Sex-dependence of synaptic depression induced by activation of metabotropic glutamate receptors in rat hippocampus.

The modulation of synaptic efficacy by group I metabotropic glutamate receptors is dysregulated in several neurodevelopmental and neurodegenerative disorders impacting cognitive function. The progression and severity of these and other disorders are affected by biological sex, and differences in metabotropic glutamate receptor signalling have been implicated in this effect. In this study, we have examined whether there are any sex-dependent differences in a form of long-term depression of synaptic responses that is triggered by application of the group I metabotropic glutamate receptor agonist 3,5-dihydroxyphenylglycine (DHPG). We studied DHPG-induced long-term depression at the Schaffer collateral-commissural pathway in area CA1 of hippocampal slices prepared from three separate age groups of Sprague Dawley rats. In both juvenile (2-week-old) and young adult (3-month-old) rats, there were no differences between sexes in the magnitude of long-term depression. However, in older adult (>1-year-old) rats, DHPG-induced long-term depression was greater in males. In contrast, there were no differences between sexes with respect to basal synaptic transmission or paired-pulse facilitation in any age group. The specific enhancement of metabotropic glutamate receptor-dependent long-term depression in older adult males, but not females, reinforces the importance of considering sex as a factor in the study and treatment of brain disorders.

Memantine: Updating a rare success story in pro-cognitive therapeutics.

The great potential for NMDA receptor modulators as druggable targets in neurodegenerative disorders has been met with limited success. Considered one of the rare exceptions, memantine has consistently demonstrated restorative and prophylactic properties in many AD models. In clinical trials memantine slows the decline in cognitive performance associated with AD. Here, we provide an overview of the basic properties including pharmacological targets, toxicology and cellular effects of memantine. Evidence demonstrating reductions in molecular, physiological and behavioural indices of AD-like impairments associated with memantine treatment are also discussed. This represents both an extension and homage to Dr. Chris Parson's considerable contributions to our fundamental understanding of a success story in the AD treatment landscape.

Amyloid-ß1-42 oligomers enhance mGlu5R-dependent synaptic weakening via NMDAR activation and complement C5aR1 signaling.

Synaptic weakening and loss are well-correlated with the pathology of Alzheimer's disease (AD). Oligomeric amyloid beta (oAß) is considered a major synaptotoxic trigger for AD. Recent studies have implicated hyperactivation of the complement cascade as the driving force for loss of synapses caused by oAß. However, the initial synaptic cues that trigger pathological complement activity remain elusive. Here, we examined a form of synaptic long-term depression (LTD) mediated by metabotropic glutamate receptors (mGluRs) that is disrupted in rodent models of AD. Exogenous application of oAß (1-42) to mouse hippocampal slices enhanced the magnitude of mGlu subtype 5 receptor (mGlu5R)-dependent LTD. We found that the enhanced synaptic weakening occurred via both N-methyl-D-aspartate receptors (NMDARs) and complement C5aR1 signaling. Our findings reveal a mechanistic interaction between mGlu5R, NMDARs, and the complement system in aberrant synaptic weakening induced by oAß, which could represent an early trigger of synaptic loss and degeneration in AD.

Time of day influences stress hormone response to ketamine.

Over 50% of depressed patients show hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis. Conventional therapy takes weeks to months to improve symptoms. Ketamine has rapid onset antidepressant effects. Yet its action on HPA axis activity is poorly understood. Here, we measured the corticosterone (CORT) response to ketamine administered at different times of day in the Wistar-Kyoto (WKY) rat. In male rats, blood was collected every 10 min for 28 h using an automated blood sampling system. Ketamine (5/10/25 mg · kg) was infused through a subcutaneous cannula at two time points-during the active and inactive period. CORT levels in blood were measured in response to ketamine using a radioimmunoassay. WKY rats displayed robust circadian secretion of corticosterone and was not overly different to Sprague Dawley rats. Ketamine (all doses) significantly increased CORT response at both infusion times. However, a dose dependent effect and marked increase over baseline was observed when ketamine was administered during the inactive phase. Ketamine has a robust and rapid effect on HPA axis function. The timing of ketamine injection may prove crucial for glucocorticoid-mediated action in depression.

Specific Role for GSK3α in Limiting Long-Term Potentiation in CA1 Pyramidal Neurons of Adult Mouse Hippocampus.

