The Dendrites of CA2 and CA1 Pyramidal Neurons Differentially Regulate Information Flow in the Cortico-Hippocampal Circuit.

The impact of a given neuronal pathway depends on the number of synapses it makes with its postsynaptic target, the strength of each individual synapse, and the integrative properties of the postsynaptic dendrites. Here we explore the cellular and synaptic mechanisms responsible for the differential excitatory drive from the entorhinal cortical pathway onto mouse CA2 compared with CA1 pyramidal neurons (PNs). Although both types of neurons receive direct input from entorhinal cortex onto their distal dendrites, these inputs produce a 5- to 6-fold larger EPSP at the soma of CA2 compared with CA1 PNs, which is sufficient to drive action potential output from CA2 but not CA1. Experimental and computational approaches reveal that dendritic propagation is more efficient in CA2 than CA1 as a result of differences in dendritic morphology and dendritic expression of the hyperpolarization-activated cation current (Ih). Furthermore, there are three times as many cortical inputs onto CA2 compared with CA1 PN distal dendrites. Using a computational model, we demonstrate that the differences in dendritic properties of CA2 compared with CA1 PNs are necessary to enable the CA2 PNs to generate their characteristically large EPSPs in response to their cortical inputs; in contrast, CA1 dendritic properties limit the size of the EPSPs they generate, even to a similar number of cortical inputs. Thus, the matching of dendritic integrative properties with the density of innervation is crucial for the differential processing of information from the direct cortical inputs by CA2 compared with CA1 PNs.SIGNIFICANCE STATEMENT Recent discoveries have shown that the long-neglected hippocampal CA2 region has distinct synaptic properties and plays a prominent role in social memory and schizophrenia. This study addresses the puzzling finding that the direct entorhinal cortical inputs to hippocampus, which target the very distal pyramidal neuron dendrites, provide an unusually strong excitatory drive at the soma of CA2 pyramidal neurons, with EPSPs that are 5-6 times larger than those in CA1 pyramidal neurons. We here elucidate synaptic and dendritic mechanisms that account quantitatively for the marked difference in EPSP size. Our findings further demonstrate the general importance of fine-tuning the integrative properties of neuronal dendrites to their density of synaptic innervation.

Medial and Lateral Entorhinal Cortex Differentially Excite Deep versus Superficial CA1 Pyramidal Neurons.

Although hippocampal CA1 pyramidal neurons (PNs) were thought to comprise a uniform population, recent evidence supports two distinct sublayers along the radial axis, with deep neurons more likely to form place cells than superficial neurons. CA1 PNs also differ along the transverse axis with regard to direct inputs from entorhinal cortex (EC), with medial EC (MEC) providing spatial information to PNs toward CA2 (proximal CA1) and lateral EC (LEC) providing non-spatial information to PNs toward subiculum (distal CA1). We demonstrate that the two inputs differentially activate the radial sublayers and that this difference reverses along the transverse axis, with MEC preferentially targeting deep PNs in proximal CA1 and LEC preferentially exciting superficial PNs in distal CA1. This differential excitation reflects differences in dendritic spine numbers. Our results reveal a heterogeneity in EC-CA1 connectivity that may help explain differential roles of CA1 PNs in spatial and non-spatial learning and memory.

Dendritic Na+ spikes enable cortical input to drive action potential output from hippocampal CA2 pyramidal neurons.

Synaptic inputs from different brain areas are often targeted to distinct regions of neuronal dendritic arbors. Inputs to proximal dendrites usually produce large somatic EPSPs that efficiently trigger action potential (AP) output, whereas inputs to distal dendrites are greatly attenuated and may largely modulate AP output. In contrast to most other cortical and hippocampal neurons, hippocampal CA2 pyramidal neurons show unusually strong excitation by their distal dendritic inputs from entorhinal cortex (EC). In this study, we demonstrate that the ability of these EC inputs to drive CA2 AP output requires the firing of local dendritic Na+ spikes. Furthermore, we find that CA2 dendritic geometry contributes to the efficient coupling of dendritic Na+ spikes to AP output. These results provide a striking example of how dendritic spikes enable direct cortical inputs to overcome unfavorable distal synaptic locale to trigger axonal AP output and thereby enable efficient cortico-hippocampal information flow.

A cortico-hippocampal learning rule shapes inhibitory microcircuit activity to enhance hippocampal information flow.

How does coordinated activity between distinct brain regions implement a set of learning rules to sculpt information processing in a given neural circuit? Using interneuron cell-type-specific optical activation and pharmacogenetic silencing in vitro, we show that temporally precise pairing of direct entorhinal perforant path (PP) and hippocampal Schaffer collateral (SC) inputs to CA1 pyramidal cells selectively suppresses SC-associated perisomatic inhibition from cholecystokinin (CCK)-expressing interneurons. The CCK interneurons provide a surprisingly strong feedforward inhibitory drive to effectively control the coincident excitation of CA1 pyramidal neurons by convergent inputs. Thus, in-phase cortico-hippocampal activity provides a powerful heterosynaptic learning rule for long-term gating of information flow through the hippocampal excitatory macrocircuit by the silencing of the CCK inhibitory microcircuit.

