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Immunocytochemical localization of extracellular signal-regulated kinases 1 and 2 phosphorylated neurons in the brainstem of rat following visceral noxious stimulation
Neuroscience Letters 349 (2003) 167–170 www.elsevier.com/locate/neulet
Immunocytochemical localization of extracellular signal-regulated kinases 1 and 2 phosphorylated neurons in the brainstem of rat following visceral noxious stimulation Magda Gioiaa,b,*, Claudia Moschenia,b, Stefania Galbiatia, Nicoletta Gaglianoa,b a
Department of Human Anatomy, LITA Segrate, University of Milan, Via Mangiagalli 31, 20133 Milan, Italy b Department of Human Anatomy-LITA, University of Milan, Via Fratelli Cervi 93, 20090 Segrate MI, Italy Received 11 June 2003; accepted 2 July 2003
Abstract The aim of the present study was to determine the activation of the extracellular signal-regulated kinases (ERKs) 1 and 2 in brainstem neurons following noxious visceral stimulation. Ether and urethane anaesthetized rats received an intraperitoneal injection of acetic acid (ENS, UNS) or were left untreated (ECT, UCT). Paraffin embedded brain sections immunoreacted with an antibody specific for phosphorylated ERKs. In noxious stimulated rats ERKs activated neuron profiles in the periaqueductal gray matter, parabrachial, dorsal raphe, solitary tract nucleus, area postrema and superior colliculus suggest that ERKs activation takes place mainly in brainstem nuclei in which nociception and visceral activities interact. The comparison between ENS and UNS rats shows that the long acting anaesthetic urethane attenuates the number of the ERKs activated neurons compared to the short acting ether. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Extracellular signal-regulated kinases; Nociception; Brainstem; Immunohistochemistry; Rat
The extracellular signal-regulated kinases (ERKs) 1 and 2 are mitogen-activated protein kinases that transduce extracellular stimuli into intracellular responses. Although in most cells ERKs activation, namely phosphorylation, is driven by growth factors, activity-dependent activation occurs in many neurons, including activities related to nociception. Following noxious stimulation ERKs phosphorylation occurs in neurons of the dorsal root ganglia [7], spinal cord [9,14], trigeminal spinal nucleus [13], nuclei of the vagus nerve [19] and tele- and diencephalon [11]. Having studied the distribution of ERKs activated neurons in the rat tele- and diencephalon following a chemical visceral noxious stimulus [11], we aimed at extending the investigation to the brainstem, where different nuclei are involved in important activities related to nociception. Using another pool of rats and the same experimental conditions, our purpose was to verify whether ERKs activation occurs in neurons of the brainstem nuclei. The localization of the ERK activated neurons in the *
Corresponding author. Tel.: þ 39-2-5031-5374; fax: þ39-2-5031-5387. E-mail address: [email protected] (M. Gioia).
