E.A. Del Bel

Neuroethological and morphological (Neo-Timm staining) correlates of limbic recruitment during the development of audiogenic kindling in seizure susceptible Wistar rats

Acute audiogenic seizures are a model of generalized tonic-clonic seizures, induced by high intensity acoustic stimulation in genetically susceptible rodents. The neural substrate are sensory motor brainstem nuclei. Recruitment of forebrain structures takes places upon repetition of acoustically evoked seizures. The term audiogenic kindling means forebrain kindling evoked by repeated brainstem seizures and has been described in several strains of genetically epilepsy-prone rats. Thus, the present work was conducted in order to test the hypothesis that audiogenic kindling recruits the forebrain, which may be behaviorally evaluated and associated with morphological changes as well. The behavioral sequences observed during the development of audiogenic kindling were assessed by neuroethological methods (cluster analysis), with the ETHOMATIC program. Seizure severity indexes (brainstem and limbic seizures) and latencies of wild running and tonic-clonic seizures were measured to quantify seizure evolution. Densitometric analysis of Neo-Timm staining was used for assessing morphological changes associated with audiogenic kindling. In group I, II resistant (R) and 16 susceptible (S) animals were stimulated (120 dB) 21 times, and allowed a 10 day recovery period prior to retesting. In group II, 22 R and 20 S were stimulated 60 times, and allowed a 2 month recovery period prior to retesting. Repetition of the acoustic stimulation in group I and group II susceptible animals led to a progressive and statistically significant attenuation of the behaviors associated with brainstem seizures and a concomitant increased expression of the behaviors associated with limbic seizures. After either a 10 day (group I) or 2 month (group II) recovery period, acoustic stimulation preferentially evoked brainstem-associated behaviors and seizures rather than limbic ones in the audiogenic susceptible animals, although in some animals overlapped brainstem and limbic seizures were detected. Latencies for the wild running and tonic seizures after acoustic stimulation significantly increased during audiogenic kindling for both group I and group II susceptible animals. The quantitative ethological evaluation in both group I and group II, illustrated by flowcharts, showed the evolution of the kindling installation by the presence of limbic seizure clusters, competing in time with the original tonic-clonic clusters. Expression of limbic seizures by group I animals, after acoustic stimulation, was not associated with changes in the mossy fiber Neo-Timm staining pattern of these animals. In group II however, Neo-Timm staining revealed mossy fiber sprouting in the ventral hippocampus (but not in the dorsal), and a significant change in the optical density of amygdaloid nuclei and perirhinal cortex in susceptible animals as compared to resistant ones. In conclusion, audiogenic kindling effectively recruits forebrain structures, responsible for the appearance of limbic seizures. It is possible that the paradigm used in group I was subthreshold for the development of clear-cut synaptic reorganization in the hippocampal mossy fiber system, since the behavioral patterns reverted ten days after the last seizure induction. In group II, however, an increased number of evoked seizures and a more prolonged time after the last chronic seizure showed structural re-arrangements in amygdala, perirhinal cortex and hippocampus, associated with permanence in terms of behavioral data (lack of regression of limbic seizures to control values).

Hippocampal 5-HT receptors and consolidation of stressful memories

It has been suggested that postsynaptic 5-HT1A receptors in the hippocampus, innervated by 5-HT neurons localized in the median raphe nucleus, mediate adaptive or coping responses to aversive events and that dysfunction of this system is related to symptoms of depression. To test this hypothesis we investigated the expression of c-fos mRNA in animals submitted to immobilization stress. The results showed that c-fos mRNA expression is significantly increased in the dentate gyrus and CA1-CA3 regions of the hippocampus after 30 min of forced restraint, suggesting that this structure is activated during stress. To investigate the role of 5-HT neurotransmission in the hippocampus on adaptation to aversive events we immobilized rats for 2 h and tested them 24 h later in an elevated plus-maze. Our results showed that the previous restraint period decreases exploration of open arms in the maze. This effect was reversed by bilateral microinjection of zimelidine (20 and 100 nmol), a 5-HT re-uptake blocker, or 8-OH-DPAT (3 nmol), a 5-HT1A agonist, into the dorsal hippocampus immediately after restraint. These results are compatible with the idea that postsynaptic 5-HT1A receptors located in the hippocampus participate in the development of tolerance to aversive events.

