Dorsomedial striatal AMPA receptor antagonism increases alcohol binge drinking in selectively bred crossed high alcohol preferring mice.

Crossed high alcohol preferring (cHAP) mice have been selectively bred to consume considerable amounts of alcohol resulting in binge drinking. The dorsomedial striatum (DMS) is a brain region involved in goal-directed action selection, and dorsolateral striatum (DLS) is a brain region involved in habitual action selection. Alcohol use disorder (AUD) may involve a disruption in the balance between the DMS and DLS. While the DLS is involved in binge drinking, the reliance on the DMS and DLS in binge drinking has not been investigated in cHAP mice. We have previously demonstrated that glutamatergic activity in the DLS is necessary for binge-like alcohol drinking in C57BL/6J mice, another high drinking mouse. Because of this, we hypothesised that DLS glutamatergic activity would gate binge-like alcohol drinking in cHAP mice. cHAP mice underwent bilateral cannulation into the DMS or DLS and were allowed free-access to 20% alcohol for 2 h each day for 11 days. Mice were microinjected with the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) antagonist, NBQX, into the DMS or DLS immediately prior to alcohol access. AMPAR protein expression was also assessed in a separate group of animals in the DMS and DLS following an 11-day drinking history. We found that intra-DMS (but not intra-DLS) NBQX alters binge alcohol drinking, with intra-DMS NBQX increasing alcohol consumption. We also found that the ratio of GluA1 to GluA2 differs across dorsal striatal subregions. Together, these findings suggest that glutamatergic activity in the DMS may serve to limit binge drinking in cHAP mice.

WWC2 modulates GABAA-receptor-mediated synaptic transmission, revealing class-specific mechanisms of synapse regulation by WWC family proteins.

The WW and C2 domain-containing protein (WWC2) is implicated in several neurological disorders. Here, we demonstrate that WWC2 interacts with inhibitory, but not excitatory, postsynaptic scaffolds, consistent with prior proteomic identification of WWC2 as a putative component of the inhibitory postsynaptic density. Using mice lacking WWC2 expression in excitatory forebrain neurons, we show that WWC2 suppresses γ-aminobutyric acid type-A receptor (GABAAR) incorporation into the plasma membrane and regulates HAP1 and GRIP1, which form a complex promoting GABAAR recycling to the membrane. Inhibitory synaptic transmission is increased in CA1 pyramidal cells lacking WWC2. Furthermore, unlike the WWC2 homolog KIBRA (kidney/brain protein; WWC1), a key regulator of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking at excitatory synapses, the deletion of WWC2 does not affect synaptic AMPAR expression. In contrast, loss of KIBRA does not affect GABAAR membrane expression. These data reveal synapse class-selective functions for WWC proteins as regulators of ionotropic neurotransmitter receptors and provide insight into mechanisms regulating GABAAR membrane expression.

Lower levels of kainate receptors, but not AMPA or NMDA receptors, in Brodmann's area (BA) 9, but not BA 10, from a subgroup of people with schizophrenia who have a marked deficit in cortical muscarinic M1 receptors.

In a previous study on ionotropic glutamate receptors, we have shown that [3H]kainate, but not [3H]AMPA or [3H]NMDA, receptor binding was lower in Brodmann's area (BA) 9 from people with schizophrenia. Subsequently, we defined a subgroup within the syndrome of schizophrenia who are termed the Muscarinic Receptor Deficit subgroup of Schizophrenia (MRDS) as they have markedly lower levels of [3H]pirenzepine binding to the muscarinic M1 receptor. The previous glutamate receptor study did not contain enough people with MRDS and other forms of schizophrenia (non-MRDS) to study any subgroup-specific differences. Hence, in this study we first measured [3H]pirenzepine binding to the muscarinic M1 receptor to confirm the MRDS subgroup, then measured [3H]kainate, [3H]AMPA and [3H]NMDA receptor binding using autoradiography in BA 9 from people with MRDS, non-MRDS and controls. We also measured binding in BA 10 as our gene expression study indicated that BA 10 is disproportionally affected by the molecular pathology of schizophrenia. As expected, due to case-selection criteria, [3H]pirenzepine binding to the M1 receptor was lower in BA 9 and BA 10 from people with MRDS, although more profound in BA 10. [3H]kainate receptor binding was lower only in BA 9 from people with MRDS, while [3H]AMPA and [3H]NMDA receptor binding was not altered in either region. Muscarinic M1 receptors and kainate receptors are both located on glutamatergic pyramidal neurons so a perturbation in both receptors could indicate altered excitatory neurotransmission in BA 9 from people with MRDS.

