Dissection of brain-wide resting-state and functional somatosensory circuits by fMRI with optogenetic silencing.

To further advance functional MRI (fMRI)-based brain science, it is critical to dissect fMRI activity at the circuit level. To achieve this goal, we combined brain-wide fMRI with neuronal silencing in well-defined regions. Since focal inactivation suppresses excitatory output to downstream pathways, intact input and suppressed output circuits can be separated. Highly specific cerebral blood volume-weighted fMRI was performed with optogenetic stimulation of local GABAergic neurons in mouse somatosensory regions. Brain-wide spontaneous somatosensory networks were found mostly in ipsilateral cortical and subcortical areas, which differed from the bilateral homotopic connections commonly observed in resting-state fMRI data. The evoked fMRI responses to somatosensory stimulation in regions of the somatosensory network were successfully dissected, allowing the relative contributions of spinothalamic (ST), thalamocortical (TC), corticothalamic (CT), corticocortical (CC) inputs, and local intracortical circuits to be determined. The ventral posterior thalamic nucleus receives ST inputs, while the posterior medial thalamic nucleus receives CT inputs from the primary somatosensory cortex (S1) with TC inputs. The secondary somatosensory cortex (S2) receives mostly direct CC inputs from S1 and a few TC inputs from the ventral posterolateral nucleus. The TC and CC input layers in cortical regions were identified by laminar-specific fMRI responses with a full width at half maximum of <150 µm. Long-range synaptic inputs in cortical areas were amplified approximately twofold by local intracortical circuits, which is consistent with electrophysiological recordings. Overall, whole-brain fMRI with optogenetic inactivation revealed brain-wide, population-based, long-range circuits, which could complement data typically collected in conventional microscopic functional circuit studies.

Using a bistable animal opsin for switchable and scalable optogenetic inhibition of neurons.

There is no consensus on the best inhibitory optogenetic tool. Since Gi/o signalling is a native mechanism of neuronal inhibition, we asked whether Lamprey Parapinopsin ("Lamplight"), a Gi/o-coupled bistable animal opsin, could be used for optogenetic silencing. We show that short (405 nm) and long (525 nm) wavelength pulses repeatedly switch Lamplight between stable signalling active and inactive states, respectively, and that combining these wavelengths can be used to achieve intermediate levels of activity. These properties can be applied to produce switchable neuronal hyperpolarisation and suppression of spontaneous spike firing in the mouse hypothalamic suprachiasmatic nucleus. Expressing Lamplight in (predominantly) ON bipolar cells can photosensitise retinas following advanced photoreceptor degeneration, with 405 and 525 nm stimuli producing responses of opposite sign in the output neurons of the retina. We conclude that bistable animal opsins can co-opt endogenous signalling mechanisms to allow optogenetic inhibition that is scalable, sustained and reversible.

Global transgenic upregulation of KCC2 confers enhanced diazepam efficacy in treating sustained seizures.

Reduced anticonvulsant efficacy of benzodiazepines is a problem in the treatment of status epilepticus, with up to 50% of patients failing to respond to their first dose. KCC2 is a neuronal K+ -Cl- co-transporter that helps set and maintain intracellular Cl- concentrations. KCC2 functional downregulation is a potential contributor to benzodiazepine resistance. We tested this idea using male and female doxycycline-inducible, conditional transgenic mice to increase the functional expression of KCC2 in pyramidal neurons. We administered mice with two doses of the chemoconvulsant kainic acid (5 mg/kg, i.p.) 60 min apart and quantified the resultant seizures with electroencephalography (EEG) recordings. Overexpression of KCC2 prior to the chemoconvulsant challenge did not affect seizure latency or other measures of seizure severity, but it did increase diazepam's efficacy in stopping EEG seizures. Spike rate, time in seizure, and EEG spectral power following diazepam (5 mg/kg, i.p) were all significantly lower in KCC2 overexpression mice as compared to control mice. Our results indicate that, in the context of benzodiazepine resistance during sustained seizures, addressing impaired Cl- homeostasis alone appreciably improves the efficacy of γ-aminobutyric acid (GABA)ergic inhibition. We therefore suggest the simultaneous targeting of KCC2 and GABAA receptors as a pathway for improving current anticonvulsant therapeutic strategies.

