Adolescent THC impacts on mPFC dopamine-mediated cognitive processes in male and female rats.

Rationale: Adolescent cannabis use is linked to later-life changes in cognition, learning, and memory. Rodent experimental studies suggest Δ9-tetrahydrocannabinol (THC) influences development of circuits underlying these processes, especially in the prefrontal cortex, which matures during adolescence. Objective: We determined how 14 daily THC injections (5mg/kg) during adolescence persistently impacts medial prefrontal cortex (mPFC) dopamine-dependent cognition. Methods: In adult Long Evans rats treated as adolescents with THC (AdoTHC), we quantify performance on two mPFC dopamine-dependent reward-based tasks-strategy set shifting and probabilistic discounting. We also determined how acute dopamine augmentation with amphetamine (0, 0.25, 0.5 mg/kg), or specific chemogenetic stimulation of ventral tegmental area (VTA) dopamine neurons and their projections to mPFC impacts probabilistic discounting. Results: AdoTHC sex-dependently impacts acquisition of cue-guided instrumental reward seeking, but has minimal effects on set-shifting or probabilistic discounting in either sex. When we challenged dopamine circuits acutely with amphetamine during probabilistic discounting, we found reduced discounting of improbable reward options, with AdoTHC rats being more sensitive to these effects than controls. In contrast, neither acute chemogenetic stimulation of VTA dopamine neurons nor pathway-specific chemogenetic stimulation of their projection to mPFC impacted probabilistic discounting in control rats, although stimulation of this cortical dopamine projection slightly disrupted choices in AdoTHC rats. Conclusions: These studies confirm a marked specificity in the cognitive processes impacted by AdoTHC exposure. They also suggest that some persistent AdoTHC effects may alter amphetamine-induced cognitive changes in a manner independent of VTA dopamine projections to mPFC, or via alterations of non-VTA dopamine neurons.

Neural circuit basis of adolescent THC-induced potentiation of opioid responses in adult mice.

Use of one drug of abuse typically influences the behavioral response to other drugs, either administered at the same time or a subsequent time point. The nature of the drugs being used, as well as the timing and dosing, also influence how these drugs interact. Here, we tested the effects of adolescent THC exposure on the development of morphine-induced behavioral adaptations following repeated morphine exposure during adulthood. We found that adolescent THC administration impacted morphine-induced behaviors across several dimensions, including potentiating reward and paradoxically impairing the development of morphine reward. We then mapped the whole-brain response to a reinstatement dose of morphine, finding that adolescent THC administration led to increased activity in the basal ganglia and increased functional connectivity between frontal cortical regions and the ventral tegmental area. Last, we show using rabies virus-based circuit mapping that adolescent THC exposure triggers a long-lasting elevation in connectivity from the frontal cortex regions onto ventral tegmental dopamine cells that has the potential to influence dopaminergic response to morphine administration during adulthood. Our study adds to the rich literature on the interaction between drugs of abuse and provides potential circuit substates by which adolescent THC exposure influences responses to morphine later in life.

Amino acid degradation pathway inhibitory by-products trigger apoptosis in CHO cells.

Chinese hamster ovary (CHO) cells are widely used to produce complex biopharmaceuticals. Improving their productivity is necessary to fulfill the growing demand for such products. One way to enhance productivity is by cultivating cells at high densities, but inhibitory by-products, such as metabolite derivatives from amino acid degradation, can hinder achieving high cell densities. This research examines the impact of these inhibitory by-products on high-density cultures. We cultured X1 and X2 CHO cell lines in a small-scale semi-perfusion system and introduced a mix of inhibitory by-products on day 10. The X1 and X2 cell lines were chosen for their varied responses to the by-products; X2 was susceptible, while X1 survived. Proteomics revealed that the X2 cell line presented changes in the proteins linked to apoptosis regulation, cell building block synthesis, cell growth, DNA repair, and energy metabolism. We later used the AB cell line, an apoptosis-resistant cell line, to validate the results. AB behaved similar to X1 under stress. We confirmed the activation of apoptosis in X2 using a caspase assay. This research provides insights into the mechanisms of cell death triggered by inhibitory by-products and can guide the optimization of CHO cell culture for biopharmaceutical manufacturing.

