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Different representations of inescapable noxious stimuli in the periaqueductal gray and upper cervical spinal cord of freely moving rats
Neuroscience Letters 313 (2001) 17–20 www.elsevier.com/locate/neulet
Different representations of inescapable noxious stimuli in the periaqueductal gray and upper cervical spinal cord of freely moving rats Kevin A. Keay a,b,*, Colin I. Clement a, Antoine Depaulis c, Richard Bandler a,b a
Department of Anatomy & Histology, The University of Sydney, Sydney, NSW 2006, Australia Pain Management & Research Centre, The University of Sydney, Sydney, NSW 2006, Australia c Neurobiologie et Neuropharmacologie des E´pilepsies Ge´ne´ralise´e, U.398 INSERM, Strasbourg, France b
Received 5 May 2001; received in revised form 20 August 2001; accepted 20 August 2001
Abstract Previous work suggested that pain of distinct tissue origins was differentially represented in the midbrain periaqueductal gray (PAG). That is, persistent pain of deep origin (muscle, joint viscera) ‘activated” ventrolateral PAG neurons and triggered quiescence, hyporeactivity and vasodepression (i.e. passive emotional coping); whereas intermittent cutaneous pain ‘activated’ lateral PAG neurons and triggered fight-flight (i.e. active emotional coping). Cutaneous noxious stimuli, if inescapable however, trigger a passive emotional coping reaction similar to that evoked by pain of deep origin. This raised the question - is it the behavioural significance (escapability versus inescapability) or the tissue origin (cutaneous versus deep) of the pain, that is represented in the PAG? In this study we used immediate-early-gene (c-Fos) expression to examine PAG and spinal activation patterns following ‘inescapable’ (persistent) pain of cutaneous versus deep origin. It was found that selective activation of the ventrolateral PAG and passive emotional coping were evoked by an inescapable cutaneous noxious stimulus (i.e. clip of the neck), as well as by a deep noxious stimulus (i.e. neck muscle pain). In the upper cervical spinal cord, however, these noxious manipulations evoked distinct patterns of Fos expression which reflected the different patterns of primary afferent termination arising from skin versus muscle. The results suggest that whereas pain representation in the spinal cord accurately reflects tissue origin, pain representation in the PAG better reflects behavioural significance. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: c-fos; Immediate early gene; Nociception; Emotion; Periaqueductal gray; Pain; Spinal cord
The study of the central representation of pain has traditionally adopted the experimental approach of the sensory physiologist, that is, to study neural responses to stimulus qualities such as tissue origin, location, duration, intensity and modality. The behavioural significance and appropriate response to a noxious event is determined by a combination of these qualities. As elegantly described by Lewis [12] short duration, mechanical stimulation of the skin triggers usually “quick protective reflexes, a rise of pulse rate and a sense of invigoration” (i.e. active emotional coping), whereas persistent pain arising from deep structures (muscles, joints, viscera) is associated usually with “quiescence, slowing of the pulse, a fall in blood pressure and loss of interest in the environment” (i.e. passive emotional
* Corresponding author. Tel.: 161-2-9351-4132; fax: 161-29351-6556. E-mail address: [email protected] (K.A. Keay).
