Laxmi Kumar Parajuli

Heterosynaptic Structural Plasticity on Local Dendritic Segments of Hippocampal CA1 Neurons

Competition between synapses contributes to activity-dependent refinement of the nervous system during development. Does local competition between neighboring synapses drive circuit remodeling during experience-dependent plasticity in the cerebral cortex? Here, we examined the role of activity-mediated competitive interactions in regulating dendritic spine structure and function on hippocampal CA1 neurons. We found that high-frequency glutamatergic stimulation at individual spines, which leads to input-specific synaptic potentiation, induces shrinkage and weakening of nearby unstimulated synapses. This heterosynaptic plasticity requires potentiation of multiple neighboring spines, suggesting that a local threshold of neural activity exists beyond which inactive synapses are punished. Notably, inhibition of calcineurin, IP3Rs, or group I metabotropic glutamate receptors (mGluRs) blocked heterosynaptic shrinkage without blocking structural potentiation, and inhibition of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) blocked structural potentiation without blocking heterosynaptic shrinkage. Our results support a model in which activity-induced shrinkage signal, and not competition for limited structural resources, drives heterosynaptic structural and functional depression during neural circuit refinement.

Quantitative regional and ultrastructural localization of the Ca(v)2.3 subunit of R-type calcium channel in mouse brain.

R-type calcium channels (RTCCs) are well known for their role in synaptic plasticity, but little is known about their subcellular distribution across various neuronal compartments. Using subtype-specific antibodies, we characterized the regional and subcellular localization of Cav2.3 in mice and rats at both light and electron microscopic levels. Cav2.3 immunogold particles were found to be predominantly presynaptic in the interpeduncular nucleus, but postsynaptic in other brain regions. Serial section analysis of electron microscopic images from the hippocampal CA1 revealed a higher density of immunogold particles in the dendritic shaft plasma membrane compared with the pyramidal cell somata. However, the labeling densities were not significantly different among the apical, oblique, or basal dendrites. Immunogold particles were also observed over the plasma membrane of dendritic spines, including both synaptic and extrasynaptic sites. Individual spine heads contained <20 immunogold particles, with an average density of ∼260 immunoparticles per μm3 spine head volume, in accordance with the density of RTCCs estimated using calcium imaging (Sabatini and Svoboda, 2000). The Cav2.3 density was variable among similar-sized spine heads and did not correlate with the density in the parent dendrite, implying that spines are individual calcium compartments operating autonomously from their parent dendrites.

Subcellular distribution of α1G subunit of T-type calcium channel in the mouse dorsal lateral geniculate nucleus.

T‐type calcium channels play a pivotal role in regulating neural membrane excitability in the nervous system. However, the precise subcellular distributions of T‐type channel subunits and their implication for membrane excitability are not well understood. Here we investigated the subcellular distribution of the α1G subunit of the calcium channel which is expressed highly in the mouse dorsal lateral geniculate nucleus (dLGN). Light microscopic analysis demonstrated that dLGN exhibits intense immunoperoxidase reactivity for the α1G subunit. Electron microscopic observation showed that the labeling was present in both the relay cells and interneurons and was found in the somatodendritic, but not axonal, domains of these cells. Most of the immunogold particles for the α1G subunit were either associated with the plasma membrane or the intracellular membranes. Reconstruction analysis of serial electron microscopic images revealed that the intensity of the intracellular labeling exhibited a gradient such that the labeling density was higher in the proximal dendrite and progressively decreased towards the distal dendrite. In contrast, the plasma membrane‐associated particles were distributed with a uniform density over the somatodendritic surface of dLGN cells. The labeling density in the relay cell plasma membrane was about 3‐fold higher than that of the interneurons. These results provide ultrastructural evidence for cell‐type‐specific expression levels and for uniform expression density of the α1G subunit over the plasma membrane of dLGN cells. J. Comp. Neurol. 518:4362–4374, 2010.

Cell type-specific spatial and functional coupling between mammalian brain Kv2.1 K+ channels and ryanodine receptors.

