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 optical neuromodulation, 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.

The rhesus macaque is an important model species for understanding neural computation, cognition, and behavior, as well as for probing the circuit-level basis of human neurological and psychiatric disorders. To resolve how complex functions emerge from the activity of diverse cell types, ideally one would be able to perturb the activity of genetically specified cell types and neural pathways in the primate brain, in a temporally precise fashion. In one recent study, adeno-associated virus (AAV) was used to deliver the Drosophila allatostatin receptor to neurons in the primate thalamus (), enabling neural silencing via intracranial delivery of the small molecule allatostatin. In general, however, the adaptation of neural control tools to the primate brain has been slow in comparison to the rapid adaptation of such tools for characterizing circuit functions in worms, flies, and mice (reviewed in). Indeed, although molecular techniques have been used to deliver genetic payloads to the primate brain (e.g.,), as well as to make transgenic primates (), no attempts have been made to target genes to genetically specified neuron types. Here we used channelrhodopsin-2 (ChR2), a genetically encoded molecular sensitizer that enables activation of neurons in response to pulses of blue light (), to assess the impact of selective activation of cortical excitatory neurons on primate cortical dynamics. We used optical fibers in conjunction with microelectrodes to perform simultaneous in vivo optical stimulation and electrical recording in the awake primate. Selectively activating ChR2-positive excitatory neurons resulted in well-timed excitatory and suppressive influences on neural activity, reflecting neural dynamics downstream of excitatory neuron activation. ChR2 was safely expressed and could mediate temporally precise optical neural stimulation of significant volumes of cortical tissue for months after viral injection, opening up the possibility for such technologies to support precise, cell-specific optical control prosthetics for patients with severe neurological and psychiatric disorders.

Results

Figure 2 Analyses of Potential Immune Responses against ChR2-GFP-Expressing Neurons in Primate Cortex Show full caption (A) Nuclear DNA staining (red; To-Pro-3 stain) of slices of monkey cortex containing ChR2-GFP-expressing neurons (green). (B) Neuronal staining (red; NeuN antibody) of slices of monkey cortex containing ChR2-GFP-expressing neurons (green). (C) Validation of ChR2-GFP expression in HEK cells via western blotting, using anti-GFP antibody. From left to right, lanes show, immunostained with anti-GFP: cytosolic fraction of HEK cells transfected with ChR2-GFP plasmid, cytosolic fraction of untransfected HEK cells, membrane fraction of HEK cells transfected with ChR2-GFP plasmid, membrane fraction (diluted 1:5) of HEK cells transfected with ChR2-GFP plasmid, and membrane fractions of untransfected HEK cells. (D) Assessment of monkey serum reaction to ChR2-GFP, for monkey N (Di) and monkey A (Dii), via western blotting, comparing preinjection (left) to postinjection (right). Membrane fractions of HEK cells transfected with ChR2-GFP (left lane), membrane fractions of untransfected HEK cells (middle lane), and monkey serum samples (right lane) were incubated with monkey serum (1:50 dilution), followed by rabbit-anti-monkey secondary antibody for visualization. Given the extended duration of nonhuman primate experiments, and the prospect of using cell-specific optical neuroprosthetics for therapy, we assessed the safety of ChR2-GFP expression in primate brain. After months of ChR2-GFP expression, during which time we repeatedly illuminated neurons with blue light and successfully made recordings, we saw widespread expression of ChR2-GFP in healthy-looking neurons, with no histological abnormalities in neurons or glia, and no immune reaction at the cellular or antibody level ( Figure 2 ; see detailed text in Supplemental Data ). These multiple lines of evidence together support the safety of ChR2-GFP expression in the brain of the nonhuman primate, and if supported by further and longer-term analyses, may provide the basis for cell-specific neuromodulation therapy in humans.

Bernstein et al., 2008 Bernstein J.G.

Han X.

Henninger M.A.

Ko E.Y.

Qian X.

Franzesi G.T.

McConnell J.P.

Stern P.

Desimone R.

