Adrián Martínez

Vagus nerve prolonged stimulation in cats: effects on epileptogenesis (amygdala electrical kindling): behavioral and electrographic changes.

Summary: Purpose: To analyze the effect of prolonged (daily) electrical vagus nerve stimulation (VNS) on daily amygdaloid kindling (AK) in freely moving cats.

Effect of electrical stimulation of the nucleus of the solitary tract on the development of electrical amygdaloid kindling in the cat.

Summary:  Purpose: This work analyzed the effect of electrical stimulation of the nucleus of the solitary tract (NTS) on the development of electrical amygdaloid kindling (AK) in freely moving cats.

Chronic stimulation of the cat vagus nerve Effect on sleep and behavior

The effect of electrical vagus nerve stimulation (VNS) on sleep and behavior was analyzed in freely moving cats. Eight cats were prepared for 23-h sleep recordings. The left vagus nerve of four of them was stimulated during 1 min, five times at 1-h intervals, for 5 days. The VNS induces: ipsilateral myosis, blinking, licking, abdominal contractions, upward gaze, swallowing, and eventually yawning and compulsive eating, as well as an increase of ponto-geniculate-occipital (PGO) wave density and of the number of stages and total amount of rapid eye movement (REM) sleep. Besides, there was a sudden transition from waking stage to REM sleep. The present results suggest that VNS modifies sleep in the cat. This effect could be explained by an activation of the areas involved in the physiological mechanisms of sleep.

Analysis of the anxiolytic-like effect of TRH and the response of amygdalar TRHergic neurons in anxiety

Thyrotropin-releasing hormone (TRH) was first described for its neuroendocrine role in controlling the hypothalamus-pituitary-thyroid axis (HPT). Anatomical and pharmacological data evidence its participation as a neuromodulator in the central nervous system. Administration of TRH induces various behavioural effects including arousal, locomotion, analepsy, and in certain paradigms, it reduces fear behaviours. In this work we studied the possible involvement of TRHergic neurons in anxiety tests. We first tested whether an ICV injection of TRH had behavioural effects on anxiety in the defensive burying test (DBT). Corticosterone serum levels were quantified to evaluate the stress response and, the activity of the HPT axis to distinguish the endocrine response of TRH injection. Compared to a saline injection, TRH reduced cumulative burying, and decreased serum corticosterone levels, supporting anxiolytic-like effects of TRH administration. The response of TRH neurons was evaluated in brain regions involved in the stress circuitry of animals submitted to the DBT and to the elevated plus maze (EPM), tests that allow to correlate biochemical parameters with anxiety-like behaviour. In the DBT, the response of Wistar rats was compared with that of the stress-hypersensitive Wistar Kyoto (WKY) strain. Behavioural parameters were analysed in recorded videos. Animals were sacrificed 30 or 60min after test completion. In various limbic areas, the relative mRNA levels of TRH, its receptors TRH-R1 and -R2, and its inactivating ectoenzyme pyroglutamyl peptidase II (PPII), were determined by RT-PCR, TRH tissue content by radioimmunoassay (RIA). The extent of the stress response was evaluated by measuring the expression profile of CRH, CRH-R1 and GR mRNA in the paraventricular nucleus (PVN) of the hypothalamus and in amygdala, corticosterone levels in serum. As these tests demand increased physical activity, the response of the HPT axis was also evaluated. Both tasks increased the levels of serum corticosterone. WKY rats showed higher anxiety-like behaviour in the DBT than Wistar, as well as increased PVN mRNA levels of CRH and GR. TRH mRNA levels increased in the PVN and TSH values remained unchanged in both strains although TRH content decreased in the medial basal hypothalamus of Wistar rats only. TRH content was measured in several limbic regions but only amygdala showed specific task-related changes after DBT exposure of both strains: increased TRH content. Expression of TRH mRNA decreased in the amygdala of Wistar, suggesting inhibition of TRHergic neuronal activity in this region. The participation of amygdalar TRH neurons in anxiety was confirmed in the EPM where TRH expression and release correlated with the number of entries, and the % of time spent in open arms, supporting an anxiolytic role of these TRH-neurons. These results contribute to the understanding of the involvement of TRH during emotionally charged situations and shed light on the participation of particular circuits in related behaviours.

