154
Brain Research, 582 (1992) 154-158 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
BRES 25204
Activity of neurons in the rostral medulla of the halothane-anesthetized rat during withdrawal from noxious heat Michael M. Morgan and Mary M. Heinricher Department of Neurology, University of California, San Francisco, CA 94143-0114 (USA) Key words: Pain modulation; Nociception; Nucleus raphe magnus; Tail flick; Barbiturate; v-Aminobutyric acid
The physiological and pharmacological properties of two classes of putative nociceptive modulatory neurons have been extensively characterized in the rostral ventromedial medulla (RVM) of the barbiturate-anesthetized rat. 'On-cells' show a burst of activity, and 'off-cells' a sudden pause immediately preceding the occurrence of nocifensor reflexes. In the present study, we have characterized the reflex-related activity of RVM neurons in halothane-anesthetized rats to determine whether the properties of these neurons are dependent on barbiturate anesthesia. Both on- and off-cells were identified in this preparation. Repeated noxious stimulation was associated with a high level of ongoing activity in on-cells, and a low level in off-cells. These data thus demonstrate that the previously described reflex-related changes in RVM neuron activity are not specific to barbiturate-anesthetized preparations, and that a failure to demonstrate off-cells in some studies may result from these neurons being inactive following repeated testing with noxious stimuli. The importance of the rostral ventromedial medulla ( R V M ) in modulating nociceptive transmission is widely acknowledged, yet the properties of the physiologically, pharmacologically and anatomically heterogeneous neuronal population found in this region have only recently come under careful scrutiny. Two classes of neurons characterized by opposite changes in activity that are temporally correlated with the execution of nocifensor reflexes have been identified, and these classes a p p e a r to have distinct and possibly opposing roles in nociceptive modulation. On-cells, which have been argued to have a permissive or even facilitating influence on nociception, and whose activity is depressed by opioids, are characterized by a sudden burst of activity that precedes the occurrence of the tail flick and other nocifensor reflexes 2'5"8A4'22. Off-cells, which are likely to exert a net inhibitory effect on nociception, and which are activated following opioid administration 5'1°, are identified by a pause in firing just prior to nocifensor reflexes 8. This pause presumably permits the reflex to occur 9. We have recently provided strong evidence that the reflex-related off-cell pause is m e d i a t e d by 7-aminobutyric acid ( G A B A ) , as it is eliminated by iontophoretic application of G A B A A receptor antagonists 15. Furthermore, microinjection of the same compounds into the R V M is antinociceptive 7'16, demonstrating that G A B A m e d i a t e d transmission is involved in the nociceptive modulating functions of this region. However, almost all of the above studies were carried
out in barbiturate-anesthetized animals. A s barbiturates are known to act at the G A B A A receptor to enhance G A B A transmission 25"29, it is possible that the physiological and pharmacological properties of R V M neurons described in these experiments were specific to rats maintained under barbiturate anesthesia, and it would be valuable to determine if these neurons can be identified in other preparations. In the present study, tail flick-related activity of R V M neurons was examined in rats anesthetized with the volatile anesthetic halothane. Male S p r a g u e - D a w l e y rats (260-320 g) were used in these experiments. Anesthesia was induced by placing the animals in a halothane chamber, and a tracheal cannula inserted for delivery of a halothane/oxygen mixture throughout the r e m a i n d e r of the experiment. H a l o t h a n e concentration was maintained at 2% during surgical preparation, and subsequently reduced (0.7-1.0%) until discrete withdrawal reflexes to noxious heat and pinch could be elicited. No spontaneous movements or signs of distress were evident when this concentration was used. Animals were allowed to breathe spontaneously, and body t e m p e r a t u r e was maintained with a 37°C circulating water blanket. A gold- and platinum-plated stainless steel microelectrode was advanced through the R V M until the activity of a single neuron could be isolated. Units were characterized as on-, off- or neutral cells based on changes in activity related to the occurrence of the tail flick reflex elicited by application of noxious radiant heat to the yen-
Correspondence: M.M. Morgan, Department of Neurology, Box 0114, University of California, San Francisco, CA 94143-0114, USA.
