[1] 13w The effect of dopamine on pain-related neurons in the parafascicular nucleus of rats

[1] 183w Dopamine (DA) regulates pain perception in the central nervous system (CNS). However, the mechanism of the action of DA in pain-related neurons of the parafascicular nucleus (Pf) is not clear. The present study aimed to determine the effect of DA and its receptor antagonist, droperidol on the pain-evoked responses of the pain-excited neurons (PEN) and pain-inhibited neurons (PIN) in the Pf of rats and to analyze the mechanisms underlying this effect. The trains of electric impulses applied to the sciatic nerve were used as noxious stimulation. The discharges of PEN and PIN in the Pf were recorded by using a glass microelectrode. The results showed that, in the Pf, intra-Pf microinjection of DA (5 lg/0.5 ll) increased the frequency of noxious stimulation-induced discharges of the PEN and decreased the frequency of those of the PIN, while the intra-Pf administration of droperidol (0.15 lg/0.5 ll) produced an opposite effect. On the basis of the above-mentioned findings, we could conclude that DA and its receptors in the Pf are involved in the modulation of the nociceptive response by regulating the discharges of PEN and PIN.

[1] 207w Dopamine (DA) is one of the most abundant catecholaminergic neurotransmitters in the central nervous system (CNS) occurring in a wide variety of animals, both vertebrates and invertebrates. Dopaminergic neurons are mainly concentrated in the substantia nigra zona compacta, ventral tegmental area, and hypothalamus. In the brain, DA has many functions, including important roles in behavior and cognition, voluntary movement, motivation and reward, inhibition of prolactin production (involved in lactation), sleep, mood, attention, and learning. Recently, DA has also been shown to play a critical role in modulating pain perception and natural analgesia within the CNS, including the spinal cord, the basal ganglia, insular cortex, cingulate cortex, thalamus, and periaqueductal gray (Wood 2008). DA receptors have been widely reported as pharmacological targets for the study of pain. The analgesic effect of DA is generally elicited when the DA D2 receptor is activated (Ansah et al. 2007;Coffeen et al. 2008;Magnusson and Fisher 2000;Meyer et al. 2009;Taylor et al. 2003). However, in the periaqueductal gray, the activation of the DA D1 receptor attenuates pain, presumably via the activation of neurons involved in descending inhibition (Flores et al. 2004). In addition, the activation of the D1 receptor in the insular cortex appears to attenuate subsequent pain-related behavior (Burkey et al. 1999).

[2] 151w The parafascicular nucleus (Pf), located in the medial thalamus, is one of the important activators in the ascending nociceptive pathway (Willis and Westlund 1997). Basal ganglia, the subthalamic nucleus, and the substantia nigra are projected to the Pf. Pallidal, cerebellar, tectal, and motor cortical inputs are received from the Pf (Ito and Craig 2005). Previous studies have indicated that the ablation of the Pf selectively reduces the emotional suffering associated with acute and chronic pain in humans (Mark et al. 1963) and reduces responses to noxious stimulation in animals (Kaebler et al. 1975). Electrophysiological recordings in animals demonstrated that noxious stimulation activates nociceptive-responsive neurons (Cheng et al. 2009;Dafny et al. 1990;Guo and Yuan 2008) in the Pf. The electrical activity of the neurons in the Pf varies with the intensity of pain stimulation and can be used as an objective criterion of pain (Sun et al. 1980;Weigel and Krauss 2004;Zhang 1973).

[3] 42w In the present study, we used extracellular electrophysiological recording techniques to determine the effects of DA and the DA receptor antagonist, droperidol on the evoked discharges of pain-related neurons in the Pf in normal rats and analyzed the mechanism underlying this effect.

[1] 197w In the DA group, the NIV of PEN was 7.92 ± 0.94 Hz, and the latency was 0.31 ± 0.08 s. Immediately after the intra-Pf administration of DA, the NIV of PEN began to increase and the latency began to shorten (Fig. 1b). At 2 min after the administration of DA, the NIV increased to 14.48 ± 2.97 Hz (F = 11.176, P = 0.0032), and the latency shortened to 0.14 ± 0.05 s (F = 25.864, P \ 0.0001). These effects reached the peak at 6 and 8 min after the administration of DA; the NIV increased to 20.42 ± 3.27 Hz (F = 46.666, P \ 0.0001) at 6 min, increasing by 157.83% compared with that before administration, and the latency shortened to 0.10 ± 0.04 s (F = 57.552, P \ 0.0001) at 8 min. During 0-12 min after the administration of DA, the NIV (F = 19.754, P \ 0.0001) and latency (F = 33.056, P \ 0.0001) of PEN showed obvious changes compared with those of the control group (Fig. 3). At 20 min after the administration of DA, the NIV and latency of PEN returned to the values observed before treatment.

