The development of the living acute brain slice preparation for analyzing synaptic function roughly a half century ago was a pivotal achievement that greatly influenced the landscape of modern ...neuroscience. Indeed, many neuroscientists regard brain slices as the gold-standard model system for detailed cellular, molecular, and circuitry level analysis and perturbation of neuronal function. A critical limitation of this model system is the difficulty in preparing slices from adult and aging animals, and over the past several decades few substantial methodological improvements have emerged to facilitate patch clamp analysis in the mature adult stage. In this chapter we describe a robust and practical protocol for preparing brain slices from mature adult mice that are suitable for patch clamp analysis. This method reduces swelling and damage in superficial layers of the slices and improves the success rate for targeted patch clamp recordings, including recordings from fluorescently labeled populations in slices derived from transgenic mice. This adult brain slice method is suitable for diverse experimental applications, including both monitoring and manipulating neuronal activity with genetically encoded calcium indicators and optogenetic actuators, respectively. We describe the application of this adult brain slice platform and associated methods for screening kinetic properties of Channelrhodopsin (ChR) variants expressed in genetically defined neuronal subtypes.
Interneurons expressing cholecystokinin (CCK) and parvalbumin (PV) constitute two key GABAergic controllers of hippocampal pyramidal cell output. Although the temporally precise and millisecond-scale ...inhibitory regulation of neuronal ensembles delivered by PV interneurons is well established, the in vivo recruitment patterns of CCK-expressing basket cell (BC) populations has remained unknown. We show in the CA1 of the mouse hippocampus that the activity of CCK BCs inversely scales with both PV and pyramidal cell activity at the behaviorally relevant timescales of seconds. Intervention experiments indicated that the inverse coupling of CCK and PV GABAergic systems arises through a mechanism involving powerful inhibitory control of CCK BCs by PV cells. The tightly coupled complementarity of two key microcircuit regulatory modules demonstrates a novel form of brain-state-specific segregation of inhibition during spontaneous behavior.
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•CA1 CCK basket cells are selectively labeled in the Sncg-Flp mouse line•CCK basket and PV cells display complementary activity during spontaneous behaviors•PV cells inhibit CCK basket cells in vivo•CCK basket cell activity inversely scales with the pyramidal cell ensemble activity
Dudok et al. show a complementary perisomatic inhibitory system consisting of CCK- and PV-expressing interneurons that are active at distinct times in an alternating fashion during spontaneous behaviors. Due to PV to CCK inhibition, CCK basket cells are the most active when PV and pyramidal cells are relatively silent.
G protein-coupled receptor kinases (GRKs) regulate numerous G protein-coupled receptors (GPCRs) by phosphorylating the intracellular domain of the active receptor, resulting in receptor ...desensitization and internalization. GRKs also regulate GPCR trafficking in a phosphorylation-independent manner via direct protein–protein interactions. Emerging evidence suggests that GRK2, the most widely studied member of this family of kinases, modulates multiple cellular responses in various physiological contexts by either phosphorylating non-receptor substrates or interacting directly with signaling molecules. In this review, we discuss traditional and newly discovered roles of GRK2 in receptor internalization and signaling as well as its impact on non-receptor substrates. We also discuss novel exciting roles of GRK2 in the regulation of dopamine receptor signaling and in the activation and trafficking of the atypical GPCR, Smoothened (Smo).
To better examine circuit mechanisms underlying perception and behavior, researchers need tools to enable temporally precise control of action-potential generation of individual cells from neuronal ...ensembles. Here we demonstrate that such precision can be achieved with two-photon (2P) temporally focused computer-generated holography to control neuronal excitability at the supragranular layers of anesthetized and awake visual cortex in both male and female mice. Using 2P-guided whole-cell or cell-attached recordings in positive neurons expressing any of the three opsins ReaChR, CoChR, or ChrimsonR, we investigated the dependence of spiking activity on the opsin's channel kinetics. We found that in all cases the use of brief illumination (≤10 ms) induces spikes of millisecond temporal resolution and submillisecond precision, which were preserved upon repetitive illuminations up to tens of hertz. To reach high temporal precision, we used a large illumination spot covering the entire cell body and an amplified laser at high peak power and low excitation intensity (on average ≤0.2 mW/μm
), thus minimizing the risk for nonlinear photodamage effects. Finally, by combining 2P holographic excitation with electrophysiological recordings and calcium imaging using GCaMP6s, we investigated the factors, including illumination shape and intensity, opsin distribution in the target cell, and cell morphology, which affect the spatial selectivity of single-cell and multicell holographic activation. Parallel optical control of neuronal activity with cellular resolution and millisecond temporal precision should make it easier to investigate neuronal connections and find further links between connectivity, microcircuit dynamics, and brain functions.
