Time Course Of Activation Research Articles

Changes Over Time in Masseter Muscle Activity, Symptoms of Discomfort, Stress Level and Salivary Flow Rate Following Intra-Oesophageal Acid Infusion.

Intra-oesophageal acid stimulation increases masseter muscle activity. However, the infusion speeds used in previous studies are significantly slow, with minimal acid volume (30 mL over 30 min). Additionally, it is unclear how masseter muscle activity, symptoms of discomfort, stress levels and saliva quantity change over time. The time course of masseter muscle activity, heartburn and discomfort symptoms, salivary cortisol concentration to assess stress, autonomic nervous system (ANS) activity and saliva quantity under the influence of intra-oesophageal acid infusion were evaluated at a faster injection rate and larger volume than in previous studies. Ten healthy adults underwent polygraphic monitoring, consisting of electromyography of the masseter muscle and electrocardiography during intra-oesophageal acid infusion (10 mL/min, 10 min). Symptoms of heartburn and discomfort were assessed using the visual analogue scale (VAS), and saliva quantity was measured. Friedman's test was used for multiple comparisons. Masseter muscle activity, VAS scores for heartburn and discomfort, as well as saliva quantity during the 10-min acid infusion, increased significantly compared with that before acid infusion (p < 0.001, 0.001, 0.019 and 0.047, respectively) and decreased 10 and 20 min after acid infusion (p = 0.004, 0.004 and 0.007, respectively). No significant changes were observed in the salivary cortisol concentration or ANS activity. Intra-oesophageal acid infusion stimulated symptoms of heartburn and discomfort and increased masseter muscle activity and saliva production, which may not be related to psychological stress.

Employing biological sex as a primary variable can restrict our understanding of behavioral diversity (and sex differences) in psychostimulant activity.

In addition to representing distinct biological sex groups, males and females are also individuals expressing behavioral diversity. Our MISSING (Mapping Intrinsic Sex Similarities as an Integral quality of Normalized Groups) model suggests that, with respect to behavior, grouping according to the individual reveals groups with differences that exceed biological sex-related differences, but this needs further clarification. We hypothesized that, compared to the current model (grouping by biological sex), the MISSING model (grouping by individual attributes) was the more effective grouping strategy to identify behavioral diversity. We conducted experiments in rats to determine the locomotor activity (in 90 min) following intraperitoneal injections of saline (n = 12 males, n = 11 females) and cocaine (n = 8 males, n = 11 females). For the current model, we compared males versus females using unpaired t-tests. For the MISSING model, we identified clusters of individuals (males and females) using normal mixtures clustering analysis of several behavioral variables and employed unpaired t-tests to compare clusters and Two-way ANOVA to determine if there were any SEX by cluster interactions. For both models, we employed linear regression analysis to compare relationships between variables and Two-way repeated measures ANOVA to analyze locomotor activity time course. For both the saline and cocaine groups, the MISSING model identified two behavioral clusters with differences that exceeded any differences due to biological sex. The MISSING model suggests that employing biological sex as a primary variable can obscure our understanding of sex and individual differences in psychostimulant activity.

Notch induces transcription by stimulating release of paused RNA polymerase II.

Notch proteins undergo ligand-induced proteolysis to release a nuclear effector that influences a wide range of cellular processes by regulating transcription. Despite years of study, however, how Notch induces the transcription of its target genes remains unclear. Here, we comprehensively examine the response to human Notch1 across a time course of activation using high-resolution genomic assays of chromatin accessibility and nascent RNA production. Our data reveal that Notch induces target gene transcription primarily by releasing paused RNA polymerase II (RNAPII). Moreover, in contrast to prevailing models suggesting that Notch acts by promoting chromatin accessibility, we found that open chromatin was established at Notch-responsive regulatory elements prior to Notch signal induction through SWI/SNF-mediated remodeling. Together, these studies show that the nuclear response to Notch signaling is dictated by the pre-existing chromatin state and RNAPII distribution at the time of signal activation.

