Paradoxical Effect of Noradrenaline-Mediated

Epilepsy & Behavior 22 (2011) 165–177

Contents lists available at ScienceDirect

Epilepsy & Behavior

journal homepage: www.elsevier.com/locate/yebeh

Paradoxical effect of noradrenaline-mediated neurotransmission in the antinociceptive phenomenon that accompanies tonic–clonic seizures: Role of locus coeruleus neurons and α2- and β-noradrenergic receptors Tatiana Tocchini Felippotti, Célio Marcos dos Reis Ferreira, Renato Leonardo de Freitas, Rithiele Cristina de Oliveira, Ricardo de Oliveira, Tatiana Paschoalin-Maurin, Norberto Cysne Coimbra ⁎

Laboratory of Neuroanatomy and Neuropsychobiology, Department of Pharmacology, University of São Paulo at Ribeirão Preto Medical School, Ribeirão Preto (SP), Brazil article info abstract

Article history: The postictal state is generally followed by antinociception. It is known that connections between the dorsal Received 13 May 2011 raphe nucleus, the periaqueductal gray matter, and the locus coeruleus, an important noradrenergic Revised 17 June 2011 brainstem nucleus, are involved in the descending control of ascending nociceptive pathways. The aim of the Accepted 20 June 2011 present study was to determine whether noradrenergic mechanisms in the locus coeruleus are involved in Available online 3 August 2011 postictal antinociception. Yohimbine (an α2-receptor antagonist) or propranolol (a β-receptor antagonist) was microinjected unilaterally into the locus coeruleus, followed by intraperitoneal administration of Keywords: – ﬂ Postictal antinociception pentylenetetrazole (PTZ), a noncompetitive antagonist that blocks GABA-mediated Cl in ux. Although the Pentylenetetrazole administration of both yohimbine and propranolol to the locus coeruleus/subcoeruleus area resulted in a γ-aminobutyric acid A receptor signiﬁcant decrease in tonic or tonic–clonic seizure-induced antinociception, the effect of yohimbine α- and β-noradrenergic receptors restricted to the locus coeruleus was more distinct compared with that of propranolol, possibly because of the Norepinephrine presynaptic localization of α2-noradrenergic receptors in locus coeruleus neurons. These effects were related Epilepsy to the modulation of noradrenergic activity in the locus coeruleus. Interestingly, microinjections of Pain noradrenaline into the locus coeruleus also decrease the postictal antinociception. The present results

suggest that the mechanism underlying postictal antinociception involves both α2- and β-noradrenergic receptors in the locus coeruleus, although the action of noradrenaline on these receptors causes a paradoxical effect, depending on the nature of the local neurotransmission. © 2011 Elsevier Inc. Open access under the Elsevier OA license.

1. Introduction that the LC represents a key structure in the organization of epilepsy-induced hypoalgesia. γ-Aminobutyric acid (GABA)-, acetylcholine-, serotonin-, and Damage to noradrenaline-containing neurons in the LC converts noradrenaline-mediated systems have often been implicated as episodic limbic seizures induced by bicuculline infusion in the neuronal bases of postictal analgesia and have been the subject anterior piriform cortex into self-sustaining status epilepticus [20]. of numerous studies concerning the neural basis of pain modulation Studying the roles of both α- and β-mediated LC mechanisms may [1–10]. The periaqueductal gray matter (PAG) and certain nuclei of elucidate the involvement of this important noradrenergic structure the reticular formation are thought to be the main structures in postictal antinociception. Descending outputs from both LC and involved in supraspinal pain modulation [6,10–16].Reciprocal subcoeruleus (SC) neurons are known to be involved in the connections between the locus coeruleus (LC), situated on both modulation of nociceptive transmission. In fact, activation of sides of the median line [17], and descending pathways from the LC descending LC/SC neurons can produce profound antinociception are crucially involved in modulating the activity of ascending [21,22], which may inhibit the nociceptive activity of spinal dorsal nociceptive pathways, which, in turn, can be modulated by sero- horn neurons [23–25] and trigeminal subnucleus caudalis neurons tonergic output from the dorsal raphe nucleus (DRN) to the dorsal [17,26]. West et al. [27] suggested that both electrical stimulation of horn of the spinal cord [18,19].Theseﬁndings lead us to postulate and administration of morphine [28] into the LC have antinociceptive effects. The interplay between the LC and serotonergic brainstem nuclei [9,17,29] may also be important for the modulation of ⁎ Corresponding author at: Laboratório de Neuroanatomia & Neuropsicobiologia, nociception. It follows that stimulation of the DRN [30] and PAG Departamento de Farmacologia, Faculdade de Medicina de Ribeirão Preto (FMRP) da [12,31–35], both structures rich in serotonin-containing neurons and Universidade de São Paulo (USP), Av. dos Bandeirantes, 3900, Ribeirão Preto (SP), ﬁ α 14049–900, Brazil. Fax: +55 16 3633 2301. bers [10,36,37], causes intense antinociception. In fact, 2-adreno- E-mail address: [email protected] (N.C. Coimbra). ceptors and 5-HT1A somatodendritic autoreceptors have important

1525-5050 © 2011 Elsevier Inc. Open access under the Elsevier OA license. doi:10.1016/j.yebeh.2011.06.028 166 T.T. Felippotti et al. / Epilepsy & Behavior 22 (2011) 165–177 regulatory functions and are involved in the interaction between Analgesia Instrument; Stoelting, IL, USA), the progressive calorimetric serotonin and noradrenaline [38]. These autoreceptors may play key elevation of which was automatically interrupted at the moment roles in modulating neurotransmission in the LC [39,40]. In addition, when the animal removed its tail from the apparatus. The current there is evidence suggesting GABAA receptor-mediated modulation of raised the temperature of the coil (Ni/Cr alloy; 26.04 cm in brainstem serotonergic and noradrenergic functions, altering the length×0.02 cm in diameter) at a rate of 9 °C/second [46], starting ﬁring rate of both the DRN [41] and the LC [42]. at room temperature (approximately 20 °C). If necessary, a small

The only evidence to date for the recruitment of both α2-andβ- current intensity adjustment was made at the start of the experiment noradrenergic receptors in the context of postictal antinociception to obtain three consecutive tail-ﬂick latencies (TFLs) between 2.5 and was obtained from peripheral administration of speciﬁcantagonists 3.5 seconds. If the animal did not remove its tail from the heater [9]. There is a lack of other studies suggesting the involvement of within 6 seconds, the apparatus was turned off to prevent skin