Boyden E.S. Prosthetic systems for therapeutic optical activation and silencing of genetically-targeted neurons. 2 radiant flux out the tip of the fiber) modulated neurons at distances over 1.2 mm away from the fiber ( Figure 3 Increases and Decreases in Neural Activity Resulting from Optical Stimulation of Excitatory Neurons Show full caption (A) Apparatus for optical activation and electrical recording. (Ai) Schematic. (Aii) Photograph, showing optical fiber (200 μm diameter) and electrode (200 μm shank diameter) in guide tubes. (B and C) Increases in spiking activity in one neuron during blue light illumination (five pulses, 20 ms duration each [B], and 1 pulse, 200 ms duration [C]). In each panel, shown at top is a spike raster plot displaying each spike as a black dot; 40 trials are shown in horizontal rows (in this and subsequent raster plots); shown at bottom is a histogram of instantaneous firing rate, averaged across all trials; bin size, 5 ms (in this and subsequent histogram plots). Periods of blue light illumination are indicated by horizontal blue dashes, in this and subsequent panels. (D and E) Decreases in spiking activity in one neuron during blue light illumination (five pulses, 20 ms duration (D), and one pulse, 200 ms duration [E]). As with (B) and (C), shown at top are spike raster plots and shown at bottom are histograms of instantaneous firing rate. (F and G) Action potential waveforms elicited during light (shown in blue, left) or occurring spontaneously in darkness (shown in black, right), for the neurons plotted in (C) and (E), respectively. (H) Instantaneous firing rate, averaged across all excited units recorded upon 200 ms blue light exposure (black line, mean; gray lines, mean ± SE; n = 50 units). (I) Relative firing rate (i.e., firing rate during the indicated period, divided by baseline firing rate) during the first 20 ms after light onset (“beginning of light”), during the period between 20 ms after light onset and 20 ms after light cessation (“steady state”), and during the 20 ms period starting 20 ms after light cessation (“after light”), for the n = 50 units shown in (H). (∗∗∗), significantly different (p < 0.0001; paired t test) from baseline rate (shown as dotted line); plotted is mean ± SE. (J) Instantaneous firing rate averaged across all suppressed units upon 200 ms blue light exposure (black line, mean; gray lines, mean ± SE; n = 20 units). (K) Relative firing rate, during the beginning of light, steady state, and after light periods, for the n = 20 units shown in (J). (L) Histogram of latencies between light onset and the earliest change in firing rate, for excited units (gray bars, n = 50 units) and suppressed units (black bars, n = 20 units); latencies longer than 50 ms were plotted in a bin labeled “>50.” (M) Histogram of time elapsed until activity recovery to baseline after light cessation, for excited (gray bars, n = 28 units) and suppressed (black bars, n = 16 units) units that had lower-than-baseline firing rates during the after light period. To assess the effect of optical activation of ChR2-expressing excitatory neurons on frontal cortical neural circuits in awake monkey, we developed a system appropriate for in vivo monkey use, coupling a fiber to a blue 473 nm laser () and assembling multiple electrodes into independently controlled drives ( Figures 3 Ai and 3Aii), which were then inserted into a single hole within the 3D printed grid ( Figure 1 C). This setup allowed us to record from neurons while exposing local cortex to pulses of blue light. In regions of cortex that were not virus labeled, we never observed light modulation of neural activity (n = 32 such sites). In regions that were virus labeled, many neurons increased their firing rate during cortical exposure to blue light ( Figures 3 B and 3C). We called these neurons “excited” units. In addition to these excited units, many neurons decreased their firing rate during cortical exposure to blue light ( Figures 3 D and 3E). We called these neurons “suppressed” units, because they did not increase firing rate during blue light exposure, but instead decreased firing rate during light delivery, even after brief illumination (i.e., a single light pulse). We hypothesized that since suppressed units decreased their firing rates without having undergone prior increases in spiking, the observed suppression was due to neural network activity, i.e., recruitment of inhibitory neurons downstream of the driven excitatory neurons. For both the excited and suppressed units, action potential waveforms elicited during light exposure were not different from waveforms observed in the dark (p > 0.1 for each of n = 15 excited single units; Kolmogorov-Smirnov test comparing waveform shapes in light versus dark; exemplars in Figures 3 F and 3G). In regions where excited or suppressed units were found, few light-nonmodulated units were observed ( Figure S5 ). These excited and suppressed units were also observed in the cortex of mice, when excitatory neurons expressing ChR2-GFP were activated by light ( Figure S3 ). Light did, however, result in a low-frequency electrical artifact on our tungsten electrodes in the brain, presumably due to the photoelectric effect; this artifact was removed from our data by high-pass filtering (see Figure S1 ). Light (80 mW/mmradiant flux out the tip of the fiber) modulated neurons at distances over 1.2 mm away from the fiber ( Figure S2 ).

