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Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (<0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.
The parallel injector array speeds up a surgery roughly by a factor equal to the number of injectors, not counting setup and recovery time, although individual times will depend on the skill of the practitioner. For a 1 microliter injection, we typically saw lentivirus expression in a sphere of approximately diameter 1mm (Fig. 1E). The precision of the injection was such that the variability in tip positioning, from trial to trial, was about 45 microns (standard deviation of the distance from the tip position to the intended tip position).
Figure 1. Design, implementation, and use of a parallel virus injector array. A, schematic of the parallel injector array system, showing a triple injector configuration, for three simultaneous injections. B, photograph of a triple parallel injector array as diagrammed in A. C,stereotaxic clamp, shown in outline from the top. D, illustration of technique for efficient, damage-minimizing, opening of holes in skull for injector insertion into brain: with a dental drill, thin the skull down to ~50 microns thickness, then use the tip of a sharp needle to open a small craniotomy. E, fluorescence image showing channelrhodopsin-2 (ChR2)-GFP-labeled cells in three mouse cortical regions, as targeted by the triple injector array shown in B.
In recent years, a number of genetically-encoded optical sensitizers have enabled neurons to be activated and silenced in vivo in a temporally-precise fashion, in response to brief pulses of light (e.g., 1,4,5,6,7,8,11). A key method with which neurons have been sensitized to light in the mammalian brain, is via viruses such as lentiviruses and adeno-associated viruses (AAV), which can deliver genes encoding for opsins to brains of animals ranging from mice to monkeys, in a safe and enduring fashion (e.g., 2,9,10). Viruses allow faster turnaround time than do transgenics, especially for organisms that are not genetic model organisms such as rats and monkeys, and for opsins may enable high expression levels that may not be possible in transgenic scenarios. Here we demonstrate a parallel injector array capable of creating, in a rapid timescale, "circuit-level transgenics," enabling entire 3-dimensional brain structures to be virally targeted with a gene, in a single surgical step. The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (<0.1 μL/min).
This technology will enable a wide variety of new kinds of experiments, such as millisecond-timescale shutdown of complexly-shaped structures (such as the hippocampus) at precise times during behavior, temporally-precise inactivation of bilateral structures that may act redundantly (such as the left and right amygdala), and the perturbation of multiple discrete brain regions (e.g., driving two connected regions out of phase to study how cross-region synchrony depends upon activity within each region, or stimulating inputs to a region while silencing a subset of the targets in order to understand which of the several targets are critical for mediating the effects of those inputs). For large brains like those in the primate, in which we have recently demonstrated optical cell-type specific neural activation3, perturbing activity in a behaviorally-relevant area may require viral labeling of large, complex structures. We note that parallel injector arrays may be used to inject almost any payload – drugs, neuromodulators, neurotransmitters, or even cells – in complex 3-D patterns in the brain, in a temporally-precise manner. Finally, from a translational standpoint, it is possible that rapid, patient-customized gene therapy or drug delivery devices may be rapidly custom-designed and fabricated to match individual brain geometries, supporting new treatments for a variety of pathologies, potentially through the use of optical control molecules.
The injector arrays are designed to be precise, both spatially and volumetrically. In the X- and Y-directions, this is accomplished by drilling very accurately placed holes using an inexpensive mini-mill, with the holes just large enough to fit the injectors, so that injectors are held parallel to one another, and in a precise location. In the Z-direction, the injectors are trimmed using a stereotaxic apparatus, allowing a level of precision equivalent to that of the stereotaxic surgery itself. The volumetric precision arises from the precision of the Hamilton pump, as well as the near-zero dead-volume connectors, adapted from the high-pressure liquid chromatography (HPLC) field. The injectors are made from fused silica capillary tubing, which is strong and rigid enough that it maintains precise shape and spacing under pressure, without the larger wall thickness of alternatives such as steel cannulas. Small modifications can easily be made to adapt the parallel injector array to a variety of experiments. For example, if a smaller volume of virus or finer spacing is required, smaller capillary tubing can be employed, along with a corresponding smaller drill bit. Future devices may utilize microfluidic channels and pumps, to increase the number of parallel injectors, to minimize the size (perhaps enabling such devices to be mounted on the heads of freely-moving animals).
ESB acknowledges funding by the NIH Director's New Innovator Award (DP2 OD002002-01), NIH Challenge Grant 1RC1MH088182-01, NIH Grand Opportunities Grant 1RC2DE020919-01, NIH 1R01NS067199-01, NSF (0835878 and 0848804), the McGovern Institute Neurotechnology Award Program, the Department of Defense, NARSAD, the Alfred P. Sloan Foundation, Jerry and Marge Burnett, the SFN Research Award for Innovation in Neuroscience, the MIT Media Lab, the Benesse Foundation, and the Wallace H. Coulter Foundation.