Mind In Vitro Platforms: Versatile, Scalable, Robust, and Open Solutions to Interfacing with Living Neurons.

Motivated by the unexplored potential of in vitro neural systems for computing and by the corresponding need of versatile, scalable interfaces for multimodal interaction, an accurate, modular, fully customizable, and portable recording/stimulation solution that can be easily fabricated, robustly operated, and broadly disseminated is presented. This approach entails a reconfigurable platform that works across multiple industry standards and that enables a complete signal chain, from neural substrates sampled through micro-electrode arrays (MEAs) to data acquisition, downstream analysis, and cloud storage. Built-in modularity supports the seamless integration of electrical/optical stimulation and fluidic interfaces. Custom MEA fabrication leverages maskless photolithography, favoring the rapid prototyping of a variety of configurations, spatial topologies, and constitutive materials. Through a dedicated analysis and management software suite, the utility and robustness of this system are demonstrated across neural cultures and applications, including embryonic stem cell-derived and primary neurons, organotypic brain slices, 3D engineered tissue mimics, concurrent calcium imaging, and long-term recording. Overall, this technology, termed "mind in vitro" to underscore the computing inspiration, provides an end-to-end solution that can be widely deployed due to its affordable (>10× cost reduction) and open-source nature, catering to the expanding needs of both conventional and unconventional electrophysiology.

A robust and compact population code for competing sounds in auditory cortex.

Cortical circuits encoding sensory information consist of populations of neurons, yet how information aggregates via pooling individual cells remains poorly understood. Such pooling may be particularly important in noisy settings where single-neuron encoding is degraded. One example is the cocktail party problem, with competing sounds from multiple spatial locations. How populations of neurons in auditory cortex code competing sounds have not been previously investigated. Here, we apply a novel information-theoretic approach to estimate information in populations of neurons in mouse auditory cortex about competing sounds from multiple spatial locations, including both summed population (SP) and labeled line (LL) codes. We find that a small subset of neurons is sufficient to nearly maximize mutual information over different spatial configurations, with the labeled line code outperforming the summed population code and approaching information levels attained in the absence of competing stimuli. Finally, information in the labeled line code increases with spatial separation between target and masker, in correspondence with behavioral results on spatial release from masking in humans and animals. Taken together, our results reveal that a compact population of neurons in auditory cortex provides a robust code for competing sounds from different spatial locations.NEW & NOTEWORTHY Little is known about how populations of neurons within cortical circuits encode sensory stimuli in the presence of competing stimuli at other spatial locations. Here, we investigate this problem in auditory cortex using a recently proposed information-theoretic approach. We find a small subset of neurons nearly maximizes information about target sounds in the presence of competing maskers, approaching information levels for isolated stimuli, and provides a noise-robust code for sounds in a complex auditory scene.

Theta and gamma rhythmic coding through two spike output modes in the hippocampus during spatial navigation.

Hippocampal CA1 neurons generate single spikes and stereotyped bursts of spikes. However, it is unclear how individual neurons dynamically switch between these output modes and whether these two spiking outputs relay distinct information. We performed extracellular recordings in spatially navigating rats and cellular voltage imaging and optogenetics in awake mice. We found that spike bursts are preferentially linked to cellular and network theta rhythms (3-12 Hz) and encode an animal's position via theta phase precession, particularly as animals are entering a place field. In contrast, single spikes exhibit additional coupling to gamma rhythms (30-100 Hz), particularly as animals leave a place field. Biophysical modeling suggests that intracellular properties alone are sufficient to explain the observed input frequency-dependent spike coding. Thus, hippocampal neurons regulate the generation of bursts and single spikes according to frequency-specific network and intracellular dynamics, suggesting that these spiking modes perform distinct computations to support spatial behavior.

Parvalbumin neurons enhance temporal coding and reduce cortical noise in complex auditory scenes.

