The pontine parabrachial region mediates some of the descending inhibitory effects of stimulating the anterior pretectal nucleus

Brain Research, 594 (1992) 205-2 ! 4 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00

205

BRES 18202

The pontine parabrachial region mediates some of the descending inhibitory effects of stimulating the anterior pretectal nucleus M a r i a n a G. Terenzi, H u w R e e s and Malcolm H.T. R o b e r t s Department of Physiology, Universityof Wales, Collegeof Cardiff, Cardzff(UK) (Accepted 26 May 1992)

Key words: Anterior pretectal nucleus; Pontine parabrachial region; Analgesia; Pain; Nociception

Electrical stimulation of the anterior pretectal nucleus (APtN) elicits antinociception by inhibiting the responses of spinal multireceptive neurones to noxious stimuli. This descending inhibition is mediated, in part, by activating cells in the ventrolateral medulla. Neuronal tract tracing has previously shown that the APtN also projects directly to the pontine parabrachial region (PPR). The PPR, investigated by Katayama et al. (Brain Res., 296 (1984) 263-283), corresponds to the cholinergic cell group Ch5 of Mesulam et al. (Neuroscience, 10 (1983) 1185-1201). In this stud~, the pathway from APtN to PPR was investigated using urethane anaesthetised rats. Electrical stimulation (single square wave 0.2 ms pulses, 1-10 V, 5 Hz) of the APtN potently excites 40% of the cells recorded in the PPR. in the reverse experiment, stimulation of the PPR at the same parameters excited 36% of the cells recorded in the APtN. The contribution of this pathway to the spinal inhibitory effects of APtN stimulation was then examined. Unanaesthetised animals received electrical stimulation to the APtN (35 ,cA r.m.s., 15 s) and the increase in tail-flick latencies was measured. Bilateral electrolytic lesions of the PPR caused a 67% reduction of the antinociceptive effect of APtN stimulation, in urethane anaesthetised rats, microinjection of tetracaine into the PPR blocked the inhibition of multireceptive dorsal horn neumnes caused by APtN stimulation (20 s train of 50 ,cA square wave 0.1 ms pulses, 100 Hz). In conclusion, these experiments strongly sugget that the PPR may be an important part of a descending antinociceptive pathway originating in the APtN.

INTRODUCTION Recent experiments have shown that low intensity electrical stimulation of the anterior pretectal nucleus (APtN) is sufficient to induce potent analgesia 42. The analgesic effects of APtN stimulation are due in part to descending inhibition of the nociceptive discharges of deep dorsal horn neurones 3~. Thus, APtN stimulation activates a descending pathway that inhibits neurones in the spinal dorsal horn. The APtN does not project directly to the spinal cord, therefore other nuclei must provide the link between these structures. In a recent study 47 we demonstrated that neurones in the ventrolateral medulla are relevant for the antinociceptive effects of stimulating the APtN. However, lesions of the ventrolateral medulla did not block the effects of APtN stimulation completely. This suggests that other sites may also be involved in the descending inhibitory effects of APtN stimulation. There is evidence for the existence of several other projections from the APtN to

areas implicated in descending inhibition ~''s'21'24'St.One of these areas is the pontine parabrachial region (PPR). The PPR is a group of cells in the pontine medial reticular formation that surrounds the superior cerebellar peduncle. It includes the rostral part of the parabrachial nucleus and the caudal third of the pedunculopontine tegmentai nucleus, This region corresponds to the cholinergic cell group Ch5 of Mesulam et a133 studied in the cat by Katayama et al. 2s'29. This region was initially described by these authors as the cholinoceptive pontine inhibitory area 28, and was subsequently named the PPR 29. Katayama et al. 2t~ (and more recently, Klamt and Prado 3°) reported that injections of the cholinomimetic carbachol into the pontine parabrachial region induce antinociception in several algesimetric tests. This effect is antagonised by systemic administration of the antimuscarinic agent atropine 29. Antagonism by atropine is also a characteristic of the antinociceptive effect of APtN stimulation 4°. In addition, nicotine and the mus-

Correspondence: H. Rees, Department of Physiology, University of Wales, College of Cardiff, P.O. Box 902, Cardiff CFI 1SS, UK.

206 carinic agonist (+)-cis-dioxolane elicit antinociception when microinjected into the pedunculopontine tegmental nucleus -'5'2~'(rostral part of the PPR). Another similarity is that the analgesia evoked by stimulating the PPR and the APtN is reduced by lesions of the dorsolateral funiculus 23'38. In the present studies, we have further investigated the existence of a reciprocal connection between the APtN and the PPR. We have also determined the relevance of PPR neurones to the antinociceptive effect of APtN stimulation in the tail-flick test in unanaesthetised rats, and on the nociceptive responses of deep dorsal horn neurones in anaesthetised rats. Some of these results have been published in the form of an abstract for the Physiological Society 4s. MATERIALS AND METHODS

