Hippocampal expression of connexin36 and connexin43 during epileptogenesis in pilocarpine model of epilepsy

Background: Gap junctions [GJs] provide direct intercellular communications that are formed by hexameric protein subunits, called connexin [Cx]. The role of Cxs in epileptogenesis has not received sufficient attention. Hippocampus with critical function in epileptogenesis has a wide network of GJs. We examined the protein expression levels of hippocampal Cx36 [the prominent Cx present between GABAergic interneurons] and Cx43 [the main Cx expressed by astrocytes] during epileptogenesis in the pilocarpine model of epilepsy

Methods: Male Wistar rats received scopolamine [1 mg/kg, s.c.]. Pilocarpine [380 mg/kg, i.p.] was administered 30 min thereafter to induce status epilepticus [SE]. SE was stopped 2 h later by diazepam [10 mg/kg, i.p.]. Cx36 and Cx43 protein expression was assessed by Western blot analysis in the hippocampus of SE-experienced rats, after injection of diazepam [F0 subgroup], after acquisition of focal seizures [F3 subgroup], and after development of generalized seizures [F5 subgroup]. The control subgroups, C0, C3, and C5, were aged-matched rats, which received saline [1 ml/kg, i.p.] instead of pilocarpine. Injection of scopolamine and diazepam, and dissection of hippocampi were carried out at the same time interval as the test subgroups

Results: SE emerged in 67.1% of pilocarpine-treated animals. Focal and generalized seizures developed 3.8 +/- 0.4 and 7.0 +/- 0.5 days after SE, respectively. Cx36 protein abundance was not significantly different between test and control groups in the three time points. However, Cx43 protein level showed 40% increase in F3 subgroup [P<0.05 compared to C3, P<0.01 compared to F0 and F5]

Conclusion: Hippocampal Cx43 is overexpressed in pilocarpine model of epileptogenesis after acquisition of focal seizures

Facilitation of hippocampal kindling and exacerbation of kindled seizures by intra-CA1 injection of quinine: a possible role of Cx36 gap junctions

Background: GABAergic interneurons in the hippocampal CA1 area are mutually communicated by gap junctions [GJs] composed of connexin36 [Cx36]. We examined the role of Cx36 in CA1 in manifestation of kindled seizures and hippocampal kindling in rats

Methods: Quinine, as the specific blocker of Cx36, was injected into CA1, and kindled seizures severity was examined 10 min afterward. Moreover, quinine was injected into CA1 once daily, and the rate of CA1 kindling was recorded

Results: Quinine 0.5 and 1 mM caused 2- and 3.5-fold increase in the duration of total seizure behavior and generalized the seizures. Primary and secondary after discharges [AD] were also significantly increased. Quinine 0.1 mM augmented the rate of kindling and the growth of secondary AD

Conclusion: Cx36 GJs in CA1 are the main components of hippocampal inhibitory circuit. Any interruption in this path by pathologic or physical damages can trigger hippocampal hyperexcitability and facilitate epileptogenesis

Generation of rat embryonic germ cells via inhibition of TGFbeta and MEK pathways

Embryonic germ [EG] cells are the results of reprogramming primordial germ cells [PGC] in vitro. Studying these cells can be of benefit in determining the mechanism by which specialized cells acquire pluripotency. Therefore in the current study we have tried to derive rat EG cells with inhibition of transforming growth factor-beta [TGFbeta] and mitogen-activated protein kinase kinase [MEK] signaling pathways. In this experimental study, rat PGCs were cultured under feeder free condition with two small molecules that inhibited the above mentioned pathways. Under this condition only two-day presence of stem cell factor [SCF] as a survival factor was applied for PGC reprogramming. Pluripotency of the resultant EG cells were further confirmed by immunofluorescent staining, directed differentiation ability to neural and cardiac cells, and their contribution to teratoma formation as well. Moreover, chromosomal stability of two different EG cells were assessed through G-banding technique. Formerly, derivation of rat EG cells were observed solely in the presence of glycogen synthase kinase-3 [GSK3beta] and MEK pathway inhibitors. Due to some drawbacks of inhibiting GSK3beta molecules such as increases in chromosomal aberrations, in the present study we have attempted to assess a feeder-free protocol that derives EG cells by the simultaneous suppression of TGFbeta signaling and the MEK pathway. We have shown that rat EG cells could be generated in the presence of two inhibitors that suppressed the above mentioned pathways. Of note, inhibition of TGFbeta instead of GSK3beta significantly maintained chromosomal integrity. The resultant EG cells demonstrated the hallmarks of pluripotency in protein expression level and also showed in vivo and in vitro differentiation capacities. Rat EG cells with higher karyotype stability establish from PGCs by inhibiting TGFbeta and MEK signaling pathways

Role of 5-HT[1] receptors of accumbens shell arena upon ACPA-induced anxiolytic-like behaviors in rat

