Heterosynaptic NMDA Receptor Plasticity in Hippocampal Dentate Granule Cells

The dentate gyrus is a key relay station that controls information transfer from the entorhinal cortex to the hippocampus proper. This process heavily relies on dendritic integration by dentate granule cells (GCs) of excitatory synaptic inputs from medial and lateral entorhinal cortex via medial and lateral perforant paths (MPP and LPP, respectively). N-methyl-D-aspartate receptors (NMDARs) can contribute significantly to the integrative properties of neurons. While early studies reported that excitatory inputs from entorhinal cortex onto GCs can undergo activity-dependent long-term plasticity of NMDAR-mediated transmission, the input-specificity of this plasticity along the dendritic axis remains unknown. Here, we examined the NMDAR plasticity rules at MPP-GC and LPP-GC synapses using physiologically relevant patterns of stimulation in acute rat hippocampal slices. We found that MPP-GC, but not LPP-GC synapses, expressed homosynaptic NMDAR-LTP. In addition, induction of NMDAR-LTP at MPP-GC synapses heterosynaptically potentiated distal LPP-GC NMDAR plasticity. The same stimulation protocol induced homosynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-LTP at MPP-GC but heterosynaptic AMPAR-LTD at distal LPP synapses, demonstrating that NMDAR and AMPAR are governed by different plasticity rules. Remarkably, heterosynaptic but not homosynaptic NMDAR-LTP required Ca2+ release from intracellular, ryanodine-dependent Ca2+ stores. Lastly, the induction and maintenance of both homo- and heterosynaptic NMDAR-LTP were blocked by GluN2D antagonism, suggesting the recruitment of GluN2D-containing receptors to the synapse. Our findings uncover a mechanism by which distinct inputs to the dentate gyrus may interact functionally and contribute to hippocampal-dependent memory formation.

GABAA Receptors in Astrocytes Are Targets for Commonly Used Intravenous and Inhalational General Anesthetic Drugs

Background: Perioperative neurocognitive disorders (PNDs) occur commonly in older patients after anesthesia and surgery. Treating astrocytes with general anesthetic drugs stimulates the release of soluble factors that increase the cell-surface expression and function of GABAA receptors in neurons. Such crosstalk may contribute to PNDs; however, the receptor targets in astrocytes for anesthetic drugs have not been identified. GABAA receptors, which are the major targets of general anesthetic drugs in neurons, are also expressed in astrocytes, raising the possibility that these drugs act on GABAA receptors in astrocytes to trigger the release of soluble factors. To date, no study has directly examined the sensitivity of GABAA receptors in astrocytes to general anesthetic drugs that are frequently used in clinical practice. Thus, the goal of this study was to determine whether the function of GABAA receptors in astrocytes was modulated by the intravenous anesthetic etomidate and the inhaled anesthetic sevoflurane.Methods: Whole-cell voltage-clamp recordings were performed in astrocytes in the stratum radiatum of the CA1 region of hippocampal slices isolated from C57BL/6 male mice. Astrocytes were identified by their morphologic and electrophysiologic properties. Focal puff application of GABA (300 μM) was applied with a Picospritzer system to evoke GABA responses. Currents were studied before and during the application of the non-competitive GABAA receptor antagonist picrotoxin (0.5 mM), or etomidate (100 μM) or sevoflurane (532 μM).Results: GABA consistently evoked inward currents that were inhibited by picrotoxin. Etomidate increased the amplitude of the peak current by 35.0 ± 24.4% and prolonged the decay time by 27.2 ± 24.3% (n = 7, P < 0.05). Sevoflurane prolonged current decay by 28.3 ± 23.1% (n = 7, P < 0.05) but did not alter the peak amplitude. Etomidate and sevoflurane increased charge transfer (area) by 71.2 ± 45.9% and 51.8 ± 48.9% (n = 7, P < 0.05), respectively.Conclusion: The function of astrocytic GABAA receptors in the hippocampus was increased by etomidate and sevoflurane. Future studies will determine whether these general anesthetic drugs act on astrocytic GABAA receptors to stimulate the release of soluble factors that may contribute to PNDs.