[2] 191w The average NIV of PIN was -4.03 ± 0.70 Hz, and the average ID of PIN was 0.31 ± 0.05 s. At 2 min after the injection of DA, the average NIV began to reduce and ID began to prolong (Fig. 2b). The average NIV was -8.94 ± 1.73 Hz (F = 18.302, P = 0.0011), and the average ID was 0.53 ± 0.19 s (F = 5.378, P = 0.04) at 2 min after the injection. These reactions reached a peak value at 6 and 8 min after the injection; the average NIV was -13.65 ± 2.01 Hz (F = 81.517, P \ 0.0001) at 8 min, reducing by 238.71% compared with that before the injection, and the ID prolonged to 0.89 ± 0.24 s (F = 28.717, P = 0.0002) at 6 min. During 0-12 min after the injection, the average NIV (F = 60.542, P \ 0.0001) and ID (F = 19.249, P = 0.001) of PIN showed obvious changes compared with those of the control group (Fig. 4). At 20 min after the injection, the NIV and ID of PIN returned to the values observed before treatment.

[3] 129w At 2 min after the administration of droperidol, the average NIV of PEN significantly decreased from 7.94 ± 0.77 to 4.63 ± 1.33 Hz (F = 57.637, P \ 0.0001), and the latency prolonged from 0.31 ± 0.08 to 0.69 ± 0.31 s (F = 8.176, P = 0.009) (Fig. 1c). These reactions reached a peak value at 6 min after the injection; the average NIV decreased to 3.28 ± 0.83 Hz (F = 202.669, P \ 0.0001), and the latency prolonged to 0.96 ± 0.37 s (F = 14.049, P = 0.0011). During 0-12 min after the injection, the NIV (F = 108.119, P \ 0.0001) and latency (F = 9.882, P = 0.005) of PEN showed obvious changes compared with those of the control group (Fig. 3).

[4] 130w At 2 min after the administration of droperidol, the average NIV of PIN significantly increased from -4.15 ± 0.74 to -2.88 ± 0.96 Hz (F = 7.308, P = 0.016), and the ID shortened from 0.31 ± 0.07 to 0.19 ± 0.04 s (F = 16.039, P = 0.0011) (Fig. 2c). These reactions also reached a peak value at 8 min after the injection; the average NIV increased to -1.61 ± 0.61 Hz (F = 93.828, P \ 0.0001), and the ID shortened to 0.12 ± 0.03 s (F = 28.396, P \ 0.0001). During 0-12 min after the injection, the NIV (F = 36.542, P \ 0.0001) and ID (F = 26.348, P \ 0.0001) of PIN showed obvious changes compared with those of the control group (Fig. 4).

[1] 210w Our study showed that in the Pf, DA potentiated the electric activities of the evoked discharges of PEN and inhibited those of PIN, while droperidol inhibited the electric activities of the evoked discharges of PEN and simultaneously potentiated those of PIN. The responses of neurons showed that DA and droperidol play different roles in pain modulation in the Pf. PEN and PIN can be considered as indexes of pain research (Bian et al. 1993). The responses of PEN and PIN are completely opposite to identical substance, which synchronously account for the effects of DA and droperidol during pain modulation. During the preliminary experiment, we used different dosages of DA and found that DA acted in a dose-dependent manner. The used dosage (5 lg/0.5 ll) of DA was both adequate and effective for recording neuronal discharges. It is interesting to note that the curves of the droperidol (5 lg/0.5 ll) group were not as distinct as those of the DA group. This could be because the used dose of droperidol was considerably high. The small dose of droperidol (0.15 lg/0.5 ll) was both adequate and effective for recording neuronal discharges. We attempted to increase the dosage of droperidol during the experiments. However, it seemed impossible to record a complete neuronal discharge.

