Epilepsia, 46(Suppl. 5):189–197, 2005 Blackwell Publishing, Inc. C International League Against Epilepsy

Proechimys guyannensis:AnAnimal Model of Resistance to Epilepsy

∗ ∗ ∗ †Ricardo Mario Arida, †Fulvio Alexandre Scorza, ‡Reinaldo de Amorim Carvalho, ∗ and Esper A. Cavalheiro

∗ Disciplina de Neurologia Experimental, Universidade Federal de Sao˜ Paulo–UNIFESP, and †Laboratorio´ de Neurociencias,ˆ Nucleo´ de Pesquisas Tecnologicas´ (NPT)–Universidade de Mogi das Cruzes-UMC, Sao˜ Paulo, and ‡Centro Nacional de Primatas, Para,´ Brazil

Summary: Purpose: The potential interest of Proechimys Wistar rats were able to induce status epilepticus (SE) in PG guyannensis (PG), a spiny rat species living in the Amazo- . Pilocarpine-induced SE in PG had a shorter duration, nian region, as an model of anticonvulsant mechanisms, rarely exceeding 2 h, in contrast to the 8- to 12- h long SE in prompted the investigation of the susceptibility of this animal the Wistar rat. Of the 61 PG animals injected with pilocarpine, species to different epileptogenic treatments. 48 presented with SE and only two presented with some sponta- Methods: Adult male Wistar and PG animals were submitted neous seizures after silent periods of 60 and 66 days. KA elicited to amygdala kindling, the pilocarpine model and the intrahip- self-sustained electrographic SE in PG animals, which lasted for pocampal kainic acid (KA) model. Electrographic, behavioral, 72 h. None of the surviving animals presented with spontaneous and neuropathological changes were compared between Wistar seizures in the long-term observation period (up to 120 days). and PG animals. Conclusions: These findings indicate that the PG animal Results: PG animals demonstrated a striking resistance to may have natural endogenous anticonvulsant mechanisms and reaching stage 5 of kindling. Of the 43 PG rats submitted to also may be an animal model that is resistant to epilepto- the kindling process, only three animals reached stage 5. In the genic treatments. KeyWords: Epilepsy—Proechimys guyan- pilocarpine and KA models, doses lower than those used in nensis—Kindling—Pilocarpine—Kainic acid.

