Optogenetic determination of dynamic and cell-type-specific inhibitory reversal potentials.

The reversal potential refers to the membrane potential at which the net current flow through a channel reverses direction. The reversal potential is determined by transmembrane ion gradients and, in turn, determines how the channel's activity will affect the membrane potential. Traditional investigation into the reversal potential of inhibitory ligand-gated ion channels (EInh) has relied upon the activation of endogenous receptors, such as the GABA-A receptor (GABAAR). There are, however, challenges associated with activating endogenous receptors, including agonist delivery, isolating channel responses, and the effects of receptor saturation and desensitization. Here we demonstrate the utility of using a light-gated anion channel, stGtACR2, to probe EInh in the rodent brain. Using mice of both sexes, we demonstrate that the properties of this optically activated channel make it a suitable proxy for studying GABAAR receptor mediated inhibition. We validate this agonist-independent optogenetic strategy in vitro and in vivo, and further show how it can accurately capture differences in EInh dynamics following manipulations of endogenous ion fluxes. This allows us to explore distinct resting EInh differences across genetically-defined neuronal subpopulations. Using this approach to challenge ion homeostasis mechanisms in neurons, we uncover cell-specific EInh dynamics that are supported by the differential expression of endogenous ion handling mechanisms. Our findings therefore establish an effective optical strategy for revealing novel aspects of inhibitory reversal potentials, and thereby expand the repertoire of optogenetics.Significance statement The strength of synaptic inhibition in the brain is determined, in part, by the reversal potential of the ionic currents that flow through inhibitory ligand-gated ion channels (EInh). Estimates of EInh have traditionally used agonists to activate receptors on the cell surface, but this has limitations. Our study presents an optogenetic strategy for performing agonist-independent measurements of EInh in the brain. We demonstrate the effectiveness of the approach in vitro, in vivo, and across different neuronal subtypes. Its excellent temporal control allows for measurements of EInh dynamics, which reveal differences between genetically-defined neuronal subpopulations. This expands the application of optogenetics and affords new opportunities to study synaptic inhibition.

Immediate reuse of patch-clamp pipettes after ultrasonic cleaning.

The patch-clamp technique has revolutionized neurophysiology by allowing to study single neuronal excitability, synaptic connectivity, morphology, and the transcriptomic profile. However, the throughput in recordings is limited because of the manual replacement of patch-pipettes after each attempt which are often also unsuccessful. This has been overcome by automated cleaning the tips in detergent solutions, allowing to reuse the pipette for further recordings. Here, we developed a novel method of automated cleaning by sonicating the tips within the bath solution wherein the cells are placed, reducing the risk of contaminating the bath solution or internal solution of the recording pipette by any detergent and avoiding the necessity of a separate chamber for cleaning. We showed that the patch-pipettes can be used consecutively at least ten times and that the cleaning process does not negatively impact neither the brain slices nor other patched neurons. This method, combined with automated patch-clamp, highly improves the throughput for single and especially multiple recordings.

Dynamic conjugate F-SHARP microscopy.

Optical microscopy is an indispensable tool in biomedical sciences, but its reach in deep tissues is limited due to aberrations and scattering. This problem can be overcome by wavefront-shaping techniques, albeit at limited fields of view (FOVs). Inspired by astronomical imaging, conjugate wavefront shaping can lead to an increased field of view in microscopy, but this correction is limited to a set depth and cannot be dynamically adapted. Here, we present a conjugate wavefront-shaping scheme based on focus scanning holographic aberration probing (F-SHARP). We combine it with a compact implementation that can be readily adapted to a variety of commercial and home-built two-photon microscopes. We demonstrate the power of the method by imaging with high resolution over extended FOV (>80 µm) deeper than 400 µm inside a mouse brain through a thinned skull.

Multiple Two-Photon Targeted Whole-Cell Patch-Clamp Recordings From Monosynaptically Connected Neurons in vivo.

