Convergence of deep somatic and visceral nociceptive information onto a discrete ventrolateral midbrain periaqueductal gray region

Neuroscience Vol. 61, No. 4, pp. 727-732, 1994 Elsetier Science Ltd Copyright Q 1994 IBRO

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CONVERGENCE OF DEEP SOMATIC AND VISCERAL ~OCICEPTIVE INFORMATION ONTO A DISCRETE VE~TROLATE~L MIDBRAIN PERIAQUEDUCTAL GRAY REGION K. A. KEAY,*t C. I. CLEMENT,* B. OWLER,* A. DEPAULISt and R. BANDLER* *Department of Anatomy and Histology,University of Sydney, NSW 2006, Australia tXNSERM Unite 398, NNEG, Centre de Neur~~mie du CNRS, Strasbourg 67084, France

Pain arising from deep structures (mush, joints, viscera) is tbe type of pain of most clinical relevance and also the type of pain about whose central represeatatkmwe have the least knowledge.In contrastto cutaneous pain Mick evokes defetive bekaviours, hypertensionand tachycardia,the physiologicalreactions to most deeppain(especiallyif persistent)usually include quiescence, hypotension, bradycardia and decressedreactivityto the enviromuent.Excitation of newroas within a discrete ventrolateralmidbrainperi~~~ gray region evokes a reaction seemingly hie&cal to that evoked by pain arising from deep structures.We reporthere, using the teclmiqueof the noxious stimulus-evokedexpressionof the immediateearly gene.,c-fos, that neuronswithinthis same ventrolateral periaqueductal gray region are selectivety activated by a rauge of deep somatic and visceral nociceptivemanipulations.Thus we have identifieda specitic braia region that both receives convergent, deepsomaticand visceralaociceptiveinpuGand which mediates the bebaviouraland physiological reactloos characteristicof most deep pain. Wall suggested in a provocative article published in 1979 that pain signals not only the existence of an injurious stimulus, but even more importantly the requirement for behavioural change.’ He proposed that pain represents first and foremost the awareness of a “need state” (more akin to hunger and thirst), rather than a sensory experience (such as hearing or seeing). Many years earlier the eminent English physician Sir Thomas Lewis, called attention to the different and distinct behavioural patterns evoked by pain arising from superficial and deep tissues, Lewis pointed out that whereas pain derived from the skin was associated with quick protective reflexes, a rise of pulse rate and a sense of in~goration, pain arising from deep structures was usually associated tTo whomcorrespondenceshould be addressed, Abbreviation:

PAG, periaqueductal gray 727

with quiescence, slowing of the pulse, a fall in blood pressure and loss of interest in the environment. He went on to conclude that the qualities of skin pain and deep pain were so distinct that it would be “unsafe to class both together under the one unqualified term pain”, and suggested, “If we are right in believing that the system of fibres subserving cutaneous pain passes to an appropriate and exclusive part of the sensorium . . . we are brought to consider whether or not fibres subserving pain derived more deeply connect to a distinct part of the sensorium , . . we should bear in mind the possible serious fallacy of regarding both types as represented in a common centre”.2 Although the study of the central representation of pain has traditionally adopted the approach of the sensory physiologist to work inward from the peripheral receptor, the views of both Wall and Lewis suggest another approach, namely the possibility of dissecting central pain circuitry by working from the response side back towards the peripheral receptor. It is this latter approach which has guided this work. The distinct behavioural and cardiovascular reactions evoked by cutaneo~ and most deep pain are remarkably simiIar to the integrated reactions evoked by excitation of neurons in the periaqueductal gray region (PAG) of the midbrain. Within the PAG, two functionally-distinct longitudinal columns of neurons are situated lateral and ventrolateral to the aqueduct (Fig. 1, row 3). Excitation of cells in the laterai PAG column evokes active defensive ~haviours (confrontation or flight), vocalisation, hypertension and tachycardia, whereas excitation of cells in the ventrolateral PAG column (Fig. 1, row 4) evokes quiescence, hyporeactivity, hypotension and bradycardia.**’ The resemblance of PAG-evoked reactions to those typical of deep and cutaneous pain responses suggests that the ventrolateral and lateral PAG might play important roles in coordinating the distinct physiological and behatioural responses to deep and cutaneous pain. In the following experiments we set

