Directory UMM :Data Elmu:jurnal:B:Brain Research:Vol888.Issue2.2001:
Brain Research 888 (2001) 314–320
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Short communication
Post-ictal analgesia: involvement of opioid, serotoninergic and
cholinergic mechanisms
a,
a
a
a
a
N.C. Coimbra *, C. Castro-Souza , E.N. Segato , J.E.P. Nora , C.F.P.S. Herrero ,
a
b
W. Tedeschi-Filho , N. Garcia-Cairasco
a
˜ Preto,
´
Laboratorio
de Neuroanatomia e Neuropsicobiologia, Departamento de Farmacologia, Faculdade de Medicina de Ribeirao
˜ Paulo ( USP), 14049 -900, Ribeirao
˜ Preto ( SP), Brazil
Universidade de Sao
b
˜ Preto ( SP), Brazil
´
Laboratorio
de Neurofisiologia e Neuroetologia Experimental, Departamento de Fisiologia, FMRP-USP, 14049 -900, Ribeirao
Accepted 10 October 2000
Abstract
The neural mechanisms involved in post-ictal analgesia remain to be elucidated. Pentylenetetrazol (PTZ) is used experimentally to
induce seizure in animal subjects. This non-competitive antagonist blocks GABA-mediated Cl 2 flux. The aim of this work is to study the
neurochemical basis of the antinociception induced by convulsions elicited by peripheral administration of PTZ (64 mg / kg). The
analgesia was measured by the tail-flick test, in eight rats per group. Convulsions were followed by significant increase in the tail-flick
latencies (TFL), at least for 30 min of the post-ictal period. Peripheral administration of naloxone (5 mg / kg and 10 mg / kg), atropine (1
mg / kg and 5 mg / kg), methysergide (1 mg / kg and 5 mg / kg) and ketanserine (1 mg / kg and 2 mg / kg) caused a significant decrease in the
TFL in seizing animals, as compared to controls. However, while naloxone antagonized analgesia 15 and 25 min post convulsions, the
other drugs caused a blockade of the post-ictal analgesia in a relatively greater period of time. These results indicate that endogenous
opioids, serotonin and acetylcholine may be involved in post-ictal analgesia. 2001 Elsevier Science B.V. All rights reserved.
Theme: Neurotransmitters, modulators, transporters and receptors
Topic: GABA
Keywords: Post-ictal analgesia; Pentylenetetrazol; GABAA receptor; 5-HT 2 receptor; Endogenous opioid; Muscarinic receptor; Tail-flick test
A recent finding in the literature, demonstrates that in
patients with temporal lobe epilepsy, the nociception
threshold seems to be spontaneously high but is not
antagonized by pretreatment with naloxone [13]. However,
the analgesia that follows experimentally induced convulsive seizures still needs to be clarified. PTZ is a GABAergic non-competitive antagonist that does not interact
directly with GABA receptors, but blocks the GABAmediated Cl 2 influx. The intraperitoneal (i.p.) injection of
PTZ, in rats, causes tonic–clonic seizures [8,19].
The study of GABAergic, opioid, serotonergic and
cholinergic mechanisms can offer elucidative data about
the neurochemistry of the post-ictal analgesia. In fact,
endogenous opioids, serotonin and acetylcholine seem to
be critically implicated in antinociceptive processes in*Corresponding author.
E-mail address: nccoimbr@fmrp.usp.br (N.C. Coimbra).
duced by stimulation of structures where it is also possible
to induce convulsive seizures [1,2,5,14,20].
In this work we tried to demonstrate whether or not
seizures induced by drugs, such as PTZ, might be followed
by antinociception. After the detection of post-ictal analgesia, the correlated neurochemistry was investigated using
some non-specific pharmacological antagonists, such as
naloxone and methysergide, and specific ones, such as
ketanserin and atropine. Thus, the involvement of some
receptors subtypes such as cholinergic muscarinic and
those from the serotonergic 5-HT 2 subfamily, were also
considered.
Wistar albino rats, weighing between 200 and 250 g,
four in a cage, had free access to food and water. All
protocols were used according to the rules for animal
experimentation of the Brazilian Society for Neuroscience
and Behavior.
All the rats had their nociception thresholds compared,
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved.
PII: S0006-8993( 00 )03103-6
N.C. Coimbra et al. / Brain Research 888 (2001) 314 – 320
using the tail flick test. Each animal was placed in a
restraining apparatus (Stoelting) with acrylic walls, and its
tail was placed in a heating sensor (tail-flick Analgesia
Instrument; Stoelting), whose calorimetric progressive
elevation was automatically interrupted, as long as the
animal took out his tail of the apparatus. A small current
intensity adjustment could be done, if necessary, in the
beginning of the experiment, aiming to obtain three
consecutive tail-flick latencies (TFL), between 2.5 s and
3.5 s. If the animal did not remove his tail out of the heater
within 6 s, the apparatus would be turned off in order to
prevent damage to the skin. All the tail flick latencies were
normalized in an index of analgesia (IA) using the
formula:
]]
(TFL test ) 2 (TFL control )
IA 5 ]]]]]]]
]]
6 2 (TFL control )
Three baselines of control tail-flick latencies were taken
at 5-min intervals. Tail-flick latencies were also measured
following seizures elicited by peripheral administrations of
PTZ.
Behavioral tests were made in the interior of a circular
arena, whose walls, made with transparent acrylic, measured 60 cm diameter and 50 cm height, and the floor was
divided in 12 equal sections. This arena was located in an
experimental compartment and illuminated by a fluorescent
lamp (350 lux at the arena floor level).
The evaluation of the effects of drugs administration
(PTZ, naloxone, methysergide, ketanserine, atropine and
saline) as well as the ethogram (analysis of motor behavior
in the open-field), and the recording of the righting
reflexes, were made in the interior of this apparatus.
In the post-ictal period, the motor performance of each
animal, in another group, was evaluated using the rota-rod
test. Animals were taken out of the arena and placed in the
rota-rod apparatus (Ugo Basile 7750 model) that consists
in an acrylic cylinder, divided in equal compartments,
placed 40 cm away from a semi-suspended plaque. This
interrupted a digital counter when activated by the rat’s
weight, during its fall in the cylinder. Latencies of the
permanence of each animal in the cylinder in movement
could be recorded after peripheral administration of either
PTZ or its vehicle.
During the tests, animals were gently placed above a
cylinder that was immediately started. Following that, the
permanence time in the cylinder in movement was also
recorded. Immediately after this recording, animals were
removed away and placed again in the arena, in order to
continue the ethological analysis.
First of all, a baseline of the tail-flick test was made in
every animal of each group. Animals of the control group
were taken out of the box and placed in the open-field,
without receiving drug injection and observed in a 15-min
time window. After this period, animals were taken out of
the arena and had their nociception threshold standardized
through the tail-flick test.
315
Two other groups received either PTZ or saline intraperitoneally (i.p.). After receiving the drug, animals were
placed in the arena, until the end of seizures (PTZ), or 15
min after saline, i.p., when their tail-flick latencies were
determined.
Independent groups of animals received peripheral
administrations of naloxone, atropine, methysergide, ketanserin or saline followed by, after 10 min, i.p. PTZ
administration. The nociceptive responses (threshold) were
measured immediately after seizures and after 5, 15, 25
and 30 min following the convulsion.
Aiming to investigate occasional motor alterations that
could interfere in the motor reflexes, another group of
animals were injected with either PTZ (i.p.) or its vehicle,
the animals were placed in the open-field, and observed up
to 30 min for the following behaviors: crossing (entrance
of the four paws in one of 12 compartments of the
open-field), turnings (3608 rotations), rearings (rearing of
the front paws, supporting in the arena’s walls), limbic
behavior (smelling out and chewing movements, with
lateral or vertical head movements), and body cleaning
(grooming). This ethological analysis was made in a 30
min period of time.