Effects of l-NOARG on Plus-Maze Performance in Rats

Nitric oxide (NO) synthase, the enzyme responsible for NO formation, is located in brain regions such as amygdala and dorsolateral central grey, regions which are known to be involved in anxiety. To investigate the possible role of NO in anxiety, rats received acute i.p. injections of NG-nitro-l-arginine (L-NOARG, 7.5-120 mg kg-1), an inhibitor of NO synthase, and were tested in the elevated plus maze, an animal model of anxiety. The drug, at doses of 30-120 mg kg-1, decreased the percentage of entries and time spent on the open arms of the maze, but these doses, with exception of 30 mg, also decreased the number of entries into enclosed arms. These effects disappeared when the animals were tested after chronic L-NOARG treatment (3.75 to 60 mg kg-1 i.p., twice a day for four days). The effects of acute i.p. injection of 30 mg kg-1 of L-NOARG were blocked by i.c.v. pretreatment with 1000 nmol of l-arginine (but not 500 nmol). Thus, inhibition of NO formation in the central nervous system seems to decrease exploration of the elevated plus maze, an effect that disappears after four days of chronic (twice a day) L-NOARG administration.

Effects of excitatory amino acids and nitric oxide on flight behavior elicited from the dorsolateral periaqueductal gray

Microinjection of excitatory amino acids (EAA) into the dorsolateral periaqueductal gray (dlPAG) induces flight reactions while EAA antagonists show anxiolytic effects. Part of the effects mediated by NMDA receptors may involve an increase in nitric oxide (NO) production. We showed that nitric oxide synthase (NOS) inhibitors injected into the dlPAG induced anxiolytic effects. Conversely, SIN-1, a NO donor, produced orientated flight reactions that resemble stimulation of the medial hypothalamus. This compound also produced extensive Fos-like immunoreactivity in this region and in other areas related to defensive reactions such as the medial amygdala and cingulate cortex. Since part of the effects of NO involves increases in guanylate cyclase levels, we found that intra-dlPAG injection of 8-Br-cGMP induced a brief flight reaction followed by increased locomotion. In another experiment, we showed that single or repeated restraint stress produced an increased expression of neuronal NOS in the dlPAG and other areas related to defense, as measured by in situ hybridization, diaphorase histochemistry and immunocytochemistry. Together, these data suggest that NO may participate in the modulation of defensive responses in the dlPAG.

Acute and delayed restraint stress-induced changes in nitric oxide producing neurons in limbic regions.

RATIONALE Microinjection into the dentate gyrus of the hippocampus of N(omega)-nitro-l-arginine methyl ester hydrochloride (l-NAME), a nitric oxide synthase (NOS) inhibitor, induces antinociceptive effect 5 days after a single restraint episode. The mechanisms of this stress-antinociceptive modulatory effect have not been investigated but may involve plastic changes in the hippocampal formation (HF). OBJECTIVE The objective of the present study was to investigate possible mechanisms of the stress-modulating effect on antinociception induced by NOS inhibition in the hippocampus. We analyzed the effects of restraint stress on neuronal NOS (nNOS) expression and nicotinamide adenine dinucleotide phosphate-diaphorase histochemical activity (NADPH-d) in the HF and related brain regions. METHODS Male Wistar rats (n=6-11/group) were submitted to a single (acute stress) or repeated (5 days) episodes of 2-h restraint. Control animals remained in their home cages being all animals daily handled during this period. In the fifth day, animals received unilateral microinjection of l-NAME (150 nmol/0.2 microl) or saline (control) into the dentate gyrus of the dorsal hippocampus (DG). Immediately before and after drug microinjection tail-flick reflex latency or hotplate licking reaction was measured. Animals were killed i. immediately; ii. 5 days after acute stress; or iii. after repeated stress. NADPH-d and nNOS expression were quantified in the HF, caudate-putamen, secondary somatosensorial, entorhinal and piriform cortices and amygdaloid complex. RESULTS Five days after one or five restraint episodes l-NAME microinjection into the DG elicited antinociceptive effect (analysis of variance [ANOVA], P<0.05). Acute restraint stress induced a significant increase in the density of neurons expressing NADPH-d and nNOS in the amygdaloid nuclei. nNOS expression increased also in the DG and piriform cortex. Five days after a single or repeated restraint stress there was an additional increase in NADPH-d- and nNOS-positive neurons in CA1, CA3, and entorhinal cortex. No changes were seen in non-limbic regions such as the caudate-putamen and secondary somatosensorial cortex. CONCLUSION The results confirm that the dorsal hippocampus participates in the modulation of stress consequences. They also show that a single stress episode causes acute changes in nitric oxide system in the amygdala complex and delayed modifications in the HF. The delayed (5 days) antinociceptive effect of NOS inhibition in the HF after a single restraint episode suggests that those latter modifications may have functional consequences. It remains to be tested if the acute amygdala and delayed hippocampal changes are causally related.

