This PDF file contains the editorial “Special Section Guest Editorial: Causal Control of Biological Systems with Light” for NPh Vol. 2 Issue 03

Attracted by the appealing advantages of optogenetics, many nonhuman primate labs are attempting to incorporate this technique in their experiments. Despite some reported successes by a few groups, many still find it difficult to develop a reliable way to transduce cells in the monkey brain and subsequently monitor light-induced neuronal activity. Here, we describe a methodology that we have developed and successfully deployed on a regular basis with multiple monkeys. All devices and accessories are easy to obtain and results using these have been proven to be highly replicable. We developed the “in-chair” viral injection system and used tapered and thinner fibers for optical stimulation, which significantly improved the efficacy and reduced tissue damage. With these methods, we have successfully transduced cells in multiple monkeys in both deep and shallow cortical areas. We could reliably obtain neural modulation for months after injection, and no light-induced artifacts were observed during recordings. Further experiments using these methods have shown that optogenetic stimulation can be used to bias spatial attention in a visual choice discrimination task in a way comparable to electrical microstimulation, which demonstrates the potential use of our methods in both fundamental research and clinical applications.

Optogenetics has revolutionized neuroscience by enabling precise control of neural excitation. The development of similar optogenetics strategies in the heart is just emerging and mainly focused on pacing with light activation of channelrhodopsin-2. Here, we aimed to develop an optogenetic approach to suppress local cardiac electrical activity by using engineered cell-grafts (HEK293-cells) transfected to express the light-sensitive hyperpolarizing proton-pump archaerhodopsin-3 (Arch3). To evaluate the ability of the engineered cells to couple and modulate the electrical activity of cardiomyocytes, we co-cultured the Arch3-HEK293 cells with neonatal rat cardiomyocytes (NRCMs) or human embryonic stem cells derived cardiomyocytes (hESC-CMs). The co-cultures’ conduction and chronotropic properties were evaluated prior, during, and following application of focused monochromatic light (590 nm) using a multielectrode array mapping system. Application of focused illumination completely silenced electrical activity at the illuminated area in all NRCM co-cultures, leading to development of localized functional conduction blocks. Similarly, illumination significantly slowed spontaneous beating-rate in the hESCs-CMs co-cultures (from 86±9 to 28±4  beats/min, p<0.001). Interestingly, a transient acceleration in beating-rate was noted immediately postillumination. In conclusion, a combined gene and cell therapy approach, using light-sensitive hyperpolarizing proteins, could be used to modulate conduction and automaticity in cardiomyocyte cultures, opening the way for future optogenetic treatments for cardiac tachyarrhythmias.

Optical tissue properties limit visible light depth penetration in tissue. Because of this, the recent development of optogenetic tools was quickly followed by the development of light delivery devices for in vivo optogenetics applications. We summarize the efforts made in the last decade to design neural probes that combine conventional electrophysiological recordings and optical channel(s) for optogenetic activation, often referred to as optodes or optrodes. Several aspects including challenges for light delivery in living brain tissue, the combination of light delivery with electrophysiological recordings, probe designs, multimodality, wireless implantable system, and practical considerations guiding the choice of configuration depending on the questions one seeks to address are presented.

Implementation of optogenetic techniques is a recent addition to the neuroscientists’ preclinical research arsenal, helping to expose the intricate connectivity of the brain and allowing for on-demand direct modulation of specific neural pathways. Developing an optogenetic system requires thorough investigation of the optogenetic technique and of previously fabricated devices, which this review accommodates. Many experiments utilize bench-top systems that are bulky, expensive, and necessitate tethering to the animal. However, these bench-top systems can make use of power-demanding technologies, such as concurrent electrical recording. Newer portable microdevices and implantable systems carried by freely moving animals are being fabricated that take advantage of wireless energy harvesting to power a system and allow for natural movements that are vital for behavioral testing and analysis. An investigation of the evolution of tethered, portable, and implantable optogenetic microdevices is presented, and an analysis of benefits and detriments of each system, including optical power output, device dimensions, electrode width, and weight is given. Opsins, light sources, and optical fiber coupling are also discussed to optimize device parameters and maximize efficiency from the light source to the fiber, respectively. These attributes are important considerations when designing and developing improved optogenetic microdevices.

