Prateep Beed

Synaptic PRG-1 Modulates Excitatory Transmission via Lipid Phosphate-Mediated Signaling

Plasticity related gene-1 (PRG-1) is a brain-specific membrane protein related to lipid phosphate phosphatases, which acts in the hippocampus specifically at the excitatory synapse terminating on glutamatergic neurons. Deletion of prg-1 in mice leads to epileptic seizures and augmentation of EPSCs, but not IPSCs. In utero electroporation of PRG-1 into deficient animals revealed that PRG-1 modulates excitation at the synaptic junction. Mutation of the extracellular domain of PRG-1 crucial for its interaction with lysophosphatidic acid (LPA) abolished the ability to prevent hyperexcitability. As LPA application in vitro induced hyperexcitability in wild-type but not in LPA(2) receptor-deficient animals, and uptake of phospholipids is reduced in PRG-1-deficient neurons, we assessed PRG-1/LPA(2) receptor-deficient animals, and found that the pathophysiology observed in the PRG-1-deficient mice was fully reverted. Thus, we propose PRG-1 as an important player in the modulatory control of hippocampal excitability dependent on presynaptic LPA(2) receptor signaling.Plasticity related gene-1 (PRG-1) is a brain-specific membrane protein related to lipid phosphate phosphatases, which acts in the hippocampus specifically at the excitatory synapse terminating on glutamatergic neurons. Deletion of prg-1 in mice leads to epileptic seizures and augmentation of EPSCs, but not IPSCs. In utero electroporation of PRG-1 into deficient animals revealed that PRG-1 modulates excitation at the synaptic junction. Mutation of the extracellular domain of PRG-1 crucial for its interaction with lysophosphatidic acid (LPA) abolished the ability to prevent hyperexcitability. As LPA application in vitro induced hyperexcitability in wild-type but not in LPA(2) receptor-deficient animals, and uptake of phospholipids is reduced in PRG-1-deficient neurons, we assessed PRG-1/LPA(2) receptor-deficient animals, and found that the pathophysiology observed in the PRG-1-deficient mice was fully reverted. Thus, we propose PRG-1 as an important player in the modulatory control of hippocampal excitability dependent on presynaptic LPA(2) receptor signaling.

Inhibitory Gradient along the Dorsoventral Axis in the Medial Entorhinal Cortex

Local inhibitory microcircuits in the medial entorhinal cortex (MEC) and their role in network activity are little investigated. Using a combination of electrophysiological, optical, and morphological circuit analysis tools, we find that layer II stellate cells are embedded in a dense local inhibitory microcircuit. Specifically, we report a gradient of inhibitory inputs along the dorsoventral axis of the MEC, with the majority of this local inhibition arising from parvalbumin positive (PV+) interneurons. Finally, the gradient of PV+ fibers is accompanied by a gradient in the power of extracellular network oscillations in the gamma range, measured both in vitro and in vivo. The reported differences in the inhibitory microcircuitry in layer II of the MEC may therefore have a profound functional impact on the computational working principles at different locations of the entorhinal network and influence the input pathways to the hippocampus.

Analysis of Excitatory Microcircuitry in the Medial Entorhinal Cortex Reveals Cell-Type-Specific Differences

Medial entorhinal cortex (MEC) plays an important role in physiological processes underlying navigation, learning, and memory. Excitatory cells in the different MEC layers project in a region-specific manner to the hippocampus. However, the intrinsic microcircuitry of the main excitatory cells in the superficial MEC layers is largely unknown. Using scanning photostimulation, we investigated the functional microcircuitry of two such cell types, stellate and pyramidal cells. We found cell-type-specific intralaminar and ascending interlaminar feedback inputs. The ascending interlaminar inputs display distinct organizational principles depending on the cell-type and its position within the superficial lamina: the spatial spread of inputs for stellate cells is narrower than for pyramidal cells, while inputs to pyramidal cells in layer 3, but not in layer 2, exhibit an asymmetric offset to the medial side of the cells main axis. Differential laminar sources of excitatory inputs might contribute to the functional diversity of stellate and pyramidal cells.

All Layers of Medial Entorhinal Cortex Receive Presubicular and Parasubicular Inputs

The medial entorhinal cortex (MEC), presubiculum (PrS), and parasubiculum (PaS) are interconnected components of the hippocampal–parahippocampal spatial-representation system. Principal cells in all layers of MEC show signs of directional tuning, overt in head direction cells present in all layers except for layer II, and covert in grid cells, which are the major spatially modulated cell type in layer II. Directional information likely originates in the head direction-vestibular system and PrS and PaS are thought to provide this information to MEC. Efferents from PaS and PrS show a selective laminar terminal distribution in MEC superficial layers II and III, respectively. We hypothesized that this anatomically determined laminar distribution does not preclude monosynaptic interaction with neurons located in deeper layers of MEC in view of the extensive apical dendrites from deeper cells reaching layers II and III. This hypothesis was tested in the rat using tilted in vitro slices in which origins and terminations of PrS and PaS fibers were maintained, as assessed using anterograde anatomical tracing. Based on voltage-sensitive dye imaging, multipatch single-cell recordings, and scanning photostimulation of caged glutamate, we report first that principal neurons in all layers of MEC receive convergent monosynaptic inputs from PrS and PaS and second, that elicited responses show layer-specific decay times and frequency-dependent facilitation. These results indicate that regardless of their selective laