Hearing ability of prairie voles (Microtus ochrogaster).

The hearing abilities of mammals are impacted by factors such as social cues, habitat, and physical characteristics. Despite being used commonly to study social behaviors, hearing of the monogamous prairie vole (Microtus ochrogaster) has never been characterized. In this study, anatomical features are measured and auditory brainstem responses (ABRs) are used to measure auditory capabilities of prairie voles, characterizing monaural and binaural hearing and hearing range. Sexually naive male and female voles were measured to characterize differences due to sex. It was found that prairie voles show a hearing range with greatest sensitivity between 8 and 32 kHz, binaural hearing across interaural time difference ranges appropriate for their head sizes. No differences are shown between the sexes in binaural hearing or hearing range (except at 1 kHz), however, female voles have increased amplitude of peripheral ABR waves I and II and longer latency of waves III and IV compared to males. The results confirm that prairie voles have a broad hearing range, binaural hearing consistent with rodents of similar size, and differences in amplitudes and thresholds of monaural physiological measures between the sexes. These data further highlight the necessity to understand sex-specific differences in neural processing that may underly variability in responses between sexes.

Double-Sided Sapphire Optrodes with Conductive Shielding Layers to Reduce Optogenetic Stimulation Artifacts.

Optrodes, which are single shaft neural probes integrated with microelectrodes and optical light sources, offer a remarkable opportunity to simultaneously record and modulate neural activities using light within an animal's brain; however, a common problem with optrodes is that stimulation artifacts can be observed in the neural recordings of microelectrodes when the light source on the optrode is activated. These stimulation artifacts are undesirable contaminants, and they cause interpretation complexity when analyzing the recorded neural activities. In this paper, we tried to mitigate the effects of the stimulation artifacts by developing a low-noise, double-sided optrode integrated with multiple Electromagnetic Shielding (EMS) layers. The LED and microelectrodes were constructed separately on the top epitaxial and bottom substrate layers, and EMS layers were used to separate the microelectrodes and LED to reduce signal cross-talks. Compared with conventional single-sided designs, in which the LED and microelectrodes are constructed on the same side, our results indicate that double-sided optrodes can significantly reduce the presence of stimulation artifacts. In addition, the presence of stimulation artifacts can further be reduced by decreasing the voltage difference and increasing the rise/fall time of the driving LED pulsed voltage. With all these strategies, the presence of stimulation artifacts was significantly reduced by ~76%. As well as stimulation suppression, the sapphire substrate also provided strong mechanical stiffness and support to the optrodes, as well as improved electronic stability, thus making the double-sided sapphire optrodes highly suitable for optogenetic neuroscience research on animal models.

Predicting the Influence of Axon Myelination on Sound Localization Precision Using a Spiking Neural Network Model of Auditory Brainstem.

Spatial hearing allows animals to rapidly detect and localize auditory events in the surrounding environment. The auditory brainstem plays a central role in processing and extracting binaural spatial cues through microsecond-precise binaural integration, especially for detecting interaural time differences (ITDs) of low-frequency sounds at the medial superior olive (MSO). A series of mechanisms exist in the underlying neural circuits for preserving accurate action potential timing across multiple fibers, synapses and nuclei along this pathway. One of these is the myelination of afferent fibers that ensures reliable and temporally precise action potential propagation in the axon. There are several reports of fine-tuned myelination patterns in the MSO circuit, but how specifically myelination influences the precision of sound localization remains incompletely understood. Here we present a spiking neural network (SNN) model of the Mongolian gerbil auditory brainstem with myelinated axons to investigate whether different axon myelination thicknesses alter the sound localization process. Our model demonstrates that axon myelin thickness along the contralateral pathways can substantially modulate ITD detection. Furthermore, optimal ITD sensitivity is reached when the MSO receives contralateral inhibition via thicker myelinated axons compared to contralateral excitation, a result that is consistent with previously reported experimental observations. Our results suggest specific roles of axon myelination for extracting temporal dynamics in ITD decoding, especially in the pathway of the contralateral inhibition.

Axonal Conduction Delay Shapes the Precision of the Spatial Hearing in A Spiking Neural Network Model of Auditory Brainstem.