coping). Our previous work found that such integrated, active versus passive coping responses were elicited by activation of lateral versus ventrolateral columns of the midbrain periaqueductal gray (PAG). These data led to the hypothesis that distinct PAG columns play specific roles in both evaluating the behavioural significance of, and implementing the appropriate response(s) to, distinct classes of pain. This hypothesis was supported by data from experiments in anaesthetised rats which demonstrated that pain arising from muscles, joints and viscera selectively activated neurons within the ventrolateral PAG, whereas noxious cutaneous stimuli [radiant heat applied to skin] preferentially activated neurones in the lateral PAG [3,10,11]. However, not all noxious cutaneous events trigger active-coping behaviors. For example, persistent mechanical stimulation of the skin (e.g. clip of the neck) evokes a passive coping response (i.e. quiescence and hyporeactivity), strikingly similar to that usually associated with persis-
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 22 6- 1
18
K.A. Keay et al. / Neuroscience Letters 313 (2001) 17–20
tent pain of deep origin [7,8]. This raises the question of whether the behavioural significance (escapability versus inescapability) or the tissue origin (cutaneous versus deep) of pain, is represented in the PAG? To answer this question we used the method of immediate-early gene expression (cFos) as a marker of neuronal activation to determine, following ‘inescapable’ pain of cutaneous versus deep origin, if PAG and spinal activation patterns reflected tissue origin or other dimensions, such as persistence or escapability. The experiments were carried out in unanaesthetised rats, with noxious-evoked behavioural responses and patterns of Fos expression being evaluated in the same animals. All procedures were carried out at U-398 INSERM, adhering to the guidelines of the International Association for the Study of Pain [18]. Data were obtained from 23 adult male Wistar rats (280–380 g). The animals were briefly anaesthetised with 5% halothane (2–3 min) and then hand-held by an experimenter while one of three noxious manipulations were performed. (i) noxious deep muscle stimulation: eight animals received a bilateral injection (50 ml each injection) of 5% formalin solution into the deep dorsal neck muscles; (ii) clip of neck: in eight animals an alligator clip was applied to the nape of the neck (see Refs. [7,8] for details); (iii) controls: seven rats were briefly anaesthetized with 5% halothane (2–3 min). Following one of the above manipulations, each rat was returned to its home cage. Forty minutes later it was placed in a neutral test cage and an untreated, weight-matched male rat was introduced for an 8 min period. Social interactions and sonic and ultrasonic (22–28 kHz) vocalizations were recorded. As described previously, the neutral test cage permitted observation of the reactions of the experimental and control rats to the non-aggressive investigative approaches of the partner rat [5,6]). Individual behavioral items, adapted from Grant and Mackintosh [9] were encoded for all rats from the video recordings, using a previously described technique and scoring system [4], which should be referred to for details. Briefly, the following behavioural categories were scored: non-social behavior (cage exploration, self grooming); social behavior (investigation/sniffing of the partner), quiescence (immobility, absence of normal spontaneous or partner-evoked behaviors) and defensive behaviors (defensive alerting/freezing, defensive sideways postures to the approach of the partner, sonic/ultrasonic vocalization). After the social interaction test, each rat was then returned to its homecage for a further 70–100 min. At the end of this period, rats were deeply anaesthetized with pentobarbital (100 mg/kg) and the brain and spinal cord fixed by perfusion. The midbrain and upper cervical spinal cord of each animals was removed, coronally sectioned and processed immunohistochemically using standard procedures for the presence of Fos protein (for details see Ref. [3]). Fos-positive cells were plotted onto standardized midbrain and spinal cord sections. Cell distributions were
quantified by taking sections at 0.5 mm distances through the midbrain PAG and every third section through spinal levels C1 and C2. Behavior: control rats (n ¼ 7) when tested (8 min period) with a weight-matched partner in the neutral test cage (40 min after brief halothane anesthesia) showed predominantly non-social behavior (281.4 ^ 18.1 s), i.e. cage exploration mixed with periods of self grooming (see Fig. 1). Periods of quiescence (93.3 ^ 35.4 s), and social behaviour (105.2 ^ 21.6 s) were also observed. Defensive behaviors (i.e. reactive immobility and active defense) and vocalization rarely occurred (note the change in scale in Fig. 1). For the rats subjected to either an inescapable ‘cutaneous’ noxious manipulation (clip of the skin of the neck), or the deep noxious manipulation (injection of formalin into neck muscles) there was a difference in the reaction to the partner during the 8 min social interaction test. Specifically, the experimental rats were more passive than the controls. Thus, as seen in Fig. 1 there was a 2–3-fold increase in the period of quiescence [clip (293.3 ^ 31.5 s), deep pain (255.2 ^ 43.1 s) versus control (93.3 ^ 35.4 s) (P , 0:01, Mann–Whitney test)] and approximately a 50% decrease in non-social behaviour [clip (122.3 ^ 22.4 s), deep pain (169.2 ^ 34.3 s) versus control (281.4 ^ 18.1 s) (P , 0:01, Mann–Whitney test)]. The duration of social and defensive behaviors were not significantly different from controls (see Fig. 1). Fos-like immunoreactivity (IR): (i) PAG: very few Foslike immunoreactive (IR) cells were observed in the PAG of controls. In contrast, there was a selective and significant increase in the numbers of Fos-like IR cells in the caudal ventrolateral PAG of animals which were subjected to (1) the inescapable ‘cutaneous’ noxious stimulus; or (2) received bilateral intramuscular injections of formalin. In
Fig. 1. Histograms showing for each manipulation: control, deep (bilateral formalin injections into deep neck muscles), clip (alligator clip on skin of dorsum of neck); the time in seconds, during an 8 min social interaction spent in each of the following behavioural categories: non-social behaviour; social behaviour, quiescence and defensive behaviour. Asterisks indicate significant changes with respect to controls (**P , 0:01).
