Inhibitory Transmission Research Articles

Norepinephrine Restores Inhibitory Tone of Spinal Lamina X Circuitry, thus Contributing to Analgesia Against Inflammatory Pain

Norepinephrine (NE) acts directly on the inhibitory interneurons of spinal lamina X and may act on spinal lamina X neurons for inhibiting nociceptive synaptic transmission against pain. We investigated this mechanism within inflammatory pain model rats. Using immunohistochemical staining and in vivo extracellular recording, the increased number of phosphorylated extracellular signal-regulated kinase profiles in lamina X (n = 6/group) and increased frequency of spontaneous neuronal firing on putative lamina X (n = 14) under the inflammatory pain were significantly suppressed by the direct application of NE (P < 0.01). Following in vivo observation of enhanced spontaneous neuronal firing, we tested the impact of NE on this discharge using an in vitro spinal slice preparation. Using in vitro patch-clamps recording, the baseline level of miniature inhibitory postsynaptic currents (mIPSCs) frequency on spinal lamina X neurons cord is decreased under inflammatory pain. Direct application of NE to spinal lamina X neurons in inflammatory pain model rats facilitates mIPSCs frequency and induces an outward current (n = 8; P < 0.05), and these responses are inhibited by α1A- and α2-receptor antagonists (n = 8; P > 0.05). Considering these results and those of our previous study (Ohashi et al., 2019), NE might act on inhibitory interneurons of spinal lamina X to facilitate inhibitory transmission and induces neurons located in or around lamina X membrane hyperpolarization. These NE-mediated responses acted through α1A- and α2-receptors. These mechanisms of NE on spinal lamina X might contribute to analgesia against inflammatory pain.

Altered excitatory and decreased inhibitory transmission in the prefrontal cortex of male mice with early developmental disruption to the ventral hippocampus.

Ventral hippocampal (vHPC)-prefrontal cortical (PFC) pathway dysfunction is a core neuroimaging feature of schizophrenia. However, mechanisms underlying impaired connectivity within this pathway remain poorly understood. The vHPC has direct projections to the PFC that help shape its maturation. Here, we wanted to investigate the effects of early developmental vHPC perturbations on long-term functional PFC organization. Using whole-cell recordings to assess PFC cellular activity in transgenic male mouse lines, we show early developmental disconnection of vHPC inputs, by excitotoxic lesion or cell-specific ablations, impairs pyramidal cell firing output and produces a persistent increase in excitatory and decrease in inhibitory synaptic inputs onto pyramidal cells. We show this effect is specific to excitatory vHPC projection cell ablation. We further identify PV-interneurons as a source of deficit in inhibitory transmission. We find PV-interneurons are reduced in density, show a reduced ability to sustain high-frequency firing, and show deficits in excitatory inputs that emerge over time. We additionally show differences in vulnerabilities to early developmental vHPC disconnection, wherein PFC PV-interneurons but not pyramidal cells show deficits in NMDA receptor-mediated current. Our results highlight mechanisms by which the PFC adapts to early developmental vHPC perturbations, providing insights into schizophrenia circuit pathology.

Lipid mediator n-3 docosapentaenoic acid-derived protectin D1 enhances synaptic inhibition of hippocampal principal neurons by interaction with a G-protein-coupled receptor.

Epilepsy is a severe neurological disease manifested by spontaneous recurrent seizures due to abnormal hyper‐synchronization of neuronal activity. Epilepsy affects about 1% of the population and up to 40% of patients experience seizures that are resistant to currently available drugs, thus highlighting an urgent need for novel treatments. In this regard, anti‐inflammatory drugs emerged as potential therapeutic candidates. In particular, specific molecules apt to resolve the neuroinflammatory response occurring in acquired epilepsies have been proven to counteract seizures in experimental models, and humans. One candidate investigational molecule has been recently identified as the lipid mediator n‐3 docosapentaenoic acid‐derived protectin D1 (PD1n‐3DPA) which significantly reduced seizures, cell loss, and cognitive deficit in a mouse model of acquired epilepsy. However, the mechanisms that mediate the PD1n‐3DPA effect remain elusive. We here addressed whether PD1n‐3DPA has direct effects on neuronal activity independent of its anti‐inflammatory action. We incubated, therefore, hippocampal slices with PD1n‐3DPA and investigated its effect on excitatory and inhibitory synaptic inputs to the CA1 pyramidal neurons. We demonstrate that inhibitory drive onto the perisomatic region of the pyramidal neurons is increased by PD1n‐3DPA, and this effect is mediated by pertussis toxin‐sensitive G‐protein coupled receptors. Our data indicate that PD1n‐3DPA acts directly on inhibitory transmission, most likely at the presynaptic site of inhibitory synapses as also supported by Xenopus oocytes and immunohistochemical experiments. Thus, in addition to its anti‐inflammatory effects, PD1n‐3DPA anti‐seizure and neuroprotective effects may be mediated by its direct action on neuronal excitability by modulating their synaptic inputs.

