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Adrenergic signaling is known to play a critical role in regulating female reproductive processes in both mammals and insects. InDrosophila, the ortholog of noradrenaline, octopamine (Oa), is required for ovulation as well as several other female reproductive processes. Loss of function studies using mutant alleles of receptors, transporters, and biosynthetic enzymes for Oa have led to a model in which disruption of octopaminergic pathways reduces egg laying. However, neither the complete expression pattern in the reproductive tract nor the role of most octopamine receptors in oviposition is known. We show that all six known Oa receptors are expressed in peripheral neurons at multiple sites within in the female fly reproductive tract as well as in non-neuronal cells within the sperm storage organs. The complex pattern of Oa receptor expression in the reproductive tract suggests the potential for influencing multiple regulatory pathways, including those known to inhibit egg-laying in unmated flies. Indeed, activation of some neurons that express Oa receptors inhibits oviposition, and neurons that express different subtypes of Oa receptor can affect different stages of egg laying. Stimulation of some Oa receptor expressing neurons (OaRNs) also induces contractions in lateral oviduct muscle and activation of non-neuronal cells in the sperm storage organs by Oa generates OAMB-dependent intracellular calcium release. Our results are consistent with a model in which adrenergic pathways play a variety of complex roles in the fly reproductive tract that includes both the stimulation and inhibition of oviposition.

Background Intermittent theta-burst stimulation priming (iTBS-P) can improve clinical outcome of patients with Major Depressive Disorder (MDD) who do not show early benefit from 10 Hz stimulation of left dorsolateral prefrontal cortex (DLPFC), also known as high-frequency left-sided (HFL) stimulation. The intensity and pulse number for iTBS-P needed to induce clinical benefit have not been systematically examined. Objective To study the effect of intensity and pulse number on the clinical efficacy of iTBS-P. Methods We conducted a retrospective review of 71 participants who received at least five sessions of HFL with limited clinical benefit and received iTBS-P augmentation for between 5 and 25 sessions. Intensity of iTBS-P priming stimuli ranged from 75 to 120% of motor threshold (MT) and pulse number ranged from 600 to 1800. Associations among intensity, pulse number, and clinical outcome were analyzed using a mixed methods linear model with change in IDS-SR as the primary outcome variable, priming stimulation intensity (subthreshold or suprathreshold), pulse number ( 1200 pulses), and gender as fixed factors, and number of iTBS-P treatments and age as continuous covariates. Results Subjects who received subthreshold intensity iTBS-P experienced greater reduction in depressive symptoms than those who received suprathreshold iTBS-P (p = 0.011) with no effect of pulse number after controlling for stimulus intensity. Conclusions Subthreshold intensity iTBS-P was associated with greater clinical improvement than suprathreshold stimulation. This finding is consistent with iTBS-P acting through homeostatic plasticity mechanisms.

The Serotonin Transporter (SERT) regulates extracellular serotonin levels and is the target of most current drugs used to treat depression. The mechanisms by which inhibition of SERT activity influences behavior are poorly understood. To address this question in the model organismDrosophila melanogaster, we developed new loss of function mutations inDrosophila SERT(dSERT).Previous studies in both flies and mammals have implicated serotonin as an important neuromodulator of sleep, and our newly generateddSERTmutants show an increase in total sleep and altered sleep architecture. Differences in daytime vs. nighttime sleep architecture as well as genetic rescue experiments unexpectedly suggest that distinct serotonergic circuits may modulate daytime versus nighttime sleep.dSERTmutants also show defects in copulation and food intake, akin to the clinical side effects of SSRIs. Starvation did not overcome the sleep drive in the mutants. Additionally in males, but not femaledSERTmutants, the drive to mate also failed to overcome sleep drive.dSERTmay be used to further explore the mechanisms by which serotonin regulates sleep and its interplay with other complex behaviors.Author SummaryMany medications used to treat depression and anxiety act by changing serotonin levels in the brain. Fruit flies also use serotonin and can be used as a model to study the brain. We have made a fly mutant for the serotonin transporter (SERT), which is the target of antidepressants in humans. The mutants sleep more, eat less, and have a decreased sex drive. These flies can be used to study the neuronal pathways by which serotonin regulates sleep, eating and sexual behaviors and may help us to understand the behavioral effects of antidepressants.

The Serotonin Transporter (SERT) regulates extracellular serotonin levels and is the target of most current drugs used to treat depression. The mechanisms by which inhibition of SERT activity influences behavior are poorly understood. To address this question in the model organism Drosophila melanogaster, we developed new loss of function mutations in Drosophila SERT (dSERT). Previous studies in both flies and mammals have implicated serotonin as an important neuromodulator of sleep, and our newly generated dSERT mutants show an increase in total sleep and altered sleep architecture that is mimicked by feeding the SSRI citalopram. Differences in daytime versus nighttime sleep architecture as well as genetic rescue experiments unexpectedly suggest that distinct serotonergic circuits may modulate daytime versus nighttime sleep. dSERT mutants also show defects in copulation and food intake, akin to the clinical side effects of SSRIs and consistent with the pleomorphic influence of serotonin on the behavior of D. melanogaster. Starvation did not overcome the sleep drive in the mutants and in male dSERT mutants, the drive to mate also failed to overcome sleep drive. dSERT may be used to further explore the mechanisms by which serotonin regulates sleep and its interplay with other complex behaviors.