155 tral surface of the tail. As defined previously 8, on-cells showed an increase, and off-cells a decrease in activity immediately preceding the occurrence of the tail flick. Neutral cells showed no change in activity associated with the tail flick. Cell activity was recorded continuously for a period of 9-27 min. The heat stimulus consisted of a linear increase at 1.8°C/s from the 35°C holding temperature until the tail was withdrawn (3.1-9.2 s), at which time the trial was terminated and the temperature returned to 35°C. Sites 3, 5 and 7 cm from the tip of the tail were tested in rotation. In one series of experiments, multiple units isolated in succession along a single dorsal-to-ventral penetration of the medulla were studied in seven animals. Tail flick trials were given at 1.5-4 min intervals. In a second series, only one cell was studied in each of 10 animals so as to avoid cumulative effects of repeated testing with a noxious stimulus. In these experiments, only spontaneously active units that displayed an increase or decrease in activity preceding withdrawal of the hindpaw to a brief noxious pinch were chosen for study. Tail flick trials were carried out at 3 min intervals. At the qompletion of the experiment, electrolytic lesion(s) were made at a recording site or sites. Animals were given a lethal injection of pentobarbital, and perfused intracardially with saline followed by formalin (10%). Brains were removed, sectioned at 50 p m and stained with Cresyl violet for histological verification of the recording site. In the first series of experiments, 3 - 6 neurons were studied in each of 7 rats. Of 30 neurons, 17 were classified as on-cells, 5 as off-cells, and 8 as neutral cells. Histograms of tail flick-related activity generated for 5 cells recorded from a single rat are displayed in Fig. 1. Cells of all 3 classes were found in this animal. The first neuron encountered (cell 1) showed no change in activity associated with the tail flick and was thus classified as a neutral cell. Two off-cells (cells 2 and 4) and two on-cells (cells 3 and 5) were also found in this animal. Each of the offcells exhibited a pause in firing beginning immediately before the tail flick, but the more dorsal (cell 2) had a much higher level of spontaneous activity, and was thus more easily identified as an off-cell. (It should be noted that two of the off-cells encountered in another animal in this series had high levels of activity (20-40 Hz), and these neurons often showed a decrease, but not a complete cessation of firing, associated with the tail flick reflex.) The two on-cells isolated in this animal also showed distinct patterns of spontaneous activity. The first on-cell encountered (cell 3) was relatively inactive, which made the marked increase in firing associated with the tail flick quite visible. The last neuron tested (cell 5) had a high
level of spontaneous activity, with a relatively small but consistent increase in firing rate associated with the tail flick. In the second series of experiments, 6 on-cells and 4 off-cells were studied in 10 rats. The tail flick-related changes in firing of these neurons were similar to those described above. The spontaneous firing of these neurons was recorded for 9 min prior to initiating tail flick testing. As reported for on- and off-cells in barbiturateanesthetized rats 3, these neurons showed fluctuations in activity during this period, with intervals of activity alternating with periods of relative quiescence (Fig. 2). The approach used in the second series of experiments also revealed an effect of repeated tail flick testing. Fig. 3 shows one neuron with a low firing rate in the intervals prior to each of the first 3 tail flick trials. However, a sustained increase in activity was induced by the third trial, and this effectively masked tail flick-related increases in firing in subsequent trials, as shown in a comparison of the two peri-event histograms generated using the first 3 and last 3 trials. The histogram constructed using the last 3 trials alone would have caused the neuron to be classified as a neutral cell. A drastic change in activity resulting from repeated testing was not seen in the other 5 on-cells tested. This transition occurs rapidly, and 4 of the neurons already had high levels of spontaneous activity when first encountered. The on-, off- and neutral cells studied in Exp. 1 and 2 were distributed throughout the RVM. The only relationship between cell class and location was an increased probability of encountering neutral cells at the dorsal or ventral borders of the RVM. There was no discernible relationship between the spontaneous activity of on- and off-cells and location within RVM. These data demonstrate that R V M neurons showing tail flick-related changes in activity can be identified in halothane-, as well as barbiturate-anesthetized rats. These neurons also exhibited the cyclic alternation between quiet and active periods reported in barbiturateanesthetized animals 3. Although the mechanisms by which halothane produces its effects are not fully understood, it is widely thought to act by non-specifically altering the properties of neuronal membranes 2°. In contrast, a receptor-mediated potentiation of G A B A transmission is thought to be central to the actions of barbiturates 25'29. Thus, taken together with the report that anatomical differences exist between these two cell classes 18'19, the present observation of both cell classes in halothane-anesthetized animals demonstrates that oncells and off-cells are not an epiphenomenon of barbiturate anesthesia. The present observations also underscore the importance of the stimulation protocol and use of a search stim-
156 25 i
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Fig. 1. Recording sites and peri-event histograms showing tail flick-related changes in activity (average of 4-6 trials; 100 ms bins) for 5 neurons encountered in a single electrode penetration. The first neuron, a neutral cell dorsal to the RVM, showed no change in activity associated with the tail flick. An off-cell was recorded at sites 2 and 4, and an on-cell at sites 3 and 5. Notice that the off-cell recorded at site 4 has a much lower level of spontaneous activity than the off-cell recorded at site 2. Conversely, the on-cell recorded at site 5 has a much higher level of spontaneous activity that the on-cell recorded at site 3. These differences presumably result from repeated nociceptive testing during characterization of cells 1-3. (-2.3 mm from the interaural line; P, pyramidal tract; VII, facial nucleus and tract.)