brainstem, that complement the data obtained in the teleand diencephalon giving a complete picture of the ERKs activation in neurons of the rat brain, could help to clarify the relationship between ERKs activation and nociceptive activities. The influence of the anaesthetic action on the ERK activation was evaluated using two anaesthetics with different action durations: ether and urethane that are, respectively, short acting and long acting anaesthetics. The housing conditions of the animals and the experimental procedures used in the present study were the same as those used in our previous investigation. The experiments were carried out according to Italian law and to the directive of the European Communities Council. Every effort was made to minimize the numbers and suffering of the animals used in the experiments. The animals, housed in individual cages with food and water ad libitum, were kept in an animal house at a constant temperature of 228C with a 12 h alternating light-dark cycle. The experiments were performed between 08:00 and 12:00 h. Special care was taken to minimize extraneous environmental stimulation or stress. The animals were made accustomed to being handled for 2
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00821-8
168
M. Gioia et al. / Neuroscience Letters 349 (2003) 167–170
weeks prior to the experimental session. Four groups (five rats for each group) of adult Wistar rats weighting 200 – 250 g were used. Two control groups (CT) of rats were exposed to ether (ECT) for less than 90 s or were intraperitoneally injected with urethane (UCT, 1– 2 g/kg); two groups of rats were anaesthetized with ether (ENS) or urethane (UNS) and noxious stimulated by an intraperitoneal injection of acetic acid (0.5 ml of 3.5% acetic acid). The intraperitoneal injection of acetic acid is generally used as a visceral noxious stimulus although it also has a somatic component. After 2 h the rats were transcardially perfused with saline followed by 1 l of 4% paraformaldehyde in 0.1 M pH 7.4 Phosphate Buffer. Paraffin embedded coronal serial 10 mm thick brain sections interspaced at 100 mm were Nissl stained or immunohistochemically processed using the mouse monoclonal antibody E10 (New England Biolabs), specific for the phosphorylated form of ERK 1 and 2 (1:600) and avidin-biotin peroxidase complex (Vector Labs). ECT, UCT, ENS and UNS brainstem sections were processed together. The control sections which had been incubated omitting the primary antibody showed no labelling. The distribution of the labelled elements of all the animals was charted with the aid of a camera lucida and transferred onto standard sections [18]. The neuron profile counts were made by two different people blinded to the treatment at a final 100 £ magnification. To asses the number of labelled neuron profiles in each considered nucleus, the number of positive profiles in each brainstem section was divided by the number of sections in which the examined nuclei were contained. Light microscopy analysis of the sections from NS rats showed labelling in the cytoplasm of the soma and, sometimes, of the dendrites of positive cells (Fig. 1). Table 1 indicates the areas of the brainstem showing ERKs neuron profiles in ECT, UCT, ENS and UNS rats; the quantitative data showing the labelled cell profiles are expressed as mean and standard deviation (SD). Only a few labelled neurons were present in ECT and UCT sections; they were almost absent in ECT. In ENS and UNS ERK activated neuron profiles were bilaterally located in similar brainstem areas (see Table 1), but in UNS they were generally less frequent and undetectable in reticular nuclei of the rostroventral medulla. In visceral noxious stimulated rats ERKs activated neuron profiles were located in nuclei which are involved in nociception and/or visceral activities. The periaqueductal gray matter (PAG), in fact, widely interacts in nociceptive transmission and in the cardiovascular and respiratory activities which accompany nociception [1,2,12]. The parabrachial nucleus (PB) plays an important role in autonomic and homeostatic reflexes [12] and its lateral part is also involved in nociception [10]. The nucleus of the solitary tract (Sol), which is reached by visceral afferents, mediates the connected vascular and respiratory responses and is involved in pain transmission [4]. In rodents, the superior colliculus (SuC) contains nociceptive neurons [16].
Fig. 1. Photomicrographs showing the distribution of ERKs activated immunoreactive neuron profiles (arrows) in PB (a); PAG (b); DR (c, d); and in the EW (d) of visceral noxious stimulated ether anaesthetized rats. Scale bar: 100 mm. Aq, aqueduct; PAG, periaqueductal gray matter; PB, lateral parabrachial nucleus; and EW, Edinger-Westphal nucleus.