Dopamine (DA) release and uptake dynamics in the nucleus accumbens (NAc) have important implications for neurological diseases and mammalian animal behaviors. We demonstrate here the use of cell-type-specific optogenetic targeting in conjunction with fast-scan cyclic voltammetry applied to brain slices prepared from specifically tailored transgenic mice, which conditionally express channelrhodopsin-2 (ChR2) through dopamine transporter (DAT)-Cre. Terminal dopaminergic dynamics and the direct manipulation of induced DA release level by controlling light intensity, pulse width, and the shape of stimulation waveforms were studied. Effective cell terminal-targeting optogenetic induction of DA release at physiological levels in NAc is demonstrated and discussed. It was found that delivering more light energy by increasing stimulation intensity and length is not the only way to control DA release; the temporal shape of the stimulus waveform at light onset is also critically related to induced DA concentrations. In addition, DA uptake dynamics as well as the recovery of the presynaptic releasable DA pool are studied and modeled. More broadly, our experimental findings provide important further evidence for effectively applying optogenetics to induce neurotransmitter release in the behaviorally relevant region of the brain in a highly cell-type selective context.

Our understanding of neural information processing could potentially be advanced by combining flexible three-dimensional (3-D) neuroimaging and stimulation. Recent developments in optogenetics suggest that neurophotonic approaches are in principle highly suited for noncontact stimulation of network activity patterns. In particular, two-photon holographic optical neural stimulation (2P-HONS) has emerged as a leading approach for multisite 3-D excitation, and combining it with temporal focusing (TF) further enables axially confined yet spatially extended light patterns. Here, we study key steps toward bidirectional cell-targeted 3-D interfacing by introducing and testing a hybrid new 2P-TF-HONS stimulation path for accurate parallel optogenetic excitation into a recently developed hybrid multiphoton 3-D imaging system. The system is shown to allow targeted all-optical probing of in vitro; cortical networks expressing channelrhodopsin-2 using a regeneratively amplified femtosecond laser source tuned to 905 nm. These developments further advance a prospective new tool for studying and achieving distributed control over 3-D neuronal circuits both in vitro and in vivo.

Viral vector-mediated expression of genes (e.g., coding for opsins and designer receptors) has grown increasingly popular. Cell-type specific expression is achieved by altering viral vector tropism through crosspackaging or by cell-specific promoters driving gene expression. Detailed information about transduction properties of most recombinant adeno-associated viral vector (rAAV) serotypes in macaque cortex is gradually becoming available. Here, we compare transduction efficiencies and expression patterns of reporter genes in two macaque neocortical areas employing different rAAV serotypes and promoters. A short version of the calmodulin-kinase-II (CaMKIIα0.4) promoter resulted in reporter gene expression in cortical neurons for all tested rAAVs, albeit with different efficiencies for spread: rAAV2/5>>rAAV2/7>rAAV2/8>rAAV2/9>>rAAV2/1 and proportion of transduced cells: rAAV2/1>rAAV2/5>rAAV2/7=rAAV2/9>rAAV2/8. In contrast to rodent studies, the cytomegalovirus (CMV) promoter appeared least efficient in macaque cortex. The human synapsin-1 promoter preceded by the CMV enhancer (enhSyn1) produced homogeneous reporter gene expression across all layers, while two variants of the CaMKIIα promoter resulted in different laminar transduction patterns and cell specificities. Finally, differences in expression patterns were observed when the same viral vector was injected in two neocortical areas. Our results corroborate previous findings that reporter-gene expression patterns and efficiency of rAAV transduction depend on serotype, promoter, cortical layer, and area.

We report a functional optical coherence tomography cross-sectional scanner to detect neural activity using unmyelinated nerves dissected from squid. The nerves, unstained or stained with a voltage-sensitive dye, were imaged in a nerve chamber. Transient phase changes from backscattered light were detected during action potential propagation. The results show that the scanner can provide high spatiotemporal resolution cross-sectional images of neural activity (15  μs/A-line; 0.25  ms/B-scan; ∼8.5×5.5  μm² in xz). The advantage of this method compared to monitoring a single depth profile z is a dramatic increase in the number of available sites that can be measured in two spatial dimensions xz with lateral scanning; therefore, the study demonstrates that two-dimensional monitoring of small-scale functional activity would also be feasible.

