Bryan M. Hooks

A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing

Cell type–specific expression of optogenetic molecules allows temporally precise manipulation of targeted neuronal activity. Here we present a toolbox of four knock-in mouse lines engineered for strong, Cre-dependent expression of channelrhodopsins ChR2-tdTomato and ChR2-EYFP, halorhodopsin eNpHR3.0 and archaerhodopsin Arch-ER2. All four transgenes mediated Cre-dependent, robust activation or silencing of cortical pyramidal neurons in vitro and in vivo upon light stimulation, with ChR2-EYFP and Arch-ER2 demonstrating light sensitivity approaching that of in utero or virally transduced neurons. We further show specific photoactivation of parvalbumin-positive interneurons in behaving ChR2-EYFP reporter mice. The robust, consistent and inducible nature of our ChR2 mice represents a significant advance over previous lines, and the Arch-ER2 and eNpHR3.0 mice are to our knowledge the first demonstration of successful conditional transgenic optogenetic silencing. When combined with the hundreds of available Cre driver lines, this optimized toolbox of reporter mice will enable widespread investigations of neural circuit function with unprecedented reliability and accuracy.

Long-Range Neuronal Circuits Underlying the Interaction between Sensory and Motor Cortex

In the rodent vibrissal system, active sensation and sensorimotor integration are mediated in part by connections between barrel cortex and vibrissal motor cortex. Little is known about how these structures interact at the level of neurons. We used Channelrhodopsin-2 (ChR2) expression, combined with anterograde and retrograde labeling, to map connections between barrel cortex and pyramidal neurons in mouse motor cortex. Barrel cortex axons preferentially targeted upper layer (L2/3, L5A) neurons in motor cortex; input to neurons projecting back to barrel cortex was particularly strong. Barrel cortex input to deeper layers (L5B, L6) of motor cortex, including neurons projecting to the brainstem, was weak, despite pronounced geometric overlap of dendrites with axons from barrel cortex. Neurons in different layers received barrel cortex input within stereotyped dendritic domains. The cortico-cortical neurons in superficial layers of motor cortex thus couple motor and sensory signals and might mediate sensorimotor integration and motor learning.

Laminar Analysis of Excitatory Local Circuits in Vibrissal Motor and Sensory Cortical Areas

Optical and electrophysiological tools were used to map out the neural circuits within and between cortical layers in three different brain regions, and the results suggest regional specializations for sensory versus motor information processing.

Ephus: Multipurpose Data Acquisition Software for Neuroscience Experiments

Physiological measurements in neuroscience experiments often involve complex stimulus paradigms and multiple data channels. Ephus (http://www.ephus.org) is an open-source software package designed for general-purpose data acquisition and instrument control. Ephus operates as a collection of modular programs, including an ephys program for standard whole-cell recording with single or multiple electrodes in typical electrophysiological experiments, and a mapper program for synaptic circuit mapping experiments involving laser scanning photostimulation based on glutamate uncaging or channelrhodopsin-2 excitation. Custom user functions allow user-extensibility at multiple levels, including on-line analysis and closed-loop experiments, where experimental parameters can be changed based on recently acquired data, such as during in vivo behavioral experiments. Ephus is compatible with a variety of data acquisition and imaging hardware. This paper describes the main features and modules of Ephus and their use in representative experimental applications.

Organization of Cortical and Thalamic Input to Pyramidal Neurons in Mouse Motor Cortex

Determining how long-range synaptic inputs engage pyramidal neurons in primary motor cortex (M1) is important for understanding circuit mechanisms involved in regulating movement. We used channelrhodopsin-2-assisted circuit mapping to characterize the long-range excitatory synaptic connections made by multiple cortical and thalamic areas onto pyramidal neurons in mouse vibrissal motor cortex (vM1). Each projection innervated vM1 pyramidal neurons with a unique laminar profile. Collectively, the profiles for different sources of input partially overlapped and spanned all cortical layers. Specifically, orbital cortex (OC) inputs primarily targeted neurons in L6. Secondary motor cortex (M2) inputs excited neurons mainly in L5B, including pyramidal tract neurons. In contrast, thalamocortical inputs from anterior motor-related thalamic regions, including VA/VL (ventral anterior thalamic nucleus/ventrolateral thalamic nucleus), targeted neurons in L2/3 through L5B, but avoided L6. Inputs from posterior sensory-related thalamic areas, including POm (posterior thalamic nuclear group), targeted neurons only in the upper layers (L2/3 and L5A), similar to inputs from somatosensory (barrel) cortex. Our results show that long-range excitatory inputs target vM1 pyramidal neurons in a layer-specific manner. Inputs from sensory-related cortical and thalamic areas preferentially target the upper-layer pyramidal neurons in vM1. In contrast, inputs from OC and M2, areas associated with volitional and cognitive aspects of movements, bypass local circuitry and have direct monosynaptic access to neurons projecting to brainstem and thalamus.

