Adeno-Associated Virus Toolkit to Target Diverse Brain Cells.

Recombinant adeno-associated viruses (AAVs) are commonly used gene delivery vehicles for neuroscience research. They have two engineerable features: the capsid (outer protein shell) and cargo (encapsulated genome). These features can be modified to enhance cell type or tissue tropism and control transgene expression, respectively. Several engineered AAV capsids with unique tropisms have been identified, including variants with enhanced central nervous system transduction, cell type specificity, and retrograde transport in neurons. Pairing these AAVs with modern gene regulatory elements and state-of-the-art reporter, sensor, and effector cargo enables highly specific transgene expression for anatomical and functional analyses of brain cells and circuits. Here, we discuss recent advances that provide a comprehensive (capsid and cargo) AAV toolkit for genetic access to molecularly defined brain cell types.

Receptor protein tyrosine phosphatases control Purkinje neuron firing.

Spinocerebellar ataxias (SCA) are a genetically heterogeneous family of cerebellar neurodegenerative diseases characterized by abnormal firing of Purkinje neurons and degeneration. We recently demonstrated the slowed firing rates seen in several SCAs share a common etiology of hyper-activation of the Src family of non-receptor tyrosine kinases (SFKs). However, the lack of clinically available neuroactive SFK inhibitors lead us to investigate alternative mechanisms to modulate SFK activity. Previous studies demonstrate that SFK activity can be enhanced by the removal of inhibitory phospho-marks by receptor-protein-tyrosine phosphatases (RPTPs). In this Extra View we show that MTSS1 inhibits SFK activity through the binding and inhibition of a subset of the RPTP family members, and lowering RPTP activity in cerebellar slices with peptide inhibitors increases the suppressed Purkinje neuron basal firing rates seen in two different SCA models. Together these results identify RPTPs as novel effectors of Purkinje neuron basal firing, extending the MTSS1/SFK regulatory circuit we previously described and expanding the therapeutic targets for SCA patients.

MTSS1/Src family kinase dysregulation underlies multiple inherited ataxias.

The genetically heterogeneous spinocerebellar ataxias (SCAs) are caused by Purkinje neuron dysfunction and degeneration, but their underlying pathological mechanisms remain elusive. The Src family of nonreceptor tyrosine kinases (SFK) are essential for nervous system homeostasis and are increasingly implicated in degenerative disease. Here we reveal that the SFK suppressor Missing-in-metastasis (MTSS1) is an ataxia locus that links multiple SCAs. MTSS1 loss results in increased SFK activity, reduced Purkinje neuron arborization, and low basal firing rates, followed by cell death. Surprisingly, mouse models for SCA1, SCA2, and SCA5 show elevated SFK activity, with SCA1 and SCA2 displaying dramatically reduced MTSS1 protein levels through reduced gene expression and protein translation, respectively. Treatment of each SCA model with a clinically approved Src inhibitor corrects Purkinje neuron basal firing and delays ataxia progression in MTSS1 mutants. Our results identify a common SCA therapeutic target and demonstrate a key role for MTSS1/SFK in Purkinje neuron survival and ataxia progression.

Progress in Understanding and Treating SCN2A-Mediated Disorders.

Advances in gene discovery for neurodevelopmental disorders have identified SCN2A dysfunction as a leading cause of infantile seizures, autism spectrum disorder, and intellectual disability. SCN2A encodes the neuronal sodium channel NaV1.2. Functional assays demonstrate strong correlation between genotype and phenotype. This insight can help guide therapeutic decisions and raises the possibility that ligands that selectively enhance or diminish channel function may improve symptoms. The well-defined function of sodium channels makes SCN2A an important test case for investigating the neurobiology of neurodevelopmental disorders more generally. Here, we discuss the progress made, through the concerted efforts of a diverse group of academic and industry scientists as well as policy advocates, in understanding and treating SCN2A-related disorders.

Optogenetic fMRI and electrophysiological identification of region-specific connectivity between the cerebellar cortex and forebrain.