Proechimys guyannensis (PG), a that lives in the to the epileptogenic treatment of the muscarinic choliner- Amazonian region, belongs to the Echimydae family, Hys- gic agonist pilocarpine (6). A previous study (7) showed tricomorpha suborder, Proechimys genus (1). PG, known that, in PG, behavioral and electrographic characteristics as Casiragua in Brazil, inhabits forests often near coasts of pilocarpine-induced status epilepticus (SE) are less in- and waterways. They are nocturnal, leaving their dens in tense than those observed in other laboratory animals (8) the evening to forage on the forest floor. The main char- and, in contrast to rats and mice, they do not develop spon- acteristic of this species is the high degree of maturity taneous seizures in the long run. shown by the newborn animal, which contrasts with the The reliability of PG as a natural animal model with majority of conventional animal species used in laborato- endogenous antiepileptic mechanisms prompted the in- ries. This animal species has received some attention as a vestigation of the susceptibility of this animal specie to natural host to infectious parasites (2), and has been ex- different experimental epilepsy paradigms. Accordingly, tensively studied in relation to their ecology and evolution the aim of the present study was to observe electrographic, (3–5). However, the organization of the PG brain has been behavioral, and neuropathological changes in PG using poorly studied. three models of temporal lobe epilepsy, i.e., the amygdala Animal studies have significantly contributed to the un- kindling, the pilocarpine model, and the intrahippocam- derstanding of the epileptogenesis and the mechanism of pal kainic acid (KA) model, and compare these findings action of several antiepileptic drugs. During a comparative with those widely reported in Wistar rats using the same investigation of the response of different rodent species to experimental protocols. proconvulsant agents, the PG was found to be resistant MATERIAL AND METHODS Address correspondence and reprint requests to Dr. R. M. Arida at Animals Disciplina de Neurologia Experimental, Universidade Federal de S˜ao Paulo–UNIFESP,Rua Botucat´u 862, Vila Clementino, CEP 04023-9000, Adult male Wistar and PG animals (2–3 months old, S˜ao Paulo, SP. Brazil. E-mail: [email protected] weighing 210–250 g) were used in these experiments. PG 189 190 R. M. ARIDA ET AL. animals, originally from the Brazilian Amazon basin, were EEG recordings bred in a colony established at the Universidade Federal For EEG recordings (pilocarpine- and KA-injected an- de S˜ao Paulo/Escola Paulista de Medicina and housed in imals), bipolar twisted wire electrodes were implanted a standard light-dark cycle with free access to food and stereotaxically in the right hippocampus and fixed to the water. Wistar rats of the same age and sex were obtained skull with dental acrylic cement. Notice that in KA experi- from the laboratory colony. ments, intrahippocampal injections were also made in the right hippocampus. Surface records were led from jew- Experimental procedures eler screws positioned bilaterally over the sensorimotor cortex. Following pilocarpine (Merck, Darmstadt, Ger- Amygdala kindling many) or KA injections, EEG activity was continuously Under deep anesthesia (Thionembutal, 50 mg/kg, i.p.), recorded for periods from 6 to 72 h. Animals that survived 43 PGs were stereotaxically implanted with twisted bipo- pilocarpine- or KA-induced SE were continuously moni- µ lar electrodes of nichrome wires (100 m), aimed at the tored during 24 h for the next 120 days for the detection right amygdala. To determine the appropriate stereotaxic of spontaneous seizures using a video-EEG system (Stel- coordinates for the amygdala in this animal species, some late System). Infrared-emitting lights were used during animals were submitted to stereotaxic surgery and histo- the dark periods to allow for video recording of the ani- logical examinations were performed to find out the ap- mals’ activity. Two independent observers were recruited propriate electrode placements. The following coordinates for EEG and behavioral analysis. were established for the PG: 2.3 mm posterior to bregma, 5.0 lateral to midline, and 8.5 deep. The same surgical Histology procedures were used for Wistar animals (n = 14) and At the end of the experiments, histological analyses stereotaxic coordinates were determined (2.5 mm poste- were performed for the three models of temporal lobe rior to bregma, 4.5 lateral to midline, and 8.5 deep) ac- epilepsy. Following variable survival times, ranging from cording to the atlas of Pellegrino and Cushman (9). The 1to120 days, animals were perfused and their brains pro- parameters of surgical techniques and stimulation were cessed for neo-Timm and Nissl methods following pro- performed following protocols similar to those described tocols described in previous studies (13,15). Naive Wis- in previous studies (10,11). tar and PG animals (n = 2) were perfused at times that matched those of experimental animals and served as Pilocarpine controls. Fourteen Wistar and 61 PG animals were used for the Statistical analysis pilocarpine procedure. Pilocarpine or vehicle injections Statistical analysis for the kindling procedure was done were performed following protocols similar to those de- using the t test and Fisher’s Exact test to analyze the after scribed in previous studies (12,13). To minimize the pe- discharge (AD) duration, number of stimuli to reach stage ripheral effects of pilocarpine, the cholinergic antagonist 5, and after discharge threshold (ADT) on PG and Wis- methyl-scopolamine (1 mg/kg, i.p.; Sigma, St. Louis, MO, tar animals. Significance was established at the p < 0.05 U.S.A.) was administered to all Wistar and PG animals. level.

