Neonatal Clonazepam Administration Induces Long-Lasting Changes in Glutamate Receptors.

γ-aminobutyric acid (GABA) pathways play an important role in neuronal circuitry formation during early postnatal development. Our previous studies revealed an increased risk for adverse neurodevelopmental consequences in animals exposed to benzodiazepines, which enhance GABA inhibition via GABAA receptors. We reported that administration of the benzodiazepine clonazepam (CZP) during postnatal days 7-11 resulted in permanent behavioral alterations. However, the mechanisms underlying these changes are unknown. We hypothesized that early CZP exposure modifies development of glutamatergic receptors and their composition due to the tight developmental link between GABAergic functions and maturation of glutamatergic signaling. These changes may alter excitatory synapses, as well as neuronal connectivity and function of the neural network. We used quantitative real-time PCR and quantitative autoradiography to examine changes in NMDA and AMPA receptor composition and binding in response to CZP (1 mg/kg/day) administration for five consecutive days, beginning on P7. Brains were collected 48 h, 1 week, or 60 days after treatment cessation, and mRNA subunit expression was assessed in the hippocampus and sensorimotor cortex. A separate group of animals was used to determine binding to NMDA in different brain regions. Patterns of CZP-induced alterations in subunit mRNA expression were dependent on brain structure, interval after CZP cessation, and receptor subunit type. In the hippocampus, upregulation of GluN1, GluN3, and GluR2 subunit mRNA was observed at the 48-h interval, and GluN2A and GluR1 mRNA expression levels were higher 1 week after CZP cessation compared to controls, while GluN2B was downregulated. CZP exposure increased GluN3 and GluR2 subunit mRNA expression levels in the sensorimotor cortex 48 h after treatment cessation. GluA3 was higher 1 week after the CZP exposure, and GluN2A and GluA4 mRNA were significantly upregulated 2 months later. Expression of other subunits was not significantly different from that of the controls. NMDA receptor binding increased 1 week after the end of exposure in most hippocampal and cortical areas, including the sensorimotor cortex at the 48-h interval. CZP exposure decreased NMDA receptor binding in most evaluated hippocampal and cortical areas 2 months after the end of administration. Overall, early CZP exposure likely results in long-term glutamatergic receptor modulation that may affect synaptic development and function, potentially causing behavioral impairment.

Behavioral effects of high frequency electrical stimulation of the hippocampus on electrical kindling in rats.

Experiments were designed to evaluate the effects of high frequency electrical stimulation (HFS) applied in ventral hippocampus during the hippocampal kindling process, as well as on the expression of fully kindled seizures and the refractoriness for subsequent convulsions during their postictal period. Male Wistar rats, stereotactically implanted in both ventral hippocampus, received daily bilateral HFS (pulses of 60 micros width at 130 Hz at subthreshold current intensity) during 1h immediately after each kindling stimulation (1s train of 60 Hz biphasic square waves, each 1 ms) during 40 days or until the kindled state was achieved. Rats were classified as follows: (a) Responder animals, who required low current intensity for HFS (208+/-38.2 microA), did not show progress of the kindling process and remained in stages II and III seizures. (b) Nonresponders rats, in which the current intensity for HFS was higher (434.5+/-51.7 microA), developed the kindling process as the kindling control group. When HFS was applied before the kindling stimulation in fully kindled rats, animals presented a reduced expression of the fully kindled seizures (nonresponders animals) and an enhanced refractoriness for subsequent seizures during the postictal period (kindling control and nonresponder animals). There was no correlation between the area where the HFS was applied and the effects induced. It was concluded that HFS at 130 Hz in ventral hippocampus is able to modify the epileptogenesis induced by the hippocampal kindling process and the refractoriness to subsequent seizures during the postictal period in rats.

5-HT1A receptor agonists modify epileptic seizures in three experimental models in rats.

The effects of two serotonergic (5-HT1A) receptor agonists (8-OH-DPAT; 0.01, 0.1, 0.3, 1 mg/kg, s.c., and Indorenate; 1, 3, 10 mg/kg, i.p.) were evaluated in three type of seizures in male Wistar rats: clonic-tonic convulsions induced by pentylenetetrazol (PTZ, 60 mg/kg, i.p.), status epilepticus (SE) of limbic seizures produced by kainic acid (KA, 10 mg/kg, i.p.) and tonic-clonic seizures by amygdala kindling. 8-OH-DPAT decreased the incidence of tonic seizures and the mortality rate induced by PTZ. Indorenate increased the latency to the PTZ-induced seizures and decreased the percentage of rats showing tonic extension and death. Concerning KA, 8-OH-DPAT augmented the latency and reduced the frequency of wet-dog shake (WDS) and generalized seizure (GS). At high doses it diminished the occurrence and delayed the establishment of SE. Indorenate augmented the latency to WDS, GS and SE, and diminished the number of GS. 8-OH-DPAT and Indorenate did not alter the expression of kindled seizures. However, Indorenate enhanced the refractoriness to subsequent seizures during the postictal depression. Some effects induced by 8-OH-DPAT and Indorenate on seizures evaluated and postictal depression were fully or partially blocked by WAY100635. These results suggest that 5-HT1A receptor agonists modify epileptic activity depending on the type of seizure.