Harnessing metabolic plasticity in CHO cells for enhanced perfusion cultivation.

Chinese Hamster Ovary (CHO) cells have rapidly become a cornerstone in biopharmaceutical production. Recently, a reinvigoration of perfusion culture mode in CHO cell cultivation has been observed. However, most cell lines currently in use have been engineered and adapted for fed-batch culture methods, and may not perform optimally under perfusion conditions. To improve the cell's resilience and viability during perfusion culture, we cultured a triple knockout CHO cell line, deficient in three apoptosis related genes BAX, BAK, and BOK in a perfusion system. After 20 days of culture, the cells exhibited a halt in cell proliferation. Interestingly, following this phase of growth arrest, the cells entered a second growth phase. During this phase, the cell numbers nearly doubled, but cell specific productivity decreased. We performed a proteomics investigation, elucidating a distinct correlation between growth arrest and cell cycle arrest and showing an upregulation of the central carbon metabolism and oxidative phosphorylation. The upregulation was partially reverted during the second growth phase, likely caused by intragenerational adaptations to stresses encountered. A phase-dependent response to oxidative stress was noted, indicating glutathione has only a secondary role during cell cycle arrest. Our data provides evidence of metabolic regulation under high cell density culturing conditions and demonstrates that cell growth arrest can be overcome. The acquired insights have the potential to not only enhance our understanding of cellular metabolism but also contribute to the development of superior cell lines for perfusion cultivation.

A head-to-head comparison of two DREADD agonists for suppressing operant behavior in rats via VTA dopamine neuron inhibition.

RATIONALE: Designer receptors exclusively activated by designer drugs (DREADDs) are a tool for "remote control" of defined neuronal populations during behavior. These receptors are inert unless bound by an experimenter-administered designer drug, commonly clozapine-n-oxide (CNO). However, questions have emerged about the suitability of CNO as a systemically administered DREADD agonist. OBJECTIVES: Second-generation agonists such as JHU37160 (J60) have been developed, which may have more favorable properties than CNO. Here we sought to directly compare effects of CNO (0, 1, 5, & 10 mg/kg, i.p.) and J60 (0, 0.03, 0.3, & 3 mg/kg, i.p.) on operant food pursuit. METHODS: Male and female TH:Cre + rats and their wildtype (WT) littermates received cre-dependent hM4Di-mCherry vector injections into ventral tegmental area (VTA), causing inhibitory DREADD expression in VTA dopamine neurons of TH:Cre + rats. All rats were trained to stably lever press for palatable food on a fixed ratio 10 schedule, and doses of both agonists were tested on separate days in counterbalanced order. RESULTS: All three CNO doses reduced operant rewards earned in rats with DREADDs, and no CNO dose had behavioral effects in WT controls. The highest J60 dose tested significantly reduced responding in DREADD rats, but this dose also increased responding in WTs, indicating non-specific effects. The magnitude of CNO and J60 effects in TH:Cre + rats were correlated and were present in both sexes. CONCLUSIONS: Findings demonstrate the usefulness of directly comparing DREADD agonists when optimizing behavioral chemogenetics, and highlight the importance of proper controls, regardless of the DREADD agonist employed.

Design of Polarity-Dependent Immunosensors Based on the Structural Analysis of Engineered Antibodies.

