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Putative nociceptive modulatory neurons in the dorsolateral pontomesencephalic reticular formation
Brain Research, 483 (1989) 272-~2 Elsevier
272 BRE 14350
Putative nociceptive modulatory neurons in the dorsolateral pontomesencephalic reticular formation* Christine M. Haws 1, Andrew M. Williamson 2, Howard L. Fields 3 Departments of 1Neurology, "-Anatomyand 3Physiology, The University of California at San Francisco, San Francisco, CA 94143 (U.S.A.) (Accepted 30 August 1988)
Key words: Antinociception; Nucleus cuneiformis; Nucleus parabrachialis; Pontomesencephalic reticular formation; Single unit recording; Rat
The present study was undertaken to confirm the dorsolateral pontomesencephalic reticular formation (an area that includes the nucleus cuneiformis (NCF) and nucleus parabrachialis (NPB)) as a sensitive site for stimulation-produced antinociception and to investigate the possibility that there are cells in this region that show a change in activity that can be correlated with the occurrence of a nocifensive reflex (the tail-flick withdrawal response from noxious heat (TF)). Such cells have been previously demonstrated in the rostral ventromedial medulla (RVM). Extracellular single unit recording studies were made at sites from which it was possible to inhibit the TF with currents of 10 ¢tA or less. Cellular response to 3 TF trials at 5 min intervals, and spontaneous activity over a 10 rain period, were monitored for each unit. Of the cells encountered, 17% displayed an increase (on-cells) and 4% a decrease (off-cells) in activity that preceded the TF. The remaining cells were unaffected by the TF (neutral cells). On- and off-cells were found throughout the region from which TF suppression was observed but were most concentrated in the areas of the NCF and NPB. The presence of on- and off-cells in the NCF/NPB region as well as the RVM suggests that the input to RVM from NCF/NPB may be important in descending nociceptive modulation.
INTRODUCTION It is well d o c u m e n t e d that nociceptive transmission through the spinal cord can be modified by supraspinal neurons 22'3s. Electrical stimulation of various brainstem sites, including the rostral ventromedial medulla ( R V M ) S'2°'46 and periaqueductal grey ( P A G ) 37'45'50, suppresses behavioral and neuronal responses e v o k e d by a noxious stimulus. Stimulation of the dorsolateral p o n t o m e s e n c e p h a l i c reticular formation adjacent to the midbrain P A G also suppresses the reflex withdrawal response to noxious stimulation and inhibits noxious-evoked excitation of spinal and trigeminal dorsal horn cells in a variety of species 14'19'25'53. M a p p i n g of this region for stimulat i o n - p r o d u c e d antinociception ( S P A ) has revealed that the most effective sites are located dorsal and
ventrolateral to the superior cerebellar peduncle (SCP), areas equivalent to the nucleus cuneiformis (NCF) and the nucleus parabrachialis (NPB) 19'53. Only scattered cells in the NPB project directly to the spinal cord dorsal hornT'32'35; it is therefore likely that spinal antinociceptive effects following stimulation of the dorsolateral pontomesencephalic region are m e d i a t e d primarily by indirect spinal projections. In fact, cells from both the N C F and NPB project to the R V M (an area that includes the nucleus r a p h e magnus and the adjacent reticular formation) 1'1°'23. R V M cells in turn provide the largest brainstem source of axons descending in the dorsolateral funiculus ( D L F ) to the dorsal horn of the spinal cord 6,7, 35,36. Electrical or chemical stimulation of R V M inhibits both spinal reflexes and neuronal responses e v o k e d by a noxious stimulus 3'18'47'51'55'63. Thus, a re-
* A preliminary report has been presented (ref. 28). Correspondence: C.M. Haws, Department of Pharmacology, Box 0450, University of California at San Francisco, San Francisco, CA 94143, U.S.A.
273 lay via neurons in the RVM would provide an anatomical substrate for the suppression of spinal transmission of nociceptive information following stimulation of the NCF/NPB area. Physiological studies have identified two classes of RVM neuron that show a change in activity closely correlated with the occurrence of a nocifensive reflex21. On-cells show an increase, and off-cells show a decrease in their activity just prior to a tail-flick (TF) withdrawal response evoked by noxious heat. Manipulations that produce antinociception can also modify the activity of on- and off-cells5'16,43. In RVM a significant percentage of cells displaying TF-related activity can be antidromically activated from the cervical spinal cord, a finding which provides additional evidence that these cells can modulate spinal nociceptive transmission 59. If a relay via RVM mediates the antinociceptive actions of dorsolateral pontomesencephalic reticular formation, then it may be anticipated that cells with characteristics similar to those of RVM on- and offcells will also be found in this area. To address this issue, extracellular single unit recording was used to study the activity of neurons in the dorsolateral pontomesencephalic reticular formation of the lightly anesthetized rat during the TF-withdrawal response. Both on- and off-cells were found in this area, most concentrated in the NCF/NPB region. MATERIALS AND METHODS Male Sprague-Dawley rats (250-300 g) were given an initial dose of pentobarbital sodium (Nembutal; 55 mg/kg i.p.). A catheter was inserted into an external jugular vein and the animal was placed into a stereotaxic frame. A discrete craniotomy was performed to reveal the cerebral cortex overlying the midbrain. Body temperature was maintained at 37 °C using a circulating hot water pad. The animal was allowed to recover from the pentobarbital to a point at which the TF could be elicited and was then maintained lightly anesthetized by the continuous infusion of methohexital (15-30 mg/kg/h). The animal showed no signs of discomfort, no spontaneous activity, nor a prolonged withdrawal response or vocalization to noxious pinch. The methods for eliciting the TF are those described by Heinricher et al. 29. Briefly, a beam from a
projector lamp was focused on the blackened ventral surface of the tail. A thermistor probe monitoring surface tail temperature provided a signal for the feedback control of the heat stimulus. Between heat stimuli tail temperature was maintained at 35 °C. The TF generally occurred at 42-45 °C with a latency of 4 - 6 s. A gold- and platinum-plated stainless-steel monopolar electrode stereotactically localised dorsal to the NCF (anteroposterior (AP) -0.3 to 1.7 from the interaural line; mediolateral (ML) 1.0 to 2.0; dorsoventral (DV) 5.0 from the cortical surface) was used for stimulation and extracellular single unit recording. The electrode was advanced through the brain in 200 p m steps until a site was reached from which the TF could be inhibited (10 s cut-off, SPA) using a current of no more than 10 p A (400 ps cathodal pulse width, 50 Hz continuous train). Advancement of the electrode in 200 p m steps was continued until SPA could no longer be elicited. The electrode was then attached to a differential pre-amplifier for extracellular recording. Action potentials were amplified, filtered and displayed on an oscilloscope where spike shape and amplitude were monitored to assure the identity of the unit being recorded. Spikes were also converted to voltage pulses using a window discriminator, the output of which was sent to an audio-amplifier and integrated and displayed on a strip chart recorder. The search for on- and off-cells was continued 1-2 mm dorsal and ventral, and along penetrations rostral and caudal, to the site from which suppression of the TF could be elicited with stimulating currents of 10pA. During the search for on- and off-cells the electrode was advanced through the brain in 3 p m steps. Noxious pinch delivered to the paw once every 100-200 p m served as a search stimulus. Each cell's response to heat (tail) and noxious pinch (hind paws and ear) were determined and the cell characterised as an on- or off-cell, or neutral cell according to the classification system applied to cells in the RVM by Fields et al. 21. For each cell, the response to 3-5 applications of noxious heat to the tail at 5 min intervals was monitored as was 10 min of spontaneous activity. After each penetration lesions were made at sites of interest (20 p A anodal current for 10 s). At completion of the experiment the animal was perfused
274 transcardially with 0.9% saline followed by 4% formaldehyde solution. Lesions were identified in 50 p m coronal sections stained with Cresyl violet and marked on diagrams derived from the stereotaxic atlas of Paxinos and Watson 48.
Single unit recording A total of 275 cells in the caudal midbrain and rostral pons in an area dorsal and ventral to the SCP were studied in 53 experiments. Spikes were generally biphasic, negative-positive in polarity. Spike amplitude ranged from 100-500/~V.
RESULTS
On-cells Stimulation produced antinociception Suppression of the TF at currents of 10 p A or less was observed following stimulation of sites ventral to the inferior colliculus, bordering on and lateral to the P A G and medial to the lateral lemniscus. This region extends from the rostral pole of the N C F (AP 1.2), to the rostral pole of the locus coeruleus ( A P - 0 . 3 ; Fig. 1). Regions more rostral and caudal to these coordinates were not systematically investigated. TF suppression could be evoked from much of the nucleus pontis oralis (NPO), the ventral border being the dorsal edges of the superior olive and the nucleus of the trapezoid body. This extensive region of the caudal midbrain and rostral pons was divided by an area, centered on the SCP, from which SPA could not be consistently evoked at the currents used.
A total of 46 (17%) cells showed an increase in activity that preceded the TF. The on-cell burst began at a variable time prior to the TF (range 200 ms to 2.4 s, mean 827 ms, S.E.M. + 90 ms). The duration of the evoked burst was also quite variable, ranging from 1 s to over 10 s (Fig. 2). Responses varied from several spikes to a vigorous and prolonged burst of activity. All cells responded to noxious pinch applied over a wide area of the body surface including the hind paw and ears bilaterally, and 17 cells (37%) could additionally be excited by non-noxious stimuli such as brushing of the fur or brisk tapping of the tail. Thirty-three (72%) on-cells demonstrated low levels of spontaneous activity (1-15 Hz) and the remaining thirteen were inactive• Spontaneous activity was generally irregular and in 11 neurons consisted of de-
0"2
Fig. 1. Drawing of coronal sections through the caudal midbrain/rostral pons illustrating regions of the electrode penetration from which the TF was inhibited with currents of less than or equal to 10 pA. Upper circle denotes the site at which SPA was first encountered while the lower circle shows the ventral limit. Regions dorsal and ventral to the SCP were the most sensitive sites for SPA. Anterior-posterior, 1.2 to -0.3 (derived from the atlas of Paxinos and Watson, 1982).