Repetitive Transcranial Magnetic Stimulation (rTMS) is an effective intervention for treatment-resistant Major Depressive Disorder (MDD). Early improvement during high-frequency left-sided (HFL) stimulation of the dorsolateral prefrontal cortex (DLPFC) is an important predictor of longer-term outcome, but most patients benefit later in their treatment course. We examined patients without early improvement with HFL to determine whether augmentation with additional stimulation approaches improved treatment outcome.139 participants received HFL in a measurement-based care paradigm. Participants who achieved20% improvement by treatment 10 could continue with HFL (N = 17) or receive one of two augmentation strategies: bilateral stimulation (BL; HFL followed by low-frequency stimulation of right DLPFC) (N = 69) or intermittent theta-burst priming of left DLPFC (iTBS-P) (N = 17) for their remaining treatment sessions. The primary outcome was the percent reduction in depressive symptoms at treatment 30.Participants who achieved20% improvement by treatment 10 and continued with HFL showed limited benefit. iTBS-P participants had significantly greater improvement, while those receiving BL trended toward improved outcomes. Ten sessions of either augmentation strategy appeared necessary to determine the likelihood of benefit.Augmentation of early non-response to HFL appears to improve rTMS outcomes, with a novel iTBS-P strategy surpassing both continued HFL or BL treatment in participants with20% improvement after 10 treatments. These findings suggest that measurement-based care with addition of augmented stimulation for those not showing early improvement may yield superior rTMS treatment outcomes.

Psychedelics (i.e., ketamine, methylenedioxymethamphetamine, psilocybin) have been effectively used globally for centuries to treat mental health and addiction issues. However, in the 1950s–1970s, a number of factors, including misuse, abuse, and poorly conceptualized and conducted clinical trials, caused the Federal Drug Administration to classify most of the psychedelic substances as having no medical value. Now, however, recent research is indicating that psychedelic-assisted therapy can significantly reduce depression and suicidal ideation in treatment-resistant clients, and it may be efficacious in treating other mental health and addiction issues as well. Researchers have also identified the critical therapeutic components that ensure effective psychedelic-assisted therapy, not least the need for mental health counseling before, during, and after treatment. The purpose of this manuscript is to share the latest psychedelic therapy research and to discuss how mental health counselors can contribute to this reemerging therapeutic trend.

BackgroundTinnitus distress is related to both the loudness and intrusiveness of the tinnitus percept. Treatment approaches targeting both attentional/limbic and auditory systems may better alleviate tinnitus distress than approaches targeting the auditory system alone.Materials and MethodsTen subjects with chronic tinnitus received sequential rTMS treatment involving: 1) excitatory stimulation administered to the left dorsolateral prefrontal cortex (DLPFC) or inhibitory stimulation administered to the right DLPFC, followed by 2) inhibitory stimulation administered to primary auditory cortex (Heschel's gyrus or HG). A systematic literature review was performed to evaluate the existing literature on sequential repetitive Transcranial Magnetic Stimulation (rTMS) treatment approaches for tinnitus. Results of the case series are interpreted in the context of tinnitus neurobiology and the extant literature.ResultsSubjects experienced a significant decrease (average 21.7%) in symptoms on the Tinnitus Functional Index (TFI). Those with tinnitus alone experienced a greater mean symptom reduction than those with comorbid MDD (27.7 vs. 17.0%, respectively). Adverse effects were transient and minor. Literature review confirmed that sequential approaches had some advantages compared to single site rTMS; in general, the addition of 1 Hz treatment at DLPFC was superior to single site rTMS in the short term (1–12 weeks), while the addition of 20 Hz treatment at DLPFC appeared superior in the long term (90–180 days).ConclusionsSequential rTMS approaches for the treatment of tinnitus—particularly those administering low-frequency treatment at left DLPFC—merit further investigation.

BackgroundSpecific phobias represent the largest category of anxiety disorders. Previous work demonstrated that stimulating the ventromedial prefrontal cortex (vmPFC) with repetitive Transcranial Magnetic Stimulation (rTMS) may improve response to exposure therapy for acrophobia.ObjectiveTo examine feasibility of accelerating extinction learning in subjects with spider phobia using intermittent Theta Burst Stimulation (iTBS) rTMS of vmPFC.MethodsIn total, 17 subjects with spider phobia determined by spider phobia questionnaires [Spider Phobia Questionnaire (SPQ) and Fear of Spiders questionnaire (FSQ)] underwent ratings of fear of spiders as well as behavioral and skin conductance data during a behavioral avoidance test (BAT). Subjects then received a sequential protocol of in vivo spider exposure followed by iTBS for three sessions administered to either active or control treatment sites (vmPFC [n = 8] or vertex [n = 9], respectively), followed 1 week later by repetition of questionnaires and BAT.ResultsAll subjects improved significantly regardless of group across both questionnaires (FSQ η2 = 0.43, p = 0.004; SPQ η2 = 0.39, p = 0.008) and skin conductance levels during BAT (Wald χ2 = 30.9, p < 0.001). Subjects in the vmPFC group tolerated lower treatment intensity than in the control group, and there was a significant correlation between treatment intensity, BAT subjective distress improvement, and physiologic measures (all ρ > 0.5).ConclusionThis proof-of-concept study provides preliminary evidence that a sequential exposure and iTBS over vmPFC is feasible and may have rTMS intensity-dependent effects on treatment outcomes, providing evidence for future areas of study in the use of rTMS for phobias.