Intrahippocampal KA RESULTS Under deep anesthesia, PG animals were placed in Behavioral and EEG observations a stereotaxic apparatus. KA injections were made with a1µl Hamilton syringe in the CA1 subregion of the Kindling hippocampus. The appropriate hippocampal coordinates All Wistar rats (n = 14) submitted to kindling process (3.5 mm posterior to bregma, 4.0 lateral to midline, and reached stage 5. The mean number of stimulations and 4.0 from the cortex surface) were determined from pre- the AD duration required for the first generalized seizure vious experiments (6). The same surgical procedure was (stage 5) and for each stage of kindling for Wistar rats used for Wistar rats and stereotaxic coordinates were de- is similar to those observed in previous studies (16,17). termined according to the atlas of Pellegrino and Cush- From 43 PG animals submitted to the kindling process, man (9). KA (Sigma, St. Louis, MO, U.S.A.) was dis- only three animals reached stage 5. Of the 40 animals solved in artificial serum, pH = 7.4, and infused in a vol- that did not reach stage 5, 16 did not get beyond stage ume of 0.2 µl overaperiod of 2 min. Different doses 1, 14 did not get beyond stage 2, 7 did not get beyond of KA were tested in PG animals: 1.0 µg(n= 3), stage 3, and 3 did not get beyond stage 4. During kindling 0.5 µg(n= 3), 0.25 µg(n= 3), 0.1 µg(n= 10), (stages 1–3), all animals presented behavioral alterations and 0.06 µg(n= 5). Wistar rats (n = 6) received the similar to those observed in Wistar rats and other species, same KA dose (1 ug/0.2 ul) as described by Cavalheiro i.e., exploratory behavior and facial movements, eye clo- et al. (14). sure, chewing, salivation, and forelimb clonus. Stages 4

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TABLE 1. Mean number of stimulations required for the first generalized seizure (stage 5) and for each stage of the kindling process for Proechimys and Wistar groups

Number of AD to first stage 5 Duration of each kindling stage (number of ADS)

Group seizure Stage 1 Stage 2 Stage 3 Stage 4 Proechimys 54.6 ± 23.0a 37.0 ± 21.7a 37.0 ± 3.4a 6.3 ± 3.0a 4.3 ± 1.5 (n = 43) (n = 3) (n = 16) (n = 15) (n = 7) (n = 3) Wistar 15.6 ± 2.6 3.3 ± 1.0 3.6 ± 1.8 3.0 ± 0.7 2.4 ± 1.6 (n = 14) (n = 14) (n = 14) (n = 14) (n = 14) (n = 14)

Data are expressed as mean ± SD. Proechimys group consisted of 43 animals and Wistar group consisted of 14 animals at the beginning of the kindling procedure. For statistical analysis, we used the number of Proechimys animals (in brackets) that evolved from one to the following stage. Different from control group for ap < 0.05 (t test). and 5 (n = 3) of kindling in PG were characterized by Pilocarpine an initial phase of catalepsy similar to that observed fol- As repeatedly reported in previous studies (12,13), lowing the administration of narcotic agonists (18). This Wistar rats injected with pilocarpine presented continu- behavior lasted 20–30 s and was followed by rearing and ous mastication, salivation, and clonic movements of the falling. The time course of development of wet dog shakes vibrissae and limbs of increasing severity. Twenty to 50 (WDS) for Wistar rats followed the pattern observed in min later, pilocarpine-injected Wistar rats presented with previous study (19), i.e., WDS occurs in relation to AD limbic convulsions with secondary generalization, simi- development and seizure stage. The timing of onset of lar to stage-5 kindled seizures, that recurred every 5–10 WDS in PG animals occurred following stage 2–3, and the min, evolving to typical limbic SE that could last 8–12 number of WDS was not altered during all the kindling h. Behavior returned progressively to normal over a 3- to procedure. 5-day period (8,12,13). Electrographic changes in Wistar Table 1 indicates the mean number of stimulations re- rats immediately after pilocarpine injection were charac- quired for the first generalized seizure (stage 5) and for terized by a theta rhythm in the hippocampus and low- each stage of the kindling process for the PG and the Wis- voltage fast activity in the cortex. This activity progressed tar groups. The number of stimulations required for each to high-voltage fast activity with spikes in the hippocam- stage, except for stage 4, was statistically higher for the pus that spread to the cortex and evolved into electro- PG group when compared to the Wistar group. In the PG graphic seizures that recurred every 2–8 min and became group, a striking decrease in the number of stimulations continuous within 50–60 min after pilocarpine injection. from stage 2 to stages 3 and 4 was also noted. The PG This pattern of electrographic activity was sustained for group showed a higher mean ADT (198.4 ± 24.3 µA) 8–12 h and then decreased gradually during the next 12–24 compared to the Wistar group (95.2 ± 12 µA). The AD h. After the silent (latent) period that lasted ∼2 weeks (8), duration from the PG group was longer from stages 1 to spontaneous recurrent seizures (SRSs; chronic period) ap- 4ofkindling development when compared to the Wistar peared (8). In PG animals, pilocarpine (350–380 mg/kg– group but not statistically different in stage 5 (Table 2). doses regularly used in Wistar rats) induced severe tonic When we computed the distribution of the ADs for differ- seizures followed by death of all animals. Doses slightly ent seizure stages we observed no significant increase in lower (300–320 mg/kg) than those previously mentioned the AD duration from stage 1 to stage 3 in PG animals. were able to induce repetitive electrographic and behav- The electrographic alterations from PG animals during ioral seizures that culminated in SE 20–30 min following the kindling process were basically the same as described pilocarpine. However, pilocarpine-induced SE in PG had in others species. During the first stage of kindling, after a shorter duration, rarely exceeding 2 h, clearly in contrast a brief period of latency (∼3–4s), epileptiform dischar- to the 8–12 h SE observed in the Wistar rat. Briefly, 15– ges of high amplitude appeared in the amygdala with 30 min after pilocarpine, PG animals exhibited behavioral no propagation to cortex. With progressive stimulation, abnormalities typical of kindling stage 2, with mastica- a subsequent propagation to the cortex could be observed tion and head nodding that rarely evolved to more severe (Fig. 1). seizures. The animals remained instead for 1–2 h with the

