Synaptopodin is required for long-term depression at Schaffer collateral-CA1 synapses.

Synaptopodin (SP), an actin-associated protein found in telencephalic neurons, affects activity-dependant synaptic plasticity and dynamic changes of dendritic spines. While being required for long-term depression (LTD) mediated by metabotropic glutamate receptor (mGluR-LTD), little is known about its role in other forms of LTD induced by low frequency stimulation (LFS-LTD) or spike-timing dependent plasticity (STDP). Using electrophysiology in ex vivo hippocampal slices from SP-deficient mice (SPKO), we show that absence of SP is associated with a deficit of LTD at Sc-CA1 synapses induced by LFS-LTD and STDP. As LTD is known to require AMPA- receptors internalization and IP3-receptors calcium signaling, we tested by western blotting and immunochemistry if there were changes in their expression which we found to be reduced. While we were not able to induce LTD, long-term potentiation (LTP), albeit diminished in SPKO, can be recovered by using a stronger stimulation protocol. In SPKO we found no differences in NMDAR, which are the primary site of calcium signalling to induce LTP. Our study shows, for the first time, the key role of the requirement of SP to allow induction of activity-dependant LTD at Sc-CA1 synapses.

Loss of synaptopodin impairs mGluR5 and protein synthesis-dependent mGluR-LTD at CA3-CA1 synapses.

Metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) is an important form of synaptic plasticity that occurs in many regions of the central nervous system and is the underlying mechanism for several learning paradigms. In the hippocampus, mGluR-LTD is manifested by the weakening of synaptic transmission and elimination of dendritic spines. Interestingly, not all spines respond or undergo plasticity equally in response to mGluR-LTD. A subset of dendritic spines containing synaptopodin (SP), an actin-associated protein is critical for mGluR-LTD and protects spines from elimination through mGluR1 activity. The precise cellular function of SP is still enigmatic and it is still unclear how SP contributes to the functional aspect of mGluR-LTD despite its modulation of the structural plasticity. In this study, we show that the lack of SP impairs mGluR-LTD by negatively affecting the mGluR5-dependent activity. Such impairment of mGluR5 activity is accompanied by a significant decrease of surface mGluR5 level in SP knockout (SPKO) mice. Intriguingly, the remaining mGluR-LTD becomes a protein synthesis-independent process in the SPKO and is mediated instead by endocannabinoid signaling. These data indicate that the postsynaptic protein SP can regulate the locus of expression of mGluR-LTD and provide insight into our understanding of spine/synapse-specific plasticity.

Invasive growth of brain metastases is linked to CHI3L1 release from pSTAT3-positive astrocytes.

BACKGROUND: Compared to minimally invasive brain metastases (MI BrM), highly invasive (HI) lesions form abundant contacts with cells in the peritumoral brain parenchyma and are associated with poor prognosis. Reactive astrocytes (RAs) labeled by phosphorylated STAT3 (pSTAT3) have recently emerged as a promising therapeutic target for BrM. Here, we explore whether BrM invasion pattern is influenced by pSTAT3+ RAs and may serve as a predictive biomarker for STAT3 inhibition. METHODS: We used immunohistochemistry to identify pSTAT3+ RAs in HI and MI human and patient-derived xenograft (PDX) BrM. Using PDX, syngeneic, and transgenic mouse models of HI and MI BrM, we assessed how pharmacological STAT3 inhibition or RA-specific STAT3 genetic ablation affected BrM growth in vivo. Cancer cell invasion was modeled in vitro using a brain slice-tumor co-culture assay. We performed single-cell RNA sequencing of human BrM and adjacent brain tissue. RESULTS: RAs expressing pSTAT3 are situated at the brain-tumor interface and drive BrM invasive growth. HI BrM invasion pattern was associated with delayed growth in the context of STAT3 inhibition or genetic ablation. We demonstrate that pSTAT3+ RAs secrete Chitinase 3-like-1 (CHI3L1), which is a known STAT3 transcriptional target. Furthermore, single-cell RNA sequencing identified CHI3L1-expressing RAs in human HI BrM. STAT3 activation, or recombinant CHI3L1 alone, induced cancer cell invasion into the brain parenchyma using a brain slice-tumor plug co-culture assay. CONCLUSIONS: Together, these data reveal that pSTAT3+ RA-derived CHI3L1 is associated with BrM invasion, implicating STAT3 and CHI3L1 as clinically relevant therapeutic targets for the treatment of HI BrM.

Loss of synaptopodin impairs mGluR5 and protein synthesis dependent mGluR-LTD at CA3-CA1 synapses.

Metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) is an important form of synaptic plasticity that occurs in many regions of the CNS and is the underlying mechanism for several learning paradigms. In the hippocampus, mGluR-LTD is manifested by the weakening of synaptic transmission and elimination of dendritic spines. Interestingly, not all spines respond or undergo plasticity equally in response to mGluR-LTD. A subset of dendritic spines containing synaptopodin (SP), an actin-associated protein, are critical for mGluR-LTD and protect spines from elimination through mGluR1 activity. The precise cellular function of SP is still enigmatic and it is still unclear how SP contributes to the functional aspect of mGluR-LTD despite of its modulation on the structural plasticity. In the present study, we show that the lack of SP impairs mGluR-LTD by negatively affecting the mGluR5-dependent activity. Such impairment of mGluR5 activity is accompanied by a significant decrease of surface mGluR5 level in SP knockout (SPKO) mice. Intriguingly, the remaining mGluR-LTD becomes a protein synthesis-independent process in the SPKO and is mediated instead by endocannabinoid signaling. These data show for the first time that the postsynaptic protein SP can regulate the locus of expression of mGluR-LTD and provide insight to our understanding of spine/synapse-specific plasticity. Significance statement: Hippocampal group I metabotropic glutamate receptor dependent long-term depression (mGluR-LTD), a form of learning and memory, is misregulated in many murine models of neurodevelopmental disorders. Despite extensive studies there is a paucity of information on the molecular mechanism underlying mGluR-LTD. Previously, we reported that loss of synaptopodin, an actin-associated protein found in a subset of mature dendritic spines, impairs mGluR-LTD. In the current study, we uncover the molecular and cellular deficits involved. We find that synaptopodin is required for the mGluR5-Homer interaction and uncover synaptopodin as a molecular switch for mGluR-LTD expression, as mGluR-LTD becomes protein synthesis-independent and relies on endocannabinoid signaling in synaptopodin knock-out. This work provides insight into synaptopodin as a gatekeeper to regulate mGluR-LTD at hippocampal synapses.

Spike timing-dependent plasticity and memory.

Spike timing-dependent plasticity (STDP) is a bidirectional form of synaptic plasticity discovered about 30 years ago and based on the relative timing of pre- and post-synaptic spiking activity with a millisecond precision. STDP is thought to be involved in the formation of memory but the millisecond-precision spike-timing required for STDP is difficult to reconcile with the much slower timescales of behavioral learning. This review therefore aims to expose and discuss recent findings about i) the multiple STDP learning rules at both excitatory and inhibitory synapses in vitro, ii) the contribution of STDP-like synaptic plasticity in the formation of memory in vivo and iii) the implementation of STDP rules in artificial neural networks and memristive devices.

Theta patterns of stimulation induce synaptic and intrinsic potentiation in O-LM interneurons.

Brain oscillations have long-lasting effects on synaptic and cellular properties. For instance, synaptic stimulation at theta (θ) frequency induces persistent depression of both excitatory synaptic transmission and intrinsic excitability in CA1 principal neurons. However, the incidence of θ activity on synaptic transmission and intrinsic excitability in hippocampal GABAergic interneurons is unclear. We report here the induction of both synaptic and intrinsic potentiation in oriens-lacunosum moleculare (O-LM) interneurons following stimulation of afferent glutamatergic inputs in the θ frequency range (∼5 Hz). Long-term synaptic potentiation (LTP) is induced by synaptic activation of calcium-permeable AMPA receptors (CP-AMPAR), whereas long-term potentiation of intrinsic excitability (LTP-IE) results from the mGluR1-dependent down-regulation of Kv7 voltage-dependent potassium channel and hyperpolarization activated and cyclic nucleotide-gated (HCN) channel through the depletion of phosphatidylinositol-4,5-biphosphate (PIP2). LTP and LTP-IE are reversible, demonstrating that both synaptic and intrinsic changes are bidirectional in O-LM cells. We conclude that synaptic activity at θ frequency induces both synaptic and intrinsic potentiation in O-LM interneurons, i.e., the opposite of what is typically seen in glutamatergic neurons.

Stabilization of Spine Synaptopodin by mGluR1 Is Required for mGluR-LTD.

