Biology subjects

Ramakrishnan, C.

Publications and source records attributed to Ramakrishnan, C..

32 records · Page 2Linked to original sources

bioRxiv2022

Direct neural perturbations reveal a dynamical mechanism for robust computation

The rich repertoire of skilled mammalian behavior is the product of neural circuits that generate robust and flexible patterns of activity distributed across populations of neurons. Decades of associative studies have linked many behaviors to specific patterns of population activity, but association alone cannot reveal the dynamical mechanisms that shape those patterns. Are local neural circuits high-dimensional dynamical reservoirs able to generate arbitrary superpositions of patterns with appropriate excitation? Or might circuit dynamics be shaped in response to behavioral context so as to generate only the low-dimensional patterns needed for the task at hand? Here, we address these questions within primate motor cortex by delivering optogenetic and electrical microstimulation perturbations during reaching behavior. We develop a novel analytic approach that relates measured activity to theoretically tractable, dynamical models of excitatory and inhibitory neurons. This computational model captures the dynamical effects of these perturbations and demonstrates that motor cortical activity during reaching is shaped by a self-contained, low-dimensional dynamical system. The subspace containing task-relevant dynamics proves to be oriented so as to be robust to strong non-normal amplification within cortical circuits. This task dynamics space exhibits a privileged causal relationship with behavior, in that stimulation in motor cortex perturb reach kinematics only to the extent that it alters neural states within this subspace. Our results resolve long-standing questions about the dynamical structure of cortical activity associated with movement, and illuminate the dynamical perturbation experiments needed to understand how neural circuits throughout the brain generate complex behavior.

neuroscience↗

bioRxiv2022

Structural basis for ion selectivity in potassium-selective channelrhodopsins

The KCR channelrhodopsins are recently-discovered light-gated ion channels with high K+ selectivity, a property that has attracted broad attention among biologists- due to intense interest in creating novel inhibitory tools for optogenetics leveraging this K+ selectivity, and due to the mystery of how this selectivity is achieved in the first place. Indeed, the molecular and structural mechanism for K+ selectivity in KCRs has remained especially puzzling since these 7-transmembrane retinal-binding proteins completely lack structural similarity with known K+ channels, which generally coordinate K+ in a precisely symmetric conduction pathway formed by a tight interface among multiple small monomeric channel subunits (presumably not an accessible mechanism for the large KCR rhodopsin proteins). Here we present the cryo-electron microscopy structures of two KCRs from Hyphochytrium catenoides with distinct spectral properties for light absorption and channel actuation, HcKCR1, and HcKCR2, at resolutions of 2.6 and 2.5 [A], respectively. Structural comparison revealed first an unusually-shaped retinal binding pocket which induces rotation of the retinal in HcKCR2, explaining the large spectral difference between HcKCR1 and 2. Next, our combined structural, electrophysiological, computational, and spectroscopic analyses revealed a new solution to the challenging problem of K+-selective transport. KCRs indeed do not exhibit the canonical tetrameric K+ selectivity filter that specifically coordinates dehydrated K+; instead, single KCR monomers form a size exclusion filter using aromatic residues at the extracellular side of the pore which inhibits passage of bulky hydrated ions. This unique feature allows KCRs to function as K+ channels under relevant physiological conditions, providing not only a novel mechanism for achieving high K+ permeability ratios in biological ion channels, but also a framework for designing the next generation of inhibitory optogenetic tools. In BriefThe first structures of K+-selective channelrhodopsins (HcKCR1 and 2) are determined, revealing a K+ selectivity mechanism distinctly different from canonical K+ channels. HighlightsO_LIThe cryo-EM structures of K+-selective channelrhodopsins, HcKCR1 and 2, in nanodisc C_LIO_LIConditions under which naturally-occurring microbial rhodopsins have a 6-s-cis retinal C_LIO_LIIdentification of key residues for high K+ permeability ratios C_LIO_LIThe unique K+ selectivity mechanism of KCRs C_LI

biophysics↗

bioRxiv2022

Dopaminergic axons track somatic signaling in behaving mice

Striatal dopamine released from the axons of midbrain dopamine neurons has been linked to a wide range of functions, including movement control and reward-based learning. Recent studies have reported functional signaling differences between axons and somas of dopamine neurons, suggesting that local modulation controls dopamine release and calling into question the classical view of somatic control. However, these experiments are technically challenging, making it difficult to ensure that axonal and somatic recordings come from the same neurons, particularly given the heterogeneity of dopaminergic cell types. Here we used genetic strategies to isolate key dopaminergic neuron subtypes and monitor their axonal and somatic signaling patterns in behaving mice. Contrary to the inferences drawn from previous studies, these experiments revealed a robust correlation between somatic and axonal signaling. Thus, by exploiting a previously unknown connection between genetic and functional diversity in dopamine neurons, we establish that subtypes must be considered to understand the mechanisms of dopamine release in striatum during behavior.