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K. A. Keay et a1

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Information out to broadly test the hypothesis

convergence onto periaqueductal gray region

that the ventrolateral PAG is an important component of the central neural circuitry underlying deep pain. We have done this by examining the distribution of Fos-like immunoreactive cells in the midbrain PAG of halothane anaesthetised rats subjected to a range of well charactedsed noxious stimuli specifically chosen to activate deep somatic (injection of algesic substances into the knee joint, triceps surae muscle) or visceral structures (peritoneum-i.p. acetic acid; or hearti.v. 5-hydroxytryptamine). The manipulations were chosen on the basis of electrophysiological and behavioural evidence of their noxious qualities.“m’3 Following a 2-2.5 h experimental or control period (no noxious manipulation), the brain and spinal cord of each rat was fixed by intracardiac perfusion, serial frozen coronal sections (50 pm) of the midbrain were cut, and standard avidin-biotin-peroxidase immunohistochemical techniques were used to detect the presence of the protein product of the c-f& immediate early gene. 16” Observation of the brain sections under the light microscope allowed us to quantify and accurately reconstruct the locations of labelled cells wlthin the midbrain PAG. For each animal, five comparable caudal midbrain sections were selected, spaced 500pm apart. These levels were chosen to agree with standard sections from the atlas of Paxinos and Watson.‘* On each section the PAG was divided into lateral and ventrolateral subdivisions (see Fig. 1, rows 3,4) according to distinct anatomical and functional criteria described by work from our laboratory and that of others.s’0 The mean number of Fospositive nuclei per 50 pm section within these lateral and ventrolateral PAG subdivisions were calculated for all animals within the experimental and control groups. As can be seen in Figs 1 and 2, when compared to anaesthetised controls, noxious stimulation of the viscera and noxious deep somatic manipulations each evoked a dramatic and significant increase in

729

the number of Fos-positive cells in a discrete caudal, ventrolateral midbrain PAG region. These results should be compared with our previous finding14 that noxious cutaneous stimulation (radiant heat of the dorsum of the neck) evoked an overall increase in the number of Fos-positive cells in the lateral PAG compared to the ventrolateral PAG (lateral PAG mean of 28 f 2.31 Fos-positive cells per section compared to vlPAG 16 f 2.24 S.E.M. labelled cells per section14). It should be noted that this difference was due mainly to the increased Fos-expression in the lateral PAG at coronal levels A2.2 and Al.7 (see Fig. 1, row 2; Fig. 2, row 1, right panel). As it is widely accepted that Fos expression following a noxious stimulus and a short survival time is indicative of neuronal activation (for further discussion see Ref. 15), these data demonstrate a selective activation of a restricted caudal ventrolateral PAG region by each of the deep noxious manipulations. In order to provide an indication of whether the “activation” of ventrolateral PAG neurons by deep painful stimuli was of “functional significance” to the animal, in a separate series of experiments (carried out at the LNBC, Centre de Neurochimie du CNRS, Strasbourg, France, adhering to the guidelines of the International Association for the Study of Pain19) the behaviour evoked by an excitatory amino acid microinjection (40 pmol kainate in 200 nl) in the caudal, ventrolateral PAG, was compared with behaviour evoked by: (i) a deep somatic noxious manipulationbilateral injection of an algesic substance 0.05 ml, 5% formalin into the deep dorsal neck muscles, n = 8; or (ii) a visceral noxious manipulation-i.p. injection of acetic acid 0.5 ml, 3.5%, n = 8. As shown in Fig. 3, each of these deep noxious manipulations evoked a significant increase in the duration of quiescence and hyporeactivity (i.e. the behavioural pattern characteristic of deep pain) in the test period. These findings suggest that deep pain evoked Fos expression in

Fig. 1. (opposite) Fig. 1. Series of coronal sections through the midbrain periaqueductal gray region A2.2-A0.2 (adapted from the atlas of Paxinos and Watson”). The approximate boundaries of the lateral and ventrolateral PAG are indicated m row 3 (see also Refs 3-6, 9, 10). For each of the noxious manipulations the distribution of Fos-like immunoreactive neurons is illustrated by filled circles. Rostra1 is to the left and caudal is to the right. The label was bilateral but has only been plotted unilaterally. Previously published plots’4 of radiant heat applied to the skin (row 2) and bilateral injections of 5% formalin into the deep dorsal neck muscles are included for comparison (row 9). Halothane anaesthetised Sprague-Dawley rats were subjected to manipulations of 2-2.5 h duration, either: (i) anaesthetic controls n = 7, [ii) a series (one injection/lOrnin) of 12 intravenous injections of 5_hydroxytryptamine, 72pg. kg-’ in O.Sml distilled water, n = 4; (iii) a single injection of 0.5 ml of 3.5% acetic acid into the peritoneal cavity, n = 6; (iv) the injection, into each knee joint, of a mechanical (0.05 ml of 4% kaolin) and 5 min later an inflammatory algesic agent (0.05 ml of 2% carrageenan type IV), n = 5; (v) a single injection into both triceps surae muscles of an intlammatory algesic agent 0.05 ml of either 2% carrageenan type IV, n = 6. Following these manipulations and perfusion, the brain and spinal cord were fixed, and 50.pm coronal sections of the midbrain were cut, and incubated at 4°C for three days in rabbit anti-Fos “Ab-2” (Oncogene Science Ltd, U.S.A.). Fos-like immunoreactivity was visual&d using standard avidin-biotin-peroxidase immunohistochemical techniques. “~28Under the light microscope, FoJ-positive cells were easily distinguished from background, using the procedure adopted by Hammond and colleague? in which cells were considered positive if the diaminohenzidine reaction within the cell nucleus was distinguishable from the background throughout a range of magnifications from x 20 to x 4. Row 4 of this figure illustrates the sites at which microinjections of kainate into the PAG evoked quiescence and hyporeactivity in the unanaesthetised and unrestrained animal.‘0