The motor performance of the animals was also investigated by means of the rota-rod test. After receiving i.p.
administrations of PTZ or saline, animals were taken to the
rota-rod, where was recorded the permanence time after
seizures (or 1 min after saline administration) and 5, 15,
25, 30 min after the drug. Putting animals in dorsal
decubit, the righting reflexes were evaluated in both
experimental groups immediately after seizures or 1 min
after saline administration. Following that, the time spent
in the righting operation was recorded.
PTZ (Sigma), naloxone (Sigma), atropine (Sigma),
methysergide (RBI) and ketanserin (RBI) were each
dissolved in physiological saline (NaCl; 0.9%) shortly
before use. Physiological saline also served as vehicle
control.
Drugs were administered in the following doses: naloxone (5 and 10 mg / kg), atropine (1 and 5 mg / kg),
methysergide (1 and 5 mg / kg), ketanserin (1 and 2 mg /
kg) and PTZ (64 mg / kg).
The data were analyzed using the Mann–Whitney Utest, because the patterns did not follow a normal distribution and the groups were independent. A level of
P,0.05 was used to confirm statistically significant differences.
PTZ induced severe tonic–clonic seizures in all animals.
Convulsions were not preceded by wild running, and lasted
from 20 s up to 80 s.
Control animals submitted to tail-flick test and placed
also in the experimental situation for 10 min, without
receiving any type of drugs afterwards, did not display
significant changes in the nociceptive threshold at any time
studied (P.0.05 in all cases).
The saline pre-treatment, used in another experimental
316
N.C. Coimbra et al. / Brain Research 888 (2001) 314 – 320
group, also did not induce any significant change in the
nociceptive threshold during the time observed, 1 min after
saline administration (Mann–Whitney: U 523.5; P.0.05),
5 (Mann–Whitney: U 514; P.0.05), 15 (Mann–Whitney:
U 515.5; P.0.05), 25 (Mann–Whitney: U 515.5; P.
0.05) and 30 min (Mann–Whitney: U 524; P.0.05) after
the drug, when compared to the control group.
However, after the generalized tonic–clonic seizures
induced by PTZ we observed a strong analgesia, supported
by increased tail-flick latencies, recorded immediately after
the end of seizures (Mann–Whitney: U 50.0; P,0.001), 5
(Mann–Whitney: U 50.0; P,0.01), 15 (Mann–Whitney:
U 50.0; P,0.001), 25 (Mann–Whitney: U 50.0; P,
0.001) and 30 min (Mann–Whitney: U 50.0; P,0.01)
after seizures. This effect was also highly significant
statistically after the saline pre-treatment, followed, after
10 min, by PTZ. Also in this experiment, which was used
as a control for the neurochemistry study, it was detected
an expressive post-ictal analgesia (Mann–Whitney: U 5
0.0; P,0.001 in all post-ictal periods studied).
Immediately (Whitney: U 520; P.0.05), and 5 min
(Mann–Whitney: U 519.5; P.0.05) after seizures, although there was a tendency to be antagonized by naloxone, this effect was not statistically significant. However,
naloxone pre-treatment (5 mg / kg) was effective in antagonizing the post-ictal analgesia recorded at 15 (Mann–
Whitney: U 513; P,0.05) and 25 (Mann–Whitney: U 5
13; P,0.05) min after convulsion. Although there was
also a tendency to antagonism in the 30 min post-seizure,
this difference was not statistically significant (Mann–
Whitney: U 513.5; P50.051). Nevertheless, the pre-treatment, with naloxone (10 mg / kg), was effective in antagonizing the post-ictal analgesia recorded immediately
(Mann–Whitney: U 55; P,0.01), 5 (Mann–Whitney: U 5
3; P,0.01), 15 (Mann–Whitney: U 55; P,0.01), 25
(Mann–Whitney: U 52; P,0.01) and 30 min (Mann–
Whitney: U 54; P,0.01) after seizures. These data followed a dose-dependent effect.
When the involvement of others transmitters in the
post-ictal analgesia were studied, we observed that, the
pre-treatment with atropine (1 mg / kg and 5 mg / kg), was
capable of antagonizing the post-ictal antinociception,
mainly at 5 (Mann–Whitney: U 51 and 11; P,0.001,
P,0.05, respectively), 15 (Mann–Whitney: U 58 and 9;
P,0.05 and P,0.02, respectively), 25 (Mann–Whitney:
U 52 and 13; P,0.01 and P,0.05, respectively) and 30
min (Mann–Whitney: U 51 and 3, respectively; P,0.01
in both cases) after convulsions. These data suggest that in
contrast to naloxone, atropine seems to have reached a
maximum effect in low doses. The data suggest the
involvement of acetylcholine in different stages of this
antinociceptive process.
The pre-treatment with methysergide (1 mg / kg), was
capable of antagonizing post-ictal antinociception at 5
(Mann–Whitney: U 513.5; P,0.05) and 25 min (Mann–
Whitney: U 50.5; P,0.001) after seizures. Although there
was a tendency to induce antagonism immediately after
convulsive reactions (Mann–Whitney: U 524; P.0.05) at
15 (Mann–Whitney: U 526; P.0.05) and 30 min (Mann–
Whitney: U 528.5; P.0.05), this effect was not statistically significant.
Methysergide pre-treatment (5 mg / kg), was capable of
antagonizing the post-ictal antinociception immediately
(Mann–Whitney: U 510; P,0.05), 5 (Mann–Whitney:
U 514; P,0.05), 25 (Mann–Whitney: U 56.5; P,0.01)
and 30 min (Mann–Whitney: U 510.5; P,0.05) after
seizures. At 15 min of the post-ictal period, however, there
was a tendency to antagonism, but this effect was not
statistically significant (Mann–Whitney: U 510.5; P.
0.05). These data follow a dose-dependent effect.
Pre-treatment with ketanserin (1 mg / kg), a specific 5
HT 2 / 5HT 1C antagonist, was effective in antagonizing the
post-ictal analgesia immediately after seizures (Mann–
Whitney: U 515; P,0.05), 25 (Mann–Whitney: U 59;
P,0.05) and 30 min (Mann–Whitney: U 57.5; P,0.01)
after seizures. However there was a tendency to antagonism at 5 (Mann–Whitney: U 524; P.0.05) and 15 min
(Mann–Whitney: U 524; P.0.05) post-convulsion, but
these differences were not statistically significant.
Kentanserin pre-treatment (2 mg / kg) was effective also
in antagonizing the post-ictal antinociception in the different stages of this antinociceptive process. This effect was
extremely consistent immediately after seizures (Mann–
Whitney: U 55; P,0.01) and at 5 (Mann–Whitney: U 59;
P,0.05), 15 (Mann–Whitney: U 52; P,0.01), 25
(Mann–Whitney: U 57; P,0.01) or 30 min (Mann–Whitney: U 54; P,0.01) after convulsion. These data follow
clearly a dose–effect curve. All psychopharmacological
effects were presented in Fig. 1.
Righting reflexes were evaluated in animals that received saline (recorded in the first minute after drug) or
PTZ (recorded immediately after seizures), detected in
100% of the animals studied in the present work; there was
no statistically significant difference in their latencies,
when compared to controls (data not shown). The evaluation of the motor performance of animals submitted to the
rota-rod test showed no statistically significant differences
between latencies observed immediately after seizures
(Mann–Whitney: U 515.5; P.0.05), at 5 (Mann–Whitney: U 515; P.0.05), 15 (Mann–Whitney: U 517.5; P.