"Reagentless" immunosensors are emerging to address the challenge of practical and sensitive detection of important biomarkers in real biological samples without the need for multistep assays and user intervention, with applications ranging from research tools to point-of-care diagnostics. Selective target binding to an affinity reagent is detected and reported in one step without the need for washing or additional reporters. In this study, we used a structure-guided approach to identify a mutation site in an antibody fragment for the polarity-dependent fluorophore, Anap, such that upon binding of the protein target cardiac troponin I, the Anap-labeled antibody would produce a detectable and dose-dependent shift in emission wavelength. We observed a significant emission wavelength shift of the Anap-labeled anti-cTnI mutant, with a blue shift of up to 37 nm, upon binding to the cTnI protein. Key differences in the resulting emission spectra between target peptides in comparison to whole proteins were also found; however, the affinity and binding characteristics remained unaffected when compared to the wild-type antibody. We also highlighted the potential flexibility of the approach by incorporating a near-infrared dye, IRDye800CW, into the same mutation site, which also resulted in a dose-dependent wavelength shift upon target incubation. These reagents can be used in experiments and devices to create simpler and more efficient biosensors across a range of research, medical laboratory, and point-of-care platforms.

A head-to-head comparison of two DREADD agonists for suppressing operant behavior in rats via VTA dopamine neuron inhibition.

Rationale: Designer receptors exclusively activated by designer drugs (DREADDs) are a tool for "remote control" of defined neuronal populations during behavior. These receptors are inert unless bound by an experimenter-administered designer drug, most commonly clozapine-n-oxide (CNO). However, questions have emerged about the suitability of CNO as a systemically administered DREADD agonist. Objectives: Second-generation agonists such as JHU37160 (J60) have been developed, which may have more favorable properties than CNO. Here we sought to directly compare effects of CNO (0, 1, 5, & 10 mg/kg, i.p.) and J60 (0, 0.03, 0.3, & 3 mg/kg, i.p.) on operant food pursuit. Methods: Male and female TH:Cre+ rats and their wildtype (WT) littermates received cre-dependent hM4Di-mCherry vector injections into ventral tegmental area (VTA), causing inhibitory DREADD expression in VTA dopamine neurons in TH:Cre+ rats. Rats were trained to stably lever press for palatable food on a fixed ratio 10 schedule, and doses of both agonists were tested on separate days in a counterbalanced order. Results: All three CNO doses reduced operant food seeking in rats with DREADDs, and no CNO dose had behavioral effects in WT controls. The highest tested J60 dose significantly reduced responding in DREADD rats, but this dose also increased responding in WTs, indicating non-specific effects. The magnitude of CNO and J60 effects in TH:Cre+ rats were correlated and were present in both sexes. Conclusions: Findings demonstrate the usefulness of directly comparing DREADD agonists when optimizing behavioral chemogenetics, and highlight the importance of proper controls, regardless of the DREADD agonist employed.

Adolescent exposure to low-dose Δ9-tetrahydrocannabinol depletes the ovarian reserve in female mice.

Cannabis use by adolescents is widespread, but its effects on the ovaries remain largely unknown. Δ9-tetrahydrocannabinol (THC) exerts its pharmacological effects by activating, and in some conditions hijacking, cannabinoid receptors (CBRs). We hypothesized that adolescent exposure to THC affects ovarian function in adulthood. Peripubertal female C57BL/6N mice were given THC (5 mg/kg) or its vehicle, once daily by intraperitoneal injection. Some mice received THC from postnatal day (PND) 30-33 and their ovaries were harvested PND34; other mice received THC from PND30-43, and their ovaries were harvested PND70. Adolescent treatment with THC depleted ovarian primordial follicle numbers by 50% at PND70, 4 weeks after the last dose. The treatment produced primordial follicle activation, which persisted until PND70. THC administration also caused DNA damage in primary follicles and increased PUMA protein expression in oocytes of primordial and primary follicles. Both CB1R and CB2R were expressed in oocytes and theca cells of ovarian follicles. Enzymes involved in the formation (N-acylphosphatidylethanolamine phospholipase D) or deactivation (fatty acid amide hydrolase) of the endocannabinoid anandamide were expressed in granulosa cells of ovarian follicles and interstitial cells. Levels of mRNA for CBR1 were significantly increased in ovaries after adolescent THC exposure, and upregulation persisted for at least 4 weeks. Our results support that adolescent exposure to THC may cause aberrant activation of the ovarian endocannabinoid system in female mice, resulting in substantial loss of ovarian reserve in adulthood. Relevance of these findings to women who frequently used cannabis during adolescence warrants investigation.