Role of Hypothalamic Reactive Astrocytes in Diet-Induced Obesity.

Hypothalamus is a brain region that controls food intake and energy expenditure while sensing signals that convey information about energy status. Within the hypothalamus, molecularly and functionally distinct neurons work in concert under physiological conditions. However, under pathological conditions such as in diet-induced obesity (DIO) model, these neurons show dysfunctional firing patterns and distorted regulation by neurotransmitters and neurohormones. Concurrently, resident glial cells including astrocytes dramatically transform into reactive states. In particular, it has been reported that reactive astrogliosis is observed in the hypothalamus, along with various neuroinflammatory signals. However, how the reactive astrocytes control and modulate DIO by influencing neighboring neurons is not well understood. Recently, new lines of evidence have emerged indicating that these reactive astrocytes directly contribute to the pathology of obesity by synthesizing and tonically releasing the major inhibitory transmitter GABA. The released GABA strongly inhibits the neighboring neurons that control energy expenditure. These surprising findings shed light on the interplay between reactive astrocytes and neighboring neurons in the hypothalamus. This review summarizes recent discoveries related to the functions of hypothalamic reactive astrocytes in obesity and raises new potential therapeutic targets against obesity.

Glucocorticoid receptors participate in epilepsy in FCDII patients and MP model rats: A potential therapeutic target for epilepsy in patients with focal cortical dysplasia II (FCDII)

ABSTRACT Background Glucocorticoid receptors (GRs) and mineralocorticoid receptors (MRs) are involved in neuronal excitability, neurogenesis, and neuroinflammation. However, the roles of GRs and MRs in epilepsy in focal cortical dysplasia II (FCDII) have not been reported. Research Design and Methods We evaluated GRs and MRs expression and distribution in FCDII patients and methylazoxymethanol-pilocarpine-induced epilepsy model rats (MP rats), and the effects of a GR agonist on neurons in human FCDII and investigated the electrophysiological properties of rats’ neurons after lentivirus-mediated GR knockdown or overexpression and GR agonist or antagonist administration. Results GR expression (not MR) was decreased in specimens from FCDII patients and model rats. GR agonist dexamethasone reduced neuronal excitatory transmission and increased neuronal inhibitory transmission in FCDII. GR knockdown increased the excitability of cultured neurons, and GR overexpression rescued the hyperexcitability of MP-treated neurons. Moreover, dexamethasone decreased neuronal excitability and excitatory transmission in MP rats, while GR antagonist exerted the opposite effects. Dexamethasone reduced the seizure number and duration by approximately 85% and 60% in MP rats within one to two hours. Conclusions These results suggested that GRs play an important role in epilepsy in FCDII and GR activation may have protective and antiepileptic effects in FCDII.