α2-andβ-noradrenergic receptors in the organization and modu- damage. As there were no statistically significant differences between lation of antinociceptive processes induced by brain epileptogenic baseline characteristics of the experimental groups, all tail-flick activity. In this study, we took into account previous evidence latencies were normalized by an index of analgesia (IA) using the suggesting the involvement of the LC in postictal antinociception following formula: [9], as well as recent robust neuroanatomical findings suggesting ð Þ−ðÞ reciprocal connections between neurons of the LC situated on both TFLtest TFLcontrol IA = −ðÞ sides of the median line [17]. There are also several reports indi- 6 TFLcontrol cating that the LC noradrenergic system plays an important role in the descending modulation of spinothalamic ascending nociceptive Three control baseline TFL readings were taken at 5-minute pathways [6,10,43–45]. In this context, the present study focused on intervals. TFLs were also measured following seizures elicited by the theroleofpresynapticα2− and postsynaptic β-noradrenergic peripheral administration of pentylenetetrazole (PTZ). receptors on LC neurons in the modulation of postictal antinociception, to better understand the oscillation of the postseizure nocicep- 2.4. Behavioral testing and seizure evaluation tive threshold, which is associated with the postictal immobility syndrome. Behavioral tests were performed by placing the rats in the interior of a circular arena with transparent acrylic walls. The arena measured 2. Materials and methods 60 cm in diameter and 50 cm in height and was located in an experimental compartment illuminated by a fluorescent lamp (350 lx 2.1. Animals at arena floor level). The effects of drug administration (PTZ, yohimbine, propranolol, and physiological saline) were evaluated We used male Wistar albino rats weighing between 200 and 250 g with the rats inside the arena. (n =5–9 per group) from the animal care facility of the Ribeirão Preto Tonic or tonic–clonic convulsive reactions induced by PTZ Campus at the University of São Paulo (USP). The animals were (64 mg/kg, ip) served as the motor parameter that was used to housed in groups of four in plexiglass cages and given free access to evaluate the effect of the blockade of GABA-mediated Cl– influx. food and water throughout the experiment. The room temperature Seizure latency was defined as the time between injection of PTZ and was controlled (22±1 °C), and a light–dark cycle (07:00– the first evidence of anterior paw myoclonia, which was considered to 19:00 hours lights on) was maintained. The experimental protocols indicate the beginning of the seizure. The severity of the convulsive were reviewed and approved (Proc. 074/2004) by the Ethics reactions was evaluated according to the following motor paramaters: Commission in Animal Experimentation (CETEA) at FMRP-USP, and index 0=exploratory behavior; index 1=jaw myoclonia and facial all experiments were performed in accordance with the recommen- musculature myoclonia, forepaw myoclonia at short duration; index dations of CETEA, based on the guidelines of the Brazilian Society for 2=head myoclonia, moderate forepaw at short duration (at least Neuroscience and Behaviour (SBNeC) and on the Animal Research 5 seconds); index 3=forepaw and/or hindpaw severe musculature Ethics standards adopted by the Brazilian College of Animal hypertonia, forepaw severe musculature hypertonia at intermediate Experimentation (COBEA). duration (at least 10 seconds); index 4=forepaw and/or hindpaw severe musculature hypertonia, rearing, and also forepaw severe 2.2. Surgical procedures myoclonia; index 5=forepaw and/or hindpaw severe musculature hypertonia, rearing and falling, and also forepaw severe myoclonia. Animals were anesthetized with sodium xilasine (0.2 mL, ip) PTZ-induced convulsive reactions were recorded using a video camera and ketamine (0.1 mL, ip) and fixed in a stereotaxic frame (David (Panasonic, M3500) focused on the open field, and the VHS tapes (JVC, Kopf, USA). A stainless-steel guide cannula (o.d. 0.6 mm, i.d. SX Gold) were subsequently evaluated to classify, characterize, and 0.4 mm) was implanted unilaterally in the brainstem, aimed at quantify the convulsive reactions. the LC. The upper incisor bar was set 3.3 mm below the interaural line, such that the skull was horizontal between bregma and 2.5. Neuropharmacological procedures lambda. The guide cannula was vertically introduced using the following coordinates, with the bregma serving as the reference for Five days after surgery, a baseline tail-flick test was performed for each plane: anteroposterior, –9.68 mm; mediolateral, 1.4 mm; dor- each animal in a given group. Animals in the control group were soventral, 6 mm. The guide cannula was fixed to the skull using removed from the box, placed in the open field, and observed for acrylic resin and two stainless-steel screws. At the end of the 15 minutes without drug injection. After this period, the animals were surgery, each guide cannula was sealed with a stainless-steel wire removed from the arena, and their nociceptive thresholds were to prevent obstruction. standardized using the tail-flick test. After that, an independent group of animals received microinjections of physiological saline (0.2 or 2.3. Nociceptive testing 0.5 μL, n=7 or 8), yohimbine (at 1.0 μg/0.2 μL, n=7, 3.0 μg/0.2 μL, n=5, 5.0 μg/0.2 μL, n=9, or 5.0 μg/0.5 μL, n=8), propranolol (at Nociception thresholds were compared using the tail-flick test. 1.0 μg/0.2 μL,n =8, 3.0 μg/0.2 μL, n =7, 5.0 μg/0.2 μL, n =9, or Each animal was placed in a restraining apparatus (Stoelting) with 5.0 μg/0.5 μL, n =8), or norepinephrine (at 1.0 μg/0.2 μL, n =8, acrylic walls, and its tail was placed in a heating sensor (tail-flick 3.0 μg/0.2 μL, n =5, 5.0 μg/0.2 μL, n =6) into the LC. The T.T. Felippotti et al. / Epilepsy & Behavior 22 (2011) 165–177 167 microinjections were done using a thin dental needle (Mizzy, o.d. washed out with cold, oxygen-enriched, Ca2+-free Tyrode's buffer 0.3 mm) introduced through the guide cannula until its lower end (40 mL at 4 °C), followed by 200 mL of ice-cold 4% (w/v) paraformal- extended 1 mm below the guide cannula. The needle was left in place dehyde in 0.1 M sodium phosphate buffer, pH 7.3, for 15 minutes at a for an additional 1 minute after the injection. Ten minutes later, the pressure of 50 mm Hg. The brainstem was quickly sectioned, animals received PTZ (64 mg/kg, ip), and nociceptive responses were removed, and immersed in fresh fixative for 4 hours at 4 °C. It was evaluated immediately after the tonic/tonic–clonic seizures and in the then rinsed in 10 and 20% sucrose dissolved in 0.1 M sodium subsequent postictal period from 10 to 180 minutes. phosphate buffer (pH 7.4) at 4 °C, for at least 12 hours in each As a control of the eventual intrinsic effect of LC neurons on the solution. Tissue pieces were immersed in 2-methylbutane (Sigma), baseline nociceptive thresholds, additional experiments were per- frozen on dry ice, embedded in Tissue Tek OCT, and cut with a cryostat formed with the following procedures: (1) microinjection of ibotenic (HM 505 Microm, Zeiss) at −22 °C. Sections were then mounted on acid (at 1.0 μg/0.2 μL) into the LC followed by intraperitoneal glass slides coated with chrome alum gelatin to prevent detachment administration of physiological saline; (2) microinjection of ibotenic and stained with hematoxylin–eosin to be viewed on a photomicro- acid (at 1.0 μg/0.2 μL) into the LC followed by intraperitoneal scope (AxioImager Z1, Zeiss). Statistical analysis was performed administration of PTZ; (3) sham procedure (introduction of an exclusively with data from the animals that presented signs of injector needle into the guide cannula inserted in the LC without microinjections of the drugs into the LC. local microinjection of the neurotoxin) followed by intraperitoneal administration of PTZ. 2.8. Analysis of results

2.6. Drugs Data from experiments to investigate the effect of seizures on nociceptive threshold and the effect of neurotoxic lesions of the LC on

Pentylenetetrazole (Sigma/Aldrich, St. Louis, MO, USA), yohimbine TFLs as well as on postictal antinociception, the involvement of α2- (Sigma), propranolol (Sigma), norepinephrine (Sigma), and ibotenic and β-receptor mediated noradrenergic mechanisms, and the role of acid (Sigma) were each dissolved in physiological saline (NaCl 0.9%) norepinephrine-mediated neurotransmission in postictal antinoci- shortly before use. Drugs were administered at the following concen- ception were submitted to a repeated-measures analysis of variance trations/doses: yohimbine and propranolol (1.0 μg/0.2 μL, 3.0 μg/0.2 μL, (ANOVA). In case of a signiﬁcant treatment versus time interaction, 5.0 μg/0.2 μL, or 5.0 μg/0.5 μL); norepinephrine (1.0 μg/0.2 μL, one-way ANOVA was performed at each time interval, followed by 3.0 μg/0.2 μL, 5.0 μg/0.2 μL); ibotenic acid (1.0 μg/0.2 μL); PTZ Duncan's post hoc test. Pb0.05 was indicative of statistically sig- (64 mg/kg). niﬁcant differences.

2.7. Histological analysis 3. Results

After testing, the rats were anesthetized with sodium pentobar- Wistar rats that had been intraperitoneally administered with PTZ bital (45 mg/kg, ip) and perfused via the left ventricle. The blood was at 64 mg/kg exhibited distinct types of motor seizures—orofacial

Table 1 Severity of generalized tonic–clonic convulsive reactions induced by intraperitoneal administration of PTZ at 64 mg/kg.

Treatment Latency (s) Duration (s) Seizure frequency Index of severitya

(A) Animals pretreated with microinjections of physiological saline, yohimbine (5.0 μg/0.5 μL), or propranolol (5.0 μg/0.5 μL) in the locus coeruleus (LC). There were no statistically signiﬁcant differences between groups Saline (NaCl 0.9%) LC+PTZ 111.0 504.0 3.0 4.6 Yohimbine 5 μg/0.5 μL LC+PTZ 111.5 460.0 3.0 4.0 Propranolol 5 μg/0.5 μL LC+PTZ 108.25 465.0 3.0 4.5

(B) Animals pretreated with microinjections of physiological saline or yohimbine (1, 3, or 5 μg/0.2 μL) in the LC nucleus Saline (NaCl 0.9%) LC+PTZ 72.2 455.2 3.0 3.3 Yohimbine 1 μg/0.2 μL LC+PTZ 61.7 835.3b 3.6 3.5 Yohimbine 3 μg/0.2 μL LC+PTZ 53.1 652.5 4.0 3.6 Yohimbine 5 μg/0.2 μL LC+PTZ 77.3 445.5c 3.0 3.4

(C) Animals pretreated with microinjections of physiological saline, propranolol (1, 3, or 5 μg/0.2 μL) in the LC nucleus Saline (NaCl 0.9%) LC+PTZ 77 478.9 3.0 3.4 Propranolol 1 μg/0.2 μL LC+PTZ 61.3 691.7b 4.12b 3.5 Propranolol 3 μg/0.2 μL LC+PTZ 62.4 878b,d. 3.14d 3.9 Propranolol 5 μg/0.2 μL LC+PTZ 57.1 433.2d 3.0d 3.6

(D) Animals pretreated with microinjections of physiological saline or norepinephrine (1, 3, or 5 μg/0.2 μL) in the LC nucleus Saline (NaCl 0.9%) LC+PTZ 75.4 474.4 3.0 3.5 Norepinephrine 1 μg/0.2 μL LC+PTZ 55.8 685.2 3.3 3.3 Norepinephrine 3 μg/0.2 μL LC+PTZ 50.9 984.3b,e 3.2 3.8 Norepinephrine 5 μg/0.2 μL LC+PTZ 60.8 1143.3b,e 3.6 3.6

a See description under Material and Methods. b Statistically significant difference (Pb0.05) compared with the saline-treated group (NaCl 0.9%, n=6). c Statistically significant difference (Pb0.05) compared with the group treated with yohimbine 1 μg/0.2 μL in the LC followed by intraperitoneal injection of PTZ at 64 mg/kg, based on repeated-measures ANOVA followed by a Duncan post hoc test. d Statistically significant difference (Pb0.05) compared with the group treated with propranolol 1 μg/0.2 μL in the LC followed by intraperitoneal injection of PTZ at 64 mg/kg, based on repeated-measures ANOVA followed by a Duncan post hoc test. e Statistically significant difference (Pb0.05) compared with the group treated with norepinephrine 1 μg/0.2 μL in the LC followed by intraperitoneal injection of PTZ at 64 mg/kg, based on repeated-measures ANOVA followed by a Duncan post hoc test. 168 T.T. Felippotti et al. / Epilepsy & Behavior 22 (2011) 165–177

in the experimental scenario for 15 minutes without receiving any type of drug afterward did not display significant changes in their nociceptive threshold, confirming previous reports [8,9]. There were no statistically significant differences in latency [F(2,20)=0.03, PN0.05]; number of each PTZ-induced seizure [F(2,20) varying from 0.05 to 0.20, PN0.05 in all cases]; total duration of the convulsive motor response time window [F(2,20)=1.07, PN0.05]; or severity of seizures [F(2,20) varying from 0.70 to 1.63, PN0.05 in all cases] between the physiological saline-, yohimbine- and propranolol- pretreated groups (Table 1A). Fig. 1 is a photomicrograph represent- ing the sites of microinjection of drugs in the LC.

Antagonism of α2- and β-noradrenergic receptors in the LC and SC area decreased postictal antinociception. There were signiﬁcant effects of treatment [F(2,20)=4.95, Pb0.0001] and time [F(9,12)= 19.65, Pb0.0001] and a treatment versus time interaction [F(18,22)= 19.65, Pb0.0001]. Repeated-measures ANOVA showed a signiﬁcant treatment effect of yohimbine from 20 to 60 minutes [F(2,20) varying from 6.1445 to 68.3952, Pb0.05]. Duncan's post hoc test (Pb0.05)

showed that blockade of α2-noradrenergic receptors in the LC caused a statistically significant decrease in postictal antinociception from 0 to 60 minutes following convulsions, whereas a β-noradrenergic antagonist decreased postictal antinociception from 10 to 60 minutes following PTZ-induced seizure [F(2,20) varying from 13.40 to 68.39, Pb0.05]. These data are illustrated in Fig. 2. The site of each drug microinjection was situated in the LC (Fig. 3). Both treatments were also conducted using a smaller volume of each solution. There were no statistically significant differences in latency to [F(3,24)=1.72, P N0.05], number of [F(3,24)=2.29, Fig. 1. Representative photomicrographs of transverse sections of the pons of Wistar PN0.05], or severity of [F(3,24)=0.08, PN0.05] seizures, but there fi rats at the level of the LC. (A) The arrow identi es the LC contralateral to the site of were statistically significant differences in duration [F(3,24)=6.27, microinjection (B) of physiological saline, norepinephrine, or noradrenergic receptor b antagonists in the LC. The bar in B corresponds to 96 μm in both (A) and (B). P 0.05] of each PTZ-induced seizure between the physiological saline- and yohimbine-pretreated groups (Table 1B). Also, there were no statistically significant differences in the latency to [F(3,27)=1.42, PN0.05], or severity of [F(3,27)=0.38, myoclonia, minimal clonus or generalized clonic reactions, tonic– PN0.05] seizures, but there were statistically significant differences clonic seizures, and tonic extension—that covered a period of 7.42– in number [F(3,27)=5.40, Pb0.05] and duration [F(3,27)=10.42, 19.05 minutes, in an average of three or four episodes of motor Pb0.05] of each PTZ-induced seizure between the physiological saline seizures in control groups. Animals that had tonic or tonic–clonic and propranolol-pretreated groups (Table 1C). seizures over this time did not resist to convulsive reactions and died Furthermore, treatment with a nonspecific agonist was per- (approximately 10% of seizing rats). Convulsions lasted from 72.2 to formed. There were no statistically significant differences in the 111 seconds in control groups and were not preceded by wild latency to [F(3,23)=2.70, P N0.05], number of [F(3,23)=0.76, running. Control animals subjected to the tail-flick test and placed P N0.05], or severity of [F(3,23)=0.27, P N0.05] seizures, but there

Fig. 2. Effect of microinjection of yohimbine (at 5.0 μg/0.5 μl) or propranolol (at 5.0 μg/0.5 μl) into the LC nucleus/subcoeruleus area (n=8 per group) on postictal antinociception. Data are presented as means±SEM. *Statistically signiﬁcant difference (Pb0.05) compared with the saline-treated group (n=8). #Statistically signiﬁcant difference (Pb0.05) compared with the group treated with propranolol in the LC followed by intraperitoneal injection of PTZ at 64 mg/kg, based on repeated-measures ANOVA followed by Duncan`s post hoc test. T.T. Felippotti et al. / Epilepsy & Behavior 22 (2011) 165–177 169

Fig. 3. Schematic representation of histologically conﬁrmed sites of microinjections (closed circles) of (A) saline (NaCl 0.9%, 0.5 μL) in the LC followed by intraperitoneal administration of PTZ at 64 mg/kg; (B) yohimbine at 5.0 μg/0.5 μL in the LC followed by intraperitoneal injection of PTZ at 64 mg/kg ip; (C) propranolol at 5.0 μg/0.5 μL in the LC followed by intraperitoneal injection of PTZ 64 mg/kg ip, shown on anagrams of Paxinos and Watson's atlas [90].