275
A
I
B
10HZI I
I
I
FP TF
TF
Fig. 2. A: single oscilloscope sweep (upper trace) shows the increase in activity that occurs approximately 1 s before the TF (lower trace). Sweep time equals 10 s. B: chart recording illustrating the increase in FR of cell in A in response to foot pinch (FP) or noxious heat (TF) as well as the low level of spontaneous activity displayed by ceils in this part of the brain. Time course as in Fig. 3. fined periods of complete silence alternating with periods of irregular spontaneous activity (Fig. 3).
R V M under similar anaesthetic conditions 4.
Neutral cells Off-cells Eleven cells (4%) showed an abrupt decrease in activity just prior to the TF withdrawal response. The onset of this reduction in activity preceded the TF by a variable period of time ranging from 400 ms to 3.0 s (mean 1.3 s, S.E.M. + 0.35 s), and lasted for a duration of 800 ms to over 10 s (Fig. 4). All cells were additionally inhibited by noxious pinch and two by nonnoxious stimuli such as light tapping or brush. Offcells showed a range of spontaneous firing rates although spontaneous activity was generally slow and irregular (3-15 Hz). Two cells showed defined periods of silence interspersed among periods of activity, a characteristic also displayed by off-cells in the A
I
5.z I I TF
I TF
I EP
The great majority of cells in the region caudal and ventral to the SCP did not show a change in firing rate that began prior to the TF (n = 218 of 275, 79%). These cells were classified as neutral cells (Fig. 5). Sixteen of these cells were excited and 5 inhibited by noxious pinch applied to the hind paws or ears. Four cells excited by noxious pinch showed an increase in firing rate which followed the TF. Similarly, 2 of the 5 cells inhibited by noxious pinch showed a decrease in activity that followed the TF. As the evoked activity displayed by these 6 cells does not conform to the original definition of on- and off-cells 21, these cells were classified as 'late-on cells' and 'late-off cells', respectively, and not included as on- and off-cells in the present analysis. All neutral cells showed some spontaneous activity. As with on- and off-cells a wide range of spontaneous firing rates and patterns was displayed by neutral cells (1-20 Hz), but none of the neutral cells displayed the alternating periods of activity and silence displayed by some on- and off-cells. In general, the spontaneous firing pattern of all 3 cell classes was sufficiently similar that it was not possible to differentiate between on-, off- and neutral cells according to spontaneous firing rate.
I min
Fig. 3. On-cell displaying spontaneous periods of activity alternating with periods of silence. A: single oscilloscope sweep (upper trace) showing on-cell burst approximately 1 s prior to the TF (lower trace). B: chart recording of spontaneous activity of cell in A. Cell responds with an increase in activity to tail-flick (TF) and ear pinch (EP).
Anatomical distribution of on- and off-cells The distribution of cells showing on- and off-cells, and neutral cells is illustrated in Figs. 6 and 7, respectively. Neurons showing a change in activity related to the
276
TF I
A
B IOHZJ I I TF FP 1 min Fig. 4. A: single oscilloscope sweep (lower trace) illustrating the pause in activity that occurs just prior to the TF (upper trace). Sweep time is 10 s. B: chart recording of same cell; tail-flick (TF), foot pinch (FP) both produce a short-lived decrease in spontaneous activity.
occurrence of the T F were found only in areas from which S P A could be elicited with currents of 10 ~ A or less. The two cell classes displayed a similar distribution, although fewer off-cells were found. On- and off-cells were found dorsal to the SCP in the N C F , and lateral and ventral to the SCP in the ventral NPB and in the adjacent N P O . Neutral cells were scatt e r e d throughout the sampled region extending into
areas outside those from which low-threshold S P A could be evoked. A total of 51 cells were located outside of the area from which stimulation with currents of 10 p A or less would suppress the TF. These cells were all neutral cells. The percentage of cells showing activity related to the occurrence of the T F dorsal and ventral to the
A
1 min Fig. 5. A: single oscilloscope trace illustrating no change in firing rate (upper trace) with the TF (lower trace). Sweep time is 10 s. B: chart recording of the spontaneous activity of the cell displayed in A.
0.2
0.7
277
a
Fig. 6. Diagrams of coronal sections through the caudal midbrain showing the location of 46 on-cells (upward pointing arrows, left) and 11 off-cells (downward pointing arrows, right). The two cell classes represented on opposite sides of the brain for clarity. On- and off-cells were only located in areas of the dorsolateral pontomesencephalic reticular formation from which the stimulation with currents of 10/~A or less would inhibit the TF. Anterior-posterior, 1.2 to 0.2. Diagrams as in Fig. 1.
S C P indicates that on- and off-cells t e n d to b e clus-
DISCUSSION
t e r e d in an a r e a r o u g h l y e q u i v a l e n t to the N C F and an a r e a i m m e d i a t e l y v e n t r a l to t h e S C P , r o u g h l y e q u i v a l e n t to the v e n t r a l N P B ( T a b l e I).
T h e w o r k d e s c r i b e d h e r e d e m o n s t r a t e s t h e prese n c e of n e u r o n s in the d o r s o l a t e r a l p o n t o m e s e n c e -
-0"3
°
/A
0.2
or'~
7
re,,
•/;Z.',,
Fig. 7. Representative coronal sections through the caudal midbrain/rostral pons showing the distribution of 218 cells that did not show a TF-related change in activity. Anterior-posterior, 1.7 to -0.3. Diagrams as in Fig. 1.
278 It is important to consider the possible influence of
TABLE I Distribution of on- and off-cells and neutral cells
The number and percentage of on- and off-cells and neutral cells located in the NCF, NPB and NPO as compared with the area of the dorsolateral pontomesencephalic region from which SPA can be elicited. Boundaries of NCF, NPB and NPO as defined by Paxinos and Watson 48. NCF
On Off Neutral
NPB
NPO
no.
%
no.
%
no.
14 4 19
37.8 10.8 51
7 3 16
27 14 11.5 1 61.5 53
Total area %
no.
%
20.5 46 17 1.5 11 4 78 218 79
phalic reticular formation that show a change in activity related to the occurrence of a nocifensive spinal reflex. As in RVM, this region contains two classes of such neurons, on-cells which show an increase, and off-cells which show a decrease in firing rate just prior to the withdrawal of the tail from noxious heat. The location of on- and off-cells was restricted to the regions of the dorsolateral pontomesencephalic reticular formation from which electrical stimulation inhibited the TF as described by this and other studies 19'53. Dorsal to the SCP and caudal to the inferior collicutus, on- and off-cells were found in the NCF. Ventral and lateral to the SCP, on- and off-cells were present in both ventral and lateral NPB and the adjacent lateral NPO. These cells were more concentrated in the N C F and NPB than in the NPO, which may correlate with the larger afferent projection to the R V M from the N C F and NPB than from the NPO1AO,23. On- and off-cells were first described in the R V M 21 and more recently have been reported to be present in the P A G 29, another brainstem area that plays an important role in nociceptive modulation 37'45'5°. Onand off-cells in the dorsolateral pontomesencephalic region more closely resemble those found in the P A G than those found in RVM. Thus, in both the NCF/ NPB region and the P A G , on- and off-cells display low irregular discharge rates and a relatively brief and/or weak discharge concomitant with nocifensive withdrawal reflexes. In contrast, on- and off-cells in R V M exhibit vigorous and long lasting changes in activity preceding the TF, and display a pattern of spontaneous activity that consists of periods of activity alternating with periods of silence 4.
experimental conditions on nociresponsivity. This is illustrated when the data obtained in our investigation is compared with data obtained from a similar study undertaken by Hardy et al. 26. They found that the majority (30/33) of neurons encountered in the 'paralemniscal reticular formation' (which includes the N C F and NPB, thus being equivalent to the area described in this report) responded to noxious pinch, approximately half showing an increase and the remainder a decrease in activity. Also, in contrast to our study, all neurons recorded from were spontaneously active. These differences may be attributed to the use of different anaesthetics (methohexital vs. chloral hydrate) or possibly to sampling bias resulting from the use of a search stimulus that would favour the selection of on-cells. The low levels of spontaneous activity that we observed may have contributed to the very small numbers of off-cells encountered as any decreases in activity associated with the TF would have been indistinguishable from spontaneous pauses. Even considering the potential effects of anaesthetic and sampling bias, it is likely that on- and offcells constitute, at most, a small percentage of the total population of neurons encountered in the dorsolateral pontomesencephalic reticular formation, a region involved in many diverse functions 11'12'17'44'52'56. Thus, neutral cells may be involved in neural mechanisms subserving a variety of visceral, neuroendocrine and behavioral functions unrelated to nociceptive modulation. Our results, however, do not rule out a possible role for neutral cells in descending nociceptive modulation. D o cells in the dorsolateral p o n t o m e s e n c e p h a l i c reticular f o r m a t i o n play a role in m o d u l a t i n g nociceptive transmission?