Electrophysiology of ionotropic GABA receptors.

GABAA receptors are ligand-gated chloride channels and ionotropic receptors of GABA, the main inhibitory neurotransmitter in vertebrates. In this review, we discuss the major and diverse roles GABAA receptors play in the regulation of neuronal communication and the functioning of the brain. GABAA receptors have complex electrophysiological properties that enable them to mediate different types of currents such as phasic and tonic inhibitory currents. Their activity is finely regulated by membrane voltage, phosphorylation and several ions. GABAA receptors are pentameric and are assembled from a diverse set of subunits. They are subdivided into numerous subtypes, which differ widely in expression patterns, distribution and electrical activity. Substantial variations in macroscopic neural behavior can emerge from minor differences in structure and molecular activity between subtypes. Therefore, the diversity of GABAA receptors widens the neuronal repertoire of responses to external signals and contributes to shaping the electrical activity of neurons and other cell types.

Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo.

Intracellular chloride ([Cl-]i) and pH (pHi) are fundamental regulators of neuronal excitability. They exert wide-ranging effects on synaptic signaling and plasticity and on development and disorders of the brain. The ideal technique to elucidate the underlying ionic mechanisms is quantitative and combined two-photon imaging of [Cl-]i and pHi, but this has never been performed at the cellular level in vivo. Here, by using a genetically encoded fluorescent sensor that includes a spectroscopic reference (an element insensitive to Cl- and pH), we show that ratiometric imaging is strongly affected by the optical properties of the brain. We have designed a method that fully corrects for this source of error. Parallel measurements of [Cl-]i and pHi at the single-cell level in the mouse cortex showed the in vivo presence of the widely discussed developmental fall in [Cl-]i and the role of the K-Cl cotransporter KCC2 in this process. Then, we introduce a dynamic two-photon excitation protocol to simultaneously determine the changes of pHi and [Cl-]i in response to hypercapnia and seizure activity.

Structural foundations of optogenetics: Determinants of channelrhodopsin ion selectivity.

The structure-guided design of chloride-conducting channelrhodopsins has illuminated mechanisms underlying ion selectivity of this remarkable family of light-activated ion channels. The first generation of chloride-conducting channelrhodopsins, guided in part by development of a structure-informed electrostatic model for pore selectivity, included both the introduction of amino acids with positively charged side chains into the ion conduction pathway and the removal of residues hypothesized to support negatively charged binding sites for cations. Engineered channels indeed became chloride selective, reversing near -65 mV and enabling a new kind of optogenetic inhibition; however, these first-generation chloride-conducting channels displayed small photocurrents and were not tested for optogenetic inhibition of behavior. Here we report the validation and further development of the channelrhodopsin pore model via crystal structure-guided engineering of next-generation light-activated chloride channels (iC++) and a bistable variant (SwiChR++) with net photocurrents increased more than 15-fold under physiological conditions, reversal potential further decreased by another ∼ 15 mV, inhibition of spiking faithfully tracking chloride gradients and intrinsic cell properties, strong expression in vivo, and the initial microbial opsin channel-inhibitor-based control of freely moving behavior. We further show that inhibition by light-gated chloride channels is mediated mainly by shunting effects, which exert optogenetic control much more efficiently than the hyperpolarization induced by light-activated chloride pumps. The design and functional features of these next-generation chloride-conducting channelrhodopsins provide both chronic and acute timescale tools for reversible optogenetic inhibition, confirm fundamental predictions of the ion selectivity model, and further elucidate electrostatic and steric structure-function relationships of the light-gated pore.

