Biology subjects

Smirnakis, S.

Publications and source records attributed to Smirnakis, S..

10 recordsLinked to original sources

bioRxiv2026

GAT1508, a selective neuronal GIRK1/2 activator, exhibits superior antiepileptic efficacy and synergizes with cannabidiol

G protein-gated inwardly rectifying potassium (GIRK; Kir3) channels are key regulators of neuronal excitability and represent promising therapeutic targets for epilepsy. Here, we characterize GAT1508, a urea-based small-molecule activator selectively targeting neuronal GIRK1/2 heterotetramers, and compare its efficacy with previously described urea compounds ML297 and GiGA1. In a pentylenetetrazole (PTZ)-induced seizure model, GAT1508 demonstrated robust antiseizure activity, outperforming both ML297 and GiGA1 and achieving efficacy comparable to valproic acid. Electrophysiological studies using two-electrode voltage clamp in Xenopus oocytes revealed that GAT1508, unlike ML297 and GiGA1, selectively activates GIRK1/2 channels without significant activation of cardiac GIRK1/4 channels, suggesting reduced risk of cardiovascular side effects. Structural and computational analyses indicate that enhanced stabilization of GAT1508 compared to ML297 within the GIRK1/2 binding pocket promotes both ion permeation and channel gating through coordinated interactions with key residues. Furthermore, GAT1508, like the two other urea drugs, exhibited synergistic activation of GIRK channels when combined with G{beta}{gamma}-mediated pathways and significantly potentiated the antiseizure effects of cannabidiol (CBD) in both hippocampal slice recordings and in vivo seizure models. These findings establish GAT1508 as a potent and selective GIRK1/2 activator and support a novel combination therapeutic strategy for epilepsy based on convergent GIRK channel modulation.

pharmacology and toxicology↗

bioRxiv2026

RNA-aware tissue preservation workflows for high-quality spatial transcriptomics

Image-based transcriptomic approaches can define, discover, and chart cell types and states within an array of tissues. However, measurement quality depends on RNA integrity, and the modern tissue preservation toolbox was not designed to protect this highly labile molecule. Here we leverage MERFISH to show that tissue-dependent differences in endogenous RNase activity can shape spatial transcriptomics data quality for different preservation methods and that RNase-activity-guided protocol optimization can improve data quality. In parallel, we introduce an RNA-aware pan-tissue preservation approach, Rapid Inhibition and Permanent Inactivation of Nucleases (RIPIN), that rapidly stabilizes samples with a broad-spectrum RNase inhibitor while permitting slow, chemical inactivation. RIPIN produces high-quality MERFISH measurements in all profiled human and mouse tissues, is compatible with clinical workflows, and is easily integrated with frozen or paraffin sectioning. By highlighting how RNA integrity can be lost during tissue processing, our work may inspire the next generation of RNA-aware histology methods.

genomics↗

bioRxiv2026

Emergence of focal cortical hyperexcitability in the murine tetanus toxin epilepsy model

