Assessing anxiety in C57BL/6J mice: a pharmacological characterization of the open-field and light/dark tests.

INTRODUCTION: In order to assess anxiety in mammals various tests and species are currently available. These current assays measure changes in anxiety-like behaviors. The open-field and the light/dark are anxiety tests based on the spontaneous behavior of the animals, with C57BL/6J mice being a frequently used strain in behavioral studies. However, the suitability of this strain as a choice in anxiety studies has been questioned. In this study, we performed two pharmacological characterizations of this strain in both the open-field and the light/dark tests. METHODS: We examined the changes in the anxiety-like behaviors of C57BL/6J mice exposed to chlordiazepoxide (CDP), an anxiolytic drug, at doses of 5 and 10 mg/kg, picrotoxine (PTX), an anxiogenic drug, at doses of 0.5 and 1 mg/kg, and methylphenidate (MPH), a psychomotor stimulant drug, at doses of 5 and 10 mg/kg, in a first experiment. In a second experiment, we tested CDP at 2.5 mg/kg, PTX at 2 mg/kg and MPH at 2.5 mg/kg. RESULTS: Results showed an absence of anxiolytic-like effects of CDP in open-field and light/dark tests. Light/dark test was more sensitive to the anxiogenic effects of PTX than the open-field test. Finally, a clear anxiogenic effect of MPH was observed in the two tests. DISCUSSION: Although C57BL/6J mice could not be a sensitive model to study anxiolytic effects in pharmacological or behavioral interventions, it might be a suitable model to test anxiogenic effects. Further studies are necessary to corroborate these results.

[Respiratory and cardiovascular responses to hypoxia under activation or blockade inhibitory transmission].

Respiratory rhythm changes and hypoxic ventilatory responses were studied in anesthetized albino rats after administration GABA and adenosine receptor antagonists. Intracisternal microinjection of picrotoxin induced pathological periodic breathing. Picrotoxin intravenous administration caused the increase of individual resistance to acute hypoxia. Aminophylline administration exerted the opposite effect. After pretreatment of hydroxybutyrate we observed different types of respiratory reactions to adenosine antagonist injection, which probably depended on the value of arterial blood pressure and cerebral hemodynamics.

Administration of the GABAA receptor antagonist picrotoxin into rat supramammillary nucleus induces c-Fos in reward-related brain structures. Supramammillary picrotoxin and c-Fos expression.

BACKGROUND: Picrotoxin blocks GABAA receptors, whose activation typically inhibits neuronal firing activity. We recently found that rats learn to selectively self-administer picrotoxin or bicuculline, another GABAA receptor antagonist, into the supramammillary nucleus (SuM), a posterior hypothalamic structure localized anterior to the ventral tegmental area. Other drugs such as nicotine or the excitatory amino acid AMPA are also self-administered into the SuM. The SuM appears to be functionally linked with the mesolimbic dopamine system and is closely connected with other brain structures that are implicated in motivational processes, including the prefrontal cortex, septal area, preoptic area, lateral hypothalamic area and dorsal raphe nucleus. Here, we hypothesized that these brain structures are activated by picrotoxin injections into the SuM. RESULTS: Picrotoxin administration into the SuM markedly facilitated locomotion and rearing. Further, it increased c-Fos expression in this region, suggesting blockade of tonic inhibition and thus the disinhibition of local neurons. This manipulation also increased c-Fos expression in structures including the ventral tegmental area, medial shell of the nucleus accumbens, medial prefrontal cortex, septal area, preoptic area, lateral hypothalamic area and dorsal raphe nucleus. CONCLUSIONS: Picrotoxin administration into the SuM appears to disinhibit local neurons and recruits activation of brain structures associated with motivational processes, including the mesolimbic dopamine system, prefrontal cortex, septal area, preoptic area, lateral hypothalamic area and dorsal raphe nucleus. These regions may be involved in mediating positive motivational effects triggered by intra-SuM picrotoxin.

Intracerebral dendritic cells critically modulate encephalitogenic versus regulatory immune responses in the CNS.