Functional brain imaging has become an important neuroimaging technique for the study of brain organization and development. Compared to other imaging techniques, diffuse optical tomography (DOT) is a portable and low-cost technique that can be applied to infants and hospitalized patients using an atlas-based light model. For DOT imaging, the accuracy of the forward model has a direct effect on the resulting recovered brain function within a field of view and so the accuracy of the spatially normalized atlas-based forward models must be evaluated. Herein, the accuracy of atlas-based DOT is evaluated on models that are spatially normalized via a number of different rigid registration methods on 24 subjects. A multileveled approach is developed to evaluate the correlation of the geometrical and sensitivity accuracies across the full field of view as well as within specific functional subregions. Results demonstrate that different registration methods are optimal for recovery of different sets of functional brain regions. However, the “nearest point to point” registration method, based on the EEG 19 landmark system, is shown to be the most appropriate registration method for image quality throughout the field of view of the high-density cap that covers the whole of the optically accessible cortex.

Optical recording facilitates monitoring the activity of a large neural network at the cellular scale, but the analysis and interpretation of the collected data remain challenging. Here, we present a MATLAB-based toolbox, named NeuroCa, for the automated processing and quantitative analysis of large-scale calcium imaging data. Our tool includes several computational algorithms to extract the calcium spike trains of individual neurons from the calcium imaging data in an automatic fashion. Two algorithms were developed to decompose the imaging data into the activity of individual cells and subsequently detect calcium spikes from each neuronal signal. Applying our method to dense networks in dissociated cultures, we were able to obtain the calcium spike trains of ∼1000 neurons in a few minutes. Further analyses using these data permitted the quantification of neuronal responses to chemical stimuli as well as functional mapping of spatiotemporal patterns in neuronal firing within the spontaneous, synchronous activity of a large network. These results demonstrate that our method not only automates time-consuming, labor-intensive tasks in the analysis of neural data obtained using optical recording techniques but also provides a systematic way to visualize and quantify the collective dynamics of a network in terms of its cellular elements.

We introduce and validate a pressure measurement paradigm that reduces extracerebral contamination from superficial tissues in optical monitoring of cerebral blood flow with diffuse correlation spectroscopy (DCS). The scheme determines subject-specific contributions of extracerebral and cerebral tissues to the DCS signal by utilizing probe pressure modulation to induce variations in extracerebral blood flow. For analysis, the head is modeled as a two-layer medium and is probed with long and short source-detector separations. Then a combination of pressure modulation and a modified Beer-Lambert law for flow enables experimenters to linearly relate differential DCS signals to cerebral and extracerebral blood flow variation without a priori anatomical information. We demonstrate the algorithm’s ability to isolate cerebral blood flow during a finger-tapping task and during graded scalp ischemia in healthy adults. Finally, we adapt the pressure modulation algorithm to ameliorate extracerebral contamination in monitoring of cerebral blood oxygenation and blood volume by near-infrared spectroscopy.

Autonomic nervous system response is known to be highly task-dependent. The sensitivity of near-infrared spectroscopy (NIRS) measurements to superficial layers, particularly to the scalp, makes it highly susceptible to systemic physiological changes. Thus, one critical step in NIRS data processing is to remove the contribution of superficial layers to the NIRS signal and to obtain the actual brain response. This can be achieved using short separation channels that are sensitive only to the hemodynamics in the scalp. We investigated the contribution of hemodynamic fluctuations due to autonomous nervous system activation during various tasks. Our results provide clear demonstrations of the critical role of using short separation channels in NIRS measurements to disentangle differing autonomic responses from the brain activation signal of interest.

Hypothermia (HT) is a potent neuroprotective therapy that is now widely used in following neurological emergencies, such as neonatal asphyxia. An important mechanism of HT-induced neuroprotection is attributed to the associated reduction in the cerebral metabolic rate of oxygen (CMRO₂). Since cerebral circulation and metabolism are tightly regulated, reduction in CMRO₂ typically results in decreased cerebral blood flow (CBF); it is only under oxidative stress, e.g., hypoxia-ischemia, that oxygen extraction fraction (OEF) deviates from its basal value, which can lead to cerebral dysfunction. As such, it is critical to measure these key physiological parameters during therapeutic HT. This report investigates a noninvasive method of measuring the coupling of CMRO₂ and CBF under HT and different anesthetic combinations of propofol/nitrous-oxide (N₂O) that may be used in clinical practice. Both CBF and CMRO₂ decreased with decreasing temperature, but the OEF remained unchanged, which indicates a tight coupling of flow and metabolism under different anesthetics and over the mild HT temperature range (38°C to 33°C).