Glycogen synthase kinase-3 (GSK3) mediates phosphorylation of several hundred proteins, and its aberrant activity is associated with an array of prevalent disorders. The two paralogs, GSK3α and GSK3ß, are expressed ubiquitously and fulfill common as well as unique tasks throughout the body. In the CNS, it is established that GSK3 is involved in synaptic plasticity. However, the relative roles of GSK3 paralogs in synaptic plasticity remains controversial. Here, we used hippocampal slices obtained from adult mice to determine the role of each paralog in CA3-CA1 long-term potentiation (LTP) of synaptic transmission, a form of plasticity critically required in learning and memory. Conditional Camk2a Cre-driven neuronal deletion of the Gsk3a gene, but not Gsk3b, resulted in enhanced LTP. There were no changes in basal synaptic function in either of the paralog-specific knockouts, including several measures of presynaptic function. Therefore, GSK3α has a specific role in serving to limit LTP in adult CA1, a postsynaptic function that is not compensated by GSK3ß.

Selective Recruitment of Presynaptic and Postsynaptic Forms of mGluR-LTD.

In area CA1 of the hippocampus, long-term depression (LTD) can be induced by activating group I metabotropic glutamate receptors (mGluRs), with the selective agonist DHPG. There is evidence that mGluR-LTD can be expressed by either a decrease in the probability of neurotransmitter release [P(r)] or by a change in postsynaptic AMPA receptor number. However, what determines the locus of expression is unknown. We investigated the expression mechanisms of mGluR-LTD using either a low (30 µM) or a high (100 µM) concentration of (RS)-DHPG. We found that 30 µM DHPG generated presynaptic LTD that required the co-activation of NMDA receptors, whereas 100 µM DHPG resulted in postsynaptic LTD that was independent of the activation of NMDA receptors. We found that both forms of LTD occur at the same synapses and that these may constitute the population with the lowest basal P(r). Our results reveal an unexpected complexity to mGluR-mediated synaptic plasticity in the hippocampus.

The continually evolving role of NMDA receptors in neurobiology and disease.

40 years ago, Jeff Watkins and Richard (Dick) Evans (Watkins and Evans, 1981) published their review on excitatory amino acids. The review, combined with the tools that they and their colleagues developed, significantly changed the field of neurobiology. This Special Issue focused on NMDA receptors is one of six that commemorate this anniversary. The broadest impact of the review, and the work of this group (Collingridge and Abraham, 2022; Evans and Watkins, 2021; Watkins, 2000), was to establish the three receptor scheme for the excitatory, l-glutamate-gated ion channels named for their selective agonists: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-d-aspartate (NMDA) (Monaghan et al., 1989; Watkins et al., 1990; Watkins, 2000; Hansen et al., 2021; Lodge, 2009; Watkins and Jane, 2006). The contribution of the Evans and Watkins team was perhaps greatest to the study of NMDA receptors - it was essential. We describe here this fundamental contribution and provide an update on NMDARs as the understanding of their function continues to grow more complex.

Synthesis and pharmacological characterisation of arctigenin analogues as antagonists of AMPA and kainate receptors.

(-)-Arctigenin and a series of new analogues have been synthesised and then tested for their potential as AMPA and kainate receptor antagonists of human homomeric GluA1 and GluK2 receptors expressed in HEK293 cells using a Ca2+ influx assay. In general, these compounds showed antagonist activity at both receptors with greater activity evident at AMPARs. Schild analysis indicates that a spirocyclic analogue 6c acts as a non-competitive antagonist. Molecular docking studies in which 6c was docked into the X-ray crystal structure of the GluA2 tetramer suggest that (-)-arctigenin and its analogues bind in the transmembrane domain in a similar manner to the known AMPA receptor non-competitive antagonists GYKI53655 and the antiepileptic drug perampanel. The arctigenin derivatives described herein may serve as novel leads for the development of drugs for the treatment of epilepsy.

Multiple roles of GluN2D-containing NMDA receptors in short-term potentiation and long-term potentiation in mouse hippocampal slices.

