Optogenetic and chemogenetic manipulation of seizure threshold in mice.

Here, we present a protocol using optogenetics or chemogenetics to assess the neuronal circuits contributing to seizure initiation. Both approaches allow for targeted control of neuronal populations in vivo and can be combined with experimental manipulations to acutely induce seizures in rodent models. We describe how to (1) introduce and (2) activate optogenetic or chemogenetic actuators while (3) inducing seizures via hyperthermia in a mouse model of epilepsy. This protocol can be adapted for use in other induced seizure models. For complete details on the use and execution of this protocol, please refer to Mattis et al. (2022).1.

Corticohippocampal circuit dysfunction in a mouse model of Dravet syndrome.

Dravet syndrome (DS) is a neurodevelopmental disorder due to pathogenic variants in SCN1A encoding the Nav1.1 sodium channel subunit, characterized by treatment-resistant epilepsy, temperature-sensitive seizures, developmental delay/intellectual disability with features of autism spectrum disorder, and increased risk of sudden death. Convergent data suggest hippocampal dentate gyrus (DG) pathology in DS (Scn1a+/-) mice. We performed two-photon calcium imaging in brain slice to uncover a profound dysfunction of filtering of perforant path input by DG in young adult Scn1a+/- mice. This was not due to dysfunction of DG parvalbumin inhibitory interneurons (PV-INs), which were only mildly impaired at this timepoint; however, we identified enhanced excitatory input to granule cells, suggesting that circuit dysfunction is due to excessive excitation rather than impaired inhibition. We confirmed that both optogenetic stimulation of entorhinal cortex and selective chemogenetic inhibition of DG PV-INs lowered seizure threshold in vivo in young adult Scn1a+/- mice. Optogenetic activation of PV-INs, on the other hand, normalized evoked responses in granule cells in vitro. These results establish the corticohippocampal circuit as a key locus of pathology in Scn1a+/- mice and suggest that PV-INs retain powerful inhibitory function and may be harnessed as a potential therapeutic approach toward seizure modulation.

Reciprocal Lateral Hypothalamic and Raphe GABAergic Projections Promote Wakefulness.

The lateral hypothalamus (LH), together with multiple neuromodulatory systems of the brain, such as the dorsal raphe nucleus (DR), is implicated in arousal, yet interactions between these systems are just beginning to be explored. Using a combination of viral tracing, circuit mapping, electrophysiological recordings from identified neurons, and combinatorial optogenetics in mice, we show that GABAergic neurons in the LH selectively inhibit GABAergic neurons in the DR, resulting in increased firing of a substantial fraction of its neurons that ultimately promotes arousal. These DRGABA neurons are wake active and project to multiple brain areas involved in the control of arousal, including the LH, where their specific activation potently influences local network activity leading to arousal from sleep. Our results show how mutual inhibitory projections between the LH and the DR promote wakefulness and suggest a complex arousal control by intimate interactions between long-range connections and local circuit dynamics.SIGNIFICANCE STATEMENT: Multiple brain systems including the lateral hypothalamus and raphe serotonergic system are involved in the regulation of the sleep/wake cycle, yet the interaction between these systems have remained elusive. Here we show that mutual disinhibition mediated by long range inhibitory projections between these brain areas can promote wakefulness. The main importance of this work relies in revealing the interaction between a brain area involved in autonomic regulation and another in controlling higher brain functions including reward, patience, mood and sensory coding.

A Guide to Creating and Testing New INTRSECT Constructs.

As the power of genetically encoded interventional and observational tools for neuroscience expands, the boundaries of experimental design are increasingly defined by limits in selectively expressing these tools in relevant cell types. Single-recombinase-dependent expression systems have been widely used as a means to restrict gene expression based on single features by combining recombinase-dependent viruses with recombinase-expressing transgenic animals. This protocol details how to create INTRSECT constructs and use multiple recombinases to achieve targeting of a desired gene to subsets of neurons that are defined by multiple genetic and/or topological features. This method includes the design and utilization of both viruses and transgenic animals: these tools are inherently flexible and modular and may be used in different combinations to achieve the desired gene expression pattern. © 2017 by John Wiley & Sons, Inc.

LSPS/Optogenetics to Improve Synaptic Connectivity Mapping: Unmasking the Role of Basket Cell-Mediated Feedforward Inhibition.

