The advent of whole exome/genome sequencing and the technology-driven reduction in the cost of next-generation sequencing as well as the introduction of diagnostic-targeted sequencing chips have resulted in an unprecedented volume of data directly linking patient genomic variability to disorders of the brain. This information has the potential to transform our understanding of neurologic disorders by improving diagnoses, illuminating the molecular heterogeneity underlying diseases, and identifying new targets for therapeutic treatment. There is a strong history of mutations in GABA receptor genes being involved in neurologic diseases, particularly the epilepsies. In addition, a substantial number of variants and mutations have been found in GABA receptor genes in patients with autism, schizophrenia, and addiction, suggesting potential links between the GABA receptors and these conditions. A new and unexpected outcome from sequencing efforts has been the surprising number of mutations found in glutamate receptor subunits, with the GRIN2A gene encoding the GluN2A N-methyl-D-aspartate receptor subunit being most often affected. These mutations are associated with multiple neurologic conditions, for which seizure disorders comprise the largest group. The GluN2A subunit appears to be a locus for epilepsy, which holds important therapeutic implications. Virtually all a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor mutations, most of which occur within GRIA3, are from patients with intellectual disabilities, suggesting a link to this condition. Similarly, the most common phenotype for kainate receptor variants is intellectual disability. Herein, we summarize the current understanding of disease-associated mutations in ionotropic GABA and glutamate receptor families, and discuss implications regarding the identification of human mutations and treatment of neurologic diseases.

Patch-clamp recording has enabled single-cell electrical, morphological and genetic studies at unparalleled resolution. Yet it remains a laborious and low-throughput technique, making it largely impractical for large-scale measurements such as cell type and connectivity characterization of neurons in the brain. Specifically, the technique is critically limited by the ubiquitous practice of manually replacing patch-clamp pipettes after each recording. To circumvent this limitation, we developed a simple, fast, and automated method for cleaning glass pipette electrodes that enables their reuse within one minute. By immersing pipette tips into Alconox, a commercially-available detergent, followed by rinsing, we were able to reuse pipettes 10 times with no degradation in signal fidelity, in experimental preparations ranging from human embryonic kidney cells to neurons in culture, slices, and in vivo. Undetectable trace amounts of Alconox remaining in the pipette after cleaning did not affect ion channel pharmacology. We demonstrate the utility of pipette cleaning by developing the first robot to perform sequential patch-clamp recordings in cell culture and in vivo without a human operator.

General anesthesia is characterized by loss of consciousness, amnesia, analgesia, and immobility. Important molecular targets of general anesthetics have been identified, but the neural circuits underlying the discrete end points of general anesthesia remain incompletely understood. General anesthesia and natural sleep share the common feature of reversible unconsciousness, and recent developments in neuroscience have enabled elegant studies that investigate the brain nuclei and neural circuits underlying this important end point. A common approach to measure cortical activity across the brain is electroencephalogram (EEG), which can reflect local neuronal activity as well as connectivity among brain regions. The EEG oscillations observed during general anesthesia depend greatly on the anesthetic agent as well as dosing, and only some resemble those observed during sleep. For example, the EEG oscillations during dexmedetomidine sedation are similar to those of stage 2 nonrapid eye movement (NREM) sleep, but high doses of propofol and ether anesthetics produce burst suppression, a pattern that is never observed during natural sleep. Sleep is primarily driven by withdrawal of subcortical excitation to the cortex, but anesthetics can directly act at both subcortical and cortical targets. While some anesthetics appear to activate specific sleep-active regions to induce unconsciousness, not all sleep-active regions play a significant role in anesthesia. Anesthetics also inhibit cortical neurons, and it is likely that each class of anesthetic drugs produces a distinct combination of subcortical and cortical effects that lead to unconsciousness. Conversely, arousal circuits that promote wakefulness are involved in anesthetic emergence and activating them can induce emergence and accelerate recovery of consciousness. Modern neuroscience techniques that enable the manipulation of specific neural circuits have led to new insights into the neural circuitry underlying general anesthesia and sleep. In the coming years, we will continue to better understand the mechanisms that generate these distinct states of reversible unconsciousness.

GABAA receptors are ligand-gated anion channels that are important regulators of neuronal inhibition. Mutations in several genes encoding receptor subunits have been identified in patients with various types of epilepsy, ranging from mild febrile seizures to severe epileptic encephalopathy. Using whole-genome sequencing, we identified a novel de novo missense variant in GABRA5 (c.880G > C, p.V294L) in a patient with severe early-onset epilepsy and developmental delay. Targeted resequencing of 279 additional epilepsy patients identified 19 rare variants from nine GABAA receptor genes, including a novel de novo missense variant in GABRA2 (c.875C > A, p.T292K) and a recurrent missense variant in GABRB3 (c.902C > T, p.P301L). Patients with the GABRA2 and GABRB3 variants also presented with severe epilepsy and developmental delay. We evaluated the effects of the GABRA5, GABRA2 and GABRB3 missense variants on receptor function using whole-cell patch-clamp recordings from human embryonic kidney 293T cells expressing appropriate α, β and γ subunits. The GABRA5 p.V294L variant produced receptors that were 10-times more sensitive to GABA but had reduced maximal GABA-evoked current due to increased receptor desensitization. The GABRA2 p.T292K variant reduced channel expression and produced mutant channels that were tonically open, even in the absence of GABA. Receptors containing the GABRB3 p.P301L variant were less sensitive to GABA and produced less GABA-evoked current. These results provide the first functional evidence that de novo variants in the GABRA5 and GABRA2 genes contribute to early-onset epilepsy and developmental delay, and demonstrate that epilepsy can result from reduced neuronal inhibition via a wide range of alterations in GABAA receptor function.