were statistically signiﬁcant differences in duration [F(3,23)=12.02, showed that blockade of β-noradrenergic receptors in the LC caused a P =0.0001] of each PTZ-induced seizure between the physiological statistically signiﬁcant decrease in postictal antinociception. These saline- and norepinephrine-pretreated groups (Table 1D). data are illustrated in Fig. 6. All sites studied were situated in the LC

Pharmacological antagonism of α2- and β-noradrenergic receptors (Fig. 7). restricted to the LC also decreased postictal antinociception. There Administration of the nonspecific agonist norepinephrine in the LC were significant effects of treatment [F(2,23)=14.02, Pb0.001], time caused an interesting depressed effect on postictal antinociception. [F(9,15)=22.55, Pb0.0001] and a treatment versus time interaction There were significant effects of treatment [F(2,23)=6.48, P=0.02], [F(9,27)=4.60, Pb0.0001]. Repeated-measures ANOVA showed that time [F(9,15)=24.11, P b0.0001] and a treatment versus time yohimbine, at the lowest and highest doses, was not able to decrease interaction [F(9,27)=3.21, Pb0.0001]. Repeated-measures ANOVA postictal antinociception during the first 30 minutes following showed that norepinephrine was able to decrease postictal anti- seizures, but there was a significant treatment effect of yohimbine nociception from 0 to 60 minutes following seizures [F(2,23) varying from 40 to 120 minutes [F(2,23) varying from 10.88 to 14.99, from 10.88 to 14.99, Pb0.001]. Moreover, repeated-measures ANOVA Pb0.001]. Moreover, repeated-measures ANOVA showed that yohim- showed that yohimbine at the intermediate dose was able to reduce bine at the intermediate dose was able to antagonize postictal postictal antinociception from 0 to 120 minutes [F(2,23) varying from antinociception from 0 to 120 minutes [F(2,23) varying from 2.15 to 3.33 to 6.67, Pb0.05]. Duncan's post hoc test (Pb0.05) showed that 14.99, Pb0.001]. Duncan's post hoc test (Pb0.05) showed that norepinephrine on noradrenergic receptors in the LC caused a blockade of α2-noradrenergic receptors in the LC caused a statistically statistically significant decrease in postictal antinociception. These significant decrease in postictal antinociception. These data are data are illustrated in Fig. 8. All sites studied were situated in the LC illustrated in Fig. 4. All sites studied were situated in the LC (Fig. 5). (Fig. 9). In addition, there were significant effects of treatment [F(2,273)= These effects were independent of epileptic seizures induced by 7.50, P=0.001], time [F(9,19)=139,39, Pb0.0001] and a treatment blockade of GABA-mediated Cl− influx, given that central adminis- versus time interaction [F(9,27)=3.69, Pb0.0001]. Repeated-mea- tration of yohimbine or propranolol at major concentrations sures ANOVA showed that propranolol was able to reduce postictal (5.0 μg/0.5 μL) targeted to the LC did not cause any statistically antinociception from 0 to 60 minutes [F(2,27) varying from 3.60 to significant effect on the nociceptive threshold (P N0.05 in all cases, as 7.18, Pb0.05] following seizures. Duncan's post hoc test (Pb0.05) compared with the control). Thus, both noradrenergic antagonists 170 T.T. Felippotti et al. / Epilepsy & Behavior 22 (2011) 165–177

Fig. 4. Effect of microinjection of yohimbine (at 1 μg/0.2 μL, n=7,3μg/0.2 μL, n=5,or5μg/0.2 μL, n=9) in the LC nucleus on postictal antinociception. Data are presented as means±SEM. *Statistically significant difference (Pb0.05) compared with the group treated with physiological saline (n=6) followed by intraperitoneal injection of PTZ at 64 mg/kg. +Statistically significant difference (Pb0.05) compared with the group treated with 1 μg/0.2 μL yohimbine in the LC followed by intraperitoneal injection of PTZ at 64 mg/kg, §Statistically significant difference (Pb0.05) compared with the group treated with 5 μg/0.2 μL yohimbine in the LC followed by intraperitoneal injection of PTZ at 64 mg/kg, based on repeated-measures ANOVA followed by Duncan`s post hoc test.

exert their effects on postictal antinociception by acting on the LC. blockade of this noradrenergic receptor in the LC would increase These data are illustrated in Fig. 10. Histological conﬁrmation of the postictal antinociception, given that the inhibition of adenylyl site of the microinjections in the LC and SC area is presented in cyclase activity, which results in decreased formation of cAMP, is

Fig. 11. an important consequence of α2-noradrenergic receptor activation Neurochemical lesion of the LC with ibotenic acid caused a [51]. The paradoxical effect of α2-noradrenergic receptor blockade decrease in postictal antinociception. There were significant effects on postictal antinociception could be easily explained by interac- of treatment [F(2,19)=124.94; P=0.001], time [F(9,11)=19.97, tions with inhibitory neurons (e.g., using GABA-mediated neuro- Pb0.001] and a treatment versus time interaction [F(9,18)=26.92, transmission) modulating the activity of LC noradrenergic outputs. Pb0.001]. Repeated-measures ANOVA showed that the neurochem- In fact, a previous neuromorphological study showed evidence of the ical lesion was able to reverse postictal antinociception immediately presence of GABAergic neurons in the LC [52,53],andLCnoradren- after seizures and 10 to 90 minutes after seizures [F(2,19) varying ergic neurons are immunoreactive for the α2 and α3 GABAA receptor from 42.49 to 156.90, Pb0.0001]. Moreover, it was shown that the LC subunits [54]. In addition, there is evidence suggesting the existence neutotoxic lesion alone was not able to cause any significant change in of a subpopulation of small GABAergic neurons in the peri-LC baseline TFLs. Duncan's post hoc test also showed statistically dendritic zone, which provide interneuronal integration for LC significant differences between the group pretreated with ibotenic noradrenergic neurons [42]. This could explain the reduction in the acid in the LC followed by IP treatment with PTZ, and the group postictal antinociception curve when yohimbine was administered pretreated with ibotenic acid in the LC followed by IP treatment with centrally in a large volume, possibly involving neurons in the peri-LC physiological saline immediately after seizures and from 10 to dendritic zone. 60 minutes after tonic–clonic convulsions. These data are illustrated Interestingly, some β-noradrenergic receptor antagonists have in Fig. 12. The site of each microinjection procedure was situated in been shown to have local anesthetic effects [55–57].Togetherwith the LC (Fig. 13). its classic nonselective β-noradrenergic receptor antagonist effect, it has been accepted that propranolol inhibits high-voltage ac- 4. Discussion tivated Ca2+ currents and a low-voltage activated Ca2+ current [58,59] at low concentrations and moderately suppresses potassi- In the present study, PTZ-induced tonic–clonic seizures were um currents at high concentrations [60]. In the present study, we accompanied by statistically significant antinociception. The present can consider the effects of propranolol on LC neurons to be due to results demonstrate the involvement of LC neurons in the organization β-noradrenergic receptor antagonism, given that there was no of postictal antinociception, considering that neurochemical lesion of intrinsic effect of central pretreatment with propranolol on baseline the LC neurons decreased the elevation of nociceptive thresholds TFLs. after tonic–clonic seizures, but not the baseline TFLs. In addition, the Surprisingly, pretreatment of the LC with norepinephrine also present work provides evidence for a paradoxical involvement of both decreased postictal antinociception, suggesting a paradoxical presynaptic α2-autoreceptors and postsynaptic β-noradrenergic re- effect of norepinephrine-mediated neurotransmission into the LC on ceptors in the LC in the mechanism of postictal antinociception. This antinociceptive outputs from this nucleus. The LC can modulate pain is the first presentation of evidence regarding the involvement of through classic noradrenergic descending pathways [25,61], but its noradrenergic receptors in the LC in antinociceptive phenomena influence on nociceptive stimuli perception can be processed through caused by seizures. Interestingly, recent findings have demonstrated an ascending pathway connecting somatosensorial and parafascicular that α- and β-noradrenergic receptors participate in other kinds of thalamic nuclei [62,63]. Ascending projections from the LC exert a hypoalgesia [47,48]. dual role at the medial thalamus, acting on parafascicular nucleus Peng et al. [49] showed that microinjections of yohimbine into with both facilitatory and inhibitory mechanisms through, respec- the dorsal horn of the spinal cord antagonize the antinociception tively, α1- and α2-noradrenergic receptors [63]. induced by electrical stimulation of the PAG. Expression of α2- Interestingly, Zhang et al. [64] showed that depending on the noradrenergic receptor mRNA in noradrenergic neurons in a dose, administration of norepinephrine in the neostriatum is previous study [50], however, indicated that this receptor subtype followed by either antinociception (at lower doses) or pronocicep- mediates presynaptic autoreceptor function. We expected that tion (at higher doses). Another possible explication for these T.T. Felippotti et al. / Epilepsy & Behavior 22 (2011) 165–177 171