Cortical representations supporting many cognitive abilities emerge from underlying circuits comprised of several different cell types. However, cell type-specific contributions to rate and timing-based cortical coding are not well-understood. Here, we investigated the role of parvalbumin neurons in cortical complex scene analysis. Many complex scenes contain sensory stimuli which are highly dynamic in time and compete with stimuli at other spatial locations. Parvalbumin neurons play a fundamental role in balancing excitation and inhibition in cortex and sculpting cortical temporal dynamics; yet their specific role in encoding complex scenes via timing-based coding, and the robustness of temporal representations to spatial competition, has not been investigated. Here, we address these questions in auditory cortex of mice using a cocktail party-like paradigm, integrating electrophysiology, optogenetic manipulations, and a family of spike-distance metrics, to dissect parvalbumin neurons' contributions towards rate and timing-based coding. We find that suppressing parvalbumin neurons degrades cortical discrimination of dynamic sounds in a cocktail party-like setting via changes in rapid temporal modulations in rate and spike timing, and over a wide range of time-scales. Our findings suggest that parvalbumin neurons play a critical role in enhancing cortical temporal coding and reducing cortical noise, thereby improving representations of dynamic stimuli in complex scenes.

Striatal cholinergic interneuron membrane voltage tracks locomotor rhythms in mice.

Rhythmic neural network activity has been broadly linked to behavior. However, it is unclear how membrane potentials of individual neurons track behavioral rhythms, even though many neurons exhibit pace-making properties in isolated brain circuits. To examine whether single-cell voltage rhythmicity is coupled to behavioral rhythms, we focused on delta-frequencies (1-4 Hz) that are known to occur at both the neural network and behavioral levels. We performed membrane voltage imaging of individual striatal neurons simultaneously with network-level local field potential recordings in mice during voluntary movement. We report sustained delta oscillations in the membrane potentials of many striatal neurons, particularly cholinergic interneurons, which organize spikes and network oscillations at beta-frequencies (20-40 Hz) associated with locomotion. Furthermore, the delta-frequency patterned cellular dynamics are coupled to animals' stepping cycles. Thus, delta-rhythmic cellular dynamics in cholinergic interneurons, known for their autonomous pace-making capabilities, play an important role in regulating network rhythmicity and movement patterning.

Hearing in Complex Environments: Auditory Gain Control, Attention, and Hearing Loss.

Listening in noisy or complex sound environments is difficult for individuals with normal hearing and can be a debilitating impairment for those with hearing loss. Extracting meaningful information from a complex acoustic environment requires the ability to accurately encode specific sound features under highly variable listening conditions and segregate distinct sound streams from multiple overlapping sources. The auditory system employs a variety of mechanisms to achieve this auditory scene analysis. First, neurons across levels of the auditory system exhibit compensatory adaptations to their gain and dynamic range in response to prevailing sound stimulus statistics in the environment. These adaptations allow for robust representations of sound features that are to a large degree invariant to the level of background noise. Second, listeners can selectively attend to a desired sound target in an environment with multiple sound sources. This selective auditory attention is another form of sensory gain control, enhancing the representation of an attended sound source while suppressing responses to unattended sounds. This review will examine both "bottom-up" gain alterations in response to changes in environmental sound statistics as well as "top-down" mechanisms that allow for selective extraction of specific sound features in a complex auditory scene. Finally, we will discuss how hearing loss interacts with these gain control mechanisms, and the adaptive and/or maladaptive perceptual consequences of this plasticity.

Dopamine depletion selectively disrupts interactions between striatal neuron subtypes and LFP oscillations.