Electrophy~tologwal ,,.Ipertmem.~ E~traeelhdar recor{hng of PPR neurones. Male Wistar rat.,, (270-300 g) were anaesthetised with urethane (1.2 g. kg- i, i.p.). The common carotid artery and the trachea were cannulated. The animal was then placed into a stereotaxic frame. Blood pressure, ECG and rectal temperature were monitored and maintained within physiological limit.,, throughout the experiment. The dorsal surface of the skull was exposed and two holes were drilled, one above the APtN and the other above the ipsilateral PPR. The incisor bar orientation and reference planes used were tile ones described in the atlas of Paxinos and Watson TM. The coordinates for the APIN were 4.5 mm posterior to bregma, 1.8 mm lateral to the midline and 4,5 mm below the brain surfitce. A monopolar stainless steel electrode wits inserted into the APIN. The indifferent electrode wits the stereotaxic incisor bar. The coordinates for tile PPR were 8.(} mm posterior to bregma, 1,8 mm laterM to the midlin~ and 0,5 to I,i,5 mm below the brMn ,~urfile¢, The extracelluhlr activity of the ncurones of the PPR wits recorded using a single barrel glass micropipette led 2-3 ~m, 2 MIJ resi,~tance) filled with 4 M NaCI. The number of action potentials occurring per 1.25 s bin wits continuously recorded with a multidumnel chart recorder. Action potentiMs were captured by an online computer which constructed peristimulus time histograms used to determine changes in activity of PPR cells when the APtN was stimulated, Oscilloscope samples of the spontaneous and evoked responses were taken with an x - y plotter, The parameters for APtN stimulation were single square wave pulses (I.0-10.0 V, (},2 ms duration) at 5 Hz, At the end of the experiment, a glass micropipette filled with 2% Pontamine sky blue dye (PSB) in 0,5 M sodium acetate was lowered into the recording area in order to mark the position of the electrode, This was done by a If} rain ejection of PSB with a current of Ill p.A. E~tracelhdar recording oj'APtN neurones. Male Wistar rats (270-300 g) were ,,ubmitted to surgical procedures identical as those described above. This time, however, the stimulating monopolar electrode was placed into the PPR (8.0 mm posterior to bregma, !.8 mm lateral to the midline and 7.5 mm ventral to the brain surface). A glass micropipette was placed into the APtN (4,5 mm posterior to bregma, 1.8 mm lateral to the midline and 4,0-5,0 mm ventral to the brain surface). Peristimulus time histograms and samples of oscilloscope tracc~ were taken as previously described, Behm tourol ev~erimt, nt,~ Male Wi,,tar rats (230-280 g) were anaestheti,,ed with sodium pentobarbitone (Sagatal 60 mg,kg-i, i,p,) and placed into a stereotaxic frame. The skull was exposed and a craniotomy performed, One hole was drilled above the APtN, The coordinates were the ~ame as previously given for the electrophysiological experiments,

Three stainless steel 10 mm length guide cannulae (G21) were positioned above the areas described and fixed with dental acrylic. Sterile obturators were inserted to keep the cannulae clear. The animals were given a topical antibiotic (terramycin) and local anaesthetic (lignocaine, xylocame gel 2%) was applied to the wound margins. Intramuscular penicillin (Mylipen) was injected into the thigh. The animals were removed from the frame and allowed to recover for at least 1 week before further experimentation. After this period, the animals were tested using the tad-flick response to noxious heat (modified by Azami etal. 3 from D'Amour and Smith14). Each rat was placed rote a ventilated plastic tube and its tail laid across a nichrome wire coil. The coil was electrically heated at the rate of 9°C •s-1 from a temperature of approximately 20°C. The tad-flick latency (in seconds) was recordt d at intervals of 5 rain until a stable baseline was reached (at least three tail-flick latencies between 2.5 and 3.5 s). The APtN was stimulated at this moment. The electrical stimulation consisted of a single 15 s period of 50 Hz alternating sine wave current applied to a monopolar electrode inserted in the APtN guide cannula. The tip of the electrode reached the centre of the dorsal part of the APtN. A saline soaked pad was held against the pinna of the ear to act as the indifferent electrode. The current flow was monitored on an oscilloscope and maintained at 35 p,A r.m.s, by observing the voltage drop across a 100 ~ resistor in series with the electrode. Immediately after the stimulation, the tail-flick latencies were measured again. If the animal failed to remove the tail from the heat source after 6 s, the current was stopped and the tail removed from the coil to prevent tissue damage, The time course of the antinociception evoked by APtN stimulation was followed for a period of 90 min. Tail-flick latencies were measured at intervals of 5 min. The latencies were normalised to an Index of Analgesia using the formula: IA=

T F L - baseline TF! 6 - baseline TFL

were TFL is the observed tail-flick latency, baseline is the mean latency obtained before stimulation, and 6 is the cut off time in seconds. One day after this first tail-flick test session, the rats were lmaestht~'tised with sodium pentobarhitone (6fl mg/kg i,p,) and placed in the siereotaxic frame, Craniotomy was performed bilaterally almve the PPR and a monopolar stainless steel electrode was placed in the area to be lesioned, The lesions were obtained by passing positive current of 10l) ~tA DC during 20 s, The holes in the skull were sealed with sterile bone wax, the skin sutured and local anaesthetic infiltrated into the wound, After I week recovery period, the rats were again subjected to the tail-flick test in order to determine changes in the effectiveness of APtN stimulation with or without lesions of the PPR, As a control, a second group of rats was submitted to identical surgical procedure but no current was passed through the electrode. The data were analysed using the non-parametric test of MannWhitney (U), The standard error bars have been included in Fig, 6 to give indication of the variability between animals,

