Neuropathic pain-induced enhancement of spontaneous and pain-evoked neuronal activity in the periaqueductal gray that is attenuated by gabapentin

Abstract

Neuropathic pain is a debilitating pathological condition that is poorly understood. Recent evidence suggests that abnormal central processing occurs during the development of neuropathic pain induced by the cancer chemotherapeutic agent, paclitaxel. Yet, it is unclear what role neurons in supraspinal pain network sites, such as the periaqueductal gray, play in altered behavioral sensitivity seen during chronic pain conditions. To elucidate these mechanisms, we studied the spontaneous and thermally evoked firing patterns of ventrolateral periaqueductal gray (vlPAG) neurons in awake-behaving rats treated with paclitaxel to induce neuropathic pain. In the present study, vlPAG neurons in naive rats exhibited either excitatory, inhibitory, or neutral responses to noxious thermal stimuli, as previously observed. However, after development of behavioral hypersensitivity induced by the chemotherapeutic agent, paclitaxel, vlPAG neurons displayed increased neuronal activity and changes in thermal pain-evoked neuronal activity. This involved elevated levels of spontaneous firing and heightened responsiveness to nonnoxious stimuli (allodynia) as well as noxious thermal stimuli (hyperalgesia) as compared with controls. Furthermore, after paclitaxel treatment, only excitatory neuronal responses were observed for both nonnoxious and noxious thermal stimuli. Systemic administration of gabapentin, a nonopioid analgesic, induced significant dose-dependent decreases in the elevated spontaneous and thermally evoked vlPAG neuronal firing to both nonnoxious and noxious thermal stimuli in rats exhibiting neuropathic pain, but not in naive rats. Thus, these results show a strong correlation between behavioral hypersensitivity to thermal stimuli and increased firing of vlPAG neurons in allodynia and hyperalgesia that occur in this neuropathic pain model.

Fig. 1. Responses of vlPAG neurons to noxious stimuli

(A) Representative vlPAG microwire recording sites indicate that most electrode placements were localized to the vlPAG at the anterior-posterior coordinates listed, according to the atlas of Paxinos and Watson [48]. Rectangles indicate the locations of vlPAG_on neurons; ovals indicate the locations of vlPAG_off neurons and triangles indicate the locations of vlPAG_neutral neurons. (B) Mean frequency of vlPAG spontaneous and evoked neuronal firing in response to noxious thermal stimulation. We observed three subclasses of neuronal responses in the vlPAG: “on,” “off,” and “neutral.” (C) A typical example of the rate meter histogram (80 ms bin width) analysis of vlPAG_on neurons that were excited by the thermal stimulus (51°C), which exhibited a significant increase in firing rate, as compared to the pre-stimulus spontaneous firing rate. (D) A typical example of the rate meter histogram (80 ms bin width) of vlPAG_off neurons that exhibited an inhibitory response to the noxious thermal stimulus characterized by significantly decreased firing rates, as compared to the spontaneous firing rate. (E) A typical example of the rate meter histogram (80 ms bin width) of vlPAG_neutral neurons that did not exhibit any detectable change in the neuronal firing in response to the thermal stimulus. *P < 0.05 (Two way ANOVA); each value is the mean ± S.D; Mean frequency (Hz) represent the mean of three trials; vlPAG_on responses (n=32), vlPAG_off responses (n=16) and vlPAG_neutral responses (n=20). The grey overlay of each histogram represents the duration of the thermal stimulus administered to the paw. Waveforms of the action potentials that were used to generate the rate histograms are shown above each histogram to the right.

Fig. 2. Response changes of vlPAG_on neurons induced by paclitaxel administration

(A) Thermal stimulation at 38°C and 42°C did not evoke any consistent paw withdrawal responses during the 30 s stimulus period in rats prior to treatment with the paclitaxel. Whereas, noxious thermal stimulation at 48°C, and 51°C evoked paw withdrawal in all the rats. The paclitaxel protocol significantly reduced paw withdrawal latencies (PWLs) as compared to pre-treatment and vehicle groups. Significant hypersensitivity to non-noxious thermal stimulus and noxious thermal stimulus after paclitaxel treatment indicates the presence of thermal allodynia and hyperalgesia. (B) Induction of neuropathic pain resulted in the emergence of spontaneous burst firing pattern, whereas vehicle treated rats did not exhibit any spontaneous burst firing pattern. Noxious thermal stimulus at 51°C resulted in a significant increase of the burst firing frequency in paclitaxel-treated rats as compared to pre-treatment and vehicle treated rats. (C) Timeline of vlPAG recordings pre and post paclitaxel treatment. (D) Changes in spontaneous neuronal firing (F(1,8)= 77.25; p< 0.0001) is directly correlated with PWLs changes observed after neuropathic pain. (E) Burst firing (F(1,22) = 101.6; p< 0.0001) observed after the paclitaxel treatment directly correlates with changes in PWLs. (F) The presentation of non-noxious thermal stimulus to the paw did not evoke any changes in the vlPAG_on neuronal responses of vehicle-treated rats. In the paclitaxel-induced hyperalgesic state vlPAG_on neurons exhibited significantly increased neuronal excitation to previously non-noxious thermal stimulus. vlPAG_on neurons showed significantly increased neuronal excitation to the noxious thermal stimulus as compared to the pre-treatment period, whereas in vehicle-treated rats we did not see any changes in neuronal response to noxious thermal stimulus compared to pretreatment. (G) Representation of the rate meter histograms (80 ms bin width) analysis of a typical vlPAG_on neuronal response during noxious thermal stimulus (51°C) from vehicle (top trace) and paclitaxel-treated rats (lower trace). vlPAG_on neurons in paclitaxel-treated rats showed significantly increased spontaneous and excitatory neuronal responses to the noxious thermal stimulus compared to vlPAG_on neuronal responses recorded from the vehicle treated rats. The grey overlay represents the thermal stimulation. Waveforms of the action potentials that were used to generate the rate histograms are shown above each histogram to the right. * P < 0.05, ** P < 0.01; compared to pre-treatment levels, # P < 0.05 compared to vehicle treated. Two-way ANOVA followed by Bonferroni post hoc test. Each value is the mean ± SD; PWLs represent the mean of three trials; N=14 rats per group.

Fig. 3. Responses from electrodes that recorded vlPAG_off neuronal responses before and from the same electrode after the neuropathic pain protocol

(A) Prior to the paclitaxel protocol (pre-paclitaxel) no detectable neuronal inhibition to the non-noxious thermal stimulus (38° and 42°C) vlPAG_off was seen (n=16). However, noxious thermal stimulus evoked vlPAG_off neuronal inhibitory responses (48° and 51°C, N=16 neurons). After paclitaxel treatment, noxious thermal stimulus did not evoke neuronal inhibition in the vlPAG neurons recorded from the same electrodes but excitatory responses were consistently seen. Prior to paclitaxel treatment, noxious thermal stimulus evoked inhibitory neuronal responses (N=16) in vlPAG_off neurons, whereas after paclitaxel treatment only excitatory neuronal responses was observed to both non-noxious thermal stimulus and noxious thermal stimulus from the same electrodes. (B) Example of the rate meter histogram (80 ms bin width) analysis of a typical vlPAG_off neuronal response during noxious thermal stimulus (51°C) from vehicle and (C) from the same electrode after paclitaxel treatment. Prior to paclitaxel treatment vlPAG_off neurons exhibited an inhibitory response to the noxious thermal stimulus. vlPAG neurons in paclitaxel-treated rats recorded from the same electrodes that previously recorded vlPAG_off neurons showed significantly increased spontaneous activity and excitatory neuronal responses to the noxious thermal stimulus. The grey overlay represents the thermal stimulation. Waveforms of the action potentials that were used to generate the rate histograms are shown above each histogram to the right. (D) Changes in thermal stimulus evoked neuronal firing (N=37 neurons, F(1,35)= 100.3; p< 0.0001) is directly correlated with PWLs changes observed after neuropathic pain. * P < 0.05, ** P < 0.01; compared to pre-treatment levels, # P < 0.05 compared to vehicle treated group. Two-way ANOVA followed by Bonferroni post hoc test. Each value is the mean ± SD; N=14 rats per group.