The Kv2.1 voltage‐gated K+ channel is widely expressed throughout mammalian brain, where it contributes to dynamic activity‐dependent regulation of intrinsic neuronal excitability. Here we show that somatic plasma membrane Kv2.1 clusters are juxtaposed to clusters of intracellular ryanodine receptor (RyR) Ca2+‐release channels in mouse brain neurons, most prominently in medium spiny neurons (MSNs) of the striatum. Electron microscopy–immunogold labeling shows that in MSNs, plasma membrane Kv2.1 clusters are adjacent to subsurface cisternae, placing Kv2.1 in close proximity to sites of RyR‐mediated Ca2+ release. Immunofluorescence labeling in transgenic mice expressing green fluorescent protein in specific MSN populations reveals the most prominent juxtaposed Kv2.1:RyR clusters in indirect pathway MSNs. Kv2.1 in both direct and indirect pathway MSNs exhibits markedly lower levels of labeling with phosphospecific antibodies directed against the S453, S563, and S603 phosphorylation site compared with levels observed in neocortical neurons, although labeling for Kv2.1 phosphorylation at S563 was significantly lower in indirect pathway MSNs compared with those in the direct pathway. Finally, acute stimulation of RyRs in heterologous cells causes a rapid hyperpolarizing shift in the voltage dependence of activation of Kv2.1, typical of Ca2+/calcineurin‐dependent Kv2.1 dephosphorylation. Together, these studies reveal that striatal MSNs are distinct in their expression of clustered Kv2.1 at plasma membrane sites juxtaposed to intracellular RyRs, as well as in Kv2.1 phosphorylation state. Differences in Kv2.1 expression and phosphorylation between MSNs in direct and indirect pathways provide a cell‐ and circuit‐specific mechanism for coupling intracellular Ca2+ release to phosphorylation‐dependent regulation of Kv2.1 to dynamically impact intrinsic excitability. J. Comp. Neurol. 522:3555–3574, 2014.

Distinct Cell- and Layer-Specific Expression Patterns and Independent Regulation of Kv2 Channel Subtypes in Cortical Pyramidal Neurons.

The Kv2 family of voltage-gated potassium channel α subunits, comprising Kv2.1 and Kv2.2, mediate the bulk of the neuronal delayed rectifier K+ current in many mammalian central neurons. Kv2.1 exhibits robust expression across many neuron types and is unique in its conditional role in modulating intrinsic excitability through changes in its phosphorylation state, which affect Kv2.1 expression, localization, and function. Much less is known of the highly related Kv2.2 subunit, especially in forebrain neurons. Here, through combined use of cortical layer markers and transgenic mouse lines, we show that Kv2.1 and Kv2.2 are localized to functionally distinct cortical cell types. Kv2.1 expression is consistently high throughout all cortical layers, especially in layer (L) 5b pyramidal neurons, whereas Kv2.2 expression is primarily limited to neurons in L2 and L5a. In addition, L4 of primary somatosensory cortex is strikingly devoid of Kv2.2 immunolabeling. The restricted pattern of Kv2.2 expression persists in Kv2.1-KO mice, suggesting distinct cell- and layer-specific functions for these two highly related Kv2 subunits. Analyses of endogenous Kv2.2 in cortical neurons in situ and recombinant Kv2.2 expressed in heterologous cells reveal that Kv2.2 is largely refractory to stimuli that trigger robust, phosphorylation-dependent changes in Kv2.1 clustering and function. Immunocytochemistry and voltage-clamp recordings from outside-out macropatches reveal distinct cellular expression patterns for Kv2.1 and Kv2.2 in intratelencephalic and pyramidal tract neurons of L5, indicating circuit-specific requirements for these Kv2 paralogs. Together, these results support distinct roles for these two Kv2 channel family members in mammalian cortex. SIGNIFICANCE STATEMENT Neurons within the neocortex are arranged in a laminar architecture and contribute to the input, processing, and/or output of sensory and motor signals in a cell- and layer-specific manner. Neurons of different cortical layers express diverse populations of ion channels and possess distinct intrinsic membrane properties. Here, we show that the Kv2 family members Kv2.1 and Kv2.2 are expressed in distinct cortical layers and pyramidal cell types associated with specific corticostriatal pathways. We find that Kv2.1 and Kv2.2 exhibit distinct responses to acute phosphorylation-dependent regulation in brain neurons in situ and in heterologous cells in vitro. These results identify a molecular mechanism that contributes to heterogeneity in cortical neuron ion channel function and regulation.