neuroscience↗

bioRxiv2021

Structural basis for channel conduction in the pump-like channelrhodopsin ChRmine

ChRmine1, a recently-discovered bacteriorhodopsin-like cation-conducting channelrhodopsin1, 2, exhibits puzzling properties (unusually-large photocurrents, exceptional red-shift in action spectrum, and extreme light-sensitivity) that have opened up new opportunities in optogenetics1, 3-5. ChRmine and its homologs function as light-gated ion channels, but by primary sequence more closely resemble ion pump rhodopsins; the molecular mechanisms for passive channel conduction in this family of proteins, as well as the unusual properties of ChRmine itself, have remained mysterious. Here we present the cryo-electron microscopy structure of ChRmine at 2.0 [A] resolution. The structure reveals striking architectural features never seen before in channelrhodopsins including trimeric assembly, a short transmembrane-helix 3 unwound in the middle of the membrane, a prominently-twisting extracellular-loop 1, remarkably-large intracellular cavities and extracellular vestibule, and an unprecedented hydrophilic pore that extends through the center of the trimer, separate from the three individual monomer pores. Electrophysiological, spectroscopic, and computational analyses provide insight into conduction and gating of light-gated channels with these distinct design features, and point the way toward structure-guided creation of novel channelrhodopsins for optogenetic applications in biology.

biophysics↗

bioRxiv2021

Similar neural and perceptual masking effects of low-power optogenetic stimulation in primate V1

Can direct stimulation of primate V1 substitute for a visual stimulus and mimic its perceptual effect? To address this question, we developed an optical-genetic toolkit to "read" neural population responses using widefield calcium imaging, while simultaneously using optogenetics to "write" neural responses into V1 of behaving macaques. We focused on the phenomenon of visual masking, where detection of a dim target is significantly reduced by a co-localized medium-brightness pedestal. Using our toolkit, we tested whether V1 optogenetic stimulation can recapitulate the perceptual masking effect of a visual pedestal. We find that, similar to a visual pedestal, low-power optostimulation can significantly reduce visual detection sensitivity, that a sublinear interaction between visual and optogenetic evoked V1 responses could account for this perceptual effect, and that these neural and behavioral effects are spatially selective. Our toolkit and results open the door for further exploration of perceptual substitutions by direct stimulation of sensory cortex.

neuroscience↗

bioRxiv2021

Optogenetic activation of Gq signaling in astrocytes yields stimulation-specific effects on basal hippocampal synaptic excitation and inhibition

Astrocytes play active roles at synapses and can monitor, respond, and adapt to local synaptic activity. To investigate this relationship, more tools that can selectively activate native G protein signaling pathways in astrocytes with both spatial and temporal precision are needed. Here, we tested AAV8-GFAP-Opto1AR-eYFP (Opto1AR), a viral vector to enable activation of Gq signaling in astrocytes via light-sensitive 1-adrenergic receptors. To determine if stimulating astrocytic Opto1AR modulates hippocampal synaptic transmission, recordings were made in CA1 pyramidal cells with surrounding astrocytes expressing Opto1AR, channelrhodopsin (ChR2), or GFP. Both high-frequency (20 Hz, 45-ms light pulses, 5 mW, 5 min) and low-frequency (0.5 Hz, 1-s pulses at increasing 1, 5, and 10 mW intensities, 90 s per intensity) blue light stimulation were tested. 20 Hz Opto1AR stimulation increased both inhibitory and excitatory postsynaptic current (IPSC and EPSC) frequency, and the mIPSC effect was largely reversible within 20 min. By contrast, low-frequency stimulation of Opto1AR did not modulate either IPSCs or EPSCs, whereas the same stimulation of astrocytic ChR2 was effective. These data demonstrate that Opto1AR activation in astrocytes changes synaptic excitation and inhibition in a stimulation-sensitive manner, demonstrating the efficacy and utility of GFAP-Opto1AR as a tool in studying astrocyte-neuron interactions.

neuroscience↗

bioRxiv2020

A unique and evolutionarily conserved retinal interneuron relays rod and cone input to the inner plexiform layer

