Summary To understand how brain states and behaviors are generated by neural circuits, it would be useful to be able to perturb precisely the activity of specific cell types and pathways in the nonhuman primate nervous system. We used lentivirus to target the light-activated cation channel channelrhodopsin-2 (ChR2) specifically to excitatory neurons of the macaque frontal cortex. Using a laser-coupled optical fiber in conjunction with a recording microelectrode, we showed that activation of excitatory neurons resulted in well-timed excitatory and suppressive influences on neocortical neural networks. ChR2 was safely expressed, and could mediate opticalneuromodulation, in primate neocortex over many months. These findings highlight a methodology for investigating the causal role of specific cell types in nonhuman primate neural computation, cognition, and behavior, and open up the possibility of a new generation of ultraprecise neurological and psychiatric therapeutics via cell-type-specific optical neural control prosthetics.

Whole-cell patch clamp electrophysiology of neurons is a gold standard technique for high-fidelity analysis of the biophysical mechanisms of neural computation and pathology but it requires great skill to perform. We have developed a robot that automatically performs patch clamping in vivo, algorithmically detecting cells by analyzing the temporal sequence of electrode impedance changes. We demonstrate good yield, throughput, and quality of automated intracellular recording in mouse cortex and hippocampus.

Microscale hydrogels of controlled sizes and shapes are useful for cell-based screening, in vitro diagnostics, tissue engineering, and drug delivery. However, the rapid cross-linking of many chemically and pH cross-linkable hydrogel materials prevents the application of existing micromolding techniques. In this work we present a method for fabricating micromolded calcium alginate and chitosan structures through controlled release of the gelling agent from a hydrogel mold. Replica molding was employed to generate patterned membranes, whereas microtransfer molding was used to produce microparticles of controlled shapes. To explore the viability of this technique for producing complex tissue engineering micro-architectures, this approach was used to generate cell-laden size- and shape-controlled 3D microgels as well as composite hydrogels with well-defined spatially segregated regions. In addition, shape-controlled microstructures that can exhibit differential release properties were loaded with macromolecules to verify the potential of this approach for drug delivery applications.

Many neural disorders are associated with aberrant activity in specific cell types or neural projection pathways embedded within the densely-wired, heterogeneous matter of the brain. An ideal therapy would permit correction of activity just in specific target neurons, while leaving other neurons unaltered. Recently our lab revealed that the naturally-occurring light-activated proteins channelrhodopsin-2 (ChR2) and halorhodopsin (Halo/NpHR) can, when genetically expressed in neurons, enable them to be safely, precisely, and reversibly activated and silenced by pulses of blue and yellow light, respectively. We here describe the ability to make specific neurons in the brain light-sensitive, using a viral approach. We also reveal the design and construction of a scalable, fully-implantable optical prosthetic capable of delivering light of appropriate intensity and wavelength to targeted neurons at arbitrary 3-D locations within the brain, enabling activation and silencing of specific neuron types at multiple locations. Finally, we demonstrate control of neural activity in the cortex of the non-human primate, a key step in the translation of such technology for human clinical use. Systems for optical targeting of specific neural circuit elements may enable a new generation of high-precision therapies for brain disorders.

Whole cell patch clamping in vivo is an important neuroscience technique that uniquely provides access to both supra-threshold spiking and sub-threshold synaptic events of single neurons in the brain. This article describes how to set up and use the autopatcher, a robot for automatically obtaining high yield and high quality whole cell patch clamp recordings in vivo. Following this protocol, a functional experimental rig for automated whole cell patch clamping can be set up in one week. High quality surgical preparation of mice takes approximately 1 hour, and each autopatching experiment can be carried out over periods lasting several hours. Autopatching should enable in vivo intracellular investigations to be accessible by a significant number of neuroscience labs, and enable labs already doing in vivo patch clamp to scale up their efforts by reducing training time for new lab members and increasing experimental durations by handling mentally intensive tasks automatically.

In networks of excitatory and inhibitory neurons with mutual synaptic coupling, specific drive to sub-ensembles of cells often leads to gamma-frequency (25–100 Hz) oscillations. When the number of driven cells is too small, however, the synaptic interactions may not be strong or homogeneous enough to support the mechanism underlying the rhythm. Using a combination of computational simulation and mathematical analysis, we study the breakdown of gamma rhythms as the driven ensembles become too small, or the synaptic interactions become too weak and heterogeneous. Heterogeneities in drives or synaptic strengths play an important role in the breakdown of the rhythms; nonetheless, we find that the analysis of homogeneous networks yields insight into the breakdown of rhythms in heterogeneous networks. In particular, if parameter values are such that in a homogeneous network, it takes several gamma cycles to converge to synchrony, then in a similar, but realistically heterogeneous network, synchrony breaks down altogether. This leads to the surprising conclusion that in a network with realistic heterogeneity, gamma rhythms based on the interaction of excitatory and inhibitory cell populations must arise either rapidly, or not at all. For given synaptic strengths and heterogeneities, there is a (soft) lower bound on the possible number of cells in an ensemble oscillating at gamma frequency, based simply on the requirement that synaptic interactions between the two cell populations be strong enough. This observation suggests explanations for recent experimental results concerning the modulation of gamma oscillations in macaque primary visual cortex by varying spatial stimulus size or attention level, and for our own experimental results, reported here, concerning the optogenetic modulation of gamma oscillations in kainate-activated hippocampal slices. We make specific predictions about the behavior of pyramidal cells and fast-spiking interneurons in these experiments.

Seconds-scale network states, affecting many neurons within a network, modulate neural activity by complementing fast integration of neuron-specific inputs that arrive in the milliseconds before spiking. Nonrhythmic subthreshold dynamics at intermediate timescales, however, are less well characterized. We found, using automated whole cell patch clamping in vivo, that spikes recorded in CA1 and barrel cortex in awake mice are often preceded not only by monotonic voltage rises lasting milliseconds but also by more gradual (lasting tens to hundreds of milliseconds) depolarizations. The latter exert a gating function on spiking, in a fashion that depends on the gradual rise duration: the probability of spiking was higher for longer gradual rises, even when controlled for the amplitude of the gradual rises. Barrel cortex double-autopatch recordings show that gradual rises are shared across some, but not all, neurons. The gradual rises may represent a new kind of state, intermediate both in timescale and in proportion of neurons participating, which gates a neuron's ability to respond to subsequent inputs. We analyzed subthreshold activity preceding spikes in hippocampus and barrel cortex of awake mice. Aperiodic voltage ramps extending over tens to hundreds of milliseconds consistently precede and facilitate spikes, in a manner dependent on both their amplitude and their duration. These voltage ramps represent a "mesoscale" activated state that gates spike production in vivo.

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