Src Family Kinases Inhibition Ameliorates Hypoxic-Ischemic Brain Injury in Immature Rats

Hypoxic-ischemic (HI) injury is one of the initial factors contributing to neonatal brain injury. Src family kinases (SFKs) are considered to act as molecular hubs for N-methyl-d-aspartate receptor (NMDAR) regulation and participate in the HI injury process. The objectives of this study were to evaluate the levels of phospho-Src (p-Src), the relationship between NMDARs and SFKs, and the effects of SFK inhibition on an immature rat HI brain injury model. The model was induced in 3-day-old Sprague–Dawley rats using the Rice-Vannucci model operation. The level of p-Src was evaluated using Western blotting. The association of NMDARs with SFKs was detected using Western blotting and coimmunoprecipitation. After intraperitoneal injection of 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyrimidine (PP2), an SFK-selective inhibitor, neuropathological changes were observed by performing H&E and immunofluorescence staining, and the neurological functions were assessed using the following behavioral tests: modified neurological severity score, open field test, and Morris water maze test. The levels of p-Src first decreased at 0 h after injury, increased at 2 h after injury, and continuously decreased from 6 h to 3 days. Along with the increased p-Src levels observed at 2 h after injury, the phosphorylation of NMDAR subunit NR2B at tyrosine 1472 was increased. Following the administration of PP2, the increased p-Src and NMDAR-2B levels detected at 2 h after injury were decreased, and tissue injury and myelin basic protein expression were improved at 7 days after injury. The PP2 intervention improved the performance of injured rats on behavioral tests. In conclusion, we determined the patterns of p-Src expression after HI brain injury in immature rats and showed a relationship with the activated NMDA receptor. The inhibition of p-Src ameliorates neuropathological changes and damages neurological functions induced by HI injury.

NMDARs Drive the Expression of Neuropsychiatric Disorder Risk Genes Within GABAergic Interneuron Subtypes in the Juvenile Brain

Medial ganglionic eminence (MGE)-derived parvalbumin (PV)+, somatostatin (SST)+and Neurogliaform (NGFC)-type cortical and hippocampal interneurons, have distinct molecular, anatomical, and physiological properties. However, the molecular mechanisms regulating their maturation remain poorly understood. Here, via single-cell transcriptomics, we show that the obligate NMDA-type glutamate receptor (NMDAR) subunit gene Grin1 mediates transcriptional regulation of gene expression in specific subtypes of MGE-derived interneurons, leading to altered subtype abundances. Notably, MGE-specific early developmental Grin1 loss results in a broad downregulation of diverse transcriptional, synaptogenic and membrane excitability regulatory programs in the juvenile brain. These widespread gene expression abnormalities mirror aberrations that are typically associated with neurodevelopmental disorders. Our study hence provides a road map for the systematic examination of NMDAR signaling in interneuron subtypes, revealing potential MGE-specific genetic targets that could instruct future therapies of psychiatric disorders.

NMDARs Containing NR2B Subunit Do Not Contribute to the LTP Form of Hippocampal Plasticity: In Vivo Pharmacological Evidence in Rats

Synaptic plasticity is the key to synaptic health, and aberrant synaptic plasticity, which in turn impairs the functioning of large-scale brain networks, has been associated with neurodegenerative and psychiatric disorders. The best known and most studied form of activity-dependent synaptic plasticity remains long-term potentiation (LTP), which is controlled by glutamatergic N-methyl-d-aspartate) receptors (NMDAR) and considered to be a mechanism crucial for cellular learning and memory. Over the past two decades, discrepancies have arisen in the literature regarding the contribution of NMDAR subunit assemblies in the direction of NMDAR-dependent synaptic plasticity. Here, the nonspecific NMDAR antagonist ketamine (5 and 10 mg/kg), and the selective NR2B antagonists CP-101606 and Ro 25-6981 (6 and 10 mg/kg), were administered intraperitoneally in Sprague Dawley rats to disentangle the contribution of NR2B subunit in the LTP induced at the Schaffer Collateral-CA1 synapse using the theta burst stimulation protocol (TBS). Ketamine reduced, while CP-101606 and Ro 25-6981 did not alter the LTP response. The administration of CP-101606 before TBS did not influence the effects of ketamine when administered half an hour after tetanization, suggesting a limited contribution of the NR2B subunit in the action of ketamine. This work confirms the role of NMDAR in the LTP form of synaptic plasticity, whereas specific blockade of the NR2B subunit was not sufficient to modify hippocampal LTP. Pharmacokinetics at the doses used may have contributed to the lack of effects with specific antagonists. The findings refute the role of the NR2B subunit in the plasticity mechanism of ketamine in the model.