ulus in studying n e u r o n s i n v o l v e d in n o c i c e p t i o n . A rev i e w o f the literature indicates that the p r o p o r t i o n s of R V M n e u r o n s r e p o r t e d to be excited o r inhibited by noxious stimulation varies greatly 1'4'6'8']1'12'17'21'23'24'26' 27,28, a l t h o u g h t h e r e has b e e n a clear p r e d o m i n a n c e of
e x c i t a t o r y responses. H o w e v e r , we h a v e s h o w n t h a t the firing of R V M n e u r o n s can be substantially a f f e c t e d by as few as 3 tail flick trials (as in Fig. 3), and c h a r a c t e r ization of o n e n e u r o n can alter the r e s p o n s i v e n e s s of n e u r o n s studied l a t e r in the e x p e r i m e n t (for e x a m p l e ,
157
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Fig. 2. Ratemeter records (i s bins) show spontaneous and tail flick-related changes in activity of an on-cell (A) and off-cell (B). Both neurons show fluctuations in spontaneous activity similar to that previously reported in barbiturate-anesthetized rats 3. The tail flick-related burst of activity in the on-cell is clearly evident only during inactive periods. Tail flick tests consistently resulted in an abrupt pause in the ongoing activity of the off-cell.
15 Hz
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TF
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Hz 15
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Fig. 3. Ratemeter record (1 s bins) shows the increase in ongoing activity of an on-cell induced by repeated tail flick testing. Very little spontaneous activity is evident prior to the first tail flick test. Following the third test, however, a marked and sustained increase in activity can be seen. A comparison of tail flick-aligned histograms generated from the first (left) and last (right) 3 trials demonstrates that the change in activity is sufficiently pronounced to obscure tail flick-related changes in activity. If tested during the period of high ongoing firing, this cell would have been incorrectly identified as a neutral cell. Interestingly, as cell activity increased, tail flick latency decreased (mean latencies for the first and last 3 tests were 7.6 and 5.1 s, resp.). This observation is consistent with previous work in the barbiturate-anesthetized rat 13.
158 t h e last t w o n e u r o n s in Fig. 1). I n g e n e r a l , o n - c e l l s be-
be activated by a search stimulus would be studied when
c o m e m o r e active, a n d off-cells less a c t i v e , w i t h r e p e a t e d
u s i n g e x t r a c e l l u l a r r e c o r d i n g t e c h n i q u e s . S u c h a bias m a y
n o x i o u s s t i m u l a t i o n . T h i s , a n d t h e lack o f a n a p p r o p r i -
u n d e r l i e t h e r e p o r t e d f a i l u r e t o o b s e r v e cells i n h i b i t e d b y n o x i o u s s t i m u l a t i o n in a w a k e r a t s 23'24.
ate s e a r c h s t i m u l u s to a c t i v a t e off-cells, c o u l d easily l e a d to i n v a l i d c o n c l u s i o n s a b o u t t h e e x i s t e n c e a n d r e l a t i v e n u m b e r s o f e a c h cell class w i t h i n t h e R V M . C l e a r l y , t h e s a m p l e s o f n e u r o n s i d e n t i f i e d w o u l d t e n d t o b e b i a s e d in only neurons that are spontaneously active or that can
This work was supported by a grant from NIDA (DA05608). M.M.M. was supported by DA05399. We thank Beth Budra for technical assistence and preparation of figures.
