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Noxious stimuli excite neurons in nucleus submedius of the normal and arthritic rat
269
Brain Research, 460 (1988) 269-280 Elsevier BRE 13925
Noxious stimuli excite neurons in nucleus submedius of the normal and arthritic rat Jonathan O. Dostrovsky 1 and Gisele Guilbaud 2 1Department of Physiology, University of Toronto, Toronto, Ont. (Canada) and 2Unit( 161, 1NSERM, Paris (France) (Accepted 5 April 1988)
Key words: Thalamus: Nucleus submedius: Nociception: Rat
Anatomical studies have revealed the existence of an ascending pathway originating in the spinal cord and medullary dorsal horn. relaying in nucleus submedius (Sin) in medial thalamus and terminating in ventrolateral orbital cortex. It has been suggested that this pathway may be involved in the transmission of nociceptive information. In the present study extracellular recordings were obtained from neurons in Sm of anesthetized arthritic and normal rats. Mechanical and thermal stimuli were delivered to various regions of the body to determine the types of somatic stimuli which could activate Sm neurons. Over 40% of the 146 neurons studied responded to somatic stimuli. In the normal rats only high intensity mechanical and thermal stimuli were effective in inducing responses. In the arthritic rats lower intensity mechanical stimuli, joint movements and high intensity thermal stimuli were effective. Such stimuli produce nociceptive reactions in the freely moving arthritic rat. Almost all the responses were excitatory and generally lasted the entire duration of the 15-s stimuli employed. In some cases after-discharges were present. The receptive fields of the neurons were in almost all cases large and bilateral. These findings support the hypothesis that Sm may be involved in mediating the affective-motivational aspects of pain.
INTRODUCTION
group and/or centralis lateralis in nociception is less well established 19,3~.47.
It is generally accepted that the spinothalamic tract is the main ascending pathway conveying sensory information related to pain and t e m p e r a t u r e sensation 3947. An analogous pathway ascends from the medullary dorsal horn conveying t e m p e r a t u r e and pain related information from the orofacial region 39. The thalamic regions where these neurons terminate are likely to be involved in mediating the sensory effects of noxious stimuli or t e m p e r a t u r e changes. In earlier anatomical studies 47, 3 main termination sites were identified in: ventrobasal complex (VBC), centrails lateralis and in the posterior group. As a consequence of many recent studies there is now good evidence implicating the ventrobasal complex and its immediately surrounding regions (the shell region in the cat) in the discriminative aspects of pain 2°'23"2s29. However. the possible involvement of the posterior
With the advent of more sensitive anatomical tracing techniques it is now possible to identify other termination sites of the spinothalamic tract in thalamus 7"34. One particularly interesting site is nucleus submedius (Sin). Nucleus submedius in the rat was first described by Krieg 31 who originally t e r m e d it gelatinosus in 1944 (it is still referred to by that name by some authors 21'3°,37) and later r e n a m e d it submedius ~-. It has also been identified in other species including cat 4"7, m o n k e y 4.7, and man 22 (see ref. 7 for a historical review of descriptions of Sin). The dorsal part of this nucleus has been shown by Craig and Burton 7 to receive a dense projection from the spinal cord and especially from the medullary dorsal horn in the cat and rat a'7"8. These findings have been confirmed by other researchers 35,4°,44,45. In the cat, Craig and Burton 7 have shown that the cells of origin of this
Correspondence: J.O. Dostrovsky, Department of Physiology. University of Toronto, Toronto, Ont. M5S lA8, Canada.
270 projection arc located cxchisiv¢lv in lamina 1, and that there is till anatomical sonlatotopic organization (within Sin). Since lamina I contairis primarily' nociceptive-specific neurons (and some temperature-responsive neurons), these findings suggest that Srn may bc involved in the processing and relay of painrelated reformation sis proposed by Craig and Burton r. The locations of the cells of origin of this pathway have not been investigated iri detail in other species. In the rat however it appears that the cells of origin nlav be located m tire deeper layers of the spinal cord, in laminsle V - V I I 15tv~v3". Elcctrophysiologiceil studies in the cat have shown that indeed there arc projections from lamina 1 of the spinal and medullary dorsal horris to the vicinity of Sm and many of lhese cells sire nociceptive-specific 'jlt. ttowever, some of the projection neurons are temperature-responsive (excited by cooling) raising the possibility that the nucleus may be involved also in the processing of innocuous temperature information. It is possible that axons of temperature-responsive neurons terminate close to but outside Sm and were activated by the Sm stimulus. Little is known concerning the relay of temperature information in the thalamus. Neurons excited by cooling the tongue m the cat have been identified slightly lateral to Sm in medial VBC 2'~3 and it is possible that this region also corltains neurons responsive to temperature changes of other body regions. Of further interest regarding the possible function of Sm are findings in the cat and rat that the cells in the dorsal part of Sin, the same region receiving the ascending afferents, have a specific projection to the ventrolateral orbital cortex (VLO) 111"34. In the rat it has been shown with electron-microscopy that the Sm cells projecting to V L O receive afferent inputs from the medullary dorsal horn 34. In the cat where this pathway has been studied in greatest detail, the termination appears to be somatotopically organized within the V L O pars alpha m. V L O has a reciprocal projection back to Sm as well as projections to parts of the primary and secondary somatosensory cortex that receive afferents from the regions of ventrobasal complex containing nociceptive neurons and to the ventrolateral periaqueductal gray matter <> which has been implicated in stimulation-produced analgesia. On the basis of the findings briefly described
above, and various other studies implicating the thedial thahunus in mediating affectivc conlponcnls el pain (see discussioris in refs, 6 and 7), ('rats has proposed that Sm may be involved in mediating affectivc aspects of pain~L It is obxiouslv inaportant to determine the functional characteristics of the neurons in Srn. This paper describes the responsiveness of neurons in the rat Sm to sensory stimulation of the body. Since previous studies have suggested that it is much easier to find thalamic neurons responding to noxious stimuli in the arthritic rat :5e(', initial studies were performed on these animals and the results compared with those obtained later in normal rats. Some of these findings have been reported briefly as abstracts l_~.13. MATERIALS AND METHODS For this study normal and arthritic SpragueDawley rats, of the same age (8-9 weeks old), weighing 250-290 and 170-240 g, respectively, were used. Arthritic rats were purchased from Charles River (France) where they had been inoculated two weeks before, by injection of Freund's adjuvant into the tail. The electrophysiological recording experiments were performed 3 - 4 weeks after this injection, tit which time the arthritic condition was at its maximum 5 u ' > . For this study the recommendations published in an Invited Editorial in Pain a(' oil ethical standards for investigations of experimental pain in animals were followed; most particularly, the number of arthritic rats was kept to a minimum. They were housed 4 - 5 to a large cage, the floor of which was covered with sawdust: this arrangement minimized the possibility of painful interactions between rats placed in close contact. They ate and drank unaided and received water and food ad libitum. The food was directly available on the sawdust in the cages, to minimize the need for the animals to make potentially painful movements to obtain food. Animals were anesthetized with halothane in a mixture of 1/3 O,, 2/3 N~O, paralyzed with an i.v. infusion of gallamine triethiodide (Flaxedil) and artificially ventilated using a Palmer pump. Frequency and volume were set in order to maintain a normal acid-base equilibrium la. The animals were maintained under deep anesthesia during the surgical procedure (2-2.5~- halothane). The percentage of halo-
271 one, and occasionally two recording sites on each side. Usually, only two tracks per animal, one on each side, were made. Single unit activity was amplified using conventional techniques and displayed on an oscilloscope. Single units were discriminated using a window discriminator whose output was utilized by a counter (2s epochs) to continuously display the frequency of firing on a rectilinear chart recorder. When a spontaneously active unit was isolated, its receptive field was roughly determined by stimulating the paws and sometimes the tail, ear and lip. The unit's responsivity to various stimuli was characterized by brushing, rubbing, pinching, stroking, tapping and moving joints. Stimuli were usually applied for 15 s. An interval of at least 1 min was left between each stimulus delivery, when applied to a different part of the body and 3 min when applied to the same part of the body. The response to heat was investigated for some units, using a hot water bath (42-52 °C), following a procedure described elsewhere in detail 41 (stimulation duration of 15 s, with intervals of at least 3 min). Encoding capacity of the neurons was tested in some cases by applying water at temperatures which were incrementally increased. Some units were also tested for possible activation by
thane was then reduced to 0.5-0.6% and maintained at this level during the recording period which began when anesthesia reached a stable level, typically 1 h after reduction of the halothane concentration. The level of anesthesia was checked throughout the experiment by monitoring the electrocorticogram (ECoG) recorded longitudinally by two silver ball electrodes on the dura mater. The ECoG consisted of theta monomorphic waves of 4 - 5 Hz, 100-120 ,uV, associated with a few spindles of 12-14 Hz. From the present and previous experiments, it was verified that this level of anesthesia was sufficient to avoid arousal reactions or alterations of the E C o G , or autonomic responses (heart rate or blood pressure changes), when noxious stimuli were applied 3z~. Body temperature was maintained at 37-37.5 °C by a thermostatically regulated heating pad, and the heart rate was continuously monitored. A craniotomy (4 x 2 mm) was 9erformed over the sagittal suture between frontal ~lanes A5.2 and A6.0 according to the Albe-Fessard atlas 1, and the dura mater was carefully removed, avoiding the longitudinal sinus. The recording electrodes were glass micropipettes filled with a mixture of 5% NaCI and Pontamine sky blue and having an impedance of 12-20 M ~ . The dye was applied iontophoreticaily ( 2 - 1 0 / ~ A , 30 min) at
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Fig. 1. This schematic diagram indicates for each Sm neuron those sites of the body where a noxious stimulus elicited responses. The regions of the body stimulated are numbered 1-8 and are identified on the rat figurine illustrated in the middle. Each line represents one cell and each column refers to the body region stimulated. Excitatory responses are indicated by a+, inhibitory responses by a-, no response by 0 and blanks indicate sites that were not tested for that particular neuron. Part A lists all the cells in normal rats, and part B all the cells in arthritic rats.
