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Tachykinin receptor inhibition and c-Fos expression in the rat brain following formalin-induced pain
Neuroscience Vol. 95, No. 3, pp. 813–820, 2000 813 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00
Brain neurokinins and pain-induced c-Fos expression
Pergamon PII: S0306-4522(99)00478-9 www.elsevier.com/locate/neuroscience
TACHYKININ RECEPTOR INHIBITION AND c-FOS EXPRESSION IN THE RAT BRAIN FOLLOWING FORMALIN-INDUCED PAIN J. BAULMANN, H. SPITZNAGEL, T. HERDEGEN, T. UNGER and J. CULMAN* Institute of Pharmacology, Christian-Albrechts-University of Kiel, 24105 Kiel, Germany and German Institute for High Blood Pressure Research, 69120 Heidelberg, Germany
Abstract—Recent pharmacological evidence has implicated substance P and neurokinin A, natural ligands for neurokinin-1 and neurokinin-2 receptors, respectively, as neurotransmitters in brain neuronal circuits activated upon noxious stimulation. The expression of the inducible transcription factor, c-Fos, was used to identify areas in the brain activated by a noxious stimulus (the subcutaneous injection of formalin), and to investigate the effects of intracerebroventricular administration of selective, nonpeptide antagonists for neurokinin-1 and neurokinin-2 tachykinin receptors on the neural activity in these areas and on the behavioural response to formalin-induced pain. Formalin (5%, 50 ml), injected subcutaneously through a chronically implanted catheter in the region of the lower hindlimb, increased c-Fos expression in a number of brain areas related to nociceptive transmission or the integration of stress responses. Grooming behaviour, licking and biting directed to the injected site, was the most frequent behavioural response. Intracerebroventricular pretreatment of rats with either RP 67580 (500 pmol), the active enantiomer of a neurokinin-1 receptor antagonist, or with SR 48968 (500 pmol), the active enantiomer of a neurokinin-2 receptor antagonist, reduced the formalin-induced c-Fos staining in the prefrontal cortex, dorsomedial and ventromedial nuclei of the hypothalamus, the locus coeruleus and the periaqueductal gray. The neurokinin-1, but not the neurokinin-2, receptor antagonist attenuated the formalin-induced activation of c-Fos in the paraventricular nucleus of the hypothalamus. Simultaneous intracerebroventricular pretreatment with both neurokinin-1 and neurokinin-2 receptor antagonists did not produce any additional inhibitory effect on the post-formalin c-Fos expression. None of the tachykinin receptor antagonists had an effect on the formalininduced c-Fos expression in the septohypothalamic nucleus, medial thalamus, parabrachial nucleus and central amygdaloid nucleus, indicating that neurotransmitters other than neurokinins are most probably responsible for the activation of these areas in respose to noxious stimulation. While both tachykinin receptor antagonists reduced the grooming behaviour to formalin, the neurokinin-1 receptor antagonist was clearly more effective than the neurokinin-2 receptor antagonist. Intracerebroventricular pretreatment of rats with the inactive enantiomers of the tachykinin receptor antagonists, RP 68651 and SR 48965, was without effect. Our results show that (i) the modified formalin test elicited an intense grooming behaviour and expression of c-Fos in numerous forebrain and brainstem areas, (ii) both tachykinin receptor antagonists were able to attenuate the behavioural response to pain and to reduce the formalin-induced c-Fos expression in some, but not all, brain areas, and (iii) the neurokinin-1 antagonist, RP 67580, was more effective in inhibiting the behavioural response to formalin and the pain-induced activation of c-Fos than the antagonist for neurokinin-2 receptors, SR 48968, indicating that neurokinin-1 receptors are preferentially activated in neurokinincontaining pathways responding to noxious stimuli. Our results demonstrate that blockade of brain tachykinin receptors, especially of the neurokinin-1 receptor, reduces the behavioural response to pain and the pain-induced c-Fos activation in distinct brain areas which are intimately linked with nociceptive neurotransmission and the initiation and integration of central stress responses. Together with the previous findings of the inhibition of hypertensive and tachycardic responses to pain, the present data indicate that tachykinin receptor antagonists can effectively inhibit the generation of an integrated cardiovascular and behavioural response pattern to noxious stimuli. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: neurokinins, substance P, tachykinin receptor antagonists, pain, brain, rat.
Substance P (SP), together with neurokinin A (NKA) and neurokinin B (NKB), is one of the most abundant neurokinin peptides in the mammalian brain. These neuropeptides interact with three receptor types, referred to as neurokinin-1 (NK1), NK2 and NK3. SP displays the highest affinity for NK1 receptors, whereas NKA and NKB interact preferentially with NK2 and NK3 receptors, respectively. 36 Data demonstrating that stimulation of rat NK1 receptors in the forebrain induces an integrated pattern of cardiovascular, endocrine and behavioural responses, which are identical to responses
of rodents to noxious stimuli and stress, gave rise to the hypothesis that SP in the brain participates in central stress reactions. 9,41 Threatening stimuli, such as pain perception, represent a classical stress event, as evidenced by cardiovascular activation and activation of the pituitary–adrenocortical axis. We have reported recently that the response pattern to formalin injected subcutaneously (s.c.) through an implanted catheter comprises an activation of the sympathoadrenal system, increases in adrenocorticotropin (ACTH) and corticosterone levels in plasma, and a characteristic behavioural manifestation. 11 Therefore, experiencing pain activates not only neuronal networks processing and modulating the nociceptive transmission per se, but also neuronal circuits initiating and regulating the cardiovascular, endocrine and behavioural responses to stress. After selective, high-affinity non-peptide antagonists for NK1 and NK2 receptors had become available, 15,18 we showed that central pretreatment of rats with NK1 receptor antagonists
*To whom correspondence should be addressed. Tel.: 149-431-597-3519 or 3500; fax: 149-431-597-3522. E-mail address: [email protected] (J. Culman) Abbreviations: ACTH, adrenocorticotropin; CeA, central amygdaloid nucleus; DMN, dorsomedial hypothalamic nucleus; LC, locus coeruleus; MTh, medial thalamus; NK, neurokinin; PAG, periaqueductal gray; PBN, parabrachial nucleus; PC, prefrontal cortex; PVN, paraventricular hypothalamic nucleus; SHy, septohypothalamic nucleus; SP, substance P; VMN, ventromedial hypothalamic nucleus. 813
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attenuated the cardiovascular and behavioural responses to a noxious stimulus. 10 These studies have demonstrated that SP in the brain most probably acts as a neurotransmitter within central pathways generating cardiovascular, endocrine and behavioural responses upon noxious stimulation. Numerous attempts have been made to study the neuronal mechanisms in the brain activated by nociceptive stimulation. Monitoring of the expression of inducible transcription factors, such as c-Fos, has been shown to be a useful tool to identify areas in the brain and spinal cord which are activated by neurotransmitters, electrical stimulation or various physiological and pathophysiological stimuli, including stress and seizures. 13,30 For instance, noxious stimulation of cutaneous sensory neurons increased c-Fos expression in the superficial layer of the dorsal horn of the spinal cord. 24,43 A number of studies has attempted to identify specific brain areas which are activated following various noxious or stressful stimuli. 1,4,5,7,8,32,34,35 In the present study, the modified formalin test 11 was used to investigate the effects of intracerebroventricular (i.c.v.) pretreatment with the selective antagonists for NK1 and NK2 receptors, RP 67580 18 and SR 48968, 16 respectively, on c-Fos expression in the brain and the behavioural response to pain. The modified formalin test represents a simple method of an s.c. application of formalin through a chronically implanted catheter. The complex response pattern to formalin is therefore brought about exclusively by formalininduced tissue injury without any additional stressful events, such as animal handling, needle insertion and restraint. 11 The tachykinin receptor antagonists were administered i.c.v. prior to exposure of the rats to the modified formalin test. The effects of the active enantiomers of the NK1 and NK2 tachykinin receptor antagonists, RP 67580 and SR 48968, respectively, on c-Fos expression and behavioural response to formalin were compared with those of their inactive enantiomers, RP 68651 and SR 48965, as a control for the nonspecific activity. EXPERIMENTAL PROCEDURES
