James H. Marshel

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.

New Rabies Virus Variants for Monitoring and Manipulating Activity and Gene Expression in Defined Neural Circuits

Glycoprotein-deleted (ΔG) rabies virus is a powerful tool for studies of neural circuit structure. Here, we describe the development and demonstrate the utility of new resources that allow experiments directly investigating relationships between the structure and function of neural circuits. New methods and reagents allowed efficient production of 12 novel ΔG rabies variants from plasmid DNA. These new rabies viruses express useful neuroscience tools, including the Ca(2+) indicator GCaMP3 for monitoring activity; Channelrhodopsin-2 for photoactivation; allatostatin receptor for inactivation by ligand application; and rtTA, ER(T2)CreER(T2), or FLPo, for control of gene expression. These new tools allow neurons targeted on the basis of their connectivity to have their function assayed or their activity or gene expression manipulated. Combining these tools with in vivo imaging and optogenetic methods and/or inducible gene expression in transgenic mice will facilitate experiments investigating neural circuit development, plasticity, and function that have not been possible with existing reagents.

Functional Specialization of Seven Mouse Visual Cortical Areas

To establish the mouse as a genetically tractable model for high-order visual processing, we characterized fine-scale retinotopic organization of visual cortex and determined functional specialization of layer 2/3 neuronal populations in seven retinotopically identified areas. Each area contains a distinct visuotopic representation and encodes a unique combination of spatiotemporal features. Areas LM, AL, RL, and AM prefer up to three times faster temporal frequencies and significantly lower spatial frequencies than V1, while V1 and PM prefer high spatial and low temporal frequencies. LI prefers both high spatial and temporal frequencies. All extrastriate areas except LI increase orientation selectivity compared to V1, and three areas are significantly more direction selective (AL, RL, and AM). Specific combinations of spatiotemporal representations further distinguish areas. These results reveal that mouse higher visual areas are functionally distinct, and separate groups of areas may be specialized for motion-related versus pattern-related computations, perhaps forming pathways analogous to dorsal and ventral streams in other species.

Closed-Loop and Activity-Guided Optogenetic Control

Advances in optical manipulation and observation of neural activity have set the stage for widespread implementation of closed-loop and activity-guided optical control of neural circuit dynamics. Closing the loop optogenetically (i.e., basing optogenetic stimulation on simultaneously observed dynamics in a principled way) is a powerful strategy for causal investigation of neural circuitry. In particular, observing and feeding back the effects of circuit interventions on physiologically relevant timescales is valuable for directly testing whether inferred models of dynamics, connectivity, and causation are accurate in vivo. Here we highlight technical and theoretical foundations as well as recent advances and opportunities in this area, and we review in detail the known caveats and limitations of optogenetic experimentation in the context of addressing these challenges with closed-loop optogenetic control in behaving animals.

Targeting Single Neuronal Networks for Gene Expression and Cell Labeling In Vivo

To understand fine-scale structure and function of single mammalian neuronal networks, we developed and validated a strategy to genetically target and trace monosynaptic inputs to a single neuron in vitro and in vivo. The strategy independently targets a neuron and its presynaptic network for specific gene expression and fine-scale labeling, using single-cell electroporation of DNA to target infection and monosynaptic retrograde spread of a genetically modifiable rabies virus. The technique is highly reliable, with transsynaptic labeling occurring in every electroporated neuron infected by the virus. Targeting single neocortical neuronal networks in vivo, we found clusters of both spiny and aspiny neurons surrounding the electroporated neuron in each case, in addition to intricately labeled distal cortical and subcortical inputs. This technique, broadly applicable for probing and manipulating single neuronal networks with single-cell resolution in vivo, may help shed new light on fundamental mechanisms underlying circuit development and information processing by neuronal networks throughout the brain.

Projections from neocortex mediate top-down control of memory retrieval.