0.05), 25 (Mann–Whitney: U 58.5; P.0.05) and 30 min
(Mann–Whitney: U 513.5; P.0.05) of the post-ictal
period. These data are presented in Fig. 2A.
In the open-field test, we detected a reduction in the
exploratory behavior in the post-ictal period, based on a
significant reduction in rearing numbers (Mann–Whitney:
U 50.0; P,0.01). There was, however, no sign of motor
deficit involvement, as confirmed by the absence of
statistically significant alterations in the number of crossing (Mann–Whitney: U 516; P.0.05), and of rotations
(Mann–Whitney: U 511.5; P.0.05). Corroborating these
data, we observed an increase in the so-called limbic
N.C. Coimbra et al. / Brain Research 888 (2001) 314 – 320
317
Fig. 1. (A) Effects of administration of PTZ (d–d) (64 mg / kg; i.p.) or saline (s–s) on nociceptive threshold (n57; **P,0.01, ***P,0.001 according
to Mann–Whitney’s U-test, as compared to the control). Control group (n–n): animals were placed in the open-field, without any pharmacological
treatment, used as general control, as well as to evaluate the aversive character of either open-field or contention cylinder. (B) Effect of administration of
PTZ (64 mg / kg), preceded by saline (s–s) administration (NaCl, 0.9%) on nociceptive threshold. (n58; ***P,0.001, according to Mann–Whitney’s
U-test, when compared with control). Control group (d–d): animals placed in the arena, without pharmacological treatment, used as general control, as
well as to evaluate the aversive character of either the open field or contention cylinder. (C) Effects of the pretreatment with naloxone in doses of 5 mg / kg
(m–m) and 10 mg / kg (j–j) or its vehicle (s–s) (NaCl, 0.9%) plus PTZ (64 mg / kg) on nociceptive threshold. (n58; *P,0.05, **P,0.01 according
to Mann–Whitney’s U-test). (D) Effects of the pretreatment with atropine, in doses of 1 mg / kg (m–m) and 5 mg / kg (j–j) or its vehicle (s–s) (NaCl,
0.9%) plus PTZ (64 mg / kg) on nociceptive threshold. (n58; *P,0,05, **P,0,01, according to Mann–Whitney’s U-test, when compared with control).
(E) Effect of the pretreatment with methysergide in doses of 1 mg / kg (m–m) and 5 mg / kg (j–j), or its vehicle (s–s) (NaCl, 0.9%) plus PTZ (64
mg / kg) on nociceptive threshold. (n58; *P,0.05, **P,0.01, ***P,0.001, according to Mann–Whitney’s U-test, when compared with control). (F)
Effect of the pretreatment with ketanserin, in doses of 1 mg / kg (m–m) and 5 mg / kg (j–j), or its vehicle (s–s) (NaCl, 0.9%) plus PTZ (64 mg / kg) on
nociceptive threshold. (n58; *P,0.05, **P,0.01, according to Mann–Whitney’s U-test, when compared with control).
318
N.C. Coimbra et al. / Brain Research 888 (2001) 314 – 320
Fig. 2. (A) Lack of effect of pretreatment with PTZ or saline (NaCl,
0.9%) on motor performance of the animals submitted to the rota-rod test.
(n58; P.0,05, when compared with control, according to Mann–
Whitney’s U-test.) Time: recorded window in which animals were on the
cylinder in movement. (B) Effect of the administration of PTZ (64
mg / kg) or saline (NaCl, 0.9%) on the motor performance of the animals
in the open-field test. The ethogram was done by observing diverse
behaviors elicited in the arena after drug’s administration, in a 30-min
period of time. The columns represent mean, and the bars, the S.E.M.
(n58; *P,0.05; **P,0.01, when compared with control, according to
Mann–Whitney’s U-test.) The open columns represent animals which
received i.p. administration of saline; the hatched, those with PTZ.
behaviors (horizontal and vertical head movement followed by smell-out and chewing movements) (Mann–
Whitney: U 53; P,0.01) at the post-ictal period, when
compared to control. However, the group of animals that
received PTZ presented reduction in grooming (Mann–
Whitney: U 57.5; P,0.05). These data are presented in
Fig. 2B.
In the current work, PTZ caused tonic–clonic seizures,
followed by statistically significant antinociception. Our
group recently described that the corpora quadrigemina
receive many GABAergic connections from substantia
nigra, pars reticulata [4,7], suggesting these structures as
strong candidates where PTZ could produce its convulsive
activity.
Since the inferior colliculus [11,12] and superior col-
liculus [17] have been involved in epileptogenic activity, a
consequent morpho-functional situation will be that neural
substrates responsible for the expression of post-ictal
analgesia may be also placed in the mesencephalic tectum
[3]. In support of this hypothesis, we also recently described the presence of neurochemical mechanisms responsible for antinociceptive processes in the mesencephalic
tectum [5,6].
Our results suggest that, although there is no evidence of
opiate involvement in the initial stage of post-ictal analgesia, these neurotransmitters seem to be recruited in a later
stage. Furthermore, at higher doses, naloxone caused a
statistically significant antagonism of post-ictal analgesia,
in all periods studied. It is possible that some opioid
receptor subtypes, such as mu-type, are involved in later
stages of post-ictal analgesia, while other types of opioid
receptors, such as kappa or delta, could be recruited in the
immediate post-ictal period. Naloxone, at higher doses,
may be reaching other receptors, antagonizing, in that
form, the post-ictal analgesia in the first steps. Corroborating these data, several works have suggested that opioid
mechanisms are involved in convulsive reactions [2,8,21].
However, a possible dissociation between endogenous
opioid effects in both analgesia and epilepsy has been
discussed elsewhere [15]. Our current results additionally
point to a cholinergic mechanism recruited in the post-ictal
analgesia. Acetylcholine seems to be crucially involved in
the initial and later stages of antinociception that follows
convulsive reactions. Corroborating these data, acetylcholine has been recently implicated in antinociceptive processes either caused by drugs that competitively inhibit
acetylcholinesterase [16] or elicited by amygdaloid complex stimulation [14], a structure also related to seizures
[12]. The lack of effect of the pre-treatment with opioid
and cholinergic antagonists on tail-flick latencies recorded
immediately after seizures can not be related to a sudden
motor deficit caused by post-ictal depression, because the
study of the motor’s performance of the animal in the
rota-rod test (with previous treatment with saline or PTZ)
did not detect statistically significant differences, immediately after seizures and some minutes later. In agreement
with these data, the ethologic analysis of these animals,
expressed by crossing and turning behavior, in the openfield test, did not detect statistically significant differences,
which could point to an eventual motor alteration caused
by the post-ictal depression period, even with a recorded
decrease of exploratory and grooming behaviors during
this period. Another result that confirms the hypothesis that
the increase in the tail-flick latencies recorded after seizures does not relate to any important motor alteration is
the observation of a significant increase of the so-called
limbic behaviors, such as smell-out and chewing movements, followed by vertical or horizontal head movement
in the post-ictal period. In addition, all the animals studied
presented normal righting reflexes, recorded immediately
after PTZ-induced seizures. This is another element that
N.C. Coimbra et al. / Brain Research 888 (2001) 314 – 320
denies a possible motor deficit that could alter spinal
reflexes.
Naloxone and atropine are opiate and cholinergic competitive antagonists, respectively, and it is possible that
their lack of effect, at lower doses, in antagonizing the
post-ictal analgesia immediately after seizures, is related to
a massive discharge of these neurotransmitters at the postictal period. They could displace blockers from their sites,
and consequently reduce their pharmacological efficiency.