Young DAPK1 knockout mice have altered pre-synaptic function

The death associated protein kinase 1 (DAPK1) has recently been shown to have a physiological function in long-term depression (LTD) of glutamatergic synapses: Acute inhibition of DAPK1 blocked the LTD that is normally seen at the hippocampal CA1 synapse in young mice, and a pharmacogenetic combination approach showed that this specifically required DAPK1-mediated suppression of post-synaptic CaMKII binding to the NMDA-type glutamate receptor (NMDAR) subunit GluN2B during LTD stimuli. Surprisingly, we found here that genetic deletion of DAPK1 (in DAPK1-/- mice) did not reduce LTD. Paired pulse facilitation experiments indicated a pre-synaptic compensation mechanism: in contrast to wild type mice, LTD stimuli in DAPK1-/- mice decreased pre-synaptic release probability. Basal synaptic strength was normal in young DAPK1-/- mice, but basal glutamate release probability was reduced, an effect that normalized with maturation.

Chronic Blockade of NMDAR Subunit 2A in the Hypothalamic Paraventricular Nucleus Alleviates Hypertension through Suppression of MEK/ERK/CREB Pathway

Background N-methyl-D-aspartate Receptor (NMDAR) in the hypothalamic paraventricular nucleus (PVN) plays critical roles in regulating sympathetic outflow. Studies showed that acute application of the antagonists of NMDAR or its subunits would reduce sympathetic nerve discharges. However, little is known about the effect of long-term management of NMDAR in hypertensive animals. Methods PEAQX, the specific antagonist of NMDAR subunit 2A (GluN2A) was injected into both side of the PVN of two-kidney, one clip (2K1C) renal hypertensive rats and control (normotensive rats) for three weeks. Results Three weeks of PEAQX infusion significantly reduced the blood pressure of the 2K1C rats. It managed to resume the balance between excitatory and inhibitory neural transmitters, reduce the level of pro-inflammatory cytokines and reactive oxygen species in the PVN, and reduce the level of norepinephrine in plasma of the 2K1C rats. PEAQX administration also largely reduced the transcription and translation levels of GluN2A and changed the expression levels of NMDAR subunit 1 and 2B (GluN1 and GluN2B). In addition, NMDAR was known to function through activating the extracellular regulated protein kinases (ERK) or phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathways. In our study we found that in the PVN of 2K1C rats treated with PEAQX, the phosphorylation levels of mitogen-activated protein kinase kinase (MEK), ERK1/2 and cAMP-response element binding protein (CREB) significantly reduced, while the phosphorylation level of PI3K didn’t change significantly. Conclusions Chronic blockade of GluN2A alleviates hypertension through suppression of MEK/ERK/CREB pathway.

CaMKII activation triggers persistent formation and segregation of postsynaptic liquid phase

Transient information input to brain leads to persistent changes in synaptic circuit, thereby forming memory engrams. Synapse undergoes coordinated functional and structural changes during this process but how such changes are achieved by its component molecules still largely remain enigmatic. We found that activated CaMKII, the central player of synaptic plasticity, undergoes liquid-liquid phase separation (LLPS) with NMDAR subunit GluN2B. Due to CaMKII autophosphorylation, the condensate stably persists even after Ca2+ is removed. The selective binding of activated CaMKII with GluN2B co-segregates AMPAR/neuroligin (NLGN) into a phase-in-phase assembly. Because postsynaptic NLGN clusters presynaptic neurexin and other active zone proteins thereby increasing the release probability of synaptic vesicles, this ensures efficient synaptic transmission. In this way, Ca2+-induced and persistent formation of LLPS by CaMKII serves as molecular basis of memory by functioning as an activity-dependent crosslinker for postsynaptic proteins and segregating trans-synaptic nanocolumns.