Symptoms of major depressive disorder (MDD) are reported to change early in treatment with repetitive transcranial magnetic stimulation (rTMS). We evaluated early changes in sleep, anxiety, and mood as predictors of nonresponse to rTMS treatment.Three hundred twenty-nine subjects with nonpsychotic MDD completed a 6-week course of rTMS treatment. Subjects were stratified by the severity of their baseline depression, and had their overall depressive symptoms recorded every week of treatment. We evaluated lack of improvement in sleep, anxiety, and mood symptoms after 1 and 2 weeks as potential predictors of eventual nonresponse, defined as50% improvement in compositive depressive symptoms after 6 weeks. This was measured as negative predictive value (NPV; the likelihood that lack of early symptom improvement accurately predicted eventual treatment nonresponse).Subjects with severe or very severe baseline depression achieving20% improvement in mood at 1 week were correctly predicted as nonresponders with NPVs largely90%. At 2 weeks, subjects with very severe baseline depression who failed to demonstrate any improvement in mood were all nonresponders. Lack of improvement in sleep at 2 weeks was also a significant predictor.Identifying a lack of early mood improvement is a practical and robust method to predict rTMS nonresponse. This suggests a treatment protocol change may be indicated in patients with more severe baseline depression showing minimal early mood improvement.

Obsessive-compulsive disorder (OCD) and major depressive disorder (MDD) are highly comorbid [1], with depressive symptoms amplifying the chronicity and severity of OCD symptoms. Comorbid illness decreases quality of life and daily functioning [2] and is associated with greater suicidality and more frequent inpatient hospitalizations [3]. Furthermore, comorbid OCD/depression is associated with poorer response to OCD-focused psychological and pharmacological treatments [4]. Epidemiologic studies have shown that OCD symptoms generally precedes the occurrence of depression, suggesting a causal interacting model in which OCD predisposes to development of depressive symptoms [5]. In line with that causal model, Tadayonnejad et al. showed aberrant effective (directional) connectivity between OCD and MDD circuits may be a potential network mechanism of depressive symptom genesis or worsening in OCD-MDD [6]. The challenging nature of this comorbidity necessitates the development of novel, more effective treatments.

Author(s): Levitt, Jennifer G; Kalender, Guldamla; O’Neill, Joseph; Diaz, Joel P; Cook, Ian A; Ginder, Nathaniel; Krantz, David; Minzenberg, Michael J; Vince-Cruz, Nikita; Nguyen, Lydia D; Alger, Jeffry R; Leuchter, Andrew F | Abstract: BackgroundThe therapeutic mechanism of repetitive transcranial magnetic stimulation (rTMS) for treatment-resistant depression (TRD) may involve modulation of γ-aminobutyric acid (GABA) levels. We used proton magnetic resonance spectroscopy (MRS) to assess changes in GABA levels at the site of rTMS in the left dorsolateral prefrontal cortex (DLPFC).MethodsIn 26 adults with TRD, we used Mescher–Garwood point-resolved spectroscopy (MEGA-PRESS) spectral-editing MRS to measure GABA in the left DLPFC before and after standard clinical treatment with rTMS. All participants but 1 were medicated, including 12 patients on GABA agonist agents.ResultsMean GABA in the DLPFC increased 10.0% (p = 0.017) post-rTMS in the overall sample. As well, GABA increased significantly in rTMS responders (n = 12; 23.6%, p = 0.015) but not in nonresponders (n = 14; 4.1%, p = not significant). Changes in GABA were not significantly affected by GABAergic agonists, but clinical response was less frequent (p = 0.005) and weaker (p = 0.035) in the 12 participants who were receiving GABA agonists concomitant with rTMS treatment.LimitationsThis study had an open-label design in a population receiving naturalistic treatment.ConclusionTreatment using rTMS was associated with increases in GABA levels at the stimulation site in the left DLPFC, and the degree of GABA change was related to clinical improvement. Participants receiving concomitant treatment with a GABA agonist were less likely to respond to rTMS. These findings were consistent with earlier studies showing the effects of rTMS on GABA levels and support a GABAergic model of depression.

Repetitive transcranial magnetic stimulation (rTMS) treatment of major depressive disorder (MDD) is associated with changes in brain functional connectivity (FC). These changes may be related to the mechanism of action of rTMS and explain the variability in clinical outcome. We examined changes in electroencephalographic FC during the first rTMS treatment in 109 subjects treated with 10 Hz stimulation to left dorsolateral prefrontal cortex. All subjects subsequently received 30 treatments and clinical response was defined as ≥40% improvement in the inventory of depressive symptomatology-30 SR score at treatment 30. Connectivity change was assessed with coherence, envelope correlation, and a novel measure, alpha spectral correlation (αSC). Machine learning was used to develop predictive models of outcome for each connectivity measure, which were compared with prediction based upon early clinical improvement. Significant connectivity changes were associated with clinical outcome (P < 0.001). Machine learning models based on αSC yielded the most accurate prediction (area under the curve, AUC = 0.83), and performance improved when combined with early clinical improvement measures (AUC = 0.91). The initial rTMS treatment session produced robust changes in FC, which were significant predictors of clinical outcome of a full course of treatment for MDD.