Lamim,c'tomy studies Male Wistar rats (3(X)-350 8) were anaesthetised with halothane (induction: 3,5% in O:, 1.5 l,min-I). The leve! of halothane was reduced to 1,5% in O, (0.5 I. rain- t ) after introducing a cannula into the trachea The surlzery was completed by placing a polythene cannula in the common carotid artery, The rat was then placed into a stereotaxic frame where the blood pressure, ECG and rectal temperature were monitored and kept within physiological limits, A laminectomy was performed in order to expose the lumbar segments of the spinal ~ r d (L 2 -S!), Warm liquid paraffin (37°C) was poured over the spinal cord to prevent drying of the tissues. The level of anaesthetic was reduced to 0,5-0,8% halothane in 0 2 for the rest of the experiment, A hole was drilled at 4.5 mm posterior to bregma and 1,8 mm lateral to the midline. A stainless steel monopolar electrode was introduced into the APtN 4.5 mm ventral to the brain surface, A glass micropipette (4 M NaCI) was used to record extracellular activity of the ipsilateral dorsal horn neurones. The number of action potentials per 1.25 s bin was continuously recorded with a

207 multichannel chart recorder, Samples of oscilloscope traces of individual spikes were taken by an x - y plotter, The receptive field of each cell was carefully mapped by applying brush or gentle pressure stimuli to the ipsilateral hind paw. The noxious stimulus was 5 s immersion of the foot in water at 50°C. The temperature of the water was monitored with a thermal probe and the stimulus was repeated every 4 rain until stable responses were obtained. Immersion of the foot in water at 37°C did not cause excitation of these neurones. The APtN was stimulated with a 20 s train of 50/tA square wave pulses (0.1 ms width, 100 Hz). The effect of APtN stimulation on the nociceptive responses of the deep dorsal horn neurones was assessed during the regular application of the noxious stimulus to the peripheral receptive field. After electrical stimulation of the APtN, a very fine glass capillary needle (o.d. 100/zm approx.) was inserted into the ipsilateral PPR. A volume of 0.5/~1 of 4% tetracame in saline was injected into the PPR. Immediately after the microinjection, the APtN was again stimulated to assess whether the inhibition of the PPR neurones would alter the antinociceptive effect of APtN stimulation on deep dorsal horn neurones. The experiment continued until the effect of APtN stimulatic 1 was recovered. Whilst waiting for recovery, the APtN was stimulated at intervals of 20 rain, These relatively long intervals were chosen in order to minimize damage to the area.

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Histological analysis At the end of each experiment, the animals were injected with pentobarbitone (Sagatal, 60 m g . k g - t i.p.) and perfused through the heart with 25% formol saline. The brains were removed and kept in the same fixative for 24 h. Sections (50-100 Izm) were taken using a freezing microtome and mounted on gelatinized slides. The slides were stained with !% Neutral red and the position of the stimulating electrodes, PSB blue dye spots and the extent of the electrolytic lesions were examined under the microscope. The lesions consisted of a hollow central core (of approx. 0.5 mm in diameter) surrounded by a dense d,,:posit of necrotic tissue (approx. 1.0 mm in diameter) darker than the aormal tissue in stained sections. All comparisons were made with the reference planes of the atlas of Paxinos and Watson 3¢'.

RESULTS

Electrophysiological study of the reciprocal connection between the APtN and the PPR Extrace!!ular recording of neurones within the pontine parabrachial region (PPR). Recordings were made from 148 neurones located in the PPR. Electrical stimulation of the APtN evoked orthodromic activation of 59 units (40%). The mean latency ( ± S.E.M.) to onset of the response was 6.0 ± 0.5 ms (range from 4 to 11 ms). Fig. 1 shows one example of a cell in PPR that was driven orthodromically by APtN stimulation. Eight PPR neurones (5% of the total recorded) were driven antidromically. The mean latency to onset was 0.8 + 0.22 ms (range 0.4 to 2 ms). The distance between the stimulating site in the APtN and the recording site in the PPR was approximately 4.0 ram. The conduction velocity was calculated as 5 m. s-i. One example of a PPR cell driven antidromically by APtN stimulation is shown in Fig. 2. The response was evoked at a constant latency, followed 300 Hz stimulation, and collision between the spontaneous and the evoked spikes occurred at a maximum interval of 3 ms

, ] 100uV 2ms 3 trials Fig, !. Response of a cell in the PPR driven orthodromically by APIN stimulatkm, Upper paneh peristimulus time histogram of the response of the cell to stimulation of the APtN with single square wave pulses of 2,4 V, 0,2 ms width, The arrow above the record indicates the time when the stimulus was applied (20 ms), The histogram shows the result of 30 trials given at intervals of 200 ms, The bin width is 1 ms, The mean latency to onset of the response was 3.4 ms. Lower panel: three oscilloscope traces of the evoked response superimposed to show the variable latency of the spike evoked by APtN stimulation. The large deflectkm to the left is three precisely superimposed stimulus artifacts,

between the spontaneous spike and the stimulation of the APtN. Fig. 3 shows the position of the cells in the PPR that were driven by stimulating the APtN. Each dot represents one neurone driven orthodromicaily, the stars show the neurones driven antidromically.

