Correction: GABAergic regulation of striatal spiny projection neurons depends upon their activity state.

[This corrects the article DOI: 10.1371/journal.pbio.3002483.].

Ca2+-dependent phosphodiesterase 1 regulates the plasticity of striatal spiny projection neuron glutamatergic synapses.

Long-term synaptic plasticity at glutamatergic synapses on striatal spiny projection neurons (SPNs) is central to learning goal-directed behaviors and habits. Our studies reveal that SPNs manifest a heterosynaptic, nitric oxide (NO)-dependent form of long-term postsynaptic depression of glutamatergic SPN synapses (NO-LTD) that is preferentially engaged at quiescent synapses. Plasticity is gated by Ca2+ entry through CaV1.3 Ca2+ channels and phosphodiesterase 1 (PDE1) activation, which blunts intracellular cyclic guanosine monophosphate (cGMP) and NO signaling. Both experimental and simulation studies suggest that this Ca2+-dependent regulation of PDE1 activity allows for local regulation of dendritic cGMP signaling. In a mouse model of Parkinson disease (PD), NO-LTD is absent because of impaired interneuronal NO release; re-balancing intrastriatal neuromodulatory signaling restores NO release and NO-LTD. Taken together, these studies provide important insights into the mechanisms governing NO-LTD in SPNs and its role in psychomotor disorders such as PD.

Rem2 interacts with CaMKII at synapses and restricts long-term potentiation in hippocampus.

Synaptic plasticity, the process whereby neuronal connections are either strengthened or weakened in response to stereotyped forms of stimulation, is widely believed to represent the molecular mechanism that underlies learning and memory. The holoenzyme calcium/calmodulin-dependent protein kinase II (CaMKII) plays a well-established and critical role in the induction of a variety of forms of synaptic plasticity such as long-term potentiation (LTP), long-term depression (LTD) and depotentiation. Previously, we identified the GTPase Rem2 as a potent, endogenous inhibitor of CaMKII. Here, we report that knock out of Rem2 enhances LTP at the Schaffer collateral to CA1 synapse in hippocampus, consistent with an inhibitory action of Rem2 on CaMKII in vivo. Further, re-expression of WT Rem2 rescues the enhanced LTP observed in slices obtained from Rem2 conditional knock out (cKO) mice, while expression of a mutant Rem2 construct that is unable to inhibit CaMKII in vitro fails to rescue increased LTP. In addition, we demonstrate that CaMKII and Rem2 interact in dendritic spines using a 2pFLIM-FRET approach. Taken together, our data lead us to propose that Rem2 serves as a brake on synaptic potentiation via inhibition of CaMKII activity. Further, the enhanced LTP phenotype we observe in Rem2 cKO slices reveals a previously unknown role for Rem2 in the negative regulation of CaMKII function.

Ca 2+ -dependent phosphodiesterase 1 regulates the plasticity of striatal spiny projection neuron glutamatergic synapses.

Long-term synaptic plasticity at glutamatergic synapses on striatal spiny projection neurons (SPNs) is central to learning goal-directed behaviors and habits. Although considerable attention has been paid to the mechanisms underlying synaptic strengthening and new learning, little scrutiny has been given to those involved in the attenuation of synaptic strength that attends suppression of a previously learned association. Our studies revealed a novel, non-Hebbian, long-term, postsynaptic depression of glutamatergic SPN synapses induced by interneuronal nitric oxide (NO) signaling (NO-LTD) that was preferentially engaged at quiescent synapses. This form of plasticity was gated by local Ca 2+ influx through CaV1.3 Ca 2+ channels and stimulation of phosphodiesterase 1 (PDE1), which degraded cyclic guanosine monophosphate (cGMP) and blunted NO signaling. Consistent with this model, mice harboring a gain-of-function mutation in the gene coding for the pore-forming subunit of CaV1.3 channels had elevated depolarization-induced dendritic Ca 2+ entry and impaired NO-LTD. Extracellular uncaging of glutamate and intracellular uncaging of cGMP suggested that this Ca 2+ -dependent regulation of PDE1 activity allowed for local regulation of dendritic NO signaling. This inference was supported by simulation of SPN dendritic integration, which revealed that dendritic spikes engaged PDE1 in a branch-specific manner. In a mouse model of Parkinson's disease (PD), NO-LTD was absent not because of a postsynaptic deficit in NO signaling machinery, but rather due to impaired interneuronal NO release. Re-balancing intrastriatal neuromodulatory signaling in the PD model restored NO release and NO-LTD. Taken together, these studies provide novel insights into the mechanisms governing NO-LTD in SPN and its role in psychomotor disorders, like PD.