Dendritic cells (DCs) appear in higher numbers within the CNS as a consequence of inflammation associated with autoimmune disorders, such as multiple sclerosis, but the contribution of these cells to the outcome of disease is not yet clear. Here, we show that stimulatory or tolerogenic functional states of intracerebral DCs regulate the systemic activation of neuroantigen-specific T cells, the recruitment of these cells into the CNS and the onset and progression of experimental autoimmune encephalomyelitis (EAE). Intracerebral microinjection of stimulatory DCs exacerbated the onset and clinical course of EAE, accompanied with an early T-cell infiltration and a decreased proportion of regulatory FoxP3-expressing cells in the brain. In contrast, the intracerebral microinjection of DCs modified by tumor necrosis factor alpha induced their tolerogenic functional state and delayed or prevented EAE onset. This triggered the generation of interleukin 10 (IL-10)-producing neuroantigen-specific lymphocytes in the periphery and restricted IL-17 production in the CNS. Our findings suggest that DCs are a rate-limiting factor for neuroinflammation.

Infralimbic cortex activation increases c-Fos expression in intercalated neurons of the amygdala.

Recently, it was reported that stimulation of the infralimbic cortex produces a feedforward inhibition of central amygdala neurons. The interest of this observation comes from the fact that the central nucleus is the main output station of the amygdala for conditioned fear responses and evidence that the infralimbic cortex plays a critical role in the extinction of conditioned fear. However, the identity of the neurons mediating this infralimbic-evoked inhibition of the central nucleus remains unknown. Likely candidates are intercalated amygdala neurons. Indeed, these cells receive glutamatergic afferents from the infralimbic cortex, use GABA as a transmitter, and project to the central amygdala. Thus, the present study was undertaken to test whether, in adult rats, the infralimbic cortex can affect the activity of intercalated neurons. To this end, disinhibition of the infralimbic cortex was induced by local infusion of the non-competitive GABA-A receptor antagonist picrotoxin. Subsequently, neuronal activation was determined bilaterally within the amygdala using induction of the immediate early gene Fos. Infralimbic disinhibition produced a significant increase in the number of Fos-immunoreactive intercalated cells bilaterally whereas no change was detected in the central nucleus. In the basolateral amygdaloid complex, increases in the number of Fos-immunoreactive cells only reached significance in the contralateral lateral nucleus. These results suggest that glutamatergic inputs from the infralimbic cortex directly activate intercalated neurons. Thus, our findings raise the possibility that the infralimbic cortex inhibits conditioned fear via the excitation of intercalated cells and the consequent inhibition of central amygdala neurons.

Participation of the lateral septal nuclei (LSN) in the antidepressant-like actions of progesterone in the forced swimming test (FST).

The possible participation of lateral septal nuclei (LSN) in the antidepressant-like actions of progesterone was evaluated. The effect of different concentrations of progesterone (0.001, 0.01 and 0.1 M) or saline solution injected directly into the LSN of ovariectomised rats was determined using the forced swimming test (FST). In addition, the temporal course of progesterone (0.1 M) antidepressant-like actions was compared with that of the classical antidepressant imipramine (0.1 M). Finally, in order to establish the possible participation of the GABA(A) receptor in the antidepressant-like action of progesterone, the effect of pre-treatment with the GABA(A) antagonist picrotoxin (0.125 mg/kg, i.p.) was evaluated. Intraseptally administered progesterone produced a concentration-dependent decrease in immobility behaviour but did not modify locomotor activity. These antidepressant-like actions lasted 4 h, while those of imipramine lasted 72 h. Finally, progesterone-induced anti-immobility effect could be blocked by the systemic injection of picrotoxin. Present results reveal that LSN play a role in the antidepressant-like actions of progesterone that appear to be mediated by the GABA(A) receptor.

Sex difference in susceptibility to epileptic seizures in rats: importance of estrous cycle.