Preclinical characterization of a water-soluble low-impact ampakine prodrug, CX1942 and its active moiety, CX1763.

Aim: AMPA-glutamate receptor (AMPAR) dysfunction mediates multiple neurological/neuropsychiatric disorders. Ampakines bind AMPARs and allosterically enhance glutamate-elicited currents. This report describes the activity of the water-soluble ampakine CX1942 prodrug and the active moiety CX1763.Results: CX1763 and CX1942 enhance synaptic transmission in hippocampi of rats. CX1763 increases attention in the 5CSRTT in rats and reduces amphetamine-induced hyperactivity in mice. CX1942 potently reverses opioid-induced respiratory depression in rats. CX1942/CX1763 was effective at 2.5-10 mg/kg. CX1763 lacked epileptogenicity up to 1500 mg/kg in rats.Conclusion: These data document that CX1942 and CX1763 are active and without prominent side effects in multiple pre-clinical assays. CX1942 could serve as a prodrug for CX1763 with the advantage of high water solubility as in an intravenous formulation.

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Safety, Tolerability, and Pharmacokinetic Profile of the Low-Impact Ampakine CX1739 in Young Healthy Volunteers.

AMPA-type glutamate receptors (AMPARs) mediate the majority of fast excitatory synaptic transmission in the mammalian brain. Ampakines, positive allosteric modulators of AMPAR, hold significant potential for the treatment of a wide range of neurological/neuropsychiatric disorders in which excitatory synaptic transmission is compromised. Low-impact ampakines are a distinct subset of ampakines that accelerate channel opening yet minimally affect receptor desensitization, which may explain their lack of seizurogenic effects at therapeutic doses in preclinical models. CX1739 is a low-impact ampakine that has shown efficacy in preclinical studies. The current clinical study examined the tolerability and pharmacokinetics of CX1739 in healthy male volunteers in a 2-part study. Part A was a single dose escalation study (100-1200 mg, 48 patients) and Part B was a multiple dose ascending study (300-600 mg BID for 7-10 days, 32 patients). CX1739 was well tolerated up to 900 mg once daily (QD) and 450 mg twice a day, with the prominent side effects being headache and nausea. Importantly, the half-life of CX1739 was 6-9 hours, and Tmax was 1-5 hours. CX1739 Cmax and AUC were dose-proportional. These findings thus set the stage for further explorations of this drug candidate in phase 2 clinical studies.

SARM1 is essential for NMDA receptor-dependent endocytosis of AMPA receptors in hippocampal neurons.

Long-term depression (LTD) is a form of synaptic plasticity thought to be the cellular basis of experience-dependent learning and memory. LTD is caused by an activity-dependent decrease in cell surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPA receptors) at the postsynaptic sites. However, the mechanism through which AMPA receptors are removed from the cell surface via neuronal activity is not fully understood. In this study, we showed that small interfering RNA (siRNA)-mediated knockdown of sterile alpha and toll/interleukin receptor motif containing 1 (SARM1) in cultured hippocampal neurons prevented the N-methyl-d-aspartate (NMDA)-induced reduction in cell surface AMPA receptors. However, the control RNA did not affect NMDA-mediated AMPA receptor trafficking. Overexpression of the siRNA-resistant form of SARM1 in SARM1-knocked-down neurons restored AMPA receptor trafficking. However, overexpression of SARM1, which lacks the mitochondrial transport signal, in the SARM1-knocked-down neurons did not restore NMDA-dependent AMPA receptor endocytosis. Moreover, the inhibition of the NADase activity of SARM1 blocked the NMDA-induced reduction of cell surface AMPA receptors. These results suggest that both the mitochondrial localization and NADase activity of SARM1 are essential for NMDA receptor-dependent AMPA receptor internalization in the hippocampal neurons.