Complex animal behavior is produced by dynamic interactions between discrete regions of the brain. As such, defining functional connections between brain regions is critical in gaining a full understanding of how the brain generates behavior. Evidence suggests that discrete regions of the cerebellar cortex functionally project to the forebrain, mediating long-range communication potentially important in motor and non-motor behaviors. However, the connectivity map remains largely incomplete owing to the challenge of driving both reliable and selective output from the cerebellar cortex, as well as the need for methods to detect region specific activation across the entire forebrain. Here we utilize a paired optogenetic and fMRI (ofMRI) approach to elucidate the downstream forebrain regions modulated by activating a region of the cerebellum that induces stereotypical, ipsilateral forelimb movements. We demonstrate with ofMRI, that activating this forelimb motor region of the cerebellar cortex results in functional activation of a variety of forebrain and midbrain areas of the brain, including the hippocampus and primary motor, retrosplenial and anterior cingulate cortices. We further validate these findings using optogenetic stimulation paired with multi-electrode array recordings and post-hoc staining for molecular markers of activated neurons (i.e. c-Fos). Together, these findings demonstrate that a single discrete region of the cerebellar cortex is capable of influencing motor output and the activity of a number of downstream forebrain as well as midbrain regions thought to be involved in different aspects of behavior.

Decreased surface expression of the δ subunit of the GABAA receptor contributes to reduced tonic inhibition in dentate granule cells in a mouse model of fragile X syndrome.

While numerous changes in the GABA system have been identified in models of Fragile X Syndrome (FXS), alterations in subunits of the GABAA receptors (GABAARs) that mediate tonic inhibition are particularly intriguing. Considering the key role of tonic inhibition in controlling neuronal excitability, reduced tonic inhibition could contribute to FXS-associated disorders such as hyperactivity, hypersensitivity, and increased seizure susceptibility. The current study has focused on the expression and function of the δ subunit of the GABAAR, a major subunit involved in tonic inhibition, in granule cells of the dentate gyrus in the Fmr1 knockout (KO) mouse model of FXS. Electrophysiological studies of dentate granule cells revealed a marked, nearly four-fold, decrease in tonic inhibition in the Fmr1 KO mice, as well as reduced effects of two δ subunit-preferring pharmacological agents, THIP and DS2, supporting the suggestion that δ subunit-containing GABAARs are compromised in the Fmr1 KO mice. Immunohistochemistry demonstrated a small but statistically significant decrease in δ subunit labeling in the molecular layer of the dentate gyrus in Fmr1 KO mice compared to wildtype (WT) littermates. The discrepancy between the large deficits in GABA-mediated tonic inhibition in granule cells in the Fmr1 KO mice and only modest reductions in immunolabeling of the δ subunit led to studies of surface expression of the δ subunit. Cross-linking experiments followed by Western blot analysis demonstrated a small, non-significant decrease in total δ subunit protein in the hippocampus of Fmr1 KO mice, but a four-fold decrease in surface expression of the δ subunit in these mice. No significant changes were observed in total or surface expression of the α4 subunit protein, a major partner of the δ subunit in the forebrain. Postembedding immunogold labeling for the δ subunit demonstrated a large, three-fold, decrease in the number of symmetric synapses with immunolabeling at perisynaptic locations in Fmr1 KO mice. While α4 immunogold particles were also reduced at perisynaptic locations in the Fmr1 KO mice, the labeling was increased at synaptic sites. Together these findings suggest that, in the dentate gyrus, altered surface expression of the δ subunit, rather than a decrease in δ subunit expression alone, could be limiting δ subunit-mediated tonic inhibition in this model of FXS. Finding ways to increase surface expression of the δ subunit of the GABAAR could be a novel approach to treatment of hyperexcitability-related alterations in FXS.

Gene co-expression network analysis for identifying modules and functionally enriched pathways in SCA2.

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant neurodegenerative disease caused by CAG repeat expansion in the ATXN2 gene. The repeat resides in an encoded region of the gene resulting in polyglutamine (polyQ) expansion which has been assumed to result in gain of function, predominantly, for the ATXN2 protein. We evaluated temporal cerebellar expression profiles by RNA sequencing of ATXN2Q127 mice versus wild-type (WT) littermates. ATXN2Q127 mice are characterized by a progressive motor phenotype onset, and have progressive cerebellar molecular and neurophysiological (Purkinje cell firing frequency) phenotypes. Our analysis revealed previously uncharacterized early and progressive abnormal patterning of cerebellar gene expression. Weighted Gene Coexpression Network Analysis revealed four gene modules that were significantly correlated with disease status, composed primarily of genes associated with GTPase signaling, calcium signaling and cell death. Of these genes, few overlapped with differentially expressed cerebellar genes that we identified in Atxn2-/- knockout mice versus WT littermates, suggesting that loss-of-function is not a significant component of disease pathology. We conclude that SCA2 is a disease characterized by gain of function for ATXN2.