TABLE 2. AD duration of each stage of kindling (in seconds)

Group Stage 1 Stage 2 Stage 3 Stage 4 Stage 5

Proechimys 62.4 ± 29.6a 66.2 ± 24.4a 65.5 ± 34.7a 93.0 ± 19.4a 78.5 ± 27.8 Wistar 8.5 ± 3.8 34.7 ± 21.3 49.5 ± 33.2 55.1 ± 28.1 64.0 ± 23.5

Data are expressed as mean ± SD. Different from Wistar group for ap < 0.05 (t test).

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FIG. 1. Electrographic recordings during amygdala kindling development in Proechimys rat.(A) After electrical stimulation (arrow) (stage 1 of kindling) it is observed a short latency period (ca 3–4 s) and then epileptiform discharges of high amplitude. Cortical recording displays no changes. (B) EEG recordings during stage 5 of kindling showing (latency of 1-2 s) epileptiform discharges in amygdala with subsequent propagation to the cortex. Theses discharges develop with high frequency and amplitude, apparently synchronised in both recordings and at the final phase, (∗), with lower frequency. four limbs on the floor, extended fingers, and tonic exten- In PG animals, one-tenth of the KA dose usually used sion of the tail, with rare clonic movements of the head in Wistar rats elicited self-sustained behavior and electro- or limbs, and then recovered to normal behavior. In PG, graphic SE, which lasted between 48–72 h. All PG animals aburst of paroxysmal spiking activity in the hippocam- that received higher KA doses (0.25–1.0 ug/0.2 µl) died pus spreading to the cortex was instead observed almost between 12 and 24 h during the SE period. Five of 10 PG immediately, 10 min after pilocarpine injection. During that received 0.1 µgofKAand four of five PG treated SE, while high-voltage spikes were observed for several with 0.06 µgofKAsurvived to the following 120 days of hours in Wistar rats, these electrographic changes were observation. Ten to 15 min after KA injection, PG pre- documented in PG only in the first3hafter pilocarpine sented tremor of vibrissae, salivation, and increased res- administration. The EEG findings we observed in PG are piratory frequency. These initial signs were followed by compared in Fig. 2 to those typically obtained in Wis- a characteristic behavior, with all four limbs on the floor, tar rats and corresponding to those reported in previous extended fingers, and tonic extension of the tail, with rare studies (12,13). Of 61 animals injected with pilocarpine, clonic movements of the head and limbs. Between 1 and 2 48 presented SE and only two PG presented some spon- h after KA injection, a cataleptic behavior was clearly ob- taneous seizures (∼1 per week for 8 weeks) after silent served in PG and was characterized by opisthotonus and periods of 60 and 66 days. an S-shaped tail. This behavior, occasionally interrupted by partial complex seizures with subsequent generaliza- tion, was variable in terms of duration and intensity and Intrahippocampal KA recurred every 15–30 min. As previously reported (14,20,21), during the first few The EEG findings in PG animals were similar to those minutes after KA injection, Wistar rats exhibited tremor typically obtained in Wistar rats and reported in previous of the vibrissae and facial muscles. Raised tails and defe- studies (14,22,24). In both animal species, EEG alterations cation were also frequent. Thereafter, when the anesthetic characterized by spikes or polyspikes of high frequency effect decreased, animals developed rotatory locomotion and large amplitude could be observed initially in the hip- toward the injected side. By 3–5 h, convulsive seizures oc- pocampus a few minutes after KA injection. This activity curred at intervals of 10–20 min, alternating with periods rapidly spread to the cortex. This pattern of electrographic of complete immobility. All Wistar rats developed lim- activity lasted for several hours for both Wistar and PG bic SE 1–2 h after KA injection that lasted up to 8–12 h. animals. In Wistar rats, the seizure activity gradually de- One out of six Wistar rats treated with intrahippocampal creased during the following 24–48 h. In PG animals, this KA died during the SE period. After the SE, a gradual pattern of electrographic activity extended until 72 h. The normalization of behavior was observed in the surviving EEG recording of PG and Wistar animals is shown in animals. The latent period for Wistar rats lasted from 5 to Fig. 3. SE developed in all PG animals with the different 20 days when the first spontaneous seizure could be ob- doses studied and lasted 48–72 h for the surviving ani- served initiating the chronic period. Spontaneous seizures mals. In contrast to Wistar rats, none of the surviving PG resembling stage 5–kindled seizures recurred three to five presented any behavioral or electrographic changes that times per week and have been extensively studied in Wis- could suggest the occurrence of epileptic manifestations tar rats (14,22–24). in the following 120 days of observation.