1 Anderson, E.G., Lobataz, M. and Proudfit, H.K., The effects of pain and opiates on unit activity in the nucleus raphe magnus. In R.W. Ryall and J.S. Kelly (Eds.), lontophoresis and Transmitter Mechanisms in the Mammalian Central Nervous System, Elsevier/North-Holland, Amsterdam, 1978, pp. 299301. 2 Barbaro, N.M., Heinricher, M.M. and Fields, H.L., Putative pain modulating neurons in the rostal ventral medulla: reflexrelated activity predicts effects of morphine, Brain Res., 366 (1985) 203-210. 3 Barbaro, N.M., Heinricher, M.M. and Fields, H.L., Putative nociceptive modulatory neurons in the rostal ventromedial medulla of the rat display highly correlated firing patterns, Somatosens. Motor Res., 6 (1989) 413-425. 4 Behbehani, M.M. and Pomeroy, S,L., Effect of morphine in periaqueductal gray on the activity of single units in the nucleus raphe magnus of the rat, Brain Res,, 149 (1978) 266-269. 5 Cheng, Z.-E, Fields, H.L. and Heinricher, M.M., Morphine microinjected into the periaqueductal gray has differential effects on 3 classes of medullary neurons, Brain Res., 375 (1986) 57-65. 6 Dickenson, A.H. and Goldsmith, G., Evidence for a role of 5-hydroxytryptamine in the responses of rat raphe magnus neurones to peripheral noxious stimuli, Neuropharmacology, 25 (1986) 863-868. 7 Drower, E.J. and Hammond, D.L., GABAergic modulation of nociceptive threshold: effects of THIP and bicuculline microinjected in the ventral medulla of the rat, Brain Res., 450 (1988) 316-324. 8 Fields, H.L., Bry, J., Hentall, I. and Zorman, G., The activity of neurons in the rostral medulla of the rat during withdrawal from noxious heat, J. Neurosci., 3 (1983) 2545-2552. 9 Fields, H.L. and Heinricher, M.M., Anatomy and physiology of a nociceptive modulatory system, Philos. Trans. R. Soc. London, Ser. B, 308 (1985) 361-374. 10 Fields, H.L., Vanegas, H., Hentall, I.D. and Zorman, G., Evidence that disinhibition of brain stem neurones contributes to morphine analgesia, Nature, 306 (1983) 648-686. 11 Friederich, M.W. and Walker, J.M., The effect of foot-shock on the noxious-evoked activity of neurons in the rostral ventral medulla, Brain Res. Bull., 24 (1990) 605-608. 12 Guilbaud, G., Peschanski, M., Gautron, M. and Binder, O., Responses of neurons of the nucleus raphe magnus to noxious stimuli, Neurosci. Lett., 17 (1980) 149-154. 13 Heinricher, M.M., Barbaro, N.M. and Fields, H.L., Putative nociceptive modulating neurons in the rostral ventromedial medulla of the rat: firing of on- and off-cells is related to nociceptive responsiveness, Somatosens. Motor Res., 6 (1989) 427-439. 14 Heinricher, M.M., Morgan, M.M. and Fields, H.L., Morphine applied iontophoretically depresses activity of on-cells in rostral ventromedial medulla of lightly anesthetized rats, Soc. Neuro-
sci. Abstr., 16 (1990) 98. 15 Heinricher, M.M., Haws, C.M. and Fields, H.L., Evidence for GABA-mediated control of putative nociceptive modulating neurons in the rostral ventromedial medulla: iontophoresis of bicuculline eliminates the off-cell pause, Somatosens. Motor Res., 8 (1991) 215-225. 16 Heinricher, M.M. and Kaplan, H.J., GABA-mediated inhibition in rostral ventromedial medulla: role in nociceptive modulation in the lightly anesthetized rat, Pain, 47 (1991) 105-113. 17 Liu, X., Zhu, B. and Zhang, S.-X., Relationship between electroacupuncture analgesia and descending pain inhibitory mechanism of nucleus raphe magnus, Pain, 24 (1986) 383-396. 18 Mason, P. and Fields, H.L., Axonal trajectories and terminations of on- and off-cells in the cat lower brainstem, J. Comp. Neurol., 288 (1989) 185-207. 19 Mason, P., Floeter, M.K. and Fields, H.L., Somatodendritic morphology of on- and off-cells in the rostral ventromedial medulla, J. Comp. Neurol., 301 (1990) 23-43. 20 Miller, K.W., The nature of the site of general anesthesia, Int. Rev. Neurobiol., 27 (1985) 1-61. 21 Mohrland, J.S. and Gebhart, G.F., Effect of morphine administered in the periaqueductal gray and at the recording locus on nociresponsive neurons in the medullary reticular formation, Brain Res., 225 (1981) 401-412. 22 Morgan, M.M., Heinricher, M.M. and Fields, H.L., Circuitry linking opioid-sensitive nociceptive modulatory systems in periaqueductal gray and spinal cord with rostral ventromedial medulla, Neuroscience, 47 (1992) 863-871. 23 Oliveras, J.L., Martin, G., Montagne, J. and Vos, B., Single unit activity at ventromedial medulla level in the awake, freely moving rat: effects of noxious heat and light tactile stimuli onto convergent neurons, Brain Res., 506 (1990) 19-30. 24 Oliveras, J.L., Vos, B., Martin, G. and Montagne, J., Electrophysiological properties of ventromedial medulla neurons in response to noxious and non-noxious stimuli in the awake, freely moving rat: a single-unit study, Brain Res., 486 (1989) 1-14. 25 Olsen, R.W., GABA-drug interactions, Prog. Drug Res., 31 (1987) 224-238. 26 Pertovaara, A. and Tukeva, T., Effect of subcutaneous formalin on responses to bulboreticular nociceptive neurons in the rat, Brain Res. Bull., 23 (1989) 457-462. 27 Rosenfeld, J.P., Huang, K.H. and Xia, L.Y., Effects of single and simultaneous combined nanoinjections of Met-enkephalin into rat midbrain and medulla on activity of differentially nociresponsive ventral medullary neurons, Brain Res., 508 (1990) 199-209. 28 Smock, T., Effects of ACTH(1-24) on single unit activity in the brainstem of the rat, Neuropharmacology, 26 (1987) 1771-1773. 29 Willow, M. and Johnston, G.A.R., Pharmacology of barbiturates: electrophysiological and neurochemical studies, Int. Rev. Neurobiol., 24 (1983) 15-49.