K.A. Keay et al. / Neuroscience Letters 313 (2001) 17–20
Fig. 2. Left panel: histograms illustrating the numbers of Fos-like IR cells in the lateral and ventrolateral PAG columns at each rostrocaudal level analyzed. Asterisks indicate significant differences with respect to controls (**P , 0:01). Right panel: schematic, representative sections through a 2.0 mm rostrocaudal extent of the PAG showing the locations of Fos-like IR neurons (black dots) on one side of a 40 mm section, following each of the (bilateral) manipulations. Similar patterns of Fos expression were observed bilaterally in the PAG. The numbers at the bottom of the panel indicate the rostrocaudal level of each section, in millimetres, with respect to bregma
contrast, within the caudal lateral PAG, the numbers of Foslike IR cells in the experimental groups did not differ from each other or from controls (see Fig. 2). (ii) Upper cervical spinal cord (UCC): in the UCC of controls, rather than a distinct distribution, small numbers of Fos-like IR cells were scattered across most laminae (see Fig. 3). In contrast, after the deep noxious manipulation (i.m. formalin) Fos-like IR cells were located in the superficial (laminae I and II) and deep (laminae IV and V) dorsal horn, and a region comprising the medial ventral horn (laminae VII and VIII) and lamina X (Fig. 3). In addition, Fos-like IR cells were found consistently in the dorsolateral fasciculus (DLF) (a region which, in the rat, includes both the lateral spinal nucleus and the lateral cervical nucleus). A different pattern was seen after the inescapable cutaneous noxious stimulus (clip of the neck), with Fos-like IR cells restricted to the superficial dorsal horn (laminae I and II) and the immediately adjacent DLF (Fig. 3). As well, a few Fos-like IR cells were located in the lateral ‘reticular region’ of lamina V. Previous studies from our own and other laboratories have reported that, in the anaesthetised rat, deep noxious manipulations (muscles, joints and viscera) evoked a selective increase in Fos-like IR within the ventrolateral PAG; whereas cutaneous noxious manipulations (e.g. brief, intermittent episodes of radiant heat) evoked increased Fos-like IR in the lateral PAG and, to a lesser extent, in the ventrolateral PAG [3,10]. The data of this study suggest, however, that the pattern of PAG Fos expression reflects dimensions
19
in addition to tissue origin. That is, the predominantly lateral PAG pattern of Fos-expression previously observed in response to an intermittent cutaneous (heat) noxious stimulus [10], was not observed when the cutaneous (mechanical) noxious event was persistent/inescapable (i.e. clip of the neck). In other words, similar to the persistence/inescapability of pain of deep origin, an inescapable cutaneous noxious stimulus evoked both a passive emotional coping response and a selective increase in ventrolateral PAG Fos-expression. In contrast, the pattern of Fos expression in the UCC reflected exclusively the tissue origin of the noxious stimulus. That is, even though each stimulus was inescapable, the noxious cutaneous stimulus activated neurons primarily in the superficial dorsal horn; whereas the noxious deep somatic stimulus activated neurons within superficial and deep dorsal horn, the medial ventral horn/lamina X and the DLF. Overall, these different spinal patterns of Fosexpression reflect the distinct UCC distributions of afferents arising from cutaneous versus deep structures of the neck [2,13,14]. That is, cutaneous afferents terminate primarily with the superficial laminae of the dorsal horn [1], which fits well with the finding of a predominantly superficial laminar pattern of Fos-like IR cells following clip of the neck. In
Fig. 3. Left panel: histograms illustrating the numbers of Fos-like IR cells in specific laminae of the upper cervical spinal cord (C1– 2). Asterisks indicate significant differences with respect to controls (**P , 0:01). Right panel: schematic, representative sections of the upper cervical spinal cord (C1–2) illustrating the location of Fos-like IR neurons (black dots) in a single 40 mm section following each of the manipulations. The laminar boundaries indicate: superficial dorsal horn (laminae I - III), deep dorsal horn and intermediate gray (laminae IV-VII), ventral horn (laminae VII and IX) and lamina X.