BackgroundMajor depressive disorder (MDD) and chronic pain are highly comorbid, and pain symptoms are associated with a poorer response to antidepressant medication treatment. It is unclear whether comorbid pain also is associated with a poorer response to treatment with repetitive transcranial magnetic stimulation (rTMS).Methods162 MDD subjects received 30 sessions of 10 Hz rTMS treatment administered to the left dorsolateral prefrontal cortex (DLPFC) with depression and pain symptoms measured before and after treatment. For a subset of 96 patients, a resting-state electroencephalogram (EEG) was recorded at baseline. Clinical outcome was compared between subjects with and without comorbid pain, and the relationships among outcome, pain severity, individual peak alpha frequency (PAF), and PAF phase-coherence in the EEG were examined.Results64.8% of all subjects reported pain, and both depressive and pain symptoms were significantly reduced after rTMS treatment, irrespective of age or gender. Patients with severe pain were 27% less likely to respond to MDD treatment than pain-free individuals. PAF was positively associated with pain severity. PAF phase-coherence in the somatosensory and default mode networks was significantly lower for MDD subjects with pain who failed to respond to MDD treatment.ConclusionsPain symptoms improved after rTMS to left DLPFC in MDD irrespective of age or gender, although the presence of chronic pain symptoms reduced the likelihood of treatment response. Individual PAF and baseline phase-coherence in the sensorimotor and midline regions may represent predictors of rTMS treatment outcome in comorbid pain and MDD.

Octopamine is essential for egg-laying in

Mammalian visceral muscles are indirectly regulated by receptors expressed in peripheral neurons and other non-contractile cells. The complex interactions between these pathways remain poorly understood. It also remains unknown whether invertebrates use pathways similar to mammals to indirectly regulate visceral contractility. To elucidate the principles of organization for the signaling pathways responsible for visceral motility and the more general logic of aminergic neuromodulation, we are using the Drosophila oviposition circuit. We find that the receptors for the adrenergic neurotransmitter octopamine as well as the metabotropic glutamate receptor are not expressed in muscle cells, but rather, in epithelial cells that line the oviduct lumen in a regionally specific pattern and in multiple subtypes of peripheral neurons, some of which co-express mechanosensory markers. We have used live imaging to demonstrate that most of the functional effects of octopamine and glutamate are mediated by indirect pathways and that contractions of the lateral and common oviducts are regulated independently. Neurons intrinsic to the lateral oviduct indirectly activate muscle cells, and mutational and optogenetic analyses indicate that Octβ2 is required for these effects. An additional indirect pathway for lateral oviduct contractions is controlled by glutamate receptors expressed on a distinct subset of neuronal processes outside of the lateral oviduct. In addition to contractions of the lateral oviduct, octopamine regulates dilation, but this occurs in both the lateral and common oviducts and is blocked by mutation of OAMB but not Octβ2. Additional, unsuspected interactions between octopamine and glutamate include contractions of the common oviduct that requires an interaction between octopamine and with glutamate, blockade of lateral oviduct contractions by preincubation with glutamate and opposing effects of octopamine and glutamate in epithelial cells. These data indicate that multiple indirect pathways regulate the oviposition circuit similar to the mechanisms described for mammalian visceral muscles. They also uncover a complex network of neuromodulatory effects that provide insight into the regulation of behavioral circuits in other systems.

Drosophila melanogaster express vesicular transporters for the storage of neurotransmitters acetylcholine, biogenic amines, GABA, and glutamate. The large array of powerful molecular-genetic tools available in Drosophila enhances the use of this model organism for studying transporter function and regulation.

Environmental toxicants have the potential to contribute to the pathophysiology of multiple complex diseases, but the underlying mechanisms remain obscure. One such toxicant is the widely used fungicide ziram, a dithiocarbamate known to have neurotoxic effects and to increase the risk of Parkinson's disease. We have used Drosophila melanogaster as an unbiased discovery tool to identify novel molecular pathways by which ziram may disrupt neuronal function. Consistent with previous results in mammalian cells, we find that ziram increases the probability of synaptic vesicle release by dysregulation of the ubiquitin signaling system. In addition, we find that ziram increases neuronal excitability. Using a combination of live imaging and electrophysiology, we find that ziram increases excitability in both aminergic and glutamatergic neurons. This increased excitability is phenocopied and occluded by null mutant animals of the ether a-go-go (eag) potassium channel. A pharmacological inhibitor of the temperature sensitive hERG (human ether-a-go-go related gene) phenocopies the excitability effects of ziram but only at elevated temperatures. seizure (sei), a fly ortholog of hERG, is thus another candidate target of ziram. Taken together, the eag family of potassium channels emerges as a candidate for mediating some of the toxic effects of ziram. We propose that ziram may contribute to the risk of complex human diseases by blockade of human eag and sei orthologs, such as hERG.

