AMPA and NMDA Receptor Trafficking at Cocaine-Generated Synapses.

Cocaine experience generates AMPA receptor (AMPAR)-silent synapses in the nucleus accumbens (NAc), which are thought to be new synaptic contacts enriched in GluN2B-containing NMDA receptors (NMDARs). After drug withdrawal, some of these synapses mature by recruiting AMPARs, strengthening the newly established synaptic transmission. Silent synapse generation and maturation are two consecutive cellular steps through which NAc circuits are profoundly remodeled to promote cue-induced cocaine seeking after drug withdrawal. However, the basic cellular processes that mediate these two critical steps remains underexplored. Using a combination of electrophysiology, viral-mediated gene transfer, and confocal imaging in male rats as well as knock-in (KI) mice of both sexes, our current study characterized the dynamic roles played by AMPARs and NMDARs in generation and maturation of silent synapses on NAc medium spiny neurons after cocaine self-administration and withdrawal. We report that cocaine-induced generation of silent synapses not only required synaptic insertion of GluN2B-containing NMDARs, but also, counterintuitively, involved insertion of AMPARs, which subsequently internalized, resulting in the AMPAR-silent state on withdrawal day 1. Furthermore, GluN2B NMDARs functioned to maintain these cocaine-generated synapses in the AMPAR-silent state during drug withdrawal, until they were replaced by nonGluN2B NMDARs, a switch that allowed AMPAR recruitment and maturation of silent synapses. These results reveal dynamic interactions between AMPARs and NMDARs during the generation and maturation of silent synapses after cocaine experience and provide a mechanistic basis through which new synaptic contacts and possibly new neural network patterns created by these synapses can be manipulated for therapeutic benefit.SIGNIFICANCE STATEMENT Studies over the past decade reveal a critical role of AMPA receptor-silent, NMDA receptor-containing synapses in forming cocaine-related memories that drive cocaine relapse. However, it remains incompletely understood how AMPA and NMDA receptors traffic at these synapses during their generation and maturation. The current study characterizes a two-step AMPA receptor trafficking cascade that contributes to the generation of silent synapses in response to cocaine experience, and a two-step NMDA receptor trafficking cascade that contributes to the maturation of these synapses after cocaine withdrawal. These results depict a highly regulated cellular procedure through which nascent glutamatergic synapses are generated in the adult brain after drug experience and provide significant insight into the roles of glutamate receptors in synapse formation and maturation.

Instant activation of TRP channels by NH4 + promotes neuronal bursting and glutamate spikes in CA1 neurons.

Ammonia ( NH 4 + ) is a by-product of cell metabolism and may elicit subcellular effects with specific physiological responses. Chronic effects have been implicated in several neurological diseases and attributed to persistent elevation in blood ammonia levels transferred to the brain. In previous studies the activities of neurons and astrocytes have been examined at ammonia concentrations an order of magnitude higher than measured in the blood. The effects developed within several minutes. Here we focused upon acute responses of neurons to ammonia and whether they may occur at much lower doses. To this end, we combined patch-clamp in CA1 neurons with glutamate imaging in hippocampal slices. Particular attention was paid to the Rett syndrome that has been originally attributed to hyperammonemia. We compared the responses in the wild-type (WT) and model Rett mice (MECP2-null, RTT) to ammonia doses from 0.3 mM on. In both preparations NH 4 + promptly depolarized neurons and increased the ambient glutamate. The bursting activity emerged in WT and it was augmented in RTT. Searching for subcellular mechanisms we examined possible modulation of ion channels by ammonia. We did not find any changes in HCN- and Ca2+ currents, which substantially contribute to the bursting activity. The non-selective cation channels were markedly potentiated by ammonia. ASIC channels had a major contribution to the augmentation of neuronal activity by ammonia. Interestingly, their general blocker amiloride (100 µM) moderately excited CA1 cells akin to NH 4 + . In its presence subsequent ammonia effects were markedly compromised. Blockade of TRPC1 channels partially occluded NH 4 + effects. ASIC and TRPC1 blockers decreased the amplitude of excitatory postsynaptic currents (EPSC) and neuronal bursts, congruent with a postsynaptic location of the channels. Inhibition of TRPV1 channels potentiated the responses to NH 4 + . EPSC amplitudes did not change, but the frequency decreased, indicating presynaptic effects. All extracellular NH 4 + actions were observed at concentrations as low as 0.3 mM and the neurons reacted immediately after ammonia arrived the slice. We propose that a brief augmentation of neuronal activity by NH 4 + may occur either spontaneously during arousal or induced by inhalation of smelling salts.