Recent developments in the field of optogenetics has enabled researchers to probe the neuronal microcircuit with light by optically actuating genetically encoded light-sensitive opsins expressed in the target cells. Here, we applied holographic light shaping and temporal focusing to simultaneously deliver axially confined holographic patterns to opsin-positive cells in the living mouse cortex. Parallel illumination efficiently induced action potentials with high temporal resolution and precision for three opsins of different kinetics. We extended the parallel optogenetic activation at low intensity to multiple neurons and concurrently monitored their calcium dynamics. These results demonstrate fast and temporally precise
control of a neuronal subpopulation, opening new opportunities for revealing circuit mechanisms underlying brain functions.
In the neocortex, subcerebral axonal projections originate largely from layer 5 (L5) extratelencephalic-projecting (ET) neurons. The unique morpho-electric properties of these neurons have been ...mainly described in rodents, where retrograde tracers or transgenic lines can label them. Similar labeling strategies are infeasible in the human neocortex, rendering the translational relevance of findings in rodents unclear. We leveraged the recent discovery of a transcriptomically defined L5 ET neuron type to study the properties of human L5 ET neurons in neocortical brain slices derived from neurosurgeries. Patch-seq recordings, where transcriptome, physiology, and morphology were assayed from the same cell, revealed many conserved morpho-electric properties of human and rodent L5 ET neurons. Divergent properties were often subtler than differences between L5 cell types within these two species. These data suggest a conserved function of L5 ET neurons in the neocortical hierarchy but also highlight phenotypic divergence possibly related to functional specialization of human neocortex.
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•Species differences in the relative density of L5 ET neurons: mouse > macaque > human•Correspondence between physiological and transcriptomic definition of L5 classes•Human L5 ET dendrites display electrogenesis during direct electrical recordings•Many conserved and divergent properties of human and rodent L5 ET neurons
Transcriptomics identifies a human L5 neuron corresponding to extratelencephalic projecting neurons in mouse neocortex. Patch-seq recordings from this cell type reveal many conserved and divergent morpho-electric features of L5 ET neurons in human cortex.
Parkinson’s disease (PD) is characterized by severe locomotor deficits and is commonly treated with the dopamine (DA) precursor l -3,4-dihydroxyphenylalanine ( l -DOPA), but its prolonged use causes ...dyskinesias referred to as l -DOPA–induced dyskinesias (LIDs). Recent studies in animal models of PD have suggested that dyskinesias are associated with the overactivation of G protein-mediated signaling through DA receptors. β-Arrestins desensitize G protein signaling at DA receptors (D1R and D2R) in addition to activating their own G protein-independent signaling events, which have been shown to mediate locomotion. Therefore, targeting β-arrestins in PD l -DOPA therapy might prove to be a desirable approach. Here we show in a bilateral DA-depletion mouse model of Parkinson’s symptoms that genetic deletion of β-arrestin2 significantly limits the beneficial locomotor effects while markedly enhancing the dyskinesia-like effects of acute or chronic l -DOPA treatment. Viral rescue or overexpression of β-arrestin2 in knockout or control mice either reverses or protects against LIDs and its key biochemical markers. In other more conventional animal models of DA neuron loss and PD, such as 6-hydroxydopamine–treated mice or rats and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine–treated nonhuman primates, β-arrestin2 overexpression significantly reduced dyskinesias while maintaining the therapeutic effect of l -DOPA. Considerable efforts are being spent in the pharmaceutical industry to identify therapeutic approaches to block LIDs in patients with PD. Our results point to a potential therapeutic approach, whereby development of either a genetic or pharmacological intervention to enhance β-arrestin2- or limit G protein-dependent D1/D2R signaling could represent a more mechanistically informed strategy.
Significance β-Arrestins are unique proteins that have multiple cellular functions such as G protein-coupled receptor signal desensitization, protein trafficking and signaling molecule scaffolding. Treatment of Parkinson’s disease (PD) motor symptoms by l -3,4-dihydroxyphenylalanine ( l -DOPA) has been hampered by abnormal involuntary movements or dyskinetic side effects. The cause of these dyskinesias has been attributed to receptor supersensitivity and uncontrolled neuronal excitability. Here we demonstrate in multiple preclinical models of l -DOPA–induced dyskinesias and PD that expression levels of β-arrestin2 can alter manifestation of these dyskinesias by reducing receptor supersensitivity while maintaining the therapeutic effect of l -DOPA. Thus novel drugs that increase β-arrestin–dependent function at dopamine receptors may be useful in ameliorating PD motor symptoms without inducing dyskinesias.