8607 Gender Affirming Hormone Therapy: Influence of Brain Network Connectivity Changes on Affect

Abstract Disclosure: J. Andreano: None. A. Lopez: None. M. Misra: None. Background: Previous research indicates that acute gonadal treatments and fluctuations in ovarian hormone levels over the course of the menstrual cycle can alter patterns of communication between brain regions, as well as producing significant changes in self-reported affect. Recent research from our laboratory indicates changes in both brain connectivity and affect with gender affirming hormone therapy (GAHT). However, the relationship between these changes with GAHT are not well understood. Additionally, it is difficult to separate influences of GAHT and transgender identity in understanding differences in connectvity and affect from cisgender controls. A better understanding of the influence of GAHT on intrinsic network connectivity could point the way towards improved GAHT treatment regimens that minimize affective side effects. Objective: To assess relationships between changes in brain network connectivity and affective changes following GAHT, identifying regions in which changes of connectivity are associated with changes in self-reported affect, and to examine differences in brain network connectivity between transgender participants and cisgender controls. Methods: 14 transgender individuals receiving GAHT (estradiol n =7, testosterone n = 7)) were scanned using functional magnetic imaging (fMRI) at rest at two timepoints: baseline, prior to treatment, and 6 months into GAHT. Additionally, 20 cisgender controls (10 male, 10 female) were scanned once each. The time course of neural activity was computed for each voxel in the brain, then tested for correlation with each other voxel to develop a whole-brain connectome for each participant. Maps of average connectivity to two seeds known to associate with two major networks in the brain (posterior cingulate cortex for Default Mode Network, and anterior insula for Salience Network) were then generated for each participant. Within transgender participants, these maps were then compared between the baseline and 6 month timepoints. Additionally, baseline scans were also compared to scans from cisgender controls corresponding to biological sex. Results: Within-subjects analyses of transgender participants showed diverse associations between changes in seed-based connectivity and changes in self-reported anxiety and depression following GAHT in both the default mode and salience networks. Between subjects comparisons showed significantly higher connectivity within the salience network in trans men relative to cis women, as well as greater inter-network connectivity in this group. In contrast, cis men showed greater connectivity within both default mode and salience networks relative to trans women. Conclusions: Changes in affect following GAHT appear related to GAHT-induced changes in network connectivity; these influences appear independent of differences between cis and transgender individuals. Presentation: 6/2/2024

Model-based translation of results from in vitro to in vivo experiments for afabicin activity against Staphylococcus aureus.

Translation of experimental data on antibiotic activity typically relies on pharmacokinetic/pharmacodynamic (PK/PD) indices. Model-based approaches, considering the full antibiotic killing time course, could be an alternative. To develop a mechanism-based modelling framework to assess the in vitro and in vivo activity of the FabI inhibitor antibiotic afabicin, and explore the ability of a model built on in vitro data to predict in vivo outcome. A PK/PD model was built to describe bacterial counts from 162 static in vitro time-kill curves evaluating the effect of afabicin desphosphono, the active moiety of the prodrug afabicin, against 21 Staphylococcus aureus strains. Combined with a mouse PK model, outcomes of afabicin doses of 0.011-190 mg/kg q6h against nine S. aureus strains in a murine thigh infection model were predicted, and thereafter refined by estimating PD parameters. A sigmoid Emax model, with EC50 scaled by the MIC described the afabicin desphosphono killing in vitro. This model predicted, without parameter re-estimation, the in vivo bacterial counts at 24 h within a ±1 log margin for most dosing groups. When parameters were allowed to be estimated, EC50 was 38%-45% lower in vivo, compared with in vitro, within the studied MIC range. The developed PK/PD model described the time course of afabicin activity across experimental conditions and bacterial strains. This model showed translational capacity as parameters estimated on in vitro time-kill data could well predict the in vivo outcome for a wide variety of doses in a mouse thigh infection model.