[2] 241w It is well known that the Pf plays a major role in nociception in animals and humans. There are two types of Pf neurons that are classified according to their response pattern following noxious stimulation. One type of Pf neurons that were excited after the stimulation is classified as PEN, and the second type of Pf neurons whose responses were inhibited after stimulation is classified as PIN. Both the types of neurons can be used as an objective criterion of nociceptive responses. Pain-evoked discharges of Pf neurons can be inhibited by stimulation of brain regions such as the central gray matter, dorsal raphe nucleus, and locus coeruleus (Li et al. 1984;Liu et al. 1993;Qiao and Dafny 1988;Zhang et al. 1998). Inhibition of pain-evoked discharges of Pf neurons can also result from analgesic treatment, including acupuncture, as well as by analgesic drugs such as morphine (Zhang 1973). In our experiment, we found that DA increased the evoked discharges of PEN and simultaneously attenuated those of PIN, and DA receptor antagonist, droperidol attenuated the evoked discharges of PEN and simultaneously increased those of PIN. Therefore, the present study provides additional electrophysiological evidence for the antinociceptive effects of droperidol and hyperalgesic response of DA by intra-Pf injection. Simultaneous recording of the responses of dopaminergic neurons stimulus artifact, empty diamond injection of saline, filled diamond injection of DA, empty triangle injection of droperidol, cross before injection; 0, 2, 4, 10, 20, time after injection (min)

[3] 235w to peripheral noxious stimuli in the substantia nigra (SN) and the lateral habenula (HbL) confirms that the responses are opposite: excitation of the neurons in the HbL corresponded to an inhibition of the neurons in the SN, and vice versa. The responses of the HbL and Pf neurons to noxious inputs are similar (Benabid 2009). These effects of DA and droperidol may be mediated by evoked dopaminergic neurons, which project to the Pf. Uparrow stimulus artifact, empty diamond injection of saline, filled diamond injection of DA, empty triangle injection of droperidol, cross before injection; 0, 2, 4, 10, 20, time after injection (min) Fig. 3 Influence of intra-Pf injection of different substance on the NIV (a) and latency (b) of PEN in the Pf. Thick line on the X axis injection of substance, cross before injection; 0, 2,…, 22, time after injection (min); values are given by means ± SEM. *P \ 0.05, **P \ 0.01, # P \ 0.05, ## P \ 0.01 when compared with saline group Fig. 4 Influence of intra-Pf injection of different substance on the NIV (a) and ID (b) of PIN in the Pf. Thick line on the X axis injection of substance, cross before injection; 0, 2,…, 22, time after injection (min); values are given by means ± SEM. *P \ 0.05, **P \ 0.01, # P \ 0.05, ## P \ 0.01 when compared with saline group

[4] 293w Results of previous studies suggest that the dopaminergic system in the brain is primarily involved in the modulation of nociception. Some studies indicated that low dopaminergic activity can be associated with high pain sensitivity, and vice versa (Cervenka et al. 2006;Ja ¨a ¨skela ¨inen et al. 2001;Treister et al. 2009;Wood et al. 2007). However, a team of researchers at the University of Michigan induced pain in their subjects and concluded that the DA system in the brain is highly active when an individual experiences pain, such as muscle pain. DA plays an important role in pain modulation, but there are some discrepancies in the reported effects of DA, ranging from excitation to inhibition of neurons. The different effects were probably induced in the different regions of the CNS: a DA D1 agonist was reported to increase hyperalgesia in the nucleus raphe magnus (Phillips et al. 1992) and the spinal cord (Gao et al. 2001), while DA D1 antagonist was reported to attenuate the analgesia in the periaqueductal gray (Flores et al. 2004), anterior cingulate cortex (Lo ´pez-Avila et al. 2004), and rostral agranular insular cortex (Burkey et al. 1999). In addition, different DA receptors or different experimental approaches were probably responsible for the different effects produced. The activation of D2 or the blockade of the D1 receptor elicited antinociception in the rostral agranular insular cortex (Coffeen et al. 2008) and the spinal cord (Gao et al. 2001). Intrathecal administration of the DA agonist, apomorphine failed to influence the tail-flick latency even at high doses, whereas it produced a dose-dependent increase in the hot plate and acetic acid writhing responses (Jensen and Yaksh 1984). Intraperitoneal injection of cocaine has been reported to activate DA receptors in the Pf producing analgesia (Shyu et al. 1992).

[5] 189w In the CNS, there are five subtypes of DA receptors, namely, D1 through D5, and the DA D2 receptors are the most important ones in pain modulation. The D2 receptors are localized in the Pf (Wamsley et al. 1989). D2 receptors sites have been reported to be present in the Pf (Rieck et al. 2009). High expression of the D2 mRNA is evident in the Pf (Hurd et al. 2001). DA exerts its effects by acting on its receptor; droperidol is a DA receptor antagonist, and it plays a primary role mostly by blocking the DA D2 receptors (Cure et al. 2004). In the present study, we found that the administration of DA produced algetic action. On the other hand, droperidol produced antinociception in the Pf. This suggests that DA directly induces hyperalgesia in the Pf. Therefore, in the Pf, DA may play a role via the activation of the DA D2 receptors, and droperidol may play a role via the blockade of the DA D2 receptors. However, whether other DA receptor subtypes also play a critical role in the analgesic action in the Pf needs to be investigated.