272
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Fig. 2, Examples of the responses of two Sm neurons to high intensity mechanical stimuli (prolonged pinch) applied to ipsilatcral (i), contralateral (c) posterior (p) and anterior (a) paws and tail. The activity of the neurons is represented as frequency of discharge histograms (2-s bin width). The neuron illustrated in A responded also to pinching the ear. The recording sites within Sm are shoxvn on the histological reconstructions at the right. A M . anteromedial nucleus: CL. centrolateral nucleus: CM. central medial nucleus: MD, mcdiodorsal nucleus: mr. mammillothalamic tract: PV. paraventricular nucleus: Re. reuniens nucleus: Rh. rhomboid nucleus: SM. suhmedius nucleus.
273 innocuous warm and cold stimuli, by applying a piece of warm (38-42 °C) or cool (18-22 °C) wet cotton to the paws or face. At the end of each experiment the rats were perfused with saline followed by 10% formalin, the brain then removed and 100 gm thick frozen sections cut and stained with Cresyl violet. Electrode tracks were reconstructed by superimposing the electrode track on a camera lucida drawing of the section containing the dye mark or marks. Recording sites were reconstructed according to the distances, derived from the micromanipulator readings, from the dye marks. From many instances where two marks were made along the same track it was clear that the errors due to tissue shrinkage or micromanipulator hysteresis were small (usually less than 200/im). Since all the sites in Sm were either those marked or close to marked locations the errors would have been smaller. Only recordings of units from tracks where dye marks could be unequivocally identified were included in the data. The data from individual experiments were pooled on thalamic transverse sections adapted from the atlas of Paxinos and Watson 37. The
location of the mammillothalamic tract relative to Sm was used as the most reliable estimate of the anteriorposterior level within Sm. RESULTS
Recordings from 146 cells subsequently confirmed histologically to be in Sm were obtained in 18 arthritic and 17 normal rats. Of these cells 80 were in arthritic rats and 66 in normals. Fifty-five of these neurons responded to peripheral stimulation, 32 (48%) in normals and 33 (41%) in arthritic. Fifty-one of these cells were excited and 4 (3 in normals, one in arthrithic) inhibited by the peripheral stimulation.
Normal rats In the normal rats responses could only be elicited by noxious stimuli. All the responding cells were excited (or inhibited) by a prolonged intense squeezing stimulus applied to one or more of the limbs and in all but 3 the response was maintained for the entire duration of the stimulus. For 19 (6/1%) of the cells the response outlasted the stimulus application, in the
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Fig. 3. An additional example of a neuron in Sm which was excited by noxious mechanical stimuli applied to the contralateral and ipsilateral posterior paws. Note the variability in afterdischarge. Other details and abbreviations as for Fig. 2.
274
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case of 8 ceils for over 30 s. In almost all cases tested the neurons responded to stimulation of both ipsilateral and contralateral hindlimb, the tail, and frequently also the forelimbs. Some (see Fig. 1) cells were also tested for inputs from the facial region (ear or lip pinch) and over 5 0 % responded. Fig, 1A shows for e~ch cell that responded to peripheral stimulation which regions were tested and whether the cell was excited or inhibited from each region. Fig. 2 shows examples of the responses of two Sm neurons to noxious stimuli applied to all 4 limbs, the tail and the contralateral ear. It is clear from these examples that the neurons do not always respgnd identically to stimuli delivered to different parts of the body. The cell in Fig, 2A had a long afterdischarge to stimulation of the contralateral ear and tail. The cell in Fig, 2B had ~ longer afterdischarge to stimulation of the tail and ~mterior paws than to the posterior paws and no response to contralateral ear pinch. In zlddition, as can be seen in Fig. 3, the responses of a neuron to repeated stimulation of the same region were not always the
by noxious stimuli :lpplicd to the p~tws, t~fil and c:lr.
same. Fig. 4 shows an example of a neuron whose spontaneous firing was inhibited by noxious pinch but not innocuous pressure. The inhibitions in this case outlasted the duration of the stimuli. In a few cases the skin overlying the a b d o m e n or thorax was pinched with forceps but no responses could be evoked. Seven out of 8 neurons tested were excited by noxious, but not innocuous heating of the paw or tail. Many of the neurons that did not respond to noxious mechanical stimulation were examined for possible activation by innocuous t e m p e r a t u r e stimuli ~pplied to the paws or face but no responsive neurons were found. A r t h r i t i c rats
In arthritic rats the responsive neurons were excited (one inhibited) by light to m o d e r a t e pressure stimuli applied to the inflamed limbs or to noxious stimuli applied to the face. These neurons could also be activated by maintained flexion or extension of the inflamed joints (usually ankle or wrist joints}.
275 The responses were maintained for the entire duration of the 15-s stimulus for all but two neurons, and outlasted the stimulus for 25 (76%) cells, in the case of 11 by over 30 s. Fig. 1B gives details on the effective sites for each neuron and indicates that most neurons responded to stimulation of both hindlimbs and frequently also to the forelimbs. Some neurons responded also to stimulation of the tail or face. Fig. 5 shows an example of a neuron that was activated by pressure applied to both anterior paws as well as to flexion of the wrist joint and pressure limited to the digits. Pressure applied to the elbow which was only slightly inflamed produced only a small response. Fig. 6 shows the responses of a different neuron to graded noxious thermal stimuli applied to the inflamed hindpaw. Clear responses were evoked at temperatures over 46 °C and to a 5 °C stimulus. Responses to noxious heating were evoked in 7 cells.
Recording sites Responsive neurons were found to be located at all rostro-caudal levels of Sm and scattered at all sites within the nucleus. No obvious differences in locations were noted between the arthritic and normal rats. The micrographs in Fig. 7 show examples of two dye marks deposited at recording sites within Sin. Fig. 8 is a composite reconstruction of all the recording sites in Sm of both normal and arthritic rats. DISCUSSION
This study provides the first description of the existence of neurons in the rat nucleus submedius that respond to somatosensory stimuli. The only other report of somatosensory responses of identified Sm neurons was the short description in the discussion section of the anatomical paper of Craig and
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Fig. 5. Responses of an Sm neuron in an arthritic rat. This neuron responded to pressure stimuli applied to the anterior paws, digits, and elbow as well as to .joint movement (flexion is illustrated) of the wrist. Other details and abbreviations as for Fig, 2: 3V, third ventricle.
276
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Fig. 6. Responses to temperature stimuli applied to the contralateral paw of a neuron in an arthritic rat. The neuron responded to temperatures over 46 °C and to 5 °C. The location of this neuron in Sm is indicated on the drawing at lower right.