Antisense oligonucleotide therapy for spinocerebellar ataxia type 2.

There are no disease-modifying treatments for adult human neurodegenerative diseases. Here we test RNA-targeted therapies in two mouse models of spinocerebellar ataxia type 2 (SCA2), an autosomal dominant polyglutamine disease. Both models recreate the progressive adult-onset dysfunction and degeneration of a neuronal network that are seen in patients, including decreased firing frequency of cerebellar Purkinje cells and a decline in motor function. We developed a potential therapy directed at the ATXN2 gene by screening 152 antisense oligonucleotides (ASOs). The most promising oligonucleotide, ASO7, downregulated ATXN2 mRNA and protein, which resulted in delayed onset of the SCA2 phenotype. After delivery by intracerebroventricular injection to ATXN2-Q127 mice, ASO7 localized to Purkinje cells, reduced cerebellar ATXN2 expression below 75% for more than 10 weeks without microglial activation, and reduced the levels of cerebellar ATXN2. Treatment of symptomatic mice with ASO7 improved motor function compared to saline-treated mice. ASO7 had a similar effect in the BAC-Q72 SCA2 mouse model, and in both mouse models it normalized protein levels of several SCA2-related proteins expressed in Purkinje cells, including Rgs8, Pcp2, Pcp4, Homer3, Cep76 and Fam107b. Notably, the firing frequency of Purkinje cells returned to normal even when treatment was initiated more than 12 weeks after the onset of the motor phenotype in BAC-Q72 mice. These findings support ASOs as a promising approach for treating some human neurodegenerative diseases.

The Roles of the Olivocerebellar Pathway in Motor Learning and Motor Control. A Consensus Paper.

For many decades, the predominant view in the cerebellar field has been that the olivocerebellar system's primary function is to induce plasticity in the cerebellar cortex, specifically, at the parallel fiber-Purkinje cell synapse. However, it has also long been proposed that the olivocerebellar system participates directly in motor control by helping to shape ongoing motor commands being issued by the cerebellum. Evidence consistent with both hypotheses exists; however, they are often investigated as mutually exclusive alternatives. In contrast, here, we take the perspective that the olivocerebellar system can contribute to both the motor learning and motor control functions of the cerebellum and might also play a role in development. We then consider the potential problems and benefits of it having multiple functions. Moreover, we discuss how its distinctive characteristics (e.g., low firing rates, synchronization, and variable complex spike waveforms) make it more or less suitable for one or the other of these functions, and why having multiple functions makes sense from an evolutionary perspective. We did not attempt to reach a consensus on the specific role(s) the olivocerebellar system plays in different types of movements, as that will ultimately be determined experimentally; however, collectively, the various contributions highlight the flexibility of the olivocerebellar system, and thereby suggest that it has the potential to act in both the motor learning and motor control functions of the cerebellum.

Cellular and circuit mechanisms underlying spinocerebellar ataxias.

Degenerative ataxias are a common form of neurodegenerative disease that affect about 20 individuals per 100,000. The autosomal dominant spinocerebellar ataxias (SCAs) are caused by a variety of protein coding mutations (single nucleotide changes, deletions and expansions) in single genes. Affected genes encode plasma membrane and intracellular ion channels, membrane receptors, protein kinases, protein phosphatases and proteins of unknown function. Although SCA-linked genes are quite diverse they share two key features: first, they are highly, although not exclusively, expressed in cerebellar Purkinje neurons (PNs), and second, when mutated they lead ultimately to the degeneration of PNs. In this review we summarize ataxia-related changes in PN neurophysiology that have been observed in various mouse knockout lines and in transgenic models of human SCA. We also highlight emerging evidence that altered metabotropic glutamate receptor signalling and disrupted calcium homeostasis in PNs form a common, early pathophysiological mechanism in SCAs. Together these findings indicate that aberrant calcium signalling and profound changes in PN neurophysiology precede PN cell loss and are likely to lead to cerebellar circuit dysfunction that explains behavioural signs of ataxia characteristic of the disease.

Ectopic Expression of α6 and δ GABAA Receptor Subunits in Hilar Somatostatin Neurons Increases Tonic Inhibition and Alters Network Activity in the Dentate Gyrus.