In the monkey cortex, we recorded 50 excited and 20 suppressed units during illumination with 200 ms blue light pulses. Out of these 70 units, 31 were single units (15 excited, 16 suppressed) and 39 were multiunits (35 excited, 4 suppressed). We pooled multiunits and single units for analysis unless otherwise indicated. Excited and suppressed units had similar baseline firing rates (p > 0.2, t test; only single units compared) and similar waveform shapes (see Supplemental Experimental Procedures ). For excited units, firing rates increased rapidly at light onset, and then settled to a lower steady-state firing level ( Figure 3 H). For suppressed units, firing rates fell sharply after a short delay, and remained low for the duration of the light pulse ( Figure 3 J). For both excited and suppressed units, after light cessation the firing rates often dipped below baseline levels for ∼100 ms. We quantified the magnitude of these changes in firing rate during three distinct periods: the first 20 ms of light exposure (“beginning of light”), the period between 20 ms after light onset and 20 ms after light cessation (“steady state”), and during the 20 ms period starting 20 ms after light cessation (“after light”). Excited units fired at 750%, 370%, and 46% of baseline firing rate during these three periods, respectively, in each case significantly different from baseline (p < 0.0001 for each, paired t test; Figure 3 I). For single units, which yield absolute values of firing rate, excited neurons fired at 37 ± 7 Hz, 16 ± 4 Hz, and 1.3 ± 1 Hz during these three periods (mean ± standard error [SE]; n = 15 single units); baseline their firing rates was 6.5 ± 1.3 Hz. In contrast to the excited units, suppressed units did not change their firing rates relative to baseline during the beginning of light period (p > 0.5, paired t test; Figure 3 K), but reduced their firing rates by 76% and 75%, respectively, during the steady state and after light periods (significantly lower than that during baseline [p < 0.0001, paired t test], but not different from each other; p > 0.8). Suppressed single units fired at 7.4 ± 1.7 Hz, 3.1 ± 1.0 Hz, and 2.5 ± 1.2 Hz during these three periods, respectively (mean ± SE; n = 16 single units); baseline firing rate was 9.9 ± 2.0 Hz.

Boyden et al., 2005 Boyden E.S.

Zhang F.

Bamberg E.

Nagel G.

Deisseroth K. Millisecond-timescale, genetically targeted optical control of neural activity. We compared the latencies to changes in firing rate between excited versus suppressed units, and found two different, but overlapping, distributions. Excited units rapidly responded to light with latencies of 8.8 ± 0.8 ms (mean ± SE; Figure 3 L). This short latency was not different from the first-spike latency of ChR2-positive cultured pyramidal neurons responding to pulses of blue light (p > 0.6, unpaired t test; compared to published data in), consistent with the idea, but not proving, that excited units were ChR2-positive pyramidal cells. In contrast to the short latencies of excited units, suppressed units began decreasing their firing rates 30.8 ± 8.0 ms after light onset (mean ± SE), a latency significantly longer than the latency for the increases in firing rates of excited units (p < 0.0001, unpaired t test). This difference is consistent with our hypothesis that suppressed units decreased their firing rates through neural network mechanisms involving inhibitory neuron recruitment, whereas excited units were directly activated by light. After light cessation (after light period), the majority of excited and suppressed units exhibited firing rates below baseline levels (28 out of 50 excited units; 16 out of 20 suppressed units). The time for this reduced firing rate to recover to baseline was similar for excited and suppressed units ( Figure 3 M, p > 0.1, unpaired t test), consistent with the idea that suppressive influences downstream of excitatory neuron activation are mediated by a neural-network-scale phenomenon such as inhibitory neuron recruitment.