f a v o r o f n e u r o n s e x c i t e d b y n o x i o u s s t i m u l a t i o n , since
Brain Research, 582 (1992) 154-158 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
BRES 25204
Activity of neurons in the rostral medulla of the halothane-anesthetized rat during withdrawal from noxious heat Michael M. Morgan and Mary M. Heinricher Department of Neurology, University of California, San Francisco, CA 94143-0114 (USA) Key words: Pain modulation; Nociception; Nucleus raphe magnus; Tail flick; Barbiturate; v-Aminobutyric acid
The physiological and pharmacological properties of two classes of putative nociceptive modulatory neurons have been extensively characterized in the rostral ventromedial medulla (RVM) of the barbiturate-anesthetized rat. 'On-cells' show a burst of activity, and 'off-cells' a sudden pause immediately preceding the occurrence of nocifensor reflexes. In the present study, we have characterized the reflex-related activity of RVM neurons in halothane-anesthetized rats to determine whether the properties of these neurons are dependent on barbiturate anesthesia. Both on- and off-cells were identified in this preparation. Repeated noxious stimulation was associated with a high level of ongoing activity in on-cells, and a low level in off-cells. These data thus demonstrate that the previously described reflex-related changes in RVM neuron activity are not specific to barbiturate-anesthetized preparations, and that a failure to demonstrate off-cells in some studies may result from these neurons being inactive following repeated testing with noxious stimuli. The importance of the rostral ventromedial medulla ( R V M ) in modulating nociceptive transmission is widely acknowledged, yet the properties of the physiologically, pharmacologically and anatomically heterogeneous neuronal population found in this region have only recently come under careful scrutiny. Two classes of neurons characterized by opposite changes in activity that are temporally correlated with the execution of nocifensor reflexes have been identified, and these classes a p p e a r to have distinct and possibly opposing roles in nociceptive modulation. On-cells, which have been argued to have a permissive or even facilitating influence on nociception, and whose activity is depressed by opioids, are characterized by a sudden burst of activity that precedes the occurrence of the tail flick and other nocifensor reflexes 2'5"8A4'22. Off-cells, which are likely to exert a net inhibitory effect on nociception, and which are activated following opioid administration 5'1°, are identified by a pause in firing just prior to nocifensor reflexes 8. This pause presumably permits the reflex to occur 9. We have recently provided strong evidence that the reflex-related off-cell pause is m e d i a t e d by 7-aminobutyric acid ( G A B A ) , as it is eliminated by iontophoretic application of G A B A A receptor antagonists 15. Furthermore, microinjection of the same compounds into the R V M is antinociceptive 7'16, demonstrating that G A B A m e d i a t e d transmission is involved in the nociceptive modulating functions of this region. However, almost all of the above studies were carried
out in barbiturate-anesthetized animals. A s barbiturates are known to act at the G A B A A receptor to enhance G A B A transmission 25"29, it is possible that the physiological and pharmacological properties of R V M neurons described in these experiments were specific to rats maintained under barbiturate anesthesia, and it would be valuable to determine if these neurons can be identified in other preparations. In the present study, tail flick-related activity of R V M neurons was examined in rats anesthetized with the volatile anesthetic halothane. Male S p r a g u e - D a w l e y rats (260-320 g) were used in these experiments. Anesthesia was induced by placing the animals in a halothane chamber, and a tracheal cannula inserted for delivery of a halothane/oxygen mixture throughout the r e m a i n d e r of the experiment. H a l o t h a n e concentration was maintained at 2% during surgical preparation, and subsequently reduced (0.7-1.0%) until discrete withdrawal reflexes to noxious heat and pinch could be elicited. No spontaneous movements or signs of distress were evident when this concentration was used. Animals were allowed to breathe spontaneously, and body t e m p e r a t u r e was maintained with a 37°C circulating water blanket. A gold- and platinum-plated stainless steel microelectrode was advanced through the R V M until the activity of a single neuron could be isolated. Units were characterized as on-, off- or neutral cells based on changes in activity related to the occurrence of the tail flick reflex elicited by application of noxious radiant heat to the yen-
Correspondence: M.M. Morgan, Department of Neurology, Box 0114, University of California, San Francisco, CA 94143-0114, USA.