MDGA1 negatively regulates amyloid precursor protein–mediated synapse inhibition in the hippocampus

Balanced synaptic inhibition, controlled by multiple synaptic adhesion proteins, is critical for proper brain function. MDGA1 (meprin, A-5 protein, and receptor protein-tyrosine phosphatase mu [MAM] domain-containing glycosylphosphatidylinositol anchor protein 1) suppresses synaptic inhibition in mammalian neurons, yet the molecular mechanisms underlying MDGA1-mediated negative regulation of GABAergic synapses remain unresolved. Here, we show that the MDGA1 MAM domain directly interacts with the extension domain of amyloid precursor protein (APP). Strikingly, MDGA1-mediated synaptic disinhibition requires the MDGA1 MAM domain and is prominent at distal dendrites of hippocampal CA1 pyramidal neurons. Down-regulation of APP in presynaptic GABAergic interneurons specifically suppressed GABAergic, but not glutamatergic, synaptic transmission strength and inputs onto both the somatic and dendritic compartments of hippocampal CA1 pyramidal neurons. Moreover, APP deletion manifested differential effects in somatostatin- and parvalbumin-positive interneurons in the hippocampal CA1, resulting in distinct alterations in inhibitory synapse numbers, transmission, and excitability. The infusion of MDGA1 MAM protein mimicked postsynaptic MDGA1 gain-of-function phenotypes that involve the presence of presynaptic APP. The overexpression of MDGA1 wild type or MAM, but not MAM-deleted MDGA1, in the hippocampal CA1 impaired novel object-recognition memory in mice. Thus, our results establish unique roles of APP-MDGA1 complexes in hippocampal neural circuits, providing unprecedented insight into trans-synaptic mechanisms underlying differential tuning of neuronal compartment-specific synaptic inhibition.

A revised mechanism of action of hyperaldosteronism-linked mutations in cytosolic domains of GIRK4 (KCNJ5).

G protein-gated, inwardly rectifying potassium channels (GIRK) mediate inhibitory transmission in brain and heart, and are present in the adrenal cortex. GIRK4 (KCNJ5) subunits are abundant in the heart and adrenal cortex. Multiple mutations of KCNJ5 cause primary aldosteronism (PA). Mutations in the pore region of GIRK4 cause loss of K+ selectivity, Na+ influx and depolarization of zona glomerulosa cells followed by hypersecretion of aldosterone. The concept of selectivity loss has been extended to mutations in cytosolic domains of GIRK4 channels, remote from the pore. We expressed aldosteronism-linked GIRK4R52H , GIRK4E246K and GIRK4G247R mutants in Xenopus oocytes. Whole-cell currents of heterotetrameric GIRK1/4R52H and GIRK1/4E246K channels were greatly reduced compared with GIRK1/4WT . Nevertheless, all heterotetrameric mutants retained full K+ selectivity and inward rectification. When expressed as homotetramers, only GIRK4WT , but none of the mutants, produced whole-cell currents. Confocal imaging, single-channel and Förster Resonance Energy Transfer (FRET) analyses showed: (1) reduction of membrane abundance of all mutated channels, especially as homotetramers, (2) impaired interaction with Gβγ subunits, and (3) reduced open probability of GIRK1/4R52H . VU0529331, a GIRK4 opener, activated homotetrameric GIRK4G247R channels, but not GIRK4R52H or GIRK4E246K . In the human adrenocortical carcinoma cell line (HAC15), VU0529331 and overexpression of heterotetrameric GIRK1/4WT , but not overexpression of GIRK1/4 mutants, reduced aldosterone secretion. Our results suggest that, contrary to pore mutants of GIRK4, non-pore mutants R52H and E246K mutants are loss-of-function rather than gain-of-function/selectivity-loss mutants. Hence, GIRK4 openers may be a potential course of treatment for patients with cytosolic N- and C-terminal mutations. KEY POINTS: Mutations in GIRK4 (KCNJ5) G protein-gated channels cause primary aldosteronism, a major cause of secondary hypertension. The primary mechanism is believed to be loss of K+ selectivity. R52H and E246K, aldosteronism-causing mutations in cytosolic N- and C- termini of GIRK4, were reported to cause loss of K+ selectivity. We show that R52H, E246K and G247R mutations render homotetrameric GIRK channels non-functional. In heterotetrameric context with GIRK1, these mutations impair membrane expression, interaction with Gβγ and open probability, but do not alter K+ selectivity or inward rectification. In the human aldosterone-secreting cell line, a GIRK4 opener and overexpression of heterotetrameric GIRK1/4WT , but not overexpression of GIRK1/4 mutants, reduced aldosterone secretion. Aldosteronism-causing mutations in the cytosolic domain of GIRK4 are loss-of-function mutations rather than gain-of-function, selectivity-loss mutations. Deciphering of exact biophysical mechanism that impairs the channel is crucial for setting the course of treatment.