AbstractFocal epilepsies are a heterogeneous group of neurological disorders that may be caused by a large variety of underlying brain lesions. Long-term quality of life prognosis is often poor, and treatment options are limited. Despite significant progress in animal model sophistication over the past decades, a deeper understanding of epileptogenic processes on a mesoscale and microscale circuit level is needed. Here we model focal epileptogenesis in mice using single intracortical tetanus toxin injections and employ chronic in vivo two-photon and widefield calcium imaging with simultaneous EEG and behavioral video recordings ("multimodal" recordings) to follow epileptogenic changes over time. We observe a moderate increase in global brain excitability on EEG but, more strikingly, a gradual emergence of brief but intense optically recorded epileptiform events ("microseizures") that are spatially limited, peaking around 20-30 days after toxin injection and waning over the next 30 days. During these events, a majority of local neurons are recruited into extreme depolarization, yet there is considerable variability in duration and spatial coverage between events, depending on the proximity to the injection focus and the number of days elapsed since injection. Notably, EEG electrodes <3 mm from the focus typically fail to detect these events, highlighting the utility of high-resolution in vivo optical imaging for capturing all events in focal epileptogenesis. Single-cell patch clamp recordings in awake animals confirm that microseizure episodes are associated with depolarization block, during which the calcium signal remains high. Using chronic multimodal recordings, we detect an initial rise of interictal activity in local cortical networks during the first day after injection, followed by a layer-specific suppression and dysregulation of interictal spontaneous network activity in local cortical circuits preceding microseizure events, and low-level suppression when microseizures started subsiding. The chronic suppression in interictal activity is accompanied by relative hypoactivity of PV+ interneurons and concurrent hyperactivity of SOM+ interneurons. This integrated imaging-electrophysiology approach provides a powerful platform for unraveling circuit-level mechanisms of focal epilepsy and potentially for identifying and testing novel therapeutic strategies in the future.

neuroscience↗

bioRxiv2024

Trial-by-trial inter-areal interactions in visual cortex in the presence or absence of visual stimulation

State-of-the-art computational models of vision largely focus on fitting trial-averaged spike counts to visual stimuli using overparameterized neural networks. However, a computational model of the visual cortex should predict the dynamic responses of neurons in single trials across different experimental conditions. In this study, we investigated trial-by-trial inter-areal interactions in the visual cortex by predicting neuronal activity in one area based on activity in another, distinguishing between stimulus-driven and non-stimulus-driven shared variability. We analyzed two datasets: calcium imaging from mouse V1 layers 2/3 and 4, and extracellular neurophysiological recordings from macaque V1 and V4. Our results show that neuronal activity can be predicted bidirectionally between L2/3 and L4 in mice, and between V1 and V4 in macaque monkeys, with the latter interaction exhibiting directional asymmetry. The predictability of neuronal responses varied with the type of visual stimulus, yet responses could also be predicted in the absence of visual stimulation. In mice, we observed a bimodal distribution of neurons, with some neurons primarily driven by visual inputs and others showing predictable activity during spontaneous activity despite lacking consistent visually evoked responses. Predictability also depended on intrinsic neuronal properties, receptive field overlap, and the relative timing of activity across areas. Our findings highlight the presence of both stimulus- and non-stimulus-related components in interactions between visual areas across diverse contexts and underscore the importance of non-visual shared variability between visual regions in both mice and macaques.

neuroscience↗

bioRxiv2022

Tug-of-peace: Visual Rivalry and Atypical Visual Motion Processing in MECP2 Duplication Syndrome of Autism

Extracting common patterns of neural circuit computations in the autism spectrum and confirming them as a cause of specific core traits of autism is the first step towards identifying cell- and circuit-level targets for effective clinical intervention. Studies in human subjects with autism have identified functional links and common anatomical substrates between core restricted behavioral repertoire, cognitive rigidity, and over-stability of visual percepts during visual rivalry. To be able to study these processes with single-cell precision and comprehensive neuronal population coverage, we developed the visual bi-stable perception paradigm for mice. Our task is based on plaid patterns consisting of two transparent gratings drifting at an angle of 120{degrees} relative to each other. This results in spontaneous reversals of the perception between local component motion (motion of the plaid perceived as two separate moving grating components) and integrated global pattern motion (motion of the plaid perceived as a fused moving texture). Furthermore, this robust paradigm does not depend on the explicit report of the mouse, since the direction of the optokinetic nystagmus (OKN, rapid eye movements driven by either pattern or component motion) is used to infer the dominant percept. Using this paradigm, we found that the rate of perceptual reversals between global and local motion interpretations of the stimulus is reduced in the MECP2 duplication mouse model of autism. Moreover, the stability of local motion percepts is greatly increased in MECP2 duplication mice at the expense of global motion percepts. Thus, our model reproduces a subclass of the core features in human autism (reduced rate of visual rivalry and atypical perception of visual motion). This further offers a well-controlled approach for dissecting neuronal circuits underlying these core features.

neuroscience↗

bioRxiv2022

A Tale of Two Trees: Modeling Apical and Basal Tree Contribution to L2/3 V1 Pyramidal Cell Orientation Selectivity

Pyramidal neurons, a mainstay of cortical regions, receive a plethora of inputs from various areas onto their morphologically distinct apical and basal trees. Both trees differentially contribute to the somatic response, defining distinct anatomical and possibly functional sub-units. To elucidate the contribution of each tree to the encoding of visual stimuli at the somatic level, we modeled the response pattern of a L2/3 V1 pyramidal neuron to orientation tuned synaptic input. Towards this goal, we used a morphologically detailed computational model of a single cell that replicates electrophysiological and two-photon imaging data. Our simulations predict a synergistic effect of apical and basal trees on somatic action potential generation: basal tree activity, in the form of either depolarization or dendritic spiking, is necessary for producing somatic activity, despite the fact that most somatic spikes are heavily driven by apical dendritic spikes. This model provides evidence for synergistic computations taking place in the basal and apical trees of the L2/3 V1 neuron along with mechanistic explanations for tree-specific contributions and emphasizes the potential role of predictive and attentional feedback input in these cells. O_FIG O_LINKSMALLFIG WIDTH=181 HEIGHT=200 SRC="FIGDIR/small/501858v2_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1b69239org.highwire.dtl.DTLVardef@13a0a32org.highwire.dtl.DTLVardef@1738a7corg.highwire.dtl.DTLVardef@88e826_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract FigureC_FLOATNO Graphical Representation of AP Generation in a L2/3 V1 Pyramidal Neuron Model Action potential generation requires the spatiotemporal coincidence of apical sodium spikes with either basal sodium spikes or significant basal depolarizations, allowing the neuron to respond to salient stimuli that may or may not be affected by attentional and/or predictive signals from higher-order areas. C_FIG

neuroscience↗

bioRxiv2018

Asynchronous suppression of visual cortex during absence seizures

Absence epilepsy is a common childhood disorder featuring frequent cortical spike-wave seizures with a loss of awareness and behavior. Using the calcium indicator GCaMP6 with in vivo 2-photon cellular microscopy and simultaneous electrocorticography, we examined the collective activity profiles of individual neurons and surrounding neuropil across all layers in V1 during spike-wave seizure activity over prolonged periods in stargazer mice. We show that most ([~]80%) neurons in all cortical layers reduce their activity during seizures, whereas a smaller pool activates or remains neutral. Unexpectedly, ictal participation of identified single unit activity is not fixed, but fluctuates on a flexible time scale across seizures. Pairwise correlation analysis of calcium activity reveals a surprising lack of synchrony among neurons and neuropil patches in all layers during seizures. Our results demonstrate an asynchronous suppression of visual cortex during absence seizures, with major implications for understanding cortical network function during EEG states of reduced awareness.

neuroscience↗

bioRxiv2018

Internal gain modulations, but not changes in stimulus contrast, preserve the neural code

Neurons in primary visual cortex (V1) are strongly modulated both by stimulus contrast and by fluctuations of internal inputs. An important question is whether population codes are preserved under these conditions. Changes in stimulus contrast are thought to leave population codes invariant, whereas the effect of internal gain modulations remains unknown. To address these questions we studied how the direction-of-motion of oriented gratings is encoded in layer 2/3 of mouse V1. Surprisingly, we found that, because contrast gain responses across cells are heterogeneous, a change in contrast alters the information distribution profile across cells leading to the failure of contrast invariance. Remarkably, internal input fluctuations that cause commensurate firing rate modulations at the single-cell level, do respect population code invariance. These observations have important implications for visual information encoding, and argue that the brain strives to maintain the stability of the neural code in the face of fluctuating internal inputs.