The GluN2 subunits of N-methyl-d-aspartate receptors (NMDARs) are key drivers of synaptic plasticity in the brain, where the particular GluN2 composition endows the NMDAR complex with distinct pharmacological and physiological properties. Compared to GluN2A and GluN2B subunits, far less is known about the role of the GluN2D subunit in synaptic plasticity. In this study, we have used a GluN2C/2D selective competitive antagonist, UBP145, in combination with a GluN2D global knockout (GluN2D KO) mouse line to study the contribution of GluN2D-containing NMDARs to short-term potentiation (STP) and long-term potentiation (LTP) in the CA1 region of mouse hippocampal slices. We made several distinct observations: First, GluN2D KO mice have higher levels of LTP compared to wild-type (WT) mice, an effect that was occluded by blockade of GABA receptor-mediated inhibition or by using a strong LTP induction protocol. Second, UBP145 partially inhibited LTP in WT but not GluN2D KO mice. Third, UBP145 inhibited a component of STP, termed STP2, in WT but not GluN2D KO mice. Taken together, these findings suggest an involvement for GluN2D-containing NMDARs in both STP and LTP in mouse hippocampus.

Further evidence that CP-AMPARs are critically involved in synaptic tag and capture at hippocampal CA1 synapses.

The synaptic tag and capture (STC) hypothesis provides an important theoretical basis for understanding the synaptic basis of associative learning. We recently provided pharmacological evidence that calcium-permeable AMPA receptors (CP-AMPARs) are a crucial component of this form of heterosynaptic metaplasticity. Here we have investigated two predictions that arise on the basis of CP-AMPARs serving as a trigger of STC. Firstly, we compared the effects of the order in which we delivered a strong theta burst stimulation (TBS) protocol (75 pulses) and a weak TBS protocol (15 pulses) to two independent inputs. We only observed significant heterosynaptic metaplasticity when the strong TBS preceded the weak TBS. Second, we found that pausing stimulation following either the sTBS or the wTBS for ~20 min largely eliminates the heterosynaptic metaplasticity. These observations are consistent with a process that is triggered by the synaptic insertion of CP-AMPARs and provide a framework for establishing the underlying molecular mechanisms.

PKA drives an increase in AMPA receptor unitary conductance during LTP in the hippocampus.

Long-term potentiation (LTP) at hippocampal CA1 synapses can be expressed by an increase either in the number (N) of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors or in their single channel conductance (γ). Here, we have established how these distinct synaptic processes contribute to the expression of LTP in hippocampal slices obtained from young adult rodents. LTP induced by compressed theta burst stimulation (TBS), with a 10 s inter-episode interval, involves purely an increase in N (LTPN). In contrast, either a spaced TBS, with a 10 min inter-episode interval, or a single TBS, delivered when PKA is activated, results in LTP that is associated with a transient increase in γ (LTPγ), caused by the insertion of calcium-permeable (CP)-AMPA receptors. Activation of CaMKII is necessary and sufficient for LTPN whilst PKA is additionally required for LTPγ. Thus, two mechanistically distinct forms of LTP co-exist at these synapses.

The GSK-3 Inhibitor CT99021 Enhances the Acquisition of Spatial Learning and the Accuracy of Spatial Memory.

Glycogen synthase kinase 3 (GSK-3) is a Ser/Thr protein kinase that regulates many cellular processes, including synaptic plasticity. Previously, we reported that inhibition of GSK-3 prevents the induction of one of the major forms of synaptic plasticity, N-methyl-D-aspartate receptor (NMDAR)-dependent long-term depression (LTD), in hippocampal slices. In the present study, we have investigated the effects of inhibiting GSK-3 on learning and memory in healthy naïve animals. Systemic administration of a highly selective GSK-3 inhibitor, CT99021, reversibly blocked NMDAR-dependent LTD in the CA1 region of the hippocampus in anesthetized adult mice. In behavioral tasks, CT99021 had no effect on locomotor activity, anxiety, hippocampus-dependent contextual fear memory, and hippocampus-dependent reversal learning. However, CT99021 facilitated the rate of learning in the Morris water maze (MWM) and T-maze and enhanced the accuracy of long-term spatial memory in the MWM. These findings suggest that GSK-3 regulates the accuracy of spatial memory acquisition and recall.

GSK-3ß regulates the synaptic expression of NMDA receptors via phosphorylation of phosphatidylinositol 4 kinase type IIα.