Visual spatial perception in the mammalian brain occurs through two parallel pathways: one reaches the primary visual cortex (V1) through the thalamus and another the superior colliculus (SC) via ...direct projections from the retina. The origin, development, and relative function of these two evolutionarily distinct pathways remain obscure. We examined the early functional development of both pathways by simultaneously imaging pre- and post-synaptic spontaneous neuronal activity. We observed that the quality of retinal activity transfer to the thalamus and superior colliculus does not change across the first two postnatal weeks. However, beginning in the second postnatal week, retinal activity does not drive V1 as strongly as earlier wave activity, suggesting that intrinsic cortical activity competes with signals from the sensory periphery as the cortex matures. Together, these findings bring new insight into the function of the SC and V1 and the role of peripheral activity in driving both circuits across development.
•Second week (“stage 3”) retinal waves are very distinct from first week (“stage 2”)•Stage 3 waves are especially sensitive to inhibitory blockade•Retinal influence on the colliculus and thalamus does not change over development•However, the cortex becomes relatively insensitive to retinal drive before eye opening
By simultaneously measuring input activity and output activity, Gribizis et al. show that peripheral spontaneous retinal activity consistently and strongly drives response in the midbrain superior colliculus during development, while the cortex becomes progressively less sensitive to peripheral drive.
Modern genetic approaches are powerful in providing access to diverse cell types in the brain and facilitating the study of their function. Here, we report a large set of driver and reporter ...transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first-generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of high-precision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.
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•23 new driver lines and 26 new reporter lines for a wide range of applications•TIGRE2.0 reporters have viral-like transgene expression level in diverse cell types•New calcium- or voltage-sensing reporters with the former functionally characterized•Comparative analysis of new optogenetic effectors with complementary properties
An expanded toolkit of transgenic mouse lines for exploring the organization, function, and development of mammalian neural circuits.
Brain circuits comprise vast numbers of interconnected neurons with diverse molecular, anatomical and physiological properties. To allow targeting of individual neurons for structural and functional ...studies, we created light-inducible site-specific DNA recombinases based on Cre, Dre and Flp (RecVs). RecVs can induce genomic modifications by one-photon or two-photon light induction in vivo. They can produce targeted, sparse and strong labeling of individual neurons by modifying multiple loci within mouse and zebrafish genomes. In combination with other genetic strategies, they allow intersectional targeting of different neuronal classes. In the mouse cortex they enable sparse labeling and whole-brain morphological reconstructions of individual neurons. Furthermore, these enzymes allow single-cell two-photon targeted genetic modifications and can be used in combination with functional optical indicators with minimal interference. In summary, RecVs enable spatiotemporally precise optogenomic modifications that can facilitate detailed single-cell analysis of neural circuits by linking genetic identity, morphology, connectivity and function.
Determining which features of the neural code drive behavior requires the ability to simultaneously read out and write in neural activity patterns with high precision across many neurons. All-optical ...systems that combine two-photon calcium imaging and targeted photostimulation enable the activation of specific, functionally defined groups of neurons. However, these techniques are unable to test how patterns of activity across a population contribute to computation because of an inability to both read and write cell-specific firing rates. To overcome this challenge, we make two advances: first, we introduce a genetic line of mice for Cre-dependent co-expression of a calcium indicator and a potent soma-targeted microbial opsin. Second, using this line, we develop a method for read-out and write-in of precise population vectors of neural activity by calibrating the photostimulation to each cell. These advances offer a powerful and convenient platform for investigating the neural codes of computation and behavior.
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•Ai203 line expresses soma-targeted ChroME opsin and GCaMP7s for all-optical studies•All-optical calibration of stimulation power per cell enables control of spike rates•Population activity vectors can be recreated on demand using this procedure
Bounds et al. develop and validate a TIGRE2 transgenic line that co-expresses an opsin and calcium indicator, facilitating two-photon all-optical read/write experiments. In this line, the authors calibrate holographic photostimulation to each targeted cell to accurately recreate visually evoked spike rates across a population of neurons.
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