Although a small percentage of on- and off-cells (37 and 18% respectively) responded to both noxious and non-noxious stimuli (brush) the majority responded exclusively to noxious stimuli (foot pinch and noxious heat). These observations correlate with anatomical studies describing afferents to NCF, NPB and NPO. N C F and NPB receive a substantial direct input from nociresponsive neurons in lamina I of both spinal and trigeminal dorsal horns 15'33'4°'42'57, and a smaller input from multireceptive neurons in laminae
279 IV, V and deeper laminae of the spinal cord 41'62. Many lamina I cells project exclusively to the mesencephalon 13,6a, others constitute collaterals of spinothalamic tract axons 33'49. This large projection to the pontomesencephalic reticular formation from nociresponsive cells in the dorsal horn points to a major role of this area in nociception. Studies of the efferent projections to the spinal cord from the NCF and NPB are also consistent with a role of this region in nociceptive modulation. Although there is a major projection via the ventral and ventrolateral funiculi that terminates in ventral regions of the cord 7'32,35, there are two minor projections that travel in the dorsolateral funiculus (DLF) to the dorsal horn: a projection from scattered neurons in an area equivalent to the ventral NPB and the adjacent nucleus Kolliker-Fuse 5s, and a second from a group of laterally placed cells adjacent to the lateral lemniscus that terminates in the contralateral dorsal horn 32'5s. These projections to the dorsal horn via the DLF provide the anatomical substrate for direct modulation of dorsal horn cell responses to noxious inputs and could underlie, at least in part, the SPA observed in the present experiments. In support of this idea is the demonstration that cells in the direct projection from the NPB and Kolliker-Fuse nucleus are immunopositive for the neurotransmitter noradrenaline 6°. There is evidence that this noradrenergic projection is a component of the pathway mediating suppression of cellular responses to noxious inputs in the dorsal horn following stimulation of the midbrain 31. Whether any on- or off-cells in the NCF/NPB are noradrenergic or spinally-projecting has yet to be determined. In addition to their possible direct effect on spinal dorsal horn neurons, dorsolateral pontomesencephalic reticular formation cells may modulate nociceptive transmission via a relay in the RVM. Anterograde and retrograde transport studies demonstrate that cells in both NCF and NPB project to R V M 1'23. Components of this projection have been demonstrated to be immunoreactive for neurotensin 1° and acetylcholine 2,54, and there is evidence that cells in the NPB may be a source of noradrenaline terminals in the RVM (A.I. Basbaum, personal communication). These anatomical observations are supported by data from both behavioural and electrophysiological studies. In the cat, Gebhart et al. 24 reported that
suppression of a heat-evoked response in dorsal horn cells following stimulation of either the P A G or dorsolateral pontomesencephalic region could be blocked by lidocaine microinjected into the RVM. Behbehani and Zemlan 9 demonstrated that stimulation of NCF produced an increase in RVM unit activity that could be mimicked by iontophoretically applied acetylcholine and partially attenuated by atropine. Furthermore, microinjection of atropine into the RVM of the rat was found to attenuate suppression of a nocifensive reflex following stimulation of the NCF/NPB area 39. Similar studies investigating the input to RVM from the NPB demonstrate that antinociception resulting from microinjection of carbachol into the NPB involves a relay with neurons in NRM and that stimulation of NPB produces excitation of raphe-spinal neurons 34. Of particular interest is the role of noradrenaline in the projection from the NPB to RVM. Microinjection of selected noradrenergic agents into the RVM produces a change in nociceptive threshold 27'51 and microiontophoretic application of noradrenergic agonists reveal an al-receptor mediated excitation and an a2-mediated inhibition restricted to on-cells 3°. These data suggest that the activity of RVM on-cells is under the influence of a noradrenergic input a component of which may originate from the NPB. In summary, there is an increasing body of evidence to support the involvement of neurons in the NCF and NPB regions in the modulation of nociceptive transmission at the dorsal horn level. This may be mediated by at least two descending pathways: one direct and one via neurons in RVM. The presence of a direct projection to the spinal cord via the ventral and ventrolateral funiculi raises the important possibility that the TF inhibition observed in the present experiments represents direct inhibition of the motor neurons involved. This possibility cannot be excluded by data obtained in the present experiments, however, several lines of evidence favor the hypothesis that inhibition of nociceptive dorsal horn neurons is at least partially involved. First, there is inhibition of dorsal horn cell activity produced by electrical stimulation of this area. Second, stimulation of this area both excites RVM neurons and inhibits withdrawal reflexes and both of these effects are blocked by RVM atropine. Since RVM projects almost exclusively to the dorsal horn,
280 that NCF/NPB on- and off-cells play a role in noci-
at least part of the SPA from the dorsolateral pontomesencephalic reticular formation is very likely via an effect on dorsal horn neurons. The evidence that projections from the NCF/NPB region are involved in antinociception relayed via R V M raises the possibility that on- and off-cells in the NCF/NPB region are connected to similar cells in
solateral pontomesencephalic reticular formation, which includes the N C F and NPB, forms part of a highly interconnected supraspinal network, includ-
the RVM. The recent observations showing a significant correlation between T F latency and the spontaneous change in R V M on- and off-cell discharge fre-
ACKNOWLEDGEMENTS
quency are important evidence implicating such cells
ceptive modulation. They also indicate that the dor-
ing R V M and P A G , that modulates the transmission of nociceptive information in the spinal cord.
in nociceptive modulation. These observations, plus the fact that dorsolateral pontomesencephalic on-
We wish to thank Pat Littlefield for histology, Mary Heinricher and Peggy Mason for helpful comments on the manuscript, and Susan Elliott and Lael
and off-cells are apparently clustered in regions projecting most densely to the R V M support the idea
Carson for secretarial assistance. This work was supported by PHS grants DA01949 and NS21445.
REFERENCES 1 Abols, I.A. and Basbaum, A.I., Afferent connections of the rostral medulla of the cat: a neural substrate for midbrain-medullary interactions in the modulation of pain, J. Comp. Neurol., 201 (1981) 285-287. 2 Amstrong, J., Saper, C.B., Levey, A.I., Wainer, B.H. and Terry, R.D., Distribution of cholinergic neurons in the rat brain: demonstrated by immunocytochemical localisation of choline acetyltransferase, J. Comp. Neurol., 216 (1983) 52-68. 3 Azami, J., Llewelyn, M.B. and Roberts, M.H.T., Spinal cord mechanisms of analgesia produced by microinjection of morphine into the nucleus raphe magnus (NRM) of the rat, J. Physiol. (Lond.), 367 (1982) 47-48P. 4 Barbaro, N.M., Fields, H.L. and Heinricher, M.M., Reciprocal activity of on- and off-cells in the rostral ventromedial medulla of the rat, Soc. Neurosci. Abstr., 11 (1985) 1180. 5 Barbaro, N.M., Heinricher, M.M. and Fields, H.L., Putative pain modulating neurons in the rostral ventral medulla: reflex related activity predicts the effects of morphine, Brain Research, 366 (1985) 203-210. 6 Basbaum, A.I., Clanton, C.H. and Fields, H.L., Three bulbospinal pathways from the rostral medulla of the cat: an autoradiographic study of pain-modulating systems, J. Comp. Neurol., 178 (1978) 209-224. 7 Basbaum, A.I. and Fields, H.L., The origin of descending pathways in the dorsolateral funiculus of the spinal cord of the cat and rat: further studies on the anatomy of pain modulation, J. Comp. Neurol., 187 (1979) 513-532. 8 Basbaum, A.I. and Fields, H.L., Endogenous pain control systems: brainstem spinal pathways and endogenous circuitry, Annu. Rev. Neurosci., 7 (1984) 309-338. 9 Behbehani, M.M. and Zemlan, F.P., Response of nucleus raphe magnus neurons to stimulation of the nucleus cuneiformis: role of acetylcholine, Brain Research, 369 (1986) 110-118. 10 Beitz, A.J., Sites of origin of brainstem neurotensin and serotonin projections to the rodent nucleus raphe magnus, J. Neurosci., 2 (1982) 829-842. 11 Bernston, G.G., Attack, grooming and threat elicited by stimulation of the pontine tegmentum in cats, Physiol. Be-