Sex difference in seizure susceptibility is one of the unresolved issues of epilepsy. It is known that estrogen is excitatory and progesterone is inhibitory to the central nervous system. Therefore, it is to be expected that seizure susceptibility may be associated with the estrous cycle, which should be tested in epilepsy research. Otherwise, different results in epilepsy studies could be an artifact of the estrous cycle. Reports in the literature are inconsistent about testosterone effects on seizures. In light of these considerations, sex differences in seizure susceptibility were restudied in rats. There was no significant sex difference in mean latencies to picrotoxin-induced seizures; prestrous-females had the shortest latencies to epileptic seizures compared to males and estrousfemales. With testosterone-injected rats, there was either no sex difference in latencies (to akinetic and focal seizures) or females had significantly shorter latencies than males (to status epilepticus, generalized tonic-clonic seizures, and myoclonic seizures). Testosterone-treated male rats had a significantly longer mean latency than controls for status epilepticus only; otherwise, these males showed no significant differences between mean latencies before and after testosterone (to focal, myoclonic, or generalized tonic-clonic seizures). In females, mean latencies to myoclonic seizures and status epilepticus were significantly shorter after testosterone than before. It was concluded that there is a sex difference in susceptibility to epileptic seizures in rats, provided that the estrous cycle is taken into account. Testosterone may increase and decrease seizure susceptibility in females and males, respectively. These effects may be important for understanding the mechanisms of epileptic phenomena and may provide some important clues to epilepsy treatment.

The effects of neurosteroids on picrotoxin-, bicuculline- and NMDA-induced seizures, and a hypnotic effect of ethanol.

The effects of intraperitoneally (IP) or intracerebroventricularly (ICV) administered neurosteroids [allopregnanolone (AP); 5beta-tetrahydrodeoxycorticosterone (5beta-THDOC); dehydroepiandrosterone sulfate (DHEAS); pregnenolone sulfate (PS)] and their precursors [progesterone (PROG), pregnanedione (PREG)] on N-methyl-D-aspartic acid (NMDA)-, picrotoxin (PTX)- and bicuculline (BIC)-induced seizures and ethanol-induced sleep were studied in mice. It was found that IP injections of (+)MK-801 most potently antagonized NMDA-, PTX- and BIC-induced seizures, as compared to diazepam (DZP), PROG and PREG. Both precursors of neurosteroids appeared only marginally active in the applied models of convulsions. ICV injections of AP selectively blocked PTX- and BIC-induced seizures, whereas 5beta-THDOC and (+)MK-801 also antagonized NMDA-induced convulsions. ICV administered DHEAS induced seizures in a dose-dependent way. ICV injections of AP and midazolam shortened the latency and prolonged the duration of sleep induced by IP injections of ethanol (5.0 g/kg). On the contrary, DHEAS and PS significantly reduced the hypnotic-like effect of ethanol. The obtained results suggest that neurosteroids may modulate in an agonistic (AP, 5beta-THDOC), or antagonistic way (PS, DHEAS), the GABA(A) receptor complex functions. Some of them (5beta-THDOC) also interact with NMDA receptors. AP appeared to be the most selectively acting compound, with its profile of action fully comparable to that of midazolam. AP also enhanced the hypnotic effect of ethanol, pointing out to the propensity to interact with centrally depressant agents. These findings, together with the possibility of conversion of some neurosteroids in the brain to other steroid hormones (testosterone, estradiol and aldosterone), indicate the limitations of their use for the treatment of neurological and psychiatric disorders.

Non-serotonergic midbrain neurons are involved in picrotoxin-induced analgesia. An immunohistochemical study in the rat.

Intravenous picrotoxin inhibits spinal nociceptive neurons through disinhibitory activation of neurons in the periaqueductal gray (PAG) and nucleus raphe dorsalis (NRD), where the descending antinociceptive system arises. We found Fos-like immunoreactivity in PAG/NRD neurons after intravenous injection of picrotoxin. This distribution of c-Fos expression is consistent with a role of PAG/NRD for antinociception; neurons with intense Fos-like immunoreactivity was also clustered in the Edinger-Westphal nucleus (EW). Double fluorescence immunohistochemistry for c-Fos and serotonin revealed that PAG/NRD/EW neurons expressing c-Fos were non-serotonergic. These data suggest that non-serotonergic PAG/NRD/EW neurons are involved in the picrotoxin-induced analgesia.

Cortically driven Fos induction in the striatum is amplified by local dopamine D2-class receptor blockade.