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contrast, deep neck muscle afferents are characterized by a strong projection into the deep dorsal horn and medial ventral horn, with a smaller superficial dorsal horn projection [2,13–15]. The pattern of Fos-expression, in deep dorsal horn and medial ventral horn/lamina X, observed after formalin injection into neck muscles is consistent with this primary afferent anatomy. The extensive Fos expression observed in the superficial dorsal horn, although greater than expected based on primary afferent anatomy alone, fits well with electrophysiological findings that the majority of superficial dorsal horn UCC neurons receive convergent cutaneous and deep (somatic and visceral) noxious inputs [16,17]. To summarize, the results of this study indicate that the patterns of neuronal activation, in two distinct parts of the neuraxis, reflect different noxious stimulus qualities. In the upper cervical spinal cord, the pattern of activation reflected primarily the tissue origin of the pain, i.e. the distinct spinal distribution patterns of primary afferent fibers arising from skin versus muscle. However, supraspinally, within the PAG, the pattern of activation reflected a quality more akin to the ‘behavioral significance’ of the noxious event. That is, the directly measurable qualities of a noxious stimulus, e.g. tissue origin, location, duration, intensity and modality, somehow combine into an ‘escapability dimension’ which is revealed both by the pattern of PAG activation and the emotional strategy (active or passive) used by the animal to cope with the noxious event. Whether ‘behavioural significance’ is represented in a similar fashion in other supraspinal regions awaits further investigation This research was supported by a grant from the Australian National Health and Medical Research Council (970688) to R.B. and K.A.K. [1] Abrahams, V.C., The distribution of muscle and cutaneous projections to the dorsal horn of the upper cervical spinal cord of the cat, In F. Cervero, G.J. Bennet and P.M. Headly (Eds.), Processing of Sensory Information in the Superficial Dorsal Horn, Plenum, New York, 1989, pp. 41–56. [2] Ammann, B., Gottschall, J. and Zenker, W., Afferent projections from rat longus capitus muscle studied by transganglionic transport of HRP, Anat. Embryol., 166 (1983) 275–289. [3] Clement, C.I., Keay, K.A., Owler, B.K. and Bandler, R., Common patterns of increased and decreased Fos expression in midbrain and pons evoked by noxious deep somatic and noxious visceral manipulations in the rat, J. Comp. Neurol., 366 (1996) 495–515.
[4] Depaulis, A., Bandler, R. and Vergnes, M., Characterisation of pretentorial periaqueductal grey neurons mediating intraspecific defensive behaviours in the rat by microinjections of kainic acid, Brain Res., 486 (1989) 121–132. [5] Depaulis, A., Keay, K.A. and Bandler, R., Longitudinal neuronal organisation of defensive reactions in the midbrain periaqueductal gray region of the rat, Exp. Brain Res., 90 (1992) 307–318. [6] Depaulis, A., Keay, K.A. and Bandler, R., Quiescence and hyporeactivity evoked by activation of cell bodies in the ventrolateral midbrain periaqueductal gray of the rat, Exp. Brain Res., 99 (1994) 75–83. [7] Fleishman, A. and Urca, G., Clip induced analgesia and immobility in the mouse: activation by different sensory modalities, Physiol. Behav., 44 (1988) 39–45. [8] Fleishman, A. and Urca, G., Clip induced analgesia: noxious neck pinch supresses spinal and mesencephalic neural responses to noxious peripheral stimulation, Physiol. Behav., 46 (1989) 151–157. [9] Grant, E.C. and Macintosh, J.H., A comparison of the social postures of some common laboratory rodents, Behaviour, 21 (1963) 246–259. [10] Keay, K.A. and Bandler, R., Deep and superficial noxious stimulation increases Fos like immunoreactivity in different regions of the midbrain periaqueductal grey of the rat, Neurosci. Lett., 154 (1993) 143–158. [11] Keay, K.A., Clement, C.I., Owler, B., Depaulis, A. and Bandler, R., Convergence of deep somatic and visceral nociceptive information onto a discrete ventrolateral midbrain periaqueductal gray region, Neuroscience, 61 (1994) 727– 732. [12] Lewis, T., Pain, McMillan, London, 1942. [13] Mysicka, A. and Zenker, W., Central projections of muscle afferents from the sternomastoid nerve in the rat, Brain Res., 211 (1981) 257–265. [14] Pfaller, K. and Arvidsson, J., Central distribution of trigeminal and upper cervical afferents in the rat studied by anterograde transport of horseradish peroxidase conjugated to wheat germ agglutinin, J. Comp. Neurol., 268 (1988) 91– 108. [15] Prihoda, M., Hiller, M.