Neocortical pyramidal cells (PYRs) receive synaptic inputs from many types of GABAergic interneurons. Connections between parvalbumin (PV)-positive, fast-spiking interneurons ("PV cells") and PYRs are characterized by perisomatic synapses and high-amplitude, short-latency IPSCs. Here, we present novel methods to study the functional influence of PV cells on layer 5 PYRs using optogenetics combined with laser-scanning photostimulation (LSPS). First, we examined the strength and spatial distribution of PV-to-PYR inputs. To that end, the fast channelrhodopsin variant AAV5-EF1α-DIO-hChR2(E123T)-eYFP (ChETA) was expressed in PV cells in somatosensory cortex of mice using an adeno-associated virus-based viral construct. Focal blue illumination (100-150 µm half-width) was directed through the microscope objective to excite PV cells along a spatial grid covering layers 2-6, while IPSCs were recorded in layer 5 PYRs. The resulting optogenetic input maps showed evoked PV cell inputs originating from an ∼500-µm-diameter area surrounding the recorded PYR. Evoked IPSCs had the short-latency/high-amplitude characteristic of PV cell inputs. Second, we investigated how PV cell activity modulates PYR output in response to synaptic excitation. We expressed halorhodopsin (eNpHR3.0) in PV cells using the same strategy as for ChETA. Yellow illumination hyperpolarized eNpHR3.0-expressing PV cells, effectively preventing action potential generation and thus decreasing the inhibition of downstream targets. Synaptic input maps onto layer 5 PYRs were acquired using standard glutamate-photolysis LSPS either with or without full-field yellow illumination to silence PV cells. The resulting IPSC input maps selectively lacked short-latency perisomatic inputs, while EPSC input maps showed increased connectivity, particularly from upper layers. This indicates that glutamate uncaging LSPS-based excitatory synaptic maps will consistently underestimate connectivity.

Hilar somatostatin interneuron loss reduces dentate gyrus inhibition in a mouse model of temporal lobe epilepsy.

OBJECTIVE: In patients with temporal lobe epilepsy, seizures usually start in the hippocampus, and dentate granule cells are hyperexcitable. Somatostatin interneurons are a major subpopulation of inhibitory neurons in the dentate gyrus, and many are lost in patients and animal models. However, surviving somatostatin interneurons sprout axon collaterals and form new synapses, so the net effect on granule cell inhibition remains unclear. METHODS: The present study uses optogenetics to activate hilar somatostatin interneurons and measure the inhibitory effect on dentate gyrus perforant path-evoked local field potential responses in a mouse model of temporal lobe epilepsy. RESULTS: In controls, light activation of hilar somatostatin interneurons inhibited evoked responses up to 40%. Epileptic pilocarpine-treated mice exhibited loss of hilar somatostatin interneurons and less light-induced inhibition of evoked responses. SIGNIFICANCE: These findings suggest that severe epilepsy-related loss of hilar somatostatin interneurons can overwhelm the surviving interneurons' capacity to compensate by sprouting axon collaterals.

Optogenetic approaches addressing extracellular modulation of neural excitability.

The extracellular ionic environment in neural tissue has the capacity to influence, and be influenced by, natural bouts of neural activity. We employed optogenetic approaches to control and investigate these interactions within and between cells, and across spatial scales. We began by developing a temporally precise means to study microdomain-scale interactions between extracellular protons and acid-sensing ion channels (ASICs). By coupling single-component proton-transporting optogenetic tools to ASICs to create two-component optogenetic constructs (TCOs), we found that acidification of the local extracellular membrane surface by a light-activated proton pump recruited a slow inward ASIC current, which required molecular proximity of the two components on the membrane. To elicit more global effects of activity modulation on 'bystander' neurons not under direct control, we used densely-expressed depolarizing (ChR2) or hyperpolarizing (eArch3.0, eNpHR3.0) tools to create a slow non-synaptic membrane current in bystander neurons, which matched the current direction seen in the directly modulated neurons. Extracellular protons played contributory role but were insufficient to explain the entire bystander effect, suggesting the recruitment of other mechanisms. Together, these findings present a new approach to the engineering of multicomponent optogenetic tools to manipulate ionic microdomains, and probe the complex neuronal-extracellular space interactions that regulate neural excitability.