Evidences have shown that the Edinger-Westphal nucleus (EW), in addition to its association with oculomotor function, has a role in autonomic modifications which accompany nociception [3]. The area postrema (AP) is implicated as a major station for visceral sensory information [6]. The dorsal raphe nucleus (DR) is involved in cardiovascular reflexes [12]. ERKs activated neuron profiles have been detected also in some brainstem areas in which noxious modulation takes place, such as the PAG [8] and the DR [5]. By contrast, labelling was scarce or absent in the raphe magnus nucleus (RMg) and in the adjacent nuclei of the rostroventral medulla, although they play a very important role in descending nociceptive modulation mediating both opioid and stimulation produced analgesia [8,15]. RMg contains cells which facilitate (on cells) and inhibit (off cells) nociceptive transmission influencing nociceptive dorsal horn neurons. Different reasons could explain the scarcity of ERKs labelled neuron profiles in these nuclei. ERKs activation might occur in RM neurons with a time course different from that considered in the present study, or a different signal transduction system might be used by the cells of these nuclei. A further possibility is that in our study ERKs activation may be mainly associated to the transduction of a signal correlated to the transmission of nociception, rather than to its descending modulation. However, it was shown that morphine treatment increases ERKs activity in the medulla and pons, which are two of the most important sites for the induction of opioid-induced antinociception [17]. On the whole, our results suggest that following visceral
M. Gioia et al. / Neuroscience Letters 349 (2003) 167–170
169
Table 1 Distribution of the phosphorylated ERK 1 and 2 labelled neuron profiles in the brainstem of anaesthetized visceral noxious stimulated and control rats
Edinger-Westphal nucleus (EW) Superior colliculus (SuC) Inferior colliculus (IC) Periaqueductal gray matter (PAG) Dorsal raphe nucleus (DR) Tegmental nucleus (Tg) Lateral parabrachial nucleus (PB) Central gray (CG) Gigantocellular reticular nucleus (Gi) Raphe magnus nucleus (RMg) Raphe pallidus nucleus (RPa) Raphe obscurus nucleus (ROb) Medial vestibular nucleus (MVe) Lateral reticular nucleus (LRt) Rostroventrolateral reticular nucleus (RVL) Nucleus of the solitay tract (Sol) Area postrema (AP) Medullary reticular nucleus (Md) Linear raphe nucleus (Li) Median raphe nucleus (MnR) Pontine reticular nucleus (PnR)
Ether þ acetic acid
Ether
Urethane þ acetic acid
Urethane
22.5 ^ 8.18 25.83 ^ 15.84 59.69 ^ 35.12 46.82 ^ 14.41 46.25 ^ 25.84 53.75 ^ 23.31 28.33 ^ 6.66 21.33 ^ 11.08 8.62 ^ 6.95 1,5 ^ 0,71 7.2 ^ 3.11 8.25 ^ 2.35 18.29 ^ 7.02 19.33 ^ 6.66 11.33 ^ 4.04 38.50 ^ 16.20 25.67 ^ 12.10 13.00 ^ 3.00 2.00 ^ 1.41 2, 10 ^ 0,82 2.40 ^ 1.67
– – – – – – – – – – – – – 2.14 ^ 1.46 – – – – – – –
13.60 ^ 4.83 13.50 ^ 4.43 11.50 ^ 5.80 6.67 ^ 0.58 11.75 ^ 5.44 11.80 ^ 5.54 11.50 ^ 2.65 4.67 ^ 2.08 3.00 ^ 1.41 1.50 ^ 0.58 3.14 ^ 1.35 – 3.60 ^ 2.30 – – 9.00 ^ 4.47 5.02 ^ 3.74 – – – –
11.33 ^ 3.06 – – 1.33 ^ 0.58 – – – 1.67 ^ 0.58 – – – – – – – – – – – – –
Data are mean ^ SD.
noxious stimulation ERKs activation takes place mainly in brainstem nuclei in which nociception and visceral activities interact. The present data and the distribution of ERKs neurons in limbic tele- and diencephalon regions [11] suggest that ERKs activation occurring in rat brain neurons following acetic acid intraperitoneal injection is mainly related to the emotional and autonomic aspect of nociception [2, 10]. This suggestion might be supported also by the different effect of ether and urethane anaesthesia on the number of ERKs activated neuron profiles after noxious stimulation. In fact, the comparison between the ENS and UNS rats shows that the ERK activation is reduced in UNS and demonstrates that in the brainstem the long acting anaesthetic urethane considerably reduces the number of ERKs activated neurons with respect to the short acting ether. In noxious stimulated rats it appears that urethane has a greater depressive effect in the brainstem than in the tele- and diencephalon [11] neurons. However, in the tele- and diencephalon of UCT rats, in contrast to what observed in the brainstem, the urethane anaesthesia alone causes considerable ERKs activation in neurons; therefore, ERKs activation triggered by noxious stimulation adds to that due to the anaesthetic. Further investigations are in course in our laboratory to clarify the relationship between ERK activation and nociception. In particular, the use of repeated noxious stimulus to determine whether ERKs activation is intensity dependent, and an analgesic administration after the noxious
stimulation to evaluate if ERKs activation is reduced, will be helpful for this purpose.
