Advanced Circuit and Cellular Imaging Methods in Nonhuman Primates

Abstract

Novel genetically encoded tools and advanced microscopy methods have revolutionized neural circuit analyses in insects and rodents over the last two decades. Whereas numerous technical hurdles originally barred these methodologies from success in nonhuman primates (NHPs), current research has started to overcome those barriers. In some cases, methodological advances developed with NHPs have even surpassed their precursors. One such advance includes new ultra-large imaging windows on NHP cortex, which are larger than the entire rodent brain and allow analysis unprecedented ultra-large-scale circuits. NHP imaging chambers now remain patent for periods longer than a mouse's lifespan, allowing for long-term all-optical interrogation of identified circuits and neurons over timeframes that are relevant to human cognitive development. Here we present some recent imaging advances brought forth by research teams using macaques and marmosets. These include technical developments in optogenetics; voltage-, calcium- and glutamate-sensitive dye imaging; two-photon and wide-field optical imaging; viral delivery; and genetic expression of indicators and light-activated proteins that result in the visualization of tens of thousands of identified cortical neurons in NHPs. We describe a subset of the many recent advances in circuit and cellular imaging tools in NHPs focusing here primarily on the research presented during the corresponding mini-symposium at the 2019 Society for Neuroscience annual meeting.

Keywords: Adeno-Associated virus; cortical mapping; optogenetics; prosthetic vision; two-photon microscopy; voltage-sensitive dye imaging.

Figure 1.

A, Experimental setup for VSDI in NHPs. B, Reconstruction of shape contours from VSD maps imaged in V1 of fixating monkeys. Left, The original shape stimuli (white contour over gray background) that were presented for a fixating monkey. Stimulus size: circle, radius = 0.8°; triangle and square were bounded within the circle. Fixation point appears in red. Middle, VSD response maps (color-coded; ΔF/F) evoked by the visual stimuli averaged over 100–200 ms after stimulus onset. The dashed line denotes the border between V1 and V2 areas. Maps were filtered with a 2D Gaussian (σ = 1 pixel) for visualization purposes only. Right, Reconstructed stimuli from the VSD maps, after applying an inverse model (Zurawel et al., 2016); baseline activity was subtracted before reconstruction.

Figure 2.

Hypothetical layout of a feature map in V4. A range of factors constrains natural object structure (including material properties, mechanical laws, growth processes, and design and construction procedures) and generally result in smooth changes in contours. The example contour (left) highlights the fact that strongly curved object elements are often flanked by straighter elements oriented 90° away from the curved part. The figure on the right reflects a hypothetical feature map that adheres to this relationship by grouping neurons (circles) first according to their contour preference (line in each circle), and second according to the co-occurrence of these shape features.

Figure 3.

A, Left, Map of orientation-selective Regions of Interest (ROIs) on dendrites of an example neuron. Orientation preferences are colored for vector sum polarity for each ROI (color bar at lower right). Right, Map of color-selective inputs. Color preferences are labeled with the dominant polarity for each ROI. B, Coexpression of RCaMP and iGluSnFR in an IT neuron. Left, Two-photon image of iGluSnFR fluorescence were acquired under a 1000 nm excitation laser with a 525 ± 35 nm filter. Right, Two-photon image of RCaMP fluorescence of the same neuron was acquired under a 1060 nm excitation laser with a 615 ± 20 nm filter, the average of 1000 frames from each recording session. C, Left, Two-photon images of iGluSnFR fluorescence of one V1 neuron. Middle, Two-photon image of RCaMP fluorescence of the same neuron. Right, Summed synaptic input signals (green line) and somatic responses (red line) as a function of stimulus (below; error bar, SEM), indicating an orientation-sharpening mechanism affecting the soma's response. Bottom, A subset from the entire set of 81 visual stimuli consisting of either 1 of 9 different color patches, or a drifting grating having 1 of 12 orientations, with 1 of 2 drift directions, and 1 of 3 spatial frequencies.