Time course of electrical activity of the diaphragm (EAdi) in the peri extubation period and its role as predictor of extubation failure in difficult to wean patients

IntroductionWeaning patients from mechanical ventilation is crucial in the management of acute respiratory failure (ARF). Spontaneous breathing trials (SBT) are used to assess readiness for extubation, but extubation failure remains a challenge. Diaphragmatic function, measured by electrical activity of the diaphragm (EAdi), may provide insights into weaning outcomes.Materials and methodsThis prospective, observational study included difficult-to-wean patients undergoing invasive mechanical ventilation. EAdi was recorded before, during, and after extubation. Patients were categorized into extubation success and failure groups based on reintubation within 48 h. Statistical analysis assessed EAdi patterns and predictive value.ResultsThirty-one patients were analyzed, with six experiencing extubation failure. Overall, EAdi increased significantly between the phases before the SBT, the SBT and post-extubation period, up to 24 h (p < 0.001). EAdi values were higher in the extubation failure group during SBT (p = 0.01). An EAdi > 30 μV during SBT predicted extubation failure with 92% sensitivity and 67% specificity. Multivariable analysis confirmed EAdi as an independent predictor of extubation failure.ConclusionsIn difficult-to-wean patients, EAdi increases significantly between the phases before the SBT, the SBT and post-extubation period and is significantly higher in patients experiencing extubation failure. An EAdi > 30 μV during SBT may enhance extubation failure prediction compared to conventional parameters. Advanced monitoring of diaphragmatic function could improve weaning outcomes in critical care settings.

TMEM9B Regulates Endosomal ClC-3 and ClC-4 Transporters.

The nine-member CLC gene family of Cl- chloride-transporting membrane proteins is divided into plasma membrane-localized Cl- channels and endo-/lysosomal Cl-/H+ antiporters. Accessory proteins have been identified for ClC-K and ClC-2 channels and for the lysosomal ClC-7, but not the other CLCs. Here, we identified TMEM9 Domain Family Member B (TMEM9B), a single-span type I transmembrane protein of unknown function, to strongly interact with the neuronal endosomal ClC-3 and ClC-4 transporters. Co-expression of TMEM9B with ClC-3 or ClC-4 dramatically reduced transporter activity in Xenopus oocytes and transfected HEK cells. For ClC-3, TMEM9B also induced a slow component in the kinetics of the activation time course, suggesting direct interaction. Currents mediated by ClC-7 were hardly affected by TMEM9B, and ClC-1 currents were only slightly reduced, demonstrating specific interaction with ClC-3 and ClC-4. We obtained strong evidence for direct interaction by detecting significant Förster Resonance Energy Transfer (FRET), exploiting fluorescence lifetime microscopy-based (FLIM-FRET) techniques between TMEM9B and ClC-3 and ClC-4, but hardly any FRET with ClC-1 or ClC-7. The discovery of TMEM9B as a novel interaction partner of ClC-3 and ClC-4 might have important implications for the physiological role of these transporters in neuronal endosomal homeostasis and for a better understanding of the pathological mechanisms in CLCN3- and CLCN4-related pathological conditions.

An Insight into the Mechanism of DNA Cleavage by DNA Endonuclease from the Hyperthermophilic Archaeon Pyrococcus furiosus.

Hyperthermophilic archaea such as Pyrococcus furiosus survive under very aggressive environmental conditions by occupying niches inaccessible to representatives of other domains of life. The ability to survive such severe living conditions must be ensured by extraordinarily efficient mechanisms of DNA processing, including repair. Therefore, in this study, we compared kinetics of conformational changes of DNA Endonuclease Q from P. furiosus during its interaction with various DNA substrates containing an analog of an apurinic/apyrimidinic site (F-site), hypoxanthine, uracil, 5,6-dihydrouracil, the α-anomer of adenosine, or 1,N6-ethenoadenosine. Our examination of DNA cleavage activity and fluorescence time courses characterizing conformational changes of the dye-labeled DNA substrates during the interaction with EndoQ revealed that the enzyme induces multiple conformational changes of DNA in the course of binding. Moreover, the obtained data suggested that the formation of the enzyme-substrate complex can proceed through dissimilar kinetic pathways, resulting in different types of DNA conformational changes, which probably allow the enzyme to perform its biological function at an extreme temperature.