CA1 Neurons Acquire Rett Syndrome Phenotype After Brief Activation of Glutamatergic Receptors: Specific Role of mGluR1/5.

Rett syndrome (RTT) is a neurological disorder caused by the mutation of the X-linked MECP2 gene. The neurophysiological hallmark of the RTT phenotype is the hyperexcitability of neurons made responsible for frequent epileptic attacks in the patients. Increased excitability in RTT might stem from impaired glutamate handling in RTT and its long-term consequences that has not been examined quantitatively. We recently reported (Balakrishnan and Mironov, 2018a,b) that the RTT hippocampus consistently demonstrates repetitive glutamate transients that parallel the burst firing in the CA1 neurons. We aimed to examine how brief stimulation of specific types of ionotropic and metabotropic glutamate receptors (GluR) can modulate the neuronal phenotype. We imaged glutamate with a fluorescence sensor (iGluSnFr) expressed in CA1 neurons in hippocampal organotypic slices from wild-type (WT) and Mecp2-/y mice (RTT). The neuronal and synaptic activities were assessed by patch-clamp and calcium imaging. In both WT and RTT slices, activation of AMPA, kainate, and NMDA receptors for 30 s first enhanced neuronal activity that induced a global release of glutamate. After transient augmentation of excitability and ambient glutamate, they subsided. After wash out of the agonists for 10 min, WT slices recovered and demonstrated repetitive glutamate transients, whose pattern resembled those observed in naïve RTT slices. Hyperpolarization-activated (HCN) decreased and voltage-sensitive calcium channel (VSCC) currents increased. The effects were long-lasting and bigger in WT. We examined the role of mGluR1/5 in more detail. The effects of the agonist (S)-3,5-dihydroxyphenylglycine (DHPG) were the same as AMPA and NMDA and occluded by mGluR1/5 antagonists. Further modifications were examined using a non-stationary noise analysis of postsynaptic currents. The mean single channel current and their number at postsynapse increased after DHPG. We identified new channels as calcium-permeable AMPARs (CP-AMPAR). We then examined back-propagating potentials (bAPs) as a measure of postsynaptic integration. After bAPs, spontaneous afterdischarges were observed that lasted for ∼2 min and were potentiated by DHPG. The effects were occluded by intracellular CP-AMPAR blocker and did not change after NMDAR blockade. We propose that brief elevations in ambient glutamate (through brief excitation with GluR agonists) specifically activate mGluR1/5. This modifies CP-AMPAR, HCN, and calcium conductances and makes neurons hyperexcitable. Induced changes can be further supported by repetitive glutamate transients established and serve to persistently maintain the aberrant neuronal RTT phenotype in the hippocampus.

Regenerative glutamate release in the hippocampus of Rett syndrome model mice.