Fig. 4. Responses from electrodes that recorded vlPAG_neutral neuronal responses before and from the same electrodes after the neuropathic pain protocol

(A) An example of a typical rate meter histogram (80 ms bin width) of a vlPAG_neutral neuronal response during noxious thermal stimulus (51°C) from vehicle and (B) paclitaxel treated rats recorded from the same electrode. Prior to paclitaxel treatment vlPAG neurons exhibited no change in neuronal activity to the noxious thermal stimulus. Paclitaxel-treated rats showed significantly increased spontaneous activity and excitatory neuronal responses to the noxious thermal stimulus unlike the vlPAG neuronal responses recorded from the vehicle treated rats. The grey overlay represents the thermal stimulation. Waveforms of the action potentials that were used to generate the rate histograms are shown above each histogram to the right. (C) Prior to paclitaxel treatment in vlPAG_neutral neurons no detectable responses to the non-noxious thermal stimulus (38°C and 42°C) or noxious thermal stimulus (48°C and 51°C) were seen. However, after the paclitaxel treatment protocol excitatory neuronal responses to non-noxious thermal stimulus and noxious thermal stimulus in vlPAG neurons were observed from the same electrodes. (D) PCA shows prior to paclitaxel treatment, there are three different clusters of single units whereas post paclitaxel treatment only one cluster of single units was observed. (E) Representative example of waveforms recorded from the vlPAG before and after paclitaxel treatment. * P < 0.05, ** P < 0.01; compared to pre-treatment levels, # P < 0.05 compared to vehicle treated group. Two-way ANOVA followed by Bonferroni post hoc test. Each value is the mean ± SD; N=14 rats per group.

Fig. 5. Effects of gabapentin on responses of vlPAG_on neurons

(A) Timeline of vlPAG recordings to evaluate effects of gabapentin in pre and post paclitaxel treated rats. (B) Systemic (intraperitoneal) administration of gabapentin resulted in dose-dependent antinociceptive effects on paw withdrawal latencies in paclitaxel treated rats but not in naïve (vehicle treated) rats. (C) Administration of gabapentin (50 mg/kg) reversed thermal hyperalgesia in paclitaxel treated rats but not in vehicle-treated rats. (D) Gabapentin administration (50 mg/kg) significantly reduced vlPAG_on spontaneous and noxious thermal stimulus (51°C)-evoked burst neuronal firing in paclitaxel-treated rats but not in vehicle-treated rat. (E) Administration of gabapentin resulted in dose-dependent decrease in vlPAG_on neuronal firing in paclitaxel treated rats but not in vehicle-treated rats. (F) Representative rate meter histograms show a significant reduction of spontaneous and noxious thermal stimulus (51°C) evoked activity in the vlPAG_on neuron by gabapentin (50 mg/kg) i.p in the PAG neurons in paclitaxel-treated rats one hr after gabapentin treatment. The grey overlay represents the thermal stimulation. (G) Systemic administration of gabapentin (50 mg/kg) resulted in significant reduction in the vlPAG_on neuronal firing evoked by the non-noxious thermal stimulus (38°C and 42°C) and noxious thermal stimulus (48°C and 51°C) in the paclitaxel-treated rats, but no effects were seen in vehicle-treated rats. (H) Gabapentin mediated attenuation of evoked neuronal firing (F(1, 37)= 56.44; p< 0.0001) and (I) burst firing (F(1,22)= 82.53; p< 0.0001) is directly correlated with elevated PWLs observed after gabapentin treatment in the paclitaxel-treated rats. * P < 0.05, ** P < 0.01; compared to pre-treatment levels. Two-way ANOVA followed by Bonferroni post hoc test. Each value is the mean ± SD; PWLs represent the mean of three trials; N=14 rats per group.