Burton 7 in the cat. They stated that "Sm neurons re-
where only high intensity noxious mechanical or
spond vigorously to mechanical and thermal noxious stimulation, are not excited by innocuous mechanical stimuli and, furthermore, display interesting integrative properties . . .'" In Fig. 17 of their paper 7, they
thermal stimuli were effective. In the arthritic rats responses could be evoked by stimuli which were of lower intensity. Previous studies in arthritic rats strongly suggest that such responses are due to acti-
show oscilloscope records of an Sm n e u r o n which reveal a long afterdischarge following a 2-s noxious
vation of nociceptive afferents whose mechanical thresholds have been reduced due to the arthritic condition ~s. Moreover, such stimuli in the arthritic
thermal stimulus. This type of response resembles those we observed in the rat. The only other reported identified Sm recordings were those of Price and Slotnick 43 in the rat. However, in that study the existence of somatosensory inputs was not tested; instead a population of neurons that were excited by electri-. cal stimulation of the olfactory bulb were identified and described. It is possible that many of those neurons in our study that were not activated from the periphery may have been neurons excited from the olfactory bulb. It should also be noted that it was not possible to deliver noxious or innocuous thermal stimuli to all the body regions and thus in the case of non-responsive neurons it is possible that had they had a small receptive field not located on the limbs it would probably have been missed. The Sm neurons in our study that responded to peripheral stimuli were only activated by noxious stimuli. This was particularly clear in the normal rats
rat are aversive to the animal 5244:. Furthermore. in the normal animal we never found units that could be activated by joint movements or lower intensity stimuli. Thus the findings of these studies indicate that those Sm neurons that respond to peripheral somatosensory stimulation only respond when these stimuli activate nociceptive afferents. There were no obvious differences in the incidence of responsive neurons and response characteristics between the normal and arthritic rats. A n o t h e r characteristic of the Sm neurons was their extensive and bilateral receptive fields. Although we did not carefully examine the entire receptive field of the neurons it would seem reasonable to assume on the basis of our findings that many of these neurons could be activated by a strong noxious stimulus applied anywhere on the skin. It may be difficult to activate a large n u m b e r of nociceptors by pinches ap-
277 difficulty in holding the units during noxious stimulation of the oral-facial region. The responses e v o k e d from Sm neurons tended to differ from those that have been r e p o r t e d for nociceptive neurons in the ventrobasal complex which tend to have graded responses frequently in the innocuous range for mechanical inputs and relatively small unilateral receptive fields 1~'2°-~2~47. The Sm neurons frequently had an afterdischarge of at least a few seconds that sometimes lasted tens of seconds. Furthermore, the responses did not a p p e a r to be well graded although in view of the high thresholds this was difficult to test. Using thermal stimuli, the responses to higher t e m p e r a t u r e stimuli were not dramatically increased with increasing t e m p e r a t u r e stimuli although the latency to onset was shorter. These findings suggest that Sm neurons respond differently than those in VBC to a noxious stimulus and are consistent with a role for this nucleus in mediating the affective and motivational aspects of a noxious stimulus. Previous studies on V B C neurons suggest that the nociceptive VBC neurons may be involved in mediating the sensory-discriminative aspects of pain t~,47. Fig. 7. Photomicrographs showing the location of nucleus submedius in medial thalamus and the loci of two Pontamine dye injections within the nucleus submedius. In A and B the midline is in the center of the photograph so that the nucleus submedius on each side can be seen. In A part of the electrode track can be seen above the dye mark. This mark, which is indicated by the arrow, was made 35(IBm below the cell illustrated in Fig. 6. In B the dye mark is clearly visible in the dorsomedial partofSm on the right hand side. Bar = 2501tm.
plied to the skin overlying the trunk due to the lower density of receptors and lack of convergent input from receptors in d e e p tissues. This may explain the failure to elicit responses from the trunk in those cases where such stimuli were delivered. The larger n u m b e r of cells responding to stimulation of the tail and hindlimb c o m p a r e d to stimulation of more rostral sites is most likely due to the fact that these were the regions usually e x a m i n e d first and in many cases the recording d e t e r i o r a t e d before it was possible to test for inputs from m o r e rostral sites. The largest input to Sm in both cat and rat appears to be from the trigeminal subnucleus caudalis TM, but activation from this region was not usually tested because of the
Anatomical studies in rats, cats, and monkeys have all shown that the ascending pathways to Sm terminate in the dorsal region of the Sm 7"34"35"4°'44"45.Furthermore, Price and Slotnick 43 have shown that Sm neurons responsive to olfactory bulb stimulation are located in the middle and ventral regions of the nucleus and not in the dorsal part. These findings suggest that the dorsal part of the nucleus is functionally quite different from the rest of the nucleus. However, the histological reconstructions of recording sites in the present study revealed that neurons responsive to noxious stimuli were located throughout Sm. It is possible that the d e e p e r located cells have dendrites extending into the dorsal region or that the dorsally located neurons have axons or axon collaterals that extend into the ventral regions of the nucleus. It is not clear where the convergence giving rise to the large and bilateral receptive fields is taking place. In the cat where the pathway has been described in greatest detail, the afferent inputs from the spinal cord and trigeminal subnucleus caudalis are almost entirely contralateral and arise exclusively from the marginal layer 7. H o w e v e r , in the rat the cells of origin appear to be located in the d e e p e r layers rather
278
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ARTHRITIC
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Fig. 8. Reconstruction of all the recording sites in Sm of normal rats (A) and arthritic rats (B). Abbreviations as in Fig. 2: VM, ~entremedial nucleus: ZI. zona inccrta.
than the superficial layers 1517z7~(' and it is possible that much of the convergence is taking place in the cord and medullary dorsal horn. In conclusion, the present study has identified a population of neurons in the rat Sm that are activated exclusively by noxious stimuli. These neurons tend to have large bilateral receptive fields, maintained responses to noxious stimuli and sometimes long afterdischarges. The responses to graded thermal stimuli are not well graded. These findings are consistent with a role of Sm in mediating affective-motivational aspects of nociception and support the original proposal of Craig and Burton v that this nucleus may be a specific pain center.
ACKNOWLEDGEMENTS
The authors wish to thank Michele Gautron for her excellent help in preparing the animals, data collection and preparing figures and acknowledge the excellent help of Jacqueline Carroue in preparing the histological sections and of Mary Teofilo in preparing some of the figures. We would also like to thank Dr. A.D. Craig for his helpful comments on a previous version of this paper. This study was supported by the French INSERM (G.G.). a joint Canadian MRCINSERM travel grant to J.O.D. and a U.S. NIH Grant DE05404 awarded to J.O.D.
279 REFERENCES I Albe-Fcssard, D., Stutinsky, F. and Libouban, S., Atlas Stereotaxique du Diencephale du Rat Bhmc, CNRS, Paris, 1966. 2 Auen, E.L., Poulos, D,A.. Hirata, H. and Molt, J.T., Location and organization of thalamic thermosensitive neurons responding to cooling the cat oral-facial regions, Brain Research. 191 (1980) 26(I-264. 3 Benoist. J.M., Kayser, V., Gautron, M. and Guilbaud, G., Letter to editor, Pain, 18 (1984) 4111-411. 4 Burton, H. and Craig Jr., A.D., Spinothalamic projections in cat, raccoon and monkey: a study based on anterograde transport of horseradish peroxidase. In G. Macchi, A. Rustioni and R. Spreafico (Eds.), Somatosenso o, Integration in the Thalauuts. Elsevier Science Publishers, Amsterdam, 1983. pp. 17-41. 5 Calvino, B.. Crepon-Bernard, M.-O. and Le Bars, D.. Parallel clinical und behavioural studies of adjuwmt-induced arthritis in the rat: possible relationship with "chronic pain', Behav. Brain Res., 24 (1987) 11-29. 6 Craig Jr.. A.D.. Medial thalamus and nociception: the nucleus submedius. In J.-M. Besson, G. Guilbaud and M. Peschanski (Eds.), Thalamus and Pain, Excerpta Medica, Amsterdam. 1987, pp. 227-243. 7 Craig Jr.. A.D. and Burton, H., Spinal and medullary lamina 1 projection to nucleus submedius in medial thalamus: a possible pain ecntcr, J. Nemophysiol.. 45 ( 1981 ) 443-465. 8 Craig Jr.. A.D. and Burton. H., The distribution and topographical orgunization in thc thalamus of antcrogradelytransported horseradish pcroxidasc after spinal injections ill c;.it ;.u)d raccoon, Exp. Brain Res.. 58 11985) 227-254. 9 Craig Jr,. A,I). and Kniffki, K.-D., Spinothalamic lumbosacral lamina I cells rcsponsivc to skin and muscle stimulation in the cat, J. Physiol. (Lond.). 365 (1985) 197-221. II) Craig Jr.. A.I).. Wiegand. S.J. and Price, J.L., The thalatoo-cortical projection of the nucleus submedius in the cat. J, ('omp. Nem'ol.. 2116(1982) 28-48. 11 Dostrovsky, J.O., Broton. J.G. and Warma, N.K., Functional properties of suhnucleus caudalis lamina I neurons projecting to nucleus submedius. In R.F. Schmidt. H.-G. Schaible and C. Vahle-Hinz (Eds.) Fine Afferent Nerve Fibers and Pain. VCH, Weinheim, 1987, pp. 355-366. 12 Dostrovskv. J.O., Guilhaud, G. and Gautron, M., Nociceptive neurons in nucleus submedius of the arthritic rat, Pain. 4 (1987) $263. 13 Dostrnvsky, J.O., Guilbaud, G. and Gautron, M., Nociceptive responses in nucleus submedius of the rat, Soc. Neurosci. Abstr.. 13 (1987) 1200. 14 Freminet. A.. Bursaux. E. and Payart, C., Mesure de la vitesse de renouvellement du lactate chez le rat par perfusion de [HCU]( l)-Iactatc +, Plhigers Arch. 334 11972) 293-3112. 15 Giesler Jr.. G,J.. Menetrev, D, and Basbaum, A.I., Differential origins of spinothal~lmic tract projections to medial and lateral thalamus in the rat, J. Comp. Neurol., 184 (1979) 1117-126. 16 Gouret, C.. Mocquct, G. and Raynaud. G., Use of Freund's adjuvant arthritis test in anti-intlammatory drug screening in the rat: value of animal selection and preparation at the breeding center, Lab. Anita. Sci.. 26 (1976) 281-287. 17 Granum. S.L.. The spinothalamic system of the rat. I. Locations of cells of origin, J. Comp. Neurol.. 247 (1986) 159-18(1.
