Physiology II

PROJECTS

Functional interaction of metabotropic receptors and ion channels in pre- and postsynaptic compartments of central neurons

Functionally diverse metabotropic GABA_B and glutamate receptors (mGluRs) control neuronal excitability and synaptic transmission by activating or inhibiting various types of ion channels, such as high voltage-activated Ca²⁺ and K⁺ channels. The impact of receptor activation on synaptic integration and regulation of transmitter release depends on the spatial relationship and coupling of receptors and their effectors in subcellular compartments of the target neurons. We have been investigating the structural and functional basis of metabotropic receptor-effector ion channel complex-mediated signaling in cortical principal cells and GABAergic interneurons, as well as studying the activity-dependent regulation of the surface dynamics of protein complexes using a combination of pharmacological and high-resolution quantitative immunoelectron microscopic approaches.

Regulation of intracellular Ca²⁺ concentration in central neurons

Calcium (Ca²⁺) plays a central role in many cellular processes in various cell types including neurons in the central nervous system (CNS). It regulates enzymatic activities and excitability, synaptic plasticity and excitation-transcription coupling, as well as controls release of neurotransmitters from presynaptic boutons (Clapham, 2007). Ca²⁺ influx into axon terminals, somata and dendrites of neurons can mainly be mediated by different high voltage-gated Ca²⁺ (Ca_v) channels (Ca_v1.2, Ca_v2.1, 2.2 and 2.3). These channels localize either to presynaptic membrane specialization of boutons of glutamatergic and GABAergic cells or distributed along the surface of somatic and dendritic membranes and are activated by action potentials and/or sub-threshold depolarizing signals. Calcium overload, however, has been associated with cell injury and cell death. Therefore, it is essential for neurons to control cytoplasmic and stored Ca²⁺ very precisely, both temporally and spatially. The Ca²⁺ extrusion from the cytosol to the extracellular millieu via membrane-bound proteins, such as plasma membrane Ca²⁺-ATPases (PMCAs) and Na⁺/Ca²⁺ exchangers (NCXs), following neuronal activation, appears to be an important way to the maintenance and precise control of cytoplasmic Ca²⁺ balance in both presynaptic and postsynaptic compartments. We combine the high-resolution SDS-FRL immunoelectron microscopy with quantitative analysis of immunoreactivity for those proteins to determine the spatial distribution and surface density of Ca_v channels, PMCAs and NCXs on somato-dendritic and axonal membranes of central neurons.

Dendritic mechanisms underlying a sparse spatial code in the dentate gyrus

The hippocampus is essential for encoding the spatial features of the environment in which our daily experience is embedded. Formation of this neuronal code depends on activity-dependent changes in synaptic strength after synchronous activation of excitatory inputs that generate local dendritic activity. This process is under tight dendritic inhibitory control, which gates the generation of dendritic non-linearities and controls the assimilation of neurons into a neuronal engram. In the dentate gyrus, only a small subset of the granule cell population takes part in a cell assembly encoding a spatial context, a necessary condition for the pattern separation function of the dentate gyrus. We hypothesize that during encoding of spatial information, granule cell dendrites undergo activity- and input-dependent changes in their morphological and physiological properties that affect the mechanism of synaptic integration and contribute to the emergence of dentate gyrus engrams. To investigate this hypothesis, we will combine the complementary expertise of Claudio Elgueta’s and my research groups to understand at the synaptic, cellular and network levels, the molecular and physiological mechanisms by which dendritic excitatory and inhibitory synaptic inputs control the emergence of dentate gyrus engrams.

Research techniques

• Conventional immunoelectron microscopic methods: pre-embedding immunoperoxidase and immunogold labeling.
• Sodium dodecyl sulfate (SDS)-digested freeze-fracture replica labeling (SDS-FRL) combined with sample analysis with transmission electron microscop (TEM).
• Three-dimensional (3D) reconstruction of immunolabeled pre- and postsynaptic compartments of central neurons.
• Focused Ion Beam Scanning Electron Microscopy (FIB/SEM).
• Pharmacological manipulation of receptors and ion channels.
• Quantitative analysis of surface density and distribution pattern of signaling molecules using automatized computational cluster analysis, smoothed distance transform analysis and bivariate extension of Ripley’ H-analysis.