[6] 35w In conclusion, the data of this study indicated that DA and Pf are involved in pain modulation. The activation of the pathway related to DA receptors may be responsible for the effect produced by DA.

[1] 130w Male and female Sprague-Dawley rats (200-260 g; Animal Centre of Second Affiliated Hospital of Harbin Medical University; Certificated No. 09-2-1) were used in this study. Rats were maintained on a lighting schedule of 14 h light and 10 h dark at a temperature of 22 ± 2°C. Standard food and water were provided ad libitum. The rats were randomly and equally divided into three groups: (1) control group, intra-Pf administration of 0.5 ll saline; (2) DA group, intra-Pf administration of 5 lg/0.5 ll DA; and (3) droperidol group, intra-Pf administration of 0.15 lg/0.5 ll droperidol. All injections were completed within 2 min by using a microliter syringe. Materials DA, droperidol, urethane, and saline were obtained from standard suppliers in China. Tubocurarine chloride was purchased from Sigma (St Louis, MO, USA).

[2] 305w Rats were anesthetized with 20% urethane (ip., 1 g/kg). The trachea was intubated for artificial respiration. Two openings were made in the skull, and they were covered with liquid paraffin. Cerebellomedullary cistern was opened to reduce the intracranial pressure. The right sciatic nerves were isolated for noxious stimulation. Rats were fixed on a stereotaxic frame (SN-2; Narishige, Japan). Warm saline was injected into the peritoneal cavity of the rats after surgery. Their body temperature was maintained at 36-37°C. After 4-10 min, the rats were paralyzed with tubocurarine chloride (1 mg/kg), and artificial ventilation of 60 times/min was maintained. Single-unit recordings were performed using a glass microelectrode (0.5-1.0 lm, DC resistance 10-30 MX) filled with KCl (3 mol/l). The glass microelectrode was inserted into the Pf (AP, -2.2 to -2.6 mm; R or L, 1.0-1.4 mm; H, 5.8-6.5 mm) by using a micromanipulator (SM-21; Narishige, Japan) (Pellegrino et al. 1979). Another glass microelectrode filled with drugs was inserted into the Pf (AP, -2.4 mm; R or L, 1.2 mm; H, 6.0 mm) by using the micromanipulator (SM-11; Narishige, Japan), and the drugs were administered using an automatic pumping note instrument. The tidal volume, heart rate and body temperature were monitored during the experiment. The electrical activity was amplified by using a microelectrical amplifier and recorded by the biological experimental system, which was simultaneously monitored using an oscilloscope (VC-10; Nihon Konden, Japan). The neural discharges were recorded concomitantly with the electrical stimulation of the sciatic nerves by using a double stainless electrode (delay 0, interval 5 ms, duration 0.3 ms, train 5) (SEN-3301; Nihon Konden, Japan) as noxious stimulation. Articular movement and hair touch were set as the non-noxious stimulation to identify the pain-related neurons. The discharge of each neuron was recorded 3 times every 2 min, and the 30-min recording was considered as a complete one.

[3] 48w Pain-related neurons refer to pain-excited neuron (PEN) and pain-inhibited neuron (PIN). Neurons that responded by increasing the discharge frequency to noxious stimulation were defined as PEN (Zhang 1973), while those that responded by decreasing the discharge frequency to noxious stimulation were defined as PIN (Sun et al. 1980).

[4] 74w (1) Net-increased value (NIV, Hz) refers to the difference of PEN or PIN between the average frequency of evoked discharges after noxious stimulation and the average frequency of discharges within 2 s before noxious stimulation. (2) Latency (s) refers to the time from the noxious stimulation to the appearance of the PEN discharges. (3) Inhibitory duration (ID, s) refers to the latency time from the noxious stimulation to the appearance of the PIN discharges.

[5] 29w At the end of the experiment, pontamine sky blue was diffused out from the microelectrode with a negative direct current (30 lA, 15 min) to identify the tip position.

[6] 62w Data were scanned into the computer with Powerlab/8sec (ADInstruments) and analyzed with Chart v5.3 software (Australian). All data were expressed a mean ± SEM and analyzed using the SPSS 16.0 software. Statistical differences were evaluated by repeated measures ANOVA, while the post hoc test was used to compare the differences between the two groups, P \ 0.05 was considered as statistically significant.

[1] 25w Significant modifications in neither PEN nor PIN of the Pf neurons were observed during the 30 min after the administration of saline (Figs. 1a, 2a).