18 Guilbaud, G., Iggo, A, and Tegner, R., Sensory receptors in anklc joint capsules of normal and arthritic rats, Exp. Brain Res., 58 (1985) 29-411. 19 Guilbaud, G., Peschanski, M. and Besson. J.-M., Experimental data related to nociception and pain at the supraspinal level. In P.D. Wall and R. Melzack (Eds.), Textbook of Pain, 2nd edn., Churchill Livingston, Edinburgh, in press. 2(1 Guilbaud, G., Peschanski, M., Gautron, M. and Bindcr, D., Neurones responding to noxious stimulation in VB complex and caudal adjacent regions in the thalamus of thc rat, Pain, 8 (19811) 3113-318. 21 Herkenham, M.. The afferent and efferent connections of the ventromedial thalamic nucleus in the rat, 2. Camp. Neurol., 183 (1979) 487-518. 22 Hirai, T., Hendry, S.H.C. and Jones, E.G,, Parccllation of human thalamic nuclei based on histochemical staining and substance P innervation, Sot'. Neurosci. Abstr., 13 (1987) 1199. 23 Honda, C.N., Mense, S. and Perl, E.R., Neurons in ventrobasal region of cat thalamus selectively responsive to noxious mechanical stimulation, J. Neurophysiol., 49 (1983) 662-673. 24 Kayser, V. and Guilbaud, G., The analgesic effects of morphine, but not those of the enkephalinase inhibitor Thiorphan, are enhanced in arthritic rats, Brain Research, 267 (1983) ]31-138. 25 Kayser, V. and Guilbaud, G., Further evidence for changes in the responsiveness of somatosensory neurons in arthritic rats: a study of the posterior intralaminar region of the thalamus, Brain Research, 323 (1984) 144- 147. 26 Gautron, M. and Guilbaud, G., Somatic responses of ventrobasal thalamic neurones in polyarthritic rats, Brain Research, 237 (1982) 459-471. 27 Kemplay, S.K. and Webster, K.E.. A qualitative and quantitative analysis of the distributions of cells in the spinal cord and spinomedullary junction projecting to the thalamus of the rat, Neuroscience, 17 (1986) 769-789. 28 Kenshalo Jr., D R . , Giesler Jr.. G.J., Leonard, R.B. and Willis, W.D., Responses of neurons in primate ventral posterior lateral nucleus to noxious stimuli. J, Neurophysiol.. 43 (19811) 1594-1614. 29 Kniffki, K.-D. and Mizumura, K,. Responses of neurons in VPL and VPL-VL region of the cat to algesic stimulation of muscle and tendon, J. Neurophysiol., 49 (1983) 649-661. 30 Konig, J.F.R. and Klippel. R.A.. The Rat Brain: a Stereotaxic Atlas of the Forebrain and Lower Parts Of the Brain Stem, Williams and Wilkins Baltimore, 1963. 31 Krieg, W.J.S,, The medial region of the thalamus of the albino rat, J. Comp. Neurol.. 81) (1944) 381-415. 32 Krieg, W.J.S., Connections of cerebral cortex, J. Comp. Neurol., 86 (1947) 267-394. 33 Landgren, S., Thalamic neurones responding to cooling of the cat's tongue, Acta Physiol. Stand.. 48 (19611) 255-267. 34 Ma, W., Aspects Structuraux et Ultrastrttctttraux des Projections Spinales et Trigeminales dons le Thalamtts e! I'Aire Parabrachiale, These de doctorat de I'Universit,5 Paris 6. 1987. 35 Mantyh, P.W., The terminations of the spinothalamic tract in the cat, Neurosci. Lett.. 38 (1983) 119-124. 36 Menetrey. D., De Pommery. J. and Roudier, F., Properties of deep spinothalamic tract cells in the rat, with special reference to ventromedial zone of lumbar dorsal horn. J. Neurophysiol., 52 (1984) 612-624. 37 Paxinos, G. and Watson, C.. The Rat Brain in Stereot,xic
28O ( rJordiHal('s. Ac~l,..Icmic, S\,,.hlc,.. I t)S2 3,"; Pearson. C,NI.. \Vaksman, B.I I. and Sharp. ,I. 1.. ,Sltldics ,,;I :nll'u'ifis alnd other lesions indtlccct ill rats b} illicctioll ,,)1nl',ctfi',actcrial adjut,mt. ",'. CllaiL£cs arlccting tile skin aild
II1LIC~.ILIS IllClll['Hilllt..'s
39
40 41
42
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process v, ith iltllllan disease. ,I. lC.~l), lh'd.. 113 (19(~1) 4S5 511~L Pcrl, E,R.. Pain and nociccption. In ,I.M. Brookhart, V,B. Mountc,iMic alld 1. [)arian-Smith (Eds.), H a n d b o o k oI t'tn'.~iolo,t:y. ,h'CCliOH 1. l'hd m'rvous Srstem. Vohlme IlL Sen,~orv l'r(~ce,s~e~. Part 2. American Physiological Society, Bcthcsda. 1')84. pp. '015-'.)75. Pcschanski. M.. Trigcnlirml allcrcnts to thc cticnccphalon I11 l h c rill. \'duro~c'i{'nc~'. 12 (1984) 4r~5-487. I'cschanski. NI.. (iuilbaud. G.. Gautron. M. and Bcsson. ,I.-M.. Encoding propcrt} of noxious heat messages in neurolls O[ tile \ CllII'OI'~ilS~IIthahmfic conlplcx o1' the rat, t~rain [~d~COvt'/I. lq7 (lOSfl) 41ll-4121. Pircio. A,\V.. Fcdclc. (2.1". and Biciw:.l,,cn M.E.. A nc~v mclh,,',d Ior the cxaluati,.m of armlgc~.ic activity using adju-
',ant-reduced arthritis in lhe rat. /;m'..I.
/'/m~;,'zm'o/.,
:~1
[1'075) 2()7 215. 43 PliCC. ,I.[+. ;llld Slotnick t},M., l)ual t~llactor} tcprcsclllalion i11 lhc rat lh:.ll,llllUS: all amltomical and clcctrophysiological Mud\..I. ('+;'lllp. N+'IlI'o/., 215 (1983) ('G 7V. 44 Ralston Ill. tt.,1.. Synaptic ,.~rg;mization ,,',1 spim:.thalamic tract projcctions tt~ the thalamus, with spccial rclcrencc to pain. In 1.. Kruger and,l.C. Licbcskind (Eds.), ,,tdvrulce+ m
Paip~ Re,earth and lT~eralw ('t. Raven. Ncvv 'l",)rk. 1984, pp. 183 Ig5.
45 Robcrtson. B.. Grant, (}. and Biorkcland. M.. Dcnlor~stration of spinoccrebcllar projections in cat using anterogradc Iranspol-t of WGA HRP, with some observations on spin(> mcsenccphalic and spinothal,m~ic projections, /:'.~p Brain Res., 52 ( 19831 99-11)4. 46 WaMcr. A.. lnvitcd editorial, Pain. 9 (198(1) 1-2. 47 Willis ,h.. W.D., The Pain System. In P.L. Gildenberg I Ed. ). Pain a*ld th'adactu', Vol. 8, Kargcr. Basel. 1985, pp. 1-346.