hav., 11 (1973) 81-87. 12 Betrand, F. and Hugelin, A., Respiratory synchronising function of the nucleus parabrachialis medialis: pneumotaxic mechanisms, J. Neurophysiol., 34 (1971) 189-207. 13 Bjorkeland, M. and Boivie, J., The termination of spinomesencephalic fibres in the cat, Anat. Embryol., 170 (1984) 265-277. 14 Carstens, E., Klumpp, D. and Zimmerman, M., Differential inhibitory effects of medial and lateral midbrain stimulation on spinal neuronal discharges to noxious skin heating in the cat, J. Neurophysiol., 43 (1980) 332-342. 15 Cechetto, D.F., Standaert, D.G. and Saper, C.P., Spinal and trigeminal dorsal horn projections to the parabrachial nucleus in the rat, J. Comp. Neurol., 240 (1985) 153-160. 16 Cheng, Z.-F., Fields, H.L. and Heinricher, M.M., Morphine microinjected into the periaqueductal gray has differential effects on 3 classes of medullary neurons, Brain Research, 375 (1986) 57-65. 17 Coote, J.H., Hilton, S.M. and Zbrozyna, W., The pontomedulla area integrating the defense reaction in the cat and its influence on muscle blood flow, J. Physiol. (Lond.), 229 (1973) 257-274. 18 Dickenson, A.H., Oliveras, J.-L. and Besson, J.-M., Role of the nucleus raphe magnus in opiate analgesia as studied by the microinjection technique in the rat, Brain Research, 170 (1979) 95-111. 19 Dostrovsky, J.O., Hu, J.W., Sessle, B.J. and Sumino, R., Stimulation sites in the periaqueductal grey, nucleus raphe magnus and adjacent regions effective in suppressing the oral-facial reflexes, Brain Research, 252 (1982) 287-297. 20 Fields, H.L. and Basbaum, A.I., Brainstem control of spinal pain-transmission neurons, Annu. Rev. Physiol., 40 (1978) 217-248. 21 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. 22 Fields, H.L. and Heinricher, M.M., Anatomy and physiology of a nociceptive modulatory system, Phil. Trans. R. Soc. Lond. Ser. B, 308 (1985) 361-374. 23 Gallagher, D.W. and Pert, A., Afferents to brainstem nu-
281 clei (brain stem raphe, nucleus reticularis caudalis and nucleus gigantocellularis) in the rat as demonstrated by microiontophoretically applied horseradish peroxidase, Brain Research, 144 (1978) 257-275. 24 Gebhart, G.F., Sandkhuler, J., Thalhammer, J.G. and Zimmerman, M., Inhibition of spinal nociceptive information by stimulation in the midbrain is blocked by lidocaine microinjected in nucleus raphe magnus and medullary reticular formation, J. Neurophysiol., 50 (1983) 1446-1459. 25 Giradot, M.N., Brennan, T.J., Martindale, M.E. and Foreman, R.D., Effects of stimulating the subcoeruleus-parabrachial region on the non-noxious and noxious responses of T1-T5 spinothalamic tract neurons in the primate, Brain Research, 409 (1987) 19-30. 26 Hardy, S.G.P., Haigler, H.J. and Leichnetz, G.R., Paralemniscal reticular formation: response of cells to a noxious stimulus, Brain Research, 267 (1983) 217-223. 27 Haws, C.M., Heinricher, M.M. and Fields, H.L., Antinociceptive action of alpha-2 adrenergic agonists in the rostroventromedial medulla of the rat may be mediated by a selective action on a single population of putative nociceptive modulatory neurons, Soc. Neurosci. Abstr., 14 (1988) in press. 28 Haws, C.M., Williamson, A.M. and Fields, H.L., Putative pain-modulating neurons in the mesencephalic and pontine reticular formation, Pain, Suppl. 4 (1987) $29. 29 Heinricher, M.M., Cheng, Z.-F. and Fields, H.L., Evidence for two classes of nociceptive modulating neurons in the periaqueductal grey, J. Neurosci., 7 (1987) 271-278. 30 Heinricher, M.M., Haws, C.M. and Fields, H.L., Opposing actions of norepinephrine and clonidine on single class of pain-modulating neurons in the rostral ventromedial medulla, Pain, Suppl. 4 (1987) $28. 31 Hodge, Jr., C.J., Apkarian, A.V. and Stevens, R.T., Inhibition of dorsal-horn cell responses by stimulation of the Kolliker-Fuse nucleus, J. Neurosurg., 65 (1986). 32 Holstege, G. and Kuypers, H.G.J.M., The anatomy of brain stem pathways to spinal cord in the cat. A labelled amino acid tracing study. In H.G.J.M. Kuypers and G.F. Martin (Eds.), Descending Pathways to the Spinal Cord, Progress in Brain Research, Vol. 57, Elsevier, Amsterdam, 1982, pp. 145-175. 33 Hylden, J.L.K., Hayashi, H., Dubner, R. and Bennett, G.J., Physiology and morphology of the lamina I spinomesencephalic projection, J. Cornp. Neurol., 247 (1986) 505-515. 34 Katayama, Y.Y., Tsubokawa, S., Maejima, S. and Yamamoto, T., Response properties of raphe-spinal neurons to stimulation of the pontine parabrachial region producing behavioural nociceptive suppression in the cat, Appl. Neurophysiol., 49 (1986) 112-120. 35 Kuypers, H.G.J.M. and Maisky, V.A., Funicular trajectories of descending brainstem pathways in the cat, Brain Research, 136 (1977) 159-165. 36 Leichnetz, G.R., Watkins, L., Griffin, G., Martin, R. and Mayer, D.J., The projection from nucleus raphe magnus and other brainstem nuclei to the spinal cord in the rat: a study using HRP blue reaction, Neurosci. Lett., 8 (1978) 19-24. 37 Mayer, D.J. and Liebeskind, J.C., Pain reduction by focal electrical stimulation of the brain: an anatomical and behavioural analysis, Brain Research, 68 (1974) 73-93. 38 Mayer, D.J. and Price, D.D., Central nervous system mechanisms of analgesia, Pain, 2 (1976) 379-404.
39 McCarthy, M.S. and Proudfit, H.K., Evidence for the cholinergic mediation of the antinociception induced by electrical stimulation of the pedunculopontine tegmental nucleus, Soc. Neurosci. Abstr., 13 (1987) 304. 40 McMahon, S.B. and Wall, P.D., Electrophysiological mapping of brainstem projections of spinal cord lamina I cells in the rat, Brain Research, 333 (1985) 19-26. 41 Menetrey, D., Chaouch, A. and Besson, J.-M., Location and properties of dorsal horn neurons at the origin of the spinoreticular tract in the lumbar enlargement of rat, J. Neurophysiol., 44 (1980) 862-877. 42 Menetrey, D., Chaouch, A., Binder, D. and Besson, J.-M., The origin of the spinomesencephalic tract in the rat: an anatomical study using the retrograde transport of horseradish peroxidase, J. Comp. Neurol., 206 (1982) 193-207. 43 Moreau, J.L. and Fields, H.L., Evidence for GABA involvement in midbrain control of medullary neurons that modulate nociceptive transmission, Brain Research, 397 (1986) 37-46. 44 Norgren, R. and Leonard, C.M., Ascending central gustatory pathways, J. Comp. Neurol., 150 (1973) 217-238. 45 Oliveras, J.L., Besson, J.-M., Guilbaud, G. and Liebeskind, J.C., Behavioural and electrophysiological evidence of pain inhibition from midbrain stimulation in the cat, Brain Research, 20 (1974) 32-45. 46 Oliveras, J.L., Guilbaud, G. and Besson, J.-M., A map of serotonergic structures involved in stimulation-produced analgesia in unrestrained freely moving cats, Brain Research, 164 (1979) 317-322. 47 Oliveras, J.L., Redjemi, F., Guilbaud, G. and Besson, J.M., Analgesia induced by electrical stimulation of the inferior central nucleus of the raphe of the cat, Pain, 1 (1975) 139-145. 48 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic, Sydney, 1982. 49 Price, D.D., Hayes, R.J., Ruda, M.A. and Dubner, R., Spatial and temporal transformations of input to spinothalamic tract neurons and their relation to sensation, J. Neurophysiol., 41 (1978) 933-947. 50 Reynolds, D.V., Surgery in the rat during analgesia induced by focal brain stimulation, Science, 164 (1969) 444-445. 51 Sagen, J. and Proudfit, H.K., Evidence for pain modulation by pre- and postsynaptic noradrenergic receptors in the medulla oblongata, Brain Research, 331 (1985) 285-293. 52 Saito, H., Sakai, K. and Jouvet, M., Discharge pattern of the nucleus parabrachialis lateralis neurons of the cat during sleep and waking, Brain Research, 134 (1977) 55-72. 53 Sandkhuler, J. and Gebhart, G.F., Characterisation of inhibition of a spinal nociceptive reflex by stimulation medially and laterally in the midbrain and medulla in the pentobarbital-anaesthetised rat, Brain Research, 305 (1984) 67-76. 54 Satoh, K., Armstrong, D.M. and Fibiger, H.C., A comparison of the distribution of central cholinergic neurons as demonstrated by acetylcholinesterase pharmacohistochemistry and choline acetyltransferase immunohistochemistry, Brain Res. Bull., 11 (1983) 693-720. 55 Satoh, M., Oku, R. and Akaike, A., Analgesia produced by microinjection of L-glutamate into the rostral ventromedial bulbar nuclei of the rat and its inhibition by intrathecal alpha-adrenergic blocking agents, Brain Research, 261 (1983) 361-364. 56 Sheard, M.H. and Flynn, J.P., Facilitation of attack by
282
57
58
59
60
stimulation in the midbrain in cats, Brain Research, 4 (1967) 324-333. Swett, J.E., McMahon, S.B. and Wall, P.D., Long ascending projections to the midbrain from cells in lamina I, J. Comp. Neurol., 238 (1985) 401-416. Tan, J. and Holstege, G., Anatomical evidence that the pontine field projects to lamina I of the caudal spinal trigeminal nucleus and the spinal cord and to the EdingerWestphal nucleus of the cat, Neurosci. Left., 65 (1986) 317-322. Vanegas, H., Barbaro, H.M. and Fields, H.L., Tail-flick related activity in medullo-spinal neurons, Brain Research, 321 (1984) 135-141. Westlund, K.N. and Coulter, J.D., Descending projections of locus coeruleus and subcoeruleus/medial parabrachial
nucleus in the monkey: axonal transport studies and dopamine-betahydroxylase neurocytochemistry, Brain Res. Rev., 2 (1980) 235-264. 61 Wiberg, R.P. and Blomquist, A., The spinomesencephalic tract in the cat: its cells of origin and termination pattern as demonstrated by the intraaxonal transport method, Brain Research, 291 (1984) 1-18. 62 Yezierski, R.P. and Schwartz, R.N., Receptive field properties of spinomesencephalic tract (SMT) cells, Pain, Suppl., 2 (1984) S127. 63 Zorman, G., Hentall, I.D., Adams, J.E. and Fields, H.L., Naloxone-reversible analgesia produced by microstimulation in the rat medulla, Brain Research, 219 (1981) 137-148.