Animals Male Wistar rats (Charles River Viga GmbH, Sulzfeld, Germany) weighing 300–350 g were used. The rats were housed at controlled temperature and humidity under a 12-h/12-h light–dark cycle, and had free access to food and water. Surgical methods For i.c.v. injections, chronic polyethylene cannulae (PP 20; LHD Heidelberg, Germany) were implanted under chloral hydrate anaesthesia (400 mg/kg body weight, i.p.) into the left lateral brain ventricle seven to 10 days before the experiment. The rats were kept in individual cages and handled daily to minimize the induction of c-Fos in the brain due to handling on the day of the experiment. Five days after the implantation of the cannulae into the lateral ventricle, angiotensin II (25 pmol) was injected i.c.v. Only rats which responded with immediate drinking were included in experiments. The animals were anaesthetized again and a PP 50 catheter (LHD Heidelberg, Germany) was implanted s.c. in the lower hindlimb, as described in detail previously. 11 Experiments were carried out 48 h after implantation of the s.c. catheter. At this time-point, rats had completely recovered from surgical stress. The basal plasma levels of ACTH, adrenaline and noradrenaline were low, and identical to those obtained in rats in which the s.c. catheter was not implanted. 11 General procedures All experiments were carried out in conscious, freely moving rats in
their home cages in a separate room with a restricted sound level. Rats were transferred into the room 16–20 h before experiments, and water and food were provided ad libitum. On the day of the experiment, the grid cage tops were removed and the animals were allowed to adapt to the new enviroment (open cage at least for 2 h). The i.c.v. cannula was connected to an extension catheter (PP 20) with a Hamilton syringe. A PP 50 catheter connected to a syringe, both filled with 5% formaldehyde solution (w/w in physiological saline), was connected to the s.c. catheter. All experiments were carried out between 9.00 a.m. and 1.00 p.m. The experiments were commenced when the animals were quietly lying on the sawdust. Vehicle or the tachykinin receptor antagonist was injected i.c.v. in a volume of 1 ml and flushed with 4 ml of physiological saline or 4 ml of phosphate-buffered saline (pH 7.4), respectively (see “Materials” section). Ten minutes later, formalin (5%, 50 ml) was injected s.c. through the implanted catheter. Ninety minutes after the formalin injection, rats were deeply anaesthetized and intracardially perfused with phosphate-buffered saline followed by ice-cold 4% paraformaldehyde in phosphate buffer. Brains were removed, postfixed for 24 h in 4% paraformaldehyde and then kept for 48 h in 30% sucrose for cryoprotection. Behavioural responses were recorded in animals placed in test cages with grid tops removed according to the 15-s sampling procedure of Gispen et al. 19 In the present experiments, the frequency of hindlimb grooming and biting was assessed over a 15-min period at 15-s intervals starting immediately after the s.c. injection of formalin. Hindlimb grooming and biting was by far the most dominant behavioural manifestation observed during the early phase of the response to formalin, which did not exceed 15 min. During the late phase of the modified formalin test, rats only exhibited increased locomotion. 11 Experimental protocols In the first set of experiments, one group of rats (n 4) bearing the s.c. catheter and one group of rats (n 4) treated i.c.v. with vehicle were killed, together with uncannulated, control rats (n 4), to assess the effects of surgery and i.c.v. injections of the vehicle on c-Fos expression in the brain. The distribution and number of c-Fos-immunoreactive nuclei throughout the brain did not differ between the uncannulated and cannulated rats, and no appreciable effects of i.c.v. injection of vehicle on c-Fos expression were detected. Therefore, in the second set of experiments, the first group of rats (n 3) received i.c.v. vehicle (controls). The second group was injected i.c.v. with the active enantiomers of the tachykinin receptor antagonists, RP 67580 (500 pmol) together with SR 48968 (500 pmol) (n 7). The third group of rats (n 7) received i.c.v. the same doses of their inactive enantiomers, RP 68651 and SR 48965, respectively, as a control for non-specific activity. Ten minutes after the i.c.v. injections, formalin (5%, 50 ml) was administered s.c. to all groups of rats. Ninety minutes later, all groups of rats were perfused together with two uncannulated rats serving as control for basal c-Fos expression. In the third set of experiments, rats were treated i.c.v. with either RP 67580 (500 pmol) alone or SR 48968 (500 pmol), 10 min prior to s.c. injection of formalin. Three separate experiments were carried out within this set of experiments; 12–15 rats were included in each. Apart from rats treated i.c.v. with RP 67580 (500 pmol, n 3–4) or SR 48698 (500 pmol, n 3–4) prior to s.c. formalin injection, each experiment also comprised uncannulated rats (absolute controls), vehicle-treated (i.c.v.) but not formalin-injected rats, vehicle-treated (i.c.v.), formalin-injected rats and rats treated i.c.v. with both inactive enantiomers of the tachykinin receptor antagonists prior to s.c. formalin injection. These additional groups of rats (two to three animals in each group in each experiment) served as controls for basal c-Fos expression in the brain and formalin-induced c-Fos expression in the brain following prior treatment of rats with vehicle or the inactive enantiomers. Effects of the tachykinin receptor antagonists on the behavioural response to s.c. formalin were investigated in the fourth set of experiments. Six groups of rats were used. Control rats (n 7) received vehicle i.c.v. and, 10 min later, without s.c. formalin injection, the behavioural response was recorded over a period of 15 min. The remaining five groups received the following i.c.v. treatment: (i) vehicle (n 8); (ii) RP 67580 (500 pmol, n 8); (iii) SR 48968 (500 pmol, n 9); (iv) RP 67580 (500 pmol)/SR 48968 (500 pmol) (n 8); (v) RP 68651 (500 pmol)/SR 48965 (500 pmol) (n 8). Ten minutes after the i.c.v. injections, formalin (5%, 50 ml) was administered
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Brain neurokinins and pain-induced c-Fos expression Table 1. Number of c-Fos-positive stained neurons in control groups of rats
Prefrontal cortex Septohypothalamic nucleus Paraventricular nucleus Medial thalamus Dorsomedial nucleus Ventromedial nucleus Parabrachial nucleus Locus coeruleus Central amygdaloid nucleus Periaqueductal gray
Basal expression
Subcutaneous catheter
Vehicle i.c.v.
RP 67580/SR 48968 i.c.v.
58 ^ 18 39 ^ 11 28 ^ 9 45 ^ 18 32 ^ 18 11 ^ 2 20 ^ 9 10 ^ 4 7^1 51 ^ 7
43 ^ 13 38 ^ 13 26 ^ 8 25 ^ 13 46 ^ 19 18 ^ 7 22 ^ 7 10 ^ 4 5^1 42 ^ 8
87 ^ 7 59 ^ 15 58 ^ 12 70 ^ 14 98 ^ 7* 18 ^ 3 15 ^ 9 12 ^ 3 17 ^ 2** 52 ^ 9
65 ^ 14 35 ^ 11 31 ^ 5 54 ^ 7 68 ^ 12 24 ^ 4* 27 ^ 6 18 ^ 2 17 ^ 4** 49 ^ 11
Values represent the numbers of c-Fos-immunoreactive neurons per section of 50 mm on both sides, expressed as mean ^ S.E.M., in uncannulated controls (basal expression), in rats bearing the subcutaneous catheter, in rats injected i.c.v. with vehicle, and with the selective NK1 and NK2 tachykinin receptor antagonists, RP 67580 and SR 48968, respectively (500 pmol each). *P , 0.05, **P , 0.01 statistical comparison to the basal expression, calculated with one-way ANOVA followed by post hoc Duncan’s test.
Table 2. Effects of central inhibition of neurokinin-1 and neurokinin-2 tachykinin receptors on the formalin-induced c-Fos expression in the rat brain Treatment (i.c.v.): Treatment (s.c.):
Vehicle —
Vehicle Formalin
RP 67580 Formalin
SR 48968 Formalin
RP 67580/SR 48968 Formalin
RP 68651/SR 48965 Formalin
Prefrontal cortex Paraventricular nucleus Dorsomedial nucleus Ventromedial nucleus Locus coeruleus Periaqueductal gray
87 ^ 7 58 ^ 12 98 ^ 7 18 ^ 3 12 ^ 3 52 ^ 9
191 ^ 23** 221 ^ 44* 270 ^ 41** 57 ^ 9* 52 ^ 9*** 138 ^ 27**
104 ^ 11††† 100 ^ 35† 103 ^ 13††† 21 ^ 4† 18 ^ 2††† 41 ^ 7†††
152 ^ 17†† 133 ^ 33 144 ^ 14††† 24 ^ 4† 28 ^ 6†† 81 ^ 14††
106 ^ 19†† 59 ^ 18† 106 ^ 24††† 23 ^ 4† 29 ^ 3† 63 ^ 15††
172 ^ 11** 214 ^ 44** 251 ^ 42** 55 ^ 17* 43 ^ 6** 129 ^ 25**
Values represent the number of c-Fos-immunoreactive neurons per section of 50 mm on both sides and are expressed as mean ^ S.E.M. Vehicle, the NK1 and NK2 tachykinin receptor antagonists, RP 67580 and SR 48968, respectively, or their inactive enantiomers, RP 68651 and SR 48965, were injected i.c.v. at a dose of 500 pmol, 10 min prior to s.c. injection of formalin (5%, 50 ml). *P , 0.05, **P , 0.01, ***P , 0.001, statistical comparison to the vehicle-treated but not formalin-injected group; †P , 0.05, ††P , 0.01, †††P , 0.001, statistical comparison to the vehicle-treated, formalin-injected group, calculated with one-way ANOVA followed by post hoc Duncan’s test.