own continuous release on noradrenergic synapses, promoting

negative feedback. The α2-noradrenergic receptor agonist clonidine, at higher doses, inhibits the release of norepinephrine through the same negative feedback mechanism in the dorsal horn of the spinal cord [67]. In addition, according to Green et al. [68], under normal conditions, the noradrenergic inhibitory feedback system can be nonresponsive, whereas under pathophysiological conditions it is commonly active. In a peripheral neuritis experimental model, injection of norepinephrine worsened the mechanical hyperalgesia, and

this was attenuated not only by an α2-noradrenergic receptor antagonist, but also by an α1-noradrenergic receptor antagonist [69,70].In addition, several studies indicate that the LC is recruited by damaging nociceptive stimuli, inﬂammation, or nerve lesion by promoting inhibitory feedback of pain [71,72]. However, although there are

studies showing the involvement of α2-noradrenergic receptors in norepinephrine-induced pain, there is also behavioral and neuro-

physiological evidence suggesting the involvement of α1-noradrenergic receptors in the facilitation of pain [70]. These ﬁndings suggest a role for LC (direct or indirectly via other structures of the brainstem) in the facilitation of pain in distinct pathophysiological processes, such as neuropathic pain [73] or postischemic chronic pain [74].In humans, injection of norepinephrine into the tegument was found to cause hyperalgesia to heat [75]. Supraspinally, the norepinephrine effect on noradrenergic receptors can both facilitate and inhibit pain depending on the supraspinal site of its release, the type of receptor subfamily activated, and the intensity and duration of painful stimuli [70].In

line with this evidence, it has been postulated that α2-noradrenergic receptors situated on noradrenergic neurons (autoreceptors) cause pronociception, whereas these same receptors situated on non-noradrenergic neurons (heteroreceptors) cause antinociception [70]. We cannot, however, rule out the possibility of involvement of other mechanisms in the elaboration of postictal antinociception. In fact, endogenous opioid peptide-, acetylcholine-, serotonin-, and norepinephrine-mediated systems have been implicated in the organization of tonic–clonic seizure-induced antinociception [9,76–78]. Yohimbine microinjected (at lower concentration) into the LC has also increased the duration of convulsive reactions, but this pretreatment did not modify the severity index of seizures. Intra-LC pretreatment with propranolol at the lower concentration caused an effect similar to that of yohimbine: increasing the duration of con- Fig. 5. Schematic representation of histologically conﬁrmed sites of microinjections vulsive reactions, but exerting no effect on the severity of convulsive μ (closed circles) of (A) saline (NaCl 0.9%, 0.5 L) into the LC followed by intraperitoneal seizures. There is evidence in the literature that both yohimbine and administration of PTZ at 64 mg/kg; (B) yohimbine 1.0 μg/0.2 μL into the LC followed by intraperitoneal injection of PTZ at 64 mg/kg; (C) yohimbine 3.0 μg/0.2 μL into the LC propranolol modulate convulsive activity [79]. In the present work followed by intraperitoneal injection of PTZ at 64 mg/kg; (D) yohimbine 5.0 μg/0.2 μL norepinephrine, at peak concentrations, increased the duration of into the LC followed by intraperitoneal injection of PTZ at 64 mg/kg, depicted on PTZ-induced seizures as evidenced by noradrenergic antagonists such anagrams of Paxinos and Watson's atlas [90]. as yohimbine and propranolol. Interestingly, it seems that norepinephrine has a dual effect on convulsive responses, modulating or mediating the elaboration of this motor phenomenon [79]. In addition to its role in the elaboration/ modulation of postictal antinociception, it is possible that norepi-

ﬁndings could be an inhibitory feedback mechanism exerted on nephrine also exerts some effect through α1-noradrenergic receptors presynaptic α2-noradrenergic receptors, or even on postsynaptic α1- in the elaboration of convulsive responses. noradrenergic receptors, already implicated in facilitatory mecha- Despite the evidence that the monoaminergic system is crucial to nisms of pain [65]. convulsive reactions [80–85], the LC is considered to be a key struc- In addition, there is other evidence suggesting that the effect of ture involved in the modulation of brain epileptic activity [20,86], norepinephrine on noradrenergic receptors can effectively either and bilateral lesions of the LC facilitate audiogenic seizures [87]. facilitate or inhibit nociceptive reﬂexes. Several behavioral in vivo These data may indicate that the LC exerts a relevant role in postictal studies showed that microinjection of an α2-noradrenergic receptor antinociceptive responses. Indeed, recent reports suggest that LC agonist or an α1-noradrenergic receptor antagonist in the nucleus neurons play an important role in determining immediate early gene raphe magnus causes spinal antinociceptive effects, whereas in- expression, even under conditions of strong pathological activation jections of α1-orα2-noradrenergic receptors agonists cause hyper- such as limbic status epilepticus [88,89], and that neurochemical algesic effects [66]. It is known that the action of norepinephrine on lesion of the LC decreases postictal analgesia [9]. The present work

α2-noradrenergic presynaptic receptors decreases about 90% of its corroborates these ﬁndings and, further, suggests involvement of the 172 T.T. Felippotti et al. / Epilepsy & Behavior 22 (2011) 165–177

Fig. 6. Effect of microinjections of propranolol (1 μg/0.2 μL, n=8, 3 μg/0.2 μL, n=7, or 5 μg/0.2 μL, n=9) into the LC nucleus on postictal antinociception. Data are presented as means±SEM. *Statistically significant difference (Pb0.05) compared with the group treated with physiological saline (n=7) followed by intraperitoneal injection of PTZ at 64 mg/kg. +Statistically significant difference (Pb0.05) compared with the group treated with 1 μg/0.2 μL propranolol into the LC followed by intraperitoneal injection of PTZ at 64 mg/kg. #Statistically significant difference (Pb0.05) compared with the group treated with 3 μg/0.2 μL propranolol into the LC followed by intraperitoneal injection of PTZ at 64 mg/kg, based on repeated-measures ANOVA followed by Duncan`s post hoc test.

Fig. 7. Schematic representation of histologically conﬁrmed sites of microinjections (closed circles) of (A) saline (NaCl 0.9%, 0.5 μL) into the LC followed by intraperitoneal administration of PTZ at 64 mg/kg; (B) propranolol at 1.0 μg/0.2 μL in the LC followed by intraperitoneal administration of PTZ at 64 mg/kg; (C) propranolol at 3.0 μg/0.2 μL into the LC followed by intraperitoneal administration of PTZ at 64 mg/kg; (D) propranolol at 5.0 μg/0.2 μL into the LC followed by intraperitoneal administration of PTZ at 64 mg/kg, depicted on anagrams of Paxinos and Watson's atlas [90]. T.T. Felippotti et al. / Epilepsy & Behavior 22 (2011) 165–177 173

Fig. 8. Microinjections of yohimbine (5.0 μg/0.5 μl) or propranolol (5.0 μg/0.5 μl) into the LC (n=8) followed by intraperitoneal saline (0.9% NaCl) had no effects on the nociceptive threshold. Data are presented as means±SEM. PN0.05 in all cases compared with the control group.