Vision Triggers an Experience-Dependent Sensitive Period at the Retinogeniculate Synapse

In the mammalian visual system, sensory experience is widely thought to sculpt cortical circuits during a precise critical period. In contrast, subcortical regions, such as the thalamus, were thought to develop at earlier ages in a vision-independent manner. Recent studies at the retinogeniculate synapse, however, have demonstrated an influence of vision on the formation of synaptic circuits in the thalamus. In mice, dark rearing from birth does not alter normal developmental maturation of the connection between retina and thalamus. However, deprivation 20 d after birth [postnatal day 20 (p20)] resulted in dramatic weakening of synaptic strength and an increase in the number of retinal inputs that innervate a thalamic relay neuron. Here, by quantifying changes in synaptic strength and connectivity in response to different time windows of deprivation, we find that several days of vision after eye opening is necessary for triggering experience-dependent plasticity. Shorter periods of visual experience do not permit similar experience-dependent synaptic reorganization. Furthermore, changes in connectivity are rapidly reversible simply by restoring normal vision. However, similar plasticity did not occur when shifting the onset of deprivation to p25. Although synapses still weakened, recruitment of additional retinal inputs no longer occurred. Therefore, synaptic circuits in the visual thalamus are unexpectedly malleable during a late developmental period, after the time when normal synapse elimination and pruning has occurred. This thalamic sensitive period overlaps temporally with experience-dependent changes in the cortex, suggesting that subcortical plasticity may influence cortical responses to sensory experience.

Distinct Balance of Excitation and Inhibition in an Interareal Feedforward and Feedback Circuit of Mouse Visual Cortex

Mouse visual cortex is subdivided into multiple distinct, hierarchically organized areas that are interconnected through feedforward (FF) and feedback (FB) pathways. The principal synaptic targets of FF and FB axons that reciprocally interconnect primary visual cortex (V1) with the higher lateromedial extrastriate area (LM) are pyramidal cells (Pyr) and parvalbumin (PV)-expressing GABAergic interneurons. Recordings in slices of mouse visual cortex have shown that layer 2/3 Pyr cells receive excitatory monosynaptic FF and FB inputs, which are opposed by disynaptic inhibition. Most notably, inhibition is stronger in the FF than FB pathway, suggesting pathway-specific organization of feedforward inhibition (FFI). To explore the hypothesis that this difference is due to diverse pathway-specific strengths of the inputs to PV neurons we have performed subcellular Channelrhodopsin-2-assisted circuit mapping in slices of mouse visual cortex. Whole-cell patch-clamp recordings were obtained from retrobead-labeled FFV1→LM- and FBLM→V1-projecting Pyr cells, as well as from tdTomato-expressing PV neurons. The results show that the FFV1→LM pathway provides on average 3.7-fold stronger depolarizing input to layer 2/3 inhibitory PV neurons than to neighboring excitatory Pyr cells. In the FBLM→V1 pathway, depolarizing inputs to layer 2/3 PV neurons and Pyr cells were balanced. Balanced inputs were also found in the FFV1→LM pathway to layer 5 PV neurons and Pyr cells, whereas FBLM→V1 inputs to layer 5 were biased toward Pyr cells. The findings indicate that FFI in FFV1→LM and FBLM→V1 circuits are organized in a pathway- and lamina-specific fashion.

A Role for Stargazin in Experience-Dependent Plasticity

During development, neurons are constantly refining their connections in response to changes in activity. Experience-dependent plasticity is a key form of synaptic plasticity, involving changes in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) accumulation at synapses. Here, we report a critical role for the AMPAR auxiliary subunit stargazin in this plasticity. We show that stargazin is functional at the retinogeniculate synapse and that in the absence of stargazin, the refinement of the retinogeniculate synapse is specifically disrupted during the experience-dependent phase. Importantly, we found that stargazin expression and phosphorylation increased with visual deprivation and led to reduced AMPAR rectification at the retinogeniculate synapse. To test whether stargazin plays a role in homeostatic plasticity, we turned to cultured neurons and found that stargazin phosphorylation is essential for synaptic scaling. Overall, our data reveal an important role for stargazin in regulating AMPAR abundance and composition at glutamatergic synapses during homeostatic and experience-dependent plasticity.

Dual-Channel Circuit Mapping Reveals Sensorimotor Convergence in the Primary Motor Cortex

Cortical cells integrate synaptic input from multiple sources, but how these different inputs are distributed across individual neurons is largely unknown. Differences in input might account for diverse responses in neighboring neurons during behavior. We present a strategy for comparing the strengths of multiple types of input onto the same neuron. We developed methods for independent dual-channel photostimulation of synaptic inputs using ChR2 together with ReaChR, a red-shifted channelrhodopsin. We used dual-channel photostimulation to probe convergence of sensory information in the mouse primary motor cortex. Input from somatosensory cortex and thalamus converges in individual neurons. Similarly, inputs from distinct somatotopic regions of the somatosensory cortex are integrated at the level of single motor cortex neurons. We next developed a ReaChR transgenic mouse under the control of both Flp- and Cre-recombinases that is an effective tool for circuit mapping. Our approach to dual-channel photostimulation enables quantitative comparison of the strengths of multiple pathways across all length scales of the brain.

Thorough GABAergic innervation of the entire axon initial segment revealed by an optogenetic ‘laserspritzer’

The laserspritzer we developed activates axo‐axonic synapses (AASs) with spatial resolutions of less than 40 μm. AASs innervate the entire length of the axon initial segment (AIS), as opposed to forming a highly concentrated cartridge. AAS‐mediated synaptic potentials are inhibitory, with reversal potentials similar to those of basket synapses. AAS inhibition impedes action potentials and epileptiform activity more robustly than perisomatic inhibitions. AAS activation alone is sufficient to inhibit action potential generation and epileptiform activities in vitro.