Biology subjects

Balasubramanian, H.

Publications and source records attributed to Balasubramanian, H..

10 recordsLinked to original sources

bioRxiv2026

Projection populations form a dorsoventral gradient of reward representations in the lateral septum

Several decades ago, the septal region was identified as the most dependable location of intracranial self-stimulation (ICSS) in the brain, but the neural substrates that mediate septal self-stimulation are still unknown. One part of the septal region, the lateral septum (LS), contributes to a number of motivated behaviors and may promote reward-seeking. However, the LS contains different anatomical subregions, complicating the characterization of its reward representations. We examine the responses of LS neurons along its dorsoventral axis using cellular resolution calcium imaging during an operant assay. We find that a greater proportion of neurons are excited in response to sucrose reward in the dorsal LS, while a greater proportion are inhibited in response to sucrose reward in the ventral LS. Additionally, we identify two distinct LS projection populations, one to the nucleus accumbens (NAc) and another to the bed nucleus of the stria terminalis (BNST), which are located in the dorsal and ventral regions of the LS, respectively. These two projection populations also have opposing reward responses and their corresponding optogenetic manipulation supports ICSS. These findings demonstrate how LS projection populations contribute to its functional heterogeneity, especially as it pertains to reward-seeking behavior.

neuroscience↗

bioRxiv2026

Mechanically-induced Septin Networks Protect Nuclear Integrity

The cytoskeleton is a key mediator of mechanical interactions in cells, but specific contributions of septins remains unclear. Septins preferentially localize with a subset of actin stress fibers positioned under the nucleus, where they are situated between the membrane and stress fibers. Removing the nucleus from the cell results in the loss of these subnuclear septin-decorated stress fibers. Surprisingly, however, their formation can be rescued using a large glass bead in place of the nucleus. Similarly, applying a compressive force to the cell via confinement, whether externally or through internally generated actomyosin forces, results in increased septin accumulation in regions where the nucleus engages the cell cortex. Finally, loss of septin filaments via knockdown of SEPT7 increases the likelihood of nuclear membrane rupture during confinement. Together these data suggest that septins act as a dynamic mechanosensitive protective mechanism to buffer mechanical forces on the nucleus.

cell biology↗

bioRxiv2025

Non-overlapping social and food reward representations in the basolateral amygdala

The ability to consider and appraise positively valenced stimuli in the environment, such as food and social interaction, to guide appropriate action is important for survival of most animals. Several studies have compared how food and social rewards are represented in different regions involved in reward processing and found either overlapping or distinct representations. In the basolateral amygdala (BLA) there seems to be opposing evidence for both shared and unique encoding of social and nonsocial stimuli. In our recent work, we found that the medial prefrontal cortex (mPFC), a region reciprocally connected to the BLA, has distinct social and food representations using a novel self-paced two-choice assay. Given the BLA and mPFCs differing roles in reward processing, it is important to understand how these two nodes may differ in their encoding reward types within the same assay. To resolve how the BLA encodes social and food information, we recorded the activity of individual BLA neurons in female and male mice during a two-choice social-sucrose operant task. We found that BLA neurons robustly and distinctly respond to social and food reward. In contrast to the mPFC, BLA neurons did not show a bias towards social reward responsiveness and instead showed equal social/sucrose representation, in males, or a sucrose reward bias, in females. BLA neurons were sensitive to internal state - water deprivation increased the proportion of sucrose reward responsive neurons. Additionally, sucrose reward responsive BLA neurons were differentially sensitive to reward omissions, such that neurons that were excited by sucrose reward were more sensitive to reward omissions compared to those inhibited by reward. Together, these findings demonstrate distinct, heterogeneous response profiles within the BLA to social and food rewards, in a manner different from the mPFC.

neuroscience↗

bioRxiv2025

Organization of brainwide inputs to discrete lateral septum projection populations

The lateral septum (LS) is anatomically positioned to play a critical role in directing information from the hippocampus and cortex to downstream subcortical structures, such as the hypothalamus. In fact, early anatomical tracing studies investigated the organization of hippocampal inputs to the LS and its hypothalamic outputs to begin to understand how its structure might relate to its function. These studies also characterized the cellular anatomy of the LS and the organization of different molecular markers within it. However, relatively little is known about the organization of other, non-hypothalamic projection populations within the LS and what types of input these different projection populations receive. Here, we used retrograde tracing to determine the organization of LS projections to six different brain regions that mediate various social behaviors. We found that these projection populations occupy discrete anatomical compartments within the LS. We then used a monosynaptic rabies tracing strategy to map brainwide inputs to these six discrete LS projection populations and examine how different brain regions innervate them. We identified unique region-dependent patterns of inputs to individual LS projection populations. In particular, we observed differences in cortical, hippocampal and thalamic innervation of the six different LS projection populations, while the hypothalamic inputs were largely similar across projection populations. Thus, this study provides insight into the anatomical connectivity that may underlie the functional heterogeneity of the LS.

neuroscience↗

bioRxiv2023

Sex differences in neural representations of social and nonsocial reward in the medial prefrontal cortex.