Acknowledgements We thank Dr Genton for the English revision of the manuscript, Silvia Celon for the immunohistochemical procedures, and Luciano Losi for the fixation by vascular perfusion and the removal of rat brains. This work was supported by a F.I.R.S.T. grant.
References [1] M.M. Behbehani, Functional characteristics of the midbrain periaqueductal gray, Prog. Neurobiol. 46 (1995) 575–605. [2] E.E. Benarroch, Pain-autonomic interactions: a selective review, Clin. Auton. Res. 11 (2001) 343 –349. [3] K. Bon, M. Lante´ri-Minet, J. de Pommery, J.F. Michiels, D. Mene´trey, Cyclophosphamide cystitis as a model of visceral pain in rats: minor effects at mesodiancephalic levels as revealed by the expression of cfos, with a note on Krox-24, Exp. Brain Res. 113 (1997) 249–264. [4] P. Boscan, A.E. Pickering, J.F.R. Paton, The nucleus of the solitary tract: an integrating station for nociceptive and cardiorespiratory afferents, Exp. Physiol. 87.2 (2002) 259–266. [5] T. Chuma, K. Taguchi, M. Kato, K. Abe, I. Utsunomiya, K. Miyamoto, T. Miyatake, Modulation of noradrenergic and serotonergic transmission by noxious stimuli and intrathecal morphine differs in the dorsal raphe nucleus of anesthetized rat: in vivo voltammetric studies, Neurosci. Res. 44 (2002) 37–44. [6] E.T. Cunningham, R.R. Miselis, P.E. Sawchenko, The relationship of efferent projections from the area postrema to vagal motor and brain stem catecholamine-containing cell groups: an axonal transport and
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Immunocytochemical localization of extracellular signal-regulated kinases 1 and 2 phosphorylated neurons in the brainstem of rat following visceral noxious stimulation Magda Gioiaa,b,*, Claudia Moschenia,b, Stefania Galbiatia, Nicoletta Gaglianoa,b a
Department of Human Anatomy, LITA Segrate, University of Milan, Via Mangiagalli 31, 20133 Milan, Italy b Department of Human Anatomy-LITA, University of Milan, Via Fratelli Cervi 93, 20090 Segrate MI, Italy Received 11 June 2003; accepted 2 July 2003
Abstract The aim of the present study was to determine the activation of the extracellular signal-regulated kinases (ERKs) 1 and 2 in brainstem neurons following noxious visceral stimulation. Ether and urethane anaesthetized rats received an intraperitoneal injection of acetic acid (ENS, UNS) or were left untreated (ECT, UCT). Paraffin embedded brain sections immunoreacted with an antibody specific for phosphorylated ERKs. In noxious stimulated rats ERKs activated neuron profiles in the periaqueductal gray matter, parabrachial, dorsal raphe, solitary tract nucleus, area postrema and superior colliculus suggest that ERKs activation takes place mainly in brainstem nuclei in which nociception and visceral activities interact. The comparison between ENS and UNS rats shows that the long acting anaesthetic urethane attenuates the number of the ERKs activated neurons compared to the short acting ether. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Extracellular signal-regulated kinases; Nociception; Brainstem; Immunohistochemistry; Rat
The extracellular signal-regulated kinases (ERKs) 1 and 2 are mitogen-activated protein kinases that transduce extracellular stimuli into intracellular responses. Although in most cells ERKs activation, namely phosphorylation, is driven by growth factors, activity-dependent activation occurs in many neurons, including activities related to nociception. Following noxious stimulation ERKs phosphorylation occurs in neurons of the dorsal root ganglia [7], spinal cord [9,14], trigeminal spinal nucleus [13], nuclei of the vagus nerve [19] and tele- and diencephalon [11]. Having studied the distribution of ERKs activated neurons in the rat tele- and diencephalon following a chemical visceral noxious stimulus [11], we aimed at extending the investigation to the brainstem, where different nuclei are involved in important activities related to nociception. Using another pool of rats and the same experimental conditions, our purpose was to verify whether ERKs activation occurs in neurons of the brainstem nuclei. The localization of the ERK activated neurons in the *
Corresponding author. Tel.: þ 39-2-5031-5374; fax: þ39-2-5031-5387. E-mail address: [email protected] (M. Gioia).
brainstem, that complement the data obtained in the teleand diencephalon giving a complete picture of the ERKs activation in neurons of the rat brain, could help to clarify the relationship between ERKs activation and nociceptive activities. The influence of the anaesthetic action on the ERK activation was evaluated using two anaesthetics with different action durations: ether and urethane that are, respectively, short acting and long acting anaesthetics. The housing conditions of the animals and the experimental procedures used in the present study were the same as those used in our previous investigation. The experiments were carried out according to Italian law and to the directive of the European Communities Council. Every effort was made to minimize the numbers and suffering of the animals used in the experiments. The animals, housed in individual cages with food and water ad libitum, were kept in an animal house at a constant temperature of 228C with a 12 h alternating light-dark cycle. The experiments were performed between 08:00 and 12:00 h. Special care was taken to minimize extraneous environmental stimulation or stress. The animals were made accustomed to being handled for 2
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00821-8
168
M. Gioia et al. / Neuroscience Letters 349 (2003) 167–170
weeks prior to the experimental session. Four groups (five rats for each group) of adult Wistar rats weighting 200 – 250 g were used. Two control groups (CT) of rats were exposed to ether (ECT) for less than 90 s or were intraperitoneally injected with urethane (UCT, 1– 2 g/kg); two groups of rats were anaesthetized with ether (ENS) or urethane (UNS) and noxious stimulated by an intraperitoneal injection of acetic acid (0.5 ml of 3.5% acetic acid). The intraperitoneal injection of acetic acid is generally used as a visceral noxious stimulus although it also has a somatic component. After 2 h the rats were transcardially perfused with saline followed by 1 l of 4% paraformaldehyde in 0.1 M pH 7.4 Phosphate Buffer. Paraffin embedded coronal serial 10 mm thick brain sections interspaced at 100 mm were Nissl stained or immunohistochemically processed using the mouse monoclonal antibody E10 (New England Biolabs), specific for the phosphorylated form of ERK 1 and 2 (1:600) and avidin-biotin peroxidase complex (Vector Labs). ECT, UCT, ENS and UNS brainstem sections were processed together. The control sections which had been incubated omitting the primary antibody showed no labelling. The distribution of the labelled elements of all the animals was charted with the aid of a camera lucida and transferred onto standard sections [18]. The neuron profile counts were made by two different people blinded to the treatment at a final 100 £ magnification. To asses the number of labelled neuron profiles in each considered nucleus, the number of positive profiles in each brainstem section was divided by the number of sections in which the examined nuclei were contained. Light microscopy analysis of the sections from NS rats showed labelling in the cytoplasm of the soma and, sometimes, of the dendrites of positive cells (Fig. 1). Table 1 indicates the areas of the brainstem showing ERKs neuron profiles in ECT, UCT, ENS and UNS rats; the quantitative data showing the labelled cell profiles are expressed as mean and standard deviation (SD). Only a few labelled neurons were present in ECT and UCT sections; they were almost absent in ECT. In ENS and UNS ERK activated neuron profiles were bilaterally located in similar brainstem areas (see Table 1), but in UNS they were generally less frequent and undetectable in reticular nuclei of the rostroventral medulla. In visceral noxious stimulated rats ERKs activated neuron profiles were located in nuclei which are involved in nociception and/or visceral activities. The periaqueductal gray matter (PAG), in fact, widely interacts in nociceptive transmission and in the cardiovascular and respiratory activities which accompany nociception [1,2,12]. The parabrachial nucleus (PB) plays an important role in autonomic and homeostatic reflexes [12] and its lateral part is also involved in nociception [10]. The nucleus of the solitary tract (Sol), which is reached by visceral afferents, mediates the connected vascular and respiratory responses and is involved in pain transmission [4]. In rodents, the superior colliculus (SuC) contains nociceptive neurons [16].