272 BRE 14350
Putative nociceptive modulatory neurons in the dorsolateral pontomesencephalic reticular formation* Christine M. Haws 1, Andrew M. Williamson 2, Howard L. Fields 3 Departments of 1Neurology, "-Anatomyand 3Physiology, The University of California at San Francisco, San Francisco, CA 94143 (U.S.A.) (Accepted 30 August 1988)
Key words: Antinociception; Nucleus cuneiformis; Nucleus parabrachialis; Pontomesencephalic reticular formation; Single unit recording; Rat
The present study was undertaken to confirm the dorsolateral pontomesencephalic reticular formation (an area that includes the nucleus cuneiformis (NCF) and nucleus parabrachialis (NPB)) as a sensitive site for stimulation-produced antinociception and to investigate the possibility that there are cells in this region that show a change in activity that can be correlated with the occurrence of a nocifensive reflex (the tail-flick withdrawal response from noxious heat (TF)). Such cells have been previously demonstrated in the rostral ventromedial medulla (RVM). Extracellular single unit recording studies were made at sites from which it was possible to inhibit the TF with currents of 10 ¢tA or less. Cellular response to 3 TF trials at 5 min intervals, and spontaneous activity over a 10 rain period, were monitored for each unit. Of the cells encountered, 17% displayed an increase (on-cells) and 4% a decrease (off-cells) in activity that preceded the TF. The remaining cells were unaffected by the TF (neutral cells). On- and off-cells were found throughout the region from which TF suppression was observed but were most concentrated in the areas of the NCF and NPB. The presence of on- and off-cells in the NCF/NPB region as well as the RVM suggests that the input to RVM from NCF/NPB may be important in descending nociceptive modulation.
INTRODUCTION It is well d o c u m e n t e d that nociceptive transmission through the spinal cord can be modified by supraspinal neurons 22'3s. Electrical stimulation of various brainstem sites, including the rostral ventromedial medulla ( R V M ) S'2°'46 and periaqueductal grey ( P A G ) 37'45'50, suppresses behavioral and neuronal responses e v o k e d by a noxious stimulus. Stimulation of the dorsolateral p o n t o m e s e n c e p h a l i c reticular formation adjacent to the midbrain P A G also suppresses the reflex withdrawal response to noxious stimulation and inhibits noxious-evoked excitation of spinal and trigeminal dorsal horn cells in a variety of species 14'19'25'53. M a p p i n g of this region for stimulat i o n - p r o d u c e d antinociception ( S P A ) has revealed that the most effective sites are located dorsal and
ventrolateral to the superior cerebellar peduncle (SCP), areas equivalent to the nucleus cuneiformis (NCF) and the nucleus parabrachialis (NPB) 19'53. Only scattered cells in the NPB project directly to the spinal cord dorsal hornT'32'35; it is therefore likely that spinal antinociceptive effects following stimulation of the dorsolateral pontomesencephalic region are m e d i a t e d primarily by indirect spinal projections. In fact, cells from both the N C F and NPB project to the R V M (an area that includes the nucleus r a p h e magnus and the adjacent reticular formation) 1'1°'23. R V M cells in turn provide the largest brainstem source of axons descending in the dorsolateral funiculus ( D L F ) to the dorsal horn of the spinal cord 6,7, 35,36. Electrical or chemical stimulation of R V M inhibits both spinal reflexes and neuronal responses e v o k e d by a noxious stimulus 3'18'47'51'55'63. Thus, a re-
* A preliminary report has been presented (ref. 28). Correspondence: C.M. Haws, Department of Pharmacology, Box 0450, University of California at San Francisco, San Francisco, CA 94143, U.S.A.
273 lay via neurons in the RVM would provide an anatomical substrate for the suppression of spinal transmission of nociceptive information following stimulation of the NCF/NPB area. Physiological studies have identified two classes of RVM neuron that show a change in activity closely correlated with the occurrence of a nocifensive reflex21. On-cells show an increase, and off-cells show a decrease in their activity just prior to a tail-flick (TF) withdrawal response evoked by noxious heat. Manipulations that produce antinociception can also modify the activity of on- and off-cells5'16,43. In RVM a significant percentage of cells displaying TF-related activity can be antidromically activated from the cervical spinal cord, a finding which provides additional evidence that these cells can modulate spinal nociceptive transmission 59. If a relay via RVM mediates the antinociceptive actions of dorsolateral pontomesencephalic reticular formation, then it may be anticipated that cells with characteristics similar to those of RVM on- and offcells will also be found in this area. To address this issue, extracellular single unit recording was used to study the activity of neurons in the dorsolateral pontomesencephalic reticular formation of the lightly anesthetized rat during the TF-withdrawal response. Both on- and off-cells were found in this area, most concentrated in the NCF/NPB region. MATERIALS AND METHODS Male Sprague-Dawley rats (250-300 g) were given an initial dose of pentobarbital sodium (Nembutal; 55 mg/kg i.p.). A catheter was inserted into an external jugular vein and the animal was placed into a stereotaxic frame. A discrete craniotomy was performed to reveal the cerebral cortex overlying the midbrain. Body temperature was maintained at 37 °C using a circulating hot water pad. The animal was allowed to recover from the pentobarbital to a point at which the TF could be elicited and was then maintained lightly anesthetized by the continuous infusion of methohexital (15-30 mg/kg/h). The animal showed no signs of discomfort, no spontaneous activity, nor a prolonged withdrawal response or vocalization to noxious pinch. The methods for eliciting the TF are those described by Heinricher et al. 29. Briefly, a beam from a
projector lamp was focused on the blackened ventral surface of the tail. A thermistor probe monitoring surface tail temperature provided a signal for the feedback control of the heat stimulus. Between heat stimuli tail temperature was maintained at 35 °C. The TF generally occurred at 42-45 °C with a latency of 4 - 6 s. A gold- and platinum-plated stainless-steel monopolar electrode stereotactically localised dorsal to the NCF (anteroposterior (AP) -0.3 to 1.7 from the interaural line; mediolateral (ML) 1.0 to 2.0; dorsoventral (DV) 5.0 from the cortical surface) was used for stimulation and extracellular single unit recording. The electrode was advanced through the brain in 200 p m steps until a site was reached from which the TF could be inhibited (10 s cut-off, SPA) using a current of no more than 10 p A (400 ps cathodal pulse width, 50 Hz continuous train). Advancement of the electrode in 200 p m steps was continued until SPA could no longer be elicited. The electrode was then attached to a differential pre-amplifier for extracellular recording. Action potentials were amplified, filtered and displayed on an oscilloscope where spike shape and amplitude were monitored to assure the identity of the unit being recorded. Spikes were also converted to voltage pulses using a window discriminator, the output of which was sent to an audio-amplifier and integrated and displayed on a strip chart recorder. The search for on- and off-cells was continued 1-2 mm dorsal and ventral, and along penetrations rostral and caudal, to the site from which suppression of the TF could be elicited with stimulating currents of 10pA. During the search for on- and off-cells the electrode was advanced through the brain in 3 p m steps. Noxious pinch delivered to the paw once every 100-200 p m served as a search stimulus. Each cell's response to heat (tail) and noxious pinch (hind paws and ear) were determined and the cell characterised as an on- or off-cell, or neutral cell according to the classification system applied to cells in the RVM by Fields et al. 21. For each cell, the response to 3-5 applications of noxious heat to the tail at 5 min intervals was monitored as was 10 min of spontaneous activity. After each penetration lesions were made at sites of interest (20 p A anodal current for 10 s). At completion of the experiment the animal was perfused
274 transcardially with 0.9% saline followed by 4% formaldehyde solution. Lesions were identified in 50 p m coronal sections stained with Cresyl violet and marked on diagrams derived from the stereotaxic atlas of Paxinos and Watson 48.
Single unit recording A total of 275 cells in the caudal midbrain and rostral pons in an area dorsal and ventral to the SCP were studied in 53 experiments. Spikes were generally biphasic, negative-positive in polarity. Spike amplitude ranged from 100-500/~V.
RESULTS
On-cells Stimulation produced antinociception Suppression of the TF at currents of 10 p A or less was observed following stimulation of sites ventral to the inferior colliculus, bordering on and lateral to the P A G and medial to the lateral lemniscus. This region extends from the rostral pole of the N C F (AP 1.2), to the rostral pole of the locus coeruleus ( A P - 0 . 3 ; Fig. 1). Regions more rostral and caudal to these coordinates were not systematically investigated. TF suppression could be evoked from much of the nucleus pontis oralis (NPO), the ventral border being the dorsal edges of the superior olive and the nucleus of the trapezoid body. This extensive region of the caudal midbrain and rostral pons was divided by an area, centered on the SCP, from which SPA could not be consistently evoked at the currents used.
A total of 46 (17%) cells showed an increase in activity that preceded the TF. The on-cell burst began at a variable time prior to the TF (range 200 ms to 2.4 s, mean 827 ms, S.E.M. + 90 ms). The duration of the evoked burst was also quite variable, ranging from 1 s to over 10 s (Fig. 2). Responses varied from several spikes to a vigorous and prolonged burst of activity. All cells responded to noxious pinch applied over a wide area of the body surface including the hind paw and ears bilaterally, and 17 cells (37%) could additionally be excited by non-noxious stimuli such as brushing of the fur or brisk tapping of the tail. Thirty-three (72%) on-cells demonstrated low levels of spontaneous activity (1-15 Hz) and the remaining thirteen were inactive• Spontaneous activity was generally irregular and in 11 neurons consisted of de-
0"2
Fig. 1. Drawing of coronal sections through the caudal midbrain/rostral pons illustrating regions of the electrode penetration from which the TF was inhibited with currents of less than or equal to 10 pA. Upper circle denotes the site at which SPA was first encountered while the lower circle shows the ventral limit. Regions dorsal and ventral to the SCP were the most sensitive sites for SPA. Anterior-posterior, 1.2 to -0.3 (derived from the atlas of Paxinos and Watson, 1982).