Rem2 interacts with CaMKII at synapses and restricts long-term potentiation in hippocampus.

Synaptic plasticity, the process whereby neuronal connections are either strengthened or weakened in response to stereotyped forms of stimulation, is widely believed to represent the molecular mechanism that underlies learning and memory. The holoenzyme CaMKII plays a well-established and critical role in the induction of a variety of forms of synaptic plasticity such as long-term potentiation (LTP), long-term depression (LTD) and depotentiation. Previously, we identified the GTPase Rem2 as a potent, endogenous inhibitor of CaMKII. Here, we report that knock out of Rem2 enhances LTP at the Schaffer collateral to CA1 synapse in hippocampus, consistent with an inhibitory action of Rem2 on CaMKII in vivo. Further, re-expression of WT Rem2 rescues the enhanced LTP observed in slices obtained from Rem2 conditional knock out (cKO) mice, while expression of a mutant Rem2 construct that is unable to inhibit CaMKII in vitro fails to rescue increased LTP. In addition, we demonstrate that CaMKII and Rem2 interact in dendritic spines using a 2pFLIM-FRET approach. Taken together, our data lead us to propose that Rem2 serves as a brake on runaway synaptic potentiation via inhibition of CaMKII activity. Further, the enhanced LTP phenotype we observe in Rem2 cKO slices reveals a previously unknown role for Rem2 in the negative regulation of CaMKII function.

GABAergic regulation of striatal spiny projection neurons depends upon their activity state.

Synaptic transmission mediated by GABAA receptors (GABAARs) in adult, principal striatal spiny projection neurons (SPNs) can suppress ongoing spiking, but its effect on synaptic integration at subthreshold membrane potentials is less well characterized, particularly those near the resting down-state. To fill this gap, a combination of molecular, optogenetic, optical, and electrophysiological approaches were used to study SPNs in mouse ex vivo brain slices, and computational tools were used to model somatodendritic synaptic integration. In perforated patch recordings, activation of GABAARs, either by uncaging of GABA or by optogenetic stimulation of GABAergic synapses, evoked currents with a reversal potential near -60 mV in both juvenile and adult SPNs. Transcriptomic analysis and pharmacological work suggested that this relatively positive GABAAR reversal potential was not attributable to NKCC1 expression, but rather to HCO3- permeability. Regardless, from down-state potentials, optogenetic activation of dendritic GABAergic synapses depolarized SPNs. This GABAAR-mediated depolarization summed with trailing ionotropic glutamate receptor (iGluR) stimulation, promoting dendritic spikes and increasing somatic depolarization. Simulations revealed that a diffuse dendritic GABAergic input to SPNs effectively enhanced the response to dendritic iGluR signaling and promoted dendritic spikes. Taken together, our results demonstrate that GABAARs can work in concert with iGluRs to excite adult SPNs when they are in the resting down-state, suggesting that their inhibitory role is limited to brief periods near spike threshold. This state-dependence calls for a reformulation for the role of intrastriatal GABAergic circuits.

Semaphorin 4D induced inhibitory synaptogenesis decreases epileptiform activity and alters progression to Status Epilepticus in mice.

Previously we demonstrated that intra-hippocampal infusion of purified, Semaphorin 4D (Sema4D) extracellular domain (ECD) into the mouse hippocampus rapidly promotes formation of GABAergic synapses and decreases seizure susceptibility in mice. Given the relatively fast action of Sema4D treatment revealed by these studies, we sought to determine the time course of Sema4D treatment on hippocampal network activity using an acute hippocampal slice preparation. We performed long-term extracellular recordings from area CA1 encompassing a 2-hour application of Sema4D and found that hippocampal excitation is suppressed for hours following treatment. We also asked if Sema4D treatment could ameliorate seizures in an acute seizure model: the kainic acid (KA) mouse model. We demonstrate that Sema4D treatment delays and suppresses ictal activity, delays the transition to Status Epilepticus (SE), and lessens the severity of SE. Lastly, we sought to explore alternative methods of Sema4D delivery to hippocampus and thus created an Adeno Associated Virus expressing the ECD of Sema4D. Our data reveal that virally delivered, chronically overexpressed Sema4D-ECD promotes GABAergic synapse formation and suppresses ictal activity and progression to SE. These results provide proof of concept that viral delivery of Sema4D is an efficacious and promising delivery method to abate epileptiform activity and progression to SE.

