BACKGROUND:Modern molecular tools make it possible to manipulate neural activity in a reversible and cell-type specific manner. For rhesus monkey research, molecular tools are generally introduced via viral vectors. New instruments designed specifically for use in monkey research are needed to enhance the efficiency and reliability of vector delivery.NEW METHOD:A suite of multi-channel injection devices was developed to permit efficient and uniform vector delivery to cortical regions of the monkey brain. Manganese was co-infused with virus to allow rapid post-surgical confirmation of targeting accuracy using MRI. A needle guide was designed to increase the accuracy of sub-cortical targeting using stereotaxic co-ordinates.RESULTS:The multi-channel injection devices produced dense, uniform coverage of dorsal surface cortex, ventral surface cortex, and intra-sulcal cortex, respectively. Co-infusion of manganese with the viral vector allowed for immediate verification of injection accuracy. The needle guide improved accuracy of targeting sub-cortical structures by preventing needle deflection.COMPARISON WITH EXISTING METHOD(S):The current methods, hand-held injections or single slow mechanical injection, for surface cortex transduction do not, in our hands, produce the density and uniformity of coverage provided by the injector arrays and associated infusion protocol.CONCLUSIONS:The efficiency and reliability of vector delivery has been considerably improved by the development of new methods and instruments. This development should facilitate the translation of chemo- and optogenetic studies performed in smaller animals to larger animals such as rhesus monkeys.

Background: Marmosets are a powerful, emerging model for human behavior and neurological disorders. However, longitudinal imaging modalities that visualize both cellular structure and function within the cortex are not available in this animal model. Hence, we implemented an approach to quantify vascular topology, hemodynamics, and neural activity in awake marmosets using two-photon microscopy (2PM).New method: Marmosets were acclimated to a custom stereotaxic system. AAV1-GCaMP5G was injected into somatosensory cortex to optically indicate neural activity, and a cranial chamber was implanted.Results: Longitudinal 2PM revealed vasculature and neurons 500 mu m below the cortical surface. Vascular response and neural activity during sensory stimulation were preserved over 5 and 3 months, respectively, before optical quality deteriorated. Vascular remodeling including increased tortuosity and branching was quantified. However, capillary connectivity from arterioles to venules remained unchanged. Further, behavioral assessment before and after surgery demonstrated no impact on cognitive and motor function. Immunohistochemistry confirmed minimal astrocyte activation with no focal damage. Over 6 months, total cortical depth visualized decreased. When under anesthesia, the most prominent isoflurane-induced vasodilation occurred in capillaries and smaller arterioles.Comparison with existing method(s): These results demonstrate the capability to repeatedly observe cortical physiology in awake marmosets over months.Conclusions: This work provides a novel and insightful technique to investigate critical mechanisms in neurological disorders in awake marmosets without introducing confounds from anesthesia. Published by Elsevier B.V.

Conventional recording methods generally preclude following the activity of the same neurons in awake animals across days. This limits our ability to systematically investigate the principles of neuronal specialization, or to study phenomena that evolve over multiple days such as experience-dependent plasticity. To redress this shortcoming, we developed a drivable, chronically implanted microwire recording preparation that allowed us to follow visual responses in inferotemporal (IT) cortex in awake behaving monkeys across multiple days, and in many cases across months. The microwire bundle and other implanted components were MRI compatible and thus permitted in the same animals both functional imaging and long-term recording from multiple neurons in deep structures within a region the approximate size of one voxel (< 1 mm). The distinct patterns of stimulus selectivity observed in IT neurons, together with stable features in spike waveforms and interspike interval distributions, allowed us to track individual neurons across weeks and sometimes months. The long-term consistency of visual responses shown here permits large-scale mappings of neuronal properties using massive image libraries presented over the course of days. We demonstrate this possibility by screening the visual responses of single neurons to a set of 10,000 stimuli.

This protocol documents the construction of a custom microscope stage system currently in widespread use by a wide variety of investigators. The current design and construction of this stage is the result of multiple iterations, integrating input from a number of electrophysiologists working with a variety of preparations. Thus, this tool is a generally applicable solution, suitable for a wide array of end-user requirements; its flexible design facilitates rapid and easy configuration, making it useful for multi-user microscopes, as individual researchers can reconfigure the stage system or have their own readily replaceable stage plates. Furthermore, the stage can be manufactured using equipment typically found in small research machine shops, and by keeping the various parts on hand, machinists can quickly satisfy new requests and/or modifications for a wide variety of applications.

Introduction: Surgically implanted chambers with removable grids are routinely used for studying patterns of neuronal activity in primate brains; however, accessing target tissues is significantly constrained by standard grid designs. Typically, grids are configured with a series of guide holes drilled vertically, parallel to the walls of the chamber, thus targeted sites are limited to those in line vertically with one of the guide holes. Methods: By using the three-dimensional modeling software, a novel grid was designed to reach the targeted sites far beyond the standard reach of the chamber. The grid was fabricated using conventional machining techniques and three-dimensional printing. Results: A pilot study involving microinjection of the magnetic resonance (MR) contrast agent gadolinium into the discrete regions of interest (ROIs) in the temporal cortex of an awake, behaving monkey demonstrated the effectiveness of this new design of the guide grid. Using multiple different angles of approach, we were readily able to access 10 injection sites, which were up to 5 mm outside the traditional, orthogonal reach of the chamber.

In order to expand the repertoire of somatosensory functions that can be effectively studied through functional MRI, we have developed a tactile stimulator which can deliver rich and varied combinations of stimulation that simulate natural tactile exploration. The system is computer controlled and compatible with an MRI environment. Complex aspects of somesthesis can thus be studied independent of confounds introduced by motor activity or problems with precision, accuracy or reproducibility of stimulus delivery.

One promising approach to studying how large numbers of neurons interact is the combination of neuronal cell cultures with multi-electrode arrays (MEAs.) Together, these allow simultaneous recording from many neurons. A particular challenge has been the long-term growth of organotypic neuronal cultures on MEAs. These cultures need alternate exposure to air and a liquid growth medium. Continuous rotary motion (of suitable closed chambers) provides this, but greatly complicates recording of large numbers of low-level electrical signals. An alternative approach is to rock chambers incorporating MEAs cyclically through a limited angle, thus facilitating direct connections. A variable speed trajectory balances cycle frequency against the shear forces acting to dislodge the tissue. Different trajectories can be used to optimize culture health during different development periods. We discuss the design of a microprocessor based life-support system that implements such a limited-angle, variable-speed cycle to culture rat brain slices on MEAs, while maintaining direct electrical connections. We show some of the neurophysiological results thus made possible, and consider improvements for a subsequent life-support system for future research.

The system described here provides a simple method of delivering anesthetic vapor to the fruit-fly Drosophila melanogaster. This system delivers known concentrations of volatile anesthetic vapor obtained from liquid anesthetics in a continuous gas stream of pure humidified air. It controls for evaporation, and absorption of volatile agents, whilst allowing for extracellular electrophysiological recordings. Recordings were made from the fly's escape muscles, the jump tergotrochanter muscle (TTM) and the flight dorsal longitudinal muscle (DLM). The system minimizes the quantity of anesthetic used, making the use of more expensive and more conventional anesthetics cost effective and practicable. It also permits monitoring the fly's movements during anesthesia.