Excess glutamate during intense neuronal activity is not instantly cleared and may accumulate in the extracellular space. This has various long-term consequences such as ectopic signaling, modulation of synaptic efficacy and excitotoxicity; the latter implicated in various neurodevelopmental and neurodegenerative diseases. In this study, the quantitative imaging of glutamate homeostasis of hippocampal slices from methyl-CpG binding protein 2 knock-out (Mecp2-/y) mice, a model of Rett syndrome (RTT), revealed unusual repetitive glutamate transients. They appeared in phase with bursts of action potentials in the CA1 neurons. Both glutamate transients and bursting activity were suppressed by the blockade of sodium, AMPA and voltage-gated calcium channels (T- and R-type), and enhanced after the inhibition of HCN channels. HCN and calcium channels in RTT and wild-type (WT) CA1 neurons displayed different voltage-dependencies and kinetics. Both channels modulated postsynaptic integration and modified the pattern of glutamate spikes in the RTT hippocampus. Spontaneous glutamate transients were much less abundant in the WT preparations, and, when observed, had smaller amplitude and frequency. The basal ambient glutamate levels in RTT were higher and transient glutamate increases (spontaneous and evoked by stimulation of Schaffer collaterals) decayed slower. Both features indicate less efficient glutamate uptake in RTT. To explain the generation of repetitive glutamate spikes, we designed a novel model of glutamate-induced glutamate release. The simulations correctly predicted the patterns of spontaneous glutamate spikes observed under different experimental conditions. We propose that pervasive spontaneous glutamate release is a hallmark of Mecp2-/y hippocampus, stemming from and modulating the hyperexcitability of neurons.

Rescue of hyperexcitability in hippocampal CA1 neurons from Mecp2 (-/y) mouse through surface potential neutralization.

Hyperventilation is a known feature of Rett syndrome (RTT). However, how hyperventilation is related to other RTT symptoms such as hyperexcitability is unknown. Intense breathing during hyperventilation induces hypocapnia and culminates in respiratory alkalosis. Alkalinization of extracellular milieu can trigger epilepsy in patients who already have neuronal hyperexcitability. By combining patch-clamp electrophysiology and quantitative glutamate imaging, we compared excitability of CA1 neurons of WT and Mecp2 (-/y) mice, and analyzed the biophysical properties of subthreshold membrane channels. The results show that Mecp2 (-/y) CA1 neurons are hyperexcitable in normal pH (7.4) and are increasingly vulnerable to alkaline extracellular pH (8.4), during which their excitability increased further. Under normal pH conditions, an abnormal negative shift in the voltage-dependencies of HCN (hyperpolarization-activated cyclic nucleotide-gated) and calcium channels in the CA1 neurons of Mecp2 (-/y) mice was observed. Alkaline pH also enhanced excitability in wild-type (WT) CA1 neurons through modulation of the voltage dependencies of HCN- and calcium channels. Additionally alkaline pH augmented spontaneous glutamate release and burst firing in WT CA1 neurons. Conversely, acidic pH (6.4) and 8 mM Mg2+ exerted the opposite effect, and diminished hyperexcitability in Mecp2 (-/y) CA1 neurons. We propose that the observed effects of pH and Mg2+ are mediated by changes in the neuronal membrane surface potential, which consecutively modulates the gating of HCN and calcium channels. The results provide insight to pivotal cellular mechanisms that can regulate neuronal excitability and help to devise treatment strategies for hyperexcitability induced symptoms of Rett syndrome.

SK3 Channel Overexpression in Mice Causes Hippocampal Shrinkage Associated with Cognitive Impairments.

The dysfunction of the small-conductance calcium-activated K+ channel SK3 has been described as one of the factors responsible for the progress of psychoneurological diseases, but the molecular basis of this is largely unknown. This report reveals through use of immunohistochemistry and computational tomography that long-term increased expression of the SK3 small-conductance calcium-activated potassium channel (SK3-T/T) in mice induces a notable bilateral reduction of the hippocampal area (more than 50 %). Histological analysis showed that SK3-T/T mice have cellular disarrangements and neuron discontinuities in the hippocampal formation CA1 and CA3 neuronal layer. SK3 overexpression resulted in cognitive loss as determined by the object recognition test. Electrophysiological examination of hippocampal slices revealed that SK3 channel overexpression induced deficiency of long-term potentiation in hippocampal microcircuits. In association with these results, there were changes at the mRNA levels of some genes involved in Alzheimer's disease and/or linked to schizophrenia, epilepsy, and autism. Taken together, these features suggest that augmenting the function of SK3 ion channel in mice may present a unique opportunity to investigate the neural basis of central nervous system dysfunctions associated with schizophrenia, Alzheimer's disease, or other neuropsychiatric/neurodegenerative disorders in this model system. As a more detailed understanding of the role of the SK3 channel in brain disorders is limited by the lack of specific SK3 antagonists and agonists, the results observed in this study are of significant interest; they suggest a new approach for the development of neuroprotective strategies in neuropsychiatric/neurodegenerative diseases with SK3 representing a potential drug target.