neuroscience↗

bioRxiv2017

The effect of single pyramidal neuron firing across and within layers in mouse V1

The influence of cortical cell spiking activity on nearby cells has been studied extensively in vitro. Less is known, however, about the impact of single cell firing on local cortical networks in vivo. In a pioneering study, Kwan et al. (Kwan et al., 2012) reported that in mouse layer 2/3 (L2/3), under anesthesia, stimulating a single pyramidal cell recruits ~1.7% of neighboring pyramidal units. Here we employ two-photon calcium imaging, in conjunction with single-cell patch clamp stimulation, to probe, in both the awake and lightly anesthetized states, how i) activating single L2/3 pyramidal neurons recruits neighboring units within L2/3 and from layer 4 (L4) to L2/3, and whether ii) activating single pyramidal neurons changes population activity in local circuit. To do this, it was essential to develop an algorithm capable of quantifying how sensitive the calcium signal is at detecting effectively recruited units (\"followers\"). This algorithm allowed us to estimate the chance of detecting a follower as a function of the probability that an epoch of stimulation elicits one extra action potential (AP) in the follower cell. Using this approach, we found only a small fraction (<0.75%) of L2/3 cells to be significantly activated within a radius of ~200 m from a stimulated neighboring L2/3 pyramidal cell. This fraction did not change significantly in the awake versus the lightly anesthetized state, nor when stimulating L2/3 versus underlying L4 pyramidal neurons. These numbers are in general agreement with, though lower than, the percentage of neighboring cells (2.1%) reported by Kwan et al. to be activated upon stimulating single L2/3 pyramidal neurons under anesthesia (Kwan et al., 2012). Interestingly, despite the small number of individual units found to be reliably driven, we did observe a modest but significant elevation in aggregate population responses compared to sham stimulation. This underscores the distributed impact that single cell stimulation has on neighboring microcircuit responses, revealing only a small minority of relatively strongly connected partners.\n\nOne Sentence SummaryPatch-clamp stimulation in conjunction with 2-photon imaging shows that activating single layer-2/3 or layer-4 pyramidal neurons produces few (<1% of local units) reliable singlecell followers in L2/3 of mouse area V1, either under light anesthesia or in quiet wakefulness: instead, single cell stimulation was found to elevate aggregate population activity in a weak but highly distributed fashion.

neuroscience↗

bioRxiv2017

Visually driven neuropil activity and information encoding in mouse area V1

Spontaneous calcium fluorescence recorded from large cortical neuropil patches strongly correlates with the electro-corticogram, and is thought to arguably reflect primarily pre-synaptic inputs. Here we used in vivo 2-photon imaging with Oregon Green Bapta (OGB) to study neuropil visual responses to moving gratings in layer 2/3 of mouse area V1. We found neuropil responses to be more reliable and more strongly modulated than neighboring somatic activity. Furthermore, stimulus independent modulations in neuropil activity, i.e. noise correlations, were highly coherent across the cortical surface, up to distances of at least 200 m. Pairwise neuropil-to-neuropil-patch noise correlation strength was much higher than cell-to-cell noise correlation strength and depended strongly on brain state, decreasing in quiet wakefulness relative to light anesthesia. The profile of neuropil noise correlation strength decreased gently with distance, dropping by ~12% at a distance of 200 m. This was comparatively slower than the profile of cell-to-cell noise correlations, which dropped by ~30% at 200 m. Interestingly, in spite of the \"salt & pepper\" organization of orientation and direction encoding across mouse V1 neurons, populations of neuropil patches, even of moderately large size (radius ~100m), showed high accuracy for discriminating perpendicularly moving gratings commensurate to the accuracy of corresponding cell populations. These observations underscore the dynamic nature of the functional organization of neuropil activity.\n\nConflict of InterestThe authors declare that no competing interests exist.

neuroscience↗