Eag1 potassium channel immunohistochemistry in the CNS of adult rat and selected regions of human brain.

Eag1 (K(V)10.1) is the founding member of an evolutionarily conserved superfamily of voltage-gated K(+) channels. In rats and humans Eag1 is preferentially expressed in adult brain but its regional distribution has only been studied at mRNA level and only in the rat at high resolution. The main aim of the present study is to describe the distribution of Eag1 protein in adult rat brain in comparison to selected regions of the human adult brain. The distribution of Eag1 protein was assessed using alkaline-phosphatase based immunohistochemistry. Eag1 immunoreactivity was widespread, although selective, throughout rat brain, especially noticeable in the perinuclear space of cells and proximal regions of the extensions, both in rat and human brain. To relate the results to the relative abundance of Eag1 transcripts in different regions of rat brain a reverse-transcription coupled to quantitative polymerase chain reaction (real time PCR) was performed. This real time PCR analysis showed high Eag1 expression in the olfactory bulb, cerebral cortex, hippocampus, hypothalamus, and cerebellum. The results indicate that Eag1 protein expression greatly overlaps with mRNA distribution in rats and humans. The physiological relevance of potassium channels in the different regions expressing Eag1 protein is discussed.

Sub-chronic inhibition of nitric-oxide synthesis modifies haloperidol-induced catalepsy and the number of NADPH-diaphorase neurons in mice

Abstract Rationale: NG-nitro-l-arginine (l-NOARG), an inhibitor of nitric-oxide synthase (NOS), induces catalepsy in mice. This effect undergoes rapid tolerance, showing a significant decrease after 2 days of sub-chronic l-NOARG treatment. Nitric oxide (NO) has been shown to influence dopaminergic neurotransmission in the striatum. Neuroleptic drugs such as haloperidol, which block dopamine receptors, also cause catalepsy in rodents. Objectives: To investigate the effects of sub-chronic l-NOARG treatment in haloperidol-induced catalepsy and the number of NOS neurons in areas related to motor control. Methods: Male albino Swiss mice were treated sub-chronically (twice a day for 4 days) with l-NOARG (40 mg/kg i.p.) or haloperidol (1 mg/kg i.p.). Catalepsy was evaluated at the beginning and the end of the treatments. Reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry was also employed to visualize NOS as an index of enzyme expression in mice brain regions related to motor control. Results:l-NOARG sub-chronic administration produced tolerance of l-NOARG and of haloperidol- induced catalepsy. It also induced an increase in the number of NADPH-d-positive cells in the dorsal part of the caudate and accumbens nuclei compared with haloperidol and in the pedunculopontine tegmental nucleus compared with saline. In contrast, there was a decrease in NADPH-d neuron number in the substantia nigra, pars compacta in both haloperidol-treated and l-NOARG-treated animals. Conclusions: The results give further support to the hypothesis that NO plays a role in motor behavior control and suggest that it may take part in the synaptic changes produced by antipsychotic treatment.