In addition, at least for naloxone, its efficiency in antagonizing the post-ictal analgesia, at a lower dose (5
mg / kg), does not seem to relate to post-ictal depression
effects, because it has been already demonstrated that
naloxone has a property to attenuate it or to abolish it
completely [10]. We should consider that during post-ictal
analgesia, muscarinic cholinergic mechanisms would be
initially recruited, while endogenous opiates such as
enkephalin and endorphin, would have a place in a later
stage of post-seizure antinociception. Meanwhile, mu
opioid receptors may be then involved in this delayed stage
of the post-ictal analgesia.
However, we cannot discard the possibility of involvement of other neurotransmitters in this process, such
as serotonin. Serotoninergic circuits are widely cited in
literature as responsible for neurochemistry of some antinociceptive processes [5,6], and as participants in the
endogenous system of pain inhibition [9,18]. Methysergide, a non-specific blocker of serotoninergic receptors,
was effective in antagonizing the post-ictal analgesia at
different steps of this antinociceptive process. Corroborating these data, peripheral administration of ketanserin, a
specific blocker of 5-HT 2 / 5HT 1C receptors, was equally
effective in antagonizing the post-ictal analgesia in all
post-convulsive periods studied. It is necessary to consider
the possibility of muscarinic cholinergic and serotoninergic
mechanisms involvement in the immediately post-ictal
period, while opioid mechanisms, mediated by mu receptors, would be recruited later on. Serotoninergic receptors in the 5-HT 2 subfamily can be negligible in this
earlier stage of the post-ictal analgesia; although there is a
clear involvement of these receptors in other post-ictal
analgesia stages. We should still consider the possibility of
other serotoninergic receptors’ involvement in this antinociceptive process, as well as other neurotransmitters.
Studies that give support to this possibility are being
conducted in our laboratory.
Acknowledgements
This work was supported by FAPESP (proc. 96 / 8574-9
and 95 / 3604-4). C. Castro-Souza (proc. 96 / 5464-8) and
E.N. Segato (proc. 98 / 07416-6) were the recipients of
fellowships from FAPESP. N.C. Coimbra and N. GarciaCairasco are recipients of Research Fellowships from
CNPq (proc. 300772 / 97 and 521697 / 96-4). The authors
319
are grateful to J.R. Espreafico, M.T. Castania, J.A.C. de
Oliveira and I.R.Violante for expert technical assistance.
J.R. Espreafico was the recipient of a fellowship from
FAPESP (proc. 97 / 10440-3).
References
˜ Defensive reactions
[1] S.H. Cardoso, N.C. Coimbra, M.L. Brandao,
evoked by activation of NMDA receptors in distinct sites of the
inferior colliculus, Behav. Brain Res. 63 (1994) 17–24.
˜ Opposite
[2] S.H. Cardoso, L.L. Mello, N.C. Coimbra, M.L. Brandao,
effects of low and high doses of morphine on neural substrates of
aversion in the inferior colliculus, Behav. Pharmacol. 3 (1992)
489–495.
[3] N.C. Coimbra, C. Castro-Souza, N. Garcia-Cairasco, Neuroanatomical and neurophysiological study of post-ictal antinociceptive processes in experimental models of epilepsy, Arquivos de NeuroPsiquiatria Resumo /Abstracts 56 (1998) 22.
˜ GABAergic nigro-collicular pathways
[4] N.C. Coimbra, M.L. Brandao,
modulate the defensive behavior elicited by midbrain tectum stimulation, Behav. Brain Res. 59 (1993) 131–139.
˜ Effects of 5-HT 2 receptors blockade
[5] N.C. Coimbra, M.L. Brandao,
on fear-induced analgesia elicited by electrical stimulation of the
deep layers of the superior colliculus and dorsal periaqueductal gray,
Behav. Brain Res. 87 (1997) 97–103.
˜ Evidence for the in[6] N.C. Coimbra, C. Tomaz, M.L. Brandao,
volvement of serotonin in the antinociception induced by electrical
or chemical stimulation of the mesencephalic tectum, Behav. Brain
Res. 50 (1992) 77–83.
[7] N.C. Coimbra, M.C. Kawasaki, J.G. Ciscato Jr., S.H. Cardoso,
ˆ Nigro-tectal pathway: neuroanatomy and role on
S.A.L. Correa,
defensive behaviour elicited by midbrain tectum stimulation, Soc.
Neurosci. Abstr. 24 (1998) 1930.
[8] T.C. De Lima, G. A Rae, Effects of cold-restraint and swim stress
on convulsions induced by penthylenetetrazol and electroshock:
influence of naloxone pretreatment, Pharmacol. Biochem. Behav. 40
(1991) 297–300.
[9] H.L. Fields, A.I. Basbaum, Endogenous pain control mechanisms,
in: P.D. Wall, R. Melzack (Eds.), Textbook of Pain, Churchill
Livingstone, Edinburgh, 1989, pp. 206–217.
[10] H. Frenk, B.C. MacCarty, J.C. Liebeskind, Different brain areas
mediate the analgesic and epileptic properties of enkephalin, Science
200 (1978) 335–337.
[11] N. Garcia-Cairasco, R.M. E Sabbatini, Possible interaction between
the inferior colliculus and the substantia nigra in audiogenic seizures
in rats, Physiol. Behav. 50 (1991) 421–427.
[12] N. Garcia-Cairasco, H. Wakamatsu, J.A.C. Oliveira, E.L.T. Gomes,
E. A Del Bel, L.E.A. M Mello, Neuroethological and morphological
(neo-Timm staining) correlates of limbic recruitment during the
development of audiogenic kindling in seizures susceptible Wistar
rats, Epilepsy Res. 26 (1996) 177–192.
[13] R. Guieu, E. Mesdjian, J. Roger, P. Dano, J. Pouget, G. Serratice,
Nociceptive threshold in patients with epilepsy, Epilepsy Res. 12
(1992) 57–61.
[14] M.A. Oliveira, W. A Prado, Antinociception and behavioral manifestations induced by intracerebroventricular or intra-amygdaloid
administration of cholinergic agonist in the rat, Pain 57 (1994)
383–391.
[15] F. Pavone, C. Castellano, A. Oliverio, Stram-dependent effects of
shock-induced release of opioids: dissociation between analgesia
and behavioral seizures, Brain Res. 366 (1986) 326–328.
[16] M.E. Pereira, J.B.T. Rocha, I. Izquierdo, Atropine reverses antinociception induced by 2,5-hexanedione in rats, Brain Res. 77
(1995) 91–94.
320
N.C. Coimbra et al. / Brain Research 888 (2001) 314 – 320
[17] C.E. Ribak, A.L. Manio, M.S. Navetta, C. M Gall, In situ hybridization for c-fos mRNA reveals the involvement of the superior
colliculus in the propagation of seizure activity in genetically
epilepsy-prone rats, Epilepsy Res. 26 (1997) 397–406.
[18] M.L.N.M. Rosa, M.A. Oliveira, R.B. Valente, N.C. Coimbra, W.A.
Prado, Pharmacological and neuroanatomical evidence for the
involvement of the anterior pretectal nucleus in the antinociception
induced by stimulation of the dorsal raphe nucleus in rats, Pain 74
(1998) 171–179.
[19] P.R. Schofield, Sequence and functional expression of the GABA-A
receptor shows a ligand-gated receptor super-family, Nature 328
(1987) 221–227.
[20] V.C. Terra, N. Garcia-Cairasco, NMDA-dependent audiogenic seizures are differentially regulated by inferior colliculus subnuclei,
Behav. Brain Res. 62 (1994) 129–139.