State-dependent GABAergic regulation of striatal spiny projection neuron excitability.

Synaptic transmission mediated by GABA A receptors (GABA A Rs) in adult, principal striatal spiny projection neurons (SPNs) can suppress ongoing spiking, but its effect on synaptic integration at sub-threshold membrane potentials is less well characterized, particularly those near the resting down-state. To fill this gap, a combination of molecular, optogenetic, optical and electrophysiological approaches were used to study SPNs in mouse ex vivo brain slices, and computational tools were used to model somatodendritic synaptic integration. Activation of GABA A Rs, either by uncaging of GABA or by optogenetic stimulation of GABAergic synapses, evoked currents with a reversal potential near -60 mV in perforated patch recordings from both juvenile and adult SPNs. Molecular profiling of SPNs suggested that this relatively positive reversal potential was not attributable to NKCC1 expression, but rather to a dynamic equilibrium between KCC2 and Cl-/HCO3-cotransporters. Regardless, from down-state potentials, optogenetic activation of dendritic GABAergic synapses depolarized SPNs. This GABAAR-mediated depolarization summed with trailing ionotropic glutamate receptor (iGluR) stimulation, promoting dendritic spikes and increasing somatic depolarization. Simulations revealed that a diffuse dendritic GABAergic input to SPNs effectively enhanced the response to coincident glutamatergic input. Taken together, our results demonstrate that GABA A Rs can work in concert with iGluRs to excite adult SPNs when they are in the resting down-state, suggesting that their inhibitory role is limited to brief periods near spike threshold. This state-dependence calls for a reformulation of the role intrastriatal GABAergic circuits.

Activity-dependent upregulation of presynaptic kainate receptors at immature CA3-CA1 synapses.

Presynaptic kainate-type glutamate receptors (KARs) regulate glutamate release probability and short-term plasticity in various areas of the brain. Here we show that long-term depression (LTD) in the area CA1 of neonatal rodent hippocampus is associated with an upregulation of tonic inhibitory KAR activity, which contributes to synaptic depression and causes a pronounced increase in short-term facilitation of transmission. This increased KAR function was mediated by high-affinity receptors and required activation of NMDA receptors, nitric oxide (NO) synthetase, and postsynaptic calcium signaling. In contrast, KAR activity was irreversibly downregulated in response to induction of long-term potentiation in a manner that depended on activation of the TrkB-receptor of BDNF. Both tonic KAR activity and its plasticity were restricted to early stages of synapse development and were lost in parallel with maturation of the network due to ongoing BDNF-TrkB signaling. These data show that presynaptic KARs are targets for activity-dependent modulation via diffusible messengers NO and BDNF, which enhance and depress tonic KAR activity at immature synapses, respectively. The plasticity of presynaptic KARs in the developing network allows nascent synapses to shape their response to incoming activity. In particular, upregulation of KAR function after LTD allows the synapse to preferentially pass high-frequency afferent activity. This can provide a potential rescue from synapse elimination by uncorrelated activity and also increase the computational dynamics of the developing CA3-CA1 circuitry.

Developmental switch in the kinase dependency of long-term potentiation depends on expression of GluA4 subunit-containing AMPA receptors.

The AMPA-receptor subunit GluA4 is expressed transiently in CA1 pyramidal neurons at the time synaptic connectivity is forming, but its physiological significance is unknown. Here we show that GluA4 expression is sufficient to alter the signaling requirements of long-term potentiation (LTP) and can fully explain the switch in the LTP kinase dependency from PKA to Ca2(+)/calmodulin-dependent protein kinase II during synapse maturation. At immature synapses, activation of PKA leads to a robust potentiation of AMPA-receptor function via the mobilization of GluA4. Analysis of GluA4-deficient mice indicates that this mechanism is critical for neonatal PKA-dependent LTP. Furthermore, lentiviral expression of GluA4 in CA1 neurons conferred a PKA-dependent synaptic potentiation and LTP regardless of the developmental stage. Thus, GluA4 defines the signaling requirements for LTP and silent synapse activation during a critical period of synapse development.