Extracellular recording of neurones within the APtN. A total of 94 neurones were recorded in the APtN. Thirty-three cells (36%) were driven orthodromically by PPR stimulation. The average latency to onset was 26 + 4 ms (range of 8-34 ms). An example is shown in Fig. 4 which is a peristimulus time histogram constructed from the response of an APtN neurone driven

208 by stimulation of the PPR. The locations of the neurones recorded in the APtN driven by stimulating the PPR are shown in Fig. 5. No cell driven antidromically was found in these studies.

Effect of lesions of the PPR on the antinociceptive effect of APtN stimulation Electrical stimulation of the APtN caused a potent and long lasting analgesia. Fig. 6 shows a graph of Index of Analgesia (normalisation of tail-flick latency) against time. The closed circles show the time course of the antinociceptive effect of APtN stimulation in a group of unanaesthetised rats (n = 6). The IA reaches maximum values immediately after the stimulation and is significantly increased for more than 30 rain. Subsequent electrolytic lesions of the PPR greatly reduced the effect of APtN st,mulation. This effect in the same rats, 1 week following lesions of the PPR, is shown by the open circles in Fig. 6. The antinociceptive effect of 40

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Fig. 2. Response of a cell in the PPR driven antidromically by APtN stimulation. Upper panel: peristimulus time histogram of the evoked r~:~ponsc of the cell, The APtN was stimulated at the time indicated by the arrow (20 ms) with a square wave of 8,8 V, 0,5 ms width, Thirty single stimuli were given at intervals of 200 ms. The bin width was I ms. The latency to onset of the response was 2 ms, Lower panel: oscilloscope traces of the response showing, from left to right, the constant latency of the evoked spike when three samples are superimposed, the ability of the cell to follow 300 Hz stimulation (the arrows show when the spike occurred) and the occurrence of collision when a spontaneous spike of the PPR cell (first deflection) triggers the APtN stimulation (second deflection) with 3 ms delay, Collision does not occur if the interval is slightly larger (4 ms).

stimulating the APtN was reduced by 67% (P ~ 0.05). This was calculated by comparing the area under the curve before and after the lesions. Animals with sham lesions in the PPR did not show any modification of the antinociceptive effect of APtN stimulation. The extent of the lesions in the PPR and the position of the tip of the stimulating electrodes in the APtN are illustrated in Fig. 7. The area lesioned was the region lateral and ventral to the periaqueductal grey (the lateral reticular formation). In this area can be found the ventral portion of the nucleus cuneiformis (dorsal to the PPR) and the PPR itself. The most effective lesions were those that destroyed the caudal third of the pedunculopontine tegmental nucleus. The lesions also extended to the rostral end of the parabrachiai nucleus.

Effect of tetracaine into the PPR on the effects of APtN stimulation on deep dorsal horn neurones A total of four complete studies were carried out with similar results. One representative example is described in Fig. 8 which shows the ratemeter record of the discharges of a deep dorsal horn neurone. This cell was potently excited by application of the noxious stimulus to its receptive field on the ipsilateral foot. This neurone was also excited by tactile (brush strokes) and light pressure non-noxious stimuli, and was therefore identified as a wide dynamic range unit. The receptive field covered the plantar surface of the foot. After a few applications of water at 50°C, the nociceptire response stabilised and the APtN was then stimulated. Brief (20 s) elcctrica[ stimulation of the APtN (50 #A, I(H) Hz) reduced th:, nociceptive response of the neurone for several minutes. Once the normal response to hot water was recovered, the glass needle was inserted into the ipsilateral parabrachial region. This caused a brief reduction of the nociceptive response of the neurone, presumably due to mechanical stimulation of the tissue, Injection of tetracaine (4%, 0,5 ~l) into the pontine parabrachial region caused a short period of abolition of the nociceptive response which recovered within 10-15 rain. The response to insertion of the microneedle and the microinjection was variable. In other studies the response to hot water was slightly and temporarily increased by these manipulations, The APtN stimulation was not effective after tetracaine had been injected into the parabrachiai region but this effect of APtN stimulation was recovered 80 min later. DISCUSSION The experiments described in this paper have shown that there is a reciprocal connection between the ante-

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Fig. 3. Diagrams of coronal sections of the rat brain showing the Iocalisation of the neurones in the PPR driven by stmmlating the anterior pretectal nucleus, A higher magnification of the area recorded is shown in the insets, Cells driven orthodromically are shown by the dots. The neurones driven antidromically are shown by the stars, The numbers indicate the distance, in millimeters, posterior to brcgma. 4n, trochlear ncrvu; Aq, aqueduct; CG, central grey; CnF, cuneiform nucleus; DR, dorsal raphe nucleus; MiTg, microcellular tegmental nucleus: PPTg, pedunculopontine tegmental nucleus; rs, rubrospinal tract; sop, superior cerebellar peduncle.

