Biology subjects

Rajadhyaksha, A. M.

Publications and source records attributed to Rajadhyaksha, A. M..

9 recordsLinked to original sources

bioRxiv2026

Reversible m6Am methylation of snRNA by FTO controls morphine reward and tolerance without altering analgesia

Mu opioids, such as morphine, are effective analgesics, but their reward and tolerance drive opioid use disorder. A major goal is to achieve analgesia without these harmful effects. Here we show that morphine reward and tolerance require the RNA demethylase FTO. Genetic depletion and pharmacologic inhibition of FTO each reduced morphine reward, measured by conditioned-place preference, and reduced antinociceptive tolerance to morphine and fentanyl, without altering analgesia. Although FTO is known to erase m6A on mRNA, we found no effect of FTO depletion on m6A sites, but markedly increased levels of m6Am on snRNA. The effects of FTO depletion were suppressed in mice that cannot make m6Am, supporting the role of m6Am in morphine reward and tolerance. We show that FTO depletion regulates a gene expression network linked to morphine signaling. FTO inhibitors may therefore provide useful adjuvants to mu opioids in pain management and treatment of opioid use disorder.

neuroscience↗

bioRxiv2026

Pharmacological Stratification of Public Bioactivity Databases: A Reusable, OECD-Anchored Curation and Benchmarking Framework Demonstrated for Opioid Receptors

Public bioactivity databases are heterogeneous not only in measurement type, where binding affinities and functional potencies are reported on different scales, but in pharmacology: the same compound and target can carry agonist, antagonist, or inhibitor records measured through binding displacement, cAMP, {beta}-arrestin, or [35S]GTP{gamma}S readouts that quantify different biological events. Pooling these records produces models whose output is detached from any coherent pharmacological claim. Prior work has standardized bioactivity at scale and quantified the noise from mixing measurement types, but pharmacological mechanism and assay-readout class have not been treated as a primary axis of large-scale curation. This study presents an auditable, OECD-anchored framework that stratifies public records by action type and assay readout before modeling, converting heterogeneous data into externally validated, interpretable QSAR tasks that compose with existing standardization resources rather than replacing them. The framework is demonstrated on the four opioid receptors (MOR, DOR, KOR, and nociceptin/orphanin FQ, NOP). Four public sources were reconciled into 72,148 merged records and 50,977 curated measurements spanning 19,585 compounds, each carrying auditable attributes for source agreement, endpoint meaning, pharmacology class, assay readout, and trust tier. Receptor-level binding tasks formed a compact benchmark with strong locked external performance, including KOR pK (R2 = 0.79, n = 798) and DOR pK (R2 = 0.77, n = 736). Pharmacology- and readout-resolved functional endpoints yielded externally validated strata that pooled labels would obscure, including a MOR antagonist functional-inhibition endpoint (R2 = 0.86, n = 110) and agonist potency endpoints for DOR, KOR, and MOR (R2 up to 0.81). Comparison against a fully pooled baseline shows that pooled models either match stratified models on coherent endpoints or reach a deceptively high R2 on functional-IC50 endpoints by training predominantly on binding-displacement records, so the pooled number predicts affinity rather than functional activity. SHAP attribution indicates that binding and functional potency encode partially distinct structure-activity signals. The dataset contract, not model performance alone, defines the validity and scope of a QSAR claim, and stratification is a precondition for a functional model to support a defensible claim. Curation logic, derived tables, frozen data, and reproducibility artifacts are released.

bioinformatics↗

bioRxiv2026

Large-scale reorganization of DNA methylation and upregulation of extracellular matrix genes in the dorsal dentate gyrus following cocaine taking

Cocaine self-administration induces neurobiological adaptations in brain circuits involved in encoding reward-associated context. The dorsal hippocampus, particularly the dorsal dentate gyrus, plays a critical role in the precise encoding of spatial and contextual information. We hypothesized that the dentate gyrus is uniquely positioned to undergo epigenomic and transcriptomic changes because of the convergence of the contextual features of reward and cocaine-enhanced dopamine and norepinephrine signals during volitional drug-taking. We report that cocaine self-administration produces significant DNA methylation changes at an unusually large number of [~]30,000 genomic regions (>10%, q<0.01) in dentate granule cells (DGCs) of male mice. Cocaine preferentially hypomethylated regions with heterogenous methylation, switching the methylation state in [~]16% of DGCs on average. The cocaine-sensitive/responsive epigenomic regions were overrepresented in enhancers and were associated with 9,833 genes, many of which were involved in diverse functions relevant to neuronal functioning. However, among the differentially methylated genes only two regulatory genes, c-fos and cartpt (known to be activated by cocaine), and a cluster of genes encoding components of the extracellular matrix (implicated in neuroplasticity) were differentially expressed (mostly upregulated) following cocaine self-administration, suggesting a gene regulatory network that is transcriptionally robust to perturbations but still specific for context-driven and reward associated neuroplasticity in DGCs. Overall, our data demonstrates that cocaine self-administration induces epigenomic and transcriptomic changes in the dorsal dentate gyrus that may contribute to dorsal hippocampal plasticity and contextual memory associated with cocaine self-administration.