s.c. to all five groups of rats and behavioural responses were recorded over a 15-min period (early phase). The experimental protocols had been approved by the Governmental Committee for Ethical Use of Animals in the German Federal State of Schleswig-Holstein. Immunohistochemistry and quantitative analysis Cryostat-cut coronal sections (50 mm) were processed for immunohistochemistry using the conventional avidin–biotin complex peroxidase reaction. Generation and specificity of the antibody have been described in detail elsewhere. 22 To quantify c-Fos immunoreactivity, sections at the same level of each brain region were taken. The level of each region was identified according to an atlas of the rat brain. 33 For quantification, the number of stained neurons of each brain area was counted by two observers in a blind manner on slices of two sections on both sides. No attempt was made to quantify the intensity of the staining. The mean number of labelled neurons on both sides per section was calculated for each brain area and group. Materials The solutions of the NK1 receptor antagonist, RP 67580, and its inactive enantiomer, RP 68651, were prepared as described elsewhere. 10 One microlitre of the solution containing either RP 67580 or RP68651 was injected i.c.v. together with 4 ml of phosphatebuffered physiological saline (pH 7.4). The final pH of the injected solution was 7.2–7.4. The NK2 receptor antagonist, SR 48968, and its inactive enantiomer, SR 48965, were dissolved in a small volume of dimethylsulphoxide (Merck, Germany), and physiological saline was added to obtain the final volume in the stock solution (5000 pmol/ml). On the day of experiment, the stock solution of the antagonist or its inactive enantiomer was further diluted with physiological saline to obtain the desired concentration of the compounds. The final solutions of SR 48968 and SR 48965 contained less than 5% dimethylsulphoxide and were injected i.c.v. in a volume of 1 ml together with 4 ml of
physiological saline. Vehicle-treated controls were injected i.c.v. with 1 ml of the vehicle for i.c.v. injections of RP 67580 containing 5% dimethylsulphoxide together with 4 ml of phosphate-buffered saline. Statistics The results are expressed as the mean ^ S.E.M. Data were analysed by one-way ANOVA. Post hoc comparisons between groups were made using Duncan’s test. A significance level of P , 0.05 was accepted. RESULTS
The basal c-Fos expression in the examined brain nuclei corresponded to that described in other reports. The chronically implantated s.c. catheter did not affect the number and distribution of c-Fos protein in these brain nuclei. Intracerebroventricular injection of vehicle moderately increased the number of positive nuclei in the dorsomedial nucleus of the hypothalamus (DMN) and slightly in the central amygdaloid nucleus (CeA). In the latter area and in the ventromedial nucleus of the hypothalamus (VMN), an increase in c-Fos expression was recorded post-i.c.v. treatment with the active enantiomers of the tachykinin antagonists (Table 1). Following s.c. injection of formalin, c-Fos expression increased in 10 brain areas (Tables 2, 3). In the septohypothalamic nucleus (SHy), medial thalamus (MTh), parabrachial nucleus (PBN) and CeA, formalin increased the number of c-Fos-positive neurons (SHy: F 6.799, P , 0.01; MTh: F 4.015, P , 0.01; PBN: F 5.761, P , 0.01; CeA:
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Table 3. Rat brain areas responding to subcutaneous formalin with increased c-Fos expression, which was not altered by inhibition of central neurokinin-1 and neurokinin-2 tachykinin receptors Pretreatment (i.c.v.): Treatment (s.c.):
Vehicle —
Vehicle Formalin
RP 67580/SR 48968 Formalin
RP 68651/SR 48965 Formalin
Septohypothalamic nucleus Medial thalamus Parabrachial nucleus Amygdaloid nucleus
59 ^ 15 70 ^ 14 15 ^ 9 17 ^ 2
177 ^ 17*** 153 ^ 25** 98 ^ 16** 31 ^ 4**
157 ^ 20** 134 ^ 9* 76 ^ 14* 26 ^ 3
169 ^ 19** 127 ^ 13* 98 ^ 12** 28 ^ 3*
Values represent the numbers of c-Fos-immunoreactive neurons per section of 50 mm on both sides, expressed as mean ^ S.E.M. *P , 0.05, **P , 0.01, ***P , 0.001, statistical comparison to the vehicle-pretreated but not formalin-injected group, calculated with one-way ANOVA followed by post hoc Duncan’s test. For further details, see footnote to Table 2.
F 3.34, P , 0.05), but neither the active enantiomers of the NK1 and NK2 tachykinin receptor antagonists, RP 67580 and SR 48968, respectively, nor their inactive enantiomers, RP 68651 and SR 48965, administered i.c.v. prior to s.c. injection of formalin, had any effects on the increased c-Fos expression (Table 3). Subcutaneous formalin also enhanced the number of c-Fospositive nuclei in the prefrontal cortex (PC; F 5.806, P , 0.001), the paraventricular nucleus (PVN; F 3.2840, P , 0.01), DMN (F 7.006, P , 0.001) and VMN (F 3.200, P , 0.05) of the hypothalamus, in the locus coeruleus (LC; F 6.120, P , 0.001) and periaqueductal gray (PAG; F 6.184, P , 0.001; Table 2). In the PVN, the number of c-Fos-positive nuclei was increased to a similar extent in both the parvocellular and magnocellular parts of the nucleus (Fig. 1). Pretreatment of rats with 500 pmol RP 67580 resulted in a significant inhibition of the increased c-Fos expression induced by formalin in all nuclei. Simultaneous pretreatment with both tachykinin receptor antagonists did not produce any additional inhibitory effects on the postformalin c-Fos induction. In the PC, DMN, VMN, LC and PAG, i.c.v. injection of the NK2 receptor antagonist, SR 48968 (500 pmol), also attenuated the formalin-induced c-Fos expression. Pretreatment of rats with the inactive enantiomers of the tachykinin receptor antagonists did not modify the number of c-Fos-positive nuclei following s.c. injection of formalin in any of these brain nuclei (Table 2). There were significant differences in the behavioural response among the experimental groups (F 47.61, P , 0.001; Table 4). In vehicle-pretreated rats, formalin injected s.c. evoked intense licking and biting at the site of application. The NK2 receptor antagonist slightly, but significantly, reduced the formalin-induced behaviour. The NK1 receptor antagonist inhibited post-formalin grooming behaviour to a greater extent than the NK2 antagonist. Pretreatment of rats with both tachykinin receptor antagonists had the same inhibitory effect as the NK1 receptor antagonist alone (Table 4).
DISCUSSION
Effect of formalin on c-Fos expression in the brain Formalin injected s.c. through the chronically implanted catheter activates primary sensory neurons and produces local pain. The pain experience includes a sensory aspect linked to the perception of potential tissue injury and an emotional, affective aspect related to the aversive component of the pain experience. Correspondingly, formalin-induced pain caused c-Fos activation in a number of forebrain and
brainstem nuclei linked to the relay, processing or modulation of nociceptive signals, such as the MTh and the PAG. 17,25 The activation of c-Fos in the CeA and in the SHy, which represents the most ventral part of the lateral septal area, may be related to control of behavioural reactions and emotional states upon noxious stimulation, although the function of the latter region in the modulation of behavioural responses to pain has not yet been clarified. Similarly to our findings, electric footshock, a pain stimulus, or swimming or restraint stress strongly increased c-Fos immunoreactivity in the ventral subdivision of the lateral septal area. 7,14,34 In addition to pain generated by formalin itself, the formalin test for nociception represents a stress event comprising the cardiovascular response brought about by the sympathoadrenal activation and endocrine responses characterized by increases in plasma levels of ACTH and corticosterone. 11 Experience of pain also caused c-Fos activation in brain regions related to the initiation and regulation of neuroendocrine and autonomic and behavioural stress reactions, such as in the PC and the PVN, which represent the major sites for the integration of these reactions. 39,40 The induction of c-Fos in the LC in response to formalin is consistent with the role of this nucleus in the processing of stress responses. 38 The activation of PBN neurons is most probably associated with the regulation of the autonomic responses to nociceptive stimuli. 28 Effect of tachykinin receptor antagonists on c-Fos expression induced by formalin We have shown recently that inhibition of forebrain NK1 receptors attenuated the cardiovascular and behavioural responses to formalin injected s.c. 10 In the present study, selective, non-peptide antagonists for NK1 and NK2 receptors were employed to determine in which brain areas the inhibition of tachykinin receptors would suppress the pain-induced c-Fos expression, an indicator of neuronal activity. 13 Intracerebroventricular treatment of rats with 500 pmol RP 67580, a dose which has been shown previously to effectively attenuate the mean arterial pressure and heart rate responses to s.c. formalin, 10 reduced formalin-induced c-Fos expression in the majority of brain nuclei activated by such a stimulus. SP serves as a mediator of nociception in the spinal cord. 31 The NK1 receptor antagonist injected i.c.v. may therefore theoretically antagonize the pain transmission at the spinal cord level, which would result in a suppression of c-Fos expression in the brain. However, the inhibition of NK1 receptors at the spinal cord level would result in attenuation of c-Fos expression in all brain nuclei which responded to s.c. formalin due to a general impairment of pain transmission by the
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Fig. 1. Frontal sections through the PVN stained for c-Fos protein of rats pretreated i.c.v. with: vehicle, no s.c. formalin injection followed (upper panel, left); vehicle, 10 min prior to s.c. formalin injection (upper panel, right); the active enantiomers of the NK1 and NK2 tachykinin receptor antagonists (500 pmol each), 10 min prior to s.c. formalin injection (lower panel, left) and the inactive enantiomers of the tachykinin receptor antagonists (500 pmol, each), 10 min prior to s.c. formalin injection (lower panel, right).
primary afferent neurons which synapse on neurons in the dorsal horn. Surprisingly, i.c.v. pretreatment with the selective NK2 receptor antagonist, SR 48968, attenuated both the c-Fos activation in some brain nuclei and the grooming behaviour in response to formalin. SR 48968 inhibits selectively and with high affinity NK2 receptors, for which NKA is the preferred natural ligand. 36 Although NKA is expressed abundantly by
neurons in the brain, and the peptide injected centrally elicits a number of distinct actions, the NK2 receptor is either not expressed in the adult rat brain or its abundance is extremely low. 31 However, recent findings indicate that NKA can also bind with relatively high affinity to the NK1 (SP) receptor and act through this receptor with high potency. Therefore, some of the actions of NKA in the brain may be mediated by the NK1 receptor. 27 However, there are no data available so far
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Table 4. Effects of central inhibition of neurokinin-1 and neurokinin-2 tachykinin receptors on hindlimb grooming (licking and biting of the lower leg) in response to subcutaneously injected formalin Treatment (i.c.v.)
Vehicle Vehicle RP 67580 SR 48968 RP 67580/SR 48968 RP 68651/SR 48965
Dose (pmol)
Formalin s.c.
n
Hindlimb grooming
— — 500 500 500 500
— 1 1 1 1 1
7 8 8 9 8 8
1.1 ^ 0.7 12.6 ^ 0.7*** 5.3 ^ 0.5*††† 9.1 ^ 1.0***† 3.6 ^ 0.5††† 14.8 ^ 0.9***
Values represent the frequency of hindlimb grooming for 15 min and are expressed as mean ^ S.E.M. Vehicle, the NK1 and NK2 tachykinin receptor antagonists, RP 67580 and SR 48968, respectively, or their inactive enantiomers, RP 68651 and SR 48965, were injected i.c.v. at a dose of 500 pmol, 10 min prior to s.c. injection of formalin (5%, 50 ml). *P , 0.05, ***P , 0.001, statistical comparison to the vehicle-treated but not formalin-injected group; †P , 0.05, †††P , 0.001, statistical comparison to the vehicle-treated, formalin-injected group, calculated with one-way ANOVA followed by post hoc Duncan’s test.
which demonstrate that the selective NK2 receptor antagonist, SR48968, can, similarly to NKA, also interact with NK1 receptors. Therefore, we assume that the attenuation of the pain-induced c-Fos expression and behaviour by SR 489689 results from inhibition of NK2 rather than NK1 receptors. SR 48968, besides exerting non-specific effects on neurotransmission, which are not related to the interaction with NK2 receptors, 26,42 exhibits a relatively high agonistic activity at the m-opioid receptor. 16 Although a low dose of SR 48968 was used in the present study, it cannot be ruled out that the interaction of SR 48968 with central opioid receptors contributed at least partly to the observed effects. All brain nuclei, besides the PC, in which tachykinin receptor antagonists reduced formalin-induced c-Fos activation, belong to regions adjacent to the ventricular system. The PVN, a key area for the integration of cardiovascular and neuroendocrine responses to stress, 39,40 contains a moderate density of SP-binding sites. 3 The PVN is innervated by SPimmunoreactive fibres originating in the A1 and C1 catecholaminergic areas, and by SP-immunoreactive neurons localized in the DMN and VMN. 2 The NK1 receptor antagonist effectively attenuated the formalin-induced c-Fos activation in the latter areas, indicating that fewer neurons were activated by noxious stimulation following the antagonist treatment. NK1 receptors are very dense in the LC and PAG. 12 Since the LC lies in close vicinity to the ventricular system and neurons of the PAG surround the cerebral aqueduct, NK1 receptors in these areas can be targeted efficiently by the tachykinin receptor antagonists. The LC belongs to the principal co-ordinators of central stress reactions. 38 The reduction in the pain-induced c-Fos activation, together with the finding of a concentration-dependent excitation of LC neurons by SP, 6 point to the involvement of the peptide in this area in the co-ordination of responses induced by noxious stimuli. The PAG appears to play a key role in the generation and control of cardiovascular responses upon stress. 21,23 Short restraint or s.c. saline injection raised the SP concentrations in this area. 37 Neuronal networks in this area are also involved in the modulation of nociceptive transmission. 17 The present data show that, following tachykinin receptor inhibition, fewer neurons in this area responded to noxious stimulation. However, owing to the diverse functions of the PAG, the
observed reduction in c-Fos induction in this area cannot be interpreted unambiguously. As the PC does not lie in the close vicinity of the ventricular system, we assume that a direct inhibition of tachykinin receptors in the PC was probably not responsible for the attenuated c-Fos activation in this area to formalin. It appears likely that blockade of tachykinin receptors in other brain region(s) was responsible for the inhibition of the formalininduced c-Fos expression in the PC. In several brain regions, treatment with the tachykinin receptor antagonists failed to affect the formalin-induced cFos expression. The most probable explanation is that the cFos activation in these areas after formalin is dependent on factors other than neurokinins. However, several other possibilities may also be considered, for instance that tachykinin receptors in these areas are not activated by noxious stimuli or that the concentrations of the tachykinin receptor antagonists in these brain areas were not high enough to effectively inhibit these receptors. Moreover, neuronal input from other brain regions, which project to the aforementioned areas but do not respond to somatic pain by expressing c-Fos, could provide a sufficiently strong signal to induce c-Fos expression, despite effective blockade of tachykinin receptors. This might be the case for the PBN, which represents an important site for the regulation of stress-related cardiovascular and respiratory responses. 28 The PBN contains a very high density of SPbinding sites, 12 which could probably be effectively targeted after treatment with the tachykinin receptor antagonists because of its localization in the vicinity of the ventricular system. This nucleus is interconnected, among others, with hypothalamic nuclei, the amygdala, nucleus tractus solitarii and area postrema. The two latter areas provide a major projection to the PBN. 20,29 The neuronal input from areas which did not respond by a reduction of neuronal activity to pain after treatment with the tachykinin receptor antagonists might provide a sufficient signal to induce c-Fos expression in the PBN, even though its tachykinin receptors were inhibited. The NK1 receptor antagonist substantially inhibited the grooming behaviour to s.c. formalin, and simultaneous treatment of rats with both tachykinin receptor antagonists did not produce an additional inhibition of the formalin-induced behaviour. In contrast to our previous findings, 10 the NK2 receptor antagonists also reduced the grooming behaviour in response to formalin. In addition to blockade of tachykinin receptors, both tachykinin receptor antagonists have been shown to interact with calcium channels and to exert nonspecific inhibitory effects on neurotransmission. 26,42 As already mentioned, SR 48968 has been reported to possess a relatively high agonistic affinity at m-opioid receptors. 16 Based on the presented results, the question as to whether these non-specific effects accounted for the reduction in the hindlimb grooming behaviour because the animals experienced less pain cannot be answered unambiguously. However, it can be argued that these non-specific effects would generally lessen the evoked activity in the neuronal pathways activated by formalin-induced pain, which would result in a widespread reduction of c-Fos expression. The finding that treatment with tachykinin receptor antagonists failed to affect the exaggerated c-Fos expression in some brain regions does not support an overall lessening of evoked activity in neuronal pathways activated by formalin-induced pain.
Brain neurokinins and pain-induced c-Fos expression CONCLUSIONS
The present data demonstrate that somatic pain also activates a number of brain areas traditionally related to the regulation of responses induced by stressful events. Blockade of
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tachykinin receptors, especially of the NK1 receptor, reduced the pain-induced neuronal activation in brain areas which are intimately linked with nociceptive neurotransmission or the initiation and integration of stress responses.
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Brain neurokinins and pain-induced c-Fos expression
Pergamon PII: S0306-4522(99)00478-9 www.elsevier.com/locate/neuroscience
TACHYKININ RECEPTOR INHIBITION AND c-FOS EXPRESSION IN THE RAT BRAIN FOLLOWING FORMALIN-INDUCED PAIN J. BAULMANN, H. SPITZNAGEL, T. HERDEGEN, T. UNGER and J. CULMAN* Institute of Pharmacology, Christian-Albrechts-University of Kiel, 24105 Kiel, Germany and German Institute for High Blood Pressure Research, 69120 Heidelberg, Germany
Abstract—Recent pharmacological evidence has implicated substance P and neurokinin A, natural ligands for neurokinin-1 and neurokinin-2 receptors, respectively, as neurotransmitters in brain neuronal circuits activated upon noxious stimulation. The expression of the inducible transcription factor, c-Fos, was used to identify areas in the brain activated by a noxious stimulus (the subcutaneous injection of formalin), and to investigate the effects of intracerebroventricular administration of selective, nonpeptide antagonists for neurokinin-1 and neurokinin-2 tachykinin receptors on the neural activity in these areas and on the behavioural response to formalin-induced pain. Formalin (5%, 50 ml), injected subcutaneously through a chronically implanted catheter in the region of the lower hindlimb, increased c-Fos expression in a number of brain areas related to nociceptive transmission or the integration of stress responses. Grooming behaviour, licking and biting directed to the injected site, was the most frequent behavioural response. Intracerebroventricular pretreatment of rats with either RP 67580 (500 pmol), the active enantiomer of a neurokinin-1 receptor antagonist, or with SR 48968 (500 pmol), the active enantiomer of a neurokinin-2 receptor antagonist, reduced the formalin-induced c-Fos staining in the prefrontal cortex, dorsomedial and ventromedial nuclei of the hypothalamus, the locus coeruleus and the periaqueductal gray. The neurokinin-1, but not the neurokinin-2, receptor antagonist attenuated the formalin-induced activation of c-Fos in the paraventricular nucleus of the hypothalamus. Simultaneous intracerebroventricular pretreatment with both neurokinin-1 and neurokinin-2 receptor antagonists did not produce any additional inhibitory effect on the post-formalin c-Fos expression. None of the tachykinin receptor antagonists had an effect on the formalininduced c-Fos expression in the septohypothalamic nucleus, medial thalamus, parabrachial nucleus and central amygdaloid nucleus, indicating that neurotransmitters other than neurokinins are most probably responsible for the activation of these areas in respose to noxious stimulation. While both tachykinin receptor antagonists reduced the grooming behaviour to formalin, the neurokinin-1 receptor antagonist was clearly more effective than the neurokinin-2 receptor antagonist. Intracerebroventricular pretreatment of rats with the inactive enantiomers of the tachykinin receptor antagonists, RP 68651 and SR 48965, was without effect. Our results show that (i) the modified formalin test elicited an intense grooming behaviour and expression of c-Fos in numerous forebrain and brainstem areas, (ii) both tachykinin receptor antagonists were able to attenuate the behavioural response to pain and to reduce the formalin-induced c-Fos expression in some, but not all, brain areas, and (iii) the neurokinin-1 antagonist, RP 67580, was more effective in inhibiting the behavioural response to formalin and the pain-induced activation of c-Fos than the antagonist for neurokinin-2 receptors, SR 48968, indicating that neurokinin-1 receptors are preferentially activated in neurokinincontaining pathways responding to noxious stimuli. Our results demonstrate that blockade of brain tachykinin receptors, especially of the neurokinin-1 receptor, reduces the behavioural response to pain and the pain-induced c-Fos activation in distinct brain areas which are intimately linked with nociceptive neurotransmission and the initiation and integration of central stress responses. Together with the previous findings of the inhibition of hypertensive and tachycardic responses to pain, the present data indicate that tachykinin receptor antagonists can effectively inhibit the generation of an integrated cardiovascular and behavioural response pattern to noxious stimuli. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: neurokinins, substance P, tachykinin receptor antagonists, pain, brain, rat.
Substance P (SP), together with neurokinin A (NKA) and neurokinin B (NKB), is one of the most abundant neurokinin peptides in the mammalian brain. These neuropeptides interact with three receptor types, referred to as neurokinin-1 (NK1), NK2 and NK3. SP displays the highest affinity for NK1 receptors, whereas NKA and NKB interact preferentially with NK2 and NK3 receptors, respectively. 36 Data demonstrating that stimulation of rat NK1 receptors in the forebrain induces an integrated pattern of cardiovascular, endocrine and behavioural responses, which are identical to responses
of rodents to noxious stimuli and stress, gave rise to the hypothesis that SP in the brain participates in central stress reactions. 9,41 Threatening stimuli, such as pain perception, represent a classical stress event, as evidenced by cardiovascular activation and activation of the pituitary–adrenocortical axis. We have reported recently that the response pattern to formalin injected subcutaneously (s.c.) through an implanted catheter comprises an activation of the sympathoadrenal system, increases in adrenocorticotropin (ACTH) and corticosterone levels in plasma, and a characteristic behavioural manifestation. 11 Therefore, experiencing pain activates not only neuronal networks processing and modulating the nociceptive transmission per se, but also neuronal circuits initiating and regulating the cardiovascular, endocrine and behavioural responses to stress. After selective, high-affinity non-peptide antagonists for NK1 and NK2 receptors had become available, 15,18 we showed that central pretreatment of rats with NK1 receptor antagonists
*To whom correspondence should be addressed. Tel.: 149-431-597-3519 or 3500; fax: 149-431-597-3522. E-mail address: [email protected] (J. Culman) Abbreviations: ACTH, adrenocorticotropin; CeA, central amygdaloid nucleus; DMN, dorsomedial hypothalamic nucleus; LC, locus coeruleus; MTh, medial thalamus; NK, neurokinin; PAG, periaqueductal gray; PBN, parabrachial nucleus; PC, prefrontal cortex; PVN, paraventricular hypothalamic nucleus; SHy, septohypothalamic nucleus; SP, substance P; VMN, ventromedial hypothalamic nucleus. 813
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attenuated the cardiovascular and behavioural responses to a noxious stimulus. 10 These studies have demonstrated that SP in the brain most probably acts as a neurotransmitter within central pathways generating cardiovascular, endocrine and behavioural responses upon noxious stimulation. Numerous attempts have been made to study the neuronal mechanisms in the brain activated by nociceptive stimulation. Monitoring of the expression of inducible transcription factors, such as c-Fos, has been shown to be a useful tool to identify areas in the brain and spinal cord which are activated by neurotransmitters, electrical stimulation or various physiological and pathophysiological stimuli, including stress and seizures. 13,30 For instance, noxious stimulation of cutaneous sensory neurons increased c-Fos expression in the superficial layer of the dorsal horn of the spinal cord. 24,43 A number of studies has attempted to identify specific brain areas which are activated following various noxious or stressful stimuli. 1,4,5,7,8,32,34,35 In the present study, the modified formalin test 11 was used to investigate the effects of intracerebroventricular (i.c.v.) pretreatment with the selective antagonists for NK1 and NK2 receptors, RP 67580 18 and SR 48968, 16 respectively, on c-Fos expression in the brain and the behavioural response to pain. The modified formalin test represents a simple method of an s.c. application of formalin through a chronically implanted catheter. The complex response pattern to formalin is therefore brought about exclusively by formalininduced tissue injury without any additional stressful events, such as animal handling, needle insertion and restraint. 11 The tachykinin receptor antagonists were administered i.c.v. prior to exposure of the rats to the modified formalin test. The effects of the active enantiomers of the NK1 and NK2 tachykinin receptor antagonists, RP 67580 and SR 48968, respectively, on c-Fos expression and behavioural response to formalin were compared with those of their inactive enantiomers, RP 68651 and SR 48965, as a control for the nonspecific activity. EXPERIMENTAL PROCEDURES
Animals Male Wistar rats (Charles River Viga GmbH, Sulzfeld, Germany) weighing 300–350 g were used. The rats were housed at controlled temperature and humidity under a 12-h/12-h light–dark cycle, and had free access to food and water. Surgical methods For i.c.v. injections, chronic polyethylene cannulae (PP 20; LHD Heidelberg, Germany) were implanted under chloral hydrate anaesthesia (400 mg/kg body weight, i.p.) into the left lateral brain ventricle seven to 10 days before the experiment. The rats were kept in individual cages and handled daily to minimize the induction of c-Fos in the brain due to handling on the day of the experiment. Five days after the implantation of the cannulae into the lateral ventricle, angiotensin II (25 pmol) was injected i.c.v. Only rats which responded with immediate drinking were included in experiments. The animals were anaesthetized again and a PP 50 catheter (LHD Heidelberg, Germany) was implanted s.c. in the lower hindlimb, as described in detail previously. 11 Experiments were carried out 48 h after implantation of the s.c. catheter. At this time-point, rats had completely recovered from surgical stress. The basal plasma levels of ACTH, adrenaline and noradrenaline were low, and identical to those obtained in rats in which the s.c. catheter was not implanted. 11 General procedures All experiments were carried out in conscious, freely moving rats in
their home cages in a separate room with a restricted sound level. Rats were transferred into the room 16–20 h before experiments, and water and food were provided ad libitum. On the day of the experiment, the grid cage tops were removed and the animals were allowed to adapt to the new enviroment (open cage at least for 2 h). The i.c.v. cannula was connected to an extension catheter (PP 20) with a Hamilton syringe. A PP 50 catheter connected to a syringe, both filled with 5% formaldehyde solution (w/w in physiological saline), was connected to the s.c. catheter. All experiments were carried out between 9.00 a.m. and 1.00 p.m. The experiments were commenced when the animals were quietly lying on the sawdust. Vehicle or the tachykinin receptor antagonist was injected i.c.v. in a volume of 1 ml and flushed with 4 ml of physiological saline or 4 ml of phosphate-buffered saline (pH 7.4), respectively (see “Materials” section). Ten minutes later, formalin (5%, 50 ml) was injected s.c. through the implanted catheter. Ninety minutes after the formalin injection, rats were deeply anaesthetized and intracardially perfused with phosphate-buffered saline followed by ice-cold 4% paraformaldehyde in phosphate buffer. Brains were removed, postfixed for 24 h in 4% paraformaldehyde and then kept for 48 h in 30% sucrose for cryoprotection. Behavioural responses were recorded in animals placed in test cages with grid tops removed according to the 15-s sampling procedure of Gispen et al. 19 In the present experiments, the frequency of hindlimb grooming and biting was assessed over a 15-min period at 15-s intervals starting immediately after the s.c. injection of formalin. Hindlimb grooming and biting was by far the most dominant behavioural manifestation observed during the early phase of the response to formalin, which did not exceed 15 min. During the late phase of the modified formalin test, rats only exhibited increased locomotion. 11 Experimental protocols In the first set of experiments, one group of rats (n 4) bearing the s.c. catheter and one group of rats (n 4) treated i.c.v. with vehicle were killed, together with uncannulated, control rats (n 4), to assess the effects of surgery and i.c.v. injections of the vehicle on c-Fos expression in the brain. The distribution and number of c-Fos-immunoreactive nuclei throughout the brain did not differ between the uncannulated and cannulated rats, and no appreciable effects of i.c.v. injection of vehicle on c-Fos expression were detected. Therefore, in the second set of experiments, the first group of rats (n 3) received i.c.v. vehicle (controls). The second group was injected i.c.v. with the active enantiomers of the tachykinin receptor antagonists, RP 67580 (500 pmol) together with SR 48968 (500 pmol) (n 7). The third group of rats (n 7) received i.c.v. the same doses of their inactive enantiomers, RP 68651 and SR 48965, respectively, as a control for non-specific activity. Ten minutes after the i.c.v. injections, formalin (5%, 50 ml) was administered s.c. to all groups of rats. Ninety minutes later, all groups of rats were perfused together with two uncannulated rats serving as control for basal c-Fos expression. In the third set of experiments, rats were treated i.c.v. with either RP 67580 (500 pmol) alone or SR 48968 (500 pmol), 10 min prior to s.c. injection of formalin. Three separate experiments were carried out within this set of experiments; 12–15 rats were included in each. Apart from rats treated i.c.v. with RP 67580 (500 pmol, n 3–4) or SR 48698 (500 pmol, n 3–4) prior to s.c. formalin injection, each experiment also comprised uncannulated rats (absolute controls), vehicle-treated (i.c.v.) but not formalin-injected rats, vehicle-treated (i.c.v.), formalin-injected rats and rats treated i.c.v. with both inactive enantiomers of the tachykinin receptor antagonists prior to s.c. formalin injection. These additional groups of rats (two to three animals in each group in each experiment) served as controls for basal c-Fos expression in the brain and formalin-induced c-Fos expression in the brain following prior treatment of rats with vehicle or the inactive enantiomers. Effects of the tachykinin receptor antagonists on the behavioural response to s.c. formalin were investigated in the fourth set of experiments. Six groups of rats were used. Control rats (n 7) received vehicle i.c.v. and, 10 min later, without s.c. formalin injection, the behavioural response was recorded over a period of 15 min. The remaining five groups received the following i.c.v. treatment: (i) vehicle (n 8); (ii) RP 67580 (500 pmol, n 8); (iii) SR 48968 (500 pmol, n 9); (iv) RP 67580 (500 pmol)/SR 48968 (500 pmol) (n 8); (v) RP 68651 (500 pmol)/SR 48965 (500 pmol) (n 8). Ten minutes after the i.c.v. injections, formalin (5%, 50 ml) was administered
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Brain neurokinins and pain-induced c-Fos expression Table 1. Number of c-Fos-positive stained neurons in control groups of rats
Prefrontal cortex Septohypothalamic nucleus Paraventricular nucleus Medial thalamus Dorsomedial nucleus Ventromedial nucleus Parabrachial nucleus Locus coeruleus Central amygdaloid nucleus Periaqueductal gray
Basal expression
Subcutaneous catheter
Vehicle i.c.v.
RP 67580/SR 48968 i.c.v.
58 ^ 18 39 ^ 11 28 ^ 9 45 ^ 18 32 ^ 18 11 ^ 2 20 ^ 9 10 ^ 4 7^1 51 ^ 7
43 ^ 13 38 ^ 13 26 ^ 8 25 ^ 13 46 ^ 19 18 ^ 7 22 ^ 7 10 ^ 4 5^1 42 ^ 8
87 ^ 7 59 ^ 15 58 ^ 12 70 ^ 14 98 ^ 7* 18 ^ 3 15 ^ 9 12 ^ 3 17 ^ 2** 52 ^ 9
65 ^ 14 35 ^ 11 31 ^ 5 54 ^ 7 68 ^ 12 24 ^ 4* 27 ^ 6 18 ^ 2 17 ^ 4** 49 ^ 11
Values represent the numbers of c-Fos-immunoreactive neurons per section of 50 mm on both sides, expressed as mean ^ S.E.M., in uncannulated controls (basal expression), in rats bearing the subcutaneous catheter, in rats injected i.c.v. with vehicle, and with the selective NK1 and NK2 tachykinin receptor antagonists, RP 67580 and SR 48968, respectively (500 pmol each). *P , 0.05, **P , 0.01 statistical comparison to the basal expression, calculated with one-way ANOVA followed by post hoc Duncan’s test.
Table 2. Effects of central inhibition of neurokinin-1 and neurokinin-2 tachykinin receptors on the formalin-induced c-Fos expression in the rat brain Treatment (i.c.v.): Treatment (s.c.):
Vehicle —
Vehicle Formalin
RP 67580 Formalin
SR 48968 Formalin
RP 67580/SR 48968 Formalin
RP 68651/SR 48965 Formalin
Prefrontal cortex Paraventricular nucleus Dorsomedial nucleus Ventromedial nucleus Locus coeruleus Periaqueductal gray
87 ^ 7 58 ^ 12 98 ^ 7 18 ^ 3 12 ^ 3 52 ^ 9
191 ^ 23** 221 ^ 44* 270 ^ 41** 57 ^ 9* 52 ^ 9*** 138 ^ 27**
104 ^ 11††† 100 ^ 35† 103 ^ 13††† 21 ^ 4† 18 ^ 2††† 41 ^ 7†††
152 ^ 17†† 133 ^ 33 144 ^ 14††† 24 ^ 4† 28 ^ 6†† 81 ^ 14††
106 ^ 19†† 59 ^ 18† 106 ^ 24††† 23 ^ 4† 29 ^ 3† 63 ^ 15††
172 ^ 11** 214 ^ 44** 251 ^ 42** 55 ^ 17* 43 ^ 6** 129 ^ 25**
Values represent the number of c-Fos-immunoreactive neurons per section of 50 mm on both sides and are expressed as mean ^ S.E.M. Vehicle, the NK1 and NK2 tachykinin receptor antagonists, RP 67580 and SR 48968, respectively, or their inactive enantiomers, RP 68651 and SR 48965, were injected i.c.v. at a dose of 500 pmol, 10 min prior to s.c. injection of formalin (5%, 50 ml). *P , 0.05, **P , 0.01, ***P , 0.001, statistical comparison to the vehicle-treated but not formalin-injected group; †P , 0.05, ††P , 0.01, †††P , 0.001, statistical comparison to the vehicle-treated, formalin-injected group, calculated with one-way ANOVA followed by post hoc Duncan’s test.
s.c. to all five groups of rats and behavioural responses were recorded over a 15-min period (early phase). The experimental protocols had been approved by the Governmental Committee for Ethical Use of Animals in the German Federal State of Schleswig-Holstein. Immunohistochemistry and quantitative analysis Cryostat-cut coronal sections (50 mm) were processed for immunohistochemistry using the conventional avidin–biotin complex peroxidase reaction. Generation and specificity of the antibody have been described in detail elsewhere. 22 To quantify c-Fos immunoreactivity, sections at the same level of each brain region were taken. The level of each region was identified according to an atlas of the rat brain. 33 For quantification, the number of stained neurons of each brain area was counted by two observers in a blind manner on slices of two sections on both sides. No attempt was made to quantify the intensity of the staining. The mean number of labelled neurons on both sides per section was calculated for each brain area and group. Materials The solutions of the NK1 receptor antagonist, RP 67580, and its inactive enantiomer, RP 68651, were prepared as described elsewhere. 10 One microlitre of the solution containing either RP 67580 or RP68651 was injected i.c.v. together with 4 ml of phosphatebuffered physiological saline (pH 7.4). The final pH of the injected solution was 7.2–7.4. The NK2 receptor antagonist, SR 48968, and its inactive enantiomer, SR 48965, were dissolved in a small volume of dimethylsulphoxide (Merck, Germany), and physiological saline was added to obtain the final volume in the stock solution (5000 pmol/ml). On the day of experiment, the stock solution of the antagonist or its inactive enantiomer was further diluted with physiological saline to obtain the desired concentration of the compounds. The final solutions of SR 48968 and SR 48965 contained less than 5% dimethylsulphoxide and were injected i.c.v. in a volume of 1 ml together with 4 ml of
physiological saline. Vehicle-treated controls were injected i.c.v. with 1 ml of the vehicle for i.c.v. injections of RP 67580 containing 5% dimethylsulphoxide together with 4 ml of phosphate-buffered saline. Statistics The results are expressed as the mean ^ S.E.M. Data were analysed by one-way ANOVA. Post hoc comparisons between groups were made using Duncan’s test. A significance level of P , 0.05 was accepted. RESULTS
The basal c-Fos expression in the examined brain nuclei corresponded to that described in other reports. The chronically implantated s.c. catheter did not affect the number and distribution of c-Fos protein in these brain nuclei. Intracerebroventricular injection of vehicle moderately increased the number of positive nuclei in the dorsomedial nucleus of the hypothalamus (DMN) and slightly in the central amygdaloid nucleus (CeA). In the latter area and in the ventromedial nucleus of the hypothalamus (VMN), an increase in c-Fos expression was recorded post-i.c.v. treatment with the active enantiomers of the tachykinin antagonists (Table 1). Following s.c. injection of formalin, c-Fos expression increased in 10 brain areas (Tables 2, 3). In the septohypothalamic nucleus (SHy), medial thalamus (MTh), parabrachial nucleus (PBN) and CeA, formalin increased the number of c-Fos-positive neurons (SHy: F 6.799, P , 0.01; MTh: F 4.015, P , 0.01; PBN: F 5.761, P , 0.01; CeA:
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Table 3. Rat brain areas responding to subcutaneous formalin with increased c-Fos expression, which was not altered by inhibition of central neurokinin-1 and neurokinin-2 tachykinin receptors Pretreatment (i.c.v.): Treatment (s.c.):
Vehicle —
Vehicle Formalin
RP 67580/SR 48968 Formalin
RP 68651/SR 48965 Formalin
Septohypothalamic nucleus Medial thalamus Parabrachial nucleus Amygdaloid nucleus
59 ^ 15 70 ^ 14 15 ^ 9 17 ^ 2
177 ^ 17*** 153 ^ 25** 98 ^ 16** 31 ^ 4**
157 ^ 20** 134 ^ 9* 76 ^ 14* 26 ^ 3
169 ^ 19** 127 ^ 13* 98 ^ 12** 28 ^ 3*
Values represent the numbers of c-Fos-immunoreactive neurons per section of 50 mm on both sides, expressed as mean ^ S.E.M. *P , 0.05, **P , 0.01, ***P , 0.001, statistical comparison to the vehicle-pretreated but not formalin-injected group, calculated with one-way ANOVA followed by post hoc Duncan’s test. For further details, see footnote to Table 2.
F 3.34, P , 0.05), but neither the active enantiomers of the NK1 and NK2 tachykinin receptor antagonists, RP 67580 and SR 48968, respectively, nor their inactive enantiomers, RP 68651 and SR 48965, administered i.c.v. prior to s.c. injection of formalin, had any effects on the increased c-Fos expression (Table 3). Subcutaneous formalin also enhanced the number of c-Fospositive nuclei in the prefrontal cortex (PC; F 5.806, P , 0.001), the paraventricular nucleus (PVN; F 3.2840, P , 0.01), DMN (F 7.006, P , 0.001) and VMN (F 3.200, P , 0.05) of the hypothalamus, in the locus coeruleus (LC; F 6.120, P , 0.001) and periaqueductal gray (PAG; F 6.184, P , 0.001; Table 2). In the PVN, the number of c-Fos-positive nuclei was increased to a similar extent in both the parvocellular and magnocellular parts of the nucleus (Fig. 1). Pretreatment of rats with 500 pmol RP 67580 resulted in a significant inhibition of the increased c-Fos expression induced by formalin in all nuclei. Simultaneous pretreatment with both tachykinin receptor antagonists did not produce any additional inhibitory effects on the postformalin c-Fos induction. In the PC, DMN, VMN, LC and PAG, i.c.v. injection of the NK2 receptor antagonist, SR 48968 (500 pmol), also attenuated the formalin-induced c-Fos expression. Pretreatment of rats with the inactive enantiomers of the tachykinin receptor antagonists did not modify the number of c-Fos-positive nuclei following s.c. injection of formalin in any of these brain nuclei (Table 2). There were significant differences in the behavioural response among the experimental groups (F 47.61, P , 0.001; Table 4). In vehicle-pretreated rats, formalin injected s.c. evoked intense licking and biting at the site of application. The NK2 receptor antagonist slightly, but significantly, reduced the formalin-induced behaviour. The NK1 receptor antagonist inhibited post-formalin grooming behaviour to a greater extent than the NK2 antagonist. Pretreatment of rats with both tachykinin receptor antagonists had the same inhibitory effect as the NK1 receptor antagonist alone (Table 4).
DISCUSSION
Effect of formalin on c-Fos expression in the brain Formalin injected s.c. through the chronically implanted catheter activates primary sensory neurons and produces local pain. The pain experience includes a sensory aspect linked to the perception of potential tissue injury and an emotional, affective aspect related to the aversive component of the pain experience. Correspondingly, formalin-induced pain caused c-Fos activation in a number of forebrain and
brainstem nuclei linked to the relay, processing or modulation of nociceptive signals, such as the MTh and the PAG. 17,25 The activation of c-Fos in the CeA and in the SHy, which represents the most ventral part of the lateral septal area, may be related to control of behavioural reactions and emotional states upon noxious stimulation, although the function of the latter region in the modulation of behavioural responses to pain has not yet been clarified. Similarly to our findings, electric footshock, a pain stimulus, or swimming or restraint stress strongly increased c-Fos immunoreactivity in the ventral subdivision of the lateral septal area. 7,14,34 In addition to pain generated by formalin itself, the formalin test for nociception represents a stress event comprising the cardiovascular response brought about by the sympathoadrenal activation and endocrine responses characterized by increases in plasma levels of ACTH and corticosterone. 11 Experience of pain also caused c-Fos activation in brain regions related to the initiation and regulation of neuroendocrine and autonomic and behavioural stress reactions, such as in the PC and the PVN, which represent the major sites for the integration of these reactions. 39,40 The induction of c-Fos in the LC in response to formalin is consistent with the role of this nucleus in the processing of stress responses. 38 The activation of PBN neurons is most probably associated with the regulation of the autonomic responses to nociceptive stimuli. 28 Effect of tachykinin receptor antagonists on c-Fos expression induced by formalin We have shown recently that inhibition of forebrain NK1 receptors attenuated the cardiovascular and behavioural responses to formalin injected s.c. 10 In the present study, selective, non-peptide antagonists for NK1 and NK2 receptors were employed to determine in which brain areas the inhibition of tachykinin receptors would suppress the pain-induced c-Fos expression, an indicator of neuronal activity. 13 Intracerebroventricular treatment of rats with 500 pmol RP 67580, a dose which has been shown previously to effectively attenuate the mean arterial pressure and heart rate responses to s.c. formalin, 10 reduced formalin-induced c-Fos expression in the majority of brain nuclei activated by such a stimulus. SP serves as a mediator of nociception in the spinal cord. 31 The NK1 receptor antagonist injected i.c.v. may therefore theoretically antagonize the pain transmission at the spinal cord level, which would result in a suppression of c-Fos expression in the brain. However, the inhibition of NK1 receptors at the spinal cord level would result in attenuation of c-Fos expression in all brain nuclei which responded to s.c. formalin due to a general impairment of pain transmission by the
Brain neurokinins and pain-induced c-Fos expression
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Fig. 1. Frontal sections through the PVN stained for c-Fos protein of rats pretreated i.c.v. with: vehicle, no s.c. formalin injection followed (upper panel, left); vehicle, 10 min prior to s.c. formalin injection (upper panel, right); the active enantiomers of the NK1 and NK2 tachykinin receptor antagonists (500 pmol each), 10 min prior to s.c. formalin injection (lower panel, left) and the inactive enantiomers of the tachykinin receptor antagonists (500 pmol, each), 10 min prior to s.c. formalin injection (lower panel, right).