LC noradrenaline-mediated system in the organization of postictal ment of noradrenergic pathways and α2- and β-noradrenaline- antinociception. mediated LC mechanisms in the organization and modulation of In summary, a decrease in LC activity impairs postictal antinoci- postictal antinociception. ception. Antagonism of the increased nociceptive threshold caused by seizures in all postictal periods was attenuated either by the Acknowledgments direction/indirect action of norepinephrine or by the blockade of α2− or β-noradrenergic receptors in LC neurons prior to tonic–clonic This research was supported by FAPESP (Proc. 96/08574-9, seizure-induced analgesia, strongly supporting a role for the involve- 02/01496-5, and 07/01174-1) and FAEPA (Proc. 537/1995 and

Fig. 9. Schematic representation of histologically conﬁrmed sites of microinjections (closed circles) of (A) physiological saline into LC followed by intraperitoneal administration of physiological saline; (B) yohimbine at 5.0 μg/0.5 μL into the LC followed by intraperitoneal administration of physiological saline; (C) propranolol at 5.0 μg/0.5 μL into the LC followed by intraperitoneal administration of physiological saline, depicted on anagrams of Paxinos and Watson's atlas [90]. 174 T.T. Felippotti et al. / Epilepsy & Behavior 22 (2011) 165–177

Fig. 10. Effect of microinjections of norepinephrine (1 μg/0.2 μL, n=8,3 μg/0.2 μL, n=5,or5μg/0.2 μL, n=6) into the LC nucleus on postictal antinociception. Data are presented as means±SEM. *Statistically significant difference (Pb0.05) compared with the group treated with physiological saline (n=8) followed by intraperitoneal injection of PTZ at 64 mg/kg, +Statistically significant difference (Pb0.05) compared with the group treated with 1 μg/0.2 μL propranolol in the LC followed by intraperitoneal administration of PTZ at 64 mg/kg. #Statistically significant difference (Pb0.05) compared with the group treated with 3 μg/0.2 μL propranolol in the LC followed by intraperitoneal administration of PTZ at 64 mg/kg, based on repeated-measures ANOVA followed by Duncan`s post hoc test.

70/2002). T.T. Felippotti was the recipient of a Master in Sciences postdoctorate fellowship, Proc. 2009/17258-5) and CAPES (Sc.D. fellowship from FAPESP (Proc. 03/09129-4) and the recipient of a fellowship). R. de Oliveira, and R.C. de Oliveira were supported by doctorate fellowship from CAPES. C.M. dos Reis Ferreira was CNPq (M.Sc. fellowships, Proc. 133330/01-0). R. de Oliveira was also supported by CAPES (M.Sc. fellowship) and CNPq (Sc.D. fellowship). supported by FAPESP (Sc.D. fellowship, Proc. 2008/09897-5). R.C. de R.L. de Freitas was supported by FAPESP (ScientiﬁcInitiation Oliveira was also supported by FAPESP (Sc.D. fellowship, Proc. Scholarship, Proc.01/03752-6; M.Sc. fellowship, Proc. 03/05256-1; 05/02989-3) and CAPES (PNPD postdoctorate fellowship, Proc.

Fig. 11. Schematic representation of histologically conﬁrmed sites of microinjections (closed circles) of (A) saline (NaCl 0.9%, 0.5 μL) into the LC, followed by intraperitoneal administration of PTZ at 64 mg/kg; (B) norepinephrine 1.0 μg/0.2 μL into the LC, followed by intraperitoneal administration of PTZ 64 mg/kg; (C) norepinephrine 3.0 μg/0.2 μL into the LC followed by intraperitoneal administration of PTZ at 64 mg/kg; (D) norepinephrine 5.0 μg/0.2 μL into the LC followed by intraperitoneal administration of PTZ 64 mg/kg, depicted on anagrams of Paxinos and Watson's atlas [90]. T.T. Felippotti et al. / Epilepsy & Behavior 22 (2011) 165–177 175

Fig. 12. Effect on postictal antinociception of neurochemical lesions of LC neurons induced by local administration of ibotenic acid at 1 μg/0.2 μL. Data are presented as means±SEM. *Statistically signiﬁcant difference (Pb0.05) compared with sham procedure group (n=8) followed by intraperitoneal injection of PTZ at 64 mg/kg. #Statistically signiﬁcant difference (Pb0.05) compared with LC neurochemically lesioned group (n=7) followed by intraperitoneal injection of physiological saline, based on repeated-measures ANOVA followed by Duncan`s post hoc test.

02518/09-0). T. Paschoalin-Maurin was supported by FAPESP (D.Sc. The authors are grateful to Daoud Hibrahim Elias Filho, Valdir fellowship, Proc. 03/07202-6) and CAPES (PNPD postdoctorate Mazzucato, and Izilda Rodrigues Violante for their expert technical fellowship, Proc. 02518/09-0). N.C. Coimbra is the recipient of a assistance. D.H. Elias-Filho was the recipient of technician scholarships research fellowship (level 1A) from CNPq (Proc. 301905/2010-0). from FAPESP (TT-2, Proc. 02/01497-1) and CNPq (Proc. 501858/2005-9, 372654/2006-1, 372810/2008-0, and 372877/2010-9).

References

[1] Dubner R. Neurophysiology of pain. Dent Clin North Am 1978;22:11–30. [2] Mason P, Strassman A, Maciewicz R. Serotonin immunocytochemistry of physiologically characterized raphe magnus neurons. Exp Brain Res 1988;73:1–7. [3] Mason P. Physiological identification of pontomedullary serotonergic neurons in the rat. J Neurophysiol 1997;11:1087–98. [4] Rosa MLNM, Oliveira MA, Valente RB, Coimbra NC, Prado WA. 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 1998;74:171–9. [5] Dunckley P, Wise RG, Fairhurst M, et al. A comparison of visceral and somatic pain processing in the human brainstem using functional magnetic resonance imaging. J Neurosci 2005;25:7333–41. [6] Wei F, Dubner R, Ren K. Nucleus reticularis gigantocellularis and hyperalgesia and spinal Fos protein expression after peripheral inflammation. Pain 1999;80: 127–41. [7] Li JL, Xiong KH, Li YQ, Kaneko T, Mizuno N. Serotoninergic innervation of mesencephalic trigeminal nucleus neurons: a light and electron microscopic study in the rat. Neurosci Res 2000;37:127–40. [8] De Freitas RL, de Oliveira RC, de Carvalho AD, et al. Role of muscarinic and nicotinic cholinergic receptors in an experimental model of epilepsy-induced analgesia. Pharmacol Biochem Behav 2004;79:367–76. [9] Freitas RL, Ferreira CMR, Ribeiro SJ, et al. Intrinsic neural circuits between dorsal midbrain neurons that control fear-induced response and seizure activity and nuclei of the pain inhibitory system elaborating postictal antinociceptive processes: a functional neuroanatomical and neuropharmacological study. Exp Neurol 2005;191:225–42. [10] Coimbra NC, De Oliveira R, Freitas RL, et al. Neuroanatomical approaches of the tectum–reticular pathways and immunohistochemical evidence for serotonin- positive perikarya on neural substrates of the superior colliculus and periaqueductal gray matter involved in the elaboration of the defensive behavior and fear- induced analgesia. Exp Neurol 2006;197:93–112. [11] Reynolds DV. Surgery in the rat during electrical analgesia induced by focal brain stimulation. Science 1969;164:444–5. [12] Nashold Jr BS, Wilson WP, Slaughter DG. Sensations evoked by stimulation in the midbrain of man. J Neurosurg 1969;30:14–24. [13] Fields HL, Basbaum AI. Endogenous pain control mechanisms. In: Wall PD, Melzack RR, editors. Textbook of pain. Edinburgh: Churchill Livingstone; 1989. p. 206–17. [14] Rosenfield JP. Interacting brain stem components of opiate-activated descending fi Fig. 13. Schematic representation of histologically con rmed sites of (A) microinjections pain-inhibition systems. Neurosci Biobehav Rev 1994;18:403–9. of ibotenic acid (1.0 μg/0.2 μL) into the LC followed by intraperitoneal administration of [15] Tracey I, Ploghaus A, Gati JS, et al. Imaging attentional modulation of pain in the PTZ at 64 mg/kg; (B) sham procedure in the LC+PTZ at 64 mg/kg; (C) microinjections of periaqueductal gray in humans. J Neurosci 2002;22:2748–52. ibotenic acid (1.0 μg/0.2 μL) into the LC followed by intraperitoneal administration of PTZ [16] Dubner R, Gold M. The neurobiology of pain. Proc Natl Acad Sci 1999;96: at 64 mg/kg, depicted on anagrams of Paxinos and Watson's atlas [90]. 7627–60. 176 T.T. Felippotti et al. / Epilepsy & Behavior 22 (2011) 165–177

