Dynamic insulin-stimulated mTOR/GSK3 signaling in peripheral immune cells: Preliminary evidence for an association with lithium response in bipolar disorder.

INTRODUCTION: A key mechanism of lithium is the inhibition of glycogen synthase kinase-3ß (GSK3ß) and activation of mammalian target of rapamycin (mTOR), two contributors to insulin signaling. We explored the relationship between these markers and clinical response to lithium in bipolar disorder (BD). METHODS: Thirty-four subjects with BD who had been taking lithium for ≥2 years and had a maintenance lithium Alda score defined as either high (≥7; n = 20) or low (≤2; n = 14) were included in the study. Baseline protein expression of GSK3ß and mTOR (total and phosphorylated (p)) was obtained from a buffy coat. Peripheral blood mononuclear cells (PBMCs) from a subset of each group (n = 11) were stimulated with insulin (10 µg) and change in protein expression was determined using Western blot. RESULTS: In buffy coat samples, significantly higher levels of pmTOR were present in subjects with an Alda score ≤2 (lithium non-responsive), relative to those with scores ≥7 (lithium-responsive). No differences were observed for pGSK3ß. In contrast, functional PBMC responses to 5 min of insulin stimulation demonstrated robust increases in pGSK3ß (87.05 ± 43.41%) and pmTOR (105.7 ± 66.48%) in the lithium responsive group only. This contrasted observed decreases in pGSK3ß (34.08 ± 16.12%) and pmTOR (37.84 ± 14.39%) 5 mins post-insulin in non-responders. CONCLUSIONS: Dynamic increases in pmTOR and pGSK3ß post-insulin stimulation may reflect an immunometabolic state that facilitates lithium response. Further prospective analyses are needed to replicate and extend these preliminary findings and further investigate the role of insulin signaling in lithium response in BD.

Lithium augmentation of ketamine increases insulin signaling and antidepressant-like active stress coping in a rodent model of treatment-resistant depression.

Lithium, a mood stabilizer and common adjunctive treatment for refractory depression, shares overlapping mechanisms of action with ketamine and enhances the duration of ketamine's antidepressant actions in rodent models at sub-therapeutic doses. Yet, in a recent clinical trial, lithium co-treatment with ketamine failed to improve antidepressant outcomes in subjects previously shown to respond to ketamine alone. The potential for lithium augmentation to improve antidepressant outcomes in ketamine nonresponders, however, has not been explored. The current study examined the behavioral, molecular and metabolic actions of lithium and ketamine co-treatment in a rodent model of antidepressant resistance. Male Wistar rats were administered adrenocorticotropic hormone (ACTH; 100 µg/day, i.p. over 14 days) and subsequently treated with ketamine (10 mg/kg; 2 days; n = 12), lithium (37 mg/kg; 2 days; n = 12), ketamine + lithium (10 mg/kg + 37 mg/kg; 2 days; n = 12), or vehicle saline (0.9%; n = 12). Rats were subjected to open field (6 min) and forced swim tests (6 min). Peripheral blood and brain prefrontal cortical (PFC) tissue was collected one hour following stress exposure. Western blotting was used to determine the effects of treatment on extracellular signal-regulated kinase (ERK); mammalian target of rapamycin (mTOR), phospho kinase B (Akt), and glycogen synthase kinase-3ß (GSK3ß) protein levels in the infralimbic (IL) and prelimbic (PL) subregions of the PFC. Prefrontal oxygen consumption rate (OCR) and extracellular acidification rates (ECAR) were also determined in anterior PFC tissue at rest and following stimulation with brain-derived neurotrophic factor (BDNF) and tumor necrosis factor α (TNFα). Blood plasma levels of mTOR and insulin were determined using enzyme-linked immunosorbent assays (ELISAs). Overall, rats receiving ketamine+lithium displayed a robust antidepressant response to the combined treatment as demonstrated through significant reductions in immobility time (p < 0.05) and latency to immobility (p < 0.01). These animals also had higher expression of plasma mTOR (p < 0.01) and insulin (p < 0.001). Tissue bioenergetics analyses revealed that combined ketamine+lithium treatment did not significantly alter the respiratory response to BDNF or TNFα. Animals receiving both ketamine and lithium had significantly higher phosphorylation (p)-to-total expression ratios of mTOR (p < 0.001) and Akt (p < 0.01), and lower ERK in the IL compared to control animals. In contrast, pmTOR/mTOR levels were reduced in the PL of ketamine+lithium treated animals, while pERK/ERK expression levels were elevated. Taken together, these data demonstrate that lithium augmentation of ketamine in antidepressant nonresponsive animals improves antidepressant-like behavioral responses under stress, together with peripheral insulin efflux and region-specific PFC insulin signaling.

