Integrins Bidirectionally Regulate the Efficacy of Inhibitory Synaptic Transmission and Control GABAergic Plasticity.

For many decades, synaptic plasticity was believed to be restricted to excitatory transmission. However, in recent years, this view started to change, and now it is recognized that GABAergic synapses show distinct forms of activity-dependent long-term plasticity, but the underlying mechanisms remain obscure. Herein, we asked whether signaling mediated by ß1 or ß3 subunit-containing integrins might be involved in regulating the efficacy of GABAergic synapses, including the NMDA receptor-dependent inhibitory long-term potentiation (iLTP) in the hippocampus. We found that activation of ß3 integrin with fibrinogen induced a stable depression, whereas inhibition of ß1 integrin potentiated GABAergic synapses at CA1 pyramidal neurons in male mice. Additionally, compounds that interfere with the interaction of ß1 or ß3 integrins with extracellular matrix blocked the induction of NMDA-iLTP. In conclusion, we provide the first evidence that integrins are key players in regulating the endogenous modulatory mechanisms of GABAergic inhibition and plasticity in the hippocampus.SIGNIFICANCE STATEMENT Epilepsy, schizophrenia, and anxiety are just a few medical conditions associated with dysfunctional inhibitory synaptic transmission. GABAergic synapses are known for their extraordinary susceptibility to modulation by endogenous factors and exogenous pharmacological agents. We describe here that integrins, adhesion proteins, play a key role in the modulation of inhibitory synaptic transmission. Specifically, we show that interference with integrin-dependent adhesion results in a variety of effects on the amplitude and frequency of GABAergic mIPSCs. Activation of ß3 subunit-containing integrins induces inhibitory long-term depression, whereas the inhibition of ß1 subunit-containing integrins induces iLTP. Our results unveil an important mechanism controlling synaptic inhibition, which opens new avenues into the usage of integrin-aimed pharmaceuticals as modulators of GABAergic synapses.

Long-term plasticity of inhibitory synapses in the hippocampus and spatial learning depends on matrix metalloproteinase 3.

Learning and memory are known to depend on synaptic plasticity. Whereas the involvement of plastic changes at excitatory synapses is well established, plasticity mechanisms at inhibitory synapses only start to be discovered. Extracellular proteolysis is known to be a key factor in glutamatergic plasticity but nothing is known about its role at GABAergic synapses. We reveal that pharmacological inhibition of MMP3 activity or genetic knockout of the Mmp3 gene abolishes induction of postsynaptic iLTP. Moreover, the application of exogenous active MMP3 mimics major iLTP manifestations: increased mIPSCs amplitude, enlargement of synaptic gephyrin clusters, and a decrease in the diffusion coefficient of synaptic GABAA receptors that favors their entrapment within the synapse. Finally, we found that MMP3 deficient mice show faster spatial learning in Morris water maze and enhanced contextual fear conditioning. We conclude that MMP3 plays a key role in iLTP mechanisms and in the behaviors that presumably in part depend on GABAergic plasticity.

Long term potentiation affects intracellular metalloproteinases activity in the mossy fiber-CA3 pathway.

Matrix Metalloproteinases (MMPs) are a family of endopeptidases known to process extracellular proteins. In the last decade, studies carried out mainly on the Schaffer collateral-CA1 hippocampal projection have provided solid evidence that MMPs regulate synaptic plasticity and learning. Recently, our group has shown that MMP blockade disrupts LTP maintenance also in the mossy fiber-CA3 (mf-CA3) projection (Wojtowicz and Mozrzymas, 2010), where LTP mechanisms are profoundly different (NMDAR-independent and presynaptic expression site). However, how plasticity of this pathway correlates with activity and expression of MMPs remains unknown. Interestingly, several potential MMP substrates (especially of gelatinases) are localized intracellularly but little is known about MMP activity in this compartment. In the present study we have asked whether LTP is associated with the expression and activity of gelatinases in apparent intra- and extracellular compartments along mf-CA3 projection. In situ zymography showed that LTP induction was associated with increased gelatinases activity in the cytoplasm of the hilar and CA3 neurons. Using gelatin zymography, immunohistochemistry and immunofluorescent staining we found that this effect was due to de novo synthesis and activation of MMP-9 which, 2-3h after LTP induction was particularly evident in the cytoplasm. In contrast, MMP-2 was localized preferentially in the nuclei and was not affected by LTP induction. In conclusion, we demonstrate that LTP induction in the mf-CA3 pathway correlates with increased expression and activity of MMP-9 and provide the first evidence that this increase is particularly evident in the neuronal cytoplasm and nucleus.

GABAergic synapses onto SST and PV interneurons in the CA1 hippocampal region show cell-specific and integrin-dependent plasticity.