Catalepsy induced by nitric oxide synthase inhibitors

1. Previous study showed that N(G)-nitro-L-arginine (L-NOARG), an inhibitor of nitric bxide synthase, induces catalepsy in a dose-dependent manner in male albino-Swiss mice. 2. The objective of the present work was to further investigate this effect, extending it to other NOS inhibitors. 3. Results showed that L-NOARG (40-80 mg/kg i.p.), N(G)-nitro-L-arginine methylester (L-NAME, 40-160 mg/kg i.p.) or N(G)-monomethyl-L-arginine (L-NMMA, 80 mg/kg i.p.) were able to induce catalepsy in mice. The effect of L-NOARG (40 mg/kg) was antagonized by pretreatment with L-arginine (300 mg/kg), but not by D-arginine (300 mg/kg). The catalepsy-inducing effect of L-NOARG suffered rapid tolerance, showing a significant decrease after two days of chronic treatment (40 mg/kg i.p., twice a day). 4. The results suggest that interference with the formation of nitric oxide induces significant motor effects in mice.

Methodological Evaluation to Analyze Functional Recovery after Sciatic Nerve Injury

The Basso, Bresnahan and Beattie (BBB) locomotor scale has not been tested to evaluate functional consequences of peripheral nerve lesions. Alternative methods to evaluate animal functional recovery after sciatic nerve injury are desirable. Male Wistar rats had a right sciatic nerve segment exposed and were divided in three experimental groups: Sham (wound open, 10 min), Sham-device (nerve segment between crushing device, 10 min), and Crush-force (nerve crushing load of 15,000 g/1,000 mm Hg/mm2, 10 min). Animals were evaluated preoperatively, 1, 7, 14, 21, and 28 days after procedure by calculation of Sciatic Functional Index (SFI), BBB score and open arena exploratory activity. The primary findings of the present study were (1) the SFI calculated by either DeMedinaceli, Carlton and Goldberg, and Bain formulae were highly correlated; (2) the BBB score evaluation was highly correlated with the SFI; (3) the BBB motor scale was able to detect functional impairments not recognized by the SFI; and (4) open are...

Nitric oxide modulation of methylphenidate-induced disruption of prepulse inhibition in Swiss mice

Drugs that facilitate dopaminergic neurotransmission induce cognitive and attentional deficits which include inability to filter sensory input measured by prepulse inhibition (PPI). Methylphenidate, an amphetamine analog is used in the treatment of attention deficit hyperactivity disorder. Given that nitric oxide (NO) modulates dopamine effect our aim is to analyze the nitric oxide synthase (NOS) and soluble guanylate cyclase (sGC) inhibitors effect on PPI disruption induced by methylphenidate. The inhibitors effects were compared to those produced by haloperidol and clozapine. Male Swiss mice received a first i.p. injection (one hour before testing), of either saline, or N(G) nitro l-arginine (10, 40 or 90 mg/kg), or 7-Nitroindazole (3, 10, 30 or 60 mg/kg), or oxadiazolo-quinoxalin (5 or 10 mg/kg), or haloperidol (1 mg/kg), or clozapine (5 mg/kg). Thirty min later mice received the second injection of either saline or methylphenidate (20 or 30 mg/kg) or amphetamine (5 or 10 mg/kg). One group of mice received intracerebroventricular 7-Nitroindazole (50 or 100 nM) followed by systemic administration of saline or methylphenidate (30 mg/kg). The results revealed a methylphenidate dose-dependent disruption of PPI comparable to amphetamine. The effect was prevented by either nitric oxide synthase or guanilate cyclase inhibitors or clozapine or haloperidol. In conclusion, methylphenidate induced a dose-dependent PPI disruption in Swiss mice modulated by dopamine and NO/sGC. The results corroborate the hypothesis of dopamine and NO interacting to modulate sensorimotor gating through central nervous system. It may be useful to understand methylphenidate and other psychostimulants effects.