155 tral surface of the tail. As defined previously 8, on-cells showed an increase, and off-cells a decrease in activity immediately preceding the occurrence of the tail flick. Neutral cells showed no change in activity associated with the tail flick. Cell activity was recorded continuously for a period of 9-27 min. The heat stimulus consisted of a linear increase at 1.8°C/s from the 35°C holding temperature until the tail was withdrawn (3.1-9.2 s), at which time the trial was terminated and the temperature returned to 35°C. Sites 3, 5 and 7 cm from the tip of the tail were tested in rotation. In one series of experiments, multiple units isolated in succession along a single dorsal-to-ventral penetration of the medulla were studied in seven animals. Tail flick trials were given at 1.5-4 min intervals. In a second series, only one cell was studied in each of 10 animals so as to avoid cumulative effects of repeated testing with a noxious stimulus. In these experiments, only spontaneously active units that displayed an increase or decrease in activity preceding withdrawal of the hindpaw to a brief noxious pinch were chosen for study. Tail flick trials were carried out at 3 min intervals. At the qompletion of the experiment, electrolytic lesion(s) were made at a recording site or sites. Animals were given a lethal injection of pentobarbital, and perfused intracardially with saline followed by formalin (10%). Brains were removed, sectioned at 50 p m and stained with Cresyl violet for histological verification of the recording site. In the first series of experiments, 3 - 6 neurons were studied in each of 7 rats. Of 30 neurons, 17 were classified as on-cells, 5 as off-cells, and 8 as neutral cells. Histograms of tail flick-related activity generated for 5 cells recorded from a single rat are displayed in Fig. 1. Cells of all 3 classes were found in this animal. The first neuron encountered (cell 1) showed no change in activity associated with the tail flick and was thus classified as a neutral cell. Two off-cells (cells 2 and 4) and two on-cells (cells 3 and 5) were also found in this animal. Each of the offcells exhibited a pause in firing beginning immediately before the tail flick, but the more dorsal (cell 2) had a much higher level of spontaneous activity, and was thus more easily identified as an off-cell. (It should be noted that two of the off-cells encountered in another animal in this series had high levels of activity (20-40 Hz), and these neurons often showed a decrease, but not a complete cessation of firing, associated with the tail flick reflex.) The two on-cells isolated in this animal also showed distinct patterns of spontaneous activity. The first on-cell encountered (cell 3) was relatively inactive, which made the marked increase in firing associated with the tail flick quite visible. The last neuron tested (cell 5) had a high
level of spontaneous activity, with a relatively small but consistent increase in firing rate associated with the tail flick. In the second series of experiments, 6 on-cells and 4 off-cells were studied in 10 rats. The tail flick-related changes in firing of these neurons were similar to those described above. The spontaneous firing of these neurons was recorded for 9 min prior to initiating tail flick testing. As reported for on- and off-cells in barbiturateanesthetized rats 3, these neurons showed fluctuations in activity during this period, with intervals of activity alternating with periods of relative quiescence (Fig. 2). The approach used in the second series of experiments also revealed an effect of repeated tail flick testing. Fig. 3 shows one neuron with a low firing rate in the intervals prior to each of the first 3 tail flick trials. However, a sustained increase in activity was induced by the third trial, and this effectively masked tail flick-related increases in firing in subsequent trials, as shown in a comparison of the two peri-event histograms generated using the first 3 and last 3 trials. The histogram constructed using the last 3 trials alone would have caused the neuron to be classified as a neutral cell. A drastic change in activity resulting from repeated testing was not seen in the other 5 on-cells tested. This transition occurs rapidly, and 4 of the neurons already had high levels of spontaneous activity when first encountered. The on-, off- and neutral cells studied in Exp. 1 and 2 were distributed throughout the RVM. The only relationship between cell class and