Fig. 1. Photomicrographs showing the distribution of ERKs activated immunoreactive neuron profiles (arrows) in PB (a); PAG (b); DR (c, d); and in the EW (d) of visceral noxious stimulated ether anaesthetized rats. Scale bar: 100 mm. Aq, aqueduct; PAG, periaqueductal gray matter; PB, lateral parabrachial nucleus; and EW, Edinger-Westphal nucleus.
Evidences have shown that the Edinger-Westphal nucleus (EW), in addition to its association with oculomotor function, has a role in autonomic modifications which accompany nociception [3]. The area postrema (AP) is implicated as a major station for visceral sensory information [6]. The dorsal raphe nucleus (DR) is involved in cardiovascular reflexes [12]. ERKs activated neuron profiles have been detected also in some brainstem areas in which noxious modulation takes place, such as the PAG [8] and the DR [5]. By contrast, labelling was scarce or absent in the raphe magnus nucleus (RMg) and in the adjacent nuclei of the rostroventral medulla, although they play a very important role in descending nociceptive modulation mediating both opioid and stimulation produced analgesia [8,15]. RMg contains cells which facilitate (on cells) and inhibit (off cells) nociceptive transmission influencing nociceptive dorsal horn neurons. Different reasons could explain the scarcity of ERKs labelled neuron profiles in these nuclei. ERKs activation might occur in RM neurons with a time course different from that considered in the present study, or a different signal transduction system might be used by the cells of these nuclei. A further possibility is that in our study ERKs activation may be mainly associated to the transduction of a signal correlated to the transmission of nociception, rather than to its descending modulation. However, it was shown that morphine treatment increases ERKs activity in the medulla and pons, which are two of the most important sites for the induction of opioid-induced antinociception [17]. On the whole, our results suggest that following visceral
M. Gioia et al. / Neuroscience Letters 349 (2003) 167–170
169
Table 1 Distribution of the phosphorylated ERK 1 and 2 labelled neuron profiles in the brainstem of anaesthetized visceral noxious stimulated and control rats
Edinger-Westphal nucleus (EW) Superior colliculus (SuC) Inferior colliculus (IC) Periaqueductal gray matter (PAG) Dorsal raphe nucleus (DR) Tegmental nucleus (Tg) Lateral parabrachial nucleus (PB) Central gray (CG) Gigantocellular reticular nucleus (Gi) Raphe magnus nucleus (RMg) Raphe pallidus nucleus (RPa) Raphe obscurus nucleus (ROb) Medial vestibular nucleus (MVe) Lateral reticular nucleus (LRt) Rostroventrolateral reticular nucleus (RVL) Nucleus of the solitay tract (Sol) Area postrema (AP) Medullary reticular nucleus (Md) Linear raphe nucleus (Li) Median raphe nucleus (MnR) Pontine reticular nucleus (PnR)
Ether þ acetic acid
Ether
Urethane þ acetic acid
Urethane
22.5 ^ 8.18 25.83 ^ 15.84 59.69 ^ 35.12 46.82 ^ 14.41 46.25 ^ 25.84 53.75 ^ 23.31 28.33 ^ 6.66 21.33 ^ 11.08 8.62 ^ 6.95 1,5 ^ 0,71 7.2 ^ 3.11 8.25 ^ 2.35 18.29 ^ 7.02 19.33 ^ 6.66 11.33 ^ 4.04 38.50 ^ 16.20 25.67 ^ 12.10 13.00 ^ 3.00 2.00 ^ 1.41 2, 10 ^ 0,82 2.40 ^ 1.67
– – – – – – – – – – – – – 2.14 ^ 1.46 – – – – – – –
13.60 ^ 4.83 13.50 ^ 4.43 11.50 ^ 5.80 6.67 ^ 0.58 11.75 ^ 5.44 11.80 ^ 5.54 11.50 ^ 2.65 4.67 ^ 2.08 3.00 ^ 1.41 1.50 ^ 0.58 3.14 ^ 1.35 – 3.60 ^ 2.30 – – 9.00 ^ 4.47 5.02 ^ 3.74 – – – –
11.33 ^ 3.06 – – 1.33 ^ 0.58 – – – 1.67 ^ 0.58 – – – – – – – – – – – – –
Data are mean ^ SD.