275
A
I
B
10HZI I
I
I
FP TF
TF
Fig. 2. A: single oscilloscope sweep (upper trace) shows the increase in activity that occurs approximately 1 s before the TF (lower trace). Sweep time equals 10 s. B: chart recording illustrating the increase in FR of cell in A in response to foot pinch (FP) or noxious heat (TF) as well as the low level of spontaneous activity displayed by ceils in this part of the brain. Time course as in Fig. 3. fined periods of complete silence alternating with periods of irregular spontaneous activity (Fig. 3).
R V M under similar anaesthetic conditions 4.
Neutral cells Off-cells Eleven cells (4%) showed an abrupt decrease in activity just prior to the TF withdrawal response. The onset of this reduction in activity preceded the TF by a variable period of time ranging from 400 ms to 3.0 s (mean 1.3 s, S.E.M. + 0.35 s), and lasted for a duration of 800 ms to over 10 s (Fig. 4). All cells were additionally inhibited by noxious pinch and two by nonnoxious stimuli such as light tapping or brush. Offcells showed a range of spontaneous firing rates although spontaneous activity was generally slow and irregular (3-15 Hz). Two cells showed defined periods of silence interspersed among periods of activity, a characteristic also displayed by off-cells in the A
I
5.z I I TF
I TF
I EP
The great majority of cells in the region caudal and ventral to the SCP did not show a change in firing rate that began prior to the TF (n = 218 of 275, 79%). These cells were classified as neutral cells (Fig. 5). Sixteen of these cells were excited and 5 inhibited by noxious pinch applied to the hind paws or ears. Four cells excited by noxious pinch showed an increase in firing rate which followed the TF. Similarly, 2 of the 5 cells inhibited by noxious pinch showed a decrease in activity that followed the TF. As the evoked activity displayed by these 6 cells does not conform to the original definition of on- and off-cells 21, these cells were classified as 'late-on cells' and 'late-off cells', respectively, and not included as on- and off-cells in the present analysis. All neutral cells showed some spontaneous activity. As with on- and off-cells a wide range of spontaneous firing rates and patterns was displayed by neutral cells (1-20 Hz), but none of the neutral cells displayed the alternating periods of activity and silence displayed by some on- and off-cells. In general, the spontaneous firing pattern of all 3 cell classes was sufficiently similar that it was not possible to differentiate between on-, off- and neutral cells according to spontaneous firing rate.
I min
Fig. 3. On-cell displaying spontaneous periods of activity alternating with periods of silence. A: single oscilloscope sweep (upper trace) showing on-cell burst approximately 1 s prior to the TF (lower trace). B: chart recording of spontaneous activity of cell in A. Cell responds with an increase in activity to tail-flick (TF) and ear pinch (EP).
Anatomical distribution of on- and off-cells The distribution of cells showing on- and off-cells, and neutral cells is illustrated in Figs. 6 and 7, respectively. Neurons showing a change in activity related to the
276
TF I
A
B IOHZJ I I TF FP 1 min Fig. 4. A: single oscilloscope sweep (lower trace) illustrating the pause in activity that occurs just prior to the TF (upper trace). Sweep time is 10 s. B: chart recording of same cell; tail-flick (TF), foot pinch (FP) both produce a short-lived decrease in spontaneous activity.
occurrence of the T F were found only in areas from which S P A could be elicited with currents of 10 ~ A or less. The two cell classes displayed a similar distribution, although fewer off-cells were found. On- and off-cells were found dorsal to the SCP in the N C F , and lateral and ventral to the SCP in the ventral NPB and in the adjacent N P O . Neutral cells were scatt e r e d throughout the sampled region extending into
areas outside those from which low-threshold S P A could be evoked. A total of 51 cells were located outside of the area from which stimulation with currents of 10 p A or less would suppress the TF. These cells were all neutral cells. The percentage of cells showing activity related to the occurrence of the T F dorsal and ventral to the
A
1 min Fig. 5. A: single oscilloscope trace illustrating no change in firing rate (upper trace) with the TF (lower trace). Sweep time is 10 s. B: chart recording of the spontaneous activity of the cell displayed in A.
0.2
0.7
277
a
Fig. 6. Diagrams of coronal sections through the caudal midbrain showing the location of 46 on-cells (upward pointing arrows, left) and 11 off-cells (downward pointing arrows, right). The two cell classes represented on opposite sides of the brain for clarity. On- and off-cells were only located in areas of the dorsolateral pontomesencephalic reticular formation from which the stimulation with currents of 10/~A or less would inhibit the TF. Anterior-posterior, 1.2 to 0.2. Diagrams as in Fig. 1.
S C P indicates that on- and off-cells t e n d to b e clus-
DISCUSSION
t e r e d in an a r e a r o u g h l y e q u i v a l e n t to the N C F and an a r e a i m m e d i a t e l y v e n t r a l to t h e S C P , r o u g h l y e q u i v a l e n t to the v e n t r a l N P B ( T a b l e I).
T h e w o r k d e s c r i b e d h e r e d e m o n s t r a t e s t h e prese n c e of n e u r o n s in the d o r s o l a t e r a l p o n t o m e s e n c e -
-0"3
°
/A
0.2
or'~
7
re,,
•/;Z.',,
Fig. 7. Representative coronal sections through the caudal midbrain/rostral pons showing the distribution of 218 cells that did not show a TF-related change in activity. Anterior-posterior, 1.7 to -0.3. Diagrams as in Fig. 1.
278 It is important to consider the possible influence of
TABLE I Distribution of on- and off-cells and neutral cells
The number and percentage of on- and off-cells and neutral cells located in the NCF, NPB and NPO as compared with the area of the dorsolateral pontomesencephalic region from which SPA can be elicited. Boundaries of NCF, NPB and NPO as defined by Paxinos and Watson 48. NCF
On Off Neutral
NPB
NPO
no.
%
no.
%
no.
14 4 19
37.8 10.8 51
7 3 16
27 14 11.5 1 61.5 53
Total area %
no.
%
20.5 46 17 1.5 11 4 78 218 79
phalic reticular formation that show a change in activity related to the occurrence of a nocifensive spinal reflex. As in RVM, this region contains two classes of such neurons, on-cells which show an increase, and off-cells which show a decrease in firing rate just prior to the withdrawal of the tail from noxious heat. The location of on- and off-cells was restricted to the regions of the dorsolateral pontomesencephalic reticular formation from which electrical stimulation inhibited the TF as described by this and other studies 19'53. Dorsal to the SCP and caudal to the inferior collicutus, on- and off-cells were found in the NCF. Ventral and lateral to the SCP, on- and off-cells were present in both ventral and lateral NPB and the adjacent lateral NPO. These cells were more concentrated in the N C F and NPB than in the NPO, which may correlate with the larger afferent projection to the R V M from the N C F and NPB than from the NPO1AO,23. On- and off-cells were first described in the R V M 21 and more recently have been reported to be present in the P A G 29, another brainstem area that plays an important role in nociceptive modulation 37'45'5°. Onand off-cells in the dorsolateral pontomesencephalic region more closely resemble those found in the P A G than those found in RVM. Thus, in both the NCF/ NPB region and the P A G , on- and off-cells display low irregular discharge rates and a relatively brief and/or weak discharge concomitant with nocifensive withdrawal reflexes. In contrast, on- and off-cells in R V M exhibit vigorous and long lasting changes in activity preceding the TF, and display a pattern of spontaneous activity that consists of periods of activity alternating with periods of silence 4.
experimental conditions on nociresponsivity. This is illustrated when the data obtained in our investigation is compared with data obtained from a similar study undertaken by Hardy et al. 26. They found that the majority (30/33) of neurons encountered in the 'paralemniscal reticular formation' (which includes the N C F and NPB, thus being equivalent to the area described in this report) responded to noxious pinch, approximately half showing an increase and the remainder a decrease in activity. Also, in contrast to our study, all neurons recorded from were spontaneously active. These differences may be attributed to the use of different anaesthetics (methohexital vs. chloral hydrate) or possibly to sampling bias resulting from the use of a search stimulus that would favour the selection of on-cells. The low levels of spontaneous activity that we observed may have contributed to the very small numbers of off-cells encountered as any decreases in activity associated with the TF would have been indistinguishable from spontaneous pauses. Even considering the potential effects of anaesthetic and sampling bias, it is likely that on- and offcells constitute, at most, a small percentage of the total population of neurons encountered in the dorsolateral pontomesencephalic reticular formation, a region involved in many diverse functions 11'12'17'44'52'56. Thus, neutral cells may be involved in neural mechanisms subserving a variety of visceral, neuroendocrine and behavioral functions unrelated to nociceptive modulation. Our results, however, do not rule out a possible role for neutral cells in descending nociceptive modulation. D o cells in the dorsolateral p o n t o m e s e n c e p h a l i c reticular f o r m a t i o n play a role in m o d u l a t i n g nociceptive transmission?