Brain Research, 460 (1988) 269-280 Elsevier BRE 13925
Noxious stimuli excite neurons in nucleus submedius of the normal and arthritic rat Jonathan O. Dostrovsky 1 and Gisele Guilbaud 2 1Department of Physiology, University of Toronto, Toronto, Ont. (Canada) and 2Unit( 161, 1NSERM, Paris (France) (Accepted 5 April 1988)
Key words: Thalamus: Nucleus submedius: Nociception: Rat
Anatomical studies have revealed the existence of an ascending pathway originating in the spinal cord and medullary dorsal horn. relaying in nucleus submedius (Sin) in medial thalamus and terminating in ventrolateral orbital cortex. It has been suggested that this pathway may be involved in the transmission of nociceptive information. In the present study extracellular recordings were obtained from neurons in Sm of anesthetized arthritic and normal rats. Mechanical and thermal stimuli were delivered to various regions of the body to determine the types of somatic stimuli which could activate Sm neurons. Over 40% of the 146 neurons studied responded to somatic stimuli. In the normal rats only high intensity mechanical and thermal stimuli were effective in inducing responses. In the arthritic rats lower intensity mechanical stimuli, joint movements and high intensity thermal stimuli were effective. Such stimuli produce nociceptive reactions in the freely moving arthritic rat. Almost all the responses were excitatory and generally lasted the entire duration of the 15-s stimuli employed. In some cases after-discharges were present. The receptive fields of the neurons were in almost all cases large and bilateral. These findings support the hypothesis that Sm may be involved in mediating the affective-motivational aspects of pain.
INTRODUCTION
group and/or centralis lateralis in nociception is less well established 19,3~.47.
It is generally accepted that the spinothalamic tract is the main ascending pathway conveying sensory information related to pain and t e m p e r a t u r e sensation 3947. An analogous pathway ascends from the medullary dorsal horn conveying t e m p e r a t u r e and pain related information from the orofacial region 39. The thalamic regions where these neurons terminate are likely to be involved in mediating the sensory effects of noxious stimuli or t e m p e r a t u r e changes. In earlier anatomical studies 47, 3 main termination sites were identified in: ventrobasal complex (VBC), centrails lateralis and in the posterior group. As a consequence of many recent studies there is now good evidence implicating the ventrobasal complex and its immediately surrounding regions (the shell region in the cat) in the discriminative aspects of pain 2°'23"2s29. However. the possible involvement of the posterior
With the advent of more sensitive anatomical tracing techniques it is now possible to identify other termination sites of the spinothalamic tract in thalamus 7"34. One particularly interesting site is nucleus submedius (Sin). Nucleus submedius in the rat was first described by Krieg 31 who originally t e r m e d it gelatinosus in 1944 (it is still referred to by that name by some authors 21'3°,37) and later r e n a m e d it submedius ~-. It has also been identified in other species including cat 4"7, m o n k e y 4.7, and man 22 (see ref. 7 for a historical review of descriptions of Sin). The dorsal part of this nucleus has been shown by Craig and Burton 7 to receive a dense projection from the spinal cord and especially from the medullary dorsal horn in the cat and rat a'7"8. These findings have been confirmed by other researchers 35,4°,44,45. In the cat, Craig and Burton 7 have shown that the cells of origin of this
Correspondence: J.O. Dostrovsky, Department of Physiology. University of Toronto, Toronto, Ont. M5S lA8, Canada.
270 projection arc located cxchisiv¢lv in lamina 1, and that there is till anatomical sonlatotopic organization (within Sin). Since lamina I contairis primarily' nociceptive-specific neurons (and some temperature-responsive neurons), these findings suggest that Srn may bc involved in the processing and relay of painrelated reformation sis proposed by Craig and Burton r. The locations of the cells of origin of this pathway have not been investigated iri detail in other species. In the rat however it appears that the cells of origin nlav be located m tire deeper layers of the spinal cord, in laminsle V - V I I 15tv~v3". Elcctrophysiologiceil studies in the cat have shown that indeed there arc projections from lamina 1 of the spinal and medullary dorsal horris to the vicinity of Sm and many of lhese cells sire nociceptive-specific 'jlt. ttowever, some of the projection neurons are temperature-responsive (excited by cooling) raising the possibility that the nucleus may be involved also in the processing of innocuous temperature information. It is possible that axons of temperature-responsive neurons terminate close to but outside Sm and were activated by the Sm stimulus. Little is known concerning the relay of temperature information in the thalamus. Neurons excited by cooling the tongue m the cat have been identified slightly lateral to Sm in medial VBC 2'~3 and it is possible that this region also corltains neurons responsive to temperature changes of other body regions. Of further interest regarding the possible function of Sm are findings in the cat and rat that the cells in the dorsal part of Sin, the same region receiving the ascending afferents, have a specific projection to the ventrolateral orbital cortex (VLO) 111"34. In the rat it has been shown with electron-microscopy that the Sm cells projecting to V L O receive afferent inputs from the medullary dorsal horn 34. In the cat where this pathway has been studied in greatest detail, the termination appears to be somatotopically organized within the V L O pars alpha m. V L O has a reciprocal projection back to Sm as well as projections to parts of the primary and secondary somatosensory cortex that receive afferents from the regions of ventrobasal complex containing nociceptive neurons and to the ventrolateral periaqueductal gray matter <> which has been implicated in stimulation-produced analgesia. On the basis of the findings briefly described
above, and various other studies implicating the thedial thahunus in mediating affectivc conlponcnls el pain (see discussioris in refs, 6 and 7), ('rats has proposed that Sm may be involved in mediating affectivc aspects of pain~L It is obxiouslv inaportant to determine the functional characteristics of the neurons in Srn. This paper describes the responsiveness of neurons in the rat Sm to sensory stimulation of the body. Since previous studies have suggested that it is much easier to find thalamic neurons responding to noxious stimuli in the arthritic rat :5e(', initial studies were performed on these animals and the results compared with those obtained later in normal rats. Some of these findings have been reported briefly as abstracts l_~.13. MATERIALS AND METHODS For this study normal and arthritic SpragueDawley rats, of the same age (8-9 weeks old), weighing 250-290 and 170-240 g, respectively, were used. Arthritic rats were purchased from Charles River (France) where they had been inoculated two weeks before, by injection of Freund's adjuvant into the tail. The electrophysiological recording experiments were performed 3 - 4 weeks after this injection, tit which time the arthritic condition was at its maximum 5 u ' > . For this study the recommendations published in an Invited Editorial in Pain a(' oil ethical standards for investigations of experimental pain in animals were followed; most particularly, the number of arthritic rats was kept to a minimum. They were housed 4 - 5 to a large cage, the floor of which was covered with sawdust: this arrangement minimized the possibility of painful interactions between rats placed in close contact. They ate and drank unaided and received water and food ad libitum. The food was directly available on the sawdust in the cages, to minimize the need for the animals to make potentially painful movements to obtain food. Animals were anesthetized with halothane in a mixture of 1/3 O,, 2/3 N~O, paralyzed with an i.v. infusion of gallamine triethiodide (Flaxedil) and artificially ventilated using a Palmer pump. Frequency and volume were set in order to maintain a normal acid-base equilibrium la. The animals were maintained under deep anesthesia during the surgical procedure (2-2.5~- halothane). The percentage of halo-
271 one, and occasionally two recording sites on each side. Usually, only two tracks per animal, one on each side, were made. Single unit activity was amplified using conventional techniques and displayed on an oscilloscope. Single units were discriminated using a window discriminator whose output was utilized by a counter (2s epochs) to continuously display the frequency of firing on a rectilinear chart recorder. When a spontaneously active unit was isolated, its receptive field was roughly determined by stimulating the paws and sometimes the tail, ear and lip. The unit's responsivity to various stimuli was characterized by brushing, rubbing, pinching, stroking, tapping and moving joints. Stimuli were usually applied for 15 s. An interval of at least 1 min was left between each stimulus delivery, when applied to a different part of the body and 3 min when applied to the same part of the body. The response to heat was investigated for some units, using a hot water bath (42-52 °C), following a procedure described elsewhere in detail 41 (stimulation duration of 15 s, with intervals of at least 3 min). Encoding capacity of the neurons was tested in some cases by applying water at temperatures which were incrementally increased. Some units were also tested for possible activation by
thane was then reduced to 0.5-0.6% and maintained at this level during the recording period which began when anesthesia reached a stable level, typically 1 h after reduction of the halothane concentration. The level of anesthesia was checked throughout the experiment by monitoring the electrocorticogram (ECoG) recorded longitudinally by two silver ball electrodes on the dura mater. The ECoG consisted of theta monomorphic waves of 4 - 5 Hz, 100-120 ,uV, associated with a few spindles of 12-14 Hz. From the present and previous experiments, it was verified that this level of anesthesia was sufficient to avoid arousal reactions or alterations of the E C o G , or autonomic responses (heart rate or blood pressure changes), when noxious stimuli were applied 3z~. Body temperature was maintained at 37-37.5 °C by a thermostatically regulated heating pad, and the heart rate was continuously monitored. A craniotomy (4 x 2 mm) was 9erformed over the sagittal suture between frontal ~lanes A5.2 and A6.0 according to the Albe-Fessard atlas 1, and the dura mater was carefully removed, avoiding the longitudinal sinus. The recording electrodes were glass micropipettes filled with a mixture of 5% NaCI and Pontamine sky blue and having an impedance of 12-20 M ~ . The dye was applied iontophoreticaily ( 2 - 1 0 / ~ A , 30 min) at
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Fig. 1. This schematic diagram indicates for each Sm neuron those sites of the body where a noxious stimulus elicited responses. The regions of the body stimulated are numbered 1-8 and are identified on the rat figurine illustrated in the middle. Each line represents one cell and each column refers to the body region stimulated. Excitatory responses are indicated by a+, inhibitory responses by a-, no response by 0 and blanks indicate sites that were not tested for that particular neuron. Part A lists all the cells in normal rats, and part B all the cells in arthritic rats.