-S. and Mayr, R., Central projections of cervical primary afferent fibres in the guinea pig: a HRP and WGA-HRP tracer study, J. Comp. Neurol., 308 (1991) 418–431. [16] Yezierski, R.P., Somatosensory input to the periaqueductal gray: a spinal relay to a descending control centre, In A. Depaulis and R. Bandler (Eds.), The Midbrain Periaqueductal Gray, Plenum, New York, 1991, pp. 365–386. [17] Yezierski, R.P. and Broton, J.G., Functional properties of spinomesencephalic cells in the upper cervical spinal cord of the cat, Pain, 45 (1991) 187–198. [18] Zimmermann, M., Ethical guidelines for investigations of experimental pain in conscious animals, Pain, 16 (1983) 109.
Different representations of inescapable noxious stimuli in the periaqueductal gray and upper cervical spinal cord of freely moving rats Kevin A. Keay a,b,*, Colin I. Clement a, Antoine Depaulis c, Richard Bandler a,b a
Department of Anatomy & Histology, The University of Sydney, Sydney, NSW 2006, Australia Pain Management & Research Centre, The University of Sydney, Sydney, NSW 2006, Australia c Neurobiologie et Neuropharmacologie des E´pilepsies Ge´ne´ralise´e, U.398 INSERM, Strasbourg, France b
Received 5 May 2001; received in revised form 20 August 2001; accepted 20 August 2001
Abstract Previous work suggested that pain of distinct tissue origins was differentially represented in the midbrain periaqueductal gray (PAG). That is, persistent pain of deep origin (muscle, joint viscera) ‘activated” ventrolateral PAG neurons and triggered quiescence, hyporeactivity and vasodepression (i.e. passive emotional coping); whereas intermittent cutaneous pain ‘activated’ lateral PAG neurons and triggered fight-flight (i.e. active emotional coping). Cutaneous noxious stimuli, if inescapable however, trigger a passive emotional coping reaction similar to that evoked by pain of deep origin. This raised the question - is it the behavioural significance (escapability versus inescapability) or the tissue origin (cutaneous versus deep) of the pain, that is represented in the PAG? In this study we used immediate-early-gene (c-Fos) expression to examine PAG and spinal activation patterns following ‘inescapable’ (persistent) pain of cutaneous versus deep origin. It was found that selective activation of the ventrolateral PAG and passive emotional coping were evoked by an inescapable cutaneous noxious stimulus (i.e. clip of the neck), as well as by a deep noxious stimulus (i.e. neck muscle pain). In the upper cervical spinal cord, however, these noxious manipulations evoked distinct patterns of Fos expression which reflected the different patterns of primary afferent termination arising from skin versus muscle. The results suggest that whereas pain representation in the spinal cord accurately reflects tissue origin, pain representation in the PAG better reflects behavioural significance. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: c-fos; Immediate early gene; Nociception; Emotion; Periaqueductal gray; Pain; Spinal cord
The study of the central representation of pain has traditionally adopted the experimental approach of the sensory physiologist, that is, to study neural responses to stimulus qualities such as tissue origin, location, duration, intensity and modality. The behavioural significance and appropriate response to a noxious event is determined by a combination of these qualities. As elegantly described by Lewis [12] short duration, mechanical stimulation of the skin triggers usually “quick protective reflexes, a rise of pulse rate and a sense of invigoration” (i.e. active emotional coping), whereas persistent pain arising from deep structures (muscles, joints, viscera) is associated usually with “quiescence, slowing of the pulse, a fall in blood pressure and loss of interest in the environment” (i.e. passive emotional
* Corresponding author. Tel.: 161-2-9351-4132; fax: 161-29351-6556. E-mail address: [email protected] (K.A. Keay).