Long-Term Imaging Reveals a Circadian Rhythm of Intracellular Chloride in Neurons of the Suprachiasmatic Nucleus.

Both inhibitory and excitatory GABA transmission exist in the mature suprachiasmatic nucleus (SCN), the master pacemaker of circadian physiology. Whether GABA is inhibitory or excitatory depends on the intracellular chloride concentration ([Cl-]i). Here, using the genetically encoded ratiometric probe Cl-Sensor, we investigated [Cl-]i in AVP and VIP-expressing SCN neurons for several days in culture. The chloride ratio (RCl) demonstrated circadian rhythmicity in AVP + neurons and VIP + neurons, but was not detected in GFAP + astrocytes. RCl peaked between ZT 7 and ZT 8 in both AVP + and VIP + neurons. RCl rhythmicity was not dependent on the activity of several transmembrane chloride carriers, action potential generation, or the L-type voltage-gated calcium channels, but was sensitive to GABA antagonists. We conclude that [Cl-]i is under circadian regulation in both AVP + and VIP + neurons.

Social isolation induces neuroinflammation and microglia overactivation, while dihydromyricetin prevents and improves them

BackgroundAnxiety disorders are the most prevalent mental illnesses in the U.S. and are estimated to consume one-third of the country’s mental health treatment cost. Although anxiolytic therapies are available, many patients still exhibit treatment resistance, relapse, or substantial side effects. Further, due to the COVID-19 pandemic and stay-at-home order, social isolation, fear of the pandemic, and unprecedented times, the incidence of anxiety has dramatically increased. Previously, we have demonstrated dihydromyricetin (DHM), the major bioactive flavonoid extracted from Ampelopsis grossedentata, exhibits anxiolytic properties in a mouse model of social isolation-induced anxiety. Because GABAergic transmission modulates the immune system in addition to the inhibitory signal transmission, we investigated the effects of short-term social isolation on the neuroimmune system.MethodsEight-week-old male C57BL/6 mice were housed under absolute social isolation for 4 weeks. The anxiety-like behaviors after DHM treatment were examined using elevated plus-maze and open field behavioral tests. Gephyrin protein expression, microglial profile changes, NF-κB pathway activation, cytokine level, and serum corticosterone were measured.ResultsSocially isolated mice showed increased anxiety levels, reduced exploratory behaviors, and reduced gephyrin levels. Also, a dynamic alteration in hippocampal microglia were detected illustrated as a decline in microglia number and overactivation as determined by significant morphological changes including decreases in lacunarity, perimeter, and cell size and increase in cell density. Moreover, social isolation induced an increase in serum corticosterone level and activation in NF-κB pathway. Notably, DHM treatment counteracted these changes.ConclusionThe results suggest that social isolation contributes to neuroinflammation, while DHM has the ability to improve neuroinflammation induced by anxiety.

Sustained Inhibitory Transmission But Dysfunctional Dopamine D2 Receptor Signaling in Dorsal Striatal Subregions Following Protracted Abstinence from Amphetamine

Sustained Inhibitory Transmission But Dysfunctional Dopamine D2 Receptor Signaling in Dorsal Striatal Subregions Following Protracted Abstinence from Amphetamine

Alteration of Effective Connectivity in the Default Mode Network of Autism After an Intervention.