One method by which the mammalian sound localization pathway localizes sound sources is by analyzing the microsecond-level difference between the arrival times of a sound at the two ears. However, how the neural circuits in the auditory brainstem precisely integrate signals from the two ears, and what the underlying mechanisms are, remains to be understood. Recent studies have reported that variations of axon myelination in the auditory brainstem produces various axonal conduction velocities and sophisticated temporal dynamics, which have not been well characterized in most existing models of sound localization circuits. Here, we present a spiking neural network model of the auditory brainstem to investigate how axon myelinations affect the precision of sound localization. Sound waves with different interaural time differences (ITDs) are encoded and used as stimuli, and the axon properties in the network are adjusted, and the corresponding axonal conduction delays are computed with a multi-compartment axon model. Through the simulation, the sensitivity of ITD perception varies with the myelin thickness of axons in the contralateral input pathways to the medial superior olive (MSO). The ITD perception becomes more precise when the contralateral inhibitory input propagates faster than the contralateral excitatory input. These results indicate that axon myelination and contralateral spike timing influence spatial hearing perception.

Influence of the Surface Material and Illumination upon the Performance of a Microelectrode/Electrolyte Interface in Optogenetics.

Integrated optrodes for optogenetics have been becoming a significant tool in neuroscience through the combination of offering accurate stimulation to target cells and recording biological signals simultaneously. This makes it not just be widely used in neuroscience researches, but also have a great potential to be employed in future treatments in clinical neurological diseases. To optimize the integrated optrodes, this paper aimed to investigate the influence of surface material and illumination upon the performance of the microelectrode/electrolyte interface and build a corresponding evaluation system. In this work, an integrated planar optrode with a blue LED and microelectrodes was designed and fabricated. The charge transfer mechanism on the interface was theoretically modeled and experimentally verified. An evaluation system for assessing microelectrodes was also built up. Using this system, the proposed model of various biocompatible surface materials on microelectrodes was further investigated under different illumination conditions. The influence of illumination on the microelectrode/electrolyte interface was the cause of optical artifacts, which interfere the biological signal recording. It was found that surface materials had a great effect on the charge transfer capacity, electrical stability and recoverability, photostability, and especially optical artifacts. The metal with better charge transfer capacity and electrical stability is highly possible to have a better performance on the optical artifacts, regardless of its electrical recoverability and photostability under the illumination conditions of optogenetics. Among the five metals used in our investigation, iridium served as the best surface material for the proposed integrated optrodes. Thus, optimizing the surface material for optrodes could reduce optical interference, enhance the quality of the neural signal recording for optogenetics, and thus help to advance the research in neuroscience.

Oxytocin modulates neural processing of mitral/tufted cells in the olfactory bulb.

AIM: Oxytocin plays an important role in social recognition in rodents, which is mediated predominantly by the olfactory system. Although oxytocin modulates neural activity in the olfactory bulb, the underlying mechanism is largely unknown. Here, we studied how direct infusion of oxytocin into the olfactory bulb affect social interactions in mice and modulate the neural activity of mitral/tufted cells in the olfactory bulb. METHODS: A three-chamber social interaction test was used in the behavioural test. For in vivo studies, single unit recordings, local field potential recordings and fibre photometry recordings were used to record the neural activity of olfactory bulb. For in vitro studies, we performed patch clamp recordings in the slice of the olfactory bulb. RESULTS: Behaviourally, direct oxytocin infusion in olfactory bulb increased performance in a social interaction task. Moreover, odour-evoked responses of mitral/tufted cells and neural discrimination of odours were both enhanced by oxytocin, whereas the spontaneous firing rate of mitral/tufted cells was reduced. At the neural network level, oxytocin decreased the amplitude of odour-evoked high gamma responses. At the cell population level, oxytocin decreased odour-evoked calcium responses (reflecting neural activity) specifically in granule cells. Moreover, in vitro slice recordings revealed that the inhibitory effect of oxytocin on mitral cell activity is mediated mainly by modulation of ATP-sensitive potassium channels and involves the oxytocin receptor-Gq-PLC-IP3 signalling pathway. CONCLUSION: Oxytocin modulates social interaction, likely by increasing the signal-to-noise ratio of odour responses in mitral cells which is partly through ATP-sensitive potassium channel.

Robotic stereotaxic system based on 3D skull reconstruction to improve surgical accuracy and speed.