Effects of Delivering Guanidinoacetic Acid or Its Prodrug to the Neural Tissue: Possible Relevance for Creatine Transporter Deficiency

The creatine precursor guanidinoacetate (GAA) was used as a dietary supplement in humans with no adverse events. Nevertheless, it has been suggested that GAA is epileptogenic or toxic to the nervous system. However, increased GAA content in rodents affected by guanidinoacetate methyltransferase (GAMT) deficiency might be responsible for their spared muscle function. Given these conflicting data, and lacking experimental evidence, we investigated whether GAA affected synaptic transmission in brain hippocampal slices. Incubation with 11.5 μM GAA (the highest concentration in the cerebrospinal fluid of GAMT-deficient patients) did not change the postsynaptic compound action potential. Even 1 or 2 mM had no effect, while 4 mM caused a reversible decrease in the potential. Guanidinoacetate increased creatine and phosphocreatine, but not after blocking the creatine transporter (also used by GAA). In an attempt to allow the brain delivery of GAA when there was a creatine transporter deficiency, we synthesized diacetyl guanidinoacetic acid ethyl ester (diacetyl-GAAE), a lipophilic derivative. In brain slices, 0.1 mM did not cause electrophysiological changes and improved tissue viability after blockage of the creatine transporter. However, diacetyl-GAAE did not increase creatine nor phosphocreatine in brain slices after blockage of the creatine transporter. We conclude that: (1) upon acute administration, GAA is neither epileptogenic nor neurotoxic; (2) Diacetyl-GAAE improves tissue viability after blockage of the creatine transporter but not through an increase in creatine or phosphocreatine. Diacetyl-GAAE might give rise to a GAA–phosphoGAA system that vicariates the missing creatine–phosphocreatine system. Our in vitro data show that GAA supplementation may be safe in the short term, and that a lipophilic GAA prodrug may be useful in creatine transporter deficiency.

DM1 Transgenic Mice Exhibit Abnormal Neurotransmitter Homeostasis and Synaptic Plasticity in Association with RNA Foci and Mis-Splicing in the Hippocampus

Myotonic dystrophy type 1 (DM1) is a severe neuromuscular disease mediated by a toxic gain of function of mutant RNAs. The neuropsychological manifestations affect multiple domains of cognition and behavior, but their etiology remains elusive. Transgenic DMSXL mice carry the DM1 mutation, show behavioral abnormalities, and express low levels of GLT1, a critical regulator of glutamate concentration in the synaptic cleft. However, the impact of glutamate homeostasis on neurotransmission in DM1 remains unknown. We confirmed reduced glutamate uptake in the DMSXL hippocampus. Patch clamp recordings in hippocampal slices revealed increased amplitude of tonic glutamate currents in DMSXL CA1 pyramidal neurons and DG granule cells, likely mediated by higher levels of ambient glutamate. Unexpectedly, extracellular GABA levels and tonic current were also elevated in DMSXL mice. Finally, we found evidence of synaptic dysfunction in DMSXL mice, suggestive of abnormal short-term plasticity, illustrated by an altered LTP time course in DG and in CA1. Synaptic dysfunction was accompanied by RNA foci accumulation in localized areas of the hippocampus and by the mis-splicing of candidate genes with relevant functions in neurotransmission. Molecular and functional changes triggered by toxic RNA may induce synaptic abnormalities in restricted brain areas that favor neuronal dysfunction.

BICS01 Mediates Reversible Anti-seizure Effects in Brain Slice Models of Epilepsy

Drug-resistant epilepsy remains a significant clinical and societal burden, with one third of people with epilepsy continuing to experience seizures despite the availability of around 30 anti-seizure drugs (ASDs). Further, ASDs often have substantial adverse effects, including impacts on learning and memory. Therefore, it is important to develop new ASDs, which may be more potent or better tolerated. Here, we report the preliminary preclinical evaluation of BICS01, a synthetic product based on a natural compound, as a potential ASD. To model seizure-like activity in vitro, we prepared hippocampal slices from adult male Sprague Dawley rats, and elicited epileptiform bursting using high extracellular potassium. BICS01 (200 μM) rapidly and reversibly reduced the frequency of epileptiform bursting but did not change broad measures of network excitability or affect short-term synaptic facilitation. BICS01 was well tolerated following systemic injection at up to 1,000 mg/kg. However, we did not observe any protective effect of systemic BICS01 injection against acute seizures evoked by pentylenetetrazol. These results indicate that BICS01 is able to acutely reduce epileptiform activity in hippocampal networks. Further preclinical development studies to enhance pharmacokinetics and accumulation in the brain, as well as studies to understand the mechanism of action, are now required.