Top-down prefrontal cortex inputs to the hippocampus have been hypothesized to be important in memory consolidation, retrieval, and the pathophysiology of major psychiatric diseases; however, no such direct projections have been identified and functionally described. Here we report the discovery of a monosynaptic prefrontal cortex (predominantly anterior cingulate) to hippocampus (CA3 to CA1 region) projection in mice, and find that optogenetic manipulation of this projection (here termed AC–CA) is capable of eliciting contextual memory retrieval. To explore the network mechanisms of this process, we developed and applied tools to observe cellular-resolution neural activity in the hippocampus while stimulating AC–CA projections during memory retrieval in mice behaving in virtual-reality environments. Using this approach, we found that learning drives the emergence of a sparse class of neurons in CA2/CA3 that are highly correlated with the local network and that lead synchronous population activity events; these neurons are then preferentially recruited by the AC–CA projection during memory retrieval. These findings reveal a sparsely implemented memory retrieval mechanism in the hippocampus that operates via direct top-down prefrontal input, with implications for the patterning and storage of salient memory representations.

Topography and Areal Organization of Mouse Visual Cortex

To guide future experiments aimed at understanding the mouse visual system, it is essential that we have a solid handle on the global topography of visual cortical areas. Ideally, the method used to measure cortical topography is objective, robust, and simple enough to guide subsequent targeting of visual areas in each subject. We developed an automated method that uses retinotopic maps of mouse visual cortex obtained with intrinsic signal imaging (Schuett et al., 2002; Kalatsky and Stryker, 2003; Marshel et al., 2011) and applies an algorithm to automatically identify cortical regions that satisfy a set of quantifiable criteria for what constitutes a visual area. This approach facilitated detailed parcellation of mouse visual cortex, delineating nine known areas (primary visual cortex, lateromedial area, anterolateral area, rostrolateral area, anteromedial area, posteromedial area, laterointermediate area, posterior area, and postrhinal area), and revealing two additional areas that have not been previously described as visuotopically mapped in mice (laterolateral anterior area and medial area). Using the topographic maps and defined area boundaries from each animal, we characterized several features of map organization, including variability in area position, area size, visual field coverage, and cortical magnification. We demonstrate that higher areas in mice often have representations that are incomplete or biased toward particular regions of visual space, suggestive of specializations for processing specific types of information about the environment. This work provides a comprehensive description of mouse visuotopic organization and describes essential tools for accurate functional localization of visual areas.

Extended field-of-view and increased-signal 3D holographic illumination with time-division multiplexing

Phase spatial light modulators (SLMs) are widely used for generating multifocal three-dimensional (3D) illumination patterns, but these are limited to a field of view constrained by the pixel count or size of the SLM. Further, with two-photon SLM-based excitation, increasing the number of focal spots penalizes the total signal linearly--requiring more laser power than is available or can be tolerated by the sample. Here we analyze and demonstrate a method of using galvanometer mirrors to time-sequentially reposition multiple 3D holograms, both extending the field of view and increasing the total time-averaged two-photon signal. We apply our approach to 3D two-photon in vivo neuronal calcium imaging.

Genetically encoded voltage sensor goes live

A genetically encoded voltage indicator enables robust optical recording of membrane voltage changes in the fly brain.

Feasibility analysis of genetically-encoded calcium indicators as a neural signal source for all-optical brain-machine interfaces

Optical techniques such as two-photon (2p) calcium imaging have the potential to transform the way we interrogate neural circuits, both in the realm of basic neuroscience and in the development of brain-machine interfaces (BMIs). This may be possible by overcoming some of the limitations of electrophysiological methods. Here we ask if optical imaging signals, in particular 2p GCaMP6 calcium imaging signals, can benefit BMIs despite their relatively long activity-response time constants, low signal-to-noise ratios (SNRs), and slow acquisition frame rates. We employed motor cortical electrode array recordings as the basis for generating synthetic 2p GCaMP signals. We then decoded movement kinematics from these surrogate data using a state-of-the-art BMI decoder algorithm. We found that it was possible to decode the position and velocity of the hand from synthetic imaging signals. We quantified the decoder performance using standard mean squared errors (MSEs) and Pearsons correlation coefficient (r) measures. Decode quality varied considerably as a function of SNR and the frame rate of data acquisition. Future computational and experimental research is required to quantify SNR more accurately and to increase the imaging frame rate while maintaining high SNR, in order to improve all-optical BMI (o-BMI) performance. This study should help establish the feasibility and design space of o-BMIs.