Brain activity implies the orchestrated functioning of interconnected brain regions. Typical in vitro models aim to mimic the brain using single human pluripotent stem cell-derived neuronal networks. However, the field is constantly evolving to model brain functions more accurately through the use of new paradigms, e.g., brain-on-a-chip models with compartmentalized structures and integrated sensors. These methods create novel data requiring more complex analysis approaches. The previously introduced circular tripartite network concept models the connectivity between spatially diverse neuronal structures. The model consists of a microfluidic device allowing axonal connectivity between separated neuronal networks with an embedded microelectrode array to record both local and global electrophysiological activity patterns in the closed circuitry. The existing tools are suboptimal for the analysis of the data produced with this model. Here, we introduce advanced tools for synchronization and functional connectivity assessment. We used our custom-designed analysis to assess the interrelations between the kainic acid (KA)-exposed proximal compartment and its nonexposed distal neighbors before and after KA. Novel multilevel circuitry bursting patterns were detected and analyzed in parallel with the inter- and intracompartmental functional connectivity. The effect of KA on the proximal compartment was captured, and the spread of this effect to the nonexposed distal compartments was revealed. KA induced divergent changes in bursting behaviors, which may be explained by distinct baseline activity and varied intra- and intercompartmental connectivity strengths. The circular tripartite network concept combined with our developed analysis advances importantly both face and construct validity in modeling human epilepsy in vitro.

The N-methyl-D-aspartate (NMDA) glutamate receptors function as plasma membrane ionic channels and take part in very tightly controlled cellular processes activating neurogenic and inflammatory pathways. In particular, the NR1 subunit (new terminology: GluN1) is required for many neuronal and non-neuronal cell functions, including plasticity, survival, and differentiation. Physiologic levels of glutamate agonists and NMDA receptor activation are required for normal neuronal functions such as neuronal development, learning, and memory. When glutamate receptor agonists are present in excess, binding to NMDA receptors produces neuronal/CNS/PNS long-term potentiation, conditions of acute pain, ongoing severe intractable pain, and potential excitotoxicity and pathology. The GluNR1 subunit (116 kD) is necessary as the anchor component directing ion channel heterodimer formation, cellular trafficking, and the nuclear localization that directs functionally specific heterodimer formation, cellular trafficking, and nuclear functions. Emerging studies report the relevance of GluN1 subunit composition and specifically that nuclear GluN1 has major physiologic potential in tissue and/or subnuclear functioning assignments. The shift of the GluN1 subunit from a surface cell membrane to nuclear localization assigns the GluN1 promoter immediate early gene behavior with access to nuclear and potentially nucleolar functions. The present narrative review addresses the nuclear translocation of GluN1, focusing particularly on examples of the role of GluN1 in nociceptive processes.

Proton-gated channels of the ASIC family are widely distributed in central neurons, suggesting their role in common neurophysiological functions. They are involved in glutamatergic neurotransmission and synaptic plasticity; however, the exact function of these channels remains unclear. One problem is that acidification of the synaptic cleft due to the acidic content of synaptic vesicles has opposite effects on ionotropic glutamate receptors and ASICs. Thus, the pH values required to activate ASICs strongly inhibit AMPA receptors and almost completely inhibit NMDA receptors. This, in turn, suggests that ASICs can provide compensation for post-synaptic responses in the case of significant acidifications. We tested this hypothesis by patch-clamp recordings of rat brain neuron responses to acidifications and glutamate receptor agonists at different pH values. Hippocampal pyramidal neurons have much lower ASICs than glutamate receptor responses, whereas striatal interneurons show the opposite ratio. Cortical pyramidal neurons and hippocampal interneurons show similar amplitudes in their responses to acidification and glutamate. Consequently, the total response to glutamate agonists at different pH levels remains rather stable up to pH 6.2. Besides these pH effects, the relationship between the responses mediated by glutamate receptors and ASICs depends on the presence of Mg2+ and the membrane voltage. Together, these factors create a complex picture that provides a framework for understanding the role of ASICs in synaptic transmission and synaptic plasticity.

