Incremental induction of NMDAR-STP and NMDAR-LTP in the CA1 area of ventral hippocampal slices relies on graded activation of discrete NMDA receptors.

N-methyl-d-aspartate receptor (NMDAR)-dependent short- and long-term types of potentiation (STP and LTP, respectively) are frequently studied in the CA1 area of dorsal hippocampal slices (DHS). Far less is known about the NMDAR dependence of STP and LTP in ventral hippocampal slices (VHS), where both types of potentiation are smaller in magnitude than in the DHS. Here, we first briefly review our knowledge about the NMDAR dependence of STP and LTP and some other forms of synaptic plasticity. We then show in new experiments that the decay of NMDAR-STP in VHS, similar to dorsal hippocampal NMDAR-STP, is not time- but activity-dependent. We also demonstrate that the induction of submaximal levels of NMDAR-STP and NMDAR-LTP in VHS differs from the induction of saturated levels of plasticity in terms of their sensitivity to subunit-preferring NMDAR antagonists. These data suggest that activation of distinct NMDAR subtypes in a population of neurons results in an incremental increase in the induction of different phases of potentiation with changing sensitivity to pharmacological agents. Differences in pharmacological sensitivity, which arise due to differences in the levels of agonist-evoked biological response, might explain the disparity of the results concerning NMDAR subunit involvement in the induction of NMDAR-dependent plasticity.This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Adiponectin rescues synaptic plasticity in the dentate gyrus of a mouse model of Fragile X Syndrome.

Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability and is the leading known single-gene cause of autism spectrum disorder. Patients with FXS display varied behavioural deficits that include mild to severe cognitive impairments in addition to mood disorders. Currently, there is no cure for this condition; however, there is an emerging focus on therapies that inhibit mechanistic target of rapamycin (mTOR)-dependent protein synthesis owing to the clinical effectiveness of metformin for alleviating some behavioural symptoms in FXS. Adiponectin (APN) is a neurohormone that is released by adipocytes and provides an alternative means to inhibit mTOR activation in the brain. In these studies, we show that Fmr1 knockout mice, like patients with FXS, show reduced levels of circulating APN and that both long-term potentiation (LTP) and long-term depression (LTD) in the dentate gyrus (DG) are impaired. Brief (20 min) incubation of hippocampal slices in APN (50 nM) was able to rescue both LTP and LTD in the DG and increased both the surface expression and phosphorylation of GluA1 receptors. These results provide evidence for reduced APN levels in FXS playing a role in decreasing bidirectional synaptic plasticity and show that therapies which enhance APN levels may have therapeutic potential for this and related conditions.This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Cage effects on synaptic plasticity and its modulation in a mouse model of fragile X syndrome.

Fragile X syndrome (FXS) is characterized by impairments in executive function including different types of learning and memory. Long-term potentiation (LTP), thought to underlie the formation of memories, has been studied in the Fmr1 mouse model of FXS. However, there have been many discrepancies in the literature with inconsistent use of littermate and non-littermate Fmr1 knockout (KO) and wild-type (WT) control mice. Here, the influence of the breeding strategy (cage effect) on short-term potentiation (STP), LTP, contextual fear conditioning (CFC), expression of N-methyl-d-aspartate receptor (NMDAR) subunits and the modulation of NMDARs, were examined. The largest deficits in STP, LTP and CFC were found in KO mice compared with non-littermate WT. However, the expression of NMDAR subunits was unchanged in this comparison. Rather, NMDAR subunit (GluN1, 2A, 2B) expression was sensitive to the cage effect, with decreased expression in both WT and KO littermates compared with non-littermates. Interestingly, an NMDAR-positive allosteric modulator, UBP714, was only effective in potentiating the induction of LTP in non-littermate KO mice and not the littermate KO mice. These results suggest that commonly studied phenotypes in Fmr1 KOs are sensitive to the cage effect and therefore the breeding strategy may contribute to discrepancies in the literature.This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

The role of calcium stores in long-term potentiation and synaptic tagging and capture in mouse hippocampus.

The roles of Ca2+-induced calcium release in synaptic plasticity and metaplasticity are poorly understood. The present study has addressed the role of intracellular Ca2+ stores in long-term potentiation (LTP) and a form of heterosynaptic metaplasticity known as synaptic tagging and capture (STC) at CA1 synapses in mouse hippocampal slices. The effects of two compounds, ryanodine and cyclopiazonic acid (CPA), were examined on LTP induced by three distinct induction protocols: weak (w), compressed (c) and spaced (s) theta-burst stimulation (TBS). These compounds did not significantly affect LTP induced by the wTBS (one episode of TBS; 25 stimuli) or cTBS (three such episodes with a 10 s inter-episode interval (IEI); 75 stimuli) but substantially inhibited LTP induced by a sTBS (10 min IEI; 75 stimuli). Ryanodine and CPA also prevented a small heterosynaptic potentiation that was observed with the sTBS protocol. Interestingly, these compounds also prevented STC when present during either the sTBS or the subsequent wTBS, applied to an independent input. All of these effects of ryanodine and CPA were similar to that of a calcium-permeable AMPA receptor blocker. In conclusion, Ca2+ stores provide one way in which signals are propagated between synaptic inputs and, by virtue of their role in STC, may be involved in associative long-term memories. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

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.

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.

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.

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.

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.

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.

(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.