Brain Research, 577 (1992) 321-325 t~) 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
321
BRES 25145
Role of the deep mesencephalic nucleus in the antinociception induced by stimulation of the anterior pretectal nucleus in rats Xiao-Min Wang, Bin Yuan and Zong-lian Hou Research Laboratory of Neurophysiology, Xi'an Medical University, Xi'an, Shaanxi (People's Republic of China)
(Accepted 21 January 1992) Key words: Anterior pretectal nucleus; Deep mesencephalic nucleus; Antinociception; Descending inhibition
The present study showed that the inhibitory effect on the tail-flick reflex (TF) of stimulating the deep mesencephalic nucleus (DpMe) was very similar to that produced by stimulation of the anterior pretectal nucleus (APtN). An electrolytic lesion of the ipsilateral DpMe greatly reduced the inhibitory effect of APtN stimulation on the TF. Furthermore, activating the neuronal cell bodies in DpMe but not the fibers of passage by microinjection of L-glutamate into this area was also shown to elicit inhibition of TF. On the other hand, inhibiting the neuronal cells in DpMe by microinjection of y-aminobutyric acid produced a marked reduction in the APtN-induced inhibition of TF, which was comparable to that produced by DpMe lesions. It is suggested that the APtN-induced antinociception is, at least in part, mediated via a relay through the DpMe. Recently, it has been suggested that the anterior pretectal nucleus (APtN) plays a role in nociceptive processing 9. Stimulation of the APtN has been reported to produce a strong and long-lasting suppression of the tailflick reflex (TF) to noxious stimuli 14-16'18'19. It has also been demonstrated that stimulation of APtN inhibits, selectively, the response to noxious skin heating of the lamina V-type spinal neurons deep in the dorsal horn 17' xs. However, little is known of the descending inhibitory pathway activated by APtN stimulation except that the inhibitory effect of APtN stimulation on the nociceptive responses of dorsal horn neurons is reported to be mediated via the dorsolateral funiculus 17. Since no direct projections are present from APtN to the spinal cord, to the nucleus raphe magnus (NRM), and to the periaqueductal gray (PAG) 1-3'2°'22, and the effects of APtN stimulation are different in some aspects from those of P A G and NRM stimulation 15-17'19, it is likely that the descending inhibitory pathway from APtN is relayed through structures other than P A G and NRM. Several morphological studies have demonstrated a dense projection from APtN to an area of the lateral mesencephalic reticular formation dorsolateral to the red nucleus (RN) 1-3'22'23, which has been named deep mesencephalic nucleus (DpMe) by Paxinos and Watson 13. It is known that stimulation of this area results in antinociception similar to that produced by pretectal stimulation 5'7A5'21, and that there exists a projection from this
area to the spinal cord 4'8A2. Therefore, it is possible that the APtN-induced antinociception might be mediated via a relay through the DpMe. This possibility was examined in the present study by: (1) comparing the antinociception produced by stimulation of APtN and DpMe in the same experimental conditions; (2) determining whether a localized electrolytic lesion of the DpMe reduces the APtN-induced antinociception; (3) observing the effects of microinjections of L-glutamate and y-aminobutyric acid (GABA) into DpMe on the TF and APtN-induced antinociception, respectively. Experiments were performed on adult Sprague-Dawley rats (200-250 g) and antinociception was assessed by analysis of changes in the tail-flick latency (TFL). Rats were initially anesthetized with sodium pentobarbital (45 mg/kg, i.p.) for surgery and a light anesthesia was maintained for the rest of the experiment by an intravenous infusion of sodium pentobarbital at a constant rate (4-5 mg/kg.h) 1°, which was sufficient to prevent spontaneous movements and yet allowed tail flick at stable latencies. The head was placed into a stereotaxic frame. Electrical stimuli (60 ms trains of 0.2 ms pulses at 300 Hz, 3/s for 15 s, 50-100 pA) were delivered to the APtN with a concentric electrode, and to the DpMe with a monopolar electrode, guided stereotaxically using the atlas of Paxinos and Watson 13. The coordinates for APtN were AP -4.6, Lat 1.7, DV 4.7 referred to bregma, and for DpMe were AP 3.2, Lat 1.8, DV 3.2 referred to interau-
Correspondence: X.-M. Wang, Research Laboratory of Neurophysiology, Xi'an Medical University, Xi'an, Shaanxi 710061, People's Republic of China.