Cordycepin Ameliorates Synaptic Dysfunction and Dendrite Morphology Damage of Hippocampal CA1 via A1R in Cerebral Ischemia

Cordycepin exerted significant neuroprotective effects and protected against cerebral ischemic damage. Learning and memory impairments after cerebral ischemia are common. Cordycepin has been proved to improve memory impairments induced by cerebral ischemia, but its underlying mechanism has not been revealed yet. The plasticity of synaptic structure and function is considered to be one of the neural mechanisms of learning and memory. Therefore, we investigated how cordycepin benefits dendritic morphology and synaptic transmission after cerebral ischemia and traced the related molecular mechanisms. The effects of cordycepin on the protection against ischemia were studied by using global cerebral ischemia (GCI) and oxygen-glucose deprivation (OGD) models. Behavioral long-term potentiation (LTP) and synaptic transmission were observed with electrophysiological recordings. The dendritic morphology and histological assessment were assessed by Golgi staining and hematoxylin-eosin (HE) staining, respectively. Adenosine A1 receptors (A1R) and adenosine A2A receptors (A2AR) were evaluated with western blotting. The results showed that cordycepin reduced the GCI-induced dendritic morphology scathing and behavioral LTP impairment in the hippocampal CA1 area, improved the learning and memory abilities, and up-regulated the level of A1R but not A2AR. In the in vitro experiments, cordycepin pre-perfusion could alleviate the hippocampal slices injury and synaptic transmission cripple induced by OGD, accompanied by increased adenosine content. In addition, the protective effect of cordycepin on OGD-induced synaptic transmission damage was eliminated by using an A1R antagonist instead of A2AR. These findings revealed that cordycepin alleviated synaptic dysfunction and dendritic injury in ischemic models by modulating A1R, which provides new insights into the pharmacological mechanisms of cordycepin for ameliorating cognitive impairment induced by cerebral ischemia.

Hormetic effect of amyloid-beta peptide in hippocampal synaptic plasticity and memory

Background: The term hormesis refers to a biphasic dose-response phenomenon characterized by low-dose stimulation and high-dose inhibition represented by a J-shaped or U-shaped curve, depending on the parameter measured (Calabrese and Baldwin, Hum Exp Toxicol, 2002). Indeed, several, if not all, physiological molecules (i.e. glutamate, glucocorticoids, nitric oxide) are likely to present a hormetic effect, exhibiting opposite effects at high or low concentrations. In the last few years, we have focused on amyloid-beta (A), a peptide widely known because it is produced in high amounts during Alzheimer’s disease (AD). A is considered a toxic fragment causing synaptic dysfunction and memory impairment (Selkoe, Science, 2002). However, the peptide is normally produced in the healthy brain and growing evidences indicate that it might have a physiologic function. Aim: Based on previous results showing that picomolar concentrations of A42 enhance synaptic plasticity and memory (Puzzo et al, J Neurosci, 2008) and that endogenous A is necessary for synaptic plasticity and memory (Puzzo et al, Ann Neurol, 2011), the aim of our study was to demonstrate the hormetic role of A in synaptic plasticity and memory. Methods: We used 3-month old wild type mice to analyze how synaptic plasticity, measured on hippocampal slices in vitro, and spatial reference memory were modified by treatment with different doses of A (from 2 pM to 20 μM). Results: We demonstrated that A has a hormetic effect (Puzzo et al, Neurobiol Aging, 2012) with low-doses (200 pM) stimulating synaptic plasticity and memory and high-doses (≥ 200 nM) inhibiting these processes. Conclusions: Our results suggest that, paradoxically, very low doses of A might serve to enhance memory at appropriate concentrations and conditions. These findings raise several issues when designing effective and safe approaches to AD therapy.

Simultaneous Monitoring of pH and Chloride (Cl−) in Brain Slices of Transgenic Mice