In the effort of developing new immunotherapies, the sentinel node (SN) has proven a promising source from which to harness an effective antitumour T cell response. However, tumour immune escape, a process in which regulatory T cells (Tregs) play a central role, remains a major limiting factor. Therefore, there is a clear need to increase the knowledge of Treg function and signalling in sentinel nodes. Here, we set out to explore whether the proteome in SN-resident T cells is altered by the tumour and to identify key proteins in SN T cell signalling, focusing on Tregs. Five patients with muscle-invasive urothelial bladder cancer were prospectively included. Mass spectrometry was performed on two patients, with validation and functional studies being performed on three additional patients and four healthy donors. At cystectomy, SN, non-SN lymph nodes and peripheral blood samples were collected from the patients and T cell subsets isolated through flow cytometry before downstream experiments. Proteomic analysis indicated that growth and immune signalling pathways are upregulated in SN-resident Tregs. Furthermore, centrality analysis identified the cytokine IL-16 to be central in the SN-Treg signalling network. We show that tumour-released factors, through activating caspase-3, increase Treg IL-16 processing into bioactive forms, reinforcing Treg suppressive capacity. In conclusion, we provide evidence that Tregs exposed to secreted factors from bladder tumours show increased immune and growth signalling and altered IL-16 processing which translates to enhanced Treg suppressive function, indicating altered IL-16 signalling as a novel tumour immune escape mechanism.

Sensory systems rely on neuromodulators, such as serotonin, to provide flexibility for information processing as stimuli vary, such as light intensity throughout the day. Serotonergic neurons broadly innervate the optic ganglia of Drosophila melanogaster, a widely used model for studying vision. It remains unclear whether serotonin modulates the physiology of interneurons in the optic ganglia. To address this question, we first mapped the expression patterns of serotonin receptors in the visual system, focusing on a subset of cells with processes in the first optic ganglion, the lamina. Serotonin receptor expression was found in several types of columnar cells in the lamina including 5-HT2B in lamina monopolar cell L2, required for spatiotemporal luminance contrast, and both 5-HT1A and 5-HT1B in T1 cells, whose function is unknown. Subcellular mapping with GFP-tagged 5-HT2B and 5-HT1A constructs indicated that these receptors localize to layer M2 of the medulla, proximal to serotonergic boutons, suggesting that the medulla neuropil is the primary site of serotonergic regulation for these neurons. Exogenous serotonin increased basal intracellular calcium in L2 terminals in layer M2 and modestly decreased the duration of visually induced calcium transients in L2 neurons following repeated dark flashes, but otherwise did not alter the calcium transients. Flies without functional 5-HT2B failed to show an increase in basal calcium in response to serotonin. 5-HT2B mutants also failed to show a change in amplitude in their response to repeated light flashes but other calcium transient parameters were relatively unaffected. While we did not detect serotonin receptor expression in L1 neurons, they, like L2, underwent serotonin-induced changes in basal calcium, presumably via interactions with other cells. These data demonstrate that serotonin modulates the physiology of interneurons involved in early visual processing in Drosophila., Author summary Serotonergic neurons innervate the Drosophila melanogaster eye, but it was not known whether serotonin signaling could induce acute physiological responses in visual interneurons. We found serotonin receptors expressed in all neuropils of the optic lobe and identified specific neurons involved in visual information processing that express serotonin receptors. Activation of these receptors increased intracellular calcium in first order interneurons L1 and L2 and may enhance visually induced calcium transients in L2 neurons. These data support a role for the serotonergic neuromodulation of interneurons in the Drosophila visual system.

Evidence indicates that neoadjuvant chemotherapy (NAC) may promote antitumour immune responses by activating T cells. The tumour-draining sentinel node (SN) is a key site to study tumour-specific T cell activation, being the primary immunological barrier against the tumour. In this prospective study, we set out to elucidate the effects of NAC on T cell subsets in the SNs of patients with muscle-invasive urothelial bladder cancer. We found that CD8