Long-term effects of THC exposure on reward learning and motivated behavior in adolescent and adult male rats.

RATIONALE: The endocannabinoid system makes critical contributions to reward processing, motivation, and behavioral control. Repeated exposure to THC or other cannabinoid drugs can cause persistent adaptions in the endocannabinoid system and associated neural circuitry. It remains unclear how such treatments affect the way rewards are processed and pursued. OBJECTIVE AND METHODS: We examined if repeated THC exposure (5 mg/kg/day for 14 days) during adolescence or adulthood led to long-term changes in rats' capacity to flexibly encode and use action-outcome associations for goal-directed decision making. Effects on hedonic feeding and progressive ratio responding were also assessed. RESULTS: THC exposure had no effect on rats' ability to flexibly select actions following reward devaluation. However, instrumental contingency degradation learning, which involves avoiding an action that is unnecessary for reward delivery, was augmented in rats with a history of adult but not adolescent THC exposure. THC-exposed rats also displayed more vigorous instrumental behavior in this study, suggesting a motivational enhancement. A separate experiment found that while THC exposure had no effect on hedonic feeding behavior, it increased rats' willingness to work for food on a progressive ratio schedule, an effect that was more pronounced when THC was administered to adults. Adolescent and adult THC exposure had opposing effects on the CB1 receptor dependence of progressive ratio performance, decreasing and increasing sensitivity to rimonabant-induced behavioral suppression, respectively. CONCLUSIONS: Our findings reveal that exposure to a translationally relevant THC exposure regimen induces long-lasting, age-dependent alterations in cognitive and motivational processes that regulate the pursuit of rewards.

Stress-induced plasticity of a CRH/GABA projection disrupts reward behaviors in mice.

Disrupted operations of the reward circuit underlie major emotional disorders, including depression, which commonly arise following early life stress / adversity (ELA). However, how ELA enduringly impacts reward circuit functions remains unclear. We characterize a stress-sensitive projection connecting basolateral amygdala (BLA) and nucleus accumbens (NAc) that co-expresses GABA and the stress-reactive neuropeptide corticotropin-releasing hormone (CRH). We identify a crucial role for this projection in executing disrupted reward behaviors provoked by ELA: chemogenetic and optogenetic stimulation of the projection in control male mice suppresses several reward behaviors, recapitulating deficits resulting from ELA and demonstrating the pathway's contributions to normal reward behaviors. In adult ELA mice, inhibiting-but not stimulating-the projection, restores typical reward behaviors yet has little effect in controls, indicating ELA-induced maladaptive plasticity of this reward-circuit component. Thus, we discover a stress-sensitive, reward inhibiting BLA → NAc projection with unique molecular features, which may provide intervention targets for disabling mental illnesses.

Nasal accumulation and metabolism of Δ9-tetrahydrocannabinol following aerosol ('vaping') administration in an adolescent rat model.

Passive aerosol exposure to Δ9-tetrahydrocannabinol (THC) in laboratory animals results in faster onset of action and less extensive liver metabolism compared to most other administration routes and might thus provide an ecologically relevant model of human cannabis inhalation. Previous studies have, however, overlooked the possibility that rodents, as obligate nose breathers, may accumulate aerosolized THC in the nasal cavity, from where the drug might directly diffuse to the brain. To test this, we administered THC (ten 5-s puffs of 100 mg/mL of THC) to adolescent (31-day-old) Sprague-Dawley rats of both sexes. We used liquid chromatography/tandem mass spectrometry to quantify the drug and its first-pass metabolites - 11-hydroxy-Δ9-THC (11-OH-THC) and 11-nor-9-carboxy-Δ9-THC (11-COOH-THC) - in nasal mucosa, lungs, plasma, and brain (olfactory bulb and cerebellum) at various time points after exposure. Apparent maximal THC concentration and area under the curve were ∼5 times higher in nasal mucosa than in lungs and 50-80 times higher than in plasma. Concentrations of 11-OH-THC were also greater in nasal mucosa and lungs than other tissues, whereas 11-COOH-THC was consistently undetectable. Experiments with microsomal preparations confirmed local metabolism of THC into 11-OH-THC (not 11-COOH-THC) in nasal mucosa and lungs. Finally, whole-body exposure to THC deposited substantial amounts of THC (∼150 mg/g) on fur but suppressed post-exposure grooming in rats of both sexes. The results indicate that THC absorption and metabolism in nasal mucosa and lungs, but probably not gastrointestinal tract, contribute to the pharmacological effects of aerosolized THC in male and female rats.