Circadian Rhythms, Sleep, and Disorders of Aging.

Sleep-wake cycles are known to be disrupted in people with neurodegenerative disorders. These findings are now supported by data from animal models for some of these disorders, raising the question of whether the disrupted sleep/circadian regulation contributes to the loss of neural function. As circadian rhythms and sleep consolidation also break down with normal aging, changes in these may be part of what makes aging a risk factor for disorders like Alzheimer's disease (AD). Mechanisms underlying the connection between circadian/sleep dysregulation and neurodegeneration remain unclear, but several recent studies provide interesting possibilities. While mechanistic analysis is under way, it is worth considering treatment of circadian/sleep disruption as a means to alleviate symptoms of neurodegenerative disorders.

Frequency-dependent, cell type-divergent signaling in the hippocamposeptal projection.

Hippocampal oscillations are critical for information processing, and are strongly influenced by inputs from the medial septum. Hippocamposeptal neurons provide direct inhibitory feedback from the hippocampus onto septal cells, and are therefore likely to also play an important role in the circuit; these neurons fire at either low or high frequency, reflecting hippocampal network activity during theta oscillations or ripple events, respectively. Here, we optogenetically target the long-range GABAergic projection from the hippocampus to the medial septum in rats, and thereby simulate hippocampal input onto downstream septal cells in an acute slice preparation. In response to optogenetic activation of hippocamposeptal fibers at theta and ripple frequencies, we elicit postsynaptic GABAergic responses in a subset (24%) of septal cells, most predominantly in fast-spiking cells. In addition, in another subset of septal cells (19%) corresponding primarily to cholinergic cells, we observe a slow hyperpolarization of the resting membrane potential and a decrease in input resistance, particularly in response to prolonged high-frequency (ripple range) stimulation. This slow response is partially sensitive to GIRK channel and D2 dopamine receptor block. Our results suggest that two independent populations of septal cells distinctly encode hippocampal feedback, enabling the septum to monitor ongoing patterns of activity in the hippocampus.

Targeting cells with single vectors using multiple-feature Boolean logic.

Precisely defining the roles of specific cell types is an intriguing frontier in the study of intact biological systems and has stimulated the rapid development of genetically encoded tools for observation and control. However, targeting these tools with adequate specificity remains challenging: most cell types are best defined by the intersection of two or more features such as active promoter elements, location and connectivity. Here we have combined engineered introns with specific recombinases to achieve expression of genetically encoded tools that is conditional upon multiple cell-type features, using Boolean logical operations all governed by a single versatile vector. We used this approach to target intersectionally specified populations of inhibitory interneurons in mammalian hippocampus and neurons of the ventral tegmental area defined by both genetic and wiring properties. This flexible and modular approach may expand the application of genetically encoded interventional and observational tools for intact-systems biology.

A unique population of ventral tegmental area neurons inhibits the lateral habenula to promote reward.

Lateral habenula (LHb) neurons convey aversive and negative reward conditions through potent indirect inhibition of ventral tegmental area (VTA) dopaminergic neurons. Although VTA dopaminergic neurons reciprocally project to the LHb, the electrophysiological properties and the behavioral consequences associated with selective manipulations of this circuit are unknown. Here, we identify an inhibitory input to the LHb arising from a unique population of VTA neurons expressing dopaminergic markers. Optogenetic activation of this circuit resulted in no detectable dopamine release in LHb brain slices. Instead, stimulation produced GABA-mediated inhibitory synaptic transmission, which suppressed the firing of postsynaptic LHb neurons in brain slices and increased the spontaneous firing rate of VTA dopaminergic neurons in vivo. Furthermore, in vivo activation of this pathway produced reward-related phenotypes that were dependent on intra-LHb GABAA receptor signaling. These results suggest that noncanonical inhibitory signaling by these hybrid dopaminergic-GABAergic neurons act to suppress LHb output under rewarding conditions.

Structural and molecular interrogation of intact biological systems.