Neuroimaging has revealed numerous atypical functional connectivity of default mode network (DMN) dedicated to social communications (SC) in autism spectrum disorder (ASD), yet their nature and directionality remain unclear. Here, preschoolers with autism received physical intervention from a 12-week mini-basketball training program (12W-MBTP). Therefore, the directionality and nature of regional interactions within the DMN after the intervention are evaluated while assessing the impact of an intervention on SC. Based on the results of independent component analysis (ICA), we applied spectral dynamic causal modeling (DCM) for participants aged 3–6 years (experimental group, N = 17, control group, N = 14) to characterize the longitudinal changes following intervention in intrinsic and extrinsic effective connectivity (EC) between core regions of the DMN. Then, we analyzed the correlation between the changes in EC and SRS-2 scores to establish symptom-based validation. We found that after the 12W-MBTP intervention, the SRS-2 score of preschoolers with ASD in the experimental group was decreased. Concurrently, the inhibitory directional connections were observed between the core regions of the DMN, including increased self-inhibition in the medial prefrontal cortex (mPFC), and the changes of EC in mPFC were significantly correlated with change in the social responsiveness scale-2 (SRS-2) score. These new findings shed light on DMN as a potential intervention target, as the inhibitory information transmission between its core regions may play a positive role in improving SC behavior in preschoolers with ASD, which may be a reliable neuroimaging biomarker for future studies.Clinical Trial Registration: This study registered with the Chinese Clinical Trial Registry (ChiCTR1900024973) on August 05, 2019.

High-Throughput Strategy for Glycine Oxidase Biosensor Development Reveals Glycine Release from Cultured Cells.

Glycine is an important biomarker in clinical analysis due to its involvement in multiple physiological processes. As such, the need for low-cost analytical tools for glycine detection is growing. As a neurotransmitter, glycine is involved in inhibitory and excitatory neurochemical transmission in the central nervous system. In this work, we present a 10 μM Pt-based electrochemical enzymatic biosensor based on the flavoenzyme glycine oxidase (GO) for localized real-time measurements of glycine. Among GO variants at position 244, the H244K variant with increased glycine turnover was selected to develop a functional biosensor. This biosensor relies on amperometric readouts and does not require additional redox mediators. The biosensor was characterized and applied for glycine detection from cells, mainly HEK 293 cells and primary rat astrocytes. We have identified an enzyme, GO H244K, with increased glycine turnover using mutagenesis but which can be developed into a functional biosensor. Noteworthy, a glycine release of 395.7 ± 123 μM from primary astrocytes was measured, which is ∼fivefold higher than glycine release from HEK 293 cells (75.4 ± 3.91 μM) using the GO H244K biosensor.

Heterogeneous GAD65 Expression in Subtypes of GABAergic Neurons Across Layers of the Cerebral Cortex and Hippocampus.

Gamma-aminobutyric acid (GABA), a major inhibitory transmitter in the central nervous system, is synthesized via either of two enzyme isoforms, GAD65 or GAD67. GAD65 is synthesized in the soma but functions at synaptic terminals in an activity-dependent manner, playing a distinct role in excitatory-inhibitory balance. However, the extent to which each GABAergic subtype expresses GAD65 in the resting state remains unclear. In this study, we compared GAD65 expression among six GABAergic subtypes: NPY+, nNOS+, PV+, SOM+, CR+, and CCK+. According to the results, the GABAergic subtypes were classified into two groups per region based on GAD65 expression levels: high-expression (NPY+ and nNOS+) and low-expression groups (PV+, SOM+, CR+, and CCK+) in the cerebral cortex and high-expression (NPY+, nNOS+, and CCK+) and low-expression groups (PV+, SOM+, and CR+) in the hippocampus. Moreover, these expression patterns revealed a distinct laminar distribution in the cerebral cortex and hippocampus. To investigate the extent of GAD65 transport from the soma to synaptic terminals, we examined GAD65 expression in colchicine-treated rats in which GAD65 was synthesized in the soma but not transported to terminals. We found a significant positive correlation in GAD65 expression across subtypes between colchicine-treated and control rats. In summary, each GABAergic subtype exhibits a distinct GAD65 expression pattern across layers of the cerebral cortex and hippocampus. In addition, the level of GAD65 expression in the soma can be used as a proxy for the amount of GAD65 in the cytoplasm. These findings suggest that exploration of the distinct profiles of GAD65 expression among GABAergic subtypes could clarify the roles that GABAergic subtypes play in maintaining the excitatory-inhibitory balance.