Background: Urinary bladder cancer is a common malignancy worldwide. Environmental factors and chronic inflammation are correlated with the disease risk. Diagnosis is performed by transurethral resection of the bladder, and patients with muscle invasive disease preferably proceed to radical cystectomy, with or without neoadjuvant chemotherapy. The anti-tumour immune responses, known to be initiated in the tumour and draining lymph nodes, may play a major role in future treatment strategies. Thus, increasing the knowledge of tumour-associated immunological processes is important. Activated CD4(+) T cells differentiate into four main separate lineages: Th1, Th2, Th17 and Treg, and they are recognized by their effector molecules IFN-gamma, IL-13, IL-17A, and the transcription factor Foxp3, respectively. We have previously demonstrated signature CpG sites predictive for lineage commitment of these four major CD4(+ )T cell lineages. Here, we investigate the lineage commitment specifically in tumour, lymph nodes and blood and relate them to the disease stage and response to neoadjuvant chemotherapy. Results: Blood, tumour and regional lymph nodes were obtained from patients at time of transurethral resection of the bladder and at radical cystectomy. Tumour-infiltrating CD4(+ )lymphocytes were significantly hypomethylated in all four investigated lineage loci compared to CD4(+) lymphocytes in lymph nodes and blood (lymph nodes vs rumour-infiltrating lymphocytes: IFNG -4229 bp p amp;lt; 0.0001, IL13 -11 bp p amp;lt; 0.05, IL17A -122 bp p amp;lt; 0.01 and FOXP3 -77 bp pamp;gt; 0.05). Examination of individual lymph nodes displayed different methylation signatures, suggesting possible correlation with future survival. More advanced post-cystectomy tumour stages correlated significantly with increased methylation at the IFNG -4229 bp locus. Patients with complete response to neoadjuvant chemotherapy displayed significant hypomethylation in CD4(+ )T cells for all four investigated loci, most prominently in IFNG p amp;lt; 0.0001. Neoadjuvant chemotherapy seemed to result in a relocation of Th1-committed CD4(+) T cells from blood, presumably to the tumour, indicated by shifts in the methylation patterns, whereas no such shifts were seen for lineages corresponding to IL13, IL17A and FOXP3. Conclusion: Increased lineage commitment in CD4(+) T cells, as determined by demethylation in predictive CpG sites, is associated with lower post-cystectomy tumour stage, complete response to neoadjuvant chemotherapy and overall better outcome, suggesting epigenetic profiling of CD4(+) T cell lineages as a useful readout for clinical staging. Funding Agencies|Swedish Cancer foundation; Wallenberg foundation; Swedish Medical Research Council; Regionala forskningsradet i Uppsala-Orebroregionen (RFR in Uppsala-Orebro); Swedish Research Council; Cancer Research Foundation in Norrland, Umea, Sweden; Stiftelsen Emil Anderssons fond for medicinsk forskning, Sundsvall, Sweden

Repetitive transcranial magnetic stimulation (rTMS) has demonstrated efficacy in major depressive disorder (MDD), although clinical outcome is variable. Change in the resting-state quantitative electroencephalogram (qEEG), particularly in theta cordance early in the course of treatment, has been linked to antidepressant medication outcomes but has not been examined extensively in clinical rTMS. This study examined change in theta cordance over the first week of clinical rTMS and sought to identify a biomarker that would predict outcome at the end of 6 weeks of treatment. Clinically stable outpatients (n = 18) received nonblinded rTMS treatment administered to the dorsolateral prefrontal cortex (DLPFC). Treatment parameters (site, intensity, number of pulses) were adjusted on an ongoing basis guided by changes in symptom severity rating scale scores. qEEGs were recorded at pretreatment baseline and after 1 week of left DLPFC (L-DLPFC) rTMS using a 21-channel dry-electrode headset. Analyses examined the association between week 1 regional changes in theta band (4-8 Hz) cordance, and week 6 patient- and physician-rated outcomes. Theta cordance change in the central brain region predicted percent change in Inventory of Depressive Symptomology–Self-Report (IDS-SR) score, and improvement versus nonimprovement on the Clinical Global Impression–Improvement Inventory (CGI-I) ( R2 = .38, P = .007; and Nagelkerke R2 = .78, P = .0001, respectively). The cordance biomarker remained significant when controlling for age, gender, and baseline severity. Treatment-emergent change in EEG theta cordance in the first week of rTMS may predict acute (6-week) treatment outcome in MDD. This oscillatory synchrony biomarker merits further study in independent samples.

Repetitive transcranial magnetic stimulation (rTMS) is an effective treatment for Major Depressive Disorder (MDD). There are clinical data that support the efficacy of many different approaches to rTMS treatment, and it remains unclear what combination of stimulation parameters is optimal to relieve depressive symptoms. Because of the costs and complexity of studies that would be necessary to explore and compare the large number of combinations of rTMS treatment parameters, it would be useful to establish reliable surrogate biomarkers of treatment efficacy that could be used to compare different approaches to treatment. This study reviews the evidence that neurophysiologic measures of cortical excitability could be used as biomarkers for screening different rTMS treatment paradigms. It examines evidence that: (1) changes in excitability are related to the mechanism of action of rTMS; (2) rTMS has consistent effects on measures of excitability that could constitute reliable biomarkers; and (3) changes in excitability are related to the outcomes of rTMS treatment of MDD. An increasing body of evidence indicates that these neurophysiologic measures have the potential to serve as reliable biomarkers for screening different approaches to rTMS treatment of MDD.

Repetitive transcranial magnetic stimulation (rTMS) treatment of major depressive disorder (MDD) is associated with changes in brain functional connectivity (FC). These changes may be related to the mechanism of action of rTMS and explain the variability in clinical outcome. We examined changes in electroencephalographic FC during the first rTMS treatment in 109 subjects treated with 10 Hz stimulation to left dorsolateral prefrontal cortex. All subjects subsequently received 30 treatments and clinical response was defined as ≥40% improvement in the inventory of depressive symptomatology-30 SR score at treatment 30. Connectivity change was assessed with coherence, envelope correlation, and a novel measure, alpha spectral correlation (αSC). Machine learning was used to develop predictive models of outcome for each connectivity measure, which were compared with prediction based upon early clinical improvement. Significant connectivity changes were associated with clinical outcome (P < 0.001). Machine learning models based on αSC yielded the most accurate prediction (area under the curve, AUC = 0.83), and performance improved when combined with early clinical improvement measures (AUC = 0.91). The initial rTMS treatment session produced robust changes in FC, which were significant predictors of clinical outcome of a full course of treatment for MDD.