Rare GABRA3 variants are associated with epileptic seizures, encephalopathy and dysmorphic features.

Genetic epilepsies are caused by mutations in a range of different genes, many of them encoding ion channels, receptors or transporters. While the number of detected variants and genes increased dramatically in the recent years, pleiotropic effects have also been recognized, revealing that clinical syndromes with various degrees of severity arise from a single gene, a single mutation, or from different mutations showing similar functional defects. Accordingly, several genes coding for GABAA receptor subunits have been linked to a spectrum of benign to severe epileptic disorders and it was shown that a loss of function presents the major correlated pathomechanism. Here, we identified six variants in GABRA3 encoding the α3-subunit of the GABAA receptor. This gene is located on chromosome Xq28 and has not been previously associated with human disease. Five missense variants and one microduplication were detected in four families and two sporadic cases presenting with a range of epileptic seizure types, a varying degree of intellectual disability and developmental delay, sometimes with dysmorphic features or nystagmus. The variants co-segregated mostly but not completely with the phenotype in the families, indicating in some cases incomplete penetrance, involvement of other genes, or presence of phenocopies. Overall, males were more severely affected and there were three asymptomatic female mutation carriers compared to only one male without a clinical phenotype. X-chromosome inactivation studies could not explain the phenotypic variability in females. Three detected missense variants are localized in the extracellular GABA-binding NH2-terminus, one in the M2-M3 linker and one in the M4 transmembrane segment of the α3-subunit. Functional studies in Xenopus laevis oocytes revealed a variable but significant reduction of GABA-evoked anion currents for all mutants compared to wild-type receptors. The degree of current reduction correlated partially with the phenotype. The microduplication disrupted GABRA3 expression in fibroblasts of the affected patient. In summary, our results reveal that rare loss-of-function variants in GABRA3 increase the risk for a varying combination of epilepsy, intellectual disability/developmental delay and dysmorphic features, presenting in some pedigrees with an X-linked inheritance pattern.

Memory formation and retrieval of neuronal silencing in the auditory cortex.

Sensory stimuli not only activate specific populations of cortical neurons but can also silence other populations. However, it remains unclear whether neuronal silencing per se leads to memory formation and behavioral expression. Here we show that mice can report optogenetic inactivation of auditory neuron ensembles by exhibiting fear responses or seeking a reward. Mice receiving pairings of footshock and silencing of a neuronal ensemble exhibited a fear response selectively to the subsequent silencing of the same ensemble. The valence of the neuronal silencing was preserved for at least 30 d and was susceptible to extinction training. When we silenced an ensemble in one side of auditory cortex for conditioning, silencing of an ensemble in another side induced no fear response. We also found that mice can find a reward based on the presence or absence of the silencing. Neuronal silencing was stored as working memory. Taken together, we propose that neuronal silencing without explicit activation in the cerebral cortex is enough to elicit a cognitive behavior.

ADCY3: the pivotal gene in classical ketogenic diet for the treatment of epilepsy.

Objective: Epilepsy is a common neurological disorder characterized by recurrent epilepsy episodes. As a non-pharmacological treatment, the ketogenic diet has been widely applied in treating epilepsy. However, the exact therapeutic mechanism of the ketogenic diet for epilepsy remains unclear. This study investigates the molecular mechanisms of the ketogenic diet in regulating fatty acid metabolism and activating the ADCY3-initiated cAMP signaling pathway to enhance neuronal inhibition and thereby treat epilepsy. Methods and results: Meta-analysis reveals that the ketogenic diet is superior to the conventional diet in treating epilepsy. Animal experiments demonstrate that the ketogenic diet is more effective than the conventional diet in treating epilepsy, with the best results achieved using the classic ketogenic diet. Transcriptome sequencing analysis identifies six essential genes, among which ADCY3 shows increased expression in the ketogenic diet. In vivo experiments confirm that the activation of the cAMP-PKA signaling pathway by ADCY3 enhances neuronal inhibition and improves epilepsy control. Conclusion: Clinical observations indicate that the ketogenic diet improves patient epilepsy episodes by regulating the ADCY3-initiated cAMP signaling pathway.