Mood Regulatory Actions of Active and Sham Nucleus Accumbens Deep Brain Stimulation in Antidepressant Resistant Rats.

The antidepressant actions of deep brain stimulation (DBS) are associated with progressive neuroadaptations within the mood network, modulated in part, by neurotrophic mechanisms. We investigated the antidepressant-like effects of chronic nucleus accumbens (NAc) DBS and its association with change in glycogen synthase kinase 3 (GSK3) and mammalian target of rapamycin (mTOR) expression in the infralimbic cortex (IL), and the dorsal (dHIP) and ventral (vHIP) subregions of the hippocampus of antidepressant resistant rats. Antidepressant resistance was induced via daily injection of adrenocorticotropic hormone (ACTH; 100 µg/day; 15 days) and confirmed by non-response to tricyclic antidepressant treatment (imipramine, 10 mg/kg). Portable microdevices provided continuous bilateral NAc DBS (130 Hz, 200 µA, 90 µs) for 7 days. A control sham electrode group was included, together with ACTH- and saline-treated control groups. Home cage monitoring, open field, sucrose preference, and, forced swim behavioral tests were performed. Post-mortem levels of GSK3 and mTOR, total and phosphorylated, were determined with Western blot. As previously reported, ACTH treatment blocked the immobility-reducing effects of imipramine in the forced swim test. In contrast, treatment with either active DBS or sham electrode placement in the NAc significantly reduced forced swim immobility time in ACTH-treated animals. This was associated with increased homecage activity in the DBS and sham groups relative to ACTH and saline groups, however, no differences in locomotor activity were observed in the open field test, nor were any group differences seen for sucrose consumption across groups. The antidepressant-like actions of NAc DBS and sham electrode placements were associated with an increase in levels of IL and vHIP phospho-GSK3ß and phospho-mTOR, however, no differences in these protein levels were observed in the dHIP region. These data suggest that early response to electrode placement in the NAc, irrespective of whether active DBS or sham, has antidepressant-like effects in the ACTH-model of antidepressant resistance associated with distal upregulation of phospho-GSK3ß and phospho-mTOR in the IL and vHIP regions of the mood network.

Automatic and Reliable Quantification of Tonic Dopamine Concentrations In Vivo Using a Novel Probabilistic Inference Method.

Dysregulation of the neurotransmitter dopamine (DA) is implicated in several neuropsychiatric conditions. Multiple-cyclic square-wave voltammetry (MCSWV) is a state-of-the-art technique for measuring tonic DA levels with high sensitivity (<5 nM), selectivity, and spatiotemporal resolution. Currently, however, analysis of MCSWV data requires manual, qualitative adjustments of analysis parameters, which can inadvertently introduce bias. Here, we demonstrate the development of a computational technique using a statistical model for standardized, unbiased analysis of experimental MCSWV data for unbiased quantification of tonic DA. The oxidation current in the MCSWV signal was predicted to follow a lognormal distribution. The DA-related oxidation signal was inferred to be present in the top 5% of this analytical distribution and was used to predict a tonic DA level. The performance of this technique was compared against the previously used peak-based method on paired in vivo and post-calibration in vitro datasets. Analytical inference of DA signals derived from the predicted statistical model enabled high-fidelity conversion of the in vivo current signal to a concentration value via in vitro post-calibration. As a result, this technique demonstrated reliable and improved estimation of tonic DA levels in vivo compared to the conventional manual post-processing technique using the peak current signals. These results show that probabilistic inference-based voltammetry signal processing techniques can standardize the determination of tonic DA concentrations, enabling progress toward the development of MCSWV as a robust research and clinical tool.

Hypoxia-Associated Changes in Striatal Tonic Dopamine Release: Real-Time in vivo Measurements With a Novel Voltammetry Technique.