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FIG. 2. Electroencephalographic recordings from the hippocampus (HPC) and cerebral cortex (CX) illustrating the sequences of alter- ations at various times following picolarpine injection to Proechimys guyannensis (right) and Wistar rat (left). Note the occurrence of spikes in the hippocampal recordings 10 min after¨pilocarpine injection, and the elec- trographic seizure at 25 min. Electrographic status epilepticus is observed at 1 h. Status epilepticus persists 2 h later in the Wistar rat, whereas a progressive normalization of the EEG can be observed in Proechimys. At 6 and 24 h after pilocarpine injection, note the pres- ence of spiking activity in the recordings from the Wistar rat, whereas those obtained from Proechimys can be considered similar to the pretreatment pattern.

Histological analysis agreement with previous studies (13), the hippocampus of PG animals that presented SRSs had fewer cells in specific Kindling areas of the hippocampal formation compared with con- The histopathological analysis to kindling in Wistar an- trols. Cell loss was seen in the CA1 area and in the hilus imals previously has been described (25). No significant of the dentate gyrus (Fig. 5). Furthermore, the pattern of cell loss in specific areas of the hippocampal formation neo-Timm staining was clearly altered in the animals that were observed in the three kindled PG animals, or in ani- showed SRSs. Neo-Timm positive granules, normally ab- mals that did not reach stage 5 when compared with con- sent in the supragranular layer, were present in the dentate trols. In addition, none of the animals subjected to the gyrus of the PG with SRSs (Fig. 5). kindling process presented altered neo-Timm staining. Pilocarpine Intrahippocampal KA Wistar rats showed cell loss in the hippocampal sub- The histopathological analysis to kainate in Wistar rats field CA1, CA3, and in the hilus of dentate gyrus as previ- has been described previously (26,27). The injected (ip- ously described (13,15). In Nissl-stained sections, all the silateral) side showed neuronal damage, predominantly classical cytoarchitectonic subdivisions of the hippocam- in the CA1/CA3 subfields and in the hilus, whereas the pus were clearly identified in the PG animals (Fig. 4). In dentate gyrus was rarely affected. In the contralateral