neuroscience↗

bioRxiv2026

Acquisition and extinction of drug-context memories are linked to distinct epigenetic and transcriptional mechanisms in the mouse dentate gyrus

Acquisition and extinction of drug-context associations both involve learning, yet whether extinction erases the original drug memory remains unresolved. As learning is associated with epigenetically mediated transcriptional plasticity, we asked whether acquisition-induced DNA methylation and gene expression changes are reversed by extinction, or whether extinction induces its own distinct methylation and transcriptional changes. Here, we show that both acquisition and extinction of cocaine conditioned place preference (CPP) preferentially hypomethylated cis-regulatory elements and upregulated transcription, but at largely non-overlapping genomic regions and genes in the dorsal dentate gyrus, a key region in contextual learning. In both learning paradigms, the number of differentially expressed genes was an order of magnitude smaller than those differentially methylated, highlighting the robustness of the transcriptional network to epigenetic modifications, and implicating a non-linear relationship between regulatory elements and transcription characteristic for gene regulatory networks (GRNs). Notably, animals that failed to extinguish cocaine CPP displayed attenuated DNA methylation changes and minimal transcriptional response, consistent with the stochastic output of GRNs to produce alternative outcomes across individuals. Acquisition-upregulated genes were enriched in neuronal cilium functions, consistent with the known role of primary cilia in hippocampal learning and the persistence of drug-context memories through stable axo-ciliary signaling. In contrast, extinction-upregulated genes were overrepresented in mitochondrial energy homeostasis functions, suggesting their role in meeting rapid energy demands during learning. Overall, acquisition and extinction engage fundamentally distinct molecular mechanisms, providing a potential mechanistic explanation for why drug-context memories are suppressed but not erased by extinction.

animal behavior and cognition↗

bioRxiv2025

Deletion of Cacna1c (CaV1.2) in D1-expressing cells elicits divergent sex-specific effects on aversive and spatial memories

Dopamine signaling is critical for cognitive and emotional regulation and is implicated in multiple neuropsychiatric disorders. One downstream effector of dopamine is the L-type calcium channel CaV1.2, encoded by the risk gene CACNA1C. Genome-wide association studies have consistently linked CACNA1C single nucleotide polymorphisms to schizophrenia, bipolar disorder, and related conditions. We previously showed that homozygous deletion of Cacna1c in dopamine receptor 1 (D1)-expressing cells enhances remote (30 days post-training) contextual fear memory in male mice. Here, we extend these findings by examining sex- and gene dosage-specific behavioral consequences of Cacna1c loss in D1 cells. We find that D1-Cacna1c deletion produces a sex- and gene dosage-dependent effect on fear memory. In males, homozygous loss of D1-Cacna1c heightens remote contextual fear at 30-days post-training, replicating prior findings, whereas partial loss had no effect. Cue-associated fear memory remained unaffected across genotypes. In contrast, females exhibited heightened contextual fear with both heterozygous and homozygous D1-Cacna1c loss at 24-hrs, 7-days, and 30-days post-training, indicating increased sensitivity to contextual aversive learning. Cue-associated fear memory was higher at 24-hrs but normalized at later time points in females. In the Water Y-maze, males with heterozygous or homozygous D1-Cacna1c loss showed impaired spatial memory at 7-days post-training, whereas females were unaffected. D1-Cacna1c deletion reduced locomotor activity selectively in females during the initial 5-mins of a 60-min session, with no genotype effects in males. Social interaction and anxiety-like behavior were unchanged across groups. Together, these findings highlight the interplay between dopamine receptor signaling and calcium channel function in shaping sex-dependent aspects of memory.

neuroscience↗

bioRxiv2024

Cerebellar output neurons impair non-motor behaviors by altering development of extracerebellar connectivity

The capacity of the brain to compensate for insults during development depends on the type of cell loss, whereas the consequences of genetic mutations in the same neurons are difficult to predict. We reveal powerful compensation from outside the cerebellum when the excitatory cerebellar output neurons are ablated embryonically and demonstrate that the minimum requirement for these neurons is for motor coordination and not learning and social behaviors. In contrast, loss of the homeobox transcription factors Engrailed1/2 (EN1/2) in the cerebellar excitatory lineage leads to additional deficits in adult learning and spatial working memory, despite half of the excitatory output neurons being intact. Diffusion MRI indicates increased thalamo-cortico-striatal connectivity in En1/2 mutants, showing that the remaining excitatory neurons lacking En1/2 exert adverse effects on extracerebellar circuits regulating motor learning and select non-motor behaviors. Thus, an absence of cerebellar output neurons is less disruptive than having cerebellar genetic mutations.