322 ral line. L-Glutamate ( m o n o s o d i u m salt, 200 m M in saline) or G A B A (100 m M in saline) were microinjected into the D p M e via an injection cannula (0.2 m m o.d.) in volumes of 0.5-1.0 ~tl for a p e r i o d of over 2 min. The ipsilateral D p M e was lesioned by passing 0.4 m A anodal D C current for 20 s. For the tail-flick test, radiant heat from a projector lamp was applied to the ventral surface of the tail in an area of 3 × 6 m m 2, 4 - 6 cm from its tip. The current was adjusted if necessary at the beginning of each experiment in o r d e r that the baseline T F L was 3.5-4.5 s. A t least three baseline T F L were tested at 5-min intervals and then the brain stimulation was applied. Testing continued at intervals of 5 min for 1 h. If the T F was greatly suppressed, a cut-off time of 7 s was a d o p t e d to minimize damage to the skin. Each T F L obtained was normalized as an 'index of analgesia' ( I A ) using the formula19:
I
1.00.8 0.6
~,
0.4 0.2 0.0 -0.2 -i0
0
I0
20
30
40
50
60
TIME (min)
B
C
I A = (test T F L - a v e r a g e baseline T F L ) / ( 7 - a v e r a g e baseline T F L ) This formula gives a value of 0.0 if there was no change from the baseline value and 1.0 if the maximal inhibition of T F was produced. Results are presented as graphs of averaged I A values (mean + S . E . M . ) plotted against time for a group of animals. Statistical comparisons were made with the Student's t-test (paired or grouped). A t the end of each experiment, the stimulation site was m a r k e d with an electrolytic lesion (40 ~ A , 20 s), and the injection site was labeled by injection of 0.5/~1 Pontamine sky blue dye via the same cannula. The animal was killed by an overdose of anaesthetic and perfused through the heart with 10% formalin containing 2% potassium ferrocyanide. 60/~m serial coronal sections were stained with Neutral red. The locations of electrolytic lesion and microinjection site were d e t e r m i n e d according to the atlas of Paxinos and Watson 13. A p p a r e n t inhibition of the T F was observed folowing A P t N stimulation in 22 out of the 29 rats tested and following D p M e stimulation in 15 out of the 21 rats tested. A comparison b e t w e e n the effects of stimulating the two structures is shown in Fig. 1A. The T F L increased to its p e a k value immediately after stimulation of A P t N and then declined slowly, with baseline latencies recovered 50-60 min later. The effect of D p M e stimulation was very similar to that of A P t N stimulation, both in the magnitude and t i m e - c o u r s e of the T F L changes. The difference between the two curves was not statistically significant at any time point (grouped-sample t-test, P > 0.05). The sites of stimulation to A P t N and D p M e are shown in Fig. 1B and C, respectively. For A P t N stimulation, all effective sites (circles) are located in the rostral and dorsal part of A P t N . F o r D p M e stimulation, all
Fig. 1. A: the time-course of the effects of stimulating APtN (Q,
n = 22) and DpMe (A, n = 15) on the normalized TFL (IA, see text). The stimulation was delivered at the time indicated by the arrow. * indicates significant difference from baseline value (P < 0.05). B and C: the location of the stimulation sites, from which the data shown in A are obtained, is shown in representative crosssections of the brain with corresponding symbols. The open circles indicate sites from which little or no effect on the TFL was produced (not included in the graph shown in A). DLG, dorsal lateral geniculate nu; SNR, substantia nigra, reticular; MGV, medial geniculate nu, ventral: R, red nu.