rior pretectal nucleus (APtN) and the pontine parabrachial region (PPR). These pathways are excitatory. More importantly, neurones localized in the PPR appear to have a role in relaying the descending inhibitory effects of APtN stimulation. The pontine parabrachial region (as defined by Katayama et al. 2s) consists of a group of neurones in the pons which surrounds the superior cerebellar peduncles (also known as the brachium conjunctivum). More precisely, it corresponds to the cholinergic cell group Ch5 of Mesulam et al) 3 extending from the rostral end of the parabrachial nucleus to the caudal third of the pedunculopontine tegmental nucleus 2'3"~. These neurones give rise to ascending cholinergic projections 2°'53 that form the dorsal tegmentai bundle of Shute and Lewis45. The ascending projections from the PPR innervate the dorsal raphe nucleus, the EdingerWestphal nucleus, the intralaminar, midline and ventromedial thalamus, the pretectum, the hypothalamus and the amygdala43'53. Although Saper and Loewy 43 reported that the APtN appears to receive virtually no

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210 PPR projection, Wilson "~3and Foster et al. ~7 presented strong evidence for a connection (originating primarily from the pedunculopontine tegmental nucleus). This anatomical evidence is supported by the electrophysiological data presented in this paper that electrical stimulation of the PPR orthodromicaily drives 36% of the cells recorded in the APtN. The parabrachial nucleus (caudal portion of the PPR) receives ascending fibres from the dorsolateral funiculus =. These fibres originate from nociceptive specific cells situated in lamina I ~J-'. The majority of the neurones in the PPR respond to peripheral noxious

stimulation ~ and it has been proposed that this area might be involved in generalized aspects of nociception H). This region, in turn, sends fibres to the APtN. Neurones in the APtN are also responsive to peripheral noxious stimulation (unpublished observations) and to electrical stimulation of the dorsolateral funiculus 39. It is possible that these effects are conveyed, at least in part, via the connection with the PPR. Thus, the PPR may act as a relay for sensory information ascending to the APtN through the dorsolateral funiculus. A high percentage (40%) of the cells in the pontine parabrachial region are driven by APtN stimulation.

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Fig. 6. "rime course (in minutes) of the antinociceptive effect of stimulating the APtN on the tail-flick response to noxious heat before (e) and after ( o ) bilateral electrolytic lesions of the PPR (n = 6). The Index of Analgesia is the normalised tail-flick 'latency calculated as described in the text. The APtN was stimulated at the time indicated by the arrow with sine wave current of 35/~A r.m.s. * P $ 0.05.

The latency to onset of the orthodromically driven response averages 6 ms. Either the fibres activated by this stimulation have a slow conduction velocity (approx. 0.7 m / s ) or the pathway is indirect involving more than one synapse. These slowb conducting fibres may be fibres of passage travelling through the APtN to make synaptic contacts with PPR neurones. Nevertheless, anatomically identified direct connections have already been described 2l'24 (however, see ref. 18). Anterograde tract tracing studies are being carried out in order to clarify further the existence of the direct connection APtN-PPR. It is also possible that a larger study examining neurones throughout the APtN may reveal restricted sites from which antidromically evoked spikes may be recorded. It is well known that electrical stimulation of the PPR elicits antinociception t5 and causes inhibition of spinal cord nociceptive neurones in the rat ~3, in cats 12 and in primates jg. It has also been reported that stimulation of the PPR elicits a more profound inhibition of nociceptive dorsal horn neurones with a lower current threshold than stimulation of the neighbouring periaqueductal grey ~3. Several of the characteristics of the

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Fig. 7. Coronal sections of the rat brain based on the reference planes of the atlas of Paxinos and Watson ~c'. The upper figures show the stimulating sites in the APtN (each closed circle is one site). The shaded areas in the bottom diagrams show the maximal extent of the bilateral lesions in the PPR. The numbers indicate the distance of the section in mm caudal to bregma.

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Fig, 8, Ratemeter record of the activity of a deep dorsal horn neurone, The trace on the top shows the number of spikes produced by a ceil which wa,~ potently excited by application of a noxious stimulus to its cutaneous receptive field, The bottom trace shows the temperature of the water applied to the foot. Brief (5 s) applica;ion of water at 50°C potently excites the neurone, The APtN was stimulated at the time indicated by the triangle.,,, with a 20 s train of 50/LA intensity square wave pulses, 0,1 ms width, 100 Hz, A mieroneedle was inserted in the PPR at the time indicated. A 4%- tetracaine solution (0,5 p,I) was injected into the PPR at the time indicated, The APtN was stimulated at 20 rain intervals as shown by the triangles,

antinociccptivc effect of stimulating the APtN 3s arc similar to those of stimulating the PPR, further indicating a common pathway tbllowed by the descending inhibition originating in the APtN and parabrachial region. Lesions of the dorsolateral funiculus impair the effects of stimulating both these areas. It is also remarkable that the antinociception elicited by APtN and PPR stimulation in the tail-flick test are both sensitive to intraperitoncal administration of atropine

(APtN, see ref. 40; PPR, see ref. 29). Serotonergic receptor activation appears not to be critical for the manifestation of these effects since antagonism of 5HT receptors does not affect the antinociception evoked by stimulating either the APtN 3~ or the PPR ~t. These studies have also shown that electrolytic lesions of the parabrachiai region greatly reduce the antinociceptive effect of stimulating the APtN. These lesions were large, affecting areas beyond the PPR.