A Sensitive Ultrahigh-Performance Liquid Chromatography/Tandem Mass Spectrometry Method for the Simultaneous Analysis of Phytocannabinoids and Endocannabinoids in Plasma and Brain.

Introduction: Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are major chemical constituents of cannabis, which may interact either directly or indirectly with the endocannabinoid and endocannabinoid-like ("paracannabinoid") systems, two lipid-based signaling complexes that play important roles in physiology. Legislative changes emphasize the need to understand how THC and CBD might impact endocannabinoid and paracannabinoid signaling, and to develop analytical approaches to study such impact. In this study, we describe a sensitive and accurate method for the simultaneous quantification of THC, its main oxidative metabolites [11-hydroxy-Δ9-THC (11-OH-THC) and 11-nor-9-carboxy-Δ9-THC (11-COOH-THC)], CBD, and a representative set of endocannabinoid [anandamide and 2-arachidonoyl-sn-glycerol (2-AG)] and paracannabinoid [palmitoylethanolamide (PEA) and oleoylethanolamide (OEA)] compounds. Analyte separation relies on the temperature-dependent shape selectivity properties of polymerically bonded C18 stationary phases. Materials and Methods: Analytes are extracted from tissues using acetonitrile precipitation followed by phospholipid removal. The ultrahigh-performance liquid chromatography/tandem mass spectrometry protocol utilizes a commercially available C18 polymeric-bonded phase column and a simple gradient elution system. Results: Ten-point calibration curves show excellent linearity (R2>0.99) over a wide range of analyte concentrations (0.02-500 ng/mL). Lowest limits of quantification are 0.05 ng/mL for anandamide, 0.1 ng/mL for 11-OH-THC and OEA, 0.2 ng/mL for THC and CBD, 0.5 ng/mL for 11-COOH-THC, 1.0 ng/mL for 2-AG, and 2.0 ng/mL for PEA. The lowest limits of detection are 0.02 ng/mL for anandamide, 0.05 ng/mL for 11-OH-THC and OEA, 0.1 ng/mL for THC and CBD, 0.2 ng/mL for 11-COOH-THC, 0.5 ng/mL for 2-AG, and 1.0 ng/mL for PEA. Conclusions: An application of the method is presented, which showed that phytocannabinoid administration elevates endocannabinoid levels in plasma and brain of adolescent male and female mice.

Ventral pallidum GABA neurons bidirectionally control opioid relapse across rat behavioral models.