Alternative translation initiation produces synaptic organizer proteoforms with distinct localization and functions.

While many mRNAs contain more than one translation initiation site (TIS), the functions of most alternative TISs and their corresponding protein isoforms (proteoforms) remain undetermined. Here, we showed that alternative usage of CUG and AUG TISs in neuronal pentraxin receptor (NPR) mRNA produced two proteoforms, of which the ratio was regulated by RNA secondary structure and neuronal activity. Downstream AUG initiation truncated the N-terminal transmembrane domain and produced a secreted NPR proteoform sufficient in promoting synaptic clustering of AMPA-type glutamate receptors. Mutations that altered the ratio of NPR proteoforms reduced AMPA receptors in parvalbumin-positive interneurons and affected learning behaviors in mice. In addition to NPR, upstream AUU-initiated N-terminal extension of C1q-like synaptic organizers anchored these otherwise secreted factors to the membrane. Together, these results uncovered the plasticity of N-terminal signal sequences regulated by alternative TIS usage as a potentially widespread mechanism in diversifying protein localization and functions.

High Impact AMPAkines Induce a Gq-Protein Coupled Endoplasmic Calcium Release in Cortical Neurons: A Possible Mechanism for Explaining the Toxicity of High Impact AMPAkines.

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) positive allosteric modulators (AMPAkines) have a multitude of promising therapeutic properties. The pharmaceutical development of high impact AMPAkines has, however, been limited by the appearance of calcium-dependent neuronal toxicity and convulsions in vivo. Such toxicity is not observed at exceptionally high concentrations of low impact AMPAkines. Because most AMPAR are somewhat impermeable to calcium, the current study sought to examine the extent to which different mechanisms contribute to the rise in intracellular calcium in the presence of high impact ampakines. In the presence of AMPA alone, cytosolic calcium elevation is shown to be sodium-dependent. In the presence of high impact AMPAkines such as cyclothiazide (CTZ) or CX614, however, AMPAR potentiation also activates an additional mechanism that induces calcium release from endoplasmic reticular (ER) stores. The pathway that connects AMPAR to the ER system involves a Gq-protein, phospholipase Cß-mediated inositol triphosphate (InsP3) formation, and ultimately stimulation of InsP3-receptors located on the ER. The same linkage was not observed using high concentrations of the low impact AMPAkines, CX516 (Ampalex), and CX717. We also demonstrate that CX614 produces neuronal hyper-excitability at therapeutic doses, whereas the newer generation low impact AMPAkine CX1739 is safe at exceedingly high doses. Although earlier studies have demonstrated a functional linkage between AMPAR and G-proteins, this report demonstrates that in the presence of high impact AMPAkines, AMPAR also couple to a Gq-protein, which triggers a secondary calcium release from the ER and provides insight into the disparate actions of high and low impact AMPAkines.

Properties of layer V pyramidal neurons in the primary motor cortex that represent acquired motor skills.

Layer V neurons in primary motor cortex (M1) are required for motor skill learning. We analyzed training-induced plasticity using a whole-cell slice patch-clamp technique with a rotor rod task, and found that training induces diverse changes in intrinsic properties and synaptic plasticity in M1 layer V neurons. Although the causal relationship between specific cellular changes and motor performance is unclear, by linking individual motor performance to cellular/synaptic functions, we identified several cellular and synaptic parameters that represent acquired motor skills. With respect to cellular properties, motor performance was positively correlated with resting membrane potential and fast afterhyperpolarization, but not with the membrane resistance, capacitance, or threshold. With respect to synaptic function, the performance was positively correlated with AMPA receptor-mediated postsynaptic currents, but not with GABAA receptor-mediated postsynaptic currents. With respect to live imaging analysis in Thy1-YFP mice, we further demonstrated a cross-correlation between motor performance, spine head volume, and self-entropy per spine. In the present study, we identified several changes in M1 layer V pyramidal neurons after motor training that represent acquired motor skills. Furthermore, training increased extracellular acetylcholine levels known to promote synaptic plasticity, which is correlated with individual motor performance. These results suggest that systematic control of specific intracellular parameters and enhancement of synaptic plasticity in M1 layer V neurons may be useful for improving motor skills.

Cell-Specific Optical Control of AMPA Glutamate Receptors with a Photoswitchable Tethered Antagonist.

AMPA receptors (AMPARs) are the main drivers of excitatory glutamatergic transmission in the brain, central to synaptic plasticity, and are key drug targets. However, AMPARs are expressed in virtually every neuron in the central nervous system and are activated with complex temporal dynamics, making it difficult to determine their functional roles with sufficient precision. Here we describe a cell specific, light-controllable competitive antagonist for the AMPA receptor called MP-GluAblock that combines the temporal precision of a photo-switchable ligand with the spatial and cellular specificity of a genetically-encoded membrane-anchor protein. This tool could pave the way for controlling endogenous AMPARs in neural circuits with cellular, spatial, and temporal specificity.

Role of α-amino-3­hydroxy-5-methyl-4-isoxazolepropionic acid receptors and the antagonist perampanel in geriatric epilepsy and status epilepticus.

The α-amino-3­hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) is an ionotropic glutamate receptor recognized for its active involvement in epilepsy. Through AMPAR functional alterations, multiple factors contribute to the increased susceptibility to seizures in the geriatric population. These factors include changes in the hippocampus, neuroinflammation, ischemic insults, amyloid deposition, previous seizures, alterations in the microenvironment, and neurovascular unit dysfunction. Perampanel, a noncompetitive AMPAR antagonist, has been approved for the treatment of focal and generalized epilepsy. However, a complete understanding of AMPAR's role in epileptogenesis and the pharmacotherapy of perampanel in the geriatric population remains elusive. To address this gap, we conducted a comprehensive literature review, screening 1557 articles and ultimately selecting 94 relevant ones. We provided insights into AMPAR functionality changes and perampanel's role in treating geriatric epilepsy. Various clinical trials and retrospective studies have demonstrated that the safety and efficacy of perampanel in the older population are comparable to those in the younger population, with overall good tolerability. It is also effective for treating focal and generalized onset seizures and possibly for managing status epilepticus. In conclusion, the existing body of evidence supports the safety and efficacy of perampanel in the geriatric population, indicating its potential as a valuable therapeutic option for focal and generalized epilepsy. Additional research is warranted to deepen our understanding of AMPAR's involvement in epileptogenesis and to refine the pharmacotherapeutic nuances in this specific demographic.

α-Bungarotoxin labelling of AMPA receptor-associated TARPs in living neurons.

In mammalian central neurons AMPARs are clustered at glutamatergic synapses where they mediate fast excitatory transmission. In addition to four pore-forming subunits (GluA1-4), AMPARs contain auxiliary transmembrane AMPAR regulatory proteins (γ2, γ3, γ4, γ5, γ7 or γ8) whose incorporation can vary between neuron types, brain regions, and stages of development. As well as modulating the functional properties of AMPARs, these auxiliary subunits play a central role in AMPAR trafficking. Directly visualizing TARPs could therefore provide a valuable insight into mechanisms underlying these processes. Although antibodies are routinely used for the detection of surface proteins, our experience suggests anti-TARP antibodies are too bulky to access their target, possibly due to close interactions between the extracellular domains of TARP and AMPAR subunits. We therefore assessed the utility of a small monovalent probe - fluorescent α-bungarotoxin (α-Btx) - for TARP labelling in living neurons. We inserted the bungarotoxin binding site (BBS) within the extracellular domain of TARPs to enable their detection in cells exposed to fluorescent α-Btx. Focusing on the prototypical TARP γ2, we demonstrate that the small size of fluorescent α-Btx allows it to bind to the BBS-tagged TARP when associated with AMPARs. Importantly, labelled γ2 enhances AMPAR function in the same way as unmodified γ2. In living neurons, fluorescent α-Btx-labelled γ2 associates with AMPAR clusters at synapses. As a proof-of-principle, we employed our method to compare the surface trafficking of γ2 and γ7 in cerebellar stellate neurons. Our approach provides a simple way to visualize TARPs within AMPARs in living cells.