A dual role for the RhoGEF Ephexin5 in regulation of dendritic spine outgrowth

Abstract The outgrowth of new dendritic spines is closely linked to the formation of new synapses, and is thought to be a vital component of the experience‐dependent circuit plasticity that supports learning. Here, we examined the role of the RhoGEF Ephexin5 in driving activity‐dependent spine outgrowth. We found that reducing Ephexin5 levels increased spine outgrowth, and increasing Ephexin5 levels decreased spine outgrowth in a GEF‐dependent manner, suggesting that Ephexin5 acts as an inhibitor of spine outgrowth. Notably, we found that increased neural activity led to a proteasome‐dependent reduction in the levels of Ephexin5 in neuronal dendrites, which could facilitate the enhanced spine outgrowth observed following increased neural activity. Surprisingly, we also found that Ephexin5‐GFP levels were elevated on the dendrite at sites of future new spines, prior to new spine outgrowth. Moreover, lowering neuronal Ephexin5 levels inhibited new spine outgrowth in response to both global increases in neural activity and local glutamatergic stimulation of the dendrite, suggesting that Ephexin5 is necessary for activity‐dependent spine outgrowth. Our data support a model in which Ephexin5 serves a dual role in spinogenesis, acting both as a brake on overall spine outgrowth and as a necessary component in the site‐specific formation of new spines. HighlightsThe RhoGEF, Ephexin5, is a negative regulator of dendritic spine outgrowth.Neural activity drives a proteasome‐dependent reduction in dendritic Ephexin5.Ephexin5 accumulates on the dendrite prior to new spine outgrowth.Ephexin5 is necessary for neural activity‐dependent spine outgrowth.Ephexin5 serves a dual role in spinogenesis.

Kv2 ion channels determine the expression and localization of the associated AMIGO-1 cell adhesion molecule in adult brain neurons

Voltage-gated K+ (Kv) channels play important roles in regulating neuronal excitability. Kv channels comprise four principal α subunits, and transmembrane and/or cytoplasmic auxiliary subunits that modify diverse aspects of channel function. AMIGO-1, which mediates homophilic cell adhesion underlying neurite outgrowth and fasciculation during development, has recently been shown to be an auxiliary subunit of adult brain Kv2.1-containing Kv channels. We show that AMIGO-1 is extensively colocalized with both Kv2.1 and its paralog Kv2.2 in brain neurons across diverse mammals, and that in adult brain, there is no apparent population of AMIGO-1 outside of that colocalized with these Kv2 α subunits. AMIGO-1 is coclustered with Kv2 α subunits at specific plasma membrane (PM) sites associated with hypolemmal subsurface cisternae at neuronal ER:PM junctions. This distinct PM clustering of AMIGO-1 is not observed in brain neurons of mice lacking Kv2 α subunit expression. Moreover, in heterologous cells, coexpression of either Kv2.1 or Kv2.2 is sufficient to drive clustering of the otherwise uniformly expressed AMIGO-1. Kv2 α subunit coexpression also increases biosynthetic intracellular trafficking and PM expression of AMIGO-1 in heterologous cells, and analyses of Kv2.1 and Kv2.2 knockout mice show selective loss of AMIGO-1 expression and localization in neurons lacking the respective Kv2 α subunit. Together, these data suggest that in mammalian brain neurons, AMIGO-1 is exclusively associated with Kv2 α subunits, and that Kv2 α subunits are obligatory in determining the correct pattern of AMIGO-1 expression, PM trafficking and clustering.