Mechanisms underlying induction of LTP-associated changes in short-term dynamics of transmission at immature synapses.

While the activity-dependent mechanisms guiding functional maturation of synaptic transmission postsynaptically are well characterized, less is known about the corresponding presynaptic mechanisms. Here we show that during the first postnatal week, a subset of CA3-CA1 synapses express postsynaptically induced LTP that is tightly associated with a robust decrease in synaptic facilitation, consistent with an increase in release probability (P(r)). The loss of facilitation is readily induced by physiologically relevant pairing protocols at immature synapses and is dependent on activation of NMDA-receptors but not L-type calcium channels. The putative pre- and postsynaptic components of neonatal LTP were distinguished in their downstream signaling requirements, PKC activity being selectively needed for the decrease in facilitation but not for synaptic potentiation per se. These data suggest that maturation of glutamatergic synapses involves a critical period during which presynaptic function is highly susceptible to activity-dependent regulation via a PKC-dependent mechanism.

Synaptic kainate receptors in CA1 interneurons gate the threshold of theta-frequency-induced long-term potentiation.

Theta oscillations (4-12 Hz) in neuronal networks are known to predispose the synapses involved to plastic changes and may underlie their association with learning behaviors. The lowered threshold for synaptic plasticity during theta oscillations is thought to be due to decreased GABAergic inhibition. Interneuronal kainate receptors (KARs) regulate GABAergic transmission and are implicated in theta activity; however, the physiological significance of this regulation is unknown. In rat hippocampus, we show that during theta activity, there is excitatory postsynaptic drive to CA1 interneurons mediated by KARs. This promotes feedforward inhibition of pyramidal neurons, raising the threshold for induction of theta-burst long-term potentiation. These results identify a novel mechanism whereby the activation of postsynaptic KARs in CA1 interneurons gate changes in synaptic efficacy to a physiologically relevant patterned stimulation.

Kainate receptor-induced ectopic spiking of CA3 pyramidal neurons initiates network bursts in neonatal hippocampus.

Kainate receptors (KARs) are expressed at high levels in the brain during early development and may be critical for the proper development of neuronal networks. Here we elucidated a physiological role of high-affinity KARs in developing hippocampal network by studying the effects of 25-100 nM kainate (KA) on intrinsic network activity in slice preparations. Whereas 100 nM KA resulted in hyperexcitability of the network and the disruption of natural activity patterns, ≤ 50 nM KA concentrations enhanced the initiation of network bursts yet preserved the characteristic patterns of endogenous activity. This was not dependent on changes in GABAergic transmission or on activation of GluK1 subunit containing KARs. However, the activation of high-affinity KARs increased glutamatergic drive by promoting spontaneous firing of CA3 pyramidal neurons without affecting action potential independent glutamate release. This was not because of changes in the intrinsic somatic properties of pyramidal neurons but seemed to reside in an electrically remote site, most probably in an axonal compartment. Although application of KAR agonists has mainly been used to study pathological type of network activities, this study provides a novel mechanism by which endogenous activity of KARs can modulate intrinsic activities of the emerging neuronal network in a physiologically relevant manner. The results support recent studies that KARs play a central role in the activity-dependent maturation of synaptic circuitries.

ACET is a highly potent and specific kainate receptor antagonist: characterisation and effects on hippocampal mossy fibre function.

Kainate receptors (KARs) are involved in both NMDA receptor-independent long-term potentiation (LTP) and synaptic facilitation at mossy fibre synapses in the CA3 region of the hippocampus. However, the identity of the KAR subtypes involved remains controversial. Here we used a highly potent and selective GluK1 (formerly GluR5) antagonist (ACET) to elucidate roles of GluK1-containing KARs in these synaptic processes. We confirmed that ACET is an extremely potent GluK1 antagonist, with a Kb value of 1.4+/-0.2 nM. In contrast, ACET was ineffective at GluK2 (formerly GluR6) receptors at all concentrations tested (up to 100 microM) and had no effect at GluK3 (formerly GluR7) when tested at 1 microM. The X-ray crystal structure of ACET bound to the ligand binding core of GluK1 was similar to the UBP310-GluK1 complex. In the CA1 region of hippocampal slices, ACET was effective at blocking the depression of both fEPSPs and monosynaptically evoked GABAergic transmission induced by ATPA, a GluK1 selective agonist. In the CA3 region of the hippocampus, ACET blocked the induction of NMDA receptor-independent mossy fibre LTP. To directly investigate the role of pre-synaptic GluK1-containing KARs we combined patch-clamp electrophysiology and 2-photon microscopy to image Ca2+ dynamics in individual giant mossy fibre boutons. ACET consistently reduced short-term facilitation of pre-synaptic calcium transients induced by 5 action potentials evoked at 20-25Hz. Taken together our data provide further evidence for a physiological role of GluK1-containing KARs in synaptic facilitation and LTP induction at mossy fibre-CA3 synapses.