[17] Couto LB, Moroni CR, Ferreira CMR, et al. Descriptive and functional neuroanatomy [47] Kawasaki Y, Kumamoto E, Furue H, Yoshimura M. Alpha 2 adrenoceptor-mediated of locus coeruleus noradrenaline-containing neuron involvement in bradykinin- presynaptic inhibition of primary afferent glutamatergic transmission in rat induced antinoception on principal sensory trigeminal nucleus. J Chem Neuroanat substantia gelatinosa neurons. Anesthesiology 2003;98:682–9. 2006;32:28–45. [48] Binder W, Mousa SA, Sitte N, Kaiser M, Stein C, Schafer M. Sympathetic activation [18] Proudfit HK, Clark FM. The projections of locus coeruleus neurons to the spinal triggers endogenous opioid release and analgesia within peripheral inflamed cord. Prog Brain Res 1991;88:123–41. tissue. Eur J Neuroci 2004;20:92–100. [19] Tjolsen A, Berge OG, Hole K. Lesion of bulbo-spinal serotonergic or noradrenergic [49] Peng YB, Lin Q, Willis WD. Involvement of alpha-2 adrenoceptors in the pathways reduces nociception as measured by formalin test. Acta Physiol Scand periaqueductal gray-induced inhibition of dorsal horn cell activity in rats. 1991;142:229–36. J Pharmacol Exp Ther 1996;278:125–35. [20] Giorgi FS, Blandini F, Cantafora E, et al. Activation of brain metabolism and fos [50] Scheinin M, Lomasney JW, Hayden-Hixson DM, et al. Distribution of alpha 2- during limbic seizures: the role of locus coeruleus. Neurobiol Dis 2008;30: adrenergic receptor subtype gene expression in rat brain. Brain Res Mol Brain Res 388–99. 1994;21:133–49. [21] Jones SL. Descending noradrenergic influences on pain. Prog Brain Res 1991;88: [51] Aantaa R, Marjamäki A, Scheinin M. Molecular pharmacology of alpha 2- 381–94. adrenoceptor subtypes. Ann Med 1995;27:439–49. [22] Jones SL, Gebhart GF. Quantitative characterization of ceruleospinal inhibition of [52]BerodA,ChatM,PautL,JappazM.CatecholaminergicandGABAergic nociceptive transmission in the rat. J Neurophysiol 1986;56:1397–410. anatomical relationship in the rat substantia nigra, locus coeruleus and [23] Margarlit D, Segal M. A pharmacologic study of analgesia produced by stimulation hypothalamic median eminence: immunohistochemical visualization of bio- of the nucleus locus coeruleus. Psychopharmacology 1979;62:169–73. synthetic enzymes on serial semithin-embeded sections. J Histochem Cytochem [24] Hodge CJ, Apkarian AV, Stevens RT. Dorsolateral pontine inhibition of dorsal horn 1984;32:1331–8. cell responses to cutaneous stimulation, lack of dependence on catecholaminergic [53] Iijima K, Kobayachi R, Kojima N. Immunohistochemical studies on GABAergic systems in cat. J Neurophysiol 1983;50:1220–35. neurons in the rat locus coeruleus, with special reference to their relationship to [25] Mokha SS, McMillan JA, Iggo A. Descending control of spinal nociceptive astrocytes. Acta Anat 1987;129:116–22. transmission: actions produced on spinal multireceptive neurones from the [54] Rodríguez-Pallares J, Caruncho HJ, Lopez-Real A, Wójcik S, Guerra MJ, Labandeira- nuclei locus coeruleus and raphe magnus. Exp Brain Res 1985;58:213–26. García JL. Rat brain cholinergic, dopaminergic, noradrenergic and serotonergic