Patients with schizophrenia show reduced NMDA glutamate receptor-dependent auditory plasticity, which is rate limiting for auditory cognitive remediation (AudRem). We evaluate the utility of behavioral and neurophysiological pharmacodynamic target engagement biomarkers, using a d-serine+AudRem combination.
Forty-five participants with schizophrenia or schizoaffective disorder were randomized to 3 once-weekly AudRem visits + double-blind d-serine (80, 100, or 120 mg/kg) or placebo in 3 dose cohorts of 12 d-serine and 3 placebo-treated participants each. In AudRem, participants indicated which paired tone was higher in pitch. The primary outcome was plasticity improvement, operationalized as change in pitch threshold between AudRem tones [(test tone Hz - reference tone Hz)/reference tone Hz] between the initial plateau pitch threshold (mean of trials 20-30 of treatment visit 1) to pitch threshold at the end of visit(s). Target engagement was assessed by electroencephalography outcomes, including mismatch negativity (pitch primary).
There was a significant overall treatment effect for plasticity improvement (p = .014). Plasticity improvement was largest within the 80 and 100 mg/kg groups (p < .001, d > 0.67), while 120 mg/kg and placebo-treated participants showed nonsignificant within-group changes. Plasticity improvement was seen after a single treatment and was sustained on subsequent treatments. Target engagement was demonstrated by significantly larger mismatch negativity (p = .049, d = 1.0) for the 100 mg/kg dose versus placebo.
Our results demonstrate sufficient proof of principle for continued development of both the d-serine+AudRem combination and our target engagement methodology. The ultimate utility is dependent on the results of an ongoing larger, longer study of the combination for clinically relevant outcomes.

Several attempts have been made to enhance N-methyl-D-aspartate (NMDA) receptor function in schizophrenia, but they have yielded mixed results. Luvadaxistat, a D-amino acid oxidase (DAAO) inhibitor that increases the glutamate co-agonist D-serine levels, is being developed for the treatment of cognitive impairment associated with schizophrenia. We conducted a biomarker study in patients, assessing several endpoints related to physiological outcomes of NMDA receptor modulation to determine whether luvadaxistat affects neural circuitry biomarkers relevant to NMDA receptor function and schizophrenia. This was a randomized, placebo-controlled, double-blind, two-period crossover phase 2a study assessing luvadaxistat 50 mg and 500 mg for 8 days in 31 patients with schizophrenia. There were no treatment effects of luvadaxistat at either dose in eyeblink conditioning, a cerebellar-dependent learning measure, compared with placebo. We observed a nominally significant improvement in mismatch negativity (MMN) and a statistical trend to improvement for auditory steady-state response at 40 Hz, in both cases with 50 mg, but not with 500 mg, compared with placebo. Although the data should be interpreted cautiously owing to the small sample size, they suggest that luvadaxistat can improve an illness-related circuitry biomarker at doses associated with partial DAAO inhibition. These results are consistent with 50 mg, but not higher doses, showing a signal of efficacy in cognitive endpoints in a larger phase 2, 12-week study conducted in parallel. Thus, MMN responses after a short treatment period may predict cognitive function improvement. MMN and ASSR should be considered as biomarkers in early trials addressing NMDA receptor hypofunction.

A core network of widely expressed proteins within the glutamatergic post-synapse mediates activity-dependent synaptic plasticity throughout the brain, but the specific proteomic composition of synapses differs between brain regions. Here, we address the question, how does proteomic composition affect activity-dependent protein-protein interaction networks (PINs) downstream of synaptic activity? Using quantitative multiplex co-immunoprecipitation, we compare the PIN response of in vivo or ex vivo neurons derived from different brain regions to activation by different agonists or different forms of eyeblink conditioning. We report that PINs discriminate between incoming stimuli using differential kinetics of overlapping and non-overlapping PIN parameters. Further, these \"molecular logic rules\" differ by brain region. We conclude that although the PIN of the glutamatergic post-synapse is expressed widely throughout the brain, its activity-dependent dynamics show remarkable stimulus-specific and brain-region-specific diversity. This diversity may help explain the challenges in developing molecule-specific drug therapies for neurological disorders.