Low-dose diazepam improves cognitive function in APP/PS1 mouse models: Involvement of AMPA receptors.

Previous studies have indicated a close association between cognitive impairment in patients with neurodegenerative diseases, such as Alzheimer's disease (AD), and synaptic damage. Diazepam (DZP), a benzodiazepine class drug, is used to control symptoms such as seizures, anxiety, and sleep disorders. These symptoms can potentially manifest throughout the entire course of AD. Therefore, DZP may be utilized in the treatment of AD to manage these symptoms. However, the specific role and mechanisms of DZP in AD remain unclear. In this study, we discovered that long-term administration of a low dose of DZP (0.5  mg/kg) improved cognitive function and protected neurons from damage in APP/PS1 mice. Mechanistic investigations revealed that DZP exerted its neuroprotective effects and reduced Aß deposition by modulating GluA1 (glutamate AMPA receptor subunit) to influence synaptic function. In conclusion, these findings highlight the potential benefits of DZP as a novel therapeutic approach, suggesting that long-term use of low-dose DZP in early-stage AD patients may be advantageous in slowing disease progression.

Plasticity-induced actin polymerization in the dendritic shaft regulates intracellular AMPA receptor trafficking.

AMPA-type receptors (AMPARs) are rapidly inserted into synapses undergoing plasticity to increase synaptic transmission, but it is not fully understood if and how AMPAR-containing vesicles are selectively trafficked to these synapses. Here, we developed a strategy to label AMPAR GluA1 subunits expressed from their endogenous loci in cultured rat hippocampal neurons and characterized the motion of GluA1-containing vesicles using single-particle tracking and mathematical modeling. We find that GluA1-containing vesicles are confined and concentrated near sites of stimulation-induced structural plasticity. We show that confinement is mediated by actin polymerization, which hinders the active transport of GluA1-containing vesicles along the length of the dendritic shaft by modulating the rheological properties of the cytoplasm. Actin polymerization also facilitates myosin-mediated transport of GluA1-containing vesicles to exocytic sites. We conclude that neurons utilize F-actin to increase vesicular GluA1 reservoirs and promote exocytosis proximal to the sites of synaptic activity.

Anti-AMPA receptor encephalitis with myasthenia gravis: A sinister combination associated with a high risk for underlying malignancy.

A rare subtype of autoimmune encephalitis consists of antibodies targetting the alpha-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid receptor in the central nervous system. We describe the clinical presentation and autoimmune profile of the first case of alpha-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid receptor encephalitis with concurrent anti-acetylcholine receptor antibodies in Pakistan. The patient was a 58-year-old male who presented with the characteristic symptoms of limbic encephalitis with memory loss, irritability, agitation, and confusion. Antibodies against the alpha-amino-3-hydroxy- 5-methyl-4-isoxazolepropionic acid receptor were detected in both serum and cerebrospinal fluid by indirect immunofluorescence. Computerised tomography of the chest showed an anterior mediastinal mass. The patient was treated with high dose Methylprednisolone and five sessions of plasma exchange. There was a short period of improvement; however, the patient now continues to exhibit irritability, aphasia, confusion, and memory loss. Video-assisted thoracoscopic surgery for mediastinal mass resection and histological testing was planned, however after review by the interventional radiologist the associated risks were deemed too high to proceed with the procedure and biopsy was not done.

Differential Alterations in the Expression of AMPA Receptor and Its Trafficking Proteins in the Hippocampus Are Associated with Recognition Memory Impairment in the Rotenone-Parkinson's Disease Mouse Model: Neuroprotective Role of Bacopa monnieri Extract CDRI 08.