Neurons in the CNS are distinguished from each other by their morphology, the types of the neurotransmitter they release, their synaptic connections, and their genetic profiles. While attempting to characterize the retinal bipolar cell (BC) input to retinal ganglion cells (RGCs), we discovered a previously undescribed type of interneuron in mice and primates. This interneuron shares some morphological, physiological, and molecular features with traditional BCs, such as having dendrites that ramify in the outer plexiform layer (OPL) and axons that ramify in the inner plexiform layer (IPL) to relay visual signals from photoreceptors to inner retinal neurons. It also shares some features with amacrine cells, particularly Aii amacrine cells, such as their axonal morphology and possibly the release of the inhibitory neurotransmitter glycine, along with the expression of some amacrine cell specific markers. Thus, we unveil an unrecognized type of interneuron, which may play unique roles in vision. Significance StatementCell types are the building blocks upon which neural circuitry is based. In the retina, it is widely believed that all neuronal types have been identified. We describe a cell type, which we call the Campana cell, that does not fit into the conventional neuronal retina categories but is evolutionarily conserved. Unlike retinal bipolar cells, the Campana cell receives synaptic input from both rods and cones, has broad axonal ramifications, and may release an inhibitory neurotransmitter. Unlike retinal amacrine cells, the Campana cell receives direct photoreceptor input has bipolar-like ribbon synapses. With this discovery, we open the possibility for new forms of visual processing in the retina.

neuroscience↗

bioRxiv2019

Dendritic calcium signals in rhesus macaque motor cortex drive an optical brain-computer interface

Calcium imaging has rapidly developed into a powerful tool for recording from large populations of neurons in vivo. Imaging in rhesus macaque motor cortex can enable the discovery of new principles of motor cortical function and can inform the design of next generation brain-computer interfaces (BCIs). Surface two-photon (2P) imaging, however, cannot presently access somatic calcium signals of neurons from all layers of macaque motor cortex due to photon scattering. Here, we demonstrate an implant and imaging system capable of chronic, motion-stabilized two-photon (2P) imaging of calcium signals from in macaques engaged in a motor task. By imaging apical dendrites, some of which originated from deep layer 5 neurons, as as well as superficial cell bodies, we achieved optical access to large populations of deep and superficial cortical neurons across dorsal premotor (PMd) and gyral primary motor (M1) cortices. Dendritic signals from individual neurons displayed tuning for different directions of arm movement, which was stable across many weeks. Combining several technical advances, we developed an optical BCI (oBCI) driven by these dendritic signals and successfully decoded movement direction online. By fusing 2P functional imaging with CLARITY volumetric imaging, we verify that an imaged dendrite, which contributed to oBCI decoding, originated from a putative Betz cell in motor cortical layer 5. This approach establishes new opportunities for studying motor control and designing BCIs.

neuroscience↗

bioRxiv2019

How neurons move during action potentials

Neurons undergo nanometer-scale deformations during action potentials, and the underlying mechanism has been actively debated for decades. Previous observations were limited to a single spot or the cell boundary, while movement across the entire neuron during the action potential remained unclear.\n\nWe report full-field imaging of cellular deformations accompanying the action potential in mammalian neuron somas (-1.8nm~1.3nm) and neurites (-0.7nm~0.9nm), using fast quantitative phase imaging with a temporal resolution of 0.1ms and an optical pathlength sensitivity of <4pm per pixel. Spike-triggered average, synchronized to electrical recording, demonstrates that the time course of the optical phase changes matches the dynamics of the electrical signal, with the optical signal revealing the intracellular potential rather than its time derivative detected via extracellular electrodes. Using 3D cellular morphology extracted via confocal microscopy, we demonstrate that the voltage-dependent changes in the membrane tension induced by ionic repulsion can explain the magnitude, time course and spatial features of the phase imaging. Our full-field observations of the spike-induced deformations in mammalian neurons opens the door to non-invasive label-free imaging of neural signaling.

neuroscience↗

bioRxiv2019

Functional maturation of human neural stem cells in a 3D bioengineered brain model enriched with fetal brain matrix

Brain extracellular matrix (ECM) is often overlooked in vitro brain tissue models, despite its instructive roles during development. Using developmental stage-sourced brain ECM in reproducible 3D bioengineered culture systems, we demonstrate enhanced functional differentiation of human induced neural stem cells (hiNSCs) into healthy neurons and astrocytes. Particularly, fetal brain tissue-derived ECM supported long-term maintenance of differentiated neurons, demonstrated by morphology, gene expression and secretome profiling. Astrocytes were evident within the second month of differentiation, and reactive astrogliosis was inhibited in brain ECM-enriched cultures when compared to unsupplemented cultures. Functional maturation of the differentiated hiNSCs within fetal ECM-enriched cultures was confirmed by calcium signaling and unsupervised cluster analysis. Additionally, the study identified native biochemical cues in decellularized ECM with notable comparisons between fetal and adult brain-derived ECMs. The development of novel brain-specific biomaterials for generating mature in vitro brain models provides an important path forward for interrogation of neuron-glia interactions.