Although a small percentage of on- and off-cells (37 and 18% respectively) responded to both noxious and non-noxious stimuli (brush) the majority responded exclusively to noxious stimuli (foot pinch and noxious heat). These observations correlate with anatomical studies describing afferents to NCF, NPB and NPO. N C F and NPB receive a substantial direct input from nociresponsive neurons in lamina I of both spinal and trigeminal dorsal horns 15'33'4°'42'57, and a smaller input from multireceptive neurons in laminae
279 IV, V and deeper laminae of the spinal cord 41'62. Many lamina I cells project exclusively to the mesencephalon 13,6a, others constitute collaterals of spinothalamic tract axons 33'49. This large projection to the pontomesencephalic reticular formation from nociresponsive cells in the dorsal horn points to a major role of this area in nociception. Studies of the efferent projections to the spinal cord from the NCF and NPB are also consistent with a role of this region in nociceptive modulation. Although there is a major projection via the ventral and ventrolateral funiculi that terminates in ventral regions of the cord 7'32,35, there are two minor projections that travel in the dorsolateral funiculus (DLF) to the dorsal horn: a projection from scattered neurons in an area equivalent to the ventral NPB and the adjacent nucleus Kolliker-Fuse 5s, and a second from a group of laterally placed cells adjacent to the lateral lemniscus that terminates in the contralateral dorsal horn 32'5s. These projections to the dorsal horn via the DLF provide the anatomical substrate for direct modulation of dorsal horn cell responses to noxious inputs and could underlie, at least in part, the SPA observed in the present experiments. In support of this idea is the demonstration that cells in the direct projection from the NPB and Kolliker-Fuse nucleus are immunopositive for the neurotransmitter noradrenaline 6°. There is evidence that this noradrenergic projection is a component of the pathway mediating suppression of cellular responses to noxious inputs in the dorsal horn following stimulation of the midbrain 31. Whether any on- or off-cells in the NCF/NPB are noradrenergic or spinally-projecting has yet to be determined. In addition to their possible direct effect on spinal dorsal horn neurons, dorsolateral pontomesencephalic reticular formation cells may modulate nociceptive transmission via a relay in the RVM. Anterograde and retrograde transport studies demonstrate that cells in both NCF and NPB project to R V M 1'23. Components of this projection have been demonstrated to be immunoreactive for neurotensin 1° and acetylcholine 2,54, and there is evidence that cells in the NPB may be a source of noradrenaline terminals in the RVM (A.I. Basbaum, personal communication). These anatomical observations are supported by data from both behavioural and electrophysiological studies. In the cat, Gebhart et al. 24 reported that
suppression of a heat-evoked response in dorsal horn cells following stimulation of either the P A G or dorsolateral pontomesencephalic region could be blocked by lidocaine microinjected into the RVM. Behbehani and Zemlan 9 demonstrated that stimulation of NCF produced an increase in RVM unit activity that could be mimicked by iontophoretically applied acetylcholine and partially attenuated by atropine. Furthermore, microinjection of atropine into the RVM of the rat was found to attenuate suppression of a nocifensive reflex following stimulation of the NCF/NPB area 39. Similar studies investigating the input to RVM from the NPB demonstrate that antinociception resulting from microinjection of carbachol into the NPB involves a relay with neurons in NRM and that stimulation of NPB produces excitation of raphe-spinal neurons 34. Of particular interest is the role of noradrenaline in the projection from the NPB to RVM. Microinjection of selected noradrenergic agents into the RVM produces a change in nociceptive threshold 27'51 and microiontophoretic application of noradrenergic agonists reveal an al-receptor mediated excitation and an a2-mediated inhibition restricted to on-cells 3°. These data suggest that the activity of RVM on-cells is under the influence of a noradrenergic input a component of which may originate from the NPB. In summary, there is an increasing body of evidence to support the involvement of neurons in the NCF and NPB regions in the modulation of nociceptive transmission at the dorsal horn level. This may be mediated by at least two descending pathways: one direct and one via neurons in RVM. The presence of a direct projection to the spinal cord via the ventral and ventrolateral funiculi raises the important possibility that the TF inhibition observed in the present experiments represents direct inhibition of the motor neurons involved. This possibility cannot be excluded by data obtained in the present experiments, however, several lines of evidence favor the hypothesis that inhibition of nociceptive dorsal horn neurons is at least partially involved. First, there is inhibition of dorsal horn cell activity produced by electrical stimulation of this area. Second, stimulation of this area both excites RVM neurons and inhibits withdrawal reflexes and both of these effects are blocked by RVM atropine. Since RVM projects almost exclusively to the dorsal horn,
280 that NCF/NPB on- and off-cells play a role in noci-
at least part of the SPA from the dorsolateral pontomesencephalic reticular formation is very likely via an effect on dorsal horn neurons. The evidence that projections from the NCF/NPB region are involved in antinociception relayed via R V M raises the possibility that on- and off-cells in the NCF/NPB region are connected to similar cells in
solateral pontomesencephalic reticular formation, which includes the N C F and NPB, forms part of a highly interconnected supraspinal network, includ-
the RVM. The recent observations showing a significant correlation between T F latency and the spontaneous change in R V M on- and off-cell discharge fre-
ACKNOWLEDGEMENTS
quency are important evidence implicating such cells
ceptive modulation. They also indicate that the dor-
ing R V M and P A G , that modulates the transmission of nociceptive information in the spinal cord.
in nociceptive modulation. These observations, plus the fact that dorsolateral pontomesencephalic on-
We wish to thank Pat Littlefield for histology, Mary Heinricher and Peggy Mason for helpful comments on the manuscript, and Susan Elliott and Lael
and off-cells are apparently clustered in regions projecting most densely to the R V M support the idea
Carson for secretarial assistance. This work was supported by PHS grants DA01949 and NS21445.
REFERENCES 1 Abols, I.A. and Basbaum, A.I., Afferent connections of the rostral medulla of the cat: a neural substrate for midbrain-medullary interactions in the modulation of pain, J. Comp. Neurol., 201 (1981) 285-287. 2 Amstrong, J., Saper, C.B., Levey, A.I., Wainer, B.H. and Terry, R.D., Distribution of cholinergic neurons in the rat brain: demonstrated by immunocytochemical localisation of choline acetyltransferase, J. Comp. Neurol., 216 (1983) 52-68. 3 Azami, J., Llewelyn, M.B. and Roberts, M.H.T., Spinal cord mechanisms of analgesia produced by microinjection of morphine into the nucleus raphe magnus (NRM) of the rat, J. Physiol. (Lond.), 367 (1982) 47-48P. 4 Barbaro, N.M., Fields, H.L. and Heinricher, M.M., Reciprocal activity of on- and off-cells in the rostral ventromedial medulla of the rat, Soc. Neurosci. Abstr., 11 (1985) 1180. 5 Barbaro, N.M., Heinricher, M.M. and Fields, H.L., Putative pain modulating neurons in the rostral ventral medulla: reflex related activity predicts the effects of morphine, Brain Research, 366 (1985) 203-210. 6 Basbaum, A.I., Clanton, C.H. and Fields, H.L., Three bulbospinal pathways from the rostral medulla of the cat: an autoradiographic study of pain-modulating systems, J. Comp. Neurol., 178 (1978) 209-224. 7 Basbaum, A.I. and Fields, H.L., The origin of descending pathways in the dorsolateral funiculus of the spinal cord of the cat and rat: further studies on the anatomy of pain modulation, J. Comp. Neurol., 187 (1979) 513-532. 8 Basbaum, A.I. and Fields, H.L., Endogenous pain control systems: brainstem spinal pathways and endogenous circuitry, Annu. Rev. Neurosci., 7 (1984) 309-338. 9 Behbehani, M.M. and Zemlan, F.P., Response of nucleus raphe magnus neurons to stimulation of the nucleus cuneiformis: role of acetylcholine, Brain Research, 369 (1986) 110-118. 10 Beitz, A.J., Sites of origin of brainstem neurotensin and serotonin projections to the rodent nucleus raphe magnus, J. Neurosci., 2 (1982) 829-842. 11 Bernston, G.G., Attack, grooming and threat elicited by stimulation of the pontine tegmentum in cats, Physiol. Be-