Changes in TRH and its degrading enzyme pyroglutamyl peptidase II, during the development of amygdaloid kindling

Pyroglutamyl peptidase II (PPII) is a neuronal ectoenzyme responsible for thyrotropin releasing hormone (TRH) degradation at the synaptic cleft. PPII, heterogeneously distributed in different brain regions and adenohypophysis, is regulated under various endocrine conditions where TRH is involved in thyrotropin or prolactin regulation but only at the adenohypophyseal level. TRH can downregulate PPII activity in cultured adenohypophyseal cells. TRH present in extrahypothalamic brain areas has been postulated to serve as a neuromodulator and levels of this peptide increase in amygdala, hippocampus and cortex after electrical stimulation (kindling or electroshock). To study whether brain PPII could be regulated in conditions that stimulate TRHergic neurons, TRH and PPII activity were determined during the development of amygdaloid kindling in the rat. TRH levels increased from stage II to V in amygdala and hippocampus in the ipsi- and contralateral side to stimulation. In n. accumbens a decrease, compared to sham was observed at stage II, but levels raised through stage V. In contrast, PPII activity was increased at stage II, in amygdala of both sides and in hipppocampus, frontal cortex, n. accumbens and hypothalamus of the contralateral side; levels decreased at stage V to sham values in most structures (except amygdala and hippocampus where the activity was 30% below controls). These results suggest that PPII activity in the central nervous system can be regulated in conditions known to affect TRHergic neurons.

Anticonvulsant Effect of Annona diversifolia Saff. and Palmitone on Penicillin‐induced Convulsive Activity. A Behavioral and EEG Study in Rats

Summary:  Purpose: To evaluate hypnotic and anticonvulsant activities of Annona diversifolia Saff. and palmitone by using behavior and electroencephalographic (EEG) analysis in an experimental model of focal seizures in rats.

Involvement of CRH-R2 receptor in eating behavior and in the response of the HPT axis in rats subjected to dehydration-induced anorexia

Wistar rats subjected to dehydration-induced anorexia (DIA), with 2.5% NaCl solution as drinking water for 7 days, decrease by 80% their food intake and present some changes common to pair-fed food restricted rats (FFR) such as: weight loss, decreased serum leptin and expression of orexigenic arcuate peptides, increasing the anorexigenic ones and serum corticosterone levels. In contrast, the response of the HPT axis differs: DIA animals have increased TRH expression in PVN and present primary as opposed to the tertiary hypothyroidism of the FFR. Exclusive to DIA is the activation of CRHergic neurons in the lateral hypothalamus (LH) that project to PVN. Since TRH neurons of the PVN contain CRH receptors, we hypothesized that the differences in the response of the HPT axis to DIA could be due to CRH regulating TRHergic neurons. CRH effect was first evaluated on TRH expression of cultured hypothalamic cells where TRH mRNA levels increased after 1h with 0.1nM of CRH. We then measured the mRNA levels of CRH receptors in the PVN of male and female rats subjected to DIA; only those of CRH-R2 were modulated (down-regulated). The CRH-R2 antagonist antisauvagine-30 was therefore injected into the PVN of male rats, during the 7 days of DIA. Antisauvagine-30 induced a higher food intake than controls, and impeded the changes produced by DIA on the HPT axis: PVN TRH mRNA, and serum TH and TSH levels were decreased to similar values of FFR animals. Results corroborate the anorexigenic effect of CRH and show its role, acting through CRH-R2 receptors, in the activation of TRHergic PVN neurons caused by DIA. These new data further supports clinical trials with CRH-R2 antagonists in anorexia nervosa patients.