It is known that GABAergic transmission onto pyramidal neurons shows different forms of plasticity. However, GABAergic cells innervate also other inhibitory interneurons and plasticity phenomena at these projections remain largely unknown. Several mechanisms underlying plastic changes, both at inhibitory and excitatory synapses, show dependence on integrins, key proteins mediating interaction between intra- and extracellular environment. We thus used hippocampal slices to address the impact of integrins on long-term plasticity of GABAergic synapses on specific inhibitory interneurons (containing parvalbumin, PV + or somatostatin, SST +) known to innervate distinct parts of principal cells. Administration of RGD sequence-containing peptide induced inhibitory long-term potentiation (iLTP) at fast-spiking (FS) PV + as well as on SST + interneurons. Interestingly, treatment with a more specific peptide GA(C)RRETAWA(C)GA (RRETAWA), affecting α5ß1 integrins, resulted in iLTP in SST + and iLTD in FS PV + interneurons. Brief exposure to NMDA is known to induce iLTP at GABAergic synapses on pyramidal cells. Intriguingly, application of this protocol for considered interneurons evoked iLTP in SST + and iLTD in PV + interneurons. Moreover, we showed that in SST + cells, NMDA-evoked iLTP depends on the incorporation of GABAA receptors containing α5 subunit to the synapses, and this iLTP is occluded by RRETAWA peptide, indicating a key role of α5ß1 integrins. Altogether, our results revealed that plasticity of inhibitory synapses at GABAergic cells shows interneuron-specificity and show differences in the underlying integrin-dependent mechanisms. This is the first evidence that neuronal disinhibition may be a highly plastic process depending on interneuron type and integrins' activity.

Induction of Inhibitory Synaptic Plasticity Enhances Tonic Current by Increasing the Content of α5-Subunit Containing GABAA Receptors in Hippocampal Pyramidal Neurons.

It is known that besides synaptic inhibition, there is a persistent component of inhibitory drive mediated by tonic currents which is believed to mediate majority of the total inhibitory charge in hippocampal neurons. Tonic currents, depending on cell types, can be mediated by a variety of GABAA receptor (GABAAR) subtypes but in pyramidal neurons, α5-subunit containing receptors were found to be predominant. Importantly, α5-GABAARs were implicated in both inhibitory and excitatory synaptic plasticity as well as in a variety of cognitive tasks. In the present study, we asked whether the protocol that evokes NMDAR-dependent GABAergic inhibitory long-term potentiation (iLTP) also induces the plasticity of tonic inhibition in hippocampal pyramidal neurons. Our whole-cell patch-clamp recordings revealed that the induction of this type of iLTP is associated with a marked increase in tonic current. By using the specific inverse agonist of α5-containing GABAARs (L-655,709) we provide evidence that this plastic change in tonic current is correlated with an increased proportion of this type of GABAARs. On the contrary, the iLTP induction did not affect the tonic current potentiated by THIP, indicating that the pool of δ subunit-containing GABAARs receptors remains unaffected. We conclude that the α5-GABAARs-dependent plasticity of tonic inhibition is a novel dimension of the neuroplasticity of the inhibitory drive in the hippocampal principal neurons. Overall, α5-containing GABAARs emerge as key players in a variety of plasticity mechanisms operating over a large span of time and spatial scales.

Spike Timing-Dependent Plasticity in the Mouse Barrel Cortex Is Strongly Modulated by Sensory Learning and Depends on Activity of Matrix Metalloproteinase 9.

Experience and learning in adult primary somatosensory cortex are known to affect neuronal circuits by modifying both excitatory and inhibitory transmission. Synaptic plasticity phenomena provide a key substrate for cognitive processes, but precise description of the cellular and molecular correlates of learning is hampered by multiplicity of these mechanisms in various projections and in different types of neurons. Herein, we investigated the impact of associative learning on neuronal plasticity in distinct types of postsynaptic neurons by checking the impact of classical conditioning (pairing whisker stroking with tail shock) on the spike timing-dependent plasticity (t-LTP and t-LTD) in the layer IV to II/III vertical pathway of the mouse barrel cortex. Learning in this paradigm practically prevented t-LTP measured in pyramidal neurons but had no effect on t-LTD. Since classical conditioning is known to affect inhibition in the barrel cortex, we examined its effect on tonic GABAergic currents and found a strong downregulation of these currents in the layer II/III interneurons but not in pyramidal cells. Matrix metalloproteinases emerged as crucial players in synaptic plasticity and learning. We report that the blockade of MMP-9 (but not MMP-3) abolished t-LTP having no effect on t-LTD. Moreover, associative learning resulted in an upregulation of gelatinolytic activity within the "trained" barrel. We conclude that LTP induced by spike timing-dependent plasticity (STDP) paradigm is strongly correlated with associative learning and critically depends on the activity of MMP-9.