hav., 11 (1973) 81-87. 12 Betrand, F. and Hugelin, A., Respiratory synchronising function of the nucleus parabrachialis medialis: pneumotaxic mechanisms, J. Neurophysiol., 34 (1971) 189-207. 13 Bjorkeland, M. and Boivie, J., The termination of spinomesencephalic fibres in the cat, Anat. Embryol., 170 (1984) 265-277. 14 Carstens, E., Klumpp, D. and Zimmerman, M., Differential inhibitory effects of medial and lateral midbrain stimulation on spinal neuronal discharges to noxious skin heating in the cat, J. Neurophysiol., 43 (1980) 332-342. 15 Cechetto, D.F., Standaert, D.G. and Saper, C.P., Spinal and trigeminal dorsal horn projections to the parabrachial nucleus in the rat, J. Comp. Neurol., 240 (1985) 153-160. 16 Cheng, Z.-F., Fields, H.L. and Heinricher, M.M., Morphine microinjected into the periaqueductal gray has differential effects on 3 classes of medullary neurons, Brain Research, 375 (1986) 57-65. 17 Coote, J.H., Hilton, S.M. and Zbrozyna, W., The pontomedulla area integrating the defense reaction in the cat and its influence on muscle blood flow, J. Physiol. (Lond.), 229 (1973) 257-274. 18 Dickenson, A.H., Oliveras, J.-L. and Besson, J.-M., Role of the nucleus raphe magnus in opiate analgesia as studied by the microinjection technique in the rat, Brain Research, 170 (1979) 95-111. 19 Dostrovsky, J.O., Hu, J.W., Sessle, B.J. and Sumino, R., Stimulation sites in the periaqueductal grey, nucleus raphe magnus and adjacent regions effective in suppressing the oral-facial reflexes, Brain Research, 252 (1982) 287-297. 20 Fields, H.L. and Basbaum, A.I., Brainstem control of spinal pain-transmission neurons, Annu. Rev. Physiol., 40 (1978) 217-248. 21 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. 22 Fields, H.L. and Heinricher, M.M., Anatomy and physiology of a nociceptive modulatory system, Phil. Trans. R. Soc. Lond. Ser. B, 308 (1985) 361-374. 23 Gallagher, D.W. and Pert, A., Afferents to brainstem nu-
281 clei (brain stem raphe, nucleus reticularis caudalis and nucleus gigantocellularis) in the rat as demonstrated by microiontophoretically applied horseradish peroxidase, Brain Research, 144 (1978) 257-275. 24 Gebhart, G.F., Sandkhuler, J., Thalhammer, J.G. and Zimmerman, M., Inhibition of spinal nociceptive information by stimulation in the midbrain is blocked by lidocaine microinjected in nucleus raphe magnus and medullary reticular formation, J. Neurophysiol., 50 (1983) 1446-1459. 25 Giradot, M.N., Brennan, T.J., Martindale, M.E. and Foreman, R.D., Effects of stimulating the subcoeruleus-parabrachial region on the non-noxious and noxious responses of T1-T5 spinothalamic tract neurons in the primate, Brain Research, 409 (1987) 19-30. 26 Hardy, S.G.P., Haigler, H.J. and Leichnetz, G.R., Paralemniscal reticular formation: response of cells to a noxious stimulus, Brain Research, 267 (1983) 217-223. 27 Haws, C.M., Heinricher, M.M. and Fields, H.L., Antinociceptive action of alpha-2 adrenergic agonists in the rostroventromedial medulla of the rat may be mediated by a selective action on a single population of putative nociceptive modulatory neurons, Soc. Neurosci. Abstr., 14 (1988) in press. 28 Haws, C.M., Williamson, A.M. and Fields, H.L., Putative pain-modulating neurons in the mesencephalic and pontine reticular formation, Pain, Suppl. 4 (1987) $29. 29 Heinricher, M.M., Cheng, Z.-F. and Fields, H.L., Evidence for two classes of nociceptive modulating neurons in the periaqueductal grey, J. Neurosci., 7 (1987) 271-278. 30 Heinricher, M.M., Haws, C.M. and Fields, H.L., Opposing actions of norepinephrine and clonidine on single class of pain-modulating neurons in the rostral ventromedial medulla, Pain, Suppl. 4 (1987) $28. 31 Hodge, Jr., C.J., Apkarian, A.V. and Stevens, R.T., Inhibition of dorsal-horn cell responses by stimulation of the Kolliker-Fuse nucleus, J. Neurosurg., 65 (1986). 32 Holstege, G. and Kuypers, H.G.J.M., The anatomy of brain stem pathways to spinal cord in the cat. A labelled amino acid tracing study. In H.G.J.M. Kuypers and G.F. Martin (Eds.), Descending Pathways to the Spinal Cord, Progress in Brain Research, Vol. 57, Elsevier, Amsterdam, 1982, pp. 145-175. 33 Hylden, J.L.K., Hayashi, H., Dubner, R. and Bennett, G.J., Physiology and morphology of the lamina I spinomesencephalic projection, J. Cornp. Neurol., 247 (1986) 505-515. 34 Katayama, Y.Y., Tsubokawa, S., Maejima, S. and Yamamoto, T., Response properties of raphe-spinal neurons to stimulation of the pontine parabrachial region producing behavioural nociceptive suppression in the cat, Appl. Neurophysiol., 49 (1986) 112-120. 35 Kuypers, H.G.J.M. and Maisky, V.A., Funicular trajectories of descending brainstem pathways in the cat, Brain Research, 136 (1977) 159-165. 36 Leichnetz, G.R., Watkins, L., Griffin, G., Martin, R. and Mayer, D.J., The projection from nucleus raphe magnus and other brainstem nuclei to the spinal cord in the rat: a study using HRP blue reaction, Neurosci. Lett., 8 (1978) 19-24. 37 Mayer, D.J. and Liebeskind, J.C., Pain reduction by focal electrical stimulation of the brain: an anatomical and behavioural analysis, Brain Research, 68 (1974) 73-93. 38 Mayer, D.J. and Price, D.D., Central nervous system mechanisms of analgesia, Pain, 2 (1976) 379-404.
39 McCarthy, M.S. and Proudfit, H.K., Evidence for the cholinergic mediation of the antinociception induced by electrical stimulation of the pedunculopontine tegmental nucleus, Soc. Neurosci. Abstr., 13 (1987) 304. 40 McMahon, S.B. and Wall, P.D., Electrophysiological mapping of brainstem projections of spinal cord lamina I cells in the rat, Brain Research, 333 (1985) 19-26. 41 Menetrey, D., Chaouch, A. and Besson, J.-M., Location and properties of dorsal horn neurons at the origin of the spinoreticular tract in the lumbar enlargement of rat, J. Neurophysiol., 44 (1980) 862-877. 42 Menetrey, D., Chaouch, A., Binder, D. and Besson, J.-M., The origin of the spinomesencephalic tract in the rat: an anatomical study using the retrograde transport of horseradish peroxidase, J. Comp. Neurol., 206 (1982) 193-207. 43 Moreau, J.L. and Fields, H.L., Evidence for GABA involvement in midbrain control of medullary neurons that modulate nociceptive transmission, Brain Research, 397 (1986) 37-46. 44 Norgren, R. and Leonard, C.M., Ascending central gustatory pathways, J. Comp. Neurol., 150 (1973) 217-238. 45 Oliveras, J.L., Besson, J.-M., Guilbaud, G. and Liebeskind, J.C., Behavioural and electrophysiological evidence of pain inhibition from midbrain stimulation in the cat, Brain Research, 20 (1974) 32-45. 46 Oliveras, J.L., Guilbaud, G. and Besson, J.-M., A map of serotonergic structures involved in stimulation-produced analgesia in unrestrained freely moving cats, Brain Research, 164 (1979) 317-322. 47 Oliveras, J.L., Redjemi, F., Guilbaud, G. and Besson, J.M., Analgesia induced by electrical stimulation of the inferior central nucleus of the raphe of the cat, Pain, 1 (1975) 139-145. 48 Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic, Sydney, 1982. 49 Price, D.D., Hayes, R.J., Ruda, M.A. and Dubner, R., Spatial and temporal transformations of input to spinothalamic tract neurons and their relation to sensation, J. Neurophysiol., 41 (1978) 933-947. 50 Reynolds, D.V., Surgery in the rat during analgesia induced by focal brain stimulation, Science, 164 (1969) 444-445. 51 Sagen, J. and Proudfit, H.K., Evidence for pain modulation by pre- and postsynaptic noradrenergic receptors in the medulla oblongata, Brain Research, 331 (1985) 285-293. 52 Saito, H., Sakai, K. and Jouvet, M., Discharge pattern of the nucleus parabrachialis lateralis neurons of the cat during sleep and waking, Brain Research, 134 (1977) 55-72. 53 Sandkhuler, J. and Gebhart, G.F., Characterisation of inhibition of a spinal nociceptive reflex by stimulation medially and laterally in the midbrain and medulla in the pentobarbital-anaesthetised rat, Brain Research, 305 (1984) 67-76. 54 Satoh, K., Armstrong, D.M. and Fibiger, H.C., A comparison of the distribution of central cholinergic neurons as demonstrated by acetylcholinesterase pharmacohistochemistry and choline acetyltransferase immunohistochemistry, Brain Res. Bull., 11 (1983) 693-720. 55 Satoh, M., Oku, R. and Akaike, A., Analgesia produced by microinjection of L-glutamate into the rostral ventromedial bulbar nuclei of the rat and its inhibition by intrathecal alpha-adrenergic blocking agents, Brain Research, 261 (1983) 361-364. 56 Sheard, M.H. and Flynn, J.P., Facilitation of attack by
282
57
58
59
60
stimulation in the midbrain in cats, Brain Research, 4 (1967) 324-333. Swett, J.E., McMahon, S.B. and Wall, P.D., Long ascending projections to the midbrain from cells in lamina I, J. Comp. Neurol., 238 (1985) 401-416. Tan, J. and Holstege, G., Anatomical evidence that the pontine field projects to lamina I of the caudal spinal trigeminal nucleus and the spinal cord and to the EdingerWestphal nucleus of the cat, Neurosci. Left., 65 (1986) 317-322. Vanegas, H., Barbaro, H.M. and Fields, H.L., Tail-flick related activity in medullo-spinal neurons, Brain Research, 321 (1984) 135-141. Westlund, K.N. and Coulter, J.D., Descending projections of locus coeruleus and subcoeruleus/medial parabrachial
nucleus in the monkey: axonal transport studies and dopamine-betahydroxylase neurocytochemistry, Brain Res. Rev., 2 (1980) 235-264. 61 Wiberg, R.P. and Blomquist, A., The spinomesencephalic tract in the cat: its cells of origin and termination pattern as demonstrated by the intraaxonal transport method, Brain Research, 291 (1984) 1-18. 62 Yezierski, R.P. and Schwartz, R.N., Receptive field properties of spinomesencephalic tract (SMT) cells, Pain, Suppl., 2 (1984) S127. 63 Zorman, G., Hentall, I.D., Adams, J.E. and Fields, H.L., Naloxone-reversible analgesia produced by microstimulation in the rat medulla, Brain Research, 219 (1981) 137-148.