BACKGROUND: Some experimental approaches in neuroscience research require the precise placement of a recording electrode, pipette or other tool into a specific brain area that can be quite small and/or located deep beneath the surface. This process is typically aided with stereotaxic methods but remains challenging due to a lack of advanced technology to aid the experimenter. Currently, procedures require a significant amount of skill, have a high failure rate, and take up a significant amount of time. NEW METHOD: We developed a next generation robotic stereotaxic platform for small rodents by combining a three-dimensional (3D) skull profiler sub-system and a full six degree-of-freedom (6DOF) robotic platform. The 3D skull profiler is based on structured illumination in which a series of horizontal and vertical line patterns are projected onto an animal skull. These patterns are captured by two two-dimensional (2D) CCD cameras which reconstruct an accurate 3D skull surface profile based on structured illumination and geometrical triangulation. Using the reconstructed 3D profile, the skull can be repositioned using a 6DOF robotic platform to accurately align a surgical tool. RESULTS: The system was evaluated using mechanical measurement techniques, and the accuracy of the platform was demonstrated using agar brain phantoms and animal skulls. Additionally, a small and deep brain nucleus (the medial nucleus of the trapezoid body) were targeted in rodents to confirm the targeting accuracy. CONCLUSIONS: The new stereotaxic system can accomplish "skull-flat" rapidly and precisely and with minimal user intervention, and thus reduces the failure rate of such experiments.

Evaluation of lensed fibers used in photodynamic therapy (PDT).

BACKGROUND: The combination of diode laser and lensed optical fiber is a commonly used technique in photodynamic therapy (PDT). The design of the lensed fiber can affect the beam quality, therefore, directly or indirectly effect treatment outcomes. In this study, several commercially available microlens and convex lens fibers used in PDT were evaluated. MATERIALS AND METHODS: A total of five types of lensed fibers were evaluated. The beam uniformity, expansion, and transmittance properties of lensed fibers to laser beams of 532 nm and 630 nm were analyzed. In addition, the spectral transmissions of lensed fibers were also examined using a broad band light source (400-1000 nm). RESULTS: Using microlens alone could significantly improve the light beam uniformity. The combination of lens and macrobendings could further improve the light beam uniformity. The beam expansion effect was determined by the numerical aperture (NA) of lens. The microlens fibers of higher NA showed better beam expansion effect than the convex lens of lower NA. The overall transmittance of tested lensed fibers ranged between 70 % and 77 %. CONCLUSIONS: Tested fibers showed good transmission property to the tested PDT wavelengths. Generally speaking, utilizing macrobending could improve the transmission quality of lens based frontal fiber-optic light distributors in PDT applications.

Low-latency single channel real-time neural spike sorting system based on template matching.

Recent technical advancements in neural engineering allow for precise recording and control of neural circuits simultaneously, opening up new opportunities for closed-loop neural control. In this work, a rapid spike sorting system was developed based on template matching to rapidly calculate instantaneous firing rates for each neuron in a multi-unit extracellular recording setting. Cluster templates were first generated by a desktop computer using a non-parameter spike sorting algorithm (Super-paramagnetic clustering) and then transferred to a field-programmable gate array digital circuit for rapid sorting through template matching. Two different matching techniques-Euclidean distance (ED) and correlational matching (CM)-were compared for the accuracy of sorting and the performance of calculating firing rates. The performance of the system was first verified using publicly available artificial data and was further confirmed with pre-recorded neural spikes from an anesthetized Mongolian gerbil. Real-time recording and sorting from an awake mouse were also conducted to confirm the system performance in a typical behavioral neuroscience experimental setting. Experimental results indicated that high sorting accuracies were achieved for both template-matching methods, but CM can better handle spikes with non-Gaussian spike distributions, making it more robust for in vivo recording. The technique was also compared to several other off-line spike sorting algorithms and the results indicated that the sorting accuracy is comparable but sorting time is significantly shorter than these other techniques. A low sorting latency of under 2 ms and a maximum spike sorting rate of 941 spikes/second have been achieved with our hybrid hardware/software system. The low sorting latency and fast sorting rate allow future system developments of neural circuit modulation through analyzing neural activities in real-time.

Computationally inexpensive enhanced growing neural gas algorithm for real-time adaptive neural spike clustering.