A study of epileptogenic network structures in rat hippocampal cultures using first spike latencies during synchronization events.

Study of hypersynchronous activity is of prime importance for combating epilepsy. Studies on network structure typically reconstruct the network by measuring various aspects of the interaction between neurons and subsequently measure the properties of the reconstructed network. In sub-sampled networks such methods lead to significant errors in reconstruction. Using rat hippocampal neurons cultured on a multi-electrode array dish and a glutamate injury model of epilepsy in vitro, we studied synchronous activity in neuronal networks. Using the first spike latencies in various neurons during a network burst, we extract various recurring spatio-temporal onset patterns in the networks. Comparing the patterns seen in control and injured networks, we observe that injured networks express a wide diversity in their foci (origin) and activation pattern, while control networks show limited diversity. Furthermore, we note that onset patterns in glutamate injured networks show a positive correlation between synchronization delay and physical distance between neurons, while control networks do not.

Epileptiform activity induces distance-dependent alterations of the Ca2+ extrusion mechanism in the apical dendrites of subicular pyramidal neurons.

The cellular and molecular mechanisms that underlie acquired changes in Ca(2+) dynamics of different neuronal compartments are important in the induction and maintenance of epileptiform activity. Simultaneous electrophysiology and Ca(2+) imaging techniques were used to understand the basic properties of dendritic Ca(2+) signaling in rat subicular pyramidal neurons during epileptiform activity. Distance-dependent changes in the Ca(2+) decay kinetics locked to spontaneous epileptiform discharges and back-propagating action potentials were observed in the apical dendrites. A decrement in the mean tau value of Ca(2+) decay was observed in distal parts (95-110 mum) of the apical dendrites compared with proximal segments (30-45 mum) in in-vitro epileptic conditions but not in control. Pharmacological agents that block Ca(2+) transporters, i.e. Na(+)/ Ca(2+) exchangers (Benzamil), plasma membrane Ca(2+)-ATPase pumps (Calmidazolium) and smooth endoplasmic reticulum Ca(2+)-ATPase pumps (Thapsigargin), were applied locally to the proximal and distal part of the apical dendrites in both experimental conditions to understand the molecular aspects of the Ca(2+) extrusion mechanisms. The relative contribution of Na(+)/Ca(2+) exchangers in Ca(2+) extrusion was higher in the distal apical dendrites in the in-vitro epileptic condition and this property modulated the excitability of the neuron in simulation. The Ca(2+) homeostatic mechanisms that restore normal Ca(2+) levels could play a major neuroprotective role in the distal dendrites that receive synaptic inputs.

Small-world network topology of hippocampal neuronal network is lost, in an in vitro glutamate injury model of epilepsy.

Neuronal network topologies and connectivity patterns were explored in control and glutamate-injured hippocampal neuronal networks, cultured on planar multielectrode arrays. Spontaneous activity was characterized by brief episodes of synchronous firing at many sites in the array (network bursts). During such assembly activity, maximum numbers of neurons are known to interact in the network. After brief glutamate exposure followed by recovery, neuronal networks became hypersynchronous and fired network bursts at higher frequency. Connectivity maps were constructed to understand how neurons communicate during a network burst. These maps were obtained by analysing the spike trains using cross-covariance analysis and graph theory methods. Analysis of degree distribution, which is a measure of direct connections between electrodes in a neuronal network, showed exponential and Gaussian distributions in control and glutamate-injured networks, respectively. Although both the networks showed random features, small-world properties in these networks were different. These results suggest that functional two-dimensional neuronal networks in vitro are not scale-free. After brief exposure to glutamate, normal hippocampal neuronal networks became hyperexcitable and fired a larger number of network bursts with altered network topology. The small-world network property was lost and this was accompanied by a change from an exponential to a Gaussian network.

High level expression of peptides and proteins using cytochrome b5 as a fusion host.

A novel fusion protein system based on the highly soluble heme-binding domain of cytochrome b5 has been designed. The ability of cytochrome b5 to increase the levels of expression and solubility of target proteins has been tested by expressing several proteins and peptides, viz., alpha hemoglobin stabilizing protein, the regulatory subunits of acetohydroxy acid synthase I (ilvM) and II (ilvN), the carboxy terminal domains of mouse neuronal kinesin and pantothenate synthatase, two peptide toxins from cone snails, and the inactivation gate from the brain voltage gated sodium channel, NaV1.2. The fusion protein system has been designed to incorporate protease cleavage sites for commonly used proteases, viz., enterokinase, Factor Xa, and Tobacco etch virus protease. Accumulation of expressed protein as a function of time may be visually ascertained by the fact that the cells take on a bright red color during the course of induction. In all the cases tested so far, the fusion protein accumulates in the soluble fraction to high levels. A novel purification protocol has been designed to purify the fusion proteins using metal affinity chromatography, without the need of a hexahistidine-tag. Mass spectral analysis has shown that the fusion proteins are of full length. CD studies have shown that the solubilized fusion proteins are structured. The proteins of interest may be cleaved from the parent protein by either chemical or enzymatic means. The results presented here demonstrate the versatility of the cytochrome b5 based fusion system for the production of peptides and small proteins (<15 kDa).