Cannabinoids induce diverse responses on anxiolytic-like behaviors. Moreover some studies postulated that there is a close relationship between this system and serotonergic system upon cognitive process formation. Thus the aim of present study is investigation the possible role of 5-HT[1] receptor on anxiolytic-like behaviors induced by ACPA in the elevated plus maze task [EPM]. In the present study rats weighting 250-300g upon surgery bilateral guide cannulae were implanted to allow microinjection of ACPA [agonist CB1 receptor], CP94253 Hcl[agonist 5-HT[1] receptor] alone and them interaction in the AcbSh. The data showed pretest AcbSh infusion of ACPA at doses of0.0002, 0.002, 0.02 and 0.2 micro g/rat increased and decreased the percentage of open-arms time [%OAT] and percentage of Enclosed-arms time [%CAT], respectively as compared to control groups. Pretest AcbSh infusion ofCP94253 Hcl at doses of 5, 0.5 and 0.05 ng/rat, did not alter anxiety-like behaviors. In addition intra-AcbSh microinjection of subthreshold dose of CP94253 Hcl did not alter ACPA-induced anxiolytic-like behaviors. Our data suggest that activation of AcbSh 5-HT[1] receptor did not involve in ACPA-induced behaviors in the EPM task

The effect of melanocortin (Mc3 and Mc4) antagonists on serotonin-induced food and water intake of broiler cockerels

The current study was designed to examine the effects of intracerebroventricular injections of SHU9119 [a nonselective melanocortin receptor (McR) antagonist] and MCL0020 (a selective McR antagonist) on the serotonin-induced eating and drinking responses of broiler cockerels deprived of food for 24 h (FD24). For Experiment 1, the chickens were intracerebroventricularly injected with 2.5, 5, and 10 microg serotonin. In Experiment 2, the chickens received 2 nmol SHU9119 before being injected with 10 microg serotonin. For Experiment 3, the chickens were given 10 microg serotonin after receiving 2 nmol MCL0020, and the level of food and water intake was determined 3 h post-injection. Results of this study showed that serotonin decreased food intake but increased water intake among the FD24 broiler cockerels and that these effects occurred in a dose-dependent manner. The inhibitory effect of serotonin on food intake was significantly attenuated by pretreatment with SHU9119 and MCL0020. However, the stimulatory effect of serotonin on water intake was not altered by this pretreatment. These results suggest that serotonin hypophagia and hyperdipsia were mediated by different mechanisms in the central nervous system, and that serotonin required downstream activation of McRs to promote hypophagia but not hyperdipsia in the FD24 chickens.

Role of 5-hydroxytryptamine 1a receptors in 6-hydroxydopamine-induced catalepsy-like immobilization in rats: a therapeutic approach for treating catalepsy of parkinson's disease

We have shown that buspirone, a partial agonist of 5-hydroxytryptamine 1A [5-HT[1A]] receptors, improves motor dysfunctions induced by 6-hydroxydopamine [6-OHDA] and haloperidol in rats. The present work extends these findings by investigating the role of 5-HT[1A] receptors on catalepsy-like immobilization in rats, a model of Parkinson's disease. Catalepsy was induced by unilateral infusion of 6-OH-dopamine [8 micro g/2 micro L/rat] into the central region of the substantia nigra, compact part [SNc] and assayed by bar-test method 5, 60, 120 and 180 min after the drugs administration. The involvement of 5-HT[1A] receptors in 6-OHDA-induced catalepsy was studied through intraperitoneal [0.25, 0.5 and 1mg/Kg IP] and intrasubstantia nigra, compact part [10 micro g/rat, intra-SNc] injection of 8-hydroxy-2-[di-n-propylamino] tetralin [8-OHDPAT] as well as administration of 1-[2-methoxyphenyl]-4-[4-[2-pthalimmido] butyl] piperazine hydrobromide [0.1, 0.5 and 1 mg/Kg, NAN-190, IP]. NAN-190 [1 mg/Kg, IP] and 8-OHDPAT [1 mg/Kg, IP and 10 micro g/rat, intra-SNc] increased and decreased 6-OHDA-induced catalepsy respectively. In normal [non 6-OHDA-lesioned] rats, NAN-190 [1 mg/Kg, IP] increased the elapsed time in bar-test while 8-OHDPAT did not produce any significant effect. The anticataleptic effect of 8-OHDPAT [1 mg/Kg, IP] was reversed markedly by co-injection with NAN-190 [1 mg/Kg, IP]. These findings suggest that 5-HT1A receptors are involved in 6-OHDA-induced catalepsy-like immobilization

Upregulation of connexins 30 and 32 gap junctions in rat hippocampus at transcription level by chronic central injection of lipopolysaccharide

Gap junctions composed of connexins [Cx] are functional in cell defense by propagation of toxic/death molecules to neighboring cells. Hippocampus, one of the brain regions with particular vulnerability to damage, has a wide network of gap junctions. Functional response of astrocytic Cx30 and neuronal Cx32 to hippocampal damage is unknown. We infused lipopolysaccharide [LPS] intracerebroventricularly [2.5 microg/rat] once daily for two weeks to create neuroinflammation. The mRNA and protein levels of the Cx were measured in the hippocampus after 1[st], 7[th] and 14[th] injection by real-time PCR and Western-blot techniques. A significant increase in Cx32 and Cx30 gene expression was observed after 7[th] and 14[th] injection of LPS with no significant change in their protein abundance. Transcriptional overexpression of hippocampal Cx30 and Cx32 could be an adaptive response to production of intracellular toxic molecules but it is not accompanied with post- transcriptional overexpression and might have no functional impact