The reinforcing nature of social interactions is necessary for the maintenance of appropriate social behavior. However, the neural substrates underlying social reward processing and how they might differ based on the sex and internal state of the animal remains unknown. It is also unclear whether these neural substrates are shared with those involved in nonsocial rewarding processing. We developed a fully automated, novel two choice (social-sucrose) operant assay in which mice choose between social and nonsocial rewards to directly compare the reward-related behaviors associated with two competing stimuli. We performed cellular resolution calcium imaging of medial prefrontal cortex (mPFC) neurons in male and female mice across varying states of water restriction and social isolation. We found that mPFC neurons maintain largely non-overlapping, flexible representations of social and nonsocial reward that vary with internal state in a sex-dependent manner. Additionally, optogenetic manipulation of mPFC activity during the reward period of the assay disrupted reward-seeking behavior across male and female mice. Thus, using a novel operant assay, we have identified sex-dependent, non-overlapping neural representations of social and nonsocial reward in the mPFC that vary with internal state and that are essential for appropriate reward-seeking behavior.

neuroscience↗

bioRxiv2022

The dependence of EGFR oligomerization on environment and structure: A camera-based N&B study

Number and Brightness analysis (N&B) is a fluorescence spectroscopy technique to quantify protein oligomerization. Accurate results, however, rely on a good knowledge of non-fluorescent states of the fluorescent labels, especially of fluorescent proteins (FP), which are widely used in biology. FPs have been characterized for confocal but not camera-based N&B, which allows in principle faster measurements over larger areas. Here, we calibrate camera-based N&B implemented on a total internal reflection fluorescence (TIRF) microscope for various fluorescent proteins by determining their propensity to be fluorescent. We then apply camera-based N&B in live CHO-K1 cells to determine the oligomerization state of the epidermal growth factor receptor (EGFR), a transmembrane receptor tyrosine kinase that is a crucial regulator of cell proliferation and survival with implications in many cancers. EGFR oligomerization in resting cells and its regulation by the plasma membrane microenvironment is still under debate. Therefore, we investigate the effects of extrinsic factors, including membrane organization, cytoskeletal structure, and ligand stimulation, and intrinsic factors, including mutations in various EGFR domains, on the receptors oligomerization. Our results demonstrate that EGFR oligomerization increases with removal of cholesterol or sphingolipids, or the disruption of GM3-EGFR interactions, indicating raft association. However, oligomerization was not significantly influenced by the cytoskeleton. Mutations in either I706/V948 residues or E685/E687/E690 residues in the kinase and juxtamembrane domains, respectively, led to a decrease in oligomerization, indicating their necessity for EGFR dimerization. Finally, EGFR phosphorylation is oligomerization-dependent involving the extracellular domain (550-580 residues). Coupled with biochemical investigations, camera-based N&B indicates that EGFR oligomerization and phosphorylation is the outcome of several molecular interactions involving the lipid content and structure of the cell membrane and multiple residues in the kinase, juxtamembrane, and extracellular domains. STATEMENT OF SIGNIFICANCENumber and brightness (N&B) analysis is a powerful tool to determine protein association but is mostly conducted in confocal microscopes. This work determines the brightness and fluorescence probability of a range of fluorescent proteins for camera-based N&B on a total internal reflection microscope, demonstrating that with proper calibration different fluorescent proteins provide the same answers on oligomerization within the margins of error. This camera-based approach allows measuring N&B values across whole cell basal membranes up to an area of ~1,000 m2 simultaneously. N&B is then used in combination with biochemical assays to investigate the oligomerization and activation of the epidermal growth factor receptor (EGFR), a prototypical receptor tyrosine kinase with importance in cell signalling, division and survival and implicated in various cancers. The results indicate that EGFR oligomerization and activation is governed by an interplay between membrane structure and composition and key amino acid residues of EGFR that span the extracellular to the intracellular domains.

biophysics↗

bioRxiv2021

Heptanol-mediated phase separation determines phase preference of molecules in live cell membranes

Plasma membranes contain diverse nanoscale assemblies of lipid and protein domains. Specific localization of lipids and proteins in these domains is often essential for membrane function and integrity. Due to the nanoscale size and dynamic nature of membrane domains, identification of molecules residing in domains either is not possible with modern imaging techniques or requires advanced methods with high spatiotemporal resolution. Such methods need expensive equipment making these approaches inaccessible and thus difficult to implement at large scale. Here, we present a novel membrane fluidizer-induced clustering (MFIC) approach to identify the phase-preference of molecules in intact cell membranes. Experiments in phase-separated bilayers and live cells on molecules with known phase preference demonstrate that heptanol hyperfluidizes the membrane and stabilizes phase separation in cell membranes. The domain stabilization results in a transition of nano-to micron-sized clusters of associated molecules and allows identification of molecules localized in domains using routine microscopy techniques. This assay can be carried out on both genetically and extrinsically labelled molecules in live cell membranes, does not require any invasive sample preparation and can be carried out in 10-15 minutes. This inexpensive and easy to implement assay can be conducted at large-scale and will allow easy identification of molecules partitioning into domains.