Although we know a great deal about monosynaptic connectivity, transmission and integration in the mammalian nervous system from in vitro studies, very little is known in vivo. This is partly because it is technically difficult to evoke action potentials and simultaneously record small amplitude subthreshold responses in closely (<150 µm) located pairs of neurons. To address this, we have developed in vivo two-photon targeted multiple (2-4) whole-cell patch clamp recordings of nearby neurons in superficial cortical layers 1-3. Here, we describe a step-by-step guide to this approach in the anesthetized mouse primary somatosensory cortex, including: the design of the setup, surgery, preparation of pipettes, targeting and acquisition of multiple whole-cell recordings, as well as in vivo and post hoc histology. The procedure takes ~4 h from start of surgery to end of recording and allows examinations both into the electrophysiological features of unitary excitatory and inhibitory monosynaptic inputs during different brain states as well as the synaptic mechanisms of correlated neuronal activity.

Dendrite-Specific Amplification of Weak Synaptic Input during Network Activity In Vivo.

Excitatory synaptic input reaches the soma of a cortical excitatory pyramidal neuron via anatomically segregated apical and basal dendrites. In vivo, dendritic inputs are integrated during depolarized network activity, but how network activity affects apical and basal inputs is not understood. Using subcellular two-photon stimulation of Channelrhodopsin2-expressing layer 2/3 pyramidal neurons in somatosensory cortex, nucleus-specific thalamic optogenetic stimulation, and paired recordings, we show that slow, depolarized network activity amplifies small-amplitude synaptic inputs targeted to basal dendrites but reduces the amplitude of all inputs from apical dendrites and the cell soma. Intracellular pharmacology and mathematical modeling suggests that the amplification of weak basal inputs is mediated by postsynaptic voltage-gated channels. Thus, network activity dynamically reconfigures the relative somatic contribution of apical and basal inputs and could act to enhance the detectability of weak synaptic inputs.

Single synaptic inputs drive high-precision action potentials in parvalbumin expressing GABA-ergic cortical neurons in vivo.

A defining feature of cortical layer 2/3 excitatory neurons is their sparse activity, often firing in singlets of action potentials. Local inhibitory neurons are thought to play a major role in regulating sparseness, but which cell types are recruited by single excitatory synaptic inputs is unknown. Using multiple, targeted, in vivo whole-cell recordings, we show that single uEPSPs have little effect on the firing rates of excitatory neurons and somatostatin-expressing GABA-ergic inhibitory neurons but evoke precisely timed action potentials in parvalbumin-expressing inhibitory neurons. Despite a uEPSP decay time of 7.8 ms, the evoked action potentials were almost completely restricted to the uEPSP rising phase (~0.5 ms). Evoked parvalbumin-expressing neuron action potentials go on to inhibit the local excitatory network, thus providing a pathway for single spike evoked disynaptic inhibition which may enforce sparse and precisely timed cortical signaling.

Precisely Timed Nicotinic Activation Drives SST Inhibition in Neocortical Circuits.

Sleep, waking, locomotion, and attention are associated with cell-type-specific changes in neocortical activity. The effect of brain state on circuit output requires understanding of how neuromodulators influence specific neuronal classes and their synapses, with normal patterns of neuromodulator release from endogenous sources. We investigated the state-dependent modulation of a ubiquitous feedforward inhibitory motif in mouse sensory cortex, local pyramidal (Pyr) inputs onto somatostatin (SST)-expressing interneurons. Paired whole-cell recordings in acute brain slices and in vivo showed that Pyr-to-SST synapses are remarkably weak, with failure rates approaching 80%. Pharmacological screening revealed that cholinergic agonists uniquely enhance synaptic efficacy. Brief, optogenetically gated acetylcholine release dramatically enhanced Pyr-to-SST input, via nicotinic receptors and presynaptic PKA signaling. Importantly, endogenous acetylcholine release preferentially activated nicotinic, not muscarinic, receptors, thus differentiating drug effects from endogenous neurotransmission. Brain state- and synapse-specific unmasking of synapses may be a powerful way to functionally rewire cortical circuits dependent on behavioral demands.

In Vivo Monosynaptic Excitatory Transmission between Layer 2 Cortical Pyramidal Neurons.