noxious stimulation ERKs activation takes place mainly in brainstem nuclei in which nociception and visceral activities interact. The present data and the distribution of ERKs neurons in limbic tele- and diencephalon regions [11] suggest that ERKs activation occurring in rat brain neurons following acetic acid intraperitoneal injection is mainly related to the emotional and autonomic aspect of nociception [2, 10]. This suggestion might be supported also by the different effect of ether and urethane anaesthesia on the number of ERKs activated neuron profiles after noxious stimulation. In fact, the comparison between the ENS and UNS rats shows that the ERK activation is reduced in UNS and demonstrates that in the brainstem the long acting anaesthetic urethane considerably reduces the number of ERKs activated neurons with respect to the short acting ether. In noxious stimulated rats it appears that urethane has a greater depressive effect in the brainstem than in the tele- and diencephalon [11] neurons. However, in the tele- and diencephalon of UCT rats, in contrast to what observed in the brainstem, the urethane anaesthesia alone causes considerable ERKs activation in neurons; therefore, ERKs activation triggered by noxious stimulation adds to that due to the anaesthetic. Further investigations are in course in our laboratory to clarify the relationship between ERK activation and nociception. In particular, the use of repeated noxious stimulus to determine whether ERKs activation is intensity dependent, and an analgesic administration after the noxious
stimulation to evaluate if ERKs activation is reduced, will be helpful for this purpose.
Acknowledgements We thank Dr Genton for the English revision of the manuscript, Silvia Celon for the immunohistochemical procedures, and Luciano Losi for the fixation by vascular perfusion and the removal of rat brains. This work was supported by a F.I.R.S.T. grant.
References [1] M.M. Behbehani, Functional characteristics of the midbrain periaqueductal gray, Prog. Neurobiol. 46 (1995) 575–605. [2] E.E. Benarroch, Pain-autonomic interactions: a selective review, Clin. Auton. Res. 11 (2001) 343 –349. [3] K. Bon, M. Lante´ri-Minet, J. de Pommery, J.F. Michiels, D. Mene´trey, Cyclophosphamide cystitis as a model of visceral pain in rats: minor effects at mesodiancephalic levels as revealed by the expression of cfos, with a note on Krox-24, Exp. Brain Res. 113 (1997) 249–264. [4] P. Boscan, A.E. Pickering, J.F.R. Paton, The nucleus of the solitary tract: an integrating station for nociceptive and cardiorespiratory afferents, Exp. Physiol. 87.2 (2002) 259–266. [5] T. Chuma, K. Taguchi, M. Kato, K. Abe, I. Utsunomiya, K. Miyamoto, T. Miyatake, Modulation of noradrenergic and serotonergic transmission by noxious stimuli and intrathecal morphine differs in the dorsal raphe nucleus of anesthetized rat: in vivo voltammetric studies, Neurosci. Res. 44 (2002) 37–44. [6] E.T. Cunningham, R.R. Miselis, P.E. Sawchenko, The relationship of efferent projections from the area postrema to vagal motor and brain stem catecholamine-containing cell groups: an axonal transport and
170
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