The role of GABAA receptor (GABAAR)-mediated tonic inhibition in interneurons remains unclear and may vary among subgroups. Somatostatin (SOM) interneurons in the hilus of the dentate gyrus show negligible expression of nonsynaptic GABAAR subunits and very low tonic inhibition. To determine the effects of ectopic expression of tonic GABAAR subtypes in these neurons, Cre-dependent viral vectors were used to express GFP-tagged GABAAR subunits (α6 and δ) selectively in hilar SOM neurons in SOM-Cre mice. In single-transfected animals, immunohistochemistry demonstrated strong expression of either the α6 or δ subunit; in cotransfected animals, both subunits were consistently expressed in the same neurons. Electrophysiology revealed a robust increase of tonic current, with progressively larger increases following transfection of δ, α6, and α6/δ subunits, respectively, indicating formation of functional receptors in all conditions and likely coassembly of the subunits in the same receptor following cotransfection. An in vitro model of repetitive bursting was used to determine the effects of increased tonic inhibition in hilar SOM interneurons on circuit activity in the dentate gyrus. Upon cotransfection, the frequency of GABAAR-mediated bursting in granule cells was reduced, consistent with a reduction in synchronous firing among hilar SOM interneurons. Moreover, in vivo studies of Fos expression demonstrated reduced activation of α6/δ-cotransfected neurons following acute seizure induction by pentylenetetrazole. The findings demonstrate that increasing tonic inhibition in hilar SOM interneurons can alter dentate gyrus circuit activity during strong stimulation and suggest that tonic inhibition of interneurons could play a role in regulating excessive synchrony within the network. SIGNIFICANCE STATEMENT: In contrast to many hippocampal interneurons, somatostatin (SOM) neurons in the hilus of the dentate gyrus have very low levels of nonsynaptic GABAARs and exhibit very little tonic inhibition. In an effort to increase tonic inhibition selectively in these interneurons, we used Cre-dependent viral vectors in SOM-Cre mice to achieve interneuron-specific expression of the nonsynaptic GABAAR subunits (α6 and δ) in vivo. We show, for the first time, that such recombinant GFP-tagged GABAAR subunits are expressed robustly, assemble to form functional receptors, substantially increase tonic inhibition in SOM interneurons, and alter circuit activity within the dentate gyrus.

Circuit mechanisms underlying motor memory formation in the cerebellum.

The cerebellum stores associative motor memories essential for properly timed movement; however, the mechanisms by which these memories form and are acted upon remain unclear. To determine how cerebellar activity relates to movement and motor learning, we used optogenetics to manipulate spontaneously firing Purkinje neurons (PNs) in mouse simplex lobe. Using high-speed videography and motion tracking, we found that altering PN activity produced rapid forelimb movement. PN inhibition drove movements time-locked to stimulus onset, whereas PN excitation drove delayed movements time-locked to stimulus offset. Pairing either PN inhibition or excitation with sensory stimuli triggered the formation of robust, associative motor memories; however, PN excitation led to learned movements whose timing more closely matched training intervals. These findings implicate inhibition of PNs as a teaching signal, consistent with a model whereby learning leads first to reductions in PN firing that subsequently instruct circuit changes in the cerebellar nucleus.

Multifocal fluorescence microscope for fast optical recordings of neuronal action potentials.

In recent years, optical sensors for tracking neural activity have been developed and offer great utility. However, developing microscopy techniques that have several kHz bandwidth necessary to reliably capture optically reported action potentials (APs) at multiple locations in parallel remains a significant challenge. To our knowledge, we describe a novel microscope optimized to measure spatially distributed optical signals with submillisecond and near diffraction-limit resolution. Our design uses a spatial light modulator to generate patterned illumination to simultaneously excite multiple user-defined targets. A galvanometer driven mirror in the emission path streaks the fluorescence emanating from each excitation point during the camera exposure, using unused camera pixels to capture time varying fluorescence at rates that are ∼1000 times faster than the camera's native frame rate. We demonstrate that this approach is capable of recording Ca(2+) transients resulting from APs in neurons labeled with the Ca(2+) sensor Oregon Green Bapta-1 (OGB-1), and can localize the timing of these events with millisecond resolution. Furthermore, optically reported APs can be detected with the voltage sensitive dye DiO-DPA in multiple locations within a neuron with a signal/noise ratio up to ∼40, resolving delays in arrival time along dendrites. Thus, the microscope provides a powerful tool for photometric measurements of dynamics requiring submillisecond sampling at multiple locations.

Targeting RNA foci in iPSC-derived motor neurons from ALS patients with a C9ORF72 repeat expansion.