Deregulation of GSK-3ß is strongly implicated in a variety of serious brain conditions, such as Alzheimer disease, bipolar disorder and schizophrenia. To understand how GSK-3ß becomes dysregulated in these conditions, it is important to understand its physiological functions in the central nervous system. In this context, GSK-3ß plays a role in the induction of NMDA receptor-dependent long-term depression (LTD) and several substrates for GSK-3ß have been identified in this form of synaptic plasticity, including KLC-2, PSD-95 and tau. Stabilization of NMDA receptors at synapses has also been shown to involve GSK-3ß, but the substrates involved are currently unknown. Recent work has identified phosphatidylinositol 4 kinase type IIα (PI4KIIα) as a neuronal GSK-3ß substrate that can potentially regulate the surface expression of AMPA receptors. In the present study, we investigated the synaptic role of PI4KIIα in organotypic rat hippocampal slices. We found that knockdown of PI4KIIα has no effect on synaptic AMPA receptor-mediated synaptic transmission but substantially reduces NMDA receptor-mediated synaptic transmission. Furthermore, the ability of the selective GSK-3 inhibitor, CT99021, to reduce the amplitude of NMDA receptor-mediated currents was occluded in shRNA-PI4KIIα transfected neurons. The effects of knocking down PI4KIIα were fully rescued by a shRNA-resistant wild-type construct, but not by a mutant construct that cannot be phosphorylated by GSK-3ß. These data suggest that GSK-3ß phosphorylates PI4KIIα to stabilize NMDA receptors at the synapse.

(2S,6S)- and (2R,6R)-hydroxynorketamine inhibit the induction of NMDA receptor-dependent LTP at hippocampal CA1 synapses in mice.

The ketamine metabolite (2R,6R)-hydroxynorketamine has been proposed to have rapid and persistent antidepressant actions in rodents, but its mechanism of action is controversial. We have compared the ability of (R,S)-ketamine with the (2S,6S)- and (2R,6R)-isomers of hydroxynorketamine to affect the induction of N-methyl-d-aspartate receptor-dependent long-term potentiation in the mouse hippocampus. Following pre-incubation of these compounds, we observed a concentration-dependent (1-10 µM) inhibition of long-term potentiation by ketamine and a similar effect of (2S,6S)-hydroxynorketamine. At a concentration of 10 µM, (2R,6R)-hydroxynorketamine also inhibited the induction of long-term potentiation. These findings raise the possibility that inhibition of N-methyl-d-aspartate receptor-mediated synaptic plasticity is a site of action of the hydroxynorketamine metabolites with respect to their rapid and long-lasting antidepressant-like effects.

Optogenetic Manipulation of Postsynaptic cAMP Using a Novel Transgenic Mouse Line Enables Synaptic Plasticity and Enhances Depolarization Following Tetanic Stimulation in the Hippocampal Dentate Gyrus.

cAMP is a positive regulator tightly involved in certain types of synaptic plasticity and related memory functions. However, its spatiotemporal roles at the synaptic and neural circuit levels remain elusive. Using a combination of a cAMP optogenetics approach and voltage-sensitive dye (VSD) imaging with electrophysiological recording, we define a novel capacity of postsynaptic cAMP in enabling dentate gyrus long-term potentiation (LTP) and depolarization in acutely prepared murine hippocampal slices. To manipulate cAMP levels at medial perforant path to granule neuron (MPP-DG) synapses by light, we generated transgenic (Tg) mice expressing photoactivatable adenylyl cyclase (PAC) in DG granule neurons. Using these Tg(CMV-Camk2a-RFP/bPAC)3Koka mice, we recorded field excitatory postsynaptic potentials (fEPSPs) from MPP-DG synapses and found that photoactivation of PAC during tetanic stimulation enabled synaptic potentiation that persisted for at least 30 min. This form of LTP was induced without the need for GABA receptor blockade that is typically required for inducing DG plasticity. The paired-pulse ratio (PPR) remained unchanged, indicating the cAMP-dependent LTP was likely postsynaptic. By employing fast fluorescent voltage-sensitive dye (VSD: di-4-ANEPPS) and fluorescence imaging, we found that photoactivation of the PAC actuator enhanced the intensity and extent of dentate gyrus depolarization triggered following tetanic stimulation. These results demonstrate that the elevation of cAMP in granule neurons is capable of rapidly enhancing synaptic strength and neuronal depolarization. The powerful actions of cAMP are consistent with this second messenger having a critical role in the regulation of synaptic function.

Illuminating Relationships Between the Pre- and Post-synapse.