coping). Our previous work found that such integrated, active versus passive coping responses were elicited by activation of lateral versus ventrolateral columns of the midbrain periaqueductal gray (PAG). These data led to the hypothesis that distinct PAG columns play specific roles in both evaluating the behavioural significance of, and implementing the appropriate response(s) to, distinct classes of pain. This hypothesis was supported by data from experiments in anaesthetised rats which demonstrated that pain arising from muscles, joints and viscera selectively activated neurons within the ventrolateral PAG, whereas noxious cutaneous stimuli [radiant heat applied to skin] preferentially activated neurones in the lateral PAG [3,10,11]. However, not all noxious cutaneous events trigger active-coping behaviors. For example, persistent mechanical stimulation of the skin (e.g. clip of the neck) evokes a passive coping response (i.e. quiescence and hyporeactivity), strikingly similar to that usually associated with persis-
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 02 22 6- 1
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K.A. Keay et al. / Neuroscience Letters 313 (2001) 17–20
tent pain of deep origin [7,8]. This raises the question of whether the behavioural significance (escapability versus inescapability) or the tissue origin (cutaneous versus deep) of pain, is represented in the PAG? To answer this question we used the method of immediate-early gene expression (cFos) as a marker of neuronal activation to determine, following ‘inescapable’ pain of cutaneous versus deep origin, if PAG and spinal activation patterns reflected tissue origin or other dimensions, such as persistence or escapability. The experiments were carried out in unanaesthetised rats, with noxious-evoked behavioural responses and patterns of Fos expression being evaluated in the same animals. All procedures were carried out at U-398 INSERM, adhering to the guidelines of the International Association for the Study of Pain [18]. Data were obtained from 23 adult male Wistar rats (280–380 g). The animals were briefly anaesthetised with 5% halothane (2–3 min) and then hand-held by an experimenter while one of three noxious manipulations were performed. (i) noxious deep muscle stimulation: eight animals received a bilateral injection (50 ml each injection) of 5% formalin solution into the deep dorsal neck muscles; (ii) clip of neck: in eight animals an alligator clip was applied to the nape of the neck (see Refs. [7,8] for details); (iii) controls: seven rats were briefly anaesthetized with 5% halothane (2–3 min). Following one of the above manipulations, each rat was returned to its home cage. Forty minutes later it was placed in a neutral test cage and an untreated, weight-matched male rat was introduced for an 8 min period. Social interactions and sonic and ultrasonic (22–28 kHz) vocalizations were recorded. As described previously, the neutral test cage permitted observation of the reactions of the experimental and control rats to the non-aggressive investigative approaches of the partner rat [5,6]). Individual behavioral items, adapted from Grant and Mackintosh [9] were encoded for all rats from the video recordings, using a previously described technique and scoring system [4], which should be referred to for details. Briefly, the following behavioural categories were scored: non-social behavior (cage exploration, self grooming); social behavior (investigation/sniffing of the partner), quiescence (immobility, absence of normal spontaneous or partner-evoked behaviors) and defensive behaviors (defensive alerting/freezing, defensive sideways postures to the approach of the partner, sonic/ultrasonic vocalization). After the social interaction test, each rat was then returned to its homecage for a further 70–100 min. At the end of this period, rats were deeply anaesthetized with pentobarbital (100 mg/kg) and the brain and spinal cord fixed by perfusion. The midbrain and upper cervical spinal cord of each animals was removed, coronally sectioned and processed immunohistochemically using standard procedures for the presence of Fos protein (for details see Ref. [3]). Fos-positive cells were plotted onto standardized midbrain and spinal cord sections. Cell distributions were