Tonic inhibition mediated by extrasynaptic GABAARs regulates various brain functions. However, the mechanisms that regulate tonic inhibition remain largely unclear. Here, we report distinct actions of GluN2A- and GluN2B-NMDA receptors (NMDARs) on tonic inhibition in hippocampal neurons under basal and high activity conditions. Specifically, overexpression of GluN2B, but not GluN2A, reduces α5-GABAAR surface expression and tonic currents. Additionally, knockout of GluN2A and GluN2B decreases and increases tonic currents, respectively. Mechanistically, GluN2A-NMDARs inhibit and GluN2B-NMDARs promote α5-GABAAR internalization, resulting in increased and decreased surface α5-GABAAR expression, respectively. Furthermore, GluN2A-NMDARs, but not GluN2B-NMDARs, are required for homeostatic potentiation of tonic inhibition induced by prolonged increase of neuronal activity. Last, tonic inhibition decreases during acute seizures, whereas it increases 24 h later, involving GluN2-NMDAR-dependent signaling. Collectively, these data reveal an NMDAR subunit-specific regulation of tonic inhibition in physiological and pathological conditions and provide mechanistic insight into activity-dependent modulation of tonic inhibition.

Evidence 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.

Human stem cell-derived neurons are increasingly considered powerful models in drug discovery and disease modeling, despite limited characterization of their molecular properties. Here, we have conducted a detailed study of the properties of a commercial human induced Pluripotent Stem Cell (iPSC)-derived neuron line, iCell [GABA] neurons, maintained for up to 3 months in vitro. We confirmed that iCell neurons display neurite outgrowth within 24 h of plating and label for the pan-neuronal marker, βIII tubulin within the first week. Our multi-electrode array (MEA) recordings clearly showed neurons generated spontaneous, spike-like activity within 2 days of plating, which peaked at one week, and rapidly decreased over the second week to remain at low levels up to one month. Extracellularly recorded spikes were reversibly inhibited by tetrodotoxin. Patch-clamp experiments showed that iCell neurons generated spontaneous action potentials and expressed voltage-gated Na and K channels with membrane capacitances, resistances and membrane potentials that are consistent with native neurons. Our single neuron recordings revealed that reduced spiking observed in the MEA after the first week results from development of a dominant inhibitory tone from GABAergic neuron circuit maturation. GABA evoked concentration-dependent currents that were inhibited by the convulsants, bicuculline and picrotoxin, and potentiated by the positive allosteric modulators, diazepam, chlordiazepoxide, phenobarbital, allopregnanolone and mefenamic acid, consistent with native neuronal GABAA receptors. We also show that glycine evoked robust concentration-dependent currents that were inhibited by the neurotoxin, strychnine. Glutamate, AMPA, Kainate and NMDA each evoked concentration-dependent currents in iCell neurons that were blocked by their selective antagonists, consistent with the expression of ionotropic glutamate receptors. The NMDA currents required the presence of the co-agonist glycine and were blocked in a highly voltage-dependent manner by Mg2+ consistent with the properties of native neuronal NMDA receptors. Together, our data suggest that such human iPSC-derived neurons may have significant value in drug discovery and development and may eventually largely replace the need for animal tissues in human biomedical research.

Cyclooxygenase (COX) is a heme-containing enzyme that produces prostaglandins (PGs) via a pathway known as the arachidonic acid (AA) cascade. Two isoforms of COX enzyme (COX-1 and COX-2) and splice variant (COX-3) have been described so far. COX-2 is a neuronal enzyme that is intensively produced during activation of the synapse and glutamate (Glu) release. The end product of COX-2 action, prostaglandin E2 (PGE2), regulates Glu level in a retrograde manner. At the same time, the level of Glu, the primary excitatory neurotransmitter, is regulated in the excitatory synapse via Glu receptors, both ionotropic and metabotropic ones. Glu receptors are known modulators of behavior, engaged in cognition and mood. So far, the interaction between ionotropic N-methyl-D-aspartate (NMDA) receptors or metabotropic glutamate (mGluRs) receptors and COX-2 was found. Here, based on literature data and own research, a new mechanism of action of COX-2 in an excitatory synapse will be presented.