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative condition characterized by loss of motor neurons in the brain and spinal cord. Expansions of a hexanucleotide repeat (GGGGCC) in the noncoding region of the C9ORF72 gene are the most common cause of the familial form of ALS (C9-ALS), as well as frontotemporal lobar degeneration and other neurological diseases. How the repeat expansion causes disease remains unclear, with both loss of function (haploinsufficiency) and gain of function (either toxic RNA or protein products) proposed. We report a cellular model of C9-ALS with motor neurons differentiated from induced pluripotent stem cells (iPSCs) derived from ALS patients carrying the C9ORF72 repeat expansion. No significant loss of C9ORF72 expression was observed, and knockdown of the transcript was not toxic to cultured human motor neurons. Transcription of the repeat was increased, leading to accumulation of GGGGCC repeat-containing RNA foci selectively in C9-ALS iPSC-derived motor neurons. Repeat-containing RNA foci colocalized with hnRNPA1 and Pur-α, suggesting that they may be able to alter RNA metabolism. C9-ALS motor neurons showed altered expression of genes involved in membrane excitability including DPP6, and demonstrated a diminished capacity to fire continuous spikes upon depolarization compared to control motor neurons. Antisense oligonucleotides targeting the C9ORF72 transcript suppressed RNA foci formation and reversed gene expression alterations in C9-ALS motor neurons. These data show that patient-derived motor neurons can be used to delineate pathogenic events in ALS.

A reorganized GABAergic circuit in a model of epilepsy: evidence from optogenetic labeling and stimulation of somatostatin interneurons.

Axonal sprouting of excitatory neurons is frequently observed in temporal lobe epilepsy, but the extent to which inhibitory interneurons undergo similar axonal reorganization remains unclear. The goal of this study was to determine whether somatostatin (SOM)-expressing neurons in stratum (s.) oriens of the hippocampus exhibit axonal sprouting beyond their normal territory and innervate granule cells of the dentate gyrus in a pilocarpine model of epilepsy. To obtain selective labeling of SOM-expressing neurons in s. oriens, a Cre recombinase-dependent construct for channelrhodopsin2 fused to enhanced yellow fluorescent protein (ChR2-eYFP) was virally delivered to this region in SOM-Cre mice. In control mice, labeled axons were restricted primarily to s. lacunosum-moleculare. However, in pilocarpine-treated animals, a rich plexus of ChR2-eYFP-labeled fibers and boutons extended into the dentate molecular layer. Electron microscopy with immunogold labeling demonstrated labeled axon terminals that formed symmetric synapses on dendritic profiles in this region, consistent with innervation of granule cells. Patterned illumination of ChR2-labeled fibers in s. lacunosum-moleculare of CA1 and the dentate molecular layer elicited GABAergic inhibitory responses in dentate granule cells in pilocarpine-treated mice but not in controls. Similar optical stimulation in the dentate hilus evoked no significant responses in granule cells of either group of mice. These findings indicate that under pathological conditions, SOM/GABAergic neurons can undergo substantial axonal reorganization beyond their normal territory and establish aberrant synaptic connections. Such reorganized circuitry could contribute to functional deficits in inhibition in epilepsy, despite the presence of numerous GABAergic terminals in the region.

A reinforcing circuit action of extrasynaptic GABAA receptor modulators on cerebellar granule cell inhibition.

GABAA receptors (GABARs) are the targets of a wide variety of modulatory drugs which enhance chloride flux through GABAR ion channels. Certain GABAR modulators appear to acutely enhance the function of δ subunit-containing GABAR subtypes responsible for tonic forms of inhibition. Here we identify a reinforcing circuit mechanism by which these drugs, in addition to directly enhancing GABAR function, also increase GABA release. Electrophysiological recordings in cerebellar slices from rats homozygous for the ethanol-hypersensitive (α6100Q) allele show that modulators and agonists selective for δ-containing GABARs such as THDOC, ethanol and THIP (gaboxadol) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in granule cells. Ethanol fails to augment granule cell sIPSC frequency in the presence of glutamate receptor antagonists, indicating that circuit mechanisms involving granule cell output contribute to ethanol-enhancement of synaptic inhibition. Additionally, GABAR antagonists decrease ethanol-induced enhancement of Golgi cell firing. Consistent with a role for glutamatergic inputs, THIP-induced increases in Golgi cell firing are abolished by glutamate receptor antagonists. Moreover, THIP enhances the frequency of spontaneous excitatory postsynaptic currents in Golgi cells. Analyses of knockout mice indicate that δ subunit-containing GABARs are required for enhancing GABA release in the presence of ethanol and THIP. The limited expression of the GABAR δ subunit protein within the cerebellar cortex suggests that an indirect, circuit mechanism is responsible for stimulating Golgi cell GABA release by drugs selective for extrasynaptic isoforms of GABARs. Such circuit effects reinforce direct actions of these positive modulators on tonic GABAergic inhibition and are likely to contribute to the potent effect of these compounds as nervous system depressants.