The Time Course of Changes in Prefrontal Cortex Activity During Walking in People With Parkinson’s Disease

Background Walking abnormalities in people with Parkinson’s disease (PD) are characterized by a shift in locomotor control from healthy automaticity to compensatory, executive control, mainly located in the prefrontal cortex (PFC). Although PFC activity during walking increases in people with PD, the time course of PFC activity during walking and its relationship to clinical or gait characteristics is unknown. Objective To identify the time course of PFC activity during walking in people with PD. To investigate whether clinical or gait variables would explain the PFC activity changes. Methods Thirty-eight people with PD tested OFF medication wore a portable, functional near-infrared spectroscopy (fNIRS) system to record relative PFC activity while walking. Wearable inertial sensors recorded spatiotemporal gait characteristics. Based on the PFC activity (fNIRS) in the late phase of the walking task (final 40 seconds), compared to the early phase (initial 40 seconds), participants were separated into 2 groups: reduced or sustained PFC activity. Results People with PD who reduced PFC activity during walking had less impaired gait (eg, faster gait speed) than those who had a sustained increase in PFC activity (P < .05). Cognitive set-shifting ability explained 18% of the PFC activation in the group with a sustained increase in PFC activity (P = .033). Conclusions The time course of reduction in PFC activity corresponds to less impaired gait performance in people with PD, while a sustained increase in PFC activity is related to worse cognitive flexibility. Reduction in PFC activity while walking may indicate a less impaired, automatic control of walking.

Conflict Processing Is Unaffected by Stimulus Duration Across Multiple Visual Tasks: Evidence for Transient over Permanent Activation Models

Permanent activation models of conflict tasks assume that the distractor constantly provides input into the response selection process as long as the stimulus is presented, whereas transient activation models assume that the influence of distractors is independent of stimulus duration. In the present study, we contrasted the prediction of these two architectures across different visual conflict tasks with manual responses. Specifically, we investigated the effects of short (150 ms) versus response-terminated stimulus duration on the delta plot (DP) slopes in a classic and accessory Simon task (Experiment 1), in a classic Simon and Eriksen flanker task (Experiment 2), and in a word-based (semantic) Simon and classic Stroop task (Experiment 3). Contrary to permanent activation models, all experiments revealed that the DP slopes were largely unaffected by stimulus duration. This result pattern is consistent with transient activation models such as the diffusion model for conflict tasks (DMC). Coherently, DMC was successfully fitted to the data. Importantly, the τ\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\ au $$\\end{document} parameter reflecting the time course of distractor activation, as well as other parameters, were largely unaffected by stimulus duration. In conclusion, our results suggest that distractor activation in visual conflict tasks with manual responses is transient rather than permanent, and stimulus duration generally has no meaningful influence on conflict processing.

The time course of Cantonese and Hong Kong Sign Language phonological activation: An ERP study of deaf bimodal bilingual readers of Chinese

This study investigated Cantonese and Hong Kong Sign Language (HKSL) phonological activation patterns in Hong Kong deaf readers using the ERP technique. Two experiments employing the error disruption paradigm were conducted while recording participants’ EEGs. Experiment 1 focused on orthographic and speech-based phonological processing, while Experiment 2 examined sign-phonological processing. ERP analyses focused on the P200 (180–220 ms) and N400 (300–500 ms) components.The results of Experiment 1 showed that hearing readers exhibited both orthographic and phonological effects in the P200 and N400 windows, consistent with previous studies on Chinese reading. In deaf readers, significant speech-based phonological effects were observed in the P200 window, and orthographic effects spanned both the P200 and N400 windows. Comparative analysis between the two groups revealed distinct spatial distributions for orthographic and speech-based phonological ERP effects, which may indicate the engagement of different neural networks during early processing stages.Experiment 2 found evidence of sign-phonological activation in both the P200 and N400 windows among deaf readers, which may reflect the involvement of sign-phonological representations in early lexical access and later semantic integration. Furthermore, exploratory analysis revealed that higher reading fluency in deaf readers correlated with stronger orthographic effects in the P200 window and diminished effects in the N400 window, indicating that efficient orthographic processing during early lexical access is a distinguishing feature of proficient deaf readers.

Phase-Dependent Visual and Sensorimotor Integration of Features for Grasp Computations before and after Effector Specification.

The simple act of viewing and grasping an object involves complex sensorimotor control mechanisms that have been shown to vary as a function of multiple object and other task features such as object size, shape, weight, and wrist orientation. However, these features have been mostly studied in isolation. In contrast, given the nonlinearity of motor control, its computations require multiple features to be incorporated concurrently. Therefore, the present study tested the hypothesis that grasp computations integrate multiple task features superadditively in particular when these features are relevant for the same action phase. We asked male and female human participants to reach-to-grasp objects of different shapes and sizes with different wrist orientations. Also, we delayed the movement onset using auditory signals to specify which effector to use. Using electroencephalography and representative dissimilarity analysis to map the time course of cortical activity, we found that grasp computations formed superadditive integrated representations of grasp features during different planning phases of grasping. Shape-by-size representations and size-by-orientation representations occurred before and after effector specification, respectively, and could not be explained by single-feature models. These observations are consistent with the brain performing different preparatory, phase-specific computations; visual object analysis to identify grasp points at abstract visual levels; and downstream sensorimotor preparatory computations for reach-to-grasp trajectories. Our results suggest the brain adheres to the needs of nonlinear motor control for integration. Furthermore, they show that examining the superadditive influence of integrated representations can serve as a novel lens to map the computations underlying sensorimotor control.

Notch induces transcription by stimulating release of paused RNA Polymerase II.

Notch proteins undergo ligand-induced proteolysis to release a nuclear effector that influences a wide range of cellular processes by regulating transcription. Despite years of study, however, how Notch induces the transcription of its target genes remains unclear. Here, we comprehensively examined the response to human Notch1 across a time course of activation using high-resolution genomic assays of chromatin accessibility and nascent RNA production. Our data reveal that Notch induces target gene transcription primarily by releasing paused RNA polymerase II (RNAPII). Moreover, in contrast to prevailing models suggesting that Notch acts by promoting chromatin accessibility, we found that open chromatin was established at Notch-responsive regulatory elements prior to Notch signal induction, through SWI/SNF-mediated remodeling. Together, these studies show that the nuclear response to Notch signaling is dictated by the pre-existing chromatin state and RNAPII distribution at the time of signal activation.

Intersubject correlations in reward and mentalizing brain circuits separately predict persuasiveness of two types of ISIS video propaganda

The Islamist group ISIS has been particularly successful at recruiting Westerners as terrorists. A hypothesized explanation is their simultaneous use of two types of propaganda: Heroic narratives, emphasizing individual glory, alongside Social narratives, which emphasize oppression against Islamic communities. In the current study, functional MRI was used to measure brain responses to short ISIS propaganda videos distributed online. Participants were shown 4 Heroic and 4 Social videos categorized as such by another independent group of subjects. Persuasiveness was measured using post-scan predictions of recruitment effectiveness. Inter-subject correlation (ISC) was used to measure commonality of brain activity time courses across individuals. ISCs in ventral striatum predicted rated persuasiveness for Heroic videos, while ISCs in mentalizing and default networks, especially in dmPFC, predicted rated persuasiveness for Social videos. This work builds on past findings that engagement of the reward circuit and of mentalizing brain regions predicts preferences and persuasion. The observed dissociation as a function of stimulus type is novel, as is the finding that intersubject synchrony in ventral striatum predicts rated persuasiveness. These exploratory results identify possible neural mechanisms by which political extremists successfully recruit prospective members and specifically support the hypothesized distinction between Heroic and Social narratives for ISIS propaganda.