location was an increased probability of encountering neutral cells at the dorsal or ventral borders of the RVM. There was no discernible relationship between the spontaneous activity of on- and off-cells and location within RVM. These data demonstrate that R V M neurons showing tail flick-related changes in activity can be identified in halothane-, as well as barbiturate-anesthetized rats. These neurons also exhibited the cyclic alternation between quiet and active periods reported in barbiturateanesthetized animals 3. Although the mechanisms by which halothane produces its effects are not fully understood, it is widely thought to act by non-specifically altering the properties of neuronal membranes 2°. In contrast, a receptor-mediated potentiation of G A B A transmission is thought to be central to the actions of barbiturates 25'29. Thus, taken together with the report that anatomical differences exist between these two cell classes 18'19, the present observation of both cell classes in halothane-anesthetized animals demonstrates that oncells and off-cells are not an epiphenomenon of barbiturate anesthesia. The present observations also underscore the importance of the stimulation protocol and use of a search stim-
156 25 i
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-10
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-10
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10
TIME (sec)
Fig. 1. Recording sites and peri-event histograms showing tail flick-related changes in activity (average of 4-6 trials; 100 ms bins) for 5 neurons encountered in a single electrode penetration. The first neuron, a neutral cell dorsal to the RVM, showed no change in activity associated with the tail flick. An off-cell was recorded at sites 2 and 4, and an on-cell at sites 3 and 5. Notice that the off-cell recorded at site 4 has a much lower level of spontaneous activity than the off-cell recorded at site 2. Conversely, the on-cell recorded at site 5 has a much higher level of spontaneous activity that the on-cell recorded at site 3. These differences presumably result from repeated nociceptive testing during characterization of cells 1-3. (-2.3 mm from the interaural line; P, pyramidal tract; VII, facial nucleus and tract.)
ulus in studying n e u r o n s i n v o l v e d in n o c i c e p t i o n . A rev i e w o f the literature indicates that the p r o p o r t i o n s of R V M n e u r o n s r e p o r t e d to be excited o r inhibited by noxious stimulation varies greatly 1'4'6'8']1'12'17'21'23'24'26' 27,28, a l t h o u g h t h e r e has b e e n a clear p r e d o m i n a n c e of
e x c i t a t o r y responses. H o w e v e r , we h a v e s h o w n t h a t the firing of R V M n e u r o n s can be substantially a f f e c t e d by as few as 3 tail flick trials (as in Fig. 3), and c h a r a c t e r ization of o n e n e u r o n can alter the r e s p o n s i v e n e s s of n e u r o n s studied l a t e r in the e x p e r i m e n t (for e x a m p l e ,
157
A
1 min
A
A
A
A
TF
TF
TF
TF
r2o Hz
B ,, ,,IL,,~,~I,r A TF
1 min
10 Hz A
A
TF
TF
Fig. 2. Ratemeter records (i s bins) show spontaneous and tail flick-related changes in activity of an on-cell (A) and off-cell (B). Both neurons show fluctuations in spontaneous activity similar to that previously reported in barbiturate-anesthetized rats 3. The tail flick-related burst of activity in the on-cell is clearly evident only during inactive periods. Tail flick tests consistently resulted in an abrupt pause in the ongoing activity of the off-cell.
15 Hz
1 min
TF
TF
TF
TF
TF
25
25
Hz 15
Hz 15
TF
5 -10
~ TF TIME (sec)
10
-10 TF TIME (sec)
Fig. 3. Ratemeter record (1 s bins) shows the increase in ongoing activity of an on-cell induced by repeated tail flick testing. Very little spontaneous activity is evident prior to the first tail flick test. Following the third test, however, a marked and sustained increase in activity can be seen. A comparison of tail flick-aligned histograms generated from the first (left) and last (right) 3 trials demonstrates that the change in activity is sufficiently pronounced to obscure tail flick-related changes in activity. If tested during the period of high ongoing firing, this cell would have been incorrectly identified as a neutral cell. Interestingly, as cell activity increased, tail flick latency decreased (mean latencies for the first and last 3 tests were 7.6 and 5.1 s, resp.). This observation is consistent with previous work in the barbiturate-anesthetized rat 13.