The pathogenic S688Y mutation in the ligand-binding domain of the GluN1 subunit regulates the properties of NMDA receptors

Although numerous pathogenic mutations have been identified in various subunits of N-methyl-D-aspartate receptors (NMDARs), ionotropic glutamate receptors that are central to glutamatergic neurotransmission, the functional effects of these mutations are often unknown. Here, we combined in silico modelling with microscopy, biochemistry, and electrophysiology in cultured HEK293 cells and hippocampal neurons to examine how the pathogenic missense mutation S688Y in the GluN1 NMDAR subunit affects receptor function and trafficking. We found that the S688Y mutation significantly increases the EC50 of both glycine and d-serine in GluN1/GluN2A and GluN1/GluN2B receptors, and significantly slows desensitisation of GluN1/GluN3A receptors. Moreover, the S688Y mutation reduces the surface expression of GluN3A-containing NMDARs in cultured hippocampal neurons, but does not affect the trafficking of GluN2-containing receptors. Finally, we found that the S688Y mutation reduces Ca2+ influx through NMDARs and reduces NMDA-induced excitotoxicity in cultured hippocampal neurons. These findings provide key insights into the molecular mechanisms that underlie the regulation of NMDAR subtypes containing pathogenic mutations.

Common synaptic phenotypes arising from diverse mutations in the human NMDA receptor subunit GluN2A

Dominant mutations in the human gene GRIN2A, encoding NMDA receptor (NMDAR) subunit GluN2A, make a significant and growing contribution to the catalogue of published single-gene epilepsies. Understanding the disease mechanism in these epilepsy patients is complicated by the surprising diversity of effects that the mutations have on NMDARs. We have examined the cell-autonomous impact of 5 severe GluN2A mutations by measuring NMDAR-mediated synaptic currents (NMDAR-EPSCs) in CA1 pyramidal neurons following rescue with human GluN2A mutants. Surprisingly, prolonged NMDAR-EPSC current decay and smaller peak amplitudes were common features of both gain- and loss-of-function mutants despite there being drastic differences between their effects on receptor function and enrichment at synapses. Modelling of NMDARs with mutant properties in CA1 neurons indicates that mutant NMDARs may contribute to broadening of depolarizations during bursts of high-frequency synaptic activity. Overall, the implication is that similar therapeutic approaches may be more widely applicable to patients with GRIN2A-related disorders irrespective of their molecular defect.

Ketamine and Ro 25-6981 Reverse Behavioral Abnormalities in Rats Subjected to Dietary Zinc Restriction

Clinical and preclinical studies indicate that zinc (Zn) is an essential factor in the development and treatment of major depressive disorder (MDD). Conventional monoamine-based antidepressants mobilize zinc in the blood and brain of depressed patients as well as rodents. N-methyl-D-aspartate acid receptor (NMDAR) antagonists exhibit antidepressant-like activity. However, not much is known about the antidepressant efficacy of NMDAR antagonists in zinc-deficient (ZnD) animals. We evaluated the antidepressant-like activity of two NMDAR antagonists (ketamine; global NMDAR antagonist and Ro 25-6981 (Ro); selective antagonist of the GluN2B NMDAR subunit) in ZnD rats using the forced swim test (FST) and sucrose intake test (SIT). A single dose of either Ro 25-6981 or ketamine normalized depressive-like behaviors in ZnD rats; however, Ro was effective in both tests, while ketamine was only effective in the FST. Additionally, we investigated the mechanism of antidepressant action of Ro at the molecular (analysis of protein expression by Western blotting) and anatomical (density of dendritic spines by Golgi Cox-staining) levels. ZnD rats exhibited decreased phosphorylation of the p70S6K protein, and enhanced density of dendritic spines in the prefrontal cortex (PFC) compared to control rats. The antidepressant-like activity of Ro was associated with the increased phosphorylation of p70S6K and ERK in the PFC. In summary, single doses of the NMDAR antagonists ketamine and Ro exhibited antidepressant-like activity in the ZnD animal model of depression. Animals were only deprived of Zn for 4 weeks and the biochemical effects of Zn deprivation and Ro were investigated in the PFC and hippocampus. The shorter duration of dietary Zn restriction may be a limitation of the study. However, future studies with longer durations of dietary Zn restriction, as well as the investigation of multiple brain structures, are encouraged as a supplement to this study.