272
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Fig. 2, Examples of the responses of two Sm neurons to high intensity mechanical stimuli (prolonged pinch) applied to ipsilatcral (i), contralateral (c) posterior (p) and anterior (a) paws and tail. The activity of the neurons is represented as frequency of discharge histograms (2-s bin width). The neuron illustrated in A responded also to pinching the ear. The recording sites within Sm are shoxvn on the histological reconstructions at the right. A M . anteromedial nucleus: CL. centrolateral nucleus: CM. central medial nucleus: MD, mcdiodorsal nucleus: mr. mammillothalamic tract: PV. paraventricular nucleus: Re. reuniens nucleus: Rh. rhomboid nucleus: SM. suhmedius nucleus.
273 innocuous warm and cold stimuli, by applying a piece of warm (38-42 °C) or cool (18-22 °C) wet cotton to the paws or face. At the end of each experiment the rats were perfused with saline followed by 10% formalin, the brain then removed and 100 gm thick frozen sections cut and stained with Cresyl violet. Electrode tracks were reconstructed by superimposing the electrode track on a camera lucida drawing of the section containing the dye mark or marks. Recording sites were reconstructed according to the distances, derived from the micromanipulator readings, from the dye marks. From many instances where two marks were made along the same track it was clear that the errors due to tissue shrinkage or micromanipulator hysteresis were small (usually less than 200/im). Since all the sites in Sm were either those marked or close to marked locations the errors would have been smaller. Only recordings of units from tracks where dye marks could be unequivocally identified were included in the data. The data from individual experiments were pooled on thalamic transverse sections adapted from the atlas of Paxinos and Watson 37. The
location of the mammillothalamic tract relative to Sm was used as the most reliable estimate of the anteriorposterior level within Sm. RESULTS
Recordings from 146 cells subsequently confirmed histologically to be in Sm were obtained in 18 arthritic and 17 normal rats. Of these cells 80 were in arthritic rats and 66 in normals. Fifty-five of these neurons responded to peripheral stimulation, 32 (48%) in normals and 33 (41%) in arthritic. Fifty-one of these cells were excited and 4 (3 in normals, one in arthrithic) inhibited by the peripheral stimulation.
Normal rats In the normal rats responses could only be elicited by noxious stimuli. All the responding cells were excited (or inhibited) by a prolonged intense squeezing stimulus applied to one or more of the limbs and in all but 3 the response was maintained for the entire duration of the stimulus. For 19 (6/1%) of the cells the response outlasted the stimulus application, in the
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Fig. 3. An additional example of a neuron in Sm which was excited by noxious mechanical stimuli applied to the contralateral and ipsilateral posterior paws. Note the variability in afterdischarge. Other details and abbreviations as for Fig. 2.
274
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case of 8 ceils for over 30 s. In almost all cases tested the neurons responded to stimulation of both ipsilateral and contralateral hindlimb, the tail, and frequently also the forelimbs. Some (see Fig. 1) cells were also tested for inputs from the facial region (ear or lip pinch) and over 5 0 % responded. Fig, 1A shows for e~ch cell that responded to peripheral stimulation which regions were tested and whether the cell was excited or inhibited from each region. Fig. 2 shows examples of the responses of two Sm neurons to noxious stimuli applied to all 4 limbs, the tail and the contralateral ear. It is clear from these examples that the neurons do not always respgnd identically to stimuli delivered to different parts of the body. The cell in Fig, 2A had a long afterdischarge to stimulation of the contralateral ear and tail. The cell in Fig, 2B had ~ longer afterdischarge to stimulation of the tail and ~mterior paws than to the posterior paws and no response to contralateral ear pinch. In zlddition, as can be seen in Fig. 3, the responses of a neuron to repeated stimulation of the same region were not always the
by noxious stimuli :lpplicd to the p~tws, t~fil and c:lr.
same. Fig. 4 shows an example of a neuron whose spontaneous firing was inhibited by noxious pinch but not innocuous pressure. The inhibitions in this case outlasted the duration of the stimuli. In a few cases the skin overlying the a b d o m e n or thorax was pinched with forceps but no responses could be evoked. Seven out of 8 neurons tested were excited by noxious, but not innocuous heating of the paw or tail. Many of the neurons that did not respond to noxious mechanical stimulation were examined for possible activation by innocuous t e m p e r a t u r e stimuli ~pplied to the paws or face but no responsive neurons were found. A r t h r i t i c rats
In arthritic rats the responsive neurons were excited (one inhibited) by light to m o d e r a t e pressure stimuli applied to the inflamed limbs or to noxious stimuli applied to the face. These neurons could also be activated by maintained flexion or extension of the inflamed joints (usually ankle or wrist joints}.
275 The responses were maintained for the entire duration of the 15-s stimulus for all but two neurons, and outlasted the stimulus for 25 (76%) cells, in the case of 11 by over 30 s. Fig. 1B gives details on the effective sites for each neuron and indicates that most neurons responded to stimulation of both hindlimbs and frequently also to the forelimbs. Some neurons responded also to stimulation of the tail or face. Fig. 5 shows an example of a neuron that was activated by pressure applied to both anterior paws as well as to flexion of the wrist joint and pressure limited to the digits. Pressure applied to the elbow which was only slightly inflamed produced only a small response. Fig. 6 shows the responses of a different neuron to graded noxious thermal stimuli applied to the inflamed hindpaw. Clear responses were evoked at temperatures over 46 °C and to a 5 °C stimulus. Responses to noxious heating were evoked in 7 cells.
Recording sites Responsive neurons were found to be located at all rostro-caudal levels of Sm and scattered at all sites within the nucleus. No obvious differences in locations were noted between the arthritic and normal rats. The micrographs in Fig. 7 show examples of two dye marks deposited at recording sites within Sin. Fig. 8 is a composite reconstruction of all the recording sites in Sm of both normal and arthritic rats. DISCUSSION
This study provides the first description of the existence of neurons in the rat nucleus submedius that respond to somatosensory stimuli. The only other report of somatosensory responses of identified Sm neurons was the short description in the discussion section of the anatomical paper of Craig and
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Fig. 5. Responses of an Sm neuron in an arthritic rat. This neuron responded to pressure stimuli applied to the anterior paws, digits, and elbow as well as to .joint movement (flexion is illustrated) of the wrist. Other details and abbreviations as for Fig, 2: 3V, third ventricle.
276
C. PAW
t l 44 ° C
t ~ 46°C
t 48°C
t
50°C
I lOHz 30s
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Fig. 6. Responses to temperature stimuli applied to the contralateral paw of a neuron in an arthritic rat. The neuron responded to temperatures over 46 °C and to 5 °C. The location of this neuron in Sm is indicated on the drawing at lower right.