Parkinson's disease (PD), an age-associated neurodegenerative motor disorder, is associated with dementia and cognitive decline. However, the precise molecular insight into PD-induced cognitive decline is not fully understood. Here, we have investigated the possible alterations in the expression of glutamate receptor and its trafficking/scaffolding/regulatory proteins underlying the memory formation and neuroprotective effects of a specialized Bacopa monnieri extract, CDRI-08 (BME) in the hippocampus of the rotenone-induced PD mouse model. Our Western blotting and qRT-PCR data reveal that the PD-induced recognition memory decline is associated with significant upregulation of the AMPA receptor subunit GluR1 and downregulation of GluR2 subunit genes in the hippocampus of rotenone-affected mice as compared to the vehicle control. Further, expressions of the trafficking proteins are significantly upregulated in the hippocampus of rotenone-affected mice compared to the vehicle control. Our results also reveal that the above alterations in the hippocampus are associated with similar expression patterns of total CREB, pCREB, and BDNF. BME (CDRI-08, 200 mg/kg BW) reverses the expression of AMPA receptor subunits, their trafficking proteins differentially, and the transcriptional modulatory proteins depending on whether the BME treatment was given before or after the rotenone treatment. Our data suggest that expression of the above genes is significantly reversed in the BME pre-treated mice subjected to rotenone treatment towards their levels in the control mice compared to its treatment after rotenone administration. Our results provide the possible molecular basis underlying the rotenone-induced recognition memory decline, conditions mimicking the PD symptoms in mouse model and neuroprotective action of bacoside A and bacoside B (58%)-enriched Bacopa monnieri extract (BME) in the hippocampus.

The Chemokine CCL2 Promotes Excitatory Synaptic Transmission in Hippocampal Neurons via GluA1 Subunit Trafficking.

The CC chemokine ligand 2 (CCL2, also known as MCP-1) and its cognate receptor CCR2 have well-characterized roles in chemotaxis. CCL2 has been previously shown to promote excitatory synaptic transmission and neuronal excitability. However, the detailed molecular mechanism underlying this process remains largely unclear. In cultured hippocampal neurons, CCL2 application rapidly upregulated surface expression of GluA1, in a CCR2-dependent manner, assayed using SEP-GluA1 live imaging, surface GluA1 antibody staining, and electrophysiology. Using pharmacology and reporter assays, we further showed that CCL2 upregulated surface GluA1 expression primarily via Gαq- and CaMKII-dependent signaling. Consistently, using i.p. injection of lipopolysaccharide to induce neuroinflammation, we found upregulated phosphorylation of S831 and S845 sites on AMPA receptor subunit GluA1 in the hippocampus, an effect blocked in Ccr2-/- mice. Together, these results provide a mechanism through which CCL2, and other secreted molecules that signal through G-protein coupled receptors, can directly regulate synaptic transmission.

Ellagic Acid Reverses Alterations in the Expression of AMPA Receptor and Its Scaffolding Proteins in the Cerebral Cortex and Memory Decline in STZ-sporadic Alzheimer' s Disease Mouse Model.

RATIONALE: Alzheimer's disease (AD), an age-dependent devastating neuropsychiatric disorder, is a leading cause of learning, memory and intellectual disabilities. Current therapeutic approaches for the amelioration of the anomalies of AD are not effective. OBJECTIVE: In the present study, the molecular mechanisms underlying sporadic AD (sAD), the memory related behavioral analysis and neuroprotective effects of Ellagic acid (EA) were investigated. METHOD: sAD mouse model was developed by intracerebroventricular (ICV) injection of Streptozotocin (STZ). The efficacy of EA, a naturally occurring polyphenol, in amelioration of anomalies associated with sAD was assessed. EA was administered once daily for 28 days at a dose of 75 mg/kg body weight followed by neurobehavioral, biochemical, molecular and neuronal count analysis to delineate the mode of action of EA. RESULT: The ICV injection of STZ in mice significantly increased the expression of AD biomarkers in addition to enhanced oxidative stress. A decline in the discrimination index in Novel Object Recognition Test was observed indicating the compromise of recognition memory in AD. Studies on the expression of genes involved in synaptic plasticity reveal the dysregulation of the α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) of the glutamate and its scaffolding proteins in the postsynaptic density and thereby synaptic plasticity in AD. ICV-STZ led to significant upregulation of apoptotic markers which led to decrease in neuronal density of the cerebral cortex. EA significantly reversed the above and improved anomalies of sAD. CONCLUSION: EA was observed to profoundly modulate the genes involved in AD pathophysiology, restored antioxidant enzymes activity, reduced lipid peroxidation and neuronal loss in the sAD brain. Further, EA was observed to effectively modulate the genes involved in apoptosis and synaptic plasticity. Therefore, EA possesses promising anti-AD properties, which may improve AD-associated anomalies by modulating synaptic plasticity via AMPAR signaling.