Dopamine degeneration in Parkinson's disease (PD) dysregulates the striatal neural network and causes motor deficits. However, it is unclear how altered striatal circuits relate to dopamine-acetylcholine chemical imbalance and abnormal local field potential (LFP) oscillations observed in PD. We perform a multimodal analysis of the dorsal striatum using cell-type-specific calcium imaging and LFP recording. We reveal that dopamine depletion selectively enhances LFP beta oscillations during impaired locomotion, supporting beta oscillations as a biomarker for PD. We further demonstrate that dynamic cholinergic interneuron activity during locomotion remains unaltered, even though cholinergic tone is implicated in PD. Instead, dysfunctional striatal output arises from elevated coordination within striatal output neurons, which is accompanied by reduced locomotor encoding of parvalbumin interneurons and transient pathological LFP high-gamma oscillations. These results identify a pathological striatal circuit state following dopamine depletion where distinct striatal neuron subtypes are selectively coordinated with LFP oscillations during locomotion.

Large-scale voltage imaging in behaving mice using targeted illumination.

Recent improvements in genetically encoded voltage indicators enabled optical imaging of action potentials and subthreshold transmembrane voltage in vivo. To perform high-speed voltage imaging of many neurons simultaneously over a large anatomical area, widefield microscopy remains an essential tool. However, the lack of optical sectioning makes widefield microscopy prone to background cross-contamination. We implemented a digital-micromirror-device-based targeted illumination strategy to restrict illumination to the cells of interest and quantified the resulting improvement both theoretically and experimentally with SomArchon expressing neurons. We found that targeted illumination increased SomArchon signal contrast, decreased photobleaching, and reduced background cross-contamination. With the use of a high-speed, large-area sCMOS camera, we routinely imaged tens of spiking neurons simultaneously over minutes in behaving mice. Thus, the targeted illumination strategy described here offers a simple solution for widefield voltage imaging of many neurons over a large field of view in behaving animals.

Region-specific effects of ultrasound on individual neurons in the awake mammalian brain.

Ultrasound modulates brain activity. However, it remains unclear how ultrasound affects individual neurons in the brain, where neural circuit architecture is intact and different brain regions exhibit distinct tissue properties. Using a high-resolution calcium imaging technique, we characterized the effect of ultrasound stimulation on thousands of individual neurons in the hippocampus and the motor cortex of awake mice. We found that brief 100-ms-long ultrasound pulses increase intracellular calcium in a large fraction of individual neurons in both brain regions. Ultrasound-evoked calcium response in hippocampal neurons exhibits a rapid onset with a latency shorter than 50 ms. The evoked response in the hippocampus is shorter in duration and smaller in magnitude than that in the motor cortex. These results demonstrate that noninvasive ultrasound stimulation transiently increases intracellular calcium in individual neurons in awake mice, and the evoked response profiles are brain region specific.

Theta-gamma coupling emerges from spatially heterogeneous cholinergic neuromodulation.

Theta and gamma rhythms and their cross-frequency coupling play critical roles in perception, attention, learning, and memory. Available data suggest that forebrain acetylcholine (ACh) signaling promotes theta-gamma coupling, although the mechanism has not been identified. Recent evidence suggests that cholinergic signaling is both temporally and spatially constrained, in contrast to the traditional notion of slow, spatially homogeneous, and diffuse neuromodulation. Here, we find that spatially constrained cholinergic stimulation can generate theta-modulated gamma rhythms. Using biophysically-based excitatory-inhibitory (E-I) neural network models, we simulate the effects of ACh on neural excitability by varying the conductance of a muscarinic receptor-regulated K+ current. In E-I networks with local excitatory connectivity and global inhibitory connectivity, we demonstrate that theta-gamma-coupled firing patterns emerge in ACh modulated network regions. Stable gamma-modulated firing arises within regions with high ACh signaling, while theta or mixed theta-gamma activity occurs at the peripheries of these regions. High gamma activity also alternates between different high-ACh regions, at theta frequency. Our results are the first to indicate a causal role for spatially heterogenous ACh signaling in the emergence of localized theta-gamma rhythmicity. Our findings also provide novel insights into mechanisms by which ACh signaling supports the brain region-specific attentional processing of sensory information.

Fast, multiplane line-scan confocal microscopy using axially distributed slits.