Dopamine D2-class receptors have been shown to control the excitability of striatal neurons in response to cortical activation. It has been unclear, however, whether such receptors could regulate the number of striatal neurons activated by cortical stimulation, and thus affect the population response of the striatum to its cortical inputs. We used Fos induction as a readout to measure the ensemble response of striatal neurons to localized stimulation of the frontal cortex and tested for the effects of D2-class dopamine receptor blockade on this response. In freely moving rats, we stimulated the frontal cortex by local epidural application of a dose of a GABAA receptor antagonist (picrotoxin) just threshold for inducing Fos in the striatum. We combined this treatment with D2-class dopamine receptor antagonist treatments at dose levels also just threshold for inducing Fos, using either (i) systemic haloperidol or (ii) intrastriatal (-)sulpiride. Both systemic and intrastriatal blockade of D2-class receptors sharply increased the numbers of striatal neurons exhibiting cortically evoked Fos induction. These findings suggest that local activation of intrastriatal D2-class dopamine receptors can regulate the number of striatal neurons responsive to cortical inputs, thus dynamically shaping the flow of information through the striatum.

Local release of GABAergic inhibition in the motor cortex induces immediate-early gene expression in indirect pathway neurons of the striatum.

The neocortex is thought to exert a powerful influence over the functions of the basal ganglia via its projection to the striatum. It is not known, however, whether corticostriatal effects are similar across different types of striatal projection neurons and interneurons or are unique for cells having different functions within striatal networks. To examine this question, we developed a method for focal synchronous activation of the primary motor cortex (MI) of freely moving rats by local release of GABAergic inhibition. With this method, we monitored cortically evoked activation of two immediate-early gene protein products, c-Fos and JunB, in phenotypically identified striatal neurons. We further studied the influence of glutamate receptor antagonists on the stimulated expression of c-Fos, JunB, FosB, and NGFI-A. Local disinhibition of MI elicited remarkably selective induction of c-Fos and JunB in enkephalinergic projection neurons. These indirect pathway neurons, through their projections to the globus pallidus, can inhibit thalamocortical motor circuits. The dynorphin-containing projection neurons of the direct pathway, with opposite effects on the thalamocortical circuits, showed very little induction of c-Fos or JunB. The gene response of striatal interneurons was also highly selective, affecting principally parvalbumin- and NADPH diaphorase-expressing interneurons. The glutamate NMDA receptor antagonist MK-801 strongly reduced the cortically evoked striatal gene expression in all cell types for each gene examined. Because the gene induction that we found followed known corticostriatal somatotopy, was dose-dependent, and was selectively sensitive to glutamate receptor antagonists, we suggest that the differential activation patterns reflect functional specialization of cortical inputs to the direct and indirect pathways of the basal ganglia and functional plasticity within these circuits.

Is perinatal picrotoxin anxiogenic?

Female Wistar rats were exposed to a subconvulsant dose of picrotoxin (0.75 mg/Kg,sc) on day 18 of pregnancy, immediately after paturition and daily during the first 5 days of lactation. In adulthood, the offspring were tested in an open-field, in an elevated plus maze and for social interaction. Results showed increased locomotor activity (75 days of age) and decreased social interaction (90 days of age) in experimental male rats compared to control male rats. No effects on behaviors related to anxiety were observed in males or females tested in the plus maze apparatus. An additional comparison of the activity male animals perinatally treated with picrotoxin showed a lack of the classical sexual dimorphic responses in the open-field (control male = 68.7 ñ 6.31; control female = 98.4 ñ 6.31; edxperimental male = 89.6 ñ 6.32; experimental female = 113.2 ñ 4.74). We suggest that perinatal picrotoxin exposure may interfere with normal male masculinization rather increasing anxiety in male rats