Presynaptic localization of R-type calcium channel in the interpeduncular nucleus

titer of GluA2 N-terminal antibody, and used for quantitative analysis of the five GluK subunits. Analytical western blots showed that amounts of the five KAR subunits were different in each brain region and subcellular fraction. In the crude fractions, GluK2 and GluK5 accounted for 2.3% and 1.8% of the total amount of AMPARs respectively in the hippocampus, while in the cerebellum GluK2 and GluK5 accounted for 2.1% and 0.4% of the total amount, respectively. These data showed that GluK2 was equally abundant in the hippocampus and cerebellum. On the other hand, GluK5 showed much less expression in the cerebellum than in the hippocampus. These results suggest that there exists a large amount of GluK2/ GluK5 heteromeric composition in the hippocampus. Furthermore, there might be other assemblies of KAR subunits rather than GluK2/GluK5 in the cerebellum.

Regional and subcellular distribution of the α1E subunit of voltage-gated calcium channel in the adult mouse brain

Calcium channels play a pivotal role in mediating the synaptic transmission. Among the six different types of calcium channels, the alpha 1A subunit of calcium channel is most abundantly expressed in the cerebellum. The purpose of this study was to investigate the precise subcellular localization of alpha 1A subunit of calcium channel by means of SDS digested freeze fracture replica labeling (SDS-FRL). Based on our previous finding with preembeding immunogold method, at the light microscopic level, immunoreactivity for the alpha 1A protein was prevalent in the molecular layer whereas immunostaining was moderate in the soma of Purkinje cells and weak in the granule cell layer. At the electron microscopic level immunogold particles for alpha 1A were found on protoplasmic face (Pface) of Purkinje cell dendrites and soma. The particles were often seen to be associated with a distinct type of intramembrane particle (IMP) which was bigger in size in compared to the surrounding IMPs. Whereas the majority of the immunogold particles in the Purkinje cell dendrite and soma were seen to form clusters, some others were rather sparsely distributed. For identification of synaptic contact, replicas were double labeled for alpha 1A and AMPA-type glutamate receptors (AMPAR). We found labeling for AMPAR in exoplasmic face (E-face) of postsynaptic site and immunogold particles for alpha 1A were found to be localized to the presynaptic membrane specialization in the adjacent protoplasmic face (P-face) of presynaptic terminals. In alpha 1A knock-out mice, the clusters of IMPs were found without alpha 1A labeling on Purkinje cell soma and dendrite and the labeling in the presynaptic active zone was also gone. These results confirm our previous findings with the preembedding immunogold labeling and further suggest hot spot formation of P/Q type calcium channels on Purkinje cell dendrites. doi:10.1016/j.neures.2010.07.2056 P1-a08 Information processing on the dendrite in hippocampal granule cells Hirofumi Hayakawa 1 , Tadanobu Kamijou 1, Makoto Yoneyama 2, Yasuhiro Fukushima 2, Takeshi Aihara 1 1 Department of Technology, Tamagawa University, Tokyo 2 Brain Science Institute, Tamagawa University, Tokyo Dentate granule cells have inputs though the lateral perforant path (LPP) with non-spatial information, smell etc., and inputs though the medial perforant path (MPP) with spatial information, place. Inputs though the LPP and those though the MPP are projected to the lateral dendrite and the medial dendrite of dentate granule cells from layer II in the entorhinal cortex (EC), respectively. In addition, it has been experimentally confirmed that memory is improved by the presence of smell information. Therefore, there is an possibility that the processing of spatial information is influenced by non-specific information in dentate granule. To investigate how input information with different modalities are integrated on the dendrite of dentate granule cells, two input stimulus were applied to lateral and medial dendrite, respectively. As the result, the responses to the LPP stimulation show different characteristics from that of the MPP stimulation depending on the stimulus frequency; sustained for LPP stimulus and transient for MPP stimulus. Furthermore, nonlinear components of superposed amplitude mediated by LPP stimulus and MPP stimulus were significantly observed only if MPP stimulus was applied before MPP stimulus. The result suggests that there is nonlinear integration on the dendrite of hippocampal dentate granule cells, in which the processing of spatial information is influenced by non-specific information with facilitatory effect. doi:10.1016/j.neures.2010.07.2057