Obtaining high-resolution information from a complex system, while maintaining the global perspective needed to understand system function, represents a key challenge in biology. Here we address this challenge with a method (termed CLARITY) for the transformation of intact tissue into a nanoporous hydrogel-hybridized form (crosslinked to a three-dimensional network of hydrophilic polymers) that is fully assembled but optically transparent and macromolecule-permeable. Using mouse brains, we show intact-tissue imaging of long-range projections, local circuit wiring, cellular relationships, subcellular structures, protein complexes, nucleic acids and neurotransmitters. CLARITY also enables intact-tissue in situ hybridization, immunohistochemistry with multiple rounds of staining and de-staining in non-sectioned tissue, and antibody labelling throughout the intact adult mouse brain. Finally, we show that CLARITY enables fine structural analysis of clinical samples, including non-sectioned human tissue from a neuropsychiatric-disease setting, establishing a path for the transmutation of human tissue into a stable, intact and accessible form suitable for probing structural and molecular underpinnings of physiological function and disease.

Diverging neural pathways assemble a behavioural state from separable features in anxiety.

Behavioural states in mammals, such as the anxious state, are characterized by several features that are coordinately regulated by diverse nervous system outputs, ranging from behavioural choice patterns to changes in physiology (in anxiety, exemplified respectively by risk-avoidance and respiratory rate alterations). Here we investigate if and how defined neural projections arising from a single coordinating brain region in mice could mediate diverse features of anxiety. Integrating behavioural assays, in vivo and in vitro electrophysiology, respiratory physiology and optogenetics, we identify a surprising new role for the bed nucleus of the stria terminalis (BNST) in the coordinated modulation of diverse anxiety features. First, two BNST subregions were unexpectedly found to exert opposite effects on the anxious state: oval BNST activity promoted several independent anxious state features, whereas anterodorsal BNST-associated activity exerted anxiolytic influence for the same features. Notably, we found that three distinct anterodorsal BNST efferent projections-to the lateral hypothalamus, parabrachial nucleus and ventral tegmental area-each implemented an independent feature of anxiolysis: reduced risk-avoidance, reduced respiratory rate, and increased positive valence, respectively. Furthermore, selective inhibition of corresponding circuit elements in freely moving mice showed opposing behavioural effects compared with excitation, and in vivo recordings during free behaviour showed native spiking patterns in anterodorsal BNST neurons that differentiated safe and anxiogenic environments. These results demonstrate that distinct BNST subregions exert opposite effects in modulating anxiety, establish separable anxiolytic roles for different anterodorsal BNST projections, and illustrate circuit mechanisms underlying selection of features for the assembly of the anxious state.

Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins.

Diverse optogenetic tools have allowed versatile control over neural activity. Many depolarizing and hyperpolarizing tools have now been developed in multiple laboratories and tested across different preparations, presenting opportunities but also making it difficult to draw direct comparisons. This challenge has been compounded by the dependence of performance on parameters such as vector, promoter, expression time, illumination, cell type and many other variables. As a result, it has become increasingly complicated for end users to select the optimal reagents for their experimental needs. For a rapidly growing field, critical figures of merit should be formalized both to establish a framework for further development and so that end users can readily understand how these standardized parameters translate into performance. Here we systematically compared microbial opsins under matched experimental conditions to extract essential principles and identify key parameters for the conduct, design and interpretation of experiments involving optogenetic techniques.

High-efficiency channelrhodopsins for fast neuronal stimulation at low light levels.

Channelrhodopsin-2 (ChR2) has become an indispensable tool in neuroscience, allowing precise induction of action potentials with short light pulses. A limiting factor for many optophysiological experiments is the relatively small photocurrent induced by ChR2. We screened a large number of ChR2 point mutants and discovered a dramatic increase in photocurrent amplitude after threonine-to-cysteine substitution at position 159. When we tested the T159C mutant in hippocampal pyramidal neurons, action potentials could be induced at very low light intensities, where currently available channelrhodopsins were unable to drive spiking. Biophysical characterization revealed that the kinetics of most ChR2 variants slows down considerably at depolarized membrane potentials. We show that the recently published E123T substitution abolishes this voltage sensitivity and speeds up channel kinetics. When we combined T159C with E123T, the resulting double mutant delivered fast photocurrents with large amplitudes and increased the precision of single action potential induction over a broad range of frequencies, suggesting it may become the standard for light-controlled activation of neurons.

Molecular and cellular approaches for diversifying and extending optogenetics.