[21] B.C. Yoburn, K. Lutfy, V. Sierra, F.C. Tortella, Tolerance develops
to spinal morphine analgesia but not morphine-induced convulsions,
Eur. J. Pharmacol. 176 (1990) 63–67.
www.elsevier.com / locate / bres
Short communication
Post-ictal analgesia: involvement of opioid, serotoninergic and
cholinergic mechanisms
a,
a
a
a
a
N.C. Coimbra *, C. Castro-Souza , E.N. Segato , J.E.P. Nora , C.F.P.S. Herrero ,
a
b
W. Tedeschi-Filho , N. Garcia-Cairasco
a
˜ Preto,
´
Laboratorio
de Neuroanatomia e Neuropsicobiologia, Departamento de Farmacologia, Faculdade de Medicina de Ribeirao
˜ Paulo ( USP), 14049 -900, Ribeirao
˜ Preto ( SP), Brazil
Universidade de Sao
b
˜ Preto ( SP), Brazil
´
Laboratorio
de Neurofisiologia e Neuroetologia Experimental, Departamento de Fisiologia, FMRP-USP, 14049 -900, Ribeirao
Accepted 10 October 2000
Abstract
The neural mechanisms involved in post-ictal analgesia remain to be elucidated. Pentylenetetrazol (PTZ) is used experimentally to
induce seizure in animal subjects. This non-competitive antagonist blocks GABA-mediated Cl 2 flux. The aim of this work is to study the
neurochemical basis of the antinociception induced by convulsions elicited by peripheral administration of PTZ (64 mg / kg). The
analgesia was measured by the tail-flick test, in eight rats per group. Convulsions were followed by significant increase in the tail-flick
latencies (TFL), at least for 30 min of the post-ictal period. Peripheral administration of naloxone (5 mg / kg and 10 mg / kg), atropine (1
mg / kg and 5 mg / kg), methysergide (1 mg / kg and 5 mg / kg) and ketanserine (1 mg / kg and 2 mg / kg) caused a significant decrease in the
TFL in seizing animals, as compared to controls. However, while naloxone antagonized analgesia 15 and 25 min post convulsions, the
other drugs caused a blockade of the post-ictal analgesia in a relatively greater period of time. These results indicate that endogenous
opioids, serotonin and acetylcholine may be involved in post-ictal analgesia. 2001 Elsevier Science B.V. All rights reserved.
Theme: Neurotransmitters, modulators, transporters and receptors
Topic: GABA
Keywords: Post-ictal analgesia; Pentylenetetrazol; GABAA receptor; 5-HT 2 receptor; Endogenous opioid; Muscarinic receptor; Tail-flick test
A recent finding in the literature, demonstrates that in
patients with temporal lobe epilepsy, the nociception
threshold seems to be spontaneously high but is not
antagonized by pretreatment with naloxone [13]. However,
the analgesia that follows experimentally induced convulsive seizures still needs to be clarified. PTZ is a GABAergic non-competitive antagonist that does not interact
directly with GABA receptors, but blocks the GABAmediated Cl 2 influx. The intraperitoneal (i.p.) injection of
PTZ, in rats, causes tonic–clonic seizures [8,19].
The study of GABAergic, opioid, serotonergic and
cholinergic mechanisms can offer elucidative data about
the neurochemistry of the post-ictal analgesia. In fact,
endogenous opioids, serotonin and acetylcholine seem to
be critically implicated in antinociceptive processes in*Corresponding author.
E-mail address: nccoimbr@fmrp.usp.br (N.C. Coimbra).
duced by stimulation of structures where it is also possible
to induce convulsive seizures [1,2,5,14,20].
In this work we tried to demonstrate whether or not
seizures induced by drugs, such as PTZ, might be followed
by antinociception. After the detection of post-ictal analgesia, the correlated neurochemistry was investigated using
some non-specific pharmacological antagonists, such as
naloxone and methysergide, and specific ones, such as
ketanserin and atropine. Thus, the involvement of some
receptors subtypes such as cholinergic muscarinic and
those from the serotonergic 5-HT 2 subfamily, were also
considered.
Wistar albino rats, weighing between 200 and 250 g,
four in a cage, had free access to food and water. All
protocols were used according to the rules for animal
experimentation of the Brazilian Society for Neuroscience
and Behavior.
All the rats had their nociception thresholds compared,
0006-8993 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved.
PII: S0006-8993( 00 )03103-6
N.C. Coimbra et al. / Brain Research 888 (2001) 314 – 320
using the tail flick test. Each animal was placed in a
restraining apparatus (Stoelting) with acrylic walls, and its
tail was placed in a heating sensor (tail-flick Analgesia
Instrument; Stoelting), whose calorimetric progressive
elevation was automatically interrupted, as long as the
animal took out his tail of the apparatus. A small current
intensity adjustment could be done, if necessary, in the
beginning of the experiment, aiming to obtain three
consecutive tail-flick latencies (TFL), between 2.5 s and
3.5 s. If the animal did not remove his tail out of the heater
within 6 s, the apparatus would be turned off in order to
prevent damage to the skin. All the tail flick latencies were
normalized in an index of analgesia (IA) using the
formula:
]]
(TFL test ) 2 (TFL control )
IA 5 ]]]]]]]
]]
6 2 (TFL control )
Three baselines of control tail-flick latencies were taken
at 5-min intervals. Tail-flick latencies were also measured
following seizures elicited by peripheral administrations of
PTZ.
Behavioral tests were made in the interior of a circular
arena, whose walls, made with transparent acrylic, measured 60 cm diameter and 50 cm height, and the floor was
divided in 12 equal sections. This arena was located in an
experimental compartment and illuminated by a fluorescent
lamp (350 lux at the arena floor level).
The evaluation of the effects of drugs administration
(PTZ, naloxone, methysergide, ketanserine, atropine and
saline) as well as the ethogram (analysis of motor behavior
in the open-field), and the recording of the righting
reflexes, were made in the interior of this apparatus.
In the post-ictal period, the motor performance of each
animal, in another group, was evaluated using the rota-rod
test. Animals were taken out of the arena and placed in the
rota-rod apparatus (Ugo Basile 7750 model) that consists
in an acrylic cylinder, divided in equal compartments,
placed 40 cm away from a semi-suspended plaque. This
interrupted a digital counter when activated by the rat’s
weight, during its fall in the cylinder. Latencies of the
permanence of each animal in the cylinder in movement
could be recorded after peripheral administration of either
PTZ or its vehicle.
During the tests, animals were gently placed above a
cylinder that was immediately started. Following that, the
permanence time in the cylinder in movement was also
recorded. Immediately after this recording, animals were
removed away and placed again in the arena, in order to
continue the ethological analysis.
First of all, a baseline of the tail-flick test was made in
every animal of each group. Animals of the control group
were taken out of the box and placed in the open-field,
without receiving drug injection and observed in a 15-min
time window. After this period, animals were taken out of
the arena and had their nociception threshold standardized
through the tail-flick test.
315
Two other groups received either PTZ or saline intraperitoneally (i.p.). After receiving the drug, animals were
placed in the arena, until the end of seizures (PTZ), or 15
min after saline, i.p., when their tail-flick latencies were
determined.
Independent groups of animals received peripheral
administrations of naloxone, atropine, methysergide, ketanserin or saline followed by, after 10 min, i.p. PTZ
administration. The nociceptive responses (threshold) were
measured immediately after seizures and after 5, 15, 25
and 30 min following the convulsion.
Aiming to investigate occasional motor alterations that
could interfere in the motor reflexes, another group of
animals were injected with either PTZ (i.p.) or its vehicle,
the animals were placed in the open-field, and observed up
to 30 min for the following behaviors: crossing (entrance
of the four paws in one of 12 compartments of the
open-field), turnings (3608 rotations), rearings (rearing of
the front paws, supporting in the arena’s walls), limbic
behavior (smelling out and chewing movements, with
lateral or vertical head movements), and body cleaning
(grooming). This ethological analysis was made in a 30
min period of time.