Changes in Purkinje cell firing and gene expression precede behavioral pathology in a mouse model of SCA2.

Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominantly inherited disorder, which is caused by a pathological expansion of a polyglutamine (polyQ) tract in the coding region of the ATXN2 gene. Like other ataxias, SCA2 most overtly affects Purkinje cells (PCs) in the cerebellum. Using a transgenic mouse model expressing a full-length ATXN2(Q127)-complementary DNA under control of the Pcp2 promoter (a PC-specific promoter), we examined the time course of behavioral, morphologic, biochemical and physiological changes with particular attention to PC firing in the cerebellar slice. Although motor performance began to deteriorate at 8 weeks of age, reductions in PC number were not seen until after 12 weeks. Decreases in the PC firing frequency first showed at 6 weeks and paralleled deterioration of motor performance with progression of disease. Transcription changes in several PC-specific genes such as Calb1 and Pcp2 mirrored the time course of changes in PC physiology with calbindin-28 K changes showing the first small, but significant decreases at 4 weeks. These results emphasize that in this model of SCA2, physiological and behavioral phenotypes precede morphological changes by several weeks and provide a rationale for future studies examining the effects of restoration of firing frequency on motor function and prevention of future loss of PCs.

Effects of climbing fiber driven inhibition on Purkinje neuron spiking.

Climbing fiber (CF) input to the cerebellum is thought to instruct associative motor memory formation through its effects on multiple sites within the cerebellar circuit. We used adeno-associated viral delivery of channelrhodopsin-2 (ChR2) to inferior olivary neurons to selectively express ChR2 in CFs, achieving nearly complete transfection of CFs in the caudal cerebellar lobules of rats. As expected, optical stimulation of ChR2-expressing CFs generates complex spike responses in individual Purkinje neurons (PNs); in addition we found that such stimulation recruits a network of inhibitory interneurons in the molecular layer. This CF-driven disynaptic inhibition prolongs the postcomplex spike pause observed when spontaneously firing PNs receive direct CF input; such inhibition also elicits pauses in spontaneously firing PNs not receiving direct CF input. Baseline firing rates of PNs are strongly suppressed by low-frequency (2 Hz) stimulation of CFs, and this suppression is partly relieved by blocking synaptic inhibition. We conclude that CF-driven, disynaptic inhibition has a major influence on PN excitability and contributes to the widely observed negative correlation between complex and simple spike rates. Because they receive input from many CFs, molecular layer interneurons are well positioned to detect the spatiotemporal patterns of CF activity believed to encode error signals. Together, our findings suggest that such inhibition may bind together groups of Purkinje neurons to provide instructive signals to downstream sites in the cerebellar circuit.

Robust photoregulation of GABA(A) receptors by allosteric modulation with a propofol analogue.

Photochemical switches represent a powerful method for improving pharmacological therapies and controlling cellular physiology. Here we report the photoregulation of GABA(A) receptors (GABA(A)Rs) by a derivative of propofol (2,6-diisopropylphenol), a GABA(A)R allosteric modulator, which we have modified to contain photoisomerizable azobenzene. Using α(1)ß(2)γ(2) GABA(A)Rs expressed in Xenopus laevis oocytes and native GABA(A)Rs of isolated retinal ganglion cells, we show that the trans-azobenzene isomer of the new compound (trans-MPC088), generated by visible light (wavelengths ~440 nm), potentiates the γ-aminobutyric acid-elicited response and, at higher concentrations, directly activates the receptors. cis-MPC088, generated from trans-MPC088 by ultraviolet light (~365 nm), produces little, if any, receptor potentiation/activation. In cerebellar slices, MPC088 co-applied with γ-aminobutyric acid affords bidirectional photomodulation of Purkinje cell membrane current and spike-firing rate. The findings demonstrate photocontrol of GABA(A)Rs by an allosteric ligand, and open new avenues for fundamental and clinically oriented research on GABA(A)Rs, a major class of neurotransmitter receptors in the central nervous system.