OBJECTIVE: Real-time closed-loop neural feedback control requires the analysis of action potential traces within several milliseconds after they have been recorded from the brain. The current generation of spike clustering algorithms were mostly designed for off-line use and also require a significant amount of computational resources. A new spike clustering algorithm, termed 'enhanced growing neural gas (EGNG)', was therefore developed that is computationally lightweight and memory conserving. The EGNG algorithm can adapt to changes of the electrophysiological recording environment and can classify both pre-recorded and streaming action potentials. APPROACH: The algorithm only uses a small number of EGNG nodes and edges to learn the neural spike distributions which eliminates the need of retaining the neural data in the system memory to conserve computational resources. Most of the computations revolve around calculating Euclidian distances, which is computationally inexpensive and can be implemented in parallel using digital circuit technology. MAIN RESULTS: EGNG was evaluated off-line using both synthetic and pre-recorded neural spikes. Streaming synthetic neural spikes were also used to evaluate the ability of EGNG to classify action potentials in real-time. The algorithm was also implemented in hardware with a Field Programming Gate Array (FPGA) chip, and the worst-case clustering latency was 3.10 µs, allowing a minimum of 322 580 neural spikes to be clustered per second. SIGNIFICANCE: The EGNG algorithm provides a viable solution to classification of neural spikes in real-time and can be implemented with limited computational resources as a front-end spike clustering unit for future tethered-free and miniaturized closed-loop neural feedback systems.

Quantitative assessment of skin swelling using optical coherence tomography.

BACKGROUND: Preclinical and clinical studies suggest that optical coherence tomography (OCT) is a useful tool to visualize inflammatory conditions. OBJECTIVE: The objective of this study was to evaluate the usefulness of combining OCT and various image processing techniques for quantitative assessment of histamine-induced tissue swelling. METHODS: Both time-domain and frequency-domain OCT were used on a mouse ear model. The ear thickness and volume before and after histamine challenge were determined from pixel locations in 2D scans and voxel number counting in 3D scans. Swelling kinetics was analyzed on 3D contour mapping. Microvessel network was visualized using speckle decorrelation analysis. RESULTS: OCT images showed that the thickness and volume changes were histamine dose and contact time dependent. The 3D mapping showed that the histamine-induced swelling spread slowly and directionally. OCT data indicated that microvessel opening and vessel dilation occurred prior to tissue swelling. CONCLUSION: OCT is a robust and quantitative non-invasive imaging tool for assessing skin swelling.

An Integrated Circuit for Simultaneous Extracellular Electrophysiology Recording and Optogenetic Neural Manipulation.

OBJECTIVE: The ability to record and to control action potential firing in neuronal circuits is critical to understand how the brain functions. The objective of this study is to develop a monolithic integrated circuit (IC) to record action potentials and simultaneously control action potential firing using optogenetics. METHODS: A low-noise and high input impedance (or low input capacitance) neural recording amplifier is combined with a high current laser/light-emitting diode (LED) driver in a single IC. RESULTS: The low input capacitance of the amplifier (9.7 pF) was achieved by adding a dedicated unity gain stage optimized for high impedance metal electrodes. The input referred noise of the amplifier is [Formula: see text], which is lower than the estimated thermal noise of the metal electrode. Thus, the action potentials originating from a single neuron can be recorded with a signal-to-noise ratio of at least 6.6. The LED/laser current driver delivers a maximum current of 330 mA, which is adequate for optogenetic control. The functionality of the IC was tested with an anesthetized Mongolian gerbil and auditory stimulated action potentials were recorded from the inferior colliculus. Spontaneous firings of fifth (trigeminal) nerve fibers were also inhibited using the optogenetic protein Halorhodopsin. Moreover, a noise model of the system was derived to guide the design. SIGNIFICANCE: A single IC to measure and control action potentials using optogenetic proteins is realized so that more complicated behavioral neuroscience research and the translational neural disorder treatments become possible in the future.

Three-Dimensional Segmentation and Quantitative Measurement of the Aqueous Outflow System of Intact Mouse Eyes Based on Spectral Two-Photon Microscopy Techniques.

PURPOSE: To visualize and quantify the three-dimensional (3D) spatial relationships of the structures of the aqueous outflow system (AOS) within intact enucleated mouse eyes using spectral two-photon microscopy (TPM) techniques. METHODS: Spectral TPM, including two-photon autofluorescence (TPAF) and second-harmonic generation (SHG), were used to image the small structures of the AOS within the limbal region of freshly enucleated mouse eyes. Long infrared excitation wavelengths (930 nm) were used to reduce optical scattering and autofluorescent background. Image stacks were collected for 3D image rendering and structural segmentation. For anatomical reference, vascular perfusion with fluorescent-conjugated dextran (150 KDa) and trans-corneal perfusion with 0.1 µm fluorescent polystyrene beads were separately performed to identify the episcleral veins (EV) and the trabecular meshwork (TM) and Schlemm's canal (SC), respectively. RESULTS: Three-dimensional rendering and segmentation of spectral two-photon images revealed detailed structures of the AOS, including SC, collector channels (CC), and aqueous veins (AV). The collagen of the TM was detected proximal to SC. The long and short axes of the SC were 82.2 ± 22.2 µm and 6.7 ± 1.4 µm. The diameters of the CC averaged 25.6 ± 7.9 µm where they originated from the SC (ostia), enlarged to 34.1 ± 13.1 µm at the midpoint, and narrowed to 18.3 ± 4.8 µm at the junction of the AV. The diameter of the AV averaged 12.5 ± 3.4 µm. CONCLUSIONS: Spectral TPM can be used to reconstruct and measure the spatial relationships of both large and small AOS structures, which will allow for better understanding of distal aqueous outflow dynamics.