Block and allosteric modulation of GABAergic currents by oenanthotoxin in rat cultured hippocampal neurons.

BACKGROUND AND PURPOSE: Oenanthotoxin (OETX), a polyacetylenic alcohol from plants of the genus Oenanthe, has recently been identified as potent inhibitor of GABA-evoked currents. However, the effects of OETX on the inhibitory postsynaptic currents (IPSCs), as well as the pharmacological mechanism(s) underlying its effects on GABA(A) receptors, remain unknown. The purpose of this study was to elucidate the mechanism underlying the inhibition of GABAergic currents by OETX. EXPERIMENTAL APPROACH: Effects of OETX on GABAergic currents were studied using the patch clamp technique on rat cultured hippocampal neurons. Miniature IPSCs (mIPSCs) were recorded in the whole-cell configuration, while the current responses were elicited by ultrafast GABA applications onto the excised patches. KEY RESULTS: OETX potently inhibited both mIPSCs and current responses, but its effect was much stronger on synaptic currents. Analysis of the effects of OETX on mIPSCs and evoked currents disclosed a complex mechanism: allosteric modulation of both GABA(A) receptor binding and gating properties and a non-competitive, probably open channel block mechanism. In particular, OETX reduced the binding rate and nearly abolished receptor desensitization. A combination of rapid clearance of synaptic GABA and OETX-induced slowing of binding kinetics is proposed to underlie the potent action of OETX on mIPSCs. CONCLUSIONS AND IMPLICATIONS: OETX shows a complex blocking mechanism of GABA(A) receptors, and the impact of this toxin is more potent on mIPSCs than on currents evoked by exogenous GABA. Such effects on GABAergic currents are compatible with the convulsions and epileptic-like activity reported for OETX.

17beta-estradiol affects GABAergic transmission in developing hippocampus.

Estrogens are potent modulators of the nervous system. In particular, 17beta-estradiol was shown to affect GABAergic synaptic transmission in hippocampus of adult animals in vivo but much less is known on the impact of this hormone on the GABAergic system in the developing brains. We have recently shown that phasic and tonic GABAergic transmissions are strongly modulated upon long-term treatment with exogenous 17beta-estradiol in hippocampal neurons developing in vitro. To check for the long-term estrogen effect in a more physiological developmental model, we have investigated the GABAergic transmission in developing brains of P7-P40 animals, injected daily with 17beta-estradiol. We have found that such a treatment clearly increased GABAergic mIPSC frequency and amplitude while the onset and decay of mIPSCs were shortened. These effects were statistically significant in the youngest considered age group (P7-P13) with a tendency to disappear in older animals. Long-term treatment with estradiol did not change the susceptibility of mIPSC amplitude to upregulation by flurazepam while mIPSC decay was prolonged by this drug to a larger extent in 17beta-estradiol-treated animals. 17beta-estradiol strongly upregulated GABAergic tonic current but again this effect was restricted to the youngest group of animals. We conclude that 17beta-estradiol strongly modulates the GABAergic synaptic transmission but this effect critically depends on the animal age being the most prominent in youngest animals.

GABA transient sets the susceptibility of mIPSCs to modulation by benzodiazepine receptor agonists in rat hippocampal neurons.

Benzodiazepines (BDZs) are known to increase the amplitude and duration of IPSCs. Moreover, at low [GABA], BDZs strongly enhance GABAergic currents suggesting the up-regulation of agonist binding while their action on gating remains a matter of debate. In the present study we have examined the impact of flurazepam and zolpidem on mIPSCs by investigating their effects on GABA(A)R binding and gating and by considering dynamic conditions of synaptic receptor activation. Flurazepam and zolpidem enhanced the amplitude and prolonged decay of mIPSCs. Both compounds strongly enhanced responses to low [GABA] but, surprisingly, decreased the currents evoked by saturating or half-saturating [GABA]. Analysis of current responses to ultrafast GABA applications indicated that these compounds enhanced binding and desensitization of GABA(A) receptors. Flurazepam and zolpidem markedly prolonged deactivation of responses to low [GABA] but had almost no effect on deactivation at saturating or half-saturating [GABA]. Moreover, at low [GABA], flurazepam enhanced desensitization-deactivation coupling but zolpidem did not. Recordings of responses to half-saturating [GABA] applications revealed that appropriate timing of agonist exposure was sufficient to reproduce either a decrease or enhancement of currents by flurazepam or zolpidem. Recordings of currents mediated by recombinant ('synaptic') alpha1beta2gamma2 receptors reproduced all major findings observed for neuronal GABA(A)Rs. We conclude that an extremely brief agonist transient renders IPSCs particularly sensitive to the up-regulation of agonist binding by BDZs.