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FIG. 3. Electroencephalographic recordings from the hippocampus (HPC) and cerebral cortex (CX) illustrating the sequences of alterations at various times following KA injection to Proechimys guyannensis (right) and Wistar rat (left). The Wistar rat data derive from laboratory archival material of the Department of Experimental Neurology of the UNIFESP-EPM (S˜ao Paulo-Brazil) and illustrate findings repeatedly reported in the literature. One hour after KA injection, it can be observed in both animals species, the occurrence of electrographic status epilepticus. Status epilepticus persisted in both animal species at 3 h. At 6, 12 and 24 h the presence of spiking activity in the recordings from both species is still maintained. Note at 24 and 48 h after KA injection the presence of spiking activity in the recordings of the Proechimys guyannensis rat, whereas a progressive normalization of the EEG can be observed in the Wistar rat 24 h after KA injection. hippocampus, the CA1 and CA3 pyramidal cells were sessment of neo-Timm–stained sections showed that all found to be vulnerable, and some subpopulations of the animals that underwent SE did present sprouting in the nonpyramidal cells were selectively damaged, mainly in supragranular layer of the dentate gyrus and in the stra- the hilus and in the CA3 region. In PG animals, at the tum radiatum of CA3 (Fig. 6). injection site, the lesion showed a complete neuronal de- struction at the injected hippocampal formation, more pro- DISCUSSION nounced in CA1/CA3 areas, and with less marked changes in the contralateral hippocampus. The pattern of cell de- The present study provides observations on behav- struction and damage around the injection site was found ioral, electrographic, and neuropathological changes in to be similar at any survival times analyzed (Fig. 6). As- PG animals after the amygdala kindling process, following

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FIG. 4. Nissl and neo-Timm staining of the hippocampal formation in a Proechimys guyannensis animal. pilocarpine and intrahippocampal KA injection. The find- propagation of the ADs from the primary focus (28). As ings pointed out a remarkable resistance of these animals kindling progresses, AD activity propagates and recruits to the three experimental models of limbic epilepsy. more neural networks (29). This new network presumably The results obtained during the amygdala kindling de- serves as a pathway for the propagation and generaliza- velopment in PG animals demonstrated that this animal tion of subsequent seizures. These observations lead us species presents significant differences in kindling devel- to believe that there must be some factors preventing the opment when compared to other animal species. To reach increased AD to gain access to structures related to the stage 5, a higher number of stimulations were necessary motor organization of the epileptic seizure. in PG animals and only three animals were fully kindled. In the pilocarpine model, only two out of 61 PG pre- These results indicate that the PG animal presents some sented rare spontaneous seizures in the chronic period af- resistance to reach a fully developed motor seizure. For ter long latent periods (6). The shorter SE duration (∼2 instance, during the course of kindling, AD duration did h), clearly in contrast to the 8- to 12-h long SE in the not change from stage 1 to stage 3 in PG animals. AD Wistar rat, could explain the very low rate of animals pre- duration in PG animals was significantly longer in stages senting spontaneous seizures. As previously demonstrated 1–4 and striking higher in stage 1 when compared to Wis- by Lemos and Cavalheiro (30), the development of long- tar rats. The rate of kindling seems to be related to the term epileptic seizures is closely related to the duration length of the initial AD, i.e., the longer the initial AD, of pilocarpine-induced SE. In this sense, this observation the more rapidly kindling appears (28). Although PG an- seems to justify the present results of few or no sponta- imals presented longer initial AD than Wistar rats, the neous seizures in PG following pilocarpine. number of stimulations to reach stage 5 was remarkably On the other hand, the same explanation can not be higher in PG animals, suggesting that different inhibitory applied to the findings obtained in PG treated with in- mechanisms may be involved in this animal species during trahippocampal KA. As observed, intrahippocampal KA the kindling process. in PG induced SE of long duration that could last, in some An augmented AD duration was also noted in PG ani- animals, >48 h and no sign of epileptic activity could mals from stages 1 to 4, but not in stage 5. The initial stages be detected in these animals during the long-term obser- of kindling (stages 1 and 2) are considered to be a phase vation. PG animals were extremely sensitive to the acute of building up activation of the stimulated site and grad- effects of the KA injection, i.e., KA doses that normally in- ual engaging of structures by the increasingly widespread duce focal spiking activity of short duration when applied

FIG. 5. Nissl and neo-Timm staining of the hippocampal formation 120 days after pilocarpine administration in a Proechimys guyannensis animal.