[26] Tsuruoka M, Matsutami K, Maeda M, Inoue T. Coeruleotrigeminal inhibition of neurons express GABAA receptors derived from alpha 3 subunit. Recept Channels nociception of nociceptive processing in the rat trigeminal subnucleus caudalis. 2001;7:471–8. Brain Res 2003;993:146–53. [55] Gill EW, Vaughan Williams EM. Local anaesthetic activity of the β-receptor [27] West WL, Yeomans DC, Proudfit HK. The function of noradrenergic neurons in antagonist, pronethalol. Nature 1964;201:199. mediating antinociception induced by electrical stimulation of the locus coeruleus [56] Morales-Aguilera A, Vaughan Williams EM. The effects on cardiac muscles of β- in two different sources of Sprague–Dawley rats. Brain Res 1993;626:127–35. receptor antagonists in relation to their activity as local anaesthetic. Br J [28] Pan YZ, Li DP, Chen SR, Pan HL. Activation of mu-opioid receptors excites a Pharmacol Chemother 1965;24:332–8. population of locus coeruleus–spinal neurons through presynaptic disinhibition. [57] Ishida H, Sasa M, Takaori S. Local anesthetic activity of the β-adrenergic blocking Brain Res 2004;997:67–78. drugs in the crayfish giant axon, with reference to calcium ion. Jpn J Pharmacol [29] Segal M. Serotonergic innervation of the locus coeruleus from the dorsal raphe and 1980;30:607–19. its action on responses to noxious stimuli. J Physiol 1979;286:401–15. [58] Akaike N, Ito H, Nishi K, Oyama Y. Further analysis of inhibitory effects of [30] Oliveiras JL, Guilbaud G, Besson JM. A map of serotonergic structures involved in propranolol and local anaethetics on the calcium current in Helix neurones. Br J stimulation producing analgesia in unrestrained freely moving cats. Brain Res Pharmacol 1982;76:37–43. 1979;164:317–22. [59] Omura T, Kobayashi T, Nishioka K, Miyae N, Akaike N. Ca2+-antagonistic action of [31] Mayer DJ, Liebeskind JC. Pain reduction by focal electrical stimulation of the brain: bevantolol on hypothalamic neurons in vitro: its comparison with those of other an anatomical and behavioral analysis. Brain Res 1974;68:73–93. β-adrenoceptor antagonists, a local anesthetic and Ca2+-antagonist. Brain Res [32] Hosobuch Y, Rossier J, Bloom FE, Guillemin R. Stimulation of human periaque- 1996;706:289–92. ductal gray for pain relief increases immunorective beta endorphin in ventricular [60] Akaike N, Nishi K, Oyama Y. Inhibitory effects of propranolol on the calcium fluid. Science 1979;203:279–81. current of Helix neurons. Br J Pharmacol 1981;73:431–4. [33] Richardson DE, Akil H. Pain reduction by electrical brain stimulation in man: Part [61] Tsuruoka M, Maeda M, Nagasawas I, Inoue T. Spinal pathways mediating 1. Acute administration in periaqueductal and periventricular sites. J Neurosurg coeruleospinal antinociception in the rat. Neurosci Lett 2004;362:236–9. 1997;47:178–80. [62] Voisin DL, Guy N, Chalus M, Dallel R. Nociceptive stimulation activates locus [34] Morgan MM, Sohn JH, Liebeskind JC. Stimulation of the periaqueductal gray matter coeruleus neurons projecting to the somatosensory thalamus in the rat. J Physiol inhibits nociception at the supraspinal as well as spinal level. Brain Res 1989;502: 2005;566:929–37. 61–6. [63] Zhang C, Guo Y-Q, Qiao J-T, Dafny N. Locus coeruleus modulates thalamic [35] Cui M, Freng Y, McAdoo DJ, Willis WD. Periaqueductal gray stimulation-induced nociceptive responses via adrenoceptors. Brain Res 1998;784:116–22. inhibition of nociceptive dorsal horn neurons in rats is associated with the release [64] Zhang G-W, Yang C-X, Gao H-R, et al. Microinjection of different doses of of norepinephrine, serotonin, and amino acids. J Pharmacol Exp Ther 1999;289: norepinephrine into the caudate putamen produces opposing effects in rats. 868–76. Neurosci Lett 2010;471:125–8. [36] Aghajanian GK, Wang RY, Baraban J. Serotonergic and non-serotonergic neurons [65] Holden JE, Shwartz EJ, Proudfit HK. Microinjection of morphine in the A7 of the dorsal raphe: reciprocal changes in firing induced by peripheral nerve catecholamine cell group produces opposing effects on nociception that are stimulation. Brain Res 1978;153:169–75. mediated by alpha1- and alpha2-adrenoceptors. Neuroscience 1999;83: [37] Jacobs BL, Gannon PJ, Azmitia EC. Atlas of serotonergic cell bodies in the cat 979–90. brainstem: an immunocytochemical analysis. Brain Res Bull 1984;13:1–31. [66] Sagen J, Proudfit HK. Evidence for pain modulation by pre- and postsynaptic [38] Mongeau M, Weiss M, de Montigny C, Blier P. Effect of acute, short- and long-term noradrenergic receptors in the medulla oblongata. Brain Res 1985;331:285–93. milnacipran administration on rat locus coeruleus noradrenergic and dorsal raphe [67] Li X, Zhao Z, Pan HL, Eisenach JC, Paqueron X. Norepinephrine release from serotonergic neurons. Neuropharmacology 1998;37:905–18. spinal synaptosomes: auto-α2-adrenergic receptor modulation. Anesthesiology [39] Haddjeri N, de Montigny C, Blier P. Modulation of the firing activity of 2000;93:393–402. noradrenergic neurones in the rat locus coeruleus by the 5-hydroxytryptamine [68] Green GM, Lyons L, Dickenson AH. Alpha2-adrenoceptor antagonists enhance system. Br J Pharmacol 1997;120:865–75. responses of dorsal horn neurons to formalin induced inflammation. Eur [40] Bandoh T, Hayashi M, Ino K, Takada S, Ushizawa D, Hoshi K. Acute effect of J Pharmacol 1998;347:201–4. milnacipran on the relationship between the locus coeruleus noradrenergic and [69] Baik E, Chung JM, Chung K. Peripheral norepinephrine exacerbates neuritis- dorsal raphe serotonergic neuronal transmitters. Eur Neuropsychopharmacol induced hyperalgesia. J Pain 2003;4:212–21. 2004;14:471–8. [70] Pertovaara A. Noradrenergic pain modulation. Prog Neurobiol 2006;80:53–83. [41] Nishikawa T, Scatton B. Evidence for a GABAergic inhibitory influence on serotonergic [71] Westlund KN, Coutler JD. Descending projections of the locus coeruleus and neurons originating from the dorsal raphe. Brain Res 1983;279:325–9. subcoeruleus/medial parabrachial nuclei in monkey: axonal transport studies [42] Aston-Jones G, Zhu Y, Card JP. Numerous GABAergic afferents to locus ceruleus in and dopamine-β-hydroxylase immunocytochemistry. Brain Res Rev 1980;2: the pericerulear dendritic zone: possible interneuronal pool. J Neurosci 2004;24: 235–64. 2313–21. [72] Kwiat GC, Basbaum AI. The origin of brainstem noradrenergic and serotonergic [43] Mohrland JS, McManus DQ, Gebhart GF. Lesions in nucleus reticularis gigan- projections to the spinal cord dorsal horn in the rat. Somatosens Mot Res 1992;9: tocellularis: effects on the antinociception produced by microinjection of 157–73. morphine and focal electrical stimulation in the periaqueductal gray matter. [73] Brightwell JJ, Taylor BK. Noradrenergic neurons in the locus coeruleus contribute Brain Res 1982;231:143–52. to neuropathic pain. Neuroscience 2009;160:174–85. [44] Fardin V, Oliveras JL, Besson JM. A reinvestigation of analgesic effects [74] Xanthos DN, Bennett GJ, Coderre TJ. Norepinephrine induced nociception and induced by stimulation of the periaqueductal gray matter in the rat I: the vasoconstrictor hypersensitivity in rats with chronic post-ischemia pain. Pain production of behavioral side effects together with analgesia. Brain Res 2008;137:640–51. 1984;306:105–23. [75] Schattschneider J, Scarano M, Binder A, Wasner G, Baron R. Modulation of [45] Zhuo M, Gebhart GF. Spinal cholinergic and monoaminergic receptors mediate sensitized C-fibers by adrenergic stimulation in human neuropathic pain. Eur J descending inhibition from the nuclei reticularis and gigantocellularis pars alpha Pain 2008;12:517–24. in the rat. Brain Res 1990;531:67–78. [76] CoimbraNC, Castro-Souza C, Segato EN, etal.Post-ictal analgesia: involvementof [46] Prado WA, Roberts MH. An assessment of the antinociceptive and aversive effects opioid, serotoninergic and cholinergic mechanisms. Brains Res 2001;888: of stimulating identified sites in the rat brain. Brain Res 1985;340:219–28. 314–20. T.T. Felippotti et al. / Epilepsy & Behavior 22 (2011) 165–177 177

[77] Coimbra NC, Freitas RL, Savoldi M, et al. Opioid neurotransmission in the post-ictal [83] Stull RE, Jobe PC, Geiger PF. Brain areas involved in the catecholamine mediated

analgesia: involvement of μ1-opioid receptor. Brain Res 2001;903:216–21. regulation of electroshock seizure intensity. J Pharm Pharmacol 1977;29:8–11. [78] De Oliveira RC, De Oliveira R, Ferreira CMR, Coimbra NC. Involvement of 5-HT2 [84] Meyer H, Frey HH. Dependence of anticonvulsant drug action on central serotonergic receptors of the nucleus raphe magnus and nucleus reticularis monoamines. Neuropharmacology 1973;12:939–47. gigantocellularis/paragigantocellularis complex neural networks in the antinoci- [85] Kellog C. Audiogenic seizures: relation to age and mechanisms of monoamine ceptive phenomenon that follows the post-ictal immobility syndrome. Exp Neurol neurotransmission. Brain Res 1976;106:87–103. 2006;201:144–53. [86] Giorgi FS, Mauceli G, Blandini F, et al. Locus coeruleus and neuronal plasticity in a [79] Giorgi FS, Pizzanelli C, Biagioni F, Murri L, Fornai F. The role of norepinephrine model of focal limbic epilepsy. Epilepsia 2006;47:21–5. in epilepsy: from the bench to the beside. Neurosci Biobehav Rev 2004;28: [87] Jerlicz M, Kostowski W, Bidzinski A, Hauptman M, Dymecki J. Audiogenic seizures 507–24. in rats: relation to noradrenergic neurons of the locus coeruleus. Acta Physiol Pol [80] El Hamdi G, Boutroy MJ, Nehlig A. Effects of pentylenetetrazol-induced seizures on 1978;29:409–12. dopamine and norepinephrine levels and on glucose utilization in various brain [88] Giorgi FS, Ferrucci M, Lazzeri G, et al. A damage to locus coeruleus neurons regions of the developing rat. Int J Dev Neurosci 1992;10:301–11. converts sporadic seizures into self-sustaining limbic status epilepticus. Eur J [81] Colasanti BK, Craig CR. Brain concentrations and synthesis rates of biogenic Neurosci 2003;17:2593–601. amines during chronic cobalt experimental epilepsy in the rat. Neuropharmacol- [89] Cox B, Lomax P. Brain amines and spontaneous epileptic seizures in the Mongolian ogy 1973;12:221–31. gerbil. Pharmacol Biochem Behav 1976;4:263–7. [82] Jobe PC, Picchioni AL, Chin L. Role of brain norepinephrine in audiogenic seizure in [90] Paxinos G, Watson C. The rat brain in stereotaxic coordinates. Sydney: Academic the rat. J Pharmacol Exp Ther 1973;184:1–10. Press; 1997.