Dendritic spines, actin-rich protrusions forming the postsynaptic sites of excitatory synapses, undergo activity-dependent molecular and structural remodeling. Activation of Group 1 metabotropic glutamate receptors (mGluR1 and mGluR5) by synaptic or pharmacological stimulation, induces LTD, but whether this is accompanied with spine elimination remains unresolved. A subset of telencephalic mushroom spines contains the spine apparatus (SA), an enigmatic organelle composed of stacks of smooth endoplasmic reticulum, whose formation depends on the expression of the actin-bundling protein Synaptopodin. Allocation of Synaptopodin to spines appears governed by cell-intrinsic mechanisms as the relative frequency of spines harboring Synaptopodin is conserved in vivo and in vitro Here we show that expression of Synaptopodin/SA in spines is required for induction of mGluR-LTD at Schaffer collateral-CA1 synapses of male mice. Post-mGluR-LTD, mushroom spines lacking Synaptopodin/SA are selectively lost, whereas spines harboring it are preserved. This process, dependent on activation of mGluR1 but not mGluR5, is conserved in mature mouse neurons and rat neurons of both sexes. Mechanistically, we find that mGluR1 supports physical retention of Synaptopodin within excitatory spine synapses during LTD while triggering lysosome-dependent degradation of the protein residing in dendritic shafts. Together, these results reveal a cellular mechanism, dependent on mGluR1, which enables selective preservation of stronger spines containing Synaptopodin/SA while eliminating weaker ones and potentially countering spurious strengthening by de novo recruitment of Synaptopodin. Overall, our results identify spines with Synaptopodin/SA as the locus of mGluR-LTD and underscore the importance of the molecular microanatomy of spines in synaptic plasticity.SIGNIFICANCE STATEMENT Long-term changes in functional synaptic strength are associated with modification of synaptic connectivity through stabilization or elimination of dendritic spines, the postsynaptic locus of excitatory synapses. How heterogeneous spine microanatomy instructs spine remodeling after long-term synaptic depression (LTD) remains unclear. Metabotropic glutamate receptors mGluR1 and mGluR5 induce a form of LTD critical to circuit function in physiological and disease conditions. Our results identify spines containing the protein Synaptopodin, which enables local assembly of a spine apparatus, as the locus of expression of mGluR-LTD and demonstrate a specific role of mGluR1 in promoting selective loss after mGluR-LTD of mature dendritic spines lacking Synaptopodin/spine apparatus. These findings highlight the fundamental contribution of spine microanatomy in selectively enabling functional and structural plasticity.

Morphological Determinants of Cell-to-Cell Variations in Action Potential Dynamics in Substantia Nigra Dopaminergic Neurons.

Action potential (AP) shape is a critical electrophysiological parameter, in particular because it strongly modulates neurotransmitter release. As it greatly varies between neuronal types, AP shape is also used to distinguish neuronal populations. For instance, AP duration ranges from hundreds of microseconds in cerebellar granule cells to 2-3 ms in SNc dopaminergic (DA) neurons. While most of this variation across cell types seems to arise from differences in the voltage- and calcium-gated ion channels expressed, a few studies suggested that dendritic morphology also affects AP shape. AP duration also displays significant variability in a same neuronal type, although the determinants of these variations are poorly known. Using electrophysiological recordings, morphological reconstructions, and realistic Hodgkin-Huxley modeling, we investigated the relationships between dendritic morphology and AP shape in rat SNc DA neurons from both sexes. In this neuronal type where the axon arises from an axon-bearing dendrite (ABD), the duration of the somatic AP could be predicted from a linear combination of the ABD and non-ABDs' complexities. Dendrotomy experiments and simulation showed that these correlations arise from the causal influence of dendritic topology on AP duration, due in particular to a high density of sodium channels in the somatodendritic compartment. Surprisingly, computational modeling suggested that this effect arises from the influence of sodium currents on the decaying phase of the AP. Consistent with previous findings, these results demonstrate that dendritic morphology plays a major role in defining the electrophysiological properties of SNc DA neurons and their cell-to-cell variations.SIGNIFICANCE STATEMENT Action potential (AP) shape is a critical electrophysiological parameter, in particular because it strongly modulates neurotransmitter release. AP shape (e.g., duration) greatly varies between neuronal types but also within a same neuronal type. While differences in ion channel expression seem to explain most of AP shape variation across cell types, the determinants of cell-to-cell variations in a same neuronal type are mostly unknown. We used electrophysiological recordings, neuronal reconstruction, and modeling to show that, because of the presence of sodium channels in the somatodendritic compartment, a large part of cell-to-cell variations in somatic AP duration in substantia nigra pars compacta dopaminergic neurons is explained by variations in dendritic topology.

Calcium and Spike Timing-Dependent Plasticity.