Measurement of colloidal phenomena during flow through refractive index matched porous media.

Colloidal phenomena in porous media, natural or engineered, are important in a breadth of science and technology applications, but fundamental understanding is hampered by the difficulty in measuring colloid deposit morphology in situ. To partially address this need, this paper describes a static light scattering apparatus using a flow cell filled with refractive index matched (RIM) porous media, allowing real-time measurement of colloidal phenomena as a function of depth within the flow cell. A laser interacts with the colloids in the pore space and their structures, but not with the RIM media. The intensity of scattered light is measured as a function of scattering angle, which allows characterization of colloid deposit morphology as a fractal dimension and a radius of gyration. In parallel, fluid discharge rate and pressure drop are recorded to determine permeability, a key parameter for any application involving flow through porous media. This apparatus should prove useful in any application requiring characterization of colloidal phenomena within porous media. Additionally, this paper describes how to use granular Nafion as RIM porous media.

A recording chamber for small volume slice electrophysiology.

Electrophysiological recordings from brain slices are typically performed in small recording chambers that allow for the superfusion of the tissue with artificial extracellular solution (ECS), while the chamber holding the tissue is mounted in the optical path of a microscope to image neurons in the tissue. ECS itself is inexpensive, and thus superfusion rates and volumes of ECS consumed during an experiment using standard ECS are not critical. However, some experiments require the addition of expensive pharmacological agents or other chemical compounds to the ECS, creating a need to build superfusion systems that operate on small volumes while still delivering appropriate amounts of oxygen and other nutrients to the tissue. We developed a closed circulation tissue chamber for slice recordings that operates with small volumes of bath solution in the range of 1.0 to 2.6 ml and a constant oxygen/carbon dioxide delivery to the solution in the bath. In our chamber, the ECS is oxygenated and recirculated directly in the recording chamber, eliminating the need for tubes and external bottles/containers to recirculate and bubble ECS and greatly reducing the total ECS volume required for superfusion. At the same time, the efficiency of tissue oxygenation and health of the section are comparable to standard superfusion methods. We also determined that the small volume of ECS contains a sufficient amount of nutrients to support the health of a standard brain slice for several hours without concern for either depletion of nutrients or accumulation of waste products.

Third harmonic generation microscopy of a mouse retina.

PURPOSE: To demonstrate lipid-specific imaging of the retina through the use of third harmonic generation (THG), a multiphoton microscopic technique in which tissue contrast is generated from optical inhomogeneities. METHODS: A custom fiber laser and multiphoton microscope was constructed and optimized for simultaneous two-photon autofluorescence (TPAF) and THG retinal imaging. Imaging was performed using fixed-frozen sections of mouse eyes without the use of exogenous fluorescent dyes. In parallel experiments, a fluorescent nuclear stain was used to verify the location of the retinal cell nuclei. RESULTS: Simultaneous THG and TPAF images revealed all retinal layers with subcellular resolution. In BALB/c strains, the THG signal stems from the lipidic organelles of the cellular and nuclear membranes. In the C57BL/6 strain, the THG signal from the RPE cells originates from the pigmented granules. CONCLUSIONS: THG microscopy can be used to image structures of the mouse retina using contrast inherent to the tissue and without the use of a fluorescent dye or exogenously expressed recombinant protein.

Circuit models and experimental noise measurements of micropipette amplifiers for extracellular neural recordings from live animals.

