point represents the mean duration ± SEM. **p < 0.01 compared with the vehicle injection. C, D, Dose dependence curves for the effects of U73122 (C) and AIDA (D) injection on the first phase (within the first 5 min after formalin injection; open squares) and the second phase (cumulative duration of the pain behavior between 15 and 45 min; filled circles), respectively. Each data point represents the mean duration ± SEM. ^**p < 0.01 compared with the vehicle injection. The number of animals is indicated for each point.

Figure 6.

Attenuation of formalin-induced nociceptive behavior in the second phase after thalamic injection with PLC inhibitors (A) and mGluR1 antagonists (B). A, Time course graphs after 10 min of pretreatment with 5.4 nmol of U73122 (n = 7) and 10 nmol of Et-18-OCH3 (n = 7) compared with that for vehicle injection (n = 12). B, Time courses after 10 min of pretreatment with 100 nmol of AIDA (n = 6) and 50 nmol of CPCCOEt (n = 9) compared with that after vehicle injection (n = 12). Each data point represents the mean time ± SEM. ^* p < 0.05; ^**p < 0.01 compared with the vehicle injection.

Figure 7.

Enhancement of formalin-induced nociceptive behavior in the second phase after thalamic injection with the mGluR1/5 agonist in wild-type mice but not in knock-out mice. The mean duration of pain behavior in the first phase (within the first 5 min after formalin injection; open bars) are shown after the second phase (cumulative duration of the pain behavior between 15 and 45 min; filled bars) in 10 min of pretreatment with vehicle or (RS)-DHPG in wild-type mice (n = 8) and knock-out mice (n = 8). *p < 0.01 compared with the vehicle injection. N.S., Not significant.

Figure 8.

Examples of spike histograms of VPL neurons of wild-type mice (A) and PLCβ4 knock-out mice (B) before (∼1 min) and after (∼60 min) formalin injection into the contralateral hindpaw. In wild-type mice, VPL neurons showed biphasic responses to formalin injection, whereas a monophasic response in the knock-out mice was observed. Arrows indicate the time when formalin was injected.

Figure 9.

Comparison of the response of VPL neurons to formalin injection between wild-type mice (n = 5) and PLCβ4 knock-out mice (n = 5). A, Total duration of the period in which the firing rate exceeded the basal activity level (see Materials and Methods) in the early phase (0-5 min) after formalin injection. B, Total number of spikes in the early phase (0-5 min) after formalin injection. C, Total duration of the period in which the firing rate exceeded the basal activity in the late phase (15-60 min). D, Total number of spikes in the late phase after formalin injection. Each data point represents the mean ± SD. *p < 0.01. N.S., Not significant.