Diazepam effect during early neonatal development correlates with neuronal Cl⁻

Abstract

ObjectiveAlthough benzodiazepines and other GABAA receptors allosteric modulators are used to treat neonatal seizures, their efficacy may derive from actions on subcortical structures. Side effects of benzodiazepines in nonseizing human neonates include myoclonus, seizures, and abnormal movements. Excitatory actions of GABA may underlie both side effects and reduced anticonvulsant activity of benzodiazepines. Neocortical organotypic slice cultures were used to study: (1) spontaneous cortical epileptiform activity during early development; (2) developmental changes in [Cl−]i and (3) whether diazepam's anticonvulsant effect correlated with neuronal [Cl−]i.MethodsEpileptiform activity in neocortical organotypic slice cultures was measured by field potential recordings. Cl− changes during development were assessed by multiphoton imaging of neurons transgenically expressing a Cl‐sensitive fluorophore. Clinically relevant concentrations of diazepam were used to test the anticonvulsant effectiveness at ages corresponding to premature neonates through early infancy.Results(1) Neocortical organotypic slices at days in vitro 5 (DIV5) exhibited spontaneous epileptiform activity. (2) Epileptiform event duration decreased with age. (3) There was a progressive decrease in [Cl−]i over the same age range. (4) Diazepam was ineffective in decreasing epileptiform activity at DIV5‐6, but progressively more effective at older ages through DIV15. (5) At DIV5‐6, diazepam worsened epileptiform activity in 50% of the slices.InterpretationThe neocortical organotypic slice is a useful model to study spontaneous epileptiform activity. Decreasing [Cl−]i during development correlates with decreasing duration of spontaneous epileptiform activity and increasing anticonvulsant efficacy of diazepam. We provide a potential explanation for the reports of seizures and myoclonus induction by benzodiazepines in newborn human neonates and the limited electrographic efficacy of benzodiazepines for the treatment of neonatal seizures.

Chloride Dysregulation, Seizures, and Cerebral Edema: A Relationship with Therapeutic Potential

“…Systematic measurements of [Cl − ] i in hundreds of neurons [44,45] with genetically-encoded ratiometric fluorophores such as Clomeleon [46], that also do not perturb the intracellular milieu, has provided additional insight into the developmental regulation of [Cl − ] i . These studies have revealed that neuronal [Cl − ] i does not decrease in all neurons simultaneously during development.…”

“…A change in mental status and seizures are among the most common presentations of HIE in neonates [171]. Importantly, the most common anti-convulsive medications used at this age (phenobarbital, phenytoin, and benzodiazepines) have poor effectiveness [37,172,173] which is correlated with high [Cl − ] i in the neocortex during early development [44,45]. This high [Cl − ] i can be further exacerbated by Cl − accumulation during ensuing seizure episodes [13,174].…”

“…The preferential effect of phenobarbital in different brain regions can explain the electro-clinical dissociation seen in so many seizing infants [44,160]. The high [Cl − ] i during early development can also explain the occasional paradoxical (pro-convulsive) effect of benzodiazepines in human neonatal neocortex [45] and the ineffectiveness of phenobarbital in the neonatal seizing hippocampus [37,172]. …”

Developmental Decrease of Neuronal Chloride Concentration Is Independent of Trauma in Thalamocortical Brain Slices

“…Measurements of neuronal [Cl - ] i in the deeper regions in both thalamus and neocortex support the developmental decrease of [Cl - ] i . Further support of the notion of a developmental decrease in [Cl - ] i comes from the intact preparations of the spinal cord [ 29 , 30 ] and retina, where sectioning trauma is not an issue [ 31 ], and the neocortical organotypic culture slice model [ 32 ]. In the neocortical organotypic culture slice, there is a progressive decrease in [Cl - ] i with no depth correlation during different days in vitro [ 32 ].…”

Songbird organotypic culture as anin vitromodel for interrogating sparse sequencing networks

“…Neuron cultures have already yielded great insights into homeostatic plasticity mechanisms, such as Long-Term Potentiation, LTP, Long-Term Depression, LTD (Karmarkar et al 2002;(Turrigiano et al 1998;Turrigiano 2008) , synaptic scaling, structural synaptic plasticity (Yasumatsu et al 2008;Tominaga et al 1994) , self-organization of neural networks (Johnson & Buonomano 2007) (Cruz-Martín & Schweizer 2008) , and the underlying mechanisms of brain disease (Glykys & Staley 2015) . Long term organotypic cultures provide a unique system where emergent properties of neural networks can be studied at the cellular and molecular level, with extremely precise experimental control.…”