Rescue of Cyclic AMP Mediated Long Term Potentiation Impairment in the Hippocampus of Mecp2 Knockout (Mecp2(-/y) ) Mice by Rolipram.

Rett syndrome (RTT) patients experience learning difficulties and memory loss. Analogous deficits of hippocampal plasticity are reported in mouse models of RTT. To elucidate the underlying pathophysiology, we studied long term potentiation (LTP) at the CA3 to CA1 synapses in the hippocampus in acute brain slices from WT and Mecp2(-/y) mice, by either activating cAMP dependent pathway or using high frequency stimulation, by means of patch clamp. We have observed that, the NMDA channel current characteristics remain unchanged in the Mecp2(-/y) mice. The adenylyl cyclase (AC) agonist forskolin evoked a long lasting potentiation of evoked EPSCs in WT CA1 neurons, but only minimally enhanced the EPSCs in the Mecp2(-/y) mice. This weaker potentiation in Mecp2 (-/) (y) mice was ameliorated by application of phosphodiesterase 4 inhibitor rolipram. The hyperpolarization activated cyclic nucleotide gated channel current (I h) was potentiated to similar extent by forskolin in both phenotypes. Multiple tetanus induced cAMP-dependent plasticity was also impaired in the Mecp2 (-/) (y) mice, and was also partially rescued by rolipram. Western blot analysis of CA region of Mecp2 (-/) (y) mice hippocampus revealed more than twofold up-regulation of protein kinase A (PKA) regulatory subunits, while the expression of the catalytic subunit remained unchanged. We hypothesize that the overexpressed PKA regulatory subunits buffer cAMP and restrict the PKA mediated phosphorylation of target proteins necessary for LTP. Blocking the degradation of cAMP, thereby saturating the regulatory subunits alleviated this defect.

Caffeine Modulates Vesicle Release and Recovery at Cerebellar Parallel Fibre Terminals, Independently of Calcium and Cyclic AMP Signalling.

BACKGROUND: Cerebellar parallel fibres release glutamate at both the synaptic active zone and at extrasynaptic sites-a process known as ectopic release. These sites exhibit different short-term and long-term plasticity, the basis of which is incompletely understood but depends on the efficiency of vesicle release and recycling. To investigate whether release of calcium from internal stores contributes to these differences in plasticity, we tested the effects of the ryanodine receptor agonist caffeine on both synaptic and ectopic transmission. METHODS: Whole cell patch clamp recordings from Purkinje neurons and Bergmann glia were carried out in transverse cerebellar slices from juvenile (P16-20) Wistar rats. KEY RESULTS: Caffeine caused complex changes in transmission at both synaptic and ectopic sites. The amplitude of postsynaptic currents in Purkinje neurons and extrasynaptic currents in Bergmann glia were increased 2-fold and 4-fold respectively, but paired pulse ratio was substantially reduced, reversing the short-term facilitation observed under control conditions. Caffeine treatment also caused synaptic sites to depress during 1 Hz stimulation, consistent with inhibition of the usual mechanisms for replenishing vesicles at the active zone. Unexpectedly, pharmacological intervention at known targets for caffeine--intracellular calcium release, and cAMP signalling--had no impact on these effects. CONCLUSIONS: We conclude that caffeine increases release probability and inhibits vesicle recovery at parallel fibre synapses, independently of known pharmacological targets. This complex effect would lead to potentiation of transmission at fibres firing at low frequencies, but depression of transmission at high frequency connections.

Ectopic release of glutamate contributes to spillover at parallel fibre synapses in the cerebellum.