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FIG. 6. Nissl and neo-Timm staining of the hippocampal formation 120 days after intrahippocampal injection of kainic acid in a Proechimys guyannensis animal. to the hippocampal formation of Wistar rats (14) were have anticonvulsant properties (32–34) and are released able to evoke severe and long-lasting seizure activity that after seizure activity (35). Rocha and colleagues (36) lasted up to 48 h in the PG. Moreover, neuropathological demonstrated that a subconvulsant amount of pentylenete- examinations of PG brains that survived KA-induced SE trazol (PTZ) enhances opioid peptide release, decreases revealed severe cytoarchitectural changes in the injected mµ receptor binding, and increases proenkephalin lev- hippocampus with mossy fiber sprouting in the dentate els in the amygdala, suggesting that activation of opioid gyrus supragranular layer. Despite the fact that PG ani- peptide system in the amygdaloid nucleus after the admin- mals have presented longer SE than usually observed in istration of a small amount of PTZ may prevent the seizure Wistar rats and neuropathological changes similar to those propagation. Other hypotheses suggest the involvement of observed in Wistar after KA injection, none of these ani- the opioid system in the initial stages of kindling develop- mals presented spontaneous seizures during the 120 days ment (36). A microdialysis study (37), after amygdaloid- of behavioral observation. kindling stimulation, verified that enkephalin-related pep- Although our results indicate that PG animals are ex- tides that suppress epileptic activity (38) are released in the tremely sensitive to the three epileptogenic treatments, amygdala during the early kindling stages. We observed several findings with our group point to the fact that these that the initial stages of the kindling process (stages 1 and animals seem unable to establish an epileptic focus with an 2) in the PG animals were strikingly longer than usually appropriate circuitry for the elaboration of chronic spon- observed in other animal species, suggesting the opiate taneous seizures. This inability to form a chronic epileptic system’s participation in this process. Thus, preliminary focus in the limbic region could be due to several intrin- data (Rocha, personal communication) have analyzed that sic processes present in both limbic and extralimbic cir- the distribution of µ opiate receptors in naive PG brain fol- cuitry preventing the local discharges to gain access to lows a different pattern from that observed in Wistar rats. structures related to the motor organization of the epilep- Among these differences, the density of mu receptors in tic seizure. Information on these intrinsic processes could PG brain was significantly increased in the dentate gyrus help us to understand how to avoid or inhibit the forma- and decreased in substantia nigra pars reticulata, two cru- tion of epileptic foci following severe early-life events, cial areas for the elaboration of limbic seizures. The reality a common condition observed in humans with temporal is that other studies are necessary to determine how these lobe epilepsy. In other words, these circumstances could changes in the PG opiate system could be related to its reflect functional changes in inhibitory and facilitory pro- resistance to develop chronic epilepsy. cesses in the PG brain that could interfere with the es- Taken together, these findings indicate that the PG an- tablishment of the epileptic circuitry. It it possible that imal may have natural endogenous antiepileptic mecha- this resistance to generate limbic epileptic foci could be nisms and also be a resistant animal model to epileptogenic due to increased expression of calcium-binding proteins in treatments. Studies to expand our knowledge on neuronal PG hippocampal formation and thalamic nuclei following networks, receptors, and neurotransmitters mediating the the pilocarpine-induced SE, as demonstrated by Fabene excitation/inhibition balance regulating seizure spread in et al. (7, 31). this animal model of resistance to epilepsy may in the Another interesting finding observed in our study was future elucidate the mechanisms of epileptogenesis. related to cataleptic manifestations observed in PG during KA- or pilocarpine-induced SE and also during amygdala- kindled seizures, characterized by opisthotonus and S tail, REFERENCES suggesting the participation of the opiate system in this 1. Emmons LH. Ecology of Proechimys (Rodentia, Echimydae) in process. There is evidence to suggest that opioid peptides South-Eastern Peru. Trop Ecol 1982;23:280–90.

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Epilepsia, Vol. 46, Suppl. 5, 2005