Optosensorics is the direction of research possessing the possibility of non-invasive monitoring of the concentration of intracellular ions or activity of intracellular components using specific biosensors. In recent years, genetically encoded proteins have been used as effective optosensory means. These probes possess fluorophore groups capable of changing fluorescence when interacting with certain ions or molecules. For monitoring of intracellular concentrations of chloride ([Cl−]i) and hydrogen ([H+] i) the construct, called ClopHensor, which consists of a H+- and Cl−-sensitive variant of the enhanced green fluorescent protein (E2GFP) fused with a monomeric red fluorescent protein (mDsRed) has been proposed [1]. We recently developed a line of transgenic mice expressing ClopHensor in neurons and obtained the map of its expression in different areas of the brain [2]. The purpose of this study was to examine the effectiveness of transgenic mice expressing ClopHensor for estimation of [H+]i and [Cl−]i concentrations in neurons of brain slices. We performed simultaneous monitoring of [H+]i and [Cl−]i under different experimental conditions including changing of external concentrations of ions (Ca2+, Cl−, K+, Na+) and synaptic stimulation of Shaffer’s collaterals of hippocampal slices. The results obtained illuminate different pathways of regulation of Cl− and pH equilibrium in neurons and demonstrate that transgenic mice expressing ClopHensor represent a reliable tool for non-invasive simultaneous monitoring of intracellular Cl− and pH.

Real-time imaging of Arc/Arg3.1 transcription ex vivo reveals input-specific immediate early gene dynamics

The ability of neurons to process and store salient environmental features underlies information processing in the brain. Long-term information storage requires synaptic plasticity and regulation of gene expression. While distinct patterns of activity have been linked to synaptic plasticity, their impact on immediate early gene (IEG) expression remains poorly understood. The activity regulated cytoskeleton associated (Arc) gene has received wide attention as an IEG implicated in synaptic plasticity and memory. Yet, to date, the transcriptional dynamics of Arc in response to compartment and input-specific activity is unclear. By developing a knock-in mouse to fluorescently tag Arc alleles, we studied real-time transcription dynamics after stimulation of dentate granule cells (GCs) in acute hippocampal slices. To our surprise, we found that Arc transcription displayed distinct temporal kinetics depending on the activation of excitatory inputs that convey functionally distinct information, i.e. medial and lateral perforant paths (MPP and LPP, respectively). Moreover, the transcriptional dynamics of Arc after synaptic stimulation was similar to direct activation of GCs, although the contribution of ionotropic glutamate receptors, L-type voltage gated calcium channel, and the endoplasmic reticulum (ER) differed. Specifically, we observed an ER-mediated synapse-to-nucleus signal that supported elevations in nuclear calcium, and rapid induction of Arc transcription following MPP stimulation. However, activation of LPP inputs displayed lower nuclear calcium rise, which could underlie the delayed transcriptional onset of Arc. Our findings highlight how input-specific activity distinctly impacts transcriptional dynamics of an IEG linked to learning and memory.

Anti-Müllerian Hormone Regulation of Synaptic Transmission in the Hippocampus Requires MAPK Signaling and Kv4.2 Potassium Channel Activity

Anti-Müllerian hormone (AMH) is a paracrine factor generated peripherally by the gonads to regulate gonadal function in adult mammals. We recently reported that AMH and AMH-specific receptor Anti-Müllerian hormone receptor 2 (AMHR2) are expressed in the hippocampus, and exogenous AMH protein rapidly increased synaptic transmission and long-term synaptic plasticity at the CA3-CA1 synapses. Here we examined the cell-specific expression of AMHR2 and the cellular mechanism of rapid boosting effect of AMH on synaptic transmission in mouse hippocampus. Immunofluorescence staining showed that AMHR2 was specifically expressed in the soma and dendrites of hippocampal pyramidal neurons, but not glial cells. Electrophysiological recordings on acute hippocampal slices showed that AMH did not affect AMPAR-mediated or N-Methyl-D-aspartic acid receptor (NMDAR)-mediated excitatory postsynaptic currents at the CA3-CA1 synapses. The small-conductance Ca2+-activated K+ channel (SK2) and A-type K+ channel (Kv4.2) contribute to shaping excitatory postsynaptic potentials (EPSPs) at the CA3-CA1 synapses. Bath application of apamin to block SK2 did not alter AMH effect on increasing EPSPs, whereas blocking Kv4.2 channel with 4-aminopyridine, or chelating internal Ca2+ with BAPTA occluded the action of AMH on boosting EPSPs. Kv4.2 activity is regulated by p38 mitogen-activated kinase (MAPK). Blocking p38 MAPK with SB203580 occluded the effect of AMH on increasing EPSPs. These results show that Kv4.2 channel contributes to the rapid action of AMH on boosting synaptic transmission in a Ca2+- and p38 MAPK-dependent manner. Our findings provide functional evidence that AMH enhances synaptic transmission through Kv4.2 channel in the hippocampus, suggesting a possible role of Kv4.2 channel in AMH-regulated neuronal process underlying learning and memory.