158 t h e last t w o n e u r o n s in Fig. 1). I n g e n e r a l , o n - c e l l s be-
be activated by a search stimulus would be studied when
c o m e m o r e active, a n d off-cells less a c t i v e , w i t h r e p e a t e d
u s i n g e x t r a c e l l u l a r r e c o r d i n g t e c h n i q u e s . S u c h a bias m a y
n o x i o u s s t i m u l a t i o n . T h i s , a n d t h e lack o f a n a p p r o p r i -
u n d e r l i e t h e r e p o r t e d f a i l u r e t o o b s e r v e cells i n h i b i t e d b y n o x i o u s s t i m u l a t i o n in a w a k e r a t s 23'24.
ate s e a r c h s t i m u l u s to a c t i v a t e off-cells, c o u l d easily l e a d to i n v a l i d c o n c l u s i o n s a b o u t t h e e x i s t e n c e a n d r e l a t i v e n u m b e r s o f e a c h cell class w i t h i n t h e R V M . C l e a r l y , t h e s a m p l e s o f n e u r o n s i d e n t i f i e d w o u l d t e n d t o b e b i a s e d in only neurons that are spontaneously active or that can
This work was supported by a grant from NIDA (DA05608). M.M.M. was supported by DA05399. We thank Beth Budra for technical assistence and preparation of figures.
1 Anderson, E.G., Lobataz, M. and Proudfit, H.K., The effects of pain and opiates on unit activity in the nucleus raphe magnus. In R.W. Ryall and J.S. Kelly (Eds.), lontophoresis and Transmitter Mechanisms in the Mammalian Central Nervous System, Elsevier/North-Holland, Amsterdam, 1978, pp. 299301. 2 Barbaro, N.M., Heinricher, M.M. and Fields, H.L., Putative pain modulating neurons in the rostal ventral medulla: reflexrelated activity predicts effects of morphine, Brain Res., 366 (1985) 203-210. 3 Barbaro, N.M., Heinricher, M.M. and Fields, H.L., Putative nociceptive modulatory neurons in the rostal ventromedial medulla of the rat display highly correlated firing patterns, Somatosens. Motor Res., 6 (1989) 413-425. 4 Behbehani, M.M. and Pomeroy, S,L., Effect of morphine in periaqueductal gray on the activity of single units in the nucleus raphe magnus of the rat, Brain Res,, 149 (1978) 266-269. 5 Cheng, Z.-E, Fields, H.L. and Heinricher, M.M., Morphine microinjected into the periaqueductal gray has differential effects on 3 classes of medullary neurons, Brain Res., 375 (1986) 57-65. 6 Dickenson, A.H. and Goldsmith, G., Evidence for a role of 5-hydroxytryptamine in the responses of rat raphe magnus neurones to peripheral noxious stimuli, Neuropharmacology, 25 (1986) 863-868. 7 Drower, E.J. and Hammond, D.L., GABAergic modulation of nociceptive threshold: effects of THIP and bicuculline microinjected in the ventral medulla of the rat, Brain Res., 450 (1988) 316-324. 8 Fields, H.L., Bry, J., Hentall, I. and Zorman, G., The activity of neurons in the rostral medulla of the rat during withdrawal from noxious heat, J. Neurosci., 3 (1983) 2545-2552. 9 Fields, H.L. and Heinricher, M.M., Anatomy and physiology of a nociceptive modulatory system, Philos. Trans. R. Soc. London, Ser. B, 308 (1985) 361-374. 10 Fields, H.L., Vanegas, H., Hentall, I.D. and Zorman, G., Evidence that disinhibition of brain stem neurones contributes to morphine analgesia, Nature, 306 (1983) 648-686. 11 Friederich, M.W. and Walker, J.M., The effect of foot-shock on the noxious-evoked activity of neurons in the rostral ventral medulla, Brain Res. Bull., 24 (1990) 605-608. 12 Guilbaud, G., Peschanski, M., Gautron, M. and Binder, O., Responses of neurons of the nucleus raphe magnus to noxious stimuli, Neurosci. Lett., 17 (1980) 149-154. 13 Heinricher, M.M., Barbaro, N.M. and Fields, H.L., Putative nociceptive modulating neurons in the rostral ventromedial medulla of the rat: firing of on- and off-cells is related to nociceptive responsiveness, Somatosens. Motor Res., 6 (1989) 427-439. 14 Heinricher, M.M., Morgan, M.M. and Fields, H.L., Morphine applied iontophoretically depresses activity of on-cells in rostral ventromedial medulla of lightly anesthetized rats, Soc. Neuro-
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f a v o r o f n e u r o n s e x c i t e d b y n o x i o u s s t i m u l a t i o n , since