Optogenetic technologies employ light to control biological processes within targeted cells in vivo with high temporal precision. Here, we show that application of molecular trafficking principles can expand the optogenetic repertoire along several long-sought dimensions. Subcellular and transcellular trafficking strategies now permit (1) optical regulation at the far-red/infrared border and extension of optogenetic control across the entire visible spectrum, (2) increased potency of optical inhibition without increased light power requirement (nanoampere-scale chloride-mediated photocurrents that maintain the light sensitivity and reversible, step-like kinetic stability of earlier tools), and (3) generalizable strategies for targeting cells based not only on genetic identity, but also on morphology and tissue topology, to allow versatile targeting when promoters are not known or in genetically intractable organisms. Together, these results illustrate use of cell-biological principles to enable expansion of the versatile fast optogenetic technologies suitable for intact-systems biology and behavior.

Major vault protein is expressed along the nucleus-neurite axis and associates with mRNAs in cortical neurons.

Major Vault Protein (MVP), the main constituent of the vault ribonucleoprotein particle, is highly conserved in eukaryotic cells and upregulated in a variety of tumors. Vaults have been speculated to function as cargo transporters in several cell lines, yet no work to date has characterized the protein in neurons. Here we first describe the cellular and subcellular expression of MVP in primate and rodent cerebral cortex, and in cortical neurons in vitro. In prefrontal, somatosensory and hippocampal cortices, MVP was predominantly expressed in pyramidal neurons. Immunogold labeled free and attached ribosomes, and structures reminiscent of vaults on the rough endoplasmic reticulum and the nuclear envelope. The nucleus was immunoreactive in association with nucleopores. Axons and particularly principal dendrites expressed MVP along individual microtubules, and in pre- and postsynaptic structures. Synapses were not labeled. Colocalization with microtubule-associated protein-2, tubulin, tau, and phalloidin was observed in neurites and growth cones in culture. Immunoprecipitation coupled with reverse transcription PCR showed that MVP associates with mRNAs that are known to be translated in response to synaptic activity. Taken together, our findings provide the first characterization of neuronal MVP along the nucleus-neurite axis and may offer new insights into its possible function(s) in the brain.

Alternative splicing of Nav1.5: an electrophysiological comparison of 'neonatal' and 'adult' isoforms and critical involvement of a lysine residue.

In developmentally regulated D1:S3 splicing of Nav1.5, there are 31 nucleotide differences between the 5'-exon ('neonatal') and the 3'-exon ('adult') forms, resulting in 7 amino acid differences in D1:S3-S3/S4 linker. In particular, splicing replaces a conserved negative aspartate residue in the 'adult' with a positive lysine. Here, 'neonatal' and 'adult' Nav1.5 alpha-subunit splice variants were stably transfected into EBNA-293 cells and their electrophysiological properties investigated by whole-cell patch-clamp recording. Compared with the 'adult' isoform, the 'neonatal' channel exhibited (1) a depolarized threshold of activation and voltage at which the current peaked; (2) much slower kinetics of activation and inactivation; (3) 50% greater transient charge (Na(+)) influx; (4) a stronger voltage dependence of time to peak; and (5) a slower recovery from inactivation. Tetrodotoxin sensitivity and VGSCbeta1-4 mRNA expression levels did not change. The significance of the charge-reversing aspartate to lysine substitution was investigated by mutating the lysine in the 'neonatal' channel back to aspartate. In this 'neonatal K211D' mutant, the electrophysiological parameters studied strongly shifted back towards the 'adult', that is the lysine residue was primarily responsible for the electrophysiological effects of Nav1.5 D1:S3 splicing. Taken together, these data suggest that the charge reversal in 'neonatal' Nav1.5 would (1) modify the channel kinetics and (2) prolong the resultant current, allowing greater intracellular Na(+) influx. Developmental and pathophysiological consequences of such differences are discussed.

Red-shifted optogenetic excitation: a tool for fast neural control derived from Volvox carteri.

The introduction of two microbial opsin-based tools, channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), to neuroscience has generated interest in fast, multimodal, cell type-specific neural circuit control. Here we describe a cation-conducting channelrhodopsin (VChR1) from Volvox carteri that can drive spiking at 589 nm, with excitation maximum red-shifted approximately 70 nm compared with ChR2. These results demonstrate fast photostimulation with yellow light, thereby defining a functionally distinct third category of microbial rhodopsin proteins.