primary afferent neurons which synapse on neurons in the dorsal horn. Surprisingly, i.c.v. pretreatment with the selective NK2 receptor antagonist, SR 48968, attenuated both the c-Fos activation in some brain nuclei and the grooming behaviour in response to formalin. SR 48968 inhibits selectively and with high affinity NK2 receptors, for which NKA is the preferred natural ligand. 36 Although NKA is expressed abundantly by
neurons in the brain, and the peptide injected centrally elicits a number of distinct actions, the NK2 receptor is either not expressed in the adult rat brain or its abundance is extremely low. 31 However, recent findings indicate that NKA can also bind with relatively high affinity to the NK1 (SP) receptor and act through this receptor with high potency. Therefore, some of the actions of NKA in the brain may be mediated by the NK1 receptor. 27 However, there are no data available so far
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Table 4. Effects of central inhibition of neurokinin-1 and neurokinin-2 tachykinin receptors on hindlimb grooming (licking and biting of the lower leg) in response to subcutaneously injected formalin Treatment (i.c.v.)
Vehicle Vehicle RP 67580 SR 48968 RP 67580/SR 48968 RP 68651/SR 48965
Dose (pmol)
Formalin s.c.
n
Hindlimb grooming
— — 500 500 500 500
— 1 1 1 1 1
7 8 8 9 8 8
1.1 ^ 0.7 12.6 ^ 0.7*** 5.3 ^ 0.5*††† 9.1 ^ 1.0***† 3.6 ^ 0.5††† 14.8 ^ 0.9***
Values represent the frequency of hindlimb grooming for 15 min and are expressed as mean ^ S.E.M. Vehicle, the NK1 and NK2 tachykinin receptor antagonists, RP 67580 and SR 48968, respectively, or their inactive enantiomers, RP 68651 and SR 48965, were injected i.c.v. at a dose of 500 pmol, 10 min prior to s.c. injection of formalin (5%, 50 ml). *P , 0.05, ***P , 0.001, statistical comparison to the vehicle-treated but not formalin-injected group; †P , 0.05, †††P , 0.001, statistical comparison to the vehicle-treated, formalin-injected group, calculated with one-way ANOVA followed by post hoc Duncan’s test.
which demonstrate that the selective NK2 receptor antagonist, SR48968, can, similarly to NKA, also interact with NK1 receptors. Therefore, we assume that the attenuation of the pain-induced c-Fos expression and behaviour by SR 489689 results from inhibition of NK2 rather than NK1 receptors. SR 48968, besides exerting non-specific effects on neurotransmission, which are not related to the interaction with NK2 receptors, 26,42 exhibits a relatively high agonistic activity at the m-opioid receptor. 16 Although a low dose of SR 48968 was used in the present study, it cannot be ruled out that the interaction of SR 48968 with central opioid receptors contributed at least partly to the observed effects. All brain nuclei, besides the PC, in which tachykinin receptor antagonists reduced formalin-induced c-Fos activation, belong to regions adjacent to the ventricular system. The PVN, a key area for the integration of cardiovascular and neuroendocrine responses to stress, 39,40 contains a moderate density of SP-binding sites. 3 The PVN is innervated by SPimmunoreactive fibres originating in the A1 and C1 catecholaminergic areas, and by SP-immunoreactive neurons localized in the DMN and VMN. 2 The NK1 receptor antagonist effectively attenuated the formalin-induced c-Fos activation in the latter areas, indicating that fewer neurons were activated by noxious stimulation following the antagonist treatment. NK1 receptors are very dense in the LC and PAG. 12 Since the LC lies in close vicinity to the ventricular system and neurons of the PAG surround the cerebral aqueduct, NK1 receptors in these areas can be targeted efficiently by the tachykinin receptor antagonists. The LC belongs to the principal co-ordinators of central stress reactions. 38 The reduction in the pain-induced c-Fos activation, together with the finding of a concentration-dependent excitation of LC neurons by SP, 6 point to the involvement of the peptide in this area in the co-ordination of responses induced by noxious stimuli. The PAG appears to play a key role in the generation and control of cardiovascular responses upon stress. 21,23 Short restraint or s.c. saline injection raised the SP concentrations in this area. 37 Neuronal networks in this area are also involved in the modulation of nociceptive transmission. 17 The present data show that, following tachykinin receptor inhibition, fewer neurons in this area responded to noxious stimulation. However, owing to the diverse functions of the PAG, the
observed reduction in c-Fos induction in this area cannot be interpreted unambiguously. As the PC does not lie in the close vicinity of the ventricular system, we assume that a direct inhibition of tachykinin receptors in the PC was probably not responsible for the attenuated c-Fos activation in this area to formalin. It appears likely that blockade of tachykinin receptors in other brain region(s) was responsible for the inhibition of the formalininduced c-Fos expression in the PC. In several brain regions, treatment with the tachykinin receptor antagonists failed to affect the formalin-induced cFos expression. The most probable explanation is that the cFos activation in these areas after formalin is dependent on factors other than neurokinins. However, several other possibilities may also be considered, for instance that tachykinin receptors in these areas are not activated by noxious stimuli or that the concentrations of the tachykinin receptor antagonists in these brain areas were not high enough to effectively inhibit these receptors. Moreover, neuronal input from other brain regions, which project to the aforementioned areas but do not respond to somatic pain by expressing c-Fos, could provide a sufficiently strong signal to induce c-Fos expression, despite effective blockade of tachykinin receptors. This might be the case for the PBN, which represents an important site for the regulation of stress-related cardiovascular and respiratory responses. 28 The PBN contains a very high density of SPbinding sites, 12 which could probably be effectively targeted after treatment with the tachykinin receptor antagonists because of its localization in the vicinity of the ventricular system. This nucleus is interconnected, among others, with hypothalamic nuclei, the amygdala, nucleus tractus solitarii and area postrema. The two latter areas provide a major projection to the PBN. 20,29 The neuronal input from areas which did not respond by a reduction of neuronal activity to pain after treatment with the tachykinin receptor antagonists might provide a sufficient signal to induce c-Fos expression in the PBN, even though its tachykinin receptors were inhibited. The NK1 receptor antagonist substantially inhibited the grooming behaviour to s.c. formalin, and simultaneous treatment of rats with both tachykinin receptor antagonists did not produce an additional inhibition of the formalin-induced behaviour. In contrast to our previous findings, 10 the NK2 receptor antagonists also reduced the grooming behaviour in response to formalin. In addition to blockade of tachykinin receptors, both tachykinin receptor antagonists have been shown to interact with calcium channels and to exert nonspecific inhibitory effects on neurotransmission. 26,42 As already mentioned, SR 48968 has been reported to possess a relatively high agonistic affinity at m-opioid receptors. 16 Based on the presented results, the question as to whether these non-specific effects accounted for the reduction in the hindlimb grooming behaviour because the animals experienced less pain cannot be answered unambiguously. However, it can be argued that these non-specific effects would generally lessen the evoked activity in the neuronal pathways activated by formalin-induced pain, which would result in a widespread reduction of c-Fos expression. The finding that treatment with tachykinin receptor antagonists failed to affect the exaggerated c-Fos expression in some brain regions does not support an overall lessening of evoked activity in neuronal pathways activated by formalin-induced pain.
Brain neurokinins and pain-induced c-Fos expression CONCLUSIONS
The present data demonstrate that somatic pain also activates a number of brain areas traditionally related to the regulation of responses induced by stressful events. Blockade of
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tachykinin receptors, especially of the NK1 receptor, reduced the pain-induced neuronal activation in brain areas which are intimately linked with nociceptive neurotransmission or the initiation and integration of stress responses.
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