Treatments for major depressive disorder (MDD) act at different hierarchical levels of biological complexity, ranging from the individual synapse to the brain as a whole. Theories of antidepressant medication action traditionally have focused on the level of cell-to-cell interaction and synaptic neurotransmission. However, recent evidence suggests that modulation of synchronized electrical activity in neuronal networks is a common effect of antidepressant treatments, including not only medications, but also neuromodulatory treatments such as repetitive transcranial magnetic stimulation. Synchronization of oscillatory network activity in particular frequency bands has been proposed to underlie neurodevelopmental and learning processes, and also may be important in the mechanism of action of antidepressant treatments. Here, we review current research on the relationship between neuroplasticity and oscillatory synchrony, which suggests that oscillatory synchrony may help mediate neuroplastic changes related to neurodevelopment, learning, and memory, as well as medication and neuromodulatory treatment for MDD. We hypothesize that medication and neuromodulation treatments may have related effects on the rate and pattern of neuronal firing, and that these effects underlie antidepressant efficacy. Elucidating the mechanisms through which oscillatory synchrony may be related to neuroplasticity could lead to enhanced treatment strategies for MDD.

Regulatory T cells (Treg) have long been considered one-sided suppressors of antitumor immune responses and hence associated with poor patient outcome in cancer. However, evidence is mounting of a paradoxical positive prognostic effect of Tregs on certain malignancies, including urinary bladder cancer (UBC). This discrepancy has partly been attributed to the shear misidentification of Tregs, but also to the inflammatory profile of the tumor. Our aim was to determine whether tumor-infiltrating Forkhead box P3+ (FOXP3+) cells confer a stable Treg phenotype and to investigate putative beneficial Treg functions, focusing on tumor-promoting inflammatory pathways in UBC. Patients (n = 52) with suspected UBC were prospectively included. We show, by using a broad range of analytical approaches, that tumor-infiltrating CD4+FOXP3+ T cells in UBC phenotypically, functionally, and epigenetically represent a true Treg population. At the invasive front of UBC tumors, we found an inverse relationship between Treg frequency and expression of matrix metalloproteinase 2 (MMP2), a key proinvasive factor induced by tumor-promoting inflammation. Correspondingly, a significant, dose-dependent Treg-mediated downregulation of MMP2 protein and mRNA expression was observed in both macrophages and UBC cells. Also, we found that Treg frequency specifically at the invasive front positively correlated with survival. Thus, we identify Treg-mediated suppression of MMP2 in the tumor microenvironment as a mechanism explaining the paradoxical positive prognostic impact of tumor-infiltrating Tregs in UBC. Cancer Immunol Res; 6(5); 528–38. ©2018 AACR.

The ability of presynaptic dopamine terminals to tune neurotransmitter release to meet the demands of neuronal activity is critical to neurotransmission. Although vesicle content has been assumed to be static, invitro data increasingly suggest that cell activity modulates vesicle content. Here, we use a coordinated genetic, pharmacological, and imaging approach in Drosophila to study the presynaptic machinery responsible for these vesicular processes invivo. We show that cell depolarization increases synaptic vesicle dopamine content prior to release via vesicular hyperacidification. This depolarization-induced hyperacidification is mediated by the vesicular glutamate transporter (VGLUT). Remarkably, both depolarization-induced dopamine vesicle hyperacidification and its dependence on VGLUT2 are seen in ventral midbrain dopamine neurons in the mouse. Together, these data suggest that in response to depolarization, dopamine vesicles utilize a cascade of vesicular transporters to dynamically increase the vesicular pH gradient, thereby increasing dopamine vesicle content.

© 2014 AICH - Servier Research Group. Major depressive disorder (MDD) is a pleomorphic illness originating from gene x environment interactions. Patients with differing symptom phenotypes receive the same diagnosis and similar treatment recommendations without regard to genomics, brain structure or function, or other physiologic or psychosocial factors. Using this present approach, only one third of patients enter remission with the first medication prescribed, and patients may take longer than 1 year to enter remission with repeated trials. Research to improve treatment effectiveness recently has focused on identification of intermediate phenotypes (IPs) that could parse the heterogeneous population of patients with MDD into subgroups with more homogeneous responses to treatment. Such IPs could be used to develop biomarkers that could be applied clinically to match patients with the treatment that would be most likely to lead to remission. Putative biomarkers include genetic polymorphisms, RNA and protein expression (transcriptome and proteome), neurotransmitter levels (metabolome), additional measures of signaling cascades, oscillatory synchrony, neuronal circuits and neural pathways (connectome), along with other possible physiologic measures. All of these measures represent components of a continuum that extends from proximity to the genome to proximity to the clinical phenotype of depression, and there are many levels along this continuum at which useful IPs may be defined. Because of the highly integrative nature of brain systems and the complex neurobiology of depression, the most useful biomarkers are likely to be those with intermediate proximity both to the genome and the clinical phenotype of MDD. Translation of findings across the spectrum from genotype to phenotype promises to better characterize the complex disruptions in signaling and neuroplasticity that accompany MDD, and ultimately to lead to greater understanding of the causes of depressive illness.