biophysics↗

bioRxiv2021

Strong and specific connections between retinal axon mosaics and midbrain neurons revealed by large scale paired recordings

The superior colliculus (SC) is a midbrain structure that plays important roles in visually guided behaviors. Neurons in the SC receive afferent inputs from retinal ganglion cells (RGC), the output cells of the retina, but how SC neurons integrate RGC activity in vivo is unknown. SC neurons might be driven by strong but sparse retinal inputs, thereby reliably transmitting specific retinal functional channels. Alternatively, SC neurons could sum numerous but weak inputs, thereby extracting new features by combining a diversity of retinal signals. Here, we discovered that high-density electrodes simultaneously capture the activity and the location of large populations of retinal axons and their postsynaptic SC target neurons, permitting us to investigate the retinocollicular circuit on a structural and functional level in vivo. We show that RGC axons in the mouse are organized in mosaics that provide a single cell precise representation of the retina as input to SC. This isomorphic mapping between retina and SC builds the scaffold for highly specific wiring in the retinocollicular circuit which we show is characterized by strong connections and limited functional convergence, established in log-normally distributed connection strength. Because our novel method of large-scale paired recordings is broadly applicable for investigating functional connectivity across brain regions, we were also able to identify retinal inputs to the avian optic tectum of the zebra finch. We found common wiring rules in mammals and birds that provide a precise and reliable representation of the visual world encoded in RGCs to neurons in retinorecipient areas. HIGHLIGHTSO_LIHigh-density electrodes capture the activity of afferent axons and target neurons in vivo C_LIO_LIRetinal ganglion cells axons are organized in mosaics C_LIO_LISingle cell precise isomorphism between dendritic and axonal RGC mosaics C_LIO_LIMidbrain neurons are driven by sparse but strong retinal inputs C_LIO_LIFunctional wiring of the retinotectal circuit is similar in mammals and birds C_LI

neuroscience↗

bioRxiv2021

Non-linear Dimensionality Reduction on Extracellular Waveforms Reveals Physiological, Functional, and Laminar Diversity in Premotor Cortex

Cortical circuits are thought to contain a large number of cell types that coordinate to produce behavior. Current in vivo methods rely on clustering of specified features of extracellular waveforms to identify putative cell types, but these capture only a small amount of variation. Here, we develop a new method (WaveMAP) that combines non-linear dimensionality reduction with graph clustering to identify putative cell types. We apply WaveMAP to extracellular waveforms recorded from dorsal premotor cortex of macaque monkeys performing a decision-making task. Using WaveMAP, we robustly establish eight waveform clusters and show that these clusters recapitulate previously identified narrow- and broad-spiking types while revealing previously unknown diversity within these subtypes. The eight clusters exhibited distinct laminar distributions, characteristic firing rate patterns, and decision-related dynamics. Such insights were weaker when using feature-based approaches. WaveMAP therefore provides a more nuanced understanding of the dynamics of cell types in cortical circuits. SignificanceHow different cell types sculpt activity patterns in brain areas associated with decision-making is a fundamentally unresolved problem in neuroscience. In monkeys, and other species where transgenic access is not yet possible, identifying physiological types in vivo relies on only a few discrete user-specified features of extracellular waveforms to identify cell types. Here, we show that non-linear dimensionality reduction with graph clustering applied to the entire extracellular waveform can delineate many different putative cell types and does so in an interpretable manner. We show that this method reveals previously undocumented physiological, functional, and laminar diversity in the dorsal premotor cortex of monkeys, a key brain area implicated in decision-making.

neuroscience↗

bioRxiv2020

SRRF 'n' TIRF - FCS: Simultaneous spatiotemporal super-resolution microscopy

Super-resolution microscopy and single molecule fluorescence spectroscopy require mutually exclusive experimental strategies optimizing either time or spatial resolution. To achieve both, we implement a GPU-supported, camera-based measurement strategy that highly resolves spatial structures (~60 nm), temporal dynamics ([≤] 2 ms), and molecular brightness from the exact same data set. We demonstrate the applicability and advantages of multi-parametric measurements to monitor the super-resolved structure and dynamics of two different biomolecules, the actin binding polypeptide LifeAct, and the epidermal growth factor receptor (EGFR). Simultaneous super-resolution of spatial and temporal details leads to an improved precision in estimating the diffusion coefficient of LifeAct in dependence of the cellular actin network. Multi-parametric analysis suggests that the domain partitioning of EGFR is primarily determined by EGFR-membrane interactions, possibly sub-resolution clustering and inter-EGFR interactions but is largely independent of EGFR-actin interactions. These results demonstrate that pixel-wise cross-correlation of parameters obtained from different techniques on the same data set enables robust physicochemical parameter estimation and provides new biological knowledge that cannot be obtained from sequential measurements.

biophysics↗