Opioid addiction is a chronic, relapsing disorder. Whether addicted individuals are forced to abstain or they decide themselves to quit using drugs, relapse rates are high-especially upon encountering contexts and stimuli associated with prior opioid use. Rodents similarly show context- and cue-induced reinstatement of drug seeking following abstinence, and intriguingly, the neural circuits underlying these relapse-like behaviors differ when abstinence is involuntarily imposed, responding is extinguished, or animals decide themselves to cease taking drug. Here, we employ two complementary rat behavioral models of relapse-like behavior for the highly reinforcing opioid drug remifentanil, and asked whether GABAergic neurons in the ventral pallidum (VPGABA) control opioid seeking under these behavioral conditions. Specifically, we asked how chemogenetically stimulating VPGABA neurons with clozapine-N-oxide (CNO) influences the ability of contextual or discrete remifentanil-paired cues to reinstate drug seeking following either voluntary abstinence (punishment-induced; GroupPunish), or extinction training (GroupExt). In GroupPunish rats, we also chemogenetically inhibited VPGABA neurons, and examined spontaneous VP activity (Fos) during cued reinstatement. In both GroupPunish and GroupExt rats, stimulating Gq-signaling in VPGABA neurons augmented remifentanil reinstatement in a cue- and context-dependent manner. Conversely, engaging inhibitory Gi-signaling in VPGABA neurons in GroupPunish suppressed cue-induced reinstatement, and cue-triggered seeking was correlated with Fos expression in rostral, but not caudal VP. Neither stimulating nor inhibiting VPGABA neurons influenced unpunished remifentanil self-administration. We conclude that VPGABA neurons bidirectionally control opioid seeking regardless of the specific relapse model employed, highlighting their fundamental role in opioid relapse-like behavior across behavioral models, and potentially across species.

Evaluation of Phage Display Biopanning Strategies for the Selection of Anti-Cell Surface Receptor Antibodies.

Monoclonal antibodies (mAbs) are one of the most successful and versatile protein-based pharmaceutical products used to treat multiple pathological conditions. The remarkable specificity of mAbs and their affinity for biological targets has led to the implementation of mAbs in the therapeutic regime of oncogenic, chronic inflammatory, cardiovascular, and infectious diseases. Thus, the discovery of novel mAbs with defined functional activities is of crucial importance to expand our ability to address current and future clinical challenges. In vitro, antigen-driven affinity selection employing phage display biopanning is a commonly used technique to isolate mAbs. The success of biopanning is dependent on the quality and the presentation format of the antigen, which is critical when isolating mAbs against membrane protein targets. Here, we provide a comprehensive investigation of two established panning strategies, surface-tethering of a recombinant extracellular domain and cell-based biopanning, to examine the impact of antigen presentation on selection outcomes with regards to the isolation of positive mAbs with functional potential against a proof-of-concept type I cell surface receptor. Based on the higher sequence diversity of the resulting antibody repertoire, presentation of a type I membrane protein in soluble form was more advantageous over presentation in cell-based format. Our results will contribute to inform and guide future antibody discovery campaigns against cell surface proteins.

Engineering death resistance in CHO cells for improved perfusion culture.

The reliable and cost-efficient manufacturing of monoclonal antibodies (mAbs) is essential to fulfil their ever-growing demand. Cell death in bioreactors reduces productivity and product quality, and is largely attributed to apoptosis. In perfusion bioreactors, this leads to the necessity of a bleed stream, which negatively affects the overall process economy. To combat this limitation, death-resistant Chinese hamster ovary cell lines were developed by simultaneously knocking out the apoptosis effector proteins Bak1, Bax, and Bok with CRISPR technology. These cell lines were cultured in fed-batch and perfusion bioreactors and compared to an unmodified control cell line. In fed-batch, the death-resistant cell lines showed higher cell densities and longer culture durations, lasting nearly a month under standard culture conditions. In perfusion, the death-resistant cell lines showed slower drops in viability and displayed an arrest in cell division after which cell size increased instead. Pertinently, the death-resistant cell lines demonstrated the ability to be cultured for several weeks without bleed, and achieved similar volumetric productivities at lower cell densities than that of the control cell line. Perfusion culture reduced fragmentation of the mAb produced, and the death-resistant cell lines showed increased glycosylation in the light chain in both bioreactor modes. These data demonstrate that rationally engineered death-resistant cell lines are ideal for mAb production in perfusion culture, negating the need to bleed the bioreactor whilst maintaining product quantity and quality.

Enduring disruption of reward and stress circuit activities by early-life adversity in male rats.