INTRODUCTION: Striatal tonic dopamine increases rapidly during global cerebral hypoxia. This phenomenon has previously been studied using microdialysis techniques which have relatively poor spatio-temporal resolution. In this study, we measured changes in tonic dopamine during hypoxia (death) in real time with high spatio-temporal resolution using novel multiple cyclic square wave voltammetry (MCSWV) and conventional fast scan cyclic voltammetry (FSCV) techniques. METHODS: MCSWV and FSCV were used to measure dopamine release at baseline and during hypoxia induced by euthanasia, with and without prior alpha-methyl-p-tyrosine (AMPT) treatment, in urethane anesthetized male Sprague-Dawley rats. RESULTS: Baseline tonic dopamine levels were found to be 274.1 ± 49.4 nM (n = 5; mean ± SEM). Following intracardiac urethane injection, the tonic levels increased to a peak concentration of 1753.8 ± 95.7 nM within 3.6 ± 0.6 min (n = 5), followed by a decline to 50.7 ± 21.5 nM (n = 4) at 20 min. AMPT pre-treatment significantly reduced this dopamine peak to 677.9 ± 185.7 nM (n = 3). FSCV showed a significantly higher (p = 0.0079) peak dopamine release of 6430.4 ± 1805.7 nM (n = 5) during euthanasia-induced cerebral hypoxia. CONCLUSION: MCSWV is a novel tool to study rapid changes in tonic dopamine release in vivo during hypoxia. We found a 6-fold increase in peak dopamine levels during hypoxia which was attenuated with AMPT pre-treatment. These changes are much lower compared to those found with microdialysis. This could be due to improved estimation of baseline tonic dopamine with MCSWV. Higher dopamine response measured with FSCV could be due to an increased oxidation current from electroactive interferents.

Clinical applications of neurochemical and electrophysiological measurements for closed-loop neurostimulation.

The development of closed-loop deep brain stimulation (DBS) systems represents a significant opportunity for innovation in the clinical application of neurostimulation therapies. Despite the highly dynamic nature of neurological diseases, open-loop DBS applications are incapable of modifying parameters in real time to react to fluctuations in disease states. Thus, current practice for the designation of stimulation parameters, such as duration, amplitude, and pulse frequency, is an algorithmic process. Ideal stimulation parameters are highly individualized and must reflect both the specific disease presentation and the unique pathophysiology presented by the individual. Stimulation parameters currently require a lengthy trial-and-error process to achieve the maximal therapeutic effect and can only be modified during clinical visits. The major impediment to the development of automated, adaptive closed-loop systems involves the selection of highly specific disease-related biomarkers to provide feedback for the stimulation platform. This review explores the disease relevance of neurochemical and electrophysiological biomarkers for the development of closed-loop neurostimulation technologies. Electrophysiological biomarkers, such as local field potentials, have been used to monitor disease states. Real-time measurement of neurochemical substances may be similarly useful for disease characterization. Thus, the introduction of measurable neurochemical analytes has significantly expanded biomarker options for feedback-sensitive neuromodulation systems. The potential use of biomarker monitoring to advance neurostimulation approaches for treatment of Parkinson's disease, essential tremor, epilepsy, Tourette syndrome, obsessive-compulsive disorder, chronic pain, and depression is examined. Further, challenges and advances in the development of closed-loop neurostimulation technology are reviewed, as well as opportunities for next-generation closed-loop platforms.

Advances in neurochemical measurements: A review of biomarkers and devices for the development of closed-loop deep brain stimulation systems.

Neurochemical recording techniques have expanded our understanding of the pathophysiology of neurological disorders, as well as the mechanisms of action of treatment modalities like deep brain stimulation (DBS). DBS is used to treat diseases such as Parkinson's disease, Tourette syndrome, and obsessive-compulsive disorder, among others. Although DBS is effective at alleviating symptoms related to these diseases and improving the quality of life of these patients, the mechanism of action of DBS is currently not fully understood. A leading hypothesis is that DBS modulates the electrical field potential by modifying neuronal firing frequencies to non-pathological rates thus providing therapeutic relief. To address this gap in knowledge, recent advances in electrochemical sensing techniques have given insight into the importance of neurotransmitters, such as dopamine, serotonin, glutamate, and adenosine, in disease pathophysiology. These studies have also highlighted their potential use in tandem with electrophysiology to serve as biomarkers in disease diagnosis and progression monitoring, as well as characterize response to treatment. Here, we provide an overview of disease-relevant neurotransmitters and their roles and implications as biomarkers, as well as innovations to the biosensors used to record these biomarkers. Furthermore, we discuss currently available neurochemical and electrophysiological recording devices, and discuss their viability to be implemented into the development of a closed-loop DBS system.