Elucidating the Potential Side Effects of Current Anti-Seizure Drugs for Epilepsy

Over the decades, various interventions have been developed and utilized to treat epilepsy. However, the majority of epileptic patients are often first prescribed anti-epileptic drugs (AED), now known as anti-seizure drugs (ASD), as the first line of defense to suppress their seizures and regain their quality of life. ASDs exert their anti-convulsant effects through various mechanisms of action, including regulation of ion channels, blocking glutamate-mediated stimulating neurotransmitter interaction, and enhancing the inhibitory GABA transmission. About one-third of epileptic patients are often resistant to anti-convulsant drugs, while others develop numerous side effects, which may lead to treatment discontinuation and further deterioration of quality of life. Common side effects of ASDs include headache, nausea and dizziness. However, more adverse effects, such as auditory and visual problems, skin problems, liver dysfunction, pancreatitis and kidney disorders may also be witnessed. Some ASDs may even result in life-threatening conditions as well as serious abnormalities, especially in patients with comorbidities and in pregnant women. Nevertheless, some clinicians had observed a reduction in the development of side effects post individualized ASD treatment. This suggests that a careful and well-informed ASD recommendation to patients may be crucial for an effective and side-effect-free control of their seizures. Therefore, this review aimed to elucidate the anticonvulsant effects of ASDs as well as their side effect profile by discussing their mechanism of action and reported adverse effects based on clinical and preclinical studies, thereby providing clinicians with a greater understanding of the safety of current ASDs.

The role of enteric inhibitory neurons in intestinal motility.

The enteric nervous system controls much of the mixing and propulsion of nutrients along the digestive tract. Enteric neural circuits involve intrinsic sensory neurons, interneurons and motor neurons. While the role of the excitatory motor neurons is well established, the role of the enteric inhibitory motor neurons (IMNs) is less clear. The discovery of inhibitory transmission in the intestine in the 1960's in the laboratory of Geoff Burnstock triggered the search for the unknown neurotransmitter. It has since emerged that most neurons including the IMNs contain and may utilise more than one transmitter substances; for IMNs these include ATP, the neuropeptide VIP/PACAP and nitric oxide. This review distinguishes the enteric neural pathways underlying the 'standing reflexes' from the pathways operating physiologically during propulsive and non-propulsive movements. Morphological evidence in small laboratory animals indicates that the IMNs are located in the myenteric plexus and project aborally to the circular muscle, where they act by relaxing the muscle. There is ongoing 'tonic' activity of these IMNs to keep the intestinal muscle relaxed. Accommodatory responses to content further activate enteric pathways that involve the IMNs as the final neural element. IMNs are activated by mechanical and chemical stimulation induced by luminal contents, which activate intrinsic sensory enteric neurons and the polarised interneuronal ascending excitatory and descending inhibitory reflex pathways. The latter relaxes the muscle ahead of the advancing bolus, thus facilitating propulsion.

MGlu1 potentiation enhances prelimbic somatostatin interneuron activity to rescue schizophrenia-like physiological and cognitive deficits.

SUMMARYEvidence for prefrontal cortical (PFC) GABAergic dysfunction is one of the most consistent findings in schizophrenia and may contribute to cognitive deficits. Recent studies suggest that the mGlu1 subtype of metabotropic glutamate receptor regulates cortical inhibition; however, understanding the mechanisms through which mGlu1 positive allosteric modulators (PAMs) regulate PFC microcircuit function and cognition is essential for advancing these potential therapeutics toward the clinic. We report a series of electrophysiology, optogenetic, pharmacological magnetic resonance imaging, and animal behavior studies demonstrating that activation of mGlu1 receptors increases inhibitory transmission in the prelimbic PFC by selective excitation of somatostatin-expressing interneurons (SST-INs). An mGlu1 PAM reverses cortical hyperactivity and concomitant cognitive deficits induced by N-methyl-d-aspartate (NMDA) receptor antagonists. Using in vivo optogenetics, we show that prelimbic SST-INs are necessary for mGlu1 PAM efficacy. Collectively, these findings suggest that mGlu1 PAMs could reverse cortical GABAergic deficits and exhibit efficacy in treating cognitive dysfunction in schizophrenia.