Burton 7 in the cat. They stated that "Sm neurons re-
where only high intensity noxious mechanical or
spond vigorously to mechanical and thermal noxious stimulation, are not excited by innocuous mechanical stimuli and, furthermore, display interesting integrative properties . . .'" In Fig. 17 of their paper 7, they
thermal stimuli were effective. In the arthritic rats responses could be evoked by stimuli which were of lower intensity. Previous studies in arthritic rats strongly suggest that such responses are due to acti-
show oscilloscope records of an Sm n e u r o n which reveal a long afterdischarge following a 2-s noxious
vation of nociceptive afferents whose mechanical thresholds have been reduced due to the arthritic condition ~s. Moreover, such stimuli in the arthritic
thermal stimulus. This type of response resembles those we observed in the rat. The only other reported identified Sm recordings were those of Price and Slotnick 43 in the rat. However, in that study the existence of somatosensory inputs was not tested; instead a population of neurons that were excited by electri-. cal stimulation of the olfactory bulb were identified and described. It is possible that many of those neurons in our study that were not activated from the periphery may have been neurons excited from the olfactory bulb. It should also be noted that it was not possible to deliver noxious or innocuous thermal stimuli to all the body regions and thus in the case of non-responsive neurons it is possible that had they had a small receptive field not located on the limbs it would probably have been missed. The Sm neurons in our study that responded to peripheral stimuli were only activated by noxious stimuli. This was particularly clear in the normal rats
rat are aversive to the animal 5244:. Furthermore. in the normal animal we never found units that could be activated by joint movements or lower intensity stimuli. Thus the findings of these studies indicate that those Sm neurons that respond to peripheral somatosensory stimulation only respond when these stimuli activate nociceptive afferents. There were no obvious differences in the incidence of responsive neurons and response characteristics between the normal and arthritic rats. A n o t h e r characteristic of the Sm neurons was their extensive and bilateral receptive fields. Although we did not carefully examine the entire receptive field of the neurons it would seem reasonable to assume on the basis of our findings that many of these neurons could be activated by a strong noxious stimulus applied anywhere on the skin. It may be difficult to activate a large n u m b e r of nociceptors by pinches ap-
277 difficulty in holding the units during noxious stimulation of the oral-facial region. The responses e v o k e d from Sm neurons tended to differ from those that have been r e p o r t e d for nociceptive neurons in the ventrobasal complex which tend to have graded responses frequently in the innocuous range for mechanical inputs and relatively small unilateral receptive fields 1~'2°-~2~47. The Sm neurons frequently had an afterdischarge of at least a few seconds that sometimes lasted tens of seconds. Furthermore, the responses did not a p p e a r to be well graded although in view of the high thresholds this was difficult to test. Using thermal stimuli, the responses to higher t e m p e r a t u r e stimuli were not dramatically increased with increasing t e m p e r a t u r e stimuli although the latency to onset was shorter. These findings suggest that Sm neurons respond differently than those in VBC to a noxious stimulus and are consistent with a role for this nucleus in mediating the affective and motivational aspects of a noxious stimulus. Previous studies on V B C neurons suggest that the nociceptive VBC neurons may be involved in mediating the sensory-discriminative aspects of pain t~,47. Fig. 7. Photomicrographs showing the location of nucleus submedius in medial thalamus and the loci of two Pontamine dye injections within the nucleus submedius. In A and B the midline is in the center of the photograph so that the nucleus submedius on each side can be seen. In A part of the electrode track can be seen above the dye mark. This mark, which is indicated by the arrow, was made 35(IBm below the cell illustrated in Fig. 6. In B the dye mark is clearly visible in the dorsomedial partofSm on the right hand side. Bar = 2501tm.
plied to the skin overlying the trunk due to the lower density of receptors and lack of convergent input from receptors in d e e p tissues. This may explain the failure to elicit responses from the trunk in those cases where such stimuli were delivered. The larger n u m b e r of cells responding to stimulation of the tail and hindlimb c o m p a r e d to stimulation of more rostral sites is most likely due to the fact that these were the regions usually e x a m i n e d first and in many cases the recording d e t e r i o r a t e d before it was possible to test for inputs from m o r e rostral sites. The largest input to Sm in both cat and rat appears to be from the trigeminal subnucleus caudalis TM, but activation from this region was not usually tested because of the
Anatomical studies in rats, cats, and monkeys have all shown that the ascending pathways to Sm terminate in the dorsal region of the Sm 7"34"35"4°'44"45.Furthermore, Price and Slotnick 43 have shown that Sm neurons responsive to olfactory bulb stimulation are located in the middle and ventral regions of the nucleus and not in the dorsal part. These findings suggest that the dorsal part of the nucleus is functionally quite different from the rest of the nucleus. However, the histological reconstructions of recording sites in the present study revealed that neurons responsive to noxious stimuli were located throughout Sm. It is possible that the d e e p e r located cells have dendrites extending into the dorsal region or that the dorsally located neurons have axons or axon collaterals that extend into the ventral regions of the nucleus. It is not clear where the convergence giving rise to the large and bilateral receptive fields is taking place. In the cat where the pathway has been described in greatest detail, the afferent inputs from the spinal cord and trigeminal subnucleus caudalis are almost entirely contralateral and arise exclusively from the marginal layer 7. H o w e v e r , in the rat the cells of origin appear to be located in the d e e p e r layers rather
278
A
B
NORMAL
ARTHRITIC
vu
* EXCITATION
o NO RESPONSE
- INHIBITION
Fig. 8. Reconstruction of all the recording sites in Sm of normal rats (A) and arthritic rats (B). Abbreviations as in Fig. 2: VM, ~entremedial nucleus: ZI. zona inccrta.
than the superficial layers 1517z7~(' and it is possible that much of the convergence is taking place in the cord and medullary dorsal horn. In conclusion, the present study has identified a population of neurons in the rat Sm that are activated exclusively by noxious stimuli. These neurons tend to have large bilateral receptive fields, maintained responses to noxious stimuli and sometimes long afterdischarges. The responses to graded thermal stimuli are not well graded. These findings are consistent with a role of Sm in mediating affective-motivational aspects of nociception and support the original proposal of Craig and Burton v that this nucleus may be a specific pain center.
ACKNOWLEDGEMENTS
The authors wish to thank Michele Gautron for her excellent help in preparing the animals, data collection and preparing figures and acknowledge the excellent help of Jacqueline Carroue in preparing the histological sections and of Mary Teofilo in preparing some of the figures. We would also like to thank Dr. A.D. Craig for his helpful comments on a previous version of this paper. This study was supported by the French INSERM (G.G.). a joint Canadian MRCINSERM travel grant to J.O.D. and a U.S. NIH Grant DE05404 awarded to J.O.D.
279 REFERENCES I Albe-Fcssard, D., Stutinsky, F. and Libouban, S., Atlas Stereotaxique du Diencephale du Rat Bhmc, CNRS, Paris, 1966. 2 Auen, E.L., Poulos, D,A.. Hirata, H. and Molt, J.T., Location and organization of thalamic thermosensitive neurons responding to cooling the cat oral-facial regions, Brain Research. 191 (1980) 26(I-264. 3 Benoist. J.M., Kayser, V., Gautron, M. and Guilbaud, G., Letter to editor, Pain, 18 (1984) 4111-411. 4 Burton, H. and Craig Jr., A.D., Spinothalamic projections in cat, raccoon and monkey: a study based on anterograde transport of horseradish peroxidase. In G. Macchi, A. Rustioni and R. Spreafico (Eds.), Somatosenso o, Integration in the Thalauuts. Elsevier Science Publishers, Amsterdam, 1983. pp. 17-41. 5 Calvino, B.. Crepon-Bernard, M.-O. and Le Bars, D.. Parallel clinical und behavioural studies of adjuwmt-induced arthritis in the rat: possible relationship with "chronic pain', Behav. Brain Res., 24 (1987) 11-29. 6 Craig Jr.. A.D.. Medial thalamus and nociception: the nucleus submedius. In J.-M. Besson, G. Guilbaud and M. Peschanski (Eds.), Thalamus and Pain, Excerpta Medica, Amsterdam. 1987, pp. 227-243. 7 Craig Jr.. A.D. and Burton, H., Spinal and medullary lamina 1 projection to nucleus submedius in medial thalamus: a possible pain ecntcr, J. Nemophysiol.. 45 ( 1981 ) 443-465. 8 Craig Jr.. A.D. and Burton. H., The distribution and topographical orgunization in thc thalamus of antcrogradelytransported horseradish pcroxidasc after spinal injections ill c;.it ;.u)d raccoon, Exp. Brain Res.. 58 11985) 227-254. 9 Craig Jr,. A,I). and Kniffki, K.-D., Spinothalamic lumbosacral lamina I cells rcsponsivc to skin and muscle stimulation in the cat, J. Physiol. (Lond.). 365 (1985) 197-221. II) Craig Jr.. A.I).. Wiegand. S.J. and Price, J.L., The thalatoo-cortical projection of the nucleus submedius in the cat. J, ('omp. Nem'ol.. 2116(1982) 28-48. 11 Dostrovsky, J.O., Broton. J.G. and Warma, N.K., Functional properties of suhnucleus caudalis lamina I neurons projecting to nucleus submedius. In R.F. Schmidt. H.-G. Schaible and C. Vahle-Hinz (Eds.) Fine Afferent Nerve Fibers and Pain. VCH, Weinheim, 1987, pp. 355-366. 12 Dostrovskv. J.O., Guilhaud, G. and Gautron, M., Nociceptive neurons in nucleus submedius of the arthritic rat, Pain. 4 (1987) $263. 13 Dostrnvsky, J.O., Guilbaud, G. and Gautron, M., Nociceptive responses in nucleus submedius of the rat, Soc. Neurosci. Abstr.. 13 (1987) 1200. 14 Freminet. A.. Bursaux. E. and Payart, C., Mesure de la vitesse de renouvellement du lactate chez le rat par perfusion de [HCU]( l)-Iactatc +, Plhigers Arch. 334 11972) 293-3112. 15 Giesler Jr.. G,J.. Menetrev, D, and Basbaum, A.I., Differential origins of spinothal~lmic tract projections to medial and lateral thalamus in the rat, J. Comp. Neurol., 184 (1979) 1117-126. 16 Gouret, C.. Mocquct, G. and Raynaud. G., Use of Freund's adjuvant arthritis test in anti-intlammatory drug screening in the rat: value of animal selection and preparation at the breeding center, Lab. Anita. Sci.. 26 (1976) 281-287. 17 Granum. S.L.. The spinothalamic system of the rat. I. Locations of cells of origin, J. Comp. Neurol.. 247 (1986) 159-18(1.