Glass micropipettes are widely used to record neural activity from single neurons or clusters of neurons extracellularly in live animals. However, to date, there has been no comprehensive study of noise in extracellular recordings with glass micropipettes. The purpose of this work was to assess various noise sources that affect extracellular recordings and to create model systems in which novel micropipette neural amplifier designs can be tested. An equivalent circuit of the glass micropipette and the noise model of this circuit, which accurately describe the various noise sources involved in extracellular recordings, have been developed. Measurement schemes using dead brain tissue as well as extracellular recordings from neurons in the inferior colliculus, an auditory brain nucleus of an anesthetized gerbil, were used to characterize noise performance and amplification efficacy of the proposed micropipette neural amplifier. According to our model, the major noise sources which influence the signal to noise ratio are the intrinsic noise of the neural amplifier and the thermal noise from distributed pipette resistance. These two types of noise were calculated and measured and were shown to be the dominating sources of background noise for in vivo experiments.

Imaging the effects of prostaglandin analogues on cultured trabecular meshwork cells by coherent anti-stokes Raman scattering.

PURPOSE: The aim of this study was to nondestructively monitor morphological changes to the lipid membranes of primary cultures of living human trabecular meshwork cells (hTMC) without the application of exogenous label. METHODS: Live hTMC were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). The hTMC were treated with a commercial formulation of latanoprost (0.5 µg/mL) for 24 hours before imaging. Untreated cells and cells treated with vehicle containing the preservative benzalkonium chloride (BAK; 2 µg/mL) were imaged as controls. After CARS/TPAF imaging, hTMC were fixed, stained with the fluorescent lipid dye Nile Red, and imaged by conventional confocal microscopy to verify lipid membrane structures. RESULTS: Analysis of CARS/TPAF images of hTMC treated with latanoprost revealed multiple intracellular lipid membranes absent from untreated or BAK-treated hTMC. Treatment of hTMC with sodium fluoride or ouabain, agents shown to cause morphological changes to hTMC, also did not induce formation of intracellular lipid membranes. CONCLUSIONS: CARS microscopy detected changes in living hTMC morphology that were validated by subsequent histological stain. Prostaglandin-induced changes to hTMC involved rearrangement of lipid membranes within these cells. These in vitro results identify a novel biological response to a class of antiglaucoma drugs, and further experiments are needed to establish how this effect is involved in the hypotensive action of prostaglandin analogues in vivo.

Imaging the intact mouse cornea using coherent anti-stokes Raman scattering (CARS).

PURPOSE: The aim of this study was to image the cellular and noncellular structures of the cornea and limbus in an intact mouse eye using the vibrational oscillation of the carbon-hydrogen bond in lipid membranes and autofluorescence as label-free contrast agents. METHODS: Freshly enucleated mouse eyes were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). Sequential images were collected through the full thickness of the cornea and limbal regions. Line scans along the transverse/sagittal axes were also performed. RESULTS: Analysis of multiple CARS/TPAF images revealed that corneal epithelial and endothelial cells could be identified by the lipid-rich plasma membrane CARS signal. The fluorescent signal from the collagen fibers of the corneal stroma was evident in the TPAF channel. The transition from the cornea to sclera at the limbus was marked by a change in collagen pattern (TPAF channel) and thickness of surface cells (CARS channel). Regions within the corneal stroma that lack collagen autofluorescence coincided with CARS signal, indicating the presence of stromal fibroblasts or nerve fibers. CONCLUSIONS: The CARS technique was successful in imaging cells in the intact mouse eye, both at the surface and within corneal tissue. Multiphoton images were comparable to histologic sections. The methods described here represent a new avenue for molecular specific imaging of the mouse eye. The lack of need for tissue fixation is unique compared with traditional histology imaging techniques.

Light scattering properties vary across different regions of the adult mouse brain.

Recently developed optogenetic tools provide powerful approaches to optically excite or inhibit neural activity. In a typical in-vivo experiment, light is delivered to deep nuclei via an implanted optical fiber. Light intensity attenuates with increasing distance from the fiber tip, determining the volume of tissue in which optogenetic proteins can successfully be activated. However, whether and how this volume of effective light intensity varies as a function of brain region or wavelength has not been systematically studied. The goal of this study was to measure and compare how light scatters in different areas of the mouse brain. We delivered different wavelengths of light via optical fibers to acute slices of mouse brainstem, midbrain and forebrain tissue. We measured light intensity as a function of distance from the fiber tip, and used the data to model the spread of light in specific regions of the mouse brain. We found substantial differences in effective attenuation coefficients among different brain areas, which lead to substantial differences in light intensity demands for optogenetic experiments. The use of light of different wavelengths additionally changes how light illuminates a given brain area. We created a brain atlas of effective attenuation coefficients of the adult mouse brain, and integrated our data into an application that can be used to estimate light scattering as well as required light intensity for optogenetic manipulation within a given volume of tissue.