Abstracts

Rationale: Benzodiazepines (BZDs) are widely used in patients of all ages including neonates. The pharmacological effects of BZDs are stable during development, but early life exposure has been found to result in alteration of many brain functions later in life. Behavioral deficits as well as alterations at the cellular or molecular level have been detected long time after treatment cessation in many studies, but possible inverse effects of early BZD exposure on brain excitability are not known.

Methods: Clonazepam (CZP) was suspended in physiological saline with addition of Tween 80 (1mg/5ml of saline with one drop of Tween 80) and injected to male rat pups in a dose of 1 mg/kg/day ip for 5 consecutive days starting at postnatal day 7 (P7) till P11. Control animals received solvent. Seizure susceptibility was assessed using PTZ-induced seizures in two experiments. In the 1st experiment, PTZ in subthreshold dose of 50 mg/kg was administered subcutaneously on P13, 15, 18, 21 and/or P25. Animals were observed for seizures for 30 min and incidence and latency of two seizure types (myoclonic, mS) and generalized tonic-clonic (GTCS) was registered. Data were analyzed using Prisma program. In the 2nd experiment, animals were implanted with cortical electrodes placed epidurally over the sensorimotor cortex at P18, 25 and 90. After the baseline EEG was recorded for 10 min, PTZ was administered in three successive doses of 20-mg/kg ip 20 minutes apart or until mS development. The latency, incidence, number and duration of episodes of RMA defined as at smallest of the three waves of 4-6 Hz frequency were evaluated between 10 and 15 min after the PTZ injections.

Results: Early life exposure to CZP resulted in significant increase of GTCS incidence p=0.0025) up to P18 and since P21 it dropped down on control level. Incidence of mS was not affected by previous CZP exposure at any interval after treatment cessation. Effects on RMA were highly dependent on the interval after the end of CZP exposure. In P90, early life CZP exposure resulted in the significant shortening of latency to the 1^st RMA (q= 0.0037) as well as to the increase of number (q=0.003) and mean duration of RMA after the 1st PTZ administration (q=0.0313). Interestingly, effect of early CZP exposure was rather opposite in younger animals and the RMA number tended to be  lower compared to vehicle treated rats in both P18 (q=0.0764) and P25 (q=0.0657) rats. Latency to the mS was significantly shorter in CZP-exposed P18 animals compared to controls (q=0.031) but not at longer intervals after treatment cessation.

Conclusions: Repeated administration of CZP in the anticonvulsant dose results in development of withdrawal phenomena associated with an increase of the GTCS incidence that lasted for 1 week. Interestingly, the increase of frequency and duration of RMAs was observed only in adult rats exposed to CZP early in life. These changes may be associated with alterations of GABA_A and NMDA receptors found in our previous studies.

Funding: The study was supported by grant 19-11931S of the Czech Science Foundation, project PHARMABRAIN, reg. no. CZ.02.1.01/0.0/0.0/16_025/0007444 (co-supported by EU) and Research Project RVO 67985823.