Inhibition of kainate receptors reduces the frequency of hippocampal theta oscillations.

We investigated the role of kainate receptors in the generation of theta oscillations using (S)-1-(2-amino-2-carboxyethyl)-3-(2-carboxythiophene-3-yl-methyl)pyrimidine-2,4-dione (UBP304), a novel, potent and highly selective antagonist of GLU(K5)-containing kainate receptors. EEG and single-unit recordings were made from the dorsal hippocampus of awake, freely moving rats trained to forage for food. Bilateral intracerebroventricular injections of UBP304 (2.0 microl, two times; 2.08 mM) caused a clear (approximately 25%) reduction in theta frequency that was dissociable from behavioral effects of the drug. The locations of firing fields of principal cells in the hippocampal formation were generally preserved, but both field firing rates and the precision of field organization decreased. UBP304 lowered the frequency of the theta modulation of hippocampal interneuron discharge, accurately matching the reduced frequency of the theta field oscillation. UBP308 [(R)-1-(2-amino-2-carboxyethyl)-3-(2-carboxythiophene-3-yl-methyl)pyrimidine-2,4-dione], the inactive enantiomer of UBP304, caused none of these effects. Our results suggest that GLU(K5) receptors have an important role in modulating theta activity. In addition, the effects on cellular responses provide both insight into the mechanisms of theta pacing, and useful information for models of temporal coding.

Characterisation of UBP296: a novel, potent and selective kainate receptor antagonist.

Willardiine derivatives with an N3-benzyl substituent bearing an acidic group have been synthesized with the aim of producing selective antagonists for GLUK5-containing kainate receptors. UBP296 was found to be a potent and selective antagonist of native GLUK5-containing kainate receptors in the spinal cord, with activity residing in the S enantiomer (UBP302). In cells expressing human kainate receptor subunits, UBP296 selectively depressed glutamate-induced calcium influx in cells containing GLUK5 in homomeric or heteromeric forms. In radioligand displacement binding studies, the willardiine analogues displaced [3H]kainate binding with IC50 values >100 microM at rat GLUK6, GLUK2 or GLUK6/GLUK2. An explanation of the GLUK5 selectivity of UBP296 was obtained using homology models of the antagonist bound forms of GLUK5 and GLUK6. In rat hippocampal slices, UBP296 reversibly blocked ATPA-induced depressions of synaptic transmission at concentrations subthreshold for affecting AMPA receptor-mediated synaptic transmission directly. UBP296 also completely blocked the induction of mossy fibre LTP, in medium containing 2 mM (but not 4 mM) Ca2+. These data provide further evidence for a role for GLUK5-containing kainate receptors in mossy fibre LTP. In conclusion, UBP296 is the most potent and selective antagonist of GLUK5-containing kainate receptors so far described.

Antagonists of GLU(K5)-containing kainate receptors prevent pilocarpine-induced limbic seizures.

Developments in the molecular biology and pharmacology of GLU(K5), a subtype of the kainate class of ionotropic glutamate receptors, have enabled insights into the roles of this subunit in synaptic transmission and plasticity. However, little is known about the possible functions of GLU(K5)-containing kainate receptors in pathological conditions. We report here that, in hippocampal slices, selective antagonists of GLU(K5)-containing kainate receptors prevented development of epileptiform activity--evoked by the muscarinic agonist, pilocarpine--and inhibited the activity when it was pre-established. In conscious rats, these GLU(K5) antagonists prevented and interrupted limbic seizures induced by intra-hippocampal pilocarpine perfusion, and attenuated accompanying rises in extracellular L-glutamate and GABA. This anticonvulsant activity occurred without overt side effects. GLU(K5) antagonism also prevented epileptiform activity induced by electrical stimulation, both in vitro and in vivo. Therefore, we propose that subtype-selective GLU(K5) kainate receptor antagonists offer a potential new therapy for epilepsy.