18 Guilbaud, G., Iggo, A, and Tegner, R., Sensory receptors in anklc joint capsules of normal and arthritic rats, Exp. Brain Res., 58 (1985) 29-411. 19 Guilbaud, G., Peschanski, M. and Besson. J.-M., Experimental data related to nociception and pain at the supraspinal level. In P.D. Wall and R. Melzack (Eds.), Textbook of Pain, 2nd edn., Churchill Livingston, Edinburgh, in press. 2(1 Guilbaud, G., Peschanski, M., Gautron, M. and Bindcr, D., Neurones responding to noxious stimulation in VB complex and caudal adjacent regions in the thalamus of thc rat, Pain, 8 (19811) 3113-318. 21 Herkenham, M.. The afferent and efferent connections of the ventromedial thalamic nucleus in the rat, 2. Camp. Neurol., 183 (1979) 487-518. 22 Hirai, T., Hendry, S.H.C. and Jones, E.G,, Parccllation of human thalamic nuclei based on histochemical staining and substance P innervation, Sot'. Neurosci. Abstr., 13 (1987) 1199. 23 Honda, C.N., Mense, S. and Perl, E.R., Neurons in ventrobasal region of cat thalamus selectively responsive to noxious mechanical stimulation, J. Neurophysiol., 49 (1983) 662-673. 24 Kayser, V. and Guilbaud, G., The analgesic effects of morphine, but not those of the enkephalinase inhibitor Thiorphan, are enhanced in arthritic rats, Brain Research, 267 (1983) ]31-138. 25 Kayser, V. and Guilbaud, G., Further evidence for changes in the responsiveness of somatosensory neurons in arthritic rats: a study of the posterior intralaminar region of the thalamus, Brain Research, 323 (1984) 144- 147. 26 Gautron, M. and Guilbaud, G., Somatic responses of ventrobasal thalamic neurones in polyarthritic rats, Brain Research, 237 (1982) 459-471. 27 Kemplay, S.K. and Webster, K.E.. A qualitative and quantitative analysis of the distributions of cells in the spinal cord and spinomedullary junction projecting to the thalamus of the rat, Neuroscience, 17 (1986) 769-789. 28 Kenshalo Jr., D R . , Giesler Jr.. G.J., Leonard, R.B. and Willis, W.D., Responses of neurons in primate ventral posterior lateral nucleus to noxious stimuli. J, Neurophysiol.. 43 (19811) 1594-1614. 29 Kniffki, K.-D. and Mizumura, K,. Responses of neurons in VPL and VPL-VL region of the cat to algesic stimulation of muscle and tendon, J. Neurophysiol., 49 (1983) 649-661. 30 Konig, J.F.R. and Klippel. R.A.. The Rat Brain: a Stereotaxic Atlas of the Forebrain and Lower Parts Of the Brain Stem, Williams and Wilkins Baltimore, 1963. 31 Krieg, W.J.S,, The medial region of the thalamus of the albino rat, J. Comp. Neurol.. 81) (1944) 381-415. 32 Krieg, W.J.S., Connections of cerebral cortex, J. Comp. Neurol., 86 (1947) 267-394. 33 Landgren, S., Thalamic neurones responding to cooling of the cat's tongue, Acta Physiol. Stand.. 48 (19611) 255-267. 34 Ma, W., Aspects Structuraux et Ultrastrttctttraux des Projections Spinales et Trigeminales dons le Thalamtts e! I'Aire Parabrachiale, These de doctorat de I'Universit,5 Paris 6. 1987. 35 Mantyh, P.W., The terminations of the spinothalamic tract in the cat, Neurosci. Lett.. 38 (1983) 119-124. 36 Menetrey. D., De Pommery. J. and Roudier, F., Properties of deep spinothalamic tract cells in the rat, with special reference to ventromedial zone of lumbar dorsal horn. J. Neurophysiol., 52 (1984) 612-624. 37 Paxinos, G. and Watson, C.. The Rat Brain in Stereot,xic
28O ( rJordiHal('s. Ac~l,..Icmic, S\,,.hlc,.. I t)S2 3,"; Pearson. C,NI.. \Vaksman, B.I I. and Sharp. ,I. 1.. ,Sltldics ,,;I :nll'u'ifis alnd other lesions indtlccct ill rats b} illicctioll ,,)1nl',ctfi',actcrial adjut,mt. ",'. CllaiL£cs arlccting tile skin aild
II1LIC~.ILIS IllClll['Hilllt..'s
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process v, ith iltllllan disease. ,I. lC.~l), lh'd.. 113 (19(~1) 4S5 511~L Pcrl, E,R.. Pain and nociccption. In ,I.M. Brookhart, V,B. Mountc,iMic alld 1. [)arian-Smith (Eds.), H a n d b o o k oI t'tn'.~iolo,t:y. ,h'CCliOH 1. l'hd m'rvous Srstem. Vohlme IlL Sen,~orv l'r(~ce,s~e~. Part 2. American Physiological Society, Bcthcsda. 1')84. pp. '015-'.)75. Pcschanski. M.. Trigcnlirml allcrcnts to thc cticnccphalon I11 l h c rill. \'duro~c'i{'nc~'. 12 (1984) 4r~5-487. I'cschanski. NI.. (iuilbaud. G.. Gautron. M. and Bcsson. ,I.-M.. Encoding propcrt} of noxious heat messages in neurolls O[ tile \ CllII'OI'~ilS~IIthahmfic conlplcx o1' the rat, t~rain [~d~COvt'/I. lq7 (lOSfl) 41ll-4121. Pircio. A,\V.. Fcdclc. (2.1". and Biciw:.l,,cn M.E.. A nc~v mclh,,',d Ior the cxaluati,.m of armlgc~.ic activity using adju-
',ant-reduced arthritis in lhe rat. /;m'..I.
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[1'075) 2()7 215. 43 PliCC. ,I.[+. ;llld Slotnick t},M., l)ual t~llactor} tcprcsclllalion i11 lhc rat lh:.ll,llllUS: all amltomical and clcctrophysiological Mud\..I. ('+;'lllp. N+'IlI'o/., 215 (1983) ('G 7V. 44 Ralston Ill. tt.,1.. Synaptic ,.~rg;mization ,,',1 spim:.thalamic tract projcctions tt~ the thalamus, with spccial rclcrencc to pain. In 1.. Kruger and,l.C. Licbcskind (Eds.), ,,tdvrulce+ m
Paip~ Re,earth and lT~eralw ('t. Raven. Ncvv 'l",)rk. 1984, pp. 183 Ig5.
45 Robcrtson. B.. Grant, (}. and Biorkcland. M.. Dcnlor~stration of spinoccrebcllar projections in cat using anterogradc Iranspol-t of WGA HRP, with some observations on spin(> mcsenccphalic and spinothal,m~ic projections, /:'.~p Brain Res., 52 ( 19831 99-11)4. 46 WaMcr. A.. lnvitcd editorial, Pain. 9 (198(1) 1-2. 47 Willis ,h.. W.D., The Pain System. In P.L. Gildenberg I Ed. ). Pain a*ld th'adactu', Vol. 8, Kargcr. Basel. 1985, pp. 1-346.