The motor performance of the animals was also investigated by means of the rota-rod test. After receiving i.p.
administrations of PTZ or saline, animals were taken to the
rota-rod, where was recorded the permanence time after
seizures (or 1 min after saline administration) and 5, 15,
25, 30 min after the drug. Putting animals in dorsal
decubit, the righting reflexes were evaluated in both
experimental groups immediately after seizures or 1 min
after saline administration. Following that, the time spent
in the righting operation was recorded.
PTZ (Sigma), naloxone (Sigma), atropine (Sigma),
methysergide (RBI) and ketanserin (RBI) were each
dissolved in physiological saline (NaCl; 0.9%) shortly
before use. Physiological saline also served as vehicle
control.
Drugs were administered in the following doses: naloxone (5 and 10 mg / kg), atropine (1 and 5 mg / kg),
methysergide (1 and 5 mg / kg), ketanserin (1 and 2 mg /
kg) and PTZ (64 mg / kg).
The data were analyzed using the Mann–Whitney Utest, because the patterns did not follow a normal distribution and the groups were independent. A level of
P,0.05 was used to confirm statistically significant differences.
PTZ induced severe tonic–clonic seizures in all animals.
Convulsions were not preceded by wild running, and lasted
from 20 s up to 80 s.
Control animals submitted to tail-flick test and placed
also in the experimental situation for 10 min, without
receiving any type of drugs afterwards, did not display
significant changes in the nociceptive threshold at any time
studied (P.0.05 in all cases).
The saline pre-treatment, used in another experimental
316
N.C. Coimbra et al. / Brain Research 888 (2001) 314 – 320
group, also did not induce any significant change in the
nociceptive threshold during the time observed, 1 min after
saline administration (Mann–Whitney: U 523.5; P.0.05),
5 (Mann–Whitney: U 514; P.0.05), 15 (Mann–Whitney:
U 515.5; P.0.05), 25 (Mann–Whitney: U 515.5; P.
0.05) and 30 min (Mann–Whitney: U 524; P.0.05) after
the drug, when compared to the control group.
However, after the generalized tonic–clonic seizures
induced by PTZ we observed a strong analgesia, supported
by increased tail-flick latencies, recorded immediately after
the end of seizures (Mann–Whitney: U 50.0; P,0.001), 5
(Mann–Whitney: U 50.0; P,0.01), 15 (Mann–Whitney:
U 50.0; P,0.001), 25 (Mann–Whitney: U 50.0; P,
0.001) and 30 min (Mann–Whitney: U 50.0; P,0.01)
after seizures. This effect was also highly significant
statistically after the saline pre-treatment, followed, after
10 min, by PTZ. Also in this experiment, which was used
as a control for the neurochemistry study, it was detected
an expressive post-ictal analgesia (Mann–Whitney: U 5
0.0; P,0.001 in all post-ictal periods studied).
Immediately (Whitney: U 520; P.0.05), and 5 min
(Mann–Whitney: U 519.5; P.0.05) after seizures, although there was a tendency to be antagonized by naloxone, this effect was not statistically significant. However,
naloxone pre-treatment (5 mg / kg) was effective in antagonizing the post-ictal analgesia recorded at 15 (Mann–
Whitney: U 513; P,0.05) and 25 (Mann–Whitney: U 5
13; P,0.05) min after convulsion. Although there was
also a tendency to antagonism in the 30 min post-seizure,
this difference was not statistically significant (Mann–
Whitney: U 513.5; P50.051). Nevertheless, the pre-treatment, with naloxone (10 mg / kg), was effective in antagonizing the post-ictal analgesia recorded immediately
(Mann–Whitney: U 55; P,0.01), 5 (Mann–Whitney: U 5
3; P,0.01), 15 (Mann–Whitney: U 55; P,0.01), 25
(Mann–Whitney: U 52; P,0.01) and 30 min (Mann–
Whitney: U 54; P,0.01) after seizures. These data followed a dose-dependent effect.
When the involvement of others transmitters in the
post-ictal analgesia were studied, we observed that, the
pre-treatment with atropine (1 mg / kg and 5 mg / kg), was
capable of antagonizing the post-ictal antinociception,
mainly at 5 (Mann–Whitney: U 51 and 11; P,0.001,
P,0.05, respectively), 15 (Mann–Whitney: U 58 and 9;
P,0.05 and P,0.02, respectively), 25 (Mann–Whitney:
U 52 and 13; P,0.01 and P,0.05, respectively) and 30
min (Mann–Whitney: U 51 and 3, respectively; P,0.01
in both cases) after convulsions. These data suggest that in
contrast to naloxone, atropine seems to have reached a
maximum effect in low doses. The data suggest the
involvement of acetylcholine in different stages of this
antinociceptive process.
The pre-treatment with methysergide (1 mg / kg), was
capable of antagonizing post-ictal antinociception at 5
(Mann–Whitney: U 513.5; P,0.05) and 25 min (Mann–
Whitney: U 50.5; P,0.001) after seizures. Although there
was a tendency to induce antagonism immediately after
convulsive reactions (Mann–Whitney: U 524; P.0.05) at
15 (Mann–Whitney: U 526; P.0.05) and 30 min (Mann–
Whitney: U 528.5; P.0.05), this effect was not statistically significant.
Methysergide pre-treatment (5 mg / kg), was capable of
antagonizing the post-ictal antinociception immediately
(Mann–Whitney: U 510; P,0.05), 5 (Mann–Whitney:
U 514; P,0.05), 25 (Mann–Whitney: U 56.5; P,0.01)
and 30 min (Mann–Whitney: U 510.5; P,0.05) after
seizures. At 15 min of the post-ictal period, however, there
was a tendency to antagonism, but this effect was not
statistically significant (Mann–Whitney: U 510.5; P.
0.05). These data follow a dose-dependent effect.
Pre-treatment with ketanserin (1 mg / kg), a specific 5
HT 2 / 5HT 1C antagonist, was effective in antagonizing the
post-ictal analgesia immediately after seizures (Mann–
Whitney: U 515; P,0.05), 25 (Mann–Whitney: U 59;
P,0.05) and 30 min (Mann–Whitney: U 57.5; P,0.01)
after seizures. However there was a tendency to antagonism at 5 (Mann–Whitney: U 524; P.0.05) and 15 min
(Mann–Whitney: U 524; P.0.05) post-convulsion, but
these differences were not statistically significant.
Kentanserin pre-treatment (2 mg / kg) was effective also
in antagonizing the post-ictal antinociception in the different stages of this antinociceptive process. This effect was
extremely consistent immediately after seizures (Mann–
Whitney: U 55; P,0.01) and at 5 (Mann–Whitney: U 59;
P,0.05), 15 (Mann–Whitney: U 52; P,0.01), 25
(Mann–Whitney: U 57; P,0.01) or 30 min (Mann–Whitney: U 54; P,0.01) after convulsion. These data follow
clearly a dose–effect curve. All psychopharmacological
effects were presented in Fig. 1.
Righting reflexes were evaluated in animals that received saline (recorded in the first minute after drug) or
PTZ (recorded immediately after seizures), detected in
100% of the animals studied in the present work; there was
no statistically significant difference in their latencies,
when compared to controls (data not shown). The evaluation of the motor performance of animals submitted to the
rota-rod test showed no statistically significant differences
between latencies observed immediately after seizures
(Mann–Whitney: U 515.5; P.0.05), at 5 (Mann–Whitney: U 515; P.0.05), 15 (Mann–Whitney: U 517.5; P.