213 The main region affected apart from the PPR was the nucleus cuneiformis (dorsal to the pedunculopontine tegmental nucleus). This nucleus projects strongly to the nucleus raphe magnus in the ventral medulla ~6.4~. The nucleus raphe magnus conveys descending inhibition of nociceptive responses to the dorsal horn (see ref. 52). Therefore, this nucleus could relay the antinociceptive effects of APtN stimulation, however, it has been demonstrated that destruction of the nucleus raphe magnus does not alter the effects of APtN stimulation 47. Furthermore, in this study we were unable to drive neurones in the nucleus cuneiformis by stimulation of the APtN. Only those cells positioned in the ventral portions immediately adjacent to the PPR were affected by APtN stimulation. Therefore, it is more likely that the PPR, and not the nucleus cuneiformis, relays the descending inhibitory effects of APtN stimulation. The behavioural experiments demonstrate that the effects of APtN stimulation are relayed to the spinal cord by the excitation of PPR neurones. These neurones project directly to the spinal cord 4'tg'3t'4~'4'~''i0. The spinal projection from the PPR originates mainly from the rostral (and not the caudal) pedunculopontine tegmentai nucleus and from the A7 cell group localized immediately ventral to the PPR. The PPR also projects to the ventrolateral medulla t'~'2z'zT''~'~'4"~and this area has been previously shown to convey part of the descending inhibitory effect of APtN stimulation 47. Electrolytic lesions of the ventrolateral medulla reduce the antinociceptive effect of APtN stimulation by up to 68%. Both the ventrolateral medulla and the PPR have been implicated in descending inhibition and both are reciprocally connected to the APtN. Thus, the neuroanatomical, physiological and pharmacological interelationships of these three areas appear extremely complex, At the pharmacological level, the effects of APtN stimulation are sensitive to alpha adrenoceptor, opioid and cholinergic antagonists 37. Less is known about the antinociception evoked from stimulating the PPR but opioid receptors appear not to be involved as this antinociception is insensitive to naloxone 2'~. However, the antinociception evoked by stimulating the PPR is sensitive to atropine z9 and nothing is known about its sensitivity to alpha adrenoceptor antagonists. The antinociception evoked by stimulating the ventrolateral medulla is sensitive to both naloxone and alpha adrenoceptor antagonists 44. It is possible that electrical stimulation of the APtN activates at least two different descending systems. One excites cells in the ventrolateral medulla and causes analgesia that is sensitive to opiates and alpha agonists. The other activates cells in the PPR causing analgesia mediated, at some point, by

cholinergic fibres. However, the two systems may have some degree of overlap. Both these areas are reciprocally connected to the APtN and we have already shown that the PPR input to the APtN is mainly excitatory. This input is integrated in the APtN to promote the activation of antinociceptive descending systems. In this way, the PPR could play an important role in both the relay of peripheral noxious input (from lamina I cells) and the modulation of this information. The neurones within the PPR that receive the projection from lamina I may be the same as those that are driven orthodromically by APtN stimulation. In this case, the PPR neurones are possibly excited in two forms: directly from the lamina I terminals or through the afferents from the APtN. In conclusion, the connection to the PPR (and presumably also to the ventrolateral medulla) may form the basis of reverberatory circuits which account for the long duration of the antinociceptive effects of APtN stimulation in unanaesthetised rats. Acknowledgements. This work was supported by the WellcomeTrust.

M.G.T. is a CAPES (Brazil) scholar. REFERENCES 1 Apkarian, A.V., Stevens, R.T. and Hedge, C.J., Funicular location of ascending axons of lamina ! cells in the cat spinal cord, Brain Res., 334 (1985) 160-164. 2 Armstrong, D,A., Saper, C.B., Levey, A.I., Wainer, B.H. and Terry, R.D., Distribution of cholinergicneurones in the rat brain demonstrated by the immunohistochemicallocalizationof choline acetyltransferase, J. Comp. Neurol., 216 (1983)53-68. 3 Azami,J., Llewclyn,M.B. and Roberts, M.H.T,, The contribution of nucleus reticularis paragigantocellularis and nucleus raphe magnus to the analgesia produced by systemically administered morphine, investigated with the microinjection technique, Pain, 12 (1982) 229-246. 4 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. 5 Berkley, K.J., Budell, R.J., Blomqvist, A. and Bull, M., Output systemsof the dorsal column nuclei in the cat, Brain Res. Rec., I I (1986) 199-225. 6 Berkley,K.J. and Mash, D.C., Somatic sensory pJojections to the pretectum in the cat, Brain Res., 158 (1978) 445-449. 7 Berkley, K.J. and Scofield, S.L., Relays from the spinal cord and solitary nucleusthrough the parabrachial nucleusto the forebrain in the cat, Brain Res., 529 (1990) 333-338. 8 Berman, N., Connections of the pretectum in the cat, J. Comp. NeuroL, 174 (1977) 227-254. 9 Bernard, J.S. and Besson, J.M.0 The spine (trigemino) ponto amygdaloid pathway: electrophysiologicalevidence for an involvement in pain processes, J. NeurophysioL, 63 (1990) 473-490. 10 Biomqvist, A., Ma, W. and Berkley, K.J., Spinal input to the parabraehial nucleus in the cat, Brain Res., 480 (1089) 29-36. 11 Carstens, E., Fraunhoffer, M. and Zimmermann, M., Serotonergic mediation of descending inhibition from midbrain periaqucductal grey, but not reticular formation, of spinal nociceptive transmission in the rat, Pain, 10 (1981) 149-167. 12 Carstens, E., Klumpp, D. and Zimmermann, M., Differential inhibitory effects of medial and lateral midbrain stimulation on