Little is known about the properties of monosynaptic connections between identified neurons in vivo. We made multiple (two to four) two-photon targeted whole-cell recordings from neighboring layer 2 mouse somatosensory barrel cortex pyramidal neurons in vivo to investigate excitatory monosynaptic transmission in the hyperpolarized downstate. We report that pyramidal neurons form a sparsely connected (6.7% connectivity) network with an overrepresentation of bidirectional connections. The majority of unitary excitatory postsynaptic potentials were small in amplitude (<0.5 mV), with a small minority >1 mV. The coefficient of variation (CV = 0.74) could largely be explained by the presence of synaptic failures (22%). Both the CV and failure rates were reduced with increasing amplitude. The mean paired-pulse ratio was 1.15 and positively correlated with the CV. Our approach will help bridge the gap between connectivity and function and allow investigations into the impact of brain state on monosynaptic transmission and integration.

Cortical fosGFP expression reveals broad receptive field excitatory neurons targeted by POm.

Neighboring cortical excitatory neurons show considerable heterogeneity in their responses to sensory stimulation. We hypothesized that a subset of layer 2 excitatory neurons in the juvenile (P18 to 27) mouse whisker somatosensory cortex, distinguished by expression of the activity-dependent fosGFP reporter gene, would be preferentially activated by whisker stimulation. In fact, two-photon targeted, dual whole-cell recordings showed that principal whisker stimulation elicits similar amplitude synaptic responses in fosGFP-expressing and fosGFP(-) neurons. FosGFP(+) neurons instead displayed shorter latency and larger amplitude subthreshold responses to surround whisker stimulation. Using optogenetic stimulation, we determined that these neurons are targeted by axons from the posteromedial nucleus (POm), a paralemniscal thalamic nucleus associated with broad receptive fields and widespread cortical projections. We conclude that fosGFP expression discriminates between single- and multi-whisker receptive field layer 2 pyramidal neurons.

Mechanisms of bi-directional modulation of thalamocortical transmission in barrel cortex by presynaptic kainate receptors.

Presynaptic kainate receptors play an important role in synaptic transmission and short-term plasticity to profoundly regulate network activity in many parts of the mammalian brain. In primary sensory neocortex, where short-term synaptic plasticity is important for receptive field structure and information processing, kainate receptors are highly expressed and regulate thalamocortical inputs, particularly during development. However, the mechanisms of the kainate receptor-dependent presynaptic regulation of thalamocortical transmission are unclear. We therefore investigated this issue using electrophysiology in neonatal thalamocortical slices of barrel cortex combined with pharmacology and biochemical analyses. We show that presynaptic kainate receptors can both facilitate or depress synaptic transmission depending on the extent of their activation. This bi-directional regulation is mediated in part by kainate receptors that directly influence thalamocortical axonal excitability, but also likely involves receptors acting at thalamocortical terminals to regulate transmitter release. The efficacy of kainate in regulating thalamocortical transmission is low compared to that reported for other inputs. Consistent with this low efficacy, our biochemical analyses indicate that the presynaptic kainate receptors regulating neonatal thalamocortical inputs likely lack the high kainate affinity GluK4 and 5 subunits. Thus thalamocortical transmission can be bi-directionally regulated by low affinity kainate receptors through two mechanisms. Such presynaptic regulation provides a potentially powerful mechanism to influence sensory processing during development of barrel cortex.

An embedded subnetwork of highly active neurons in the neocortex.

VIDEO ABSTRACT: Unbiased methods to assess the firing activity of individual neurons in the neocortex have revealed that a large proportion of cells fire at extremely low rates (<0.1 Hz), both in their spontaneous and evoked activity. Thus, firing in neocortical networks appears to be dominated by a small population of highly active neurons. Here, we use a fosGFP transgenic mouse to examine the properties of cells with a recent history of elevated activity. FosGFP-expressing layer 2/3 pyramidal cells fired at higher rates compared to fosGFP(-) neurons, both in vivo and in vitro. Elevated activity could be attributed to increased excitatory and decreased inhibitory drive to fosGFP(+) neurons. Paired-cell recordings indicated that fosGFP(+) neurons had a greater likelihood of being connected to each other. These findings indicate that highly active, interconnected neuronal ensembles are present in the neocortex and suggest these cells may play a role in the encoding of sensory information.