In the rat cerebellar molecular layer, spillover of glutamate between parallel fibre synapses can lead to activation of perisynaptic receptors that mediate short- and long-term plasticity. This effect is greatest when clusters of fibres are stimulated at high frequencies, suggesting that glutamate clearance mechanisms must be overwhelmed before spillover can occur. However, parallel fibres can also release transmitter directly into the extracellular space, from 'ectopic' release sites. Ectopic transmission activates AMPA receptors on the Bergmann glial cell processes that envelop parallel fibre synapses, but the possible contribution of this extrasynaptic release to intersynaptic communication has not been explored. We exploited long-term depression of ectopic transmission, and selective pharmacology, to investigate the impact of these release sites on the time course of Purkinje neuron excitatory postsynaptic currents (EPSCs). Depletion of ectopic release pools by activity-dependent long-term depression decreased EPSC decay time, revealing a 'late' current that is present when fibres are stimulated at low frequencies. This effect was enhanced when glutamate transporters were inhibited, and reduced when extracellular diffusion was impeded. Blockade of N-type Ca(2+) channels inhibited ectopic transmission to Bergmann glia and decreased EPSC decay time. Similarly, perfusion of the Ca(2+) chelator EGTA-AM into the slice progressively eliminated ectopic transmission to glia and decreased EPSC decay time with closely similar time courses. Collectively, this evidence suggests that ectopically released glutamate contributes to spillover transmission, and that ectopic release therefore degrades the spatial precision of synapses that fire infrequently, and may make them more prone to exhibit plasticity.

Ectopic release sites lack fast vesicle recycling mechanisms, causing long-term depression of neuron-glial transmission in rat cerebellum.

Classical synaptic transmission occurs at active zones within the synaptic cleft, but increasing evidence suggests that vesicle fusion can also occur outside of these zones, releasing transmitter directly into the extrasynaptic space. The role of such "ectopic" release is unclear, but in the cerebellar molecular layer it is thought to guide the processes of Bergmann glia toward synaptic terminals through activation of glial α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) receptors. Once surrounding the terminal, the glial process is presumed to limit spillover of neurotransmitter between synapses by rapid uptake of glutamate. We have previously reported that this route for neuron-glial transmission exhibits long-term depression following repetitive stimulation at frequencies in the 0.1-1 Hz range, in ex vivo slices from rat cerebellum. Here, we present evidence that LTD arises because ectopic sites lack the fast recycling mechanisms that operate at the active zone. Consequently, ectopic vesicles constitute an exhaustible pool that is depleted at normal synaptic firing rates and only recovers slowly. This effect is cumulative, meaning that the strength of ectopic transmission provides a read-out of the average frequency of presynaptic firing over several minutes. Glial processes are therefore likely to interact most closely with terminals that fire infrequently; conditions that may promote elimination of, rather than support for, the connection.

Depression of parallel and climbing fiber transmission to Bergmann glia is input specific and correlates with increased precision of synaptic transmission.

In the cerebellar cortex, Bergmann glia enclose the synapses of both parallel and climbing fiber inputs to the Purkinje neuron. The glia express Ca(2+)-permeable AMPA receptors, and the GLAST and GLT-1 classes of glutamate transporter, which are activated by glutamate released during synaptic transmission. We have previously reported that parallel fiber to Bergmann glial transmission in rat cerebellar slices exhibits a form of frequency-dependent plasticity, namely long-term depression, following repetitive stimulation at 0.1-1 Hz. Here, we report that this form of plasticity is also present at the climbing fiber input, that climbing and parallel fibers can be depressed independently, that discrete parallel fiber inputs can also be depressed independently, and that depression is maintained when a distributed array of parallel fibers are stimulated (in contrast to several forms of synaptic plasticity at the Purkinje neuron). Depression of glutamate transporter currents does not correlate with a decrease in the stringency with which Purkinje neuron synapses are isolated. Rather, postsynaptic currents in Purkinje neurons decay more rapidly and perisynaptic metabotropic glutamate receptors are activated less effectively after stimulation at 0.2 and 1 Hz, suggesting that depression arises from a decrease in extrasynaptic glutamate concentration and not from impairment of glutamate clearance in and around the synapse. These results indicate that neuron-glial plasticity is activity dependent, input specific and does not require spillover between adjacent synapses to manifest. They also argue against a withdrawal of the glial sheath from synaptic regions as the putative mechanism of plasticity.

