Protein kinase D promotes activity-dependent AMPA receptor endocytosis in hippocampal neurons.

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors (AMPARs) mediate the majority of fast excitatory neurotransmission in the brain. The continuous trafficking of AMPARs into and out of synapses is a core feature of synaptic plasticity, which is considered as the cellular basis of learning and memory. The molecular mechanisms underlying the postsynaptic AMPAR trafficking, however, are still not fully understood. In this work, we demonstrate that the protein kinase D (PKD) family promotes basal and activity-induced AMPAR endocytosis in primary hippocampal neurons. Pharmacological inhibition of PKD increased synaptic levels of GluA1-containing AMPARs, slowed down their endocytic trafficking and increased neuronal network activity. By contrast, ectopic expression of constitutive active PKD decreased the synaptic level of AMPARs, while increasing their colocalization with early endosomes. Our results thus establish an important role for PKD in the regulation of postsynaptic AMPAR trafficking during synaptic plasticity.

Homeostatic plasticity and burst activity are mediated by hyperpolarization-activated cation currents and T-type calcium channels in neuronal cultures.

Homeostatic plasticity stabilizes neuronal networks by adjusting the responsiveness of neurons according to their global activity and the intensity of the synaptic inputs. We investigated the homeostatic regulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) and T-type calcium (CaV3) channels in dissociated and organotypic slice cultures. After 48 h blocking of neuronal activity by tetrodotoxin (TTX), our patch-clamp experiments revealed an increase in the depolarizing voltage sag and post-inhibitory rebound mediated by HCN and CaV3 channels, respectively. All HCN subunits (HCN1 to 4) and T-type Ca-channel subunits (CaV3.1, 3.2 and 3.3) were expressed in both control and activity-deprived hippocampal cultures. Elevated expression levels of CaV3.1 mRNA and a selective increase in the expression of TRIP8b exon 4 isoforms, known to regulate HCN channel localization, were also detected in TTX-treated cultured hippocampal neurons. Immunohistochemical staining in TTX-treated organotypic slices verified a more proximal translocation of HCN1 channels in CA1 pyramidal neurons. Computational modeling also implied that HCN and T-type calcium channels have important role in the regulation of synchronized bursting evoked by previous activity-deprivation. Thus, our findings indicate that HCN and T-type Ca-channels contribute to the homeostatic regulation of excitability and integrative properties of hippocampal neurons.

Learning to Sort: Few-shot Spike Sorting with Adversarial Representation Learning.

Spike sorting has long been used to obtain activities of single neurons from multi-unit recordings by extracting spikes from continuous data and assigning them to putative neurons. A large body of spike sorting algorithms have been developed that typically project spikes into a low-dimensional feature space and cluster them through iterative computations. However, there is no reached consensus on the optimal feature space or the best way of segmenting spikes into clusters, which often leads to the requirement of human intervention. It is hence desirable to effectively and efficiently utilize human knowledge in spike sorting while keeping a minimum level of manual intervention. Furthermore, the iterative computations that are commonly involved during clustering are inherently slow and hinder real-time processing of large-scale recordings. In this paper, we propose a novel few-shot spike sorting paradigm that employs a deep adversarial representation neural network to learn from a handful of annotated spikes and robustly classify unseen spikes sharing similar properties to the labeled ones. Once trained, the deep neural network can implement a parametric function that encodes analytically the categorical distribution of spike clusters, which can be significantly accelerated by GPUs and support processing hundreds of thousands of recording channels in real time. The paradigm also includes a clustering routine termed DidacticSortto aid users for labeling spikes that will be used to train the deep neural network. We have validated the performance of the proposed paradigm with both synthetic and in vitro datasets.

Frequency-dependent regulation of intrinsic excitability by voltage-activated membrane conductances, computational modeling and dynamic clamp.

As one of the most unique properties of nerve cells, their intrinsic excitability allows them to transform synaptic inputs into action potentials. This process reflects a complex interplay between the synaptic inputs and the voltage-dependent membrane currents of the postsynaptic neuron. While neurons in natural conditions mostly fire under the action of intense synaptic bombardment and receive fluctuating patterns of excitation and inhibition, conventional techniques to characterize intrinsic excitability mainly utilize static means of stimulation. Recently, we have shown that voltage-gated membrane currents regulate the firing responses under current step stimulation and under physiologically more realistic inputs in a differential manner. At the same time, a multitude of neuron types have been shown to exhibit some form of subthreshold resonance that potentially allows them to respond to synaptic inputs in a frequency-selective manner. In this study, we performed virtual experiments in computational models of neurons to examine how specific voltage-gated currents regulate their excitability under simulated frequency-modulated synaptic inputs. The model simulations and subsequent dynamic clamp experiments on mouse hippocampal pyramidal neurons revealed that the impact of voltage-gated currents in regulating the firing output is strongly frequency-dependent and mostly affecting the synaptic integration at theta frequencies. Notably, robust frequency-dependent regulation of intrinsic excitability was observed even when conventional analysis of membrane impedance suggested no such tendency. Consequently, plastic or homeostatic regulation of intrinsic membrane properties can tune the frequency selectivity of neuron populations in a way that is not readily expected from subthreshold impedance measurements.

Ras and Rab interactor 1 controls neuronal plasticity by coordinating dendritic filopodial motility and AMPA receptor turnover.

Ras and Rab interactor 1 (RIN1) is predominantly expressed in the nervous system. RIN1-knockout animals have deficits in latent inhibition and fear extinction in the amygdala, suggesting a critical role for RIN1 in preventing the persistence of unpleasant memories. At the molecular level, RIN1 signals through Rab5 GTPases that control endocytosis of cell-surface receptors and Abl nonreceptor tyrosine kinases that participate in actin cytoskeleton remodeling. Here we report that RIN1 controls the plasticity of cultured mouse hippocampal neurons. Our results show that RIN1 affects the morphology of dendritic protrusions and accelerates dendritic filopodial motility through an Abl kinase-dependent pathway. Lack of RIN1 results in enhanced mEPSC amplitudes, indicating an increase in surface AMPA receptor levels compared with wild-type neurons. We further provide evidence that the Rab5 GEF activity of RIN1 regulates surface GluA1 subunit endocytosis. Consequently loss of RIN1 blocks surface AMPA receptor down-regulation evoked by chemically induced long-term depression. Our findings indicate that RIN1 destabilizes synaptic connections and is a key player in postsynaptic AMPA receptor endocytosis, providing multiple ways of negatively regulating memory stabilization during neuronal plasticity.

Protein kinase D promotes plasticity-induced F-actin stabilization in dendritic spines and regulates memory formation.

Actin turnover in dendritic spines influences spine development, morphology, and plasticity, with functional consequences on learning and memory formation. In nonneuronal cells, protein kinase D (PKD) has an important role in stabilizing F-actin via multiple molecular pathways. Using in vitro models of neuronal plasticity, such as glycine-induced chemical long-term potentiation (LTP), known to evoke synaptic plasticity, or long-term depolarization block by KCl, leading to homeostatic morphological changes, we show that actin stabilization needed for the enlargement of dendritic spines is dependent on PKD activity. Consequently, impaired PKD functions attenuate activity-dependent changes in hippocampal dendritic spines, including LTP formation, cause morphological alterations in vivo, and have deleterious consequences on spatial memory formation. We thus provide compelling evidence that PKD controls synaptic plasticity and learning by regulating actin stability in dendritic spines.