CGP7930 - An allosteric modulator of GABABRs, GABAARs and inwardly-rectifying potassium channels.

Type-A and -B GABA receptors (GABAARs/GABABRs) control brain function and behaviour by fine tuning neurotransmission. Over-time these receptors have become important therapeutic targets for treating neurodevelopmental and neuropsychiatric disorders. Several positive allosteric modulators (PAMs) of GABARs have reached the clinic and selective targeting of receptor subtypes is crucial. For GABABRs, CGP7930 is a widely used PAM for in vivo studies, but its full pharmacological profile has not yet been established. Here, we reveal that CGP7930 has multiple effects not only on GABABRs but also GABAARs, which for the latter involves potentiation of GABA currents, direct receptor activation, and also inhibition. Furthermore, at higher concentrations, CGP7930 also blocks G protein-coupled inwardly-rectifying K+ (GIRK) channels diminishing GABABR signalling in HEK 293 cells. In male and female rat hippocampal neuron cultures, CGP7930 allosteric effects on GABAARs caused prolonged rise and decay times and reduced the frequency of inhibitory postsynaptic currents and potentiated GABAAR-mediated tonic inhibition. Additional comparison between predominant synaptic- and extrasynaptic-isoforms of GABAAR indicated no evident subtype selectivity for CGP7930. In conclusion, our study of CGP7930 modulation of GABAARs, GABABRs and GIRK channels, indicates this compound is unsuitable for use as a specific GABABR PAM.

Using Inhibitory DREADDs to Silence LC Neurons in Monkeys.

Understanding the role of the noradrenergic nucleus locus coeruleus (LC) in cognition and behavior is critical: It is involved in several key behavioral functions such as stress and vigilance, as well as in cognitive processes such as attention and decision making. In recent years, the development of viral tools has provided a clear insight into numerous aspects of brain functions in rodents. However, given the specificity of primate brains and the key benefit of monkey research for translational applications, developing viral tools to study the LC in monkeys is essential for understanding its function and exploring potential clinical strategies. Here, we describe a pharmacogenetics approach that allows to selectively and reversibly inactivate LC neurons using Designer Receptors Exclusively Activated by Designer Drugs (DREADD). We show that the expression of the hM4Di DREADD can be restricted to noradrenergic LC neurons and that the amount of LC inhibition can be adjusted by adapting the dose of the specific DREADD activator deschloroclozapine (DCZ). Indeed, even if high doses (>0.3 mg/kg) induce a massive inhibition of LC neurons and a clear decrease in vigilance, smaller doses (<0.3 mg/kg) induce a more moderate decrease in LC activity, but it does not affect vigilance, which is more compatible with an assessment of subtle cognitive functions such as decision making and attention.

A photoswitchable GPCR-based opsin for presynaptic inhibition.

Optical manipulations of genetically defined cell types have generated significant insights into the dynamics of neural circuits. While optogenetic activation has been relatively straightforward, rapid and reversible synaptic inhibition has proven more elusive. Here, we leveraged the natural ability of inhibitory presynaptic GPCRs to suppress synaptic transmission and characterize parapinopsin (PPO) as a GPCR-based opsin for terminal inhibition. PPO is a photoswitchable opsin that couples to Gi/o signaling cascades and is rapidly activated by pulsed blue light, switched off with amber light, and effective for repeated, prolonged, and reversible inhibition. PPO rapidly and reversibly inhibits glutamate, GABA, and dopamine release at presynaptic terminals. Furthermore, PPO alters reward behaviors in a time-locked and reversible manner in vivo. These results demonstrate that PPO fills a significant gap in the neuroscience toolkit for rapid and reversible synaptic inhibition and has broad utility for spatiotemporal control of inhibitory GPCR signaling cascades.