quantified by taking sections at 0.5 mm distances through the midbrain PAG and every third section through spinal levels C1 and C2. Behavior: control rats (n ¼ 7) when tested (8 min period) with a weight-matched partner in the neutral test cage (40 min after brief halothane anesthesia) showed predominantly non-social behavior (281.4 ^ 18.1 s), i.e. cage exploration mixed with periods of self grooming (see Fig. 1). Periods of quiescence (93.3 ^ 35.4 s), and social behaviour (105.2 ^ 21.6 s) were also observed. Defensive behaviors (i.e. reactive immobility and active defense) and vocalization rarely occurred (note the change in scale in Fig. 1). For the rats subjected to either an inescapable ‘cutaneous’ noxious manipulation (clip of the skin of the neck), or the deep noxious manipulation (injection of formalin into neck muscles) there was a difference in the reaction to the partner during the 8 min social interaction test. Specifically, the experimental rats were more passive than the controls. Thus, as seen in Fig. 1 there was a 2–3-fold increase in the period of quiescence [clip (293.3 ^ 31.5 s), deep pain (255.2 ^ 43.1 s) versus control (93.3 ^ 35.4 s) (P , 0:01, Mann–Whitney test)] and approximately a 50% decrease in non-social behaviour [clip (122.3 ^ 22.4 s), deep pain (169.2 ^ 34.3 s) versus control (281.4 ^ 18.1 s) (P , 0:01, Mann–Whitney test)]. The duration of social and defensive behaviors were not significantly different from controls (see Fig. 1). Fos-like immunoreactivity (IR): (i) PAG: very few Foslike immunoreactive (IR) cells were observed in the PAG of controls. In contrast, there was a selective and significant increase in the numbers of Fos-like IR cells in the caudal ventrolateral PAG of animals which were subjected to (1) the inescapable ‘cutaneous’ noxious stimulus; or (2) received bilateral intramuscular injections of formalin. In
Fig. 1. Histograms showing for each manipulation: control, deep (bilateral formalin injections into deep neck muscles), clip (alligator clip on skin of dorsum of neck); the time in seconds, during an 8 min social interaction spent in each of the following behavioural categories: non-social behaviour; social behaviour, quiescence and defensive behaviour. Asterisks indicate significant changes with respect to controls (**P , 0:01).
K.A. Keay et al. / Neuroscience Letters 313 (2001) 17–20
Fig. 2. Left panel: histograms illustrating the numbers of Fos-like IR cells in the lateral and ventrolateral PAG columns at each rostrocaudal level analyzed. Asterisks indicate significant differences with respect to controls (**P , 0:01). Right panel: schematic, representative sections through a 2.0 mm rostrocaudal extent of the PAG showing the locations of Fos-like IR neurons (black dots) on one side of a 40 mm section, following each of the (bilateral) manipulations. Similar patterns of Fos expression were observed bilaterally in the PAG. The numbers at the bottom of the panel indicate the rostrocaudal level of each section, in millimetres, with respect to bregma
contrast, within the caudal lateral PAG, the numbers of Foslike IR cells in the experimental groups did not differ from each other or from controls (see Fig. 2). (ii) Upper cervical spinal cord (UCC): in the UCC of controls, rather than a distinct distribution, small numbers of Fos-like IR cells were scattered across most laminae (see Fig. 3). In contrast, after the deep noxious manipulation (i.m. formalin) Fos-like IR cells were located in the superficial (laminae I and II) and deep (laminae IV and V) dorsal horn, and a region comprising the medial ventral horn (laminae VII and VIII) and lamina X (Fig. 3). In addition, Fos-like IR cells were found consistently in the dorsolateral fasciculus (DLF) (a region which, in the rat, includes both the lateral spinal nucleus and the lateral cervical nucleus). A different pattern was seen after the inescapable cutaneous noxious stimulus (clip of the neck), with Fos-like IR cells restricted to the superficial dorsal horn (laminae I and II) and the immediately adjacent DLF (Fig. 3). As well, a few Fos-like IR cells were located in the lateral ‘reticular region’ of lamina V. Previous studies from our own and other laboratories have reported that, in the anaesthetised rat, deep noxious manipulations (muscles, joints and viscera) evoked a selective increase in Fos-like IR within the ventrolateral PAG; whereas cutaneous noxious manipulations (e.g. brief, intermittent episodes of radiant heat) evoked increased Fos-like IR in the lateral PAG and, to a lesser extent, in the ventrolateral PAG [3,10]. The data of this study suggest, however, that the pattern of PAG Fos expression reflects dimensions
19