Expression of a Cu,Zn superoxide dismutase typical for familial amyotrophic lateral sclerosis increases the vulnerability of neuroblastoma cells to infectious injury.

BACKGROUND: Infections can aggravate the course of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Mutations in the anti-oxidant enzyme Cu,Zn superoxide dismutase (EC 1.15.1.1, SOD1) are associated with familial ALS. Streptococcus pneumoniae, the most frequent respiratory pathogen, causes damage by the action of the cholesterol-binding virulence factor pneumolysin and by stimulation of the innate immune system, particularly via Toll-like-receptor 2. METHODS: SH-SY5Y neuroblastoma cells transfected with the G93A mutant of SOD1 typical for familial ALS (G93A-SOD1) and SH-SY5Y neuroblastoma cells transfected with wildtype SOD1 were both exposed to pneumolysin and in co-cultures with cultured human macrophages treated with the Toll like receptor 2 agonist N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine x 3 HCl (Pam3CSK4). Cell viability and apoptotic cell death were compared morphologically and by in-situ tailing. With the help of the WST-1 test, cell viability was quantified, and by measurement of neuron-specific enolase in the culture supernatant neuronal damage in co-cultures was investigated. Intracellular calcium levels were measured by fluorescence analysis using fura-2 AM. RESULTS: SH-SY5Y neuroblastoma cells transfected with the G93A mutant of SOD1 typical for familial ALS (G93A-SOD1) were more vulnerable to the neurotoxic action of pneumolysin and to the attack of monocytes stimulated by Pam3CSK4 than SH-SY5Y cells transfected with wild-type human SOD1. The enhanced pneumolysin toxicity in G93A-SOD1 neuronal cells depended on the inability of these cells to cope with an increased calcium influx caused by pores formed by pneumolysin. This inability was caused by an impaired capacity of the mitochondria to remove cytoplasmic calcium. Treatment of G93A-SOD1 SH-SY5Y neuroblastoma cells with the antioxidant N-acetylcysteine reduced the toxicity of pneumolysin. CONCLUSION: The particular vulnerability of G93A-SOD1 neuronal cells to hemolysins and inflammation may be partly responsible for the clinical deterioration of ALS patients during infections. These findings link infection and motor neuron disease and suggest early treatment of respiratory infections in ALS patients.

Localization of the mouse 5-hydroxytryptamine(1A) receptor in lipid microdomains depends on its palmitoylation and is involved in receptor-mediated signaling.

In the present study, we have used wild-type and palmitoylation-deficient mouse 5-hydroxytryptamine(1A) receptor (5-HT1A) receptors fused to the yellow fluorescent protein- and the cyan fluorescent protein (CFP)-tagged alpha(i3) subunit of heterotrimeric G-protein to study spatiotemporal distribution of the 5-HT1A-mediated signaling in living cells. We also addressed the question on the molecular mechanisms by which receptor palmitoylation may regulate communication between receptors and G(i)-proteins. Our data demonstrate that activation of the 5-HT1A receptor caused a partial release of Galpha(i) protein into the cytoplasm and that this translocation is accompanied by a significant increase of the intracellular Ca(2+) concentration. In contrast, acylation-deficient 5-HT1A mutants failed to reproduce both Galpha(i3)-CFP relocation and changes in [Ca(2+)](i) upon agonist stimulation. By using gradient centrifugation and copatching assays, we also demonstrate that a significant fraction of the 5-HT1A receptor resides in membrane rafts, whereas the yield of the palmitoylation-deficient receptor in these membrane microdomains is reduced considerably. Our results suggest that receptor palmitoylation serves as a targeting signal responsible for the retention of the 5-HT1A receptor in membrane rafts. More importantly, the raft localization of the 5-HT1A receptor seems to be involved in receptor-mediated signaling.