Pumilio2 and Staufen2 selectively balance the synaptic proteome.

Neurons have the capacity to adapt to environmental stimuli, a phenomenon termed cellular plasticity. The underlying processes are controlled by a network of RNA-binding proteins (RBPs). Their precise impact, however, is largely unknown. To address this important question, we chose Pumilio2 (Pum2) and Staufen2 (Stau2), which both regulate synaptic transmission. Surprisingly, even though both RBPs dynamically interact with each other in neurons, their respective impact on the transcriptome and proteome is highly selective. Although Pum2 deficiency leads to reduced translation and protein expression, Stau2 depletion preferentially impacts RNA levels and increases protein abundance. Furthermore, we show that Pum2 activates expression of key GABAergic synaptic components, e.g., the GABAA receptor scaffold protein Gephyrin. Consequently, Pum2 depletion selectively reduced the amplitude of miniature inhibitory postsynaptic currents. Together, our data argue for an important role of RBPs to maintain proteostasis in order to control distinct aspects of synaptic transmission.

Molecular Tools for Targeted Control of Nerve Cell Electrical Activity. Part I.

In modern life sciences, the issue of a specific, exogenously directed manipulation of a cell's biochemistry is a highly topical one. In the case of electrically excitable cells, the aim of the manipulation is to control the cells' electrical activity, with the result being either excitation with subsequent generation of an action potential or inhibition and suppression of the excitatory currents. The techniques of electrical activity stimulation are of particular significance in tackling the most challenging basic problem: figuring out how the nervous system of higher multicellular organisms functions. At this juncture, when neuroscience is gradually abandoning the reductionist approach in favor of the direct investigation of complex neuronal systems, minimally invasive methods for brain tissue stimulation are becoming the basic element in the toolbox of those involved in the field. In this review, we describe three approaches that are based on the delivery of exogenous, genetically encoded molecules sensitive to external stimuli into the nervous tissue. These approaches include optogenetics (Part I) as well as chemogenetics and thermogenetics (Part II), which are significantly different not only in the nature of the stimuli and structure of the appropriate effector proteins, but also in the details of experimental applications. The latter circumstance is an indication that these are rather complementary than competing techniques.

Potential Application of Optogenetic Stimulation in the Treatment of Pain and Migraine Headache: A Perspective from Animal Studies.

Optogenetic manipulation is uniquely useful in unraveling the functional organization of neuronal circuits in the central nervous system by enabling reversible gain- or loss-of-function of discrete populations of neurons within restricted brain regions. This state-of-the-art technology can produce circuit-specific neuromodulation by overexpressing light-sensitive proteins (opsins) in particular cell types of interest. Here, we discuss the principle of optogenetic manipulation and its application in pain research using animal models, and we also discuss how to potentially use optogenetic stimulation in the treatment of migraine headache in the future.

Adaptive sparse coding based on memristive neural network with applications.

Memristor is a nanoscale circuit element with nonvolatile, binary, multilevel and analog states. Its conductance (resistance) plasticity is similar to biological synapses. Information sparse coding is considered as the key mechanism of biological neural systems to process mass complex perception data, which is applied in the fields of signal processing, computer vision and so on. This paper proposes a soft-threshold adaptive sparse coding algorithm named MMN-SLCA based on the memristor, neural network and sparse coding theory. Specifically, the memristor crossbar array is used to realize the dictionary set. And by leveraging its unique vector-matrix operation advantages and biological synaptic characteristic, two key compositions of the sparse coding, namely, pattern matching and lateral neuronal inhibition are realized conveniently and efficiently. Besides, threshold variability further enhances the adaptive ability of the intelligent sparse coding. Furthermore, a hardware implementation framework of the sparse coding algorithm is designed to provide feasible solutions for hardware acceleration, real-time processing and embedded applications. Finally, the application of MMN-SLCA in image super-resolution reconstruction is discussed. Experimental simulations and result analysis verify the effectiveness of the proposed scheme and show its superior potentials in large-scale low-power intelligent information coding and processing.