in addition to tissue origin. That is, the predominantly lateral PAG pattern of Fos-expression previously observed in response to an intermittent cutaneous (heat) noxious stimulus [10], was not observed when the cutaneous (mechanical) noxious event was persistent/inescapable (i.e. clip of the neck). In other words, similar to the persistence/inescapability of pain of deep origin, an inescapable cutaneous noxious stimulus evoked both a passive emotional coping response and a selective increase in ventrolateral PAG Fos-expression. In contrast, the pattern of Fos expression in the UCC reflected exclusively the tissue origin of the noxious stimulus. That is, even though each stimulus was inescapable, the noxious cutaneous stimulus activated neurons primarily in the superficial dorsal horn; whereas the noxious deep somatic stimulus activated neurons within superficial and deep dorsal horn, the medial ventral horn/lamina X and the DLF. Overall, these different spinal patterns of Fosexpression reflect the distinct UCC distributions of afferents arising from cutaneous versus deep structures of the neck [2,13,14]. That is, cutaneous afferents terminate primarily with the superficial laminae of the dorsal horn [1], which fits well with the finding of a predominantly superficial laminar pattern of Fos-like IR cells following clip of the neck. In
Fig. 3. Left panel: histograms illustrating the numbers of Fos-like IR cells in specific laminae of the upper cervical spinal cord (C1– 2). Asterisks indicate significant differences with respect to controls (**P , 0:01). Right panel: schematic, representative sections of the upper cervical spinal cord (C1–2) illustrating the location of Fos-like IR neurons (black dots) in a single 40 mm section following each of the manipulations. The laminar boundaries indicate: superficial dorsal horn (laminae I - III), deep dorsal horn and intermediate gray (laminae IV-VII), ventral horn (laminae VII and IX) and lamina X.
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contrast, deep neck muscle afferents are characterized by a strong projection into the deep dorsal horn and medial ventral horn, with a smaller superficial dorsal horn projection [2,13–15]. The pattern of Fos-expression, in deep dorsal horn and medial ventral horn/lamina X, observed after formalin injection into neck muscles is consistent with this primary afferent anatomy. The extensive Fos expression observed in the superficial dorsal horn, although greater than expected based on primary afferent anatomy alone, fits well with electrophysiological findings that the majority of superficial dorsal horn UCC neurons receive convergent cutaneous and deep (somatic and visceral) noxious inputs [16,17]. To summarize, the results of this study indicate that the patterns of neuronal activation, in two distinct parts of the neuraxis, reflect different noxious stimulus qualities. In the upper cervical spinal cord, the pattern of activation reflected primarily the tissue origin of the pain, i.e. the distinct spinal distribution patterns of primary afferent fibers arising from skin versus muscle. However, supraspinally, within the PAG, the pattern of activation reflected a quality more akin to the ‘behavioral significance’ of the noxious event. That is, the directly measurable qualities of a noxious stimulus, e.g. tissue origin, location, duration, intensity and modality, somehow combine into an ‘escapability dimension’ which is revealed both by the pattern of PAG activation and the emotional strategy (active or passive) used by the animal to cope with the noxious event. Whether ‘behavioural significance’ is represented in a similar fashion in other supraspinal regions awaits further investigation This research was supported by a grant from the Australian National Health and Medical Research Council (970688) to R.B. and K.A.K. [1] Abrahams, V.C., The distribution of muscle and cutaneous projections to the dorsal horn of the upper cervical spinal cord of the cat, In F. Cervero, G.J. Bennet and P.M. Headly (Eds.), Processing of Sensory Information in the Superficial Dorsal Horn, Plenum, New York, 1989, pp. 41–56. [2] Ammann, B., Gottschall, J. and Zenker, W., Afferent projections from rat longus capitus muscle studied by transganglionic transport of HRP, Anat. Embryol., 166 (1983) 275–289. [3] Clement, C.I., Keay, K.A., Owler, B.K. and Bandler, R., Common patterns of increased and decreased Fos expression in midbrain and pons evoked by noxious deep somatic and noxious visceral manipulations in the rat, J. Comp. Neurol., 366 (1996) 495–515.
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