Repeated penicillin-induced amygdala epileptic focus in freely moving cats. EEG, polysomnographic (23-h recording), and brain mapping study

The effect of repeated Na-penicillin (PCN) microinjections in the temporal lobe amygdala (AM) of free-moving cats was investigated in order to establish if kindling epileptogenesis is possible with this procedure. The cortical propagation of the PCN-induced post-discharge in AM and the sequence of behavioral changes induced by PCN were similar to those of AM electrical kindling. Nevertheless, the epileptogenic effect of PCN had a different evolution from that of electrical kindling, since some PCN habituation was observed after several doses. Repeated microinjections of PCN did not produce lasting alterations in sleep onset and organization. The only mild changes recorded in the 23 h following PCN microinjections were an increased latency of the first rapid eye movement (REM) sleep episode, a SWS II total time and percentage increase, and, with the highest PCN doses, a not very significant diminution of REM sleep total time. Another finding was the occurrence of REM sleep ponto-geniculo-occipital (PGO) waves, coinciding with a depression of the frequency and amplitude of interictal amygdaloid and cortical spikes. The results showed that a microinjection of PCN in the AM produced a reliable model of interictal spikes, paroxysms and generalized convulsive seizures. Nevertheless, long lasting kindling effect was not observed.

Amygdala kindling differentially regulates the expression of the elements involved in TRH transmission

Subthreshold electrical stimulation of the amygdala (kindling) activates neuronal pathways increasing the expression of several neuropeptides including thyrotropin releasing-hormone (TRH). Partial kindling enhances TRH expression and the activity or its inactivating ectoenzyme; once kindling is established (stage V), TRH and its mRNA levels are further increased but TRH-binding and pyroglutamyl aminopeptidase II (PPII) activity decreased in epileptogenic areas. To determine whether variations in TRH receptor binding or PPII activity are due to regulation of their synthesis, mRNA levels of TRH receptors (R1, R2) and PPII were semi-quantified by RT-PCR in amygdala, frontal cortex and hippocampus of kindled rats sacrificed at stage II or V. Increased mRNA levels of PPII were found at stage II in amygdala and frontal cortex, and of pro-TRH and TRH-R2, in amygdala and hippocampus. At stage V, pro-TRH mRNA levels increased and those of PPII, decreased in the three regions; TRH-R2 mRNA levels diminished in amygdala and frontal cortex and of TRH-R1 only in amygdala. In situ hybridization analyses revealed, at stage II, enhanced TRH-R1 mRNA levels in dentate gyrus and amygdala while decreased in piriform cortex; those of TRH-R2 increased in amygdala, CA2, dentate gyrus, piriform cortex, thalamus and subiculum and of PPII, in CAs and piriform cortex. In contrast, at stage V decreased expression of TRH-R1 occurred in amygdala, CA2/3, dentate gyrus and piriform cortex; of TRH-R2 in CA2, thalamus and piriform cortex, and of PPII in CA2, and amygdala. The magnitude of changes differed between ipsi and contralateral side. These results support a trans-synaptic modulation of all elements involved in TRH transmission in conditions that stimulate the activity of TRHergic neurons. They show that reported changes in PPII activity or TRH-binding caused by kindling relate to regulation of the expression of TRH receptors and degrading enzyme.

Effect of repeated administration of Annona diversifolia Saff. (ilama) extracts and palmitone on rat amygdala kindling

Annonas are consumed as fresh fruits, but, because of their effects on the central nervous system, are also used in folk medicine. The effect on rat amygdala kindling of repeated administration of Annona diversifolia hexane (100mg/kg IP or PO) and ethanol (100mg/kg, PO) leaf extracts and palmitone (10mg/kg, IP) was determined. Electrographic and/or behavioral changes were monitored during kindling-induced seizures 60minutes after treatments. Antiepileptic efficacy was evaluated with respect to afterdischarge (AD) duration, spike frequency, and/or behavioral seizure activity. Oral administration of both extracts significantly decreased spike frequency, whereas intraperitoneally administered hexane extract and palmitone only reduced AD duration. Hexane extract and palmitone exhibited anticonvulsant properties and delayed establishment of a kindling state as observed with diazepam (0.3mg/kg IP). These results reinforce the anticonvulsant properties of this plant, and palmitone and other constituents are responsible for the pharmacological effects.