Extending the Time Domain of Neuronal Silencing with Cryptophyte Anion Channelrhodopsins.

Optogenetic inhibition of specific neuronal types in the brain enables analysis of neural circuitry and is promising for the treatment of a number of neurological disorders. Anion channelrhodopsins (ACRs) from the cryptophyte alga Guillardia theta generate larger photocurrents than other available inhibitory optogenetic tools, but more rapid channels are needed for temporally precise inhibition, such as single-spike suppression, of high-frequency firing neurons. Faster ACRs have been reported, but their potential advantages for time-resolved inhibitory optogenetics have not so far been verified in neurons. We report RapACR, nicknamed so for "rapid," an ACR from Rhodomonas salina, that exhibits channel half-closing times below 10 ms and achieves equivalent inhibition at 50-fold lower light intensity in lentivirally transduced cultured mouse hippocampal neurons as the second-generation engineered Cl--conducting channelrhodopsin iC++. The upper limit of the time resolution of neuronal silencing with RapACR determined by measuring the dependence of spiking recovery after photoinhibition on the light intensity was calculated to be 100 Hz, whereas that with the faster of the two G. theta ACRs was 13 Hz. Further acceleration of RapACR channel kinetics was achieved by site-directed mutagenesis of a single residue in transmembrane helix 3 (Thr111 to Cys). We also show that mutation of another ACR (Cys to Ala at the same position) with a greatly extended lifetime of the channel open state acts as a bistable photochromic tool in mammalian neurons. These molecules extend the time domain of optogenetic neuronal silencing while retaining the high light sensitivity of Guillardia ACRs.

Implications of GABAergic Neurotransmission in Alzheimer's Disease.

Alzheimer's disease (AD) is characterized pathologically by the deposition of ß-amyloid peptides (Aß) and the accumulation of neurofibrillary tangles (NFTs) composed of hyper-phosphorylated tau. Regardless of the pathological hallmarks, synaptic dysfunction is widely accepted as a causal event in AD. Of the two major types of synapses in the central nervous system (CNS): glutamatergic and GABAergic, which provide excitatory and inhibitory outputs respectively, abundant data implicate an impaired glutamatergic system during disease progression. However, emerging evidence supports the notion that disrupted default neuronal network underlies impaired memory, and that alterations of GABAergic circuits, either plays a primary role or as a compensatory response to excitotoxicity, may also contribute to AD by disrupting the overall network function. The goal of this review is to provide an overview of the involvement of Aß, tau and apolipoprotein E4 (apoE4), the major genetic risk factor in late-onset AD (LOAD), in GABAergic neurotransmission and the potential of modulating the GABAergic function as AD therapy.

Pathophysiological aspects of diversity in neuronal inhibition: a new benzodiazepine pharmacology.

Inhibitory interneurons in the brain provide the balance to excitatory signaling. On the basis of brain imaging and human genetics, a deficit in GABAergic inhibition (GABA, γ-aminobuiyric acid) has been identified as contributing to the pathophysiology of anxiety disorders, epilepsy, and schizophrenia. Therapeutically, GABA(A) receptors play a major role as targets for benzodiazepine drugs. The therapeutic relevance of the multitude of structurally diverse GABA(A) receptor subtypes has only recently been identified. α(1)-GABA(A) receptors were found to mediate sedation, anterograde amnesia, and part of the seizure protection of these drugs, whereas α(2)-GABA(A) receptors, but not α(3)-GABA(A) receptors, mediate anxiolysis. Rational drug targeting to specific receptor subtypes has now become possible. Only restricted neuronal networks will be modulated by the upcoming subtype-selective drugs. For instance, anxiolytics devoid of drowsiness and sedation promise more sophisticated interventions in anxiety disorders. A new pharmacology of the benzodiazepine site is on the horizon.