The inherent constraints on resolution, speed and field of view have hindered the development of high-speed, three-dimensional microscopy techniques over large scales. Here, we present a multiplane line-scan imaging strategy, which uses a series of axially distributed reflecting slits to probe different depths within a sample volume. Our technique enables the simultaneous imaging of an optically sectioned image stack with a single camera at frame rates of hundreds of hertz, without the need for axial scanning. We demonstrate the applicability of our system to monitor fast dynamics in biological samples by performing calcium imaging of neuronal activity in mouse brains and voltage imaging of cardiomyocytes in cardiac samples.

Distinct neuronal populations contribute to trace conditioning and extinction learning in the hippocampal CA1.

Trace conditioning and extinction learning depend on the hippocampus, but it remains unclear how neural activity in the hippocampus is modulated during these two different behavioral processes. To explore this question, we performed calcium imaging from a large number of individual CA1 neurons during both trace eye-blink conditioning and subsequent extinction learning in mice. Our findings reveal that distinct populations of CA1 cells contribute to trace conditioned learning versus extinction learning, as learning emerges. Furthermore, we examined network connectivity by calculating co-activity between CA1 neuron pairs and found that CA1 network connectivity patterns also differ between conditioning and extinction, even though the overall connectivity density remains constant. Together, our results demonstrate that distinct populations of hippocampal CA1 neurons, forming different sub-networks with unique connectivity patterns, encode different aspects of learning.

A Viral Toolbox of Genetically Encoded Fluorescent Synaptic Tags.

Fibronectin intrabodies generated with mRNA display (FingRs) are a recently developed tool for labeling excitatory or inhibitory synapses, with the benefit of not altering endogenous synaptic protein expression levels or synaptic transmission. Here, we generated a viral vector FingR toolbox that allows for multi-color, neuron-type-specific labeling of excitatory or inhibitory synapses in multiple brain regions. We screened various fluorophores, FingR fusion configurations, and transcriptional control regulations in adeno-associated virus (AAV) and retrovirus vector designs. We report the development of a red FingR variant and demonstrated dual labeling of excitatory and inhibitory synapses in the same cells. Furthermore, we developed cre-inducible FingR AAV variants and demonstrated their utility, finding that the density of inhibitory synapses in aspiny striatal cholinergic interneurons remained unchanged in response to dopamine depletion. Finally, we generated FingR retroviral vectors, which enabled us to track the development of excitatory and inhibitory synapses in hippocampal adult-born granule cells.

Precision Calcium Imaging of Dense Neural Populations via a Cell-Body-Targeted Calcium Indicator.

Methods for one-photon fluorescent imaging of calcium dynamics can capture the activity of hundreds of neurons across large fields of view at a low equipment complexity and cost. In contrast to two-photon methods, however, one-photon methods suffer from higher levels of crosstalk from neuropil, resulting in a decreased signal-to-noise ratio and artifactual correlations of neural activity. We address this problem by engineering cell-body-targeted variants of the fluorescent calcium indicators GCaMP6f and GCaMP7f. We screened fusions of GCaMP to natural, as well as artificial, peptides and identified fusions that localized GCaMP to within 50 µm of the cell body of neurons in mice and larval zebrafish. One-photon imaging of soma-targeted GCaMP in dense neural circuits reported fewer artifactual spikes from neuropil, an increased signal-to-noise ratio, and decreased artifactual correlation across neurons. Thus, soma-targeting of fluorescent calcium indicators facilitates usage of simple, powerful, one-photon methods for imaging neural calcium dynamics.

High-contrast multifocus microscopy with a single camera and z-splitter prism.