Excitatory synapses in the mammalian cortex are highly diverse, both in terms of their structure and function. However, relationships between synaptic features indicate they are highly coordinated entities. Imaging techniques, that enable physiology at the resolution of individual synapses to be investigated, have allowed the presynaptic activity level of the synapse to be related to postsynaptic function. This approach has revealed that neuronal activity induces the pre- and post-synapse to be functionally correlated and that subsets of synapses are more susceptible to certain forms of synaptic plasticity. As presynaptic function is often examined in isolation from postsynaptic properties, the effect it has on the post-synapse is not fully understood. However, since postsynaptic receptors at excitatory synapses respond to release of glutamate, it follows that they may be differentially regulated depending on the frequency of its release. Therefore, examining postsynaptic properties in the context of presynaptic function may be a useful way to approach a broad range of questions on synaptic physiology. In this review, we focus on how optophysiology tools have been utilized to study relationships between the pre- and the post-synapse. Multiple imaging techniques have revealed correlations in synaptic properties from the submicron to the dendritic level. Optical tools together with advanced imaging techniques are ideally suited to illuminate this area further, due to the spatial resolution and control they allow.

Autism-Misregulated eIF4G Microexons Control Synaptic Translation and Higher Order Cognitive Functions.

Microexons represent the most highly conserved class of alternative splicing, yet their functions are poorly understood. Here, we focus on closely related neuronal microexons overlapping prion-like domains in the translation initiation factors, eIF4G1 and eIF4G3, the splicing of which is activity dependent and frequently disrupted in autism. CRISPR-Cas9 deletion of these microexons selectively upregulates synaptic proteins that control neuronal activity and plasticity and further triggers a gene expression program mirroring that of activated neurons. Mice lacking the Eif4g1 microexon display social behavior, learning, and memory deficits, accompanied by altered hippocampal synaptic plasticity. We provide evidence that the eIF4G microexons function as a translational brake by causing ribosome stalling, through their propensity to promote the coalescence of cytoplasmic granule components associated with translation repression, including the fragile X mental retardation protein FMRP. The results thus reveal an autism-disrupted mechanism by which alternative splicing specializes neuronal translation to control higher order cognitive functioning.

Structural basis of subtype-selective competitive antagonism for GluN2C/2D-containing NMDA receptors.

N-Methyl-D-aspartate receptors (NMDARs) play critical roles in the central nervous system. Their heterotetrameric composition generates subtypes with distinct functional properties and spatio-temporal distribution in the brain, raising the possibility for subtype-specific targeting by pharmacological means for treatment of neurological diseases. While specific compounds for GluN2A and GluN2B-containing NMDARs are well established, those that target GluN2C and GluN2D are currently underdeveloped with low potency and uncharacterized binding modes. Here, using electrophysiology and X-ray crystallography, we show that UBP791 ((2S*,3R*)-1-(7-(2-carboxyethyl)phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid) inhibits GluN2C/2D with 40-fold selectivity over GluN2A-containing receptors, and that a methionine and a lysine residue in the ligand binding pocket (GluN2D-Met763/Lys766, GluN2C-Met736/Lys739) are the critical molecular elements for the subtype-specific binding. These findings led to development of UBP1700 ((2S*,3R*)-1-(7-(2-carboxyvinyl)phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid) which shows over 50-fold GluN2C/2D-selectivity over GluN2A with potencies in the low nanomolar range. Our study shows that the L-glutamate binding site can be targeted for GluN2C/2D-specific inhibition.

Mice lacking neuronal calcium sensor-1 show social and cognitive deficits.

Neuronal calcium sensor-1 or Frequenin is a calcium sensor widely expressed in the nervous system, with roles in neurotransmission, neurite outgrowth, synaptic plasticity, learning, and motivated behaviours. Neuronal calcium sensor-1 has been implicated in neuropsychiatric disorders including autism spectrum disorder, schizophrenia, and bipolar disorder. However, the role of neuronal calcium sensor-1 in behavioural phenotypes and brain changes relevant to autism spectrum disorder have not been evaluated. We show that neuronal calcium sensor-1 deletion in the mouse leads to a mild deficit in social approach and impaired displaced object recognition without affecting social interactions, behavioural flexibility, spatial reference memory, anxiety-like behaviour, or sensorimotor gating. Morphologically, neuronal calcium sensor-1 deletion leads to increased dendritic arbour complexity in the frontal cortex. At the level of hippocampal synaptic plasticity, neuronal calcium sensor-1 deletion leads to a reduction in long-term potentiation in the dentate gyrus, but not area Cornu Ammonis 1. Metabotropic glutamate receptor-induced long-term depression was unaffected in both dentate and Cornu Ammonis 1. These studies identify roles for neuronal calcium sensor-1 in specific subregions of the brain including a phenotype relevant to neuropsychiatric disorders.