Low-Dose Augmentation With Buprenorphine for Treatment-Resistant Depression: A Multisite Randomized Controlled Trial With Multimodal Assessment of Target Engagement

BackgroundThe experimental therapeutics approach that combines a placebo-controlled clinical trial with translational neuroscience methods can provide a better understanding of both the clinical and physiological effects of pharmacotherapy. We aimed to test the efficacy and tolerability of low-dose augmentation with buprenorphine (BPN) for treatment-resistant depression, combined with multimodal assessment of target engagement. MethodsIn this multisite randomized clinical trial, 85 participants ≥50 years of age with a major depressive episode that had not responded to venlafaxine extended release were randomized to augmentation with BPN or placebo for 8 weeks. The primary outcome measure was the Montgomery–Åsberg Depression Rating Scale. In addition, three linked experiments were conducted to test target engagement: 1) functional magnetic resonance imaging using the monetary incentive delay task, 2) brain positron emission tomography of healthy participants using a novel kappa opioid receptor antagonist tracer [11C]LY2795050, and 3) transcranial magnetic stimulation measure of cortical transmission after daily BPN administration. ResultsThe mean ± SD dosage of BPN was 0.59 ± 0.33 mg/day. There were no significant differences between the BPN and placebo groups in Montgomery–Åsberg Depression Rating Scale changes over time or adverse effects. BPN administration had minimal effects on functional magnetic resonance imaging blood oxygen level–dependent responses in regions involved in reward anticipation and response, no significant displacement of kappa opioid receptor radioligand in positron emission tomography imaging, and no significant changes in transcranial magnetic stimulation measures of inhibitory and excitatory cortical transmission. ConclusionsOur findings suggest a lack of clinical effect of low-dose BPN augmentation and lack of target engagement with this dosage and physiological probes.

Upregulation of the mGlu5 receptor and COX-2 protein in the mouse brain after imipramine and NS398, searching for mechanisms of regulation

Imipramine belongs to a group of tricyclic antidepressants (TCAs). It has been also documented that its antidepressant activity connects with the modulation of cytosolic phospholipase A2 (cPLA2) and arachidonic acid (AA) turnover. Through this mechanism, imipramine can indirectly modify glutamate (Glu) transmission. Additionally, it has been shown that chronic treatment with imipramine results in the upregulation of the metabotropic glutamate receptor subtype 5 (mGlu5 receptor) in the hippocampus of rats. Our previous study revealed that manipulation of the AA pathway via inhibition of cyclooxygenase-2 (COX-2) by selective COX-2 inhibitor (NS398) could effectively modulate the behavior of mice treated with imipramine. Here, we hypothesized that COX-2 inhibition could similarly to imipramine influence mGlu5 receptor, and thus NS398 can modulate the effect of imipramine on Glu. Moreover, such regulation changes should correspond with alterations in neurotransmission. Increased cPLA activity after imipramine administration may change the activity of the AA pathway and the endocannabinoid metabolism, e.g., 2-Arachidonyl-glycerol (2-AG). To verify the idea, mGlu5 receptor level was investigated in the hippocampus (HC) and prefrontal cortex (PFC) of mice treated for 7 or 14 days with imipramine and/or COX-2 inhibitor: NS398. Western blot and PCR analyses were conducted. Moreover, the excitatory (Glu) and inhibitory (gamma-aminobutyric acid; GABA) neurotransmitters were measured using HPLC and 2-AG using ELISA.A time-dependent change in mGlu5 receptor and COX-2 protein level, COX-2 expression, and 2-AG level in the PFC after imipramine administration was found. Up-regulation of mGlu5 receptor after NS398 was found in HC and PFC. A structure-dependent shift between excitatory vs. inhibitory transmission was detected when NS398 and imipramine were co-administered.

Novel Functional Properties of Missense Mutations in the Glycine Receptor β Subunit in Startle Disease.