Optical microscopy has been an indispensable tool for studying complex biological systems, but is often hampered by problems of speed and complexity when performing 3D volumetric imaging. Here, we present a multifocus imaging strategy based on the use of a simple z-splitter prism that can be assembled from off-the-shelf components. Our technique enables a widefield image stack to be distributed onto a single camera and recorded simultaneously. We exploit the volumetric nature of our image acquisition by further introducing a novel extended-volume 3D deconvolution strategy to suppress far-out-of-focus fluorescence background to significantly improve the contrast of our recorded images, conferring to our system a capacity for quasi-optical sectioning. By swapping in different z-splitter configurations, we can prioritize high speed or large 3D field-of-view imaging depending on the application of interest. Moreover, our system can be readily applied to a variety of imaging modalities in addition to fluorescence, such as phase-contrast and darkfield imaging. Because of its simplicity, versatility, and performance, we believe our system will be a useful tool for general biological or biomedical imaging applications.

Population imaging of neural activity in awake behaving mice.

A longstanding goal in neuroscience has been to image membrane voltage across a population of individual neurons in an awake, behaving mammal. Here we describe a genetically encoded fluorescent voltage indicator, SomArchon, which exhibits millisecond response times and is compatible with optogenetic control, and which increases the sensitivity, signal-to-noise ratio, and number of neurons observable several-fold over previously published fully genetically encoded reagents1-8. Under conventional one-photon microscopy, SomArchon enables the routine population analysis of around 13 neurons at once, in multiple brain regions (cortex, hippocampus, and striatum) of head-fixed, awake, behaving mice. Using SomArchon, we detected both positive and negative responses of striatal neurons during movement, as previously reported by electrophysiology but not easily detected using modern calcium imaging techniques9-11, highlighting the power of voltage imaging to reveal bidirectional modulation. We also examined how spikes relate to the subthreshold theta oscillations of individual hippocampal neurons, with SomArchon showing that the spikes of individual neurons are more phase-locked to their own subthreshold theta oscillations than to local field potential theta oscillations. Thus, SomArchon reports both spikes and subthreshold voltage dynamics in awake, behaving mice.

A Teensy microcontroller-based interface for optical imaging camera control during behavioral experiments.

BACKGROUND: Systems neuroscience experiments often require the integration of precisely timed data acquisition and behavioral monitoring. While specialized commercial systems have been designed to meet various needs of data acquisition and device control, they often fail to offer flexibility to interface with new instruments and variable behavioral experimental designs. NEW METHOD: We developed a Teensy 3.2 microcontroller-based interface that is easily programmable, and offers high-speed, precisely timed behavioral data acquisition and digital and analog outputs for controlling sCMOS cameras and other devices. RESULTS: We demonstrate the flexibility and the temporal precision of the Teensy interface in two experimental settings. In one example, we used the Teensy interface to record an animal's directional movement on a spherical treadmill, while delivering repeated digital pulses that can be used to control image acquisition from a sCMOS camera. In another example, we used the Teensy interface to deliver an auditory stimulus and a gentle eye puff at precise times in a trace conditioning eye blink behavioral paradigm, while delivering repeated digital pulses to trigger camera image acquisition. COMPARISON WITH EXISTING METHODS: This interface allows high-speed and temporally precise digital data acquisition and device control during diverse behavioral experiments. CONCLUSION: The Teensy interface, consisting of a Teensy 3.2 and custom software functions, provides a temporally precise, low-cost, and flexible platform to integrate sCMOS camera control into behavioral experiments.

Unique contributions of parvalbumin and cholinergic interneurons in organizing striatal networks during movement.

Striatal parvalbumin (PV) and cholinergic interneurons (CHIs) are poised to play major roles in behavior by coordinating the networks of medium spiny cells that relay motor output. However, the small numbers and scattered distribution of these cells have hindered direct assessment of their contribution to activity in networks of medium spiny neurons (MSNs) during behavior. Here, we build on recent improvements in single-cell calcium imaging combined with optogenetics to test the capacity of PVs and CHIs to affect MSN activity and behavior in mice engaged in voluntary locomotion. We find that PVs and CHIs have unique effects on MSN activity and dissociable roles in supporting movement. PV cells facilitate movement by refining the activation of MSN networks responsible for movement execution. CHIs, in contrast, synchronize activity within MSN networks to signal the end of a movement bout. These results provide new insights into the striatal network activity that supports movement.