214 ~pinal neuronal discharges to noxious skin heating in the cat, J. Neurophyslol, 43 (1980) 332-342, 13 Carstens, E. and Watkins, L.R., Inhibition of responses of new runes m the rat spinal cord to noxious ~kin heating by stimulation m midhrain penaqueductal grey or lateral reticular formation, Brain Res.. 382 (1986) 256-277. 14 D'Amour, F,E, and Smith, D.L., A method for determining loss of pain sensation, J. Phannacol E.~p. Ther., 72 (1941) 74-79. 15 Desalles, A.F., Katayama, Y., Becker, M.P. and Hayes, R.L., Pare suppression induced by electrical stimulation of the pontine parabrachial region, I. Neurosurg., 62 (1985) 397-407. 16 Edwards, S,B., Autoradiographic studies of the projections of the midbrain reticular formation: descending projections of the nucleus cuneiformis, J. Comp. Neurol., 161 (1o75) 341-358. 17 Foster, G.A., Sizer, A.R., Rues, H. and Roberts, M.H.T., Afferent projections to the rostral anterior pretectal nucleus of the rat: a possible role in the processing of noxious stimuli, Neurosctence, 29 (1989) 685-694. 18 Fulwiller, C.E. and Saper, C.B., Subnuclear organization of the efferent connectmns of the parabrachial nucleus in the rat, Brain Re~, Rer., 7 (1984) 229-259. 19 Gerhart, K.D., Yezierski, R.P., Wilcox, T.K. and Willis, W.D., Inhibition of primate spinothalamic tract neurons by stimulation in periaqueductal gray on adjacent midbrain reticular formation, J. Neurophy~tol., 51 (1984) 450-466. 20 Goldsmith, M. and Van der Kooy, D.. Separate non-cholinergic descending projection~ and cholinergic ascending projections from the nucleus tegmenti pedunculopontinus, Brain Res., 445 (1988) 386-391. 21 Graybiel, A.M., Some efferents of the pretectal region in the cat, AmH Rec., 178 (1974) 365. 22 Grofiwa, !. and Keane, S., Descending brainstem projections of the pedunculopontine tegmental nucleus in the rat, Anat. Era. b~'ol,, 184 ( 1991 ) 275-2t){}. 23 tl,'lyes, R.L,, Katayama, Y., Watkins, L,R., and Bucker, D.P., Bilateral lesions of the DLF of the cat spinal cord: effects on ba,~al ,ociccptive reflexes and nociceptive suppression produced by cholin~rgic activation of the Dentine parabrachial region, Brain Re,~,, 311 (1984) 267-280, 2,l Itoh, K,, Elferent projections of the prefecture in the cat, F.t'p, Brain Res,, 30 (1977) 89-105, 25 Iwamoto, E,T,, Anlinociccplion after nicotine administration into the mesoponttne tcgmentum of rats: evidence for muscarinic actions, J. lqmrmacol, EATs. 7her., 251 (1989) 412-421. 2f~ Iwamt,to, E.T., Charactertsation of the antinociception induced by nicotine in the peduneulopontine legmental nucleus and the nucleus raphe magnus, J. Phammeol, Exp, Ttler., 257 (1991) 12t;- 133. 27 Jones, B.E,, lmmunohistochemical study of choline acetyltransferase.immunoreactive processes and cells innervating the Dontomedullary reticular formation in the rat, J. Comp. NeuroL, 295 (1990) 485-514, 28 Katayama. Y., De Wilt, D,S,, Bucker, D,P, and Itayes, R,L,, Behavioural evidence for a cholinoceptive Dentine inhibitory area: descending control of spinal motor output and sensory input, Brain Res,, 296 (1984) 241-262, 29 Katayama, Y., Watkins, L,R., Bucker, D.P, and Hayes, R,L,, Non-opiate analgesia induced by carbachol microinjection into the pontiac parabrachial region of the cat, Brain Res,, 296 (1984) 2¢~3-283, 30 Klamt, J.G, and Prado, W,A,, Antinociception and behavioural ch;mge,~ induced by carbachol microinjectcd into identified sites of the rat brain, Brain Re,~,, 549 (1991) 9-18, 31 Kuypers, H.GJ.M. and Maisky, V.A., Funicular trajectories of descending brain stem pathways in the cat, Brah~ Res,, 136 (1977) 159-165. 32 McMahon, S.B. and Wall, P,D.. Electrophysiological mapping of brain.qem projections of spinal cord lamina i cells in the rat, Brain Rt'~ . 333 (1985) 19-26, 33 Mesulam, M,M,, Mufson, E.J,, Walnut, B,H, and Levey, A.i,, Central cholinergic pathways in the rat: an overview based on an alternative nomenclature (Chl-Ch6), Neurosclence, I0 (1983) 1185-12()1.