Low-frequency ERK and Akt activity dynamics are predictive of stochastic cell division events

Understanding the dynamics of intracellular signaling pathways, such as ERK1/2 (ERK) and Akt1/2 (Akt), in the context of cell fate decisions is important for advancing our knowledge of cellular processes and diseases, particularly cancer. While previous studies have established associations between ERK and Akt activities and proliferative cell fate, the heterogeneity of single-cell responses adds complexity to this understanding. This study employed a data-driven approach to address this challenge, developing machine learning models trained on a dataset of growth factor-induced ERK and Akt activity time courses in single cells, to predict cell division events. The most predictive models were developed by applying discrete wavelet transforms (DWTs) to extract low-frequency features from the time courses, followed by using Ensemble Integration, a data integration and predictive modeling framework. The results demonstrated that these models effectively predicted cell division events in MCF10A cells (F-measure=0.524, AUC=0.726). ERK dynamics were found to be more predictive than Akt, but the combination of both measurements further enhanced predictive performance. The ERK model`s performance also generalized to predicting division events in RPE cells, indicating the potential applicability of these models and our data-driven methodology for predicting cell division across different biological contexts. Interpretation of these models suggested that ERK dynamics throughout the cell cycle, rather than immediately after growth factor stimulation, were associated with the likelihood of cell division. Overall, this work contributes insights into the predictive power of intra-cellular signaling dynamics for cell fate decisions, and highlights the potential of machine learning approaches in unraveling complex cellular behaviors.

Coherent photosynthetic activity between deciduous arboreal trees.

The long-term time course of photosynthetic activity in arboreal trees of aspen Populus tremula L. and birch Betula pendula Roth species native for Kola Subarctic was observed through 2014–2017 yr vegetation seasons in terms of PAM (Pulse Amplitude Modulated) foliage chlorophyll fluorescence measurements in situ. The temporal pair- wise correlation chlorophyll fluorescence pattern between any leaf blades couple for the whole any single crone as well as for proximate tree crones was found. The abrupt temperature drops and other environmental changes promote transition to blades coherence, the most synchronicity between trees was observed in 2014th fall. The global synchronization between any leaf couple was often preceded by transition of the foliar blades photosynthetic activity in common coherent quasi-cyclic rhythm. It is suggested that mutual crone’s intra and inter temporal organization and cooperative assimilation promote resistance and optimal realization of adaptive species potential.

Idiosyncratic patterns of interhemispheric connectivity in the face and scene networks of the human brain

Abstract Neuroimaging studies have revealed a network of regions in both hemispheres of the human brain that respond selectively to faces. Neural models of face processing have typically focused on functional connectivity between regions in the same hemisphere (intrahemispheric), with a particular bias toward the right hemisphere. Here, we explored the role of interhemispheric connectivity using fMRI. We used three datasets to compare functional connectivity, as shown by correlations between the time-courses of neural activity of face regions during different natural viewing paradigms. We found higher correlations of neural activity between corresponding interhemispheric regions (e.g., rFFA–lFFA) than between intrahemispheric regions (e.g., rFFA–rOFA), indicating a bias towards higher interhemispheric than intrahemispheric connectivity. A similar interhemispheric bias was evident in scene-selective regions. In contrast, we did not find an interhemispheric bias in early visual regions (V1–V3), where intrahemispheric connectivity between corresponding regions was generally higher than interhemispheric connectivity. Next, we asked whether the higher interhemispheric connectivity in the face and scene networks between corresponding regions was consistent across participants. We found that the interhemispheric bias was significantly attenuated when we compared the time-course of response across participants. This shows that interhemispheric bias in connectivity between corresponding regions in the face and scene networks is specific to the individual. These findings raise the possibility that idiosyncratic variation in interhemispheric connectivity may explain individual differences in perception.