effective sites (triangles) are located in a region of D p M e dorsolateral to the RN. The ineffective stimulation sites (open circles) are also shown, but the data are not included in Fig. 1A. Some animals were stimulated on two occasions, with the site of the second stimulation 0.5 m m d e e p e r than the first one. Some of them showed antinociception on the first occasion but not on the second and some gave the opposite results. This suggests that the effective spread of current from the electrode tip probably does not exceed 0.5 mm. In order to verify whether the descending inhibition produced by A P t N stimulation is m e d i a t e d with a relay in the D p M e , an electrolytic lesion was m a d e in the ipsilateral D p M e and the effect on the T F of A P t N stimulation was tested both before and after the lesion was made. In 13 out of the 20 rats tested, the lesions were histologically identified to be in the region of D p M e comparable to that where electrical stimulation was ef-
323
321
BRES 25145
Role of the deep mesencephalic nucleus in the antinociception induced by stimulation of the anterior pretectal nucleus in rats Xiao-Min Wang, Bin Yuan and Zong-lian Hou Research Laboratory of Neurophysiology, Xi'an Medical University, Xi'an, Shaanxi (People's Republic of China)
(Accepted 21 January 1992) Key words: Anterior pretectal nucleus; Deep mesencephalic nucleus; Antinociception; Descending inhibition
The present study showed that the inhibitory effect on the tail-flick reflex (TF) of stimulating the deep mesencephalic nucleus (DpMe) was very similar to that produced by stimulation of the anterior pretectal nucleus (APtN). An electrolytic lesion of the ipsilateral DpMe greatly reduced the inhibitory effect of APtN stimulation on the TF. Furthermore, activating the neuronal cell bodies in DpMe but not the fibers of passage by microinjection of L-glutamate into this area was also shown to elicit inhibition of TF. On the other hand, inhibiting the neuronal cells in DpMe by microinjection of y-aminobutyric acid produced a marked reduction in the APtN-induced inhibition of TF, which was comparable to that produced by DpMe lesions. It is suggested that the APtN-induced antinociception is, at least in part, mediated via a relay through the DpMe. Recently, it has been suggested that the anterior pretectal nucleus (APtN) plays a role in nociceptive processing 9. Stimulation of the APtN has been reported to produce a strong and long-lasting suppression of the tailflick reflex (TF) to noxious stimuli 14-16'18'19. It has also been demonstrated that stimulation of APtN inhibits, selectively, the response to noxious skin heating of the lamina V-type spinal neurons deep in the dorsal horn 17' xs. However, little is known of the descending inhibitory pathway activated by APtN stimulation except that the inhibitory effect of APtN stimulation on the nociceptive responses of dorsal horn neurons is reported to be mediated via the dorsolateral funiculus 17. Since no direct projections are present from APtN to the spinal cord, to the nucleus raphe magnus (NRM), and to the periaqueductal gray (PAG) 1-3'2°'22, and the effects of APtN stimulation are different in some aspects from those of P A G and NRM stimulation 15-17'19, it is likely that the descending inhibitory pathway from APtN is relayed through structures other than P A G and NRM. Several morphological studies have demonstrated a dense projection from APtN to an area of the lateral mesencephalic reticular formation dorsolateral to the red nucleus (RN) 1-3'22'23, which has been named deep mesencephalic nucleus (DpMe) by Paxinos and Watson 13. It is known that stimulation of this area results in antinociception similar to that produced by pretectal stimulation 5'7A5'21, and that there exists a projection from this
area to the spinal cord 4'8A2. Therefore, it is possible that the APtN-induced antinociception might be mediated via a relay through the DpMe. This possibility was examined in the present study by: (1) comparing the antinociception produced by stimulation of APtN and DpMe in the same experimental conditions; (2) determining whether a localized electrolytic lesion of the DpMe reduces the APtN-induced antinociception; (3) observing the effects of microinjections of L-glutamate and y-aminobutyric acid (GABA) into DpMe on the TF and APtN-induced antinociception, respectively. Experiments were performed on adult Sprague-Dawley rats (200-250 g) and antinociception was assessed by analysis of changes in the tail-flick latency (TFL). Rats were initially anesthetized with sodium pentobarbital (45 mg/kg, i.p.) for surgery and a light anesthesia was maintained for the rest of the experiment by an intravenous infusion of sodium pentobarbital at a constant rate (4-5 mg/kg.h) 1°, which was sufficient to prevent spontaneous movements and yet allowed tail flick at stable latencies. The head was placed into a stereotaxic frame. Electrical stimuli (60 ms trains of 0.2 ms pulses at 300 Hz, 3/s for 15 s, 50-100 pA) were delivered to the APtN with a concentric electrode, and to the DpMe with a monopolar electrode, guided stereotaxically using the atlas of Paxinos and Watson 13. The coordinates for APtN were AP -4.6, Lat 1.7, DV 4.7 referred to bregma, and for DpMe were AP 3.2, Lat 1.8, DV 3.2 referred to interau-
Correspondence: X.-M. Wang, Research Laboratory of Neurophysiology, Xi'an Medical University, Xi'an, Shaanxi 710061, People's Republic of China.