Figure 4.

Plan for the OBServe. The Macknik and Martinez-Conde laboratories (and their partners) have developed cortical optogenetic and bioluminescence techniques in area V1 circuits of NHPs with the long-term goal of restoring vision using naturalistic stereoscopic foveal input from a video camera at the highest attainable visual acuity and contrast; in area V1 of NHPs for subsequent translation to the brains of blind human patients. In its envisioned final use in humans, blind patients will wear eye-tracking glasses that monitor the center of gaze on the visual scene, and transmit encrypted visual information to OBServe via a built-in encrypted ultra-wide band transceiver module. A, To get the information from the glasses into the brain, adeno-associated virus will be injected into the LGN, where it will deliver genetically encoded optogenetic light-sensitive proteins. These channelrhodopsins will percolate up through the optic radiations into the boutons in V1, where they can be optically stimulated with precise targeting of purely excitatory glutamatergic inputs; using the same synaptic mechanisms as normal vision. Preliminary studies in the Macknik and Martinez-Conde laboratories show that V1 cells are indeed stimulated by LGN optical stimulation from the surface of the cortex. B, Because the cortex varies its response to stable inputs in an ongoing fashion (through a number of mechanisms), prosthetic activation must be correspondingly adjusted to achieve stable function. To read out the responses of V1 and make these real-time adjustments, in a way that can be enclosed within a human skull, the system will use a hyperspectral photometer to readout the activity from cortical neurons transfected with a multicolor array of bioluminescent calcium indicators. Results from the foveal region of NHP V1 indicate that five different fluorophores provide at least 4752 distinguishable colors in >60,000 uniquely identified and targeted neurons (Chanovas et al., 2019). C, OBServe's coplanar imager/emitter chip will have individual twenty-five 1 μm² pixels in a 400 × 400 1 cm² square array, each made up of an LED to stimulate LGN boutons, surrounded by five monochromatic cameras with colored microfilters to optimize their chromatic sensitivity to the different transfected bioluminescent colors. D, The human implant will contain the emitter/imager chip and its supporting electronics in a 1 cm³ case that is fully implantable with no percutaneous wiring. E, Novel method to mount the implant within the skull. F, Both left and right fovea will receive an implant.

Figure 5.

Exploded view of the pressure-regulating implant for OBServe testing. *Indicates part is made of PEEK plastic, chosen for its radiolucent properties, strength, and ability to be sterilized. Component listing with description:

1. Cap and cap screws: removable cap for imaging and cleaning.

2. Silicone O-ring: ∼0.4 mm thick, prevents bacterial movement between chamber and cap.

3. Thin securing ring: Secures rotating ring against the bottom shelf of the chamber and prevents it from moving up.

4. Stabilizing screws: pushes against cup and prevents the imaging cup from moving closer to the chamber. Threaded into the bottom securing ring and penetrates silicone.

5. Guide cannulas: cannulas are threaded into the rotating ring at 3 different locations and sealed with silicone glue. These cannulas allow 18G CED needles to penetrate the cylinder.

6. CED injection needles: convection enhanced delivery needles. Designed for cortical injections but can also be used to deliver drugs or imaging contrast agents into the soft tissue.

7. Rotating ring: multiple threaded holes for the height adjusting screws and can rotate to adjust the positions of the screws. Sits between shelf in chamber and thin securing ring.

8. Chamber: Has holes for bone screws that are perpendicular to the surface of the bone to increase the strength of the bond between the chamber and bone. It also includes three threaded holes on the top to allow attachments and to secure the cap.

9. Resistance member: silicone is preferred for its ease in manufacturing, control of mechanical properties, and ability to be sterilized. Serves as a spring to adjust for pressure changes caused by the variations in swelling in the brain. Although not indicated in the drawing, the silicone connects to the lip of the imaging cup and rotating ring to create a sealed environment.

10. Imaging cup and glass cover slip: the glass is glued to the cup. Together they create a bowl that can hold liquid for a water-immersion objective.