0.05), 25 (Mann–Whitney: U 58.5; P.0.05) and 30 min
(Mann–Whitney: U 513.5; P.0.05) of the post-ictal
period. These data are presented in Fig. 2A.
In the open-field test, we detected a reduction in the
exploratory behavior in the post-ictal period, based on a
significant reduction in rearing numbers (Mann–Whitney:
U 50.0; P,0.01). There was, however, no sign of motor
deficit involvement, as confirmed by the absence of
statistically significant alterations in the number of crossing (Mann–Whitney: U 516; P.0.05), and of rotations
(Mann–Whitney: U 511.5; P.0.05). Corroborating these
data, we observed an increase in the so-called limbic
N.C. Coimbra et al. / Brain Research 888 (2001) 314 – 320
317
Fig. 1. (A) Effects of administration of PTZ (d–d) (64 mg / kg; i.p.) or saline (s–s) on nociceptive threshold (n57; **P,0.01, ***P,0.001 according
to Mann–Whitney’s U-test, as compared to the control). Control group (n–n): animals were placed in the open-field, without any pharmacological
treatment, used as general control, as well as to evaluate the aversive character of either open-field or contention cylinder. (B) Effect of administration of
PTZ (64 mg / kg), preceded by saline (s–s) administration (NaCl, 0.9%) on nociceptive threshold. (n58; ***P,0.001, according to Mann–Whitney’s
U-test, when compared with control). Control group (d–d): animals placed in the arena, without pharmacological treatment, used as general control, as
well as to evaluate the aversive character of either the open field or contention cylinder. (C) Effects of the pretreatment with naloxone in doses of 5 mg / kg
(m–m) and 10 mg / kg (j–j) or its vehicle (s–s) (NaCl, 0.9%) plus PTZ (64 mg / kg) on nociceptive threshold. (n58; *P,0.05, **P,0.01 according
to Mann–Whitney’s U-test). (D) Effects of the pretreatment with atropine, in doses of 1 mg / kg (m–m) and 5 mg / kg (j–j) or its vehicle (s–s) (NaCl,
0.9%) plus PTZ (64 mg / kg) on nociceptive threshold. (n58; *P,0,05, **P,0,01, according to Mann–Whitney’s U-test, when compared with control).
(E) Effect of the pretreatment with methysergide in doses of 1 mg / kg (m–m) and 5 mg / kg (j–j), or its vehicle (s–s) (NaCl, 0.9%) plus PTZ (64
mg / kg) on nociceptive threshold. (n58; *P,0.05, **P,0.01, ***P,0.001, according to Mann–Whitney’s U-test, when compared with control). (F)
Effect of the pretreatment with ketanserin, in doses of 1 mg / kg (m–m) and 5 mg / kg (j–j), or its vehicle (s–s) (NaCl, 0.9%) plus PTZ (64 mg / kg) on
nociceptive threshold. (n58; *P,0.05, **P,0.01, according to Mann–Whitney’s U-test, when compared with control).
318
N.C. Coimbra et al. / Brain Research 888 (2001) 314 – 320
Fig. 2. (A) Lack of effect of pretreatment with PTZ or saline (NaCl,
0.9%) on motor performance of the animals submitted to the rota-rod test.
(n58; P.0,05, when compared with control, according to Mann–
Whitney’s U-test.) Time: recorded window in which animals were on the
cylinder in movement. (B) Effect of the administration of PTZ (64
mg / kg) or saline (NaCl, 0.9%) on the motor performance of the animals
in the open-field test. The ethogram was done by observing diverse
behaviors elicited in the arena after drug’s administration, in a 30-min
period of time. The columns represent mean, and the bars, the S.E.M.
(n58; *P,0.05; **P,0.01, when compared with control, according to
Mann–Whitney’s U-test.) The open columns represent animals which
received i.p. administration of saline; the hatched, those with PTZ.
behaviors (horizontal and vertical head movement followed by smell-out and chewing movements) (Mann–
Whitney: U 53; P,0.01) at the post-ictal period, when
compared to control. However, the group of animals that
received PTZ presented reduction in grooming (Mann–
Whitney: U 57.5; P,0.05). These data are presented in
Fig. 2B.
In the current work, PTZ caused tonic–clonic seizures,
followed by statistically significant antinociception. Our
group recently described that the corpora quadrigemina
receive many GABAergic connections from substantia
nigra, pars reticulata [4,7], suggesting these structures as
strong candidates where PTZ could produce its convulsive
activity.
Since the inferior colliculus [11,12] and superior col-
liculus [17] have been involved in epileptogenic activity, a
consequent morpho-functional situation will be that neural
substrates responsible for the expression of post-ictal
analgesia may be also placed in the mesencephalic tectum
[3]. In support of this hypothesis, we also recently described the presence of neurochemical mechanisms responsible for antinociceptive processes in the mesencephalic
tectum [5,6].
Our results suggest that, although there is no evidence of
opiate involvement in the initial stage of post-ictal analgesia, these neurotransmitters seem to be recruited in a later
stage. Furthermore, at higher doses, naloxone caused a
statistically significant antagonism of post-ictal analgesia,
in all periods studied. It is possible that some opioid
receptor subtypes, such as mu-type, are involved in later
stages of post-ictal analgesia, while other types of opioid
receptors, such as kappa or delta, could be recruited in the
immediate post-ictal period. Naloxone, at higher doses,
may be reaching other receptors, antagonizing, in that
form, the post-ictal analgesia in the first steps. Corroborating these data, several works have suggested that opioid
mechanisms are involved in convulsive reactions [2,8,21].
However, a possible dissociation between endogenous
opioid effects in both analgesia and epilepsy has been
discussed elsewhere [15]. Our current results additionally
point to a cholinergic mechanism recruited in the post-ictal
analgesia. Acetylcholine seems to be crucially involved in
the initial and later stages of antinociception that follows
convulsive reactions. Corroborating these data, acetylcholine has been recently implicated in antinociceptive processes either caused by drugs that competitively inhibit
acetylcholinesterase [16] or elicited by amygdaloid complex stimulation [14], a structure also related to seizures
[12]. The lack of effect of the pre-treatment with opioid
and cholinergic antagonists on tail-flick latencies recorded
immediately after seizures can not be related to a sudden
motor deficit caused by post-ictal depression, because the
study of the motor’s performance of the animal in the
rota-rod test (with previous treatment with saline or PTZ)
did not detect statistically significant differences, immediately after seizures and some minutes later. In agreement
with these data, the ethologic analysis of these animals,
expressed by crossing and turning behavior, in the openfield test, did not detect statistically significant differences,
which could point to an eventual motor alteration caused
by the post-ictal depression period, even with a recorded
decrease of exploratory and grooming behaviors during
this period. Another result that confirms the hypothesis that
the increase in the tail-flick latencies recorded after seizures does not relate to any important motor alteration is
the observation of a significant increase of the so-called
limbic behaviors, such as smell-out and chewing movements, followed by vertical or horizontal head movement
in the post-ictal period. In addition, all the animals studied
presented normal righting reflexes, recorded immediately
after PTZ-induced seizures. This is another element that
N.C. Coimbra et al. / Brain Research 888 (2001) 314 – 320
denies a possible motor deficit that could alter spinal
reflexes.
Naloxone and atropine are opiate and cholinergic competitive antagonists, respectively, and it is possible that
their lack of effect, at lower doses, in antagonizing the
post-ictal analgesia immediately after seizures, is related to
a massive discharge of these neurotransmitters at the postictal period. They could displace blockers from their sites,
and consequently reduce their pharmacological efficiency.