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Fig. 2. Histograms illustrating numbers of Fos-like immunoreactive cells in the lateral and the ventrolateral PAG at five different anterior-posterior levels (A2.2-A0.2) of the midbrain PAG following either: (i) anaesthetised control; (ii) cutaneous noxious manipulation radiant heat of the neck; or (iii) deep noxious manipulation either: (a) i.v. Shydroxytryptamine; (b) i.p. acetic acid; (c) knee joint injection of 4% kaolin/2% carrageenan; (d) triceps surae muscle injection 2% carrageenan type IV. Previously published findings of deep dorsal neck muscle injections 5% formalin are included for comparison. Significant differences between lateral and ventrolateral PAG, unpaired “[“-test, ‘P < 0.01.

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Fig. 3. Histograms showing for each manipulation the time (s) spent in each of the following behavioural categories: QUIESCENCE, hyporeactive and immobile; NON-SOCIAL BEHAVIOIJR, cage exploration, self grooming; SOCIAL BEHAVIOUR, investigation or grooming of partner; DEFENSIVE BEHAVIOUR, defensive alerting, defensive uprights, backing and forward locomotion away from the partner and reactive immobility (freezing) (for details see Refs 4, 10). The manipulations were: CONTROL, no noxious manipulation, n = 14; DEEP, bilateral injection 0.05 ml of 5% formalin into the deep dorsal neck muscles, n = 8; ACETIC ACID, intra-peritoneal injection 05 ml of 3.5% acetic acid, n = 8; KAINATE, microinjection of 40 pmol of kainate into the ventrolateral PAG, n = 48. For detailed description of methods and results see Refs &IO. For the injection of algesic substances each rat was briefly anaesthetised (l-2 min) with halothane and hand held by the experimenter for the manipulation, and then returned to the home cage. Control rats (n = 14)were

briefly anaesthetised with halothane and then returned to their home cage. Forty minutes after the injection of the algesic substance, the anaesthetic control, or immediately after kainate microinjection into the ventrolateral PAG, the rat was placed in a neutral test cage and 2 min later a weight-matched, untreated male rat was introduced for an 8 min period. The resultant interactions were videotape recorded and later analysed.

ventrolateral PAG neurons is indicative of a functionally significant pattern of activation, not dissimilar to that evoked by an injection of excitatory amino acid into the same ventrolateral PAG region. Considered together, the Fos and behavioural data: (i) provide compelling initial evidence for a convergence of deep somatic and visceral nociceptive input onto a discrete ventrolateral midbrain PAG region which, in turn, mediates the behavioural and physiological reaction characteristic of deep pain and; (ii) establish a fundamental separability of the central processing of deep and cutaneous pain. In contrast to cutaneous pain which often can be controlled to some extent by the animal, and from which an animal may be able to escape, deep pain is both inescapable and usually impossible for an animal to actively behaviourally control. A quiescent and hyporeactive response may represent one way for the

animal to reduce discomfort and limit interactions which might increase pain (e.g. touch, pressure or movement of the “painful” part of the body). It is interesting to note that a similar reaction of quiescence and loss of interest in the environment is characteristic of chronic pain’ and commonly observed in animals that have been defeated or injured in a social encounter.20J’ In each of these contexts, such a passive coping reaction, perhaps coordinated by the caudal ventrolateral PAG, may serve a similar function, to lessen the physiological/emotional impact of the situation and to promote healing and later recovery. It should be noted that the passive coping reaction (quiescence, hyporeactivity, hypotension and bradycardia) evoked from the caudal, ventrolatera1 PAG is strikingly similar to the conservation-withdrawal response to social interactions described in the classical stress literature.22-24

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K. A. Keay et al.

As well, the caudal, is the midbrain region

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period is the time, during which an animal is usually quiescent and hyporeactive, and during which opioids are present in brain and spinal cord in high quantities?’

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deep pain, but also evoke a strong inhibition of motor systems;*’ (ii) that the coupling of an opioid-mediated analgesia and a behavioural reaction of quiescence and hyporeactivity has been observed in the mouse following defeat in a social encounter;*’ and (iii) that

Acknowledgements-This research was supported by grants to R. Bandler from the Australian National Health and Medical Research Council, the New South Wales Government Employees Medical Research Fund, the National Heart Foundation of Australia and the C’live and Vera Ramaciotti Foundation.

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(Accepted 23 March 1994)