neuroscience↗

bioRxiv2024

Elevating levels of the endocannabinoid 2-arachidonoylglycerol blunts opioid reward but not analgesia

Converging findings have established that the endocannabinoid (eCB) system serves as a possible target for the development of new treatments for pain as a complement to opioid-based treatments. Here we show in male and female mice that enhancing levels of the eCB, 2-arachidonoylglycerol (2-AG), through pharmacological inhibition of its catabolic enzyme, monoacylglycerol lipase (MAGL), either systemically or in the ventral tegmental area (VTA) with JZL184, leads to a substantial attenuation of the rewarding effects of opioids in male and female mice using conditioned place preference and self-administration paradigms, without altering their analgesic properties. These effects are driven by CB1 receptors (CB1Rs) within the VTA as VTA CB1R conditional knockout, counteracts JZL184s effects. Conversely, pharmacologically enhancing the levels of the other eCB, anandamide (AEA), by inhibition of fatty acid amide hydrolase (FAAH) has no effect on opioid reward or analgesia. Using fiber photometry with fluorescent sensors for calcium and dopamine (DA), we find that enhancing 2-AG levels diminishes opioid reward-related nucleus accumbens (NAc) activity and DA neurotransmission. Together these findings reveal that 2-AG counteracts the rewarding properties of opioids and provides a potential adjunctive therapeutic strategy for opioid-related analgesic treatments.

neuroscience↗

bioRxiv2023

A Genetically Encoded Actuator Selectively Boosts L-type Calcium Channels in Diverse Physiological Settings

L-type Ca2+ channels (CaV1.2/1.3) convey influx of calcium ions (Ca2+) that orchestrate a bevy of biological responses including muscle contraction and gene transcription. Deficits in CaV1 function play a vital role in cardiac and neurodevelopmental disorders. Yet conventional pharmacological approaches to upregulate CaV1 are limited, as excessive Ca2+ influx leads to cytotoxicity. Here, we develop a genetically encoded enhancer of CaV1.2/1.3 channels (GeeC) to manipulate Ca2+ entry in distinct physiological settings. Specifically, we functionalized a nanobody that targets the CaV macromolecular complex by attaching a minimal effector domain from a CaV enhancer--leucine rich repeat containing protein 10 (Lrrc10). In cardiomyocytes, GeeC evoked a 3-fold increase in L-type current amplitude. In neurons, GeeC augmented excitation-transcription (E-T) coupling. In all, GeeC represents a powerful strategy to boost CaV1.2/1.3 function in distinct physiological settings and, in so doing, lays the groundwork to illuminate new insights on neuronal and cardiac physiology and disease.

physiology↗

bioRxiv2023

Repeat investigation during social preference behavior is suppressed in male mice with prefrontal cortex cacna1c (Cav1.2)-deficiency through the dysregulation of neural dynamics

Impairments in social behavior are observed in a range of neuropsychiatric disorders and several lines of evidence have demonstrated that dysfunction of the prefrontal cortex (PFC) plays a central role in social deficits. We have previously shown that loss of neuropsychiatric risk gene Cacna1c that codes for the Cav1.2 isoform of L-type calcium channels (LTCCs) in the PFC result in impaired sociability as tested using the three-chamber social approach test. In this study we aimed to further characterize the nature of the social deficit associated with a reduction in PFC Cav1.2 channels (Cav1.2PFCKO mice) by testing male mice in a range of social and non-social tests while examining PFC neural activity using in vivo GCaMP6s fiber photometry. We found that during the first investigation of the social and non-social stimulus in the three-chamber test, both Cav1.2PFCKO male mice and Cav1.2PFCGFP controls spent significantly more time with the social stimulus compared to a non-social object. In contrast, during repeat investigations while Cav1.2PFCWT mice continued to spend more time with the social stimulus, Cav1.2PFCKO mice spent equal amount of time with both social and non-social stimuli. Neural activity recordings paralleled social behavior with increase in PFC population activity in Cav1.2PFCWT mice during first and repeat investigations, which was predictive of social preference behavior. In Cav1.2PFCKO mice, there was an increase in PFC activity during first social investigation but not during repeat investigations. These behavioral and neural differences were not observed during a reciprocal social interaction test nor during a forced alternation novelty test. To evaluate a potential deficit in reward-related processes, we tested mice in a three-chamber test wherein the social stimulus was replaced by food. Behavioral testing revealed that both Cav1.2PFCWT and Cav1.2PFCKO mice showed a preference for food over object with significantly greater preference during repeat investigation. Interestingly, there was no increase in PFC activity when Cav1.2PFCWT or Cav1.2PFCKO first investigated the food however activity significantly increased in Cav1.2PFCWT mice during repeat investigations of the food. This was not observed in Cav1.2PFCKO mice. In summary, a reduction in Cav1.2 channels in the PFC suppresses the development of a sustained social preference in mice that is associated with lack of PFC neuronal population activity that may be related to deficits in social reward.

neuroscience↗