In addition, at least for naloxone, its efficiency in antagonizing the post-ictal analgesia, at a lower dose (5
mg / kg), does not seem to relate to post-ictal depression
effects, because it has been already demonstrated that
naloxone has a property to attenuate it or to abolish it
completely [10]. We should consider that during post-ictal
analgesia, muscarinic cholinergic mechanisms would be
initially recruited, while endogenous opiates such as
enkephalin and endorphin, would have a place in a later
stage of post-seizure antinociception. Meanwhile, mu
opioid receptors may be then involved in this delayed stage
of the post-ictal analgesia.
However, we cannot discard the possibility of involvement of other neurotransmitters in this process, such
as serotonin. Serotoninergic circuits are widely cited in
literature as responsible for neurochemistry of some antinociceptive processes [5,6], and as participants in the
endogenous system of pain inhibition [9,18]. Methysergide, a non-specific blocker of serotoninergic receptors,
was effective in antagonizing the post-ictal analgesia at
different steps of this antinociceptive process. Corroborating these data, peripheral administration of ketanserin, a
specific blocker of 5-HT 2 / 5HT 1C receptors, was equally
effective in antagonizing the post-ictal analgesia in all
post-convulsive periods studied. It is necessary to consider
the possibility of muscarinic cholinergic and serotoninergic
mechanisms involvement in the immediately post-ictal
period, while opioid mechanisms, mediated by mu receptors, would be recruited later on. Serotoninergic receptors in the 5-HT 2 subfamily can be negligible in this
earlier stage of the post-ictal analgesia; although there is a
clear involvement of these receptors in other post-ictal
analgesia stages. We should still consider the possibility of
other serotoninergic receptors’ involvement in this antinociceptive process, as well as other neurotransmitters.
Studies that give support to this possibility are being
conducted in our laboratory.
Acknowledgements
This work was supported by FAPESP (proc. 96 / 8574-9
and 95 / 3604-4). C. Castro-Souza (proc. 96 / 5464-8) and
E.N. Segato (proc. 98 / 07416-6) were the recipients of
fellowships from FAPESP. N.C. Coimbra and N. GarciaCairasco are recipients of Research Fellowships from
CNPq (proc. 300772 / 97 and 521697 / 96-4). The authors
319
are grateful to J.R. Espreafico, M.T. Castania, J.A.C. de
Oliveira and I.R.Violante for expert technical assistance.
J.R. Espreafico was the recipient of a fellowship from
FAPESP (proc. 97 / 10440-3).
References
˜ Defensive reactions
[1] S.H. Cardoso, N.C. Coimbra, M.L. Brandao,
evoked by activation of NMDA receptors in distinct sites of the
inferior colliculus, Behav. Brain Res. 63 (1994) 17–24.
˜ Opposite
[2] S.H. Cardoso, L.L. Mello, N.C. Coimbra, M.L. Brandao,
effects of low and high doses of morphine on neural substrates of
aversion in the inferior colliculus, Behav. Pharmacol. 3 (1992)
489–495.
[3] N.C. Coimbra, C. Castro-Souza, N. Garcia-Cairasco, Neuroanatomical and neurophysiological study of post-ictal antinociceptive processes in experimental models of epilepsy, Arquivos de NeuroPsiquiatria Resumo /Abstracts 56 (1998) 22.
˜ GABAergic nigro-collicular pathways
[4] N.C. Coimbra, M.L. Brandao,
modulate the defensive behavior elicited by midbrain tectum stimulation, Behav. Brain Res. 59 (1993) 131–139.
˜ Effects of 5-HT 2 receptors blockade
[5] N.C. Coimbra, M.L. Brandao,
on fear-induced analgesia elicited by electrical stimulation of the
deep layers of the superior colliculus and dorsal periaqueductal gray,
Behav. Brain Res. 87 (1997) 97–103.
˜ Evidence for the in[6] N.C. Coimbra, C. Tomaz, M.L. Brandao,
volvement of serotonin in the antinociception induced by electrical
or chemical stimulation of the mesencephalic tectum, Behav. Brain
Res. 50 (1992) 77–83.
[7] N.C. Coimbra, M.C. Kawasaki, J.G. Ciscato Jr., S.H. Cardoso,
ˆ Nigro-tectal pathway: neuroanatomy and role on
S.A.L. Correa,
defensive behaviour elicited by midbrain tectum stimulation, Soc.
Neurosci. Abstr. 24 (1998) 1930.
[8] T.C. De Lima, G. A Rae, Effects of cold-restraint and swim stress
on convulsions induced by penthylenetetrazol and electroshock:
influence of naloxone pretreatment, Pharmacol. Biochem. Behav. 40
(1991) 297–300.
[9] H.L. Fields, A.I. Basbaum, Endogenous pain control mechanisms,
in: P.D. Wall, R. Melzack (Eds.), Textbook of Pain, Churchill
Livingstone, Edinburgh, 1989, pp. 206–217.
[10] H. Frenk, B.C. MacCarty, J.C. Liebeskind, Different brain areas
mediate the analgesic and epileptic properties of enkephalin, Science
200 (1978) 335–337.
[11] N. Garcia-Cairasco, R.M. E Sabbatini, Possible interaction between
the inferior colliculus and the substantia nigra in audiogenic seizures
in rats, Physiol. Behav. 50 (1991) 421–427.
[12] N. Garcia-Cairasco, H. Wakamatsu, J.A.C. Oliveira, E.L.T. Gomes,
E. A Del Bel, L.E.A. M Mello, Neuroethological and morphological
(neo-Timm staining) correlates of limbic recruitment during the
development of audiogenic kindling in seizures susceptible Wistar
rats, Epilepsy Res. 26 (1996) 177–192.
[13] R. Guieu, E. Mesdjian, J. Roger, P. Dano, J. Pouget, G. Serratice,
Nociceptive threshold in patients with epilepsy, Epilepsy Res. 12
(1992) 57–61.
[14] M.A. Oliveira, W. A Prado, Antinociception and behavioral manifestations induced by intracerebroventricular or intra-amygdaloid
administration of cholinergic agonist in the rat, Pain 57 (1994)
383–391.
[15] F. Pavone, C. Castellano, A. Oliverio, Stram-dependent effects of
shock-induced release of opioids: dissociation between analgesia
and behavioral seizures, Brain Res. 366 (1986) 326–328.
[16] M.E. Pereira, J.B.T. Rocha, I. Izquierdo, Atropine reverses antinociception induced by 2,5-hexanedione in rats, Brain Res. 77
(1995) 91–94.
320
N.C. Coimbra et al. / Brain Research 888 (2001) 314 – 320
[17] C.E. Ribak, A.L. Manio, M.S. Navetta, C. M Gall, In situ hybridization for c-fos mRNA reveals the involvement of the superior
colliculus in the propagation of seizure activity in genetically
epilepsy-prone rats, Epilepsy Res. 26 (1997) 397–406.
[18] M.L.N.M. Rosa, M.A. Oliveira, R.B. Valente, N.C. Coimbra, W.A.
Prado, Pharmacological and neuroanatomical evidence for the
involvement of the anterior pretectal nucleus in the antinociception
induced by stimulation of the dorsal raphe nucleus in rats, Pain 74
(1998) 171–179.
[19] P.R. Schofield, Sequence and functional expression of the GABA-A
receptor shows a ligand-gated receptor super-family, Nature 328
(1987) 221–227.
[20] V.C. Terra, N. Garcia-Cairasco, NMDA-dependent audiogenic seizures are differentially regulated by inferior colliculus subnuclei,
Behav. Brain Res. 62 (1994) 129–139.
[21] B.C. Yoburn, K. Lutfy, V. Sierra, F.C. Tortella, Tolerance develops
to spinal morphine analgesia but not morphine-induced convulsions,
Eur. J. Pharmacol. 176 (1990) 63–67.