In humans, early-life adversity (ELA) such as trauma, poverty, and chaotic environment is linked to increased risk of later-life emotional disorders including depression and substance abuse. These disorders involve underlying disruption of reward circuits and likely vary by sex. Accordingly, we previously found that ELA leads to anhedonia for natural rewards and cocaine in male rodents, whereas in females ELA instead increases vulnerability to addiction-like use of opioid drugs and palatable food. While these findings suggest that ELA-induced disruption of reward circuitry may differ between the sexes, the specific circuit nodes that are influenced by ELA in either sex remain poorly understood. Here, in adult male Sprague-Dawley rats, we ask how ELA impacts opioid addiction-relevant behaviors that we previously tested after ELA in females. We probe potential circuit mechanisms in males by assessing opioid-associated neuronal activation in stress and reward circuit nodes including nucleus accumbens (NAc), amygdala, medial prefrontal cortex (mPFC), and paraventricular thalamus. We find that ELA diminishes opioid-seeking behaviors in males, and alters heroin-induced activation of NAc, PFC, and amygdala, suggesting a potential circuit-based mechanism. These studies demonstrate that ELA leads to behavioral and neurobiological disruptions consistent with anhedonia in male rodents, unlike the increased opioid seeking we previously saw in females. Our findings, taken together with our prior work, suggest that men and women could face qualitatively different mental health consequences of ELA, which may be essential for individually tailoring future intervention strategies.

Comparative Pharmacokinetics of Δ9-Tetrahydrocannabinol in Adolescent and Adult Male and Female Rats.

Introduction: Studies in rodent models have shown that adolescent exposure to Δ9-THC, the psychotropic constituent of cannabis, produces long-lasting alterations in brain function and behavior. However, our understanding of how age and sex might influence the distribution and metabolism of THC in laboratory rodents is still incomplete. In the present report, we provide a comparative analysis of the pharmacokinetic (PK) properties of THC in adolescent and adult rats of both sexes, and outline several dissimilarities across these groups. Materials and Methods: A single (acute) or 2-week daily (subchronic) administration of THC (0.5 or 5 mg/kg, acute; 5 mg/kg, subchronic; intraperitoneal) was given to adolescent (33-day-old, acute; 30-44-day-old, subchronic) and young adult (70-day-old, acute only) male and female rats. THC and its first-pass metabolites-11-hydroxy-Δ9-THC (11-OH-THC) and 11-nor-9-carboxy-Δ9-THC (11-COOH-THC)-were quantified in plasma and brain tissue using a selective isotope-dilution liquid chromatography/tandem mass spectrometry assay. Changes in body temperature were measured using abdominally implanted microchips. Biotransformation of THC to its metabolites using freshly prepared liver microsomes was assessed. Results: At the acute 5 mg/kg dose, maximal plasma concentrations of THC were twice as high in adult than in adolescent rats. Conversely, in adults, brain concentrations and brain-to-plasma ratios for THC were substantially lower (25-50%) than those measured in adolescents. Similarly, plasma and brain concentrations of THC metabolites were higher in adolescent male rats compared with adult males. Interestingly, plasma and brain concentrations of the psychoactive THC metabolite 11-OH-THC were twofold to sevenfold higher in female animals of both ages compared with males. Moreover, liver microsomes from adolescent males and adolescent and adult females converted THC to 11-OH-THC twice as fast as adult male microsomes. A dose-dependent hypothermic response to THC was observed in females with 0.5 and 5 mg/kg THC, whereas only the highest dose elicited a response in males. Finally, subchronic administration of THC during adolescence did not significantly affect the drug's PK profile. Conclusions: The results reveal the existence of multiple age and sex differences in the distribution and metabolism of THC in rats, which might influence the pharmacological response to the drug.

Understanding nanomedicine treatment in an aggressive spontaneous brain cancer model at the stage of early blood brain barrier disruption.