Insulin-stimulated mTOR activation in peripheral blood mononuclear cells associated with early treatment response to lithium augmentation in rodent model of antidepressant-resistance.

Lithium has been shown to have some therapeutic efficacy as an adjunctive treatment for intractable forms of major depression. Activation of mammalian target of rapamycin (mTOR) and inhibition of glycogen synthase kinase-3ß (GSK3ß) have been implicated in its putative mechanisms of action. These proteins are integral components of the insulin signaling pathway, which may serve as a critical co-regulator of drug action. Utilizing an animal model of tricyclic antidepressant resistance, we investigated the relationship between insulin signaling and antidepressant response to lithium augmentation. Pre-treatment with adrenocorticotropic hormone (ACTH 100 µg/day i.p.) for 14 days effectively blocked the immobility-reducing effects of an acute dose of imipramine (10 mg/kg i.p.) in the forced swim test (FST). Lithium augmentation (100 mg/kg i.p.) rescued the antidepressant-like effects of imipramine in this model. Total and phosphorylated (p) levels of protein kinase B (Akt), mTOR, and GSK3ß protein were quantified in the infralimbic cortex (ILPFC) following FST stress via Western blot. Levels of mTOR and pmTOR were further quantified in isolated peripheral blood mononuclear cells (PBMCs) following insulin stimulation (10 mg/mL for 5 min) via ELISA. Elevated levels of phosphorylated insulin signaling proteins were present in the ILPFC of ACTH-pretreated animals that received both imipramine and lithium, together with a concurrent increase in mTOR activation in PBMCs. Large correlations were observed between immobility time and insulin-stimulated mTOR levels in PBMCs. We propose that PBMC insulin challenge may be a useful probe for predicting antidepressant response to lithium administration, and potentially other therapies acting via similar mechanisms of action.

Bioenergetics and synaptic plasticity as potential targets for individualizing treatment for depression.

Disruptions of bioenergetic signaling and neurogenesis are hallmarks of depression physiology and are often the product of dysregulation of the inflammatory, stress-response, and metabolic systems. These systems are extensively interrelated at the physiological level, yet the bulk of the literature to date addresses pathophysiological mechanisms in isolation. A more integrated understanding of the etiology, progression, and treatment response profiles of depression is possible through wider consideration of relevant preclinical and clinical studies that examine the result of disruptions in these systems. Here, we review recent data demonstrating the critical effects of bioenergetic disruption on neuroplasticity and the development and progression of depressive illness. We further highlight the interactive and dynamic nature of the inflammatory and stress response systems and how disruption of these systems influences bioenergetic signaling pathways critical to treatment outcomes. In so doing, we underscore the pressing need to reconsider the implications of treatment resistance and present a framework for developing novel, personalized treatment approaches for depression.

Stress, Inflammation, and Cellular Vulnerability during Early Stages of Affective Disorders: Biomarker Strategies and Opportunities for Prevention and Intervention.

The mood disorder prodrome is conceptualized as a symptomatic, but not yet clinically diagnosable stage of an affective disorder. Although a growing area, more focused research is needed in the pediatric population to better characterize psychopathological symptoms and biological markers that can reliably identify this very early stage in the evolution of mood disorder pathology. Such information will facilitate early prevention and intervention, which has the potential to affect a person's disease course. This review focuses on the prodromal characteristics, risk factors, and neurobiological mechanisms of mood disorders. In particular, we consider the influence of early-life stress, inflammation, and allostatic load in mediating neural mechanisms of neuroprogression. These inherently modifiable factors have known neuroadaptive and neurodegenerative implications, and consequently may provide useful biomarker targets. Identification of these factors early in the course of the disease will accordingly allow for the introduction of early interventions which augment an individual's capacity for psychological resilience through maintenance of synaptic integrity and cellular resilience. A targeted and complementary approach to boosting both psychological and physiological resilience simultaneously during the prodromal stage of mood disorder pathology has the greatest promise for optimizing the neurodevelopmental potential of those individuals at risk of disabling mood disorders.