The GluN2B subunit of NMDA receptors represents a perspective therapeutic target in various CNS pathologies, including epilepsy. Because of its predominant expression in the immature brain, selective GluN2B antagonists are expected to be more effective early in postnatal development. The aim of this study was to identify age-dependent differences in the anticonvulsant activity of the GluN2B-selective antagonist Ro 25-6981 and assess the safety of this drug for the developing brain. Anticonvulsant activity of Ro 25-6981 (1, 3, and 10 mg/kg) was tested in a pentylenetetrazol (PTZ) model in infantile (12-day-old, P12) and juvenile (25-day-old, P25) rats. Ro 25-6981 (1 or 3 mg/kg/day) was administered from P7 till P11 to assess safety for the developing brain. Animals were then tested repeatedly in a battery of behavioral tests focusing on sensorimotor development, cognition, and emotionality till adulthood. Effects of early exposure to Ro 25-6981 on later seizure susceptibility were tested in the PTZ model. Ro 25-6981 was effective against PTZ-induced seizures in infantile rats, specifically suppressing the tonic phase of the generalized tonic-clonic seizures, but it failed in juveniles. Neither sensorimotor development nor cognitive abilities and emotionality were affected by early-life exposure to Ro 25-6981. Treatment cessation did not affect later seizure susceptibility. Our data are in line with the maturational gradient of the GluN2B-subunit of NMDA receptors and demonstrate developmental differences in the anti-seizure activity of the GluN2B-selective antagonist and its safety for the developing brain.

• Keywords
• GluN2B-selective antagonist, Ro 25-6981, anti-seizure effects, development, immature rats, memory, motor performance,
• Publication type
• Journal Article MeSH

Benzodiazepines (BZDs) are widely used in patients of all ages. Unlike adults, neonatal animals treated with BZDs exhibit a variety of behavioral deficits later in life; however, the mechanisms underlying these deficits are poorly understood. This study aims to examine whether administration of clonazepam (CZP; 1 mg/kg/day) in 7-11-day-old rats affects Gama aminobutyric acid (GABA)ergic receptors in both the short and long terms. Using RT-PCR and quantitative autoradiography, we examined the expression of the selected GABAA receptor subunits (α1, α2, α4, γ2, and δ) and the GABAB B2 subunit, and GABAA, benzodiazepine, and GABAB receptor binding 48 h, 1 week, and 2 months after treatment discontinuation. Within one week after CZP cessation, the expression of the α2 subunit was upregulated, whereas that of the δ subunit was downregulated in both the hippocampus and cortex. In the hippocampus, the α4 subunit was downregulated after the 2-month interval. Changes in receptor binding were highly dependent on the receptor type, the interval after treatment cessation, and the brain structure. GABAA receptor binding was increased in almost all of the brain structures after the 48-h interval. BZD-binding was decreased in many brain structures involved in the neuronal networks associated with emotional behavior, anxiety, and cognitive functions after the 2-month interval. Binding of the GABAB receptors changed depending on the interval and brain structure. Overall, the described changes may affect both synaptic development and functioning and may potentially cause behavioral impairment.

• Keywords
• GABAA/BZD receptor binding, GABAB receptor binding, clonazepam, neonatal rat, subunit mRNA expression,
• MeSH
• Benzodiazepines pharmacology   MeSH
• gamma-Aminobutyric Acid metabolism   MeSH
• Hippocampus drug effects   metabolism   MeSH
• Clonazepam pharmacology   MeSH
• Rats MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Brain drug effects   metabolism   MeSH
• Animals, Newborn MeSH
• Rats, Inbred WF MeSH
• Receptors, GABA-A metabolism   MeSH
• Receptors, GABA-B metabolism   MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Benzodiazepines MeSH
• gamma-Aminobutyric Acid MeSH
• Clonazepam MeSH
• Receptors, GABA-A MeSH
• Receptors, GABA-B MeSH

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.

• Keywords
• AMPA receptor subunits, NMDA receptor autoradiography, NMDA receptor subunits, [3H] MK-801, benzodiazepine, clonazepam, mRNA expression, neonatal rat,
• Publication type
• Journal Article MeSH