34 Miura, M. and Takayama, K., Circulatory and respiratory responses to glutamate stimulation of the lateral parabrachial nucleus of the cat, J. Auton. Nercous System, 32 (1991) 121-134. 35 Moga, M.M., Herbert, H., Hurley, K.M., Yasui, Y., Gray, T.S. and Saper, C.B., Organization of cortical, basal forebrain, and hypothalamic afferents to the parabrachial nucleus in the rat, J. Comp. Neurol., 295 (1990) 624-661. 36 Paxinos, G. and Watson, C., The Rat Brmn in Stereotaxlc Coordlnates, 2nd edn., Academlc Press, New York, 1986. 37 Rees, H., Prado, W.A., Rawlings, S. and Roberts, M.H.T., The effects of intraperitoneal administration of antagonists and development of morphine tolerance on the antinociception produced by stimulating the APtN of the rat, Br. J. PharmacoL, 92 (1987) 769-779. 38 Rees, H. and Roberts, M.H.T., Anterior pretectal stimulation alters the responses of spinal dorsal horn neurones to cutaneous stimulation in the rat, J. PhysioL, 385 (1987) 415-436, 39 Rues, H. and Roberts, M.H.T., Excitation of superficial dorsal horn neurones by electrical stimulation of the anterior pretectal nucleus in the anaesthetised rat, J. Physiol., in press. 40 Rees, H., Terenzi, M.G. and Roberts, M.H.T., The involvement of acetylcholine in antinociception evoked by electrical stimulation of the anterior pretectal nucleus, Br, J, Pharmacol,, in press, 41 Richter, R.C. and Behbehani, M.M., Evidence for glutamic acid as a posstble neurotransmitter between the mesencepnalic nucleus cuneiformis and the medullary nucleus raphe magnus in the lightly anaesthetised rat, Brain Res., 544 (1991) 279-286, 42 Roberts, M.H.T. and Rues, H, The antinociceptive effects of stimulating the pretectal nucleus of the rat, Pain, 25 (1986) 83-93. 43 Saper, C.B. and Loewy, A,D., Efferent connections of the parabrachial nucleus in the rat, Brain Res., 197 (1980) 291-317. 44 Satoh, M., Akaike, A., Nakazawa, T. and Takagi, H., Evidence for involvement of separate mechanisms in the production of analgesia by electrical stimulation of the nucleus reticularis paragigantocelhdaris and nucleus raphe magnus in the cat, Brain Rt,s., 194 (1980) 525-529. 45 Shute, C.C.D, and Lewis, P,R,, The ascending cholinergie reticular system' neocortical, olfactory and subcortieal projections, Brain, 90 (1967) 497-520, 46 Span,, B,M, and Orofova, I., Origin of ascending and spinal pathways from the nucleus tegmenti pedunculopontinus in the rat, J. Comp. Nel,ml,, 283 (1989) 13-27, 47 Terenzi, M,G,, Rues, H,, Morgan, S,J,S,, Foster, O,A, and Roberts, M,H,T., The antinociceptton evoked by anterior pretectal nucleu,: stimulation is partially dependent upon ventrolateral medullary neurones, Pain, 47 (1991)231-239, 48 Terenzi, M,O,, Rues, H, and Roberts, M.H,T., The inhibitory effects of APtN stimulation are partly mediated via a relay in the pontine parabrachial region in anaesthetised and unanaesthestised rats, 1. Physiol., 435 (1991) 66P. 49 Tohyama, M,, Sakai, K,, Touter, M., Salvert, D, and Jouvet, M., Spinal projections from the lower brain stem in the cats as demonstrated by the HRP technique, !1, Projections front the dotsolateral ~ntine tegmentum and raphe nuclei, Brain Res., 176 (1979) 215-231, 50 Watkins, L,R,, Grifffin, G., Leichnetz, O.R. and Mayer, DJ., Identification and somatotopic organisation of nuclei projecting via the DLF in rats: a retrograde tracing study using HRP slow release gels, Bra#l Res,, 223 O981) 237-255, 51 Weber, J,T. and Hartin8, J,K,, The efferent projections of the pretectal complex: an autoradiographic and horseradish peroxidase analysis, Brain Res,, 194 (1980) 1-28. 52 Willis, W.D,, Anatomy and philology of descending control of nociceptive responses of dorsal horn neurones: comprehensive review, Pro& Brain Res,, 77 (1988) 1-29, 53 Wilson, P,M,, A photographic perspective on the origins, form, course, and relations of the acetylcholinesterase containing fibres of the dorsal tegmental pathway in the rat brain, Brain Res., 10 (1985) 85-118,