Since its discovery, spike timing-dependent synaptic plasticity (STDP) has been thought to be a primary mechanism underlying the brain's ability to learn and to form new memories. However, despite the enormous interest in both the experimental and theoretical neuroscience communities in activity-dependent plasticity, it is still unclear whether plasticity rules inferred from in vitro experiments apply to in vivo conditions. Among the multiple reasons why plasticity rules in vivo might differ significantly from in vitro studies is that extracellular calcium concentration use in most studies is higher than concentrations estimated in vivo. STDP, like many forms of long-term synaptic plasticity, strongly depends on intracellular calcium influx for its induction. Here, we discuss the importance of considering physiological levels of extracellular calcium concentration to study functional plasticity.

Endocannabinoids Tune Intrinsic Excitability in O-LM Interneurons by Direct Modulation of Postsynaptic Kv7 Channels.

KCNQ-Kv7 channels are found at the axon initial segment of pyramidal neurons, where they control cell firing and membrane potential. In oriens lacunosum moleculare (O-LM) interneurons, these channels are mainly expressed in the dendrites, suggesting a peculiar function of Kv7 channels in these neurons. Here, we show that Kv7 channel activity is upregulated following induction of presynaptic long-term synaptic depression (LTD) in O-LM interneurons from rats of both sex, thus resulting in a synergistic long-term depression of intrinsic excitability (LTD-IE). Both LTD and LTD-IE involve endocannabinoid (eCB) biosynthesis for induction. However, although LTD is dependent on cannabinoid type 1 receptors, LTD-IE is not. Molecular modeling shows a strong interaction of eCBs with Kv7.2/3 channel, suggesting a persistent action of these lipids on Kv7 channel activity. Our data thus unveil a major role for eCB synthesis in triggering both synaptic and intrinsic depression in O-LM interneurons.SIGNIFICANCE STATEMENT In principal cells, Kv7 channels are essentially located at the axon initial segment. In contrast, in O-LM interneurons, Kv7 channels are highly expressed in the dendrites, suggesting a singular role of these channels in O-LM cell function. Here, we show that LTD of excitatory inputs in O-LM interneurons is associated with an upregulation of Kv7 channels, thus resulting in a synergistic LTD of LTD-IE. Both forms of plasticity are mediated by the biosynthesis of eCBs. Stimulation of CB1 receptors induces LTD, whereas the direct interaction of eCBs with Kv7 channels induces LTD-IE. Our results thus provide a previously unexpected involvement of eCBs in long-lasting plasticity of intrinsic excitability in GABAergic interneurons.

Synaptic plasticity rules with physiological calcium levels.

Spike-timing-dependent plasticity (STDP) is considered as a primary mechanism underlying formation of new memories during learning. Despite the growing interest in activity-dependent plasticity, it is still unclear whether synaptic plasticity rules inferred from in vitro experiments are correct in physiological conditions. The abnormally high calcium concentration used in in vitro studies of STDP suggests that in vivo plasticity rules may differ significantly from in vitro experiments, especially since STDP depends strongly on calcium for induction. We therefore studied here the influence of extracellular calcium on synaptic plasticity. Using a combination of experimental (patch-clamp recording and Ca2+ imaging at CA3-CA1 synapses) and theoretical approaches, we show here that the classic STDP rule in which pairs of single pre- and postsynaptic action potentials induce synaptic modifications is not valid in the physiological Ca2+ range. Rather, we found that these pairs of single stimuli are unable to induce any synaptic modification in 1.3 and 1.5 mM calcium and lead to depression in 1.8 mM. Plasticity can only be recovered when bursts of postsynaptic spikes are used, or when neurons fire at sufficiently high frequency. In conclusion, the STDP rule is profoundly altered in physiological Ca2+, but specific activity regimes restore a classical STDP profile.

Axonal Na+ channels detect and transmit levels of input synchrony in local brain circuits.

Sensory processing requires mechanisms of fast coincidence detection to discriminate synchronous from asynchronous inputs. Spike threshold adaptation enables such a discrimination but is ineffective in transmitting this information to the network. We show here that presynaptic axonal sodium channels read and transmit precise levels of input synchrony to the postsynaptic cell by modulating the presynaptic action potential (AP) amplitude. As a consequence, synaptic transmission is facilitated at cortical synapses when the presynaptic spike is produced by synchronous inputs. Using dual soma-axon recordings, imaging, and modeling, we show that this facilitation results from enhanced AP amplitude in the axon due to minimized inactivation of axonal sodium channels. Quantifying local circuit activity and using network modeling, we found that spikes induced by synchronous inputs produced a larger effect on network activity than spikes induced by asynchronous inputs. Therefore, this input synchrony-dependent facilitation may constitute a powerful mechanism, regulating synaptic transmission at proximal synapses.

Plasticity of intrinsic neuronal excitability.