developmental biology↗

bioRxiv2018

Reversible temporally-specific inhibition of muscle using a light-activated chloride channel

Achieving reversible, temporally specific inhibition of motor neurons has the potential to revolutionize treatment of disorders marked by muscle hyperactivity. Current treatment strategies are inadequate - surgical interventions are irreversible and pharmaceutical interventions have off-target effects. Optogenetic strategies for inhibiting muscle activity have theoretical promise; however, trafficking and expression problems have prevented translatable optogenetic suppression of muscle activity. Here, we exploit recent innovations in opsin engineering to demonstrate virally mediated, temporally specific optogenetic inhibition of motor neurons and muscle activity in vivo. We show that intra-muscular injection of adeno-associated virus serotype 6 can drive expression of the inhibitory channelrhodopsin mutant iC++ in both immunodeficient and wild-type mice. Illumination of the sciatic nerve in wild-type mice resulted in 64.8% inhibition of evoked twitch force. Optical excitation during tetanic stimulation in wild-type mice resulted in 59.1% inhibition at 10 Hz and 55.4% at 25 Hz. The extent of optogenetic inhibition was titratable, and ranged from 0% to 78.4% as illumination intensity was changed from 1 mW to 20 mW/mm2. These results could have therapeutic applicability to disorders such as spasticity, hypertonia and urinary incontinence, and provide a new tool to neuroscientists and muscle physiologists wishing to reversibly inhibit motor neuron activity in vivo.

neuroscience↗

bioRxiv2018

A Proof-of-Principle of Nanoscale Optogenetics

Optogenetics has revolutionized the study of circuit function in the brain, by allowing activation of specific ensembles of neurons by light. However, this technique has not yet been exploited extensively at the subcellular level. Here we propose a novel focal stimulation approach using STED/RESOLFT-like illumination, whereby switchable light-gated channels are focally activated by a laser beam of one wavelength and deactivated by an overlapping donut-shaped beam of a different wavelength, confining activation to a center focal region. We demonstrate the utility of current optogenetic tools to achieve highly focal depolarization using this method and further examine a proof-of-principle of nanoscale optogenetic activation using an initial macroscale approach. When employed at the nanoscale, this approach will allow unprecedented optogenetic control of nanodomains within cells.

neuroscience↗

bioRxiv2017

Identification Of Cellular-Activity Dynamics Across Large Tissue Volumes In The Mammalian Brain

Tracking the coordinated activity of cellular events through volumes of intact tissue is a major challenge in biology that has inspired significant technological innovation. Yet scanless measurement of the high-speed activity of individual neurons across three dimensions in scattering mammalian tissue remains an open problem. Here we develop and validate a computational imaging approach (SWIFT) that integrates high-dimensional, structured statistics with light field microscopy to allow the synchronous acquisition of single-neuron resolution activity throughout intact tissue volumes as fast as a camera can capture images (currently up to 100 Hz at full camera resolution), attaining rates needed to keep pace with emerging fast calcium and voltage sensors. We demonstrate that this large field-of-view, single-snapshot volume acquisition method--which requires only a simple and inexpensive modification to a standard fluorescence microscope--enables scanless capture of coordinated activity patterns throughout mammalian neural volumes. Further, the volumetric nature of SWIFT also allows fast in vivo imaging, motion correction, and cell identification throughout curved subcortical structures like the dorsal hippocampus, where cellular-resolution dynamics spanning hippocampal subfields can be simultaneously observed during a virtual context learning task in a behaving animal. SWIFTs ability to rapidly and easily record from volumes of many cells across layers opens the door to widespread identification of dynamical motifs and timing dependencies among coordinated cell assemblies during adaptive, modulated, or maladaptive physiological processes in neural systems.

neuroscience↗

bioRxiv2017

The central amygdala controls learning in the lateral amygdala

Both the lateral and the central nuclei of the amygdala are required for adaptive behavioral responses to environmental cues predicting threats. While experience-driven synaptic plasticity in the lateral amygdala is thought to underlie the formation of association between a sensory stimulus and an ensuing threat, how the central amygdala participates in such learning process remains unclear. Here we show that a specific class of central amygdala neurons, the protein kinase C-{delta}-expressing neurons, is essential for the synaptic plasticity underlying learning in the lateral amygdala, as it is required for lateral amygdala neurons to respond to unconditioned stimulus, and furthermore carries information about the unconditioned stimulus to instruct learning. Our results uncover an amygdala functional organization that may play a key role in diverse learning processes.

neuroscience↗