Effect of manipulation of the GABA system on dopamine-related behaviors

The interaction between GABAergic and dopaminergic system within the central nervous system was investigated in rats using the open-field apparatus and apomorphine-induced stereotypy, and in mice using haloperidol-induced catalepsy. The single intraperitoneal adminsitration of baclofen 3.0 mg/kg, 4,5,6,7-tetrahydroisoxasolo-(5,4-c) piridin-3-ol (THIP) 10.0 mg/kg and picrotoxin 2.0 mg/kg decreased both ambulation and rearing frequencies of the rats in the open-field; only the GABA agonists increased the duration of animal immobility. THIP (10.0 mg/kg) increased the duration of haloperidol-induced catalepsy. For apomorphine-induced stereotypy, baclofen 3.0 mg/kg and picrotoxin 1.0 mg/kg induced a significant leftward displacement of the control dose-response curve constructed for apomorphine (0.1-10 mg/kg) in relation to the control. In addition, baclofen, THIP, picrotoxin and 3-mercaptopropionic acid (3-MPA) 10.0 mg/kg decreased both rearing and sniffing behaviors elicited by apomorphine and increased licking and/ or gnawing. Different mechanisms seem to be involved in the similar effects induced by GABA agonists and antagonists. Picrotoxin induced stereotyped movements per se with a dose-dependent effect, but baclofen and THIP did not. The present data suggest that GABA manipulation facilitates the progressive activation of the different dopaminergic pathways involved in stereotyped behaviors, thus increasing those stereotyped components (gnawing and licking) that appear after a high level of activation of dopaminergic pathways

Inhibitory effect of 5 beta-pregnan-3 alpha-o1-20-one on gonadotropin-releasing hormone gene expression in the male rat.

Neuroanatomical data have documented the existence of synaptic contacts between gamma-aminobutyric acid (GABA) terminals and preoptic gonadotropin-releasing hormone (GnRH) neurons in the rat anterior hypothalamus. In addition, pharmacological studies have suggested that the GABAergic system may be involved in the control of gonadotropin release. Moreover, it has been shown that some progesterone metabolites such as 5 beta-pregnan-3 alpha-ol-20-one (5 beta 3 alpha P) are able to interact with the GABAA receptor complex. In the present study, we have investigated the effects of chronic (5 days) treatment with the GABAA-positive ligand 5 beta 3 alpha P (20 mg/kg body weight i.p., twice a day) or with the GABAA agonist muscimol (1 mg/kg body weight i.p., twice a day) alone or in combination on GnRH mRNA levels in the preoptic area of the male anterior hypothalamus as measured by in situ hybridization. Treatment with 5 beta 3 alpha P produced a 30% decrease in the number of grains overlying labelled cells, while muscimol treatment decreased the hybridization signal by 36%. The concomitant administration of 5 beta 3 alpha P and muscimol resulted in a 46% decrease in the GnRH mRNA levels. This inhibitory effect was completely antagonized by the concomitant administration of picrotoxin (4 mg/kg body weight i.p., twice a day). These data suggest that the GABAA receptor complex and steroids that interact positively with this GABAA receptor complex may play an important role in the regulation of GnRH biosynthesis by hypothalamic neurons.

Central effects of picrotoxin when acting from the liquor spaces in anaesthetized cats.

1. Picrotoxin was perfused through different parts of the cerebral ventricles in cats anaesthetized with chloralose, and applied topically to the upper cervical cord in cats anaesthetized with intraperitoneal pentobarbitone sodium. With both methods, picrotoxin produced effects similar to those produced by tubocurarine; but it was active in weaker concentrations.2. Perfused through the third ventricle, picrotoxin caused shivering resulting in a rise of rectal temperature, increased motor excitability, muscle jerks, tachypnoea, mydriasis, and withdrawal of the nictitating membranes. The effects were due to an action on structures in the walls of the ventral half of the ventricle, because they occurred only on perfusion of tubocurarine through this half and not on its perfusion through the dorsal half of the third ventricle. Noradrenaline perfused through the third ventricle abolished the shivering and the rise in temperature, but did not affect the motor hyperexcitability or the muscle jerks, whereas pentobarbitone sodium similarly perfused abolished these effects as well.3. Perfused through the inferior horn of a lateral ventricle, picrotoxin caused excitation of the hippocampus. This resulted in a rhythmic discharge of high voltage negative spikes in the electrocorticogram taken from the occipital cortices. The spikes developed after-positivity with after-discharges and were interrupted from time to time by bursts of fast activity termed episodes. This abnormal discharge was recorded also from the perfusion cannula which was inserted into the inferior horn and acted as a lead from the surface of the hippocampus.4. Perfused through the anterior horn of a lateral ventricle picrotoxin caused a rhythmic discharge of negative spikes which was recorded from the anterior horn cannula and resulted from excitation of the grey matter in the anterior limbic area which forms part of the medial wall of the horn.5. Applied to the surface of the upper cervical cord, picrotoxin produced scratching movements.