322 ral line. L-Glutamate ( m o n o s o d i u m salt, 200 m M in saline) or G A B A (100 m M in saline) were microinjected into the D p M e via an injection cannula (0.2 m m o.d.) in volumes of 0.5-1.0 ~tl for a p e r i o d of over 2 min. The ipsilateral D p M e was lesioned by passing 0.4 m A anodal D C current for 20 s. For the tail-flick test, radiant heat from a projector lamp was applied to the ventral surface of the tail in an area of 3 × 6 m m 2, 4 - 6 cm from its tip. The current was adjusted if necessary at the beginning of each experiment in o r d e r that the baseline T F L was 3.5-4.5 s. A t least three baseline T F L were tested at 5-min intervals and then the brain stimulation was applied. Testing continued at intervals of 5 min for 1 h. If the T F was greatly suppressed, a cut-off time of 7 s was a d o p t e d to minimize damage to the skin. Each T F L obtained was normalized as an 'index of analgesia' ( I A ) using the formula19:
I
1.00.8 0.6
~,
0.4 0.2 0.0 -0.2 -i0
0
I0
20
30
40
50
60
TIME (min)
B
C
I A = (test T F L - a v e r a g e baseline T F L ) / ( 7 - a v e r a g e baseline T F L ) This formula gives a value of 0.0 if there was no change from the baseline value and 1.0 if the maximal inhibition of T F was produced. Results are presented as graphs of averaged I A values (mean + S . E . M . ) plotted against time for a group of animals. Statistical comparisons were made with the Student's t-test (paired or grouped). A t the end of each experiment, the stimulation site was m a r k e d with an electrolytic lesion (40 ~ A , 20 s), and the injection site was labeled by injection of 0.5/~1 Pontamine sky blue dye via the same cannula. The animal was killed by an overdose of anaesthetic and perfused through the heart with 10% formalin containing 2% potassium ferrocyanide. 60/~m serial coronal sections were stained with Neutral red. The locations of electrolytic lesion and microinjection site were d e t e r m i n e d according to the atlas of Paxinos and Watson 13. A p p a r e n t inhibition of the T F was observed folowing A P t N stimulation in 22 out of the 29 rats tested and following D p M e stimulation in 15 out of the 21 rats tested. A comparison b e t w e e n the effects of stimulating the two structures is shown in Fig. 1A. The T F L increased to its p e a k value immediately after stimulation of A P t N and then declined slowly, with baseline latencies recovered 50-60 min later. The effect of D p M e stimulation was very similar to that of A P t N stimulation, both in the magnitude and t i m e - c o u r s e of the T F L changes. The difference between the two curves was not statistically significant at any time point (grouped-sample t-test, P > 0.05). The sites of stimulation to A P t N and D p M e are shown in Fig. 1B and C, respectively. For A P t N stimulation, all effective sites (circles) are located in the rostral and dorsal part of A P t N . F o r D p M e stimulation, all
Fig. 1. A: the time-course of the effects of stimulating APtN (Q,
n = 22) and DpMe (A, n = 15) on the normalized TFL (IA, see text). The stimulation was delivered at the time indicated by the arrow. * indicates significant difference from baseline value (P < 0.05). B and C: the location of the stimulation sites, from which the data shown in A are obtained, is shown in representative crosssections of the brain with corresponding symbols. The open circles indicate sites from which little or no effect on the TFL was produced (not included in the graph shown in A). DLG, dorsal lateral geniculate nu; SNR, substantia nigra, reticular; MGV, medial geniculate nu, ventral: R, red nu.
effective sites (triangles) are located in a region of D p M e dorsolateral to the RN. The ineffective stimulation sites (open circles) are also shown, but the data are not included in Fig. 1A. Some animals were stimulated on two occasions, with the site of the second stimulation 0.5 m m d e e p e r than the first one. Some of them showed antinociception on the first occasion but not on the second and some gave the opposite results. This suggests that the effective spread of current from the electrode tip probably does not exceed 0.5 mm. In order to verify whether the descending inhibition produced by A P t N stimulation is m e d i a t e d with a relay in the D p M e , an electrolytic lesion was m a d e in the ipsilateral D p M e and the effect on the T F of A P t N stimulation was tested both before and after the lesion was made. In 13 out of the 20 rats tested, the lesions were histologically identified to be in the region of D p M e comparable to that where electrical stimulation was ef-
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