Effective suppression of Ih and INa caused by capsazepine, known to be a blocker of TRPV1 receptor

A synthetic inhibitor of capsaicin-induced TRPV1 channel activation is called capsazepine (CPZ). In this study, we aimed to explore the effects of CPZ on hyperpolarization-activated cationic current (Ih) and voltage-gated Na + current (INa) in pituitary tumor (GH3) cells. Through patch-clamp recordings, we found that CPZ concentration-dependently inhibited Ih amplitude and slowed its activation time course. The IC50 and KD values were 3.1 and 3.16 μM, respectively. CPZ also shifted the steady-state activation curve of Ih towards a more hyperpolarized potential. However, there was no change in the gating charge of the curve. A modified Markovian model predicted the CPZ-induced decrease in the voltage-dependent hysteresis of Ih. CPZ suppressed INa in GH3 cells, without altering its activation or inactivation time course. Additionally, exposure to CPZ reduced spontaneous firing. These findings suggest that CPZ’s inhibitory effects on Ih and INa are direct and not dependent on vanilloid receptor binding. This could provide light on an unidentified ionic mechanism influencing the membrane excitability of neurons and endocrine or neuroendocrine cells in vivo.

In vitro and in vivo stability of a highly efficient long-acting cocaine hydrolase

It is recognized as a promising therapeutic strategy for cocaine use disorder to develop an efficient enzyme which can rapidly convert cocaine to physiologically inactive metabolites. We have designed and discovered a series of highly efficient cocaine hydrolases, including CocH5-Fc(M6) which is the currently known as the most efficient cocaine hydrolase with both the highest catalytic activity against (−)-cocaine and the longest biological half-life in rats. In the present study, we characterized the time courses of protein appearance, pH, structural integrity, and catalytic activity against cocaine in vitro and in vivo of a CocH5-Fc(M6) bulk drug substance produced in a bioreactor for its in vitro and in vivo stability after long-time storage under various temperatures (− 80, − 20, 4, 25, or 37 °C). Specifically, all the tested properties of the CocH5-Fc(M6) protein did not significantly change after the protein was stored at any of four temperatures including − 80, − 20, 4, and 25 °C for ~ 18 months. In comparison, at 37 °C, the protein was less stable, with a half-life of ~ 82 days for cocaine hydrolysis activity. Additionally, the in vivo studies further confirmed the linear elimination PK profile of CocH5-Fc(M6) with an elimination half-life of ~ 9 days. All the in vitro and in vivo data on the efficacy and stability of CocH5-Fc(M6) have consistently demonstrated that CocH5-Fc(M6) has the desired in vitro and in vivo stability as a promising therapeutic candidate for treatment of cocaine use disorder.

Progressive Circuit Hyperexcitability in Mouse Neocortical Slice Cultures with Increasing Duration of Activity Silencing.

Forebrain neurons deprived of activity become hyperactive when activity is restored. Rebound activity has been linked to spontaneous seizures in vivo following prolonged activity blockade. Here, we measured the time course of rebound activity and the contributing circuit mechanisms using calcium imaging, synaptic staining, and whole-cell patch clamp in organotypic slice cultures of mouse neocortex. Calcium imaging revealed hypersynchronous activity increasing in intensity with longer periods of deprivation. While activity partially recovered 3 d after slices were released from 5 d of deprivation, they were less able to recover after 10 d of deprivation. However, even after the longer period of deprivation, activity patterns eventually returned to baseline levels. The degree of deprivation-induced rebound was age-dependent, with the greatest effects occurring when silencing began in the second week. Pharmacological blockade of NMDA receptors indicated that hypersynchronous rebound activity did not require activation of Hebbian plasticity. In single-neuron recordings, input resistance roughly doubled with a concomitant increase in intrinsic excitability. Synaptic imaging of pre- and postsynaptic proteins revealed dramatic reductions in the number of presumptive synapses with a larger effect on inhibitory than excitatory synapses. Putative excitatory synapses colocalizing PSD-95 and Bassoon declined by 39 and 56% following 5 and 10 d of deprivation, but presumptive inhibitory synapses colocalizing gephyrin and VGAT declined by 55 and 73%, respectively. The results suggest that with prolonged deprivation, a progressive reduction in synapse number is accompanied by a shift in the balance between excitation and inhibition and increased cellular excitability.