Improved Radiosynthesis and Automation of [11C]2-(2,6-Difluoro-4-((2-(N-methylphenylsulfonamido)ethyl)thio)phenoxy)acetamide ([11C]K2) for Positron Emission Tomography of the Glutamate α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid (AMPA) Receptor.

A new automated radiosynthesis of [11C]2-(2,6-difluoro-4-((2-(N-methylphenylsulfonamido)ethyl)thio)phenoxy)acetamide ([11C]K2), a radiopharmaceutical for the glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor, is reported. Although manual syntheses have been described, these are unsuitable for routine production of larger batches of [11C]K2 for (pre)clinical PET imaging applications. To meet demands for the imaging agent from our functional neuroimaging collaborators, herein, we report a current good manufacturing practice (cGMP)-compliant synthesis of [11C]K2 using a commercial synthesis module. The new synthesis is fully automated and has been validated for clinical use. The total synthesis time is 33 min from end of bombardment, and the production method provides 2.66 ± 0.3 GBq (71.9 ± 8.6 mCi) of [11C]K2 in 97.7 ± 0.5% radiochemical purity and 754.1 ± 231.5 TBq/mmol (20,382.7 ± 6256.1 Ci/mmol) molar activity (n = 3). Batches passed all requisite quality control testing confirming suitability for clinical use.

Enhanced hippocampal LTP but normal NMDA receptor and AMPA receptor function in a rat model of CDKL5 deficiency disorder.

BACKGROUND: Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) cause a severe neurological disorder characterised by early-onset epileptic seizures, autism and intellectual disability (ID). Impaired hippocampal function has been implicated in other models of monogenic forms of autism spectrum disorders and ID and is often linked to epilepsy and behavioural abnormalities. Many individuals with CDKL5 deficiency disorder (CDD) have null mutations and complete loss of CDKL5 protein, therefore in the current study we used a Cdkl5-/y rat model to elucidate the impact of CDKL5 loss on cellular excitability and synaptic function of CA1 pyramidal cells (PCs). We hypothesised abnormal pre and/or post synaptic function and plasticity would be observed in the hippocampus of Cdkl5-/y rats. METHODS: To allow cross-species comparisons of phenotypes associated with the loss of CDKL5, we generated a loss of function mutation in exon 8 of the rat Cdkl5 gene and assessed the impact of the loss of CDLK5 using a combination of extracellular and whole-cell electrophysiological recordings, biochemistry, and histology. RESULTS: Our results indicate that CA1 hippocampal long-term potentiation (LTP) is enhanced in slices prepared from juvenile, but not adult, Cdkl5-/y rats. Enhanced LTP does not result from changes in NMDA receptor function or subunit expression as these remain unaltered throughout development. Furthermore, Ca2+ permeable AMPA receptor mediated currents are unchanged in Cdkl5-/y rats. We observe reduced mEPSC frequency accompanied by increased spine density in basal dendrites of CA1 PCs, however we find no evidence supporting an increase in silent synapses when assessed using a minimal stimulation protocol in slices. Additionally, we found no change in paired-pulse ratio, consistent with normal release probability at Schaffer collateral to CA1 PC synapses. CONCLUSIONS: Our data indicate a role for CDKL5 in hippocampal synaptic function and raise the possibility that altered intracellular signalling rather than synaptic deficits contribute to the altered plasticity. LIMITATIONS: This study has focussed on the electrophysiological and anatomical properties of hippocampal CA1 PCs across early postnatal development. Studies involving other brain regions, older animals and behavioural phenotypes associated with the loss of CDKL5 are needed to understand the pathophysiology of CDD.