The model genetic organism Drosophila melanogaster, commonly known as the fruit fly, uses many of the same neurotransmitters as mammals and very similar mechanisms of neurotransmitter storage, release and recycling. This system offers a variety of powerful molecular-genetic methods for the study of transporters, many of which would be difficult in mammalian models. We review here progress made using Drosophila to understand the function and regulation of neurotransmitter transporters and discuss future directions for its use.

Monoamine neurotransmitters are stored in both synaptic vesicles (SVs), which are required for release at the synapse, and large dense-core vesicles (LDCVs), which mediate extrasynaptic release. The contributions of each type of vesicular release to specific behaviors are not known. To address this issue, we generated mutations in the C-terminal trafficking domain of theDrosophilavesicular monoamine transporter (DVMAT), which is required for the vesicular storage of monoamines in both SVs and LDCVs. Deletion of the terminal 23 aa (DVMAT-Δ3) reduced the rate of endocytosis and localization of DVMAT to SVs, but supported localization to LDCVs. An alanine substitution mutation in a tyrosine-based motif (DVMAT-Y600A) also reduced sorting to SVs and showed an endocytic deficit specific to aminergic nerve terminals. Redistribution of DVMAT-Y600A from SV to LDCV fractions was also enhanced in aminergic neurons. To determine how these changes might affect behavior, we expressed DVMAT-Δ3 and DVMAT-Y600A in adVMATnull genetic background that lacks endogenousdVMATactivity. When expressed ubiquitously, DVMAT-Δ3 showed a specific deficit in female fertility, whereas DVMAT-Y600A rescued behavior similarly to DVMAT-wt. In contrast, when expressed more specifically in octopaminergic neurons, both DVMAT-Δ3 and DVMAT-Y600A failed to rescue female fertility, and DVMAT-Y600A showed deficits in larval locomotion. DVMAT-Y600A also showed more severe dominant effects than either DVMAT-wt or DVMAT-Δ3. We propose that these behavioral deficits result from the redistribution of DVMAT from SVs to LDCVs. By extension, our data suggest that the balance of amine release from SVs versus that from LDCVs is critical for the function of some aminergic circuits.

Author(s): Hunter, Aimee M; Minzenberg, Michael J; Cook, Ian A; Krantz, David E; Levitt, Jennifer G; Rotstein, Natalie M; Chawla, Shweta A; Leuchter, Andrew F | Abstract: BackgroundRepetitive Transcranial Magnetic Stimulation (rTMS) is commonly administered to Major Depressive Disorder (MDD) patients taking psychotropic medications, yet the effects on treatment outcomes remain unknown. We explored how concomitant medication use relates to clinical response to a standard course of rTMS.MethodsMedications were tabulated for 181 MDD patients who underwent a six-week rTMS treatment course. All patients received 10nHz rTMS administered to left dorsolateral prefrontal cortex (DLPFC), with 1nHz administered to right DLPFC in patients with inadequate response to and/or intolerance of left-sided stimulation. Primary outcomes were change in Inventory of Depressive Symptomatology Self Report (IDS-SR30) total score after 2, 4, and 6nweeks.ResultsUse of benzodiazepines was associated with less improvement at week 2, whereas use of psychostimulants was associated with greater improvement at week 2 and across 6nweeks. These effects were significant controlling for baseline variables including age, overall symptom severity, and severity of anxiety symptoms. Response rates at week 6 were lower in benzodiazepine users versus non-users (16.4% vs. 35.5%, pn=n0.008), and higher in psychostimulant users versus non-users (39.2% vs. 22.0%, pn=n0.02).ConclusionsConcomitant medication use may impact rTMS treatment outcome. While the differences reported here could be considered clinically significant, results were not corrected for multiple comparisons and findings should be replicated before clinicians incorporate the evidence into clinical practice. Prospective, hypothesis-based treatment studies will aid in determining causal relationships between medication treatments and outcome.

Summary Submarine groundwater discharge (SGD) can be an important pathway for transport of nutrients and contaminants to estuaries. A better understanding of the geologic and hydrologic controls on these fluxes is critical for their estimation and management. We examined geologic features, porewater salinity, and SGD rates and patterns at an estuarine study site. Seismic data showed the existence of paleovalleys infilled with estuarine mud and peat that extend hundreds of meters offshore. A low-salinity groundwater plume beneath this low-permeability fill was mapped with continuous resistivity profiling. Extensive direct SGD measurements with seepage meters (n = 551) showed fresh groundwater discharge patterns that correlated well with shallow porewater salinity and the hydrogeophysical framework. Small-scale variability in fresh and saline discharge indicates influence of meter-scale geologic heterogeneity, while site-scale discharge patterns are evidence of the influence of the paleovalley feature. Beneath the paleovalley fill, fresh groundwater flows offshore and mixes with saltwater before discharging along paleovalley flanks. On the adjacent drowned interfluve where low-permeability fill is absent, fresh groundwater discharge is focused at the shoreline. Shallow saltwater exchange was greatest across sandy sediments and where fresh SGD was low. The geologic control of groundwater flowpaths and discharge salinity demonstrated in this work are likely to affect geochemical reactions and the chemical loads delivered by SGD to coastal surface waters. Because similar processes are likely to exist in other estuaries where drowned paleovalleys commonly cross modern shorelines, the existence and implications of complex hydrogeology are important considerations for studies of groundwater fluxes and related management decisions.