Long-term synaptic modification is not the exclusive mode of memory storage, and persistent regulation of voltage-gated ion channels also participates in memory formation. Intrinsic plasticity is expressed in virtually all neuronal types including principal cells and interneurons. Activation of synaptic glutamate receptors initiates long-lasting changes in neuronal excitability at presynaptic and postsynaptic side. As synaptic plasticity, intrinsic plasticity is bi-directional and expresses a certain level of input-specificity or cell-specificity. We discuss here the nature of the learning rules shared by intrinsic and synaptic plasticity and the impact of intrinsic plasticity on temporal processing.

Plasticity of intrinsic excitability during LTD is mediated by bidirectional changes in h-channel activity.

The polarity of excitability changes associated with induction of Long-Term synaptic Depression (LTD) in CA1 pyramidal neurons is a contentious issue. Postsynaptic neuronal excitability after LTD induction is found to be reduced in certain cases (i.e. synergistic changes) but enhanced in others (i.e. compensatory or homeostatic). We examined here whether these divergent findings could result from the activation of two separate mechanisms converging onto a single learning rule linking synergistic and homeostatic plasticity. We show that the magnitude of LTD induced with low frequency stimulation (LFS) of the Schaffer collaterals determines the polarity of intrinsic changes in CA1 pyramidal neurons. Apparent input resistance (Rin) is reduced following induction of moderate LTD (<20-30%). In contrast, Rin is increased after induction of large LTD (>40%) induced by repetitive episodes of LFS. The up-regulation of I h observed after moderate LTD results from the activation of NMDA receptors whereas the down-regulation of I h is due to activation of mGluR1 receptors. These changes in Rin were associated with changes in intrinsic excitability. In conclusion, our study indicates that changes in excitability after LTD induction follow a learning rule describing a continuum linking synergistic and compensatory changes in excitability.

Homeostatic regulation of h-conductance controls intrinsic excitability and stabilizes the threshold for synaptic modification in CA1 neurons.

KEY POINTS: We determined the contribution of the hyperpolarization-activated cationic (h) current (Ih ) to the homeostatic regulation of CA1 pyramidal cells in vitro using chronic treatments (48 h) that either increase (picrotoxin) or decrease (kynurenate) neuronal activity. The h-conductance was found to be up- or down-regulated following chronic activity enhancement or activity deprivation, respectively. This bidirectional plasticity of Ih was found to subsequently alter both apparent input resistance and intrinsic neuronal excitability. Bidirectional homeostatic plasticity of Ih also determined EPSP waveform and EPSP summation tested at 5-30 Hz. Long-term synaptic modification induced by repetitive stimulation of the Schaffer collaterals was found to be constant across treatments in the presence of Ih but not when Ih was blocked pharmacologically. Thus, bidirectional homeostatic regulation of Ih stabilizes induction of long-term synaptic modification in CA1 pyramidal neurons that depends on EPSP summation. ABSTRACT: The hyperpolarization-activated cationic (h) current is a voltage-shock absorber, highly expressed in the dendrites of CA1 pyramidal neurons. Up-regulation of Ih has been reported following episodes of intense network activity but the effect of activity deprivation on Ih and the functional consequence of homeostatic regulation of Ih remain unclear. We determined here the contribution of Ih to the homeostatic regulation of CA1 pyramidal cell excitability. Intrinsic neuronal excitability was decreased in neurons treated for 2-3 days with the GABAA channel blocker picrotoxin (PiTx) but increased in neurons treated (2-3 days) with the glutamate receptor antagonist kynurenate (Kyn). Membrane capacitance remained unchanged after treatment but the apparent input resistance was reduced for PiTx-treated neurons and enhanced for Kyn-treated neurons. Maximal Ih conductance was up-regulated after chronic hyperactivity but down-regulated following chronic hypoactivity. Up-regulation of Ih in PiTx-treated cultures was found to accelerate EPSP kinetics and reduce temporal summation of EPSPs whereas opposite effects were observed in Kyn-treated cultures, indicating that homeostatic regulation of Ih may control the induction of synaptic modification depending on EPSP summation. In fact, stimulation of the Schaffer collaterals at 3-10 Hz induced differential levels of plasticity in PiTx-treated and Kyn-treated neurons when Ih was blocked pharmacologically but not in control conditions. These data indicate that homeostatic regulation of Ih normalizes the threshold for long-term synaptic modification that depends on EPSP summation. In conclusion, bidirectional homeostatic regulation of Ih not only controls spiking activity but also stabilizes the threshold for long-term potentiation induced in CA1 pyramidal neurons by repetitive stimulation.