Startle disease is a rare disorder associated with mutations in GLRA1 and GLRB, encoding glycine receptor (GlyR) α1 and β subunits, which enable fast synaptic inhibitory transmission in the spinal cord and brainstem. The GlyR β subunit is important for synaptic localization via interactions with gephyrin and contributes to agonist binding and ion channel conductance. Here, we have studied three GLRB missense mutations, Y252S, S321F, and A455P, identified in startle disease patients. For Y252S in M1 a disrupted stacking interaction with surrounding aromatic residues in M3 and M4 is suggested which is accompanied by an increased EC50 value. By contrast, S321F in M3 might stabilize stacking interactions with aromatic residues in M1 and M4. No significant differences in glycine potency or efficacy were observed for S321F. The A455P variant was not predicted to impact on subunit folding but surprisingly displayed increased maximal currents which were not accompanied by enhanced surface expression, suggesting that A455P is a gain-of-function mutation. All three GlyR β variants are trafficked effectively with the α1 subunit through intracellular compartments and inserted into the cellular membrane. In vivo, the GlyR β subunit is transported together with α1 and the scaffolding protein gephyrin to synaptic sites. The interaction of these proteins was studied using eGFP-gephyrin, forming cytosolic aggregates in non-neuronal cells. eGFP-gephyrin and β subunit co-expression resulted in the recruitment of both wild-type and mutant GlyR β subunits to gephyrin aggregates. However, a significantly lower number of GlyR β aggregates was observed for Y252S, while for mutants S321F and A455P, the area and the perimeter of GlyR β subunit aggregates was increased in comparison to wild-type β. Transfection of hippocampal neurons confirmed differences in GlyR-gephyrin clustering with Y252S and A455P, leading to a significant reduction in GlyR β-positive synapses. Although none of the mutations studied is directly located within the gephyrin-binding motif in the GlyR β M3-M4 loop, we suggest that structural changes within the GlyR β subunit result in differences in GlyR β-gephyrin interactions. Hence, we conclude that loss- or gain-of-function, or alterations in synaptic GlyR clustering may underlie disease pathology in startle disease patients carrying GLRB mutations.

Serotonin activates paraventricular thalamic neurons through direct depolarization and indirect disinhibition from zona incerta.

Paraventricular thalamus (PVT) is a midline thalamic area that receives dense GABA projections from zona incerta (ZI) for the regulation of feeding behaviours. Activation of central serotonin (5-HT) signalling is known to inhibit food intake. Although previous studies have reported both 5-HT fibres and receptors in the PVT, it remains unknown how 5-HT regulates PVT neurons and whether PVT 5-HT signalling is involved in the control of food intake. Using slice patch-clamp recordings in combination with optogenetics, we found that 5-HT not only directly excited PVT neurons by activating 5-HT7 receptors to modulate hyperpolarization-activated cyclic nucleotide-gated (HCN) channels but also disinhibited these neurons by acting on presynaptic 5-HT1A receptors to reduce GABA inhibition. Specifically, 5-HT depressed photostimulation-evoked inhibitory postsynaptic currents (eIPSCs) in PVT neurons innervated by channelrhodopsin-2-positive GABA axons from ZI. Using paired-pulse photostimulation, we found 5-HT increased paired-pulse ratios of eIPSCs, suggesting 5-HT decreases ZI-PVT GABA release. Furthermore, we found that exposure to a high-fat-high-sucrose diet for 2weeks impairedboth 5-HT inhibition of ZI-PVT GABA transmission and 5-HT excitation of PVT neurons. Using retrograde tracer in combination with immunocytochemistry and slice electrophysiology, we found that PVT-projecting dorsal raphe neurons expressed 5-HT and were inhibited by food deprivation. Together, our study reveals the mechanism by which 5-HT activates PVT neurons through both direct excitation and indirect disinhibition from the ZI. The downregulation in 5-HT excitation and disinhibition of PVT neurons may contribute to the development of overeating and obesity after chronic high-fat diet. KEY POINTS: Serotonin (5-HT) depolarizes and excites paraventricular thalamus (PVT) neurons. 5-HT7 receptors are responsible for 5-HT excitation of PVT neurons and the coupling of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels to 5-HT receptors in part mediates the excitatory effect of 5-HT. 5-HT depresses the frequency of spontaneous inhibitory but not excitatory postsynaptic currents in PVT neurons. 5-HT1A receptors contribute to the depressive effect of 5-HT on inhibitory transmissions. 5-HT inhibits GABA release from zona incerta (ZI) GABA terminals in PVT. Chronic high-fat diet not only impairs 5-HT inhibition of the ZI-PVT GABA transmission but also downregulates 5-HT excitation of PVT neurons. PVT-projecting dorsal raphe neurons express 5-HT and are inhibited by food deprivation.