Personalised nanomedicine is an advancing field which has developed significant improvements for targeting therapeutics to aggressive cancer and with fewer side effects. The treatment of gliomas such as glioblastoma (or other brain tumours), with nanomedicine is complicated by a commonly poor accumulation of drugs in tumour tissue owing to the partially intact blood-brain barrier (BBB). Nonetheless, the BBB becomes compromised following surgical intervention, and gradually with disease progression. Increased vasculature permeability generated by a tumour, combined with decreased BBB integrity, offers a mechanism to enhance therapeutic outcomes. We monitored a spontaneous glioma tumour model in immunocompetent mice with ongoing T2-weighted and contrast-enhanced T1-weighted magnetic resonance imaging gradient echo and spin echo sequences to predict an optimal "leakiness" stage for nanomedicine injections. To ascertain the effectiveness of targeted nanomedicines in treating brain tumours, subsequent systemic administration of targeted hyperbranched polymers was then utislised, to deliver the therapeutic payload when both the tumour and brain vascularity had become sufficiently susceptible to allow drug accumulation. Treatment with either doxorubicin-loaded hyperbranched polymer, or the same nanomedicine targeted to an ephrin receptor (EphA2) using a bispecific antibody, resulted in uptake of chemotherapeutic doxorubicin in the tumour and in reduced tumour growth. Compared to vehicle and doxorubicin only, nanoparticle delivered doxorubicin resulted in increased tumour apoptosis, while averting cardiotoxicity. This suggests that polyethylene based (PEGylated)-nanoparticle delivered doxorubicin could provide a more efficient treatment in tumours with a disrupted BBB, and that treatment should commence immediately following detection of gadolinium permeability, with early detection and ongoing 'leakiness' monitoring in susceptible patients being a key factor.

Developmental Trajectories of Anhedonia in Preclinical Models.

This chapter discusses how the complex concept of anhedonia can be operationalized and studied in preclinical models. It provides information about the development of anhedonia in the context of early-life adversity, and the power of preclinical models to tease out the diverse molecular, epigenetic, and network mechanisms that are responsible for anhedonia-like behaviors.Specifically, we first discuss the term anhedonia, reviewing the conceptual components underlying reward-related behaviors and distinguish anhedonia pertaining to deficits in motivational versus consummatory behaviors. We then describe the repertoire of experimental approaches employed to study anhedonia-like behaviors in preclinical models, and the progressive refinement over the past decade of both experimental instruments (e.g., chemogenetics, optogenetics) and conceptual constructs (salience, valence, conflict). We follow with an overview of the state of current knowledge of brain circuits, nodes, and projections that execute distinct aspects of hedonic-like behaviors, as well as neurotransmitters, modulators, and receptors involved in the generation of anhedonia-like behaviors. Finally, we discuss the special case of anhedonia that arises following early-life adversity as an eloquent example enabling the study of causality, mechanisms, and sex dependence of anhedonia.Together, this chapter highlights the power, potential, and limitations of using preclinical models to advance our understanding of the origin and mechanisms of anhedonia and to discover potential targets for its prevention and mitigation.

Modeling apoptosis resistance in CHO cells with CRISPR-mediated knockouts of Bak1, Bax, and Bok.

Chinese hamster ovary (CHO) cells are the primary platform for the production of biopharmaceuticals. To increase yields, many CHO cell lines have been genetically engineered to resist cell death. However, the kinetics that governs cell fate in bioreactors are confounded by many variables associated with batch processes. Here, we used CRISPR-Cas9 to create combinatorial knockouts of the three known BCL-2 family effector proteins: Bak1, Bax, and Bok. To assess the response to apoptotic stimuli, cell lines were cultured in the presence of four cytotoxic compounds with different mechanisms of action. A population-based model was developed to describe the behavior of the resulting viable cell dynamics as a function of genotype and treatment. Our results validated the synergistic antiapoptotic nature of Bak1 and Bax, while the deletion of Bok had no significant impact. Importantly, the uniform application of apoptotic stresses permitted direct observation and quantification of a delay in the onset of cell death through Bayesian inference of meaningful model parameters. In addition to the classical death rate, a delay function was found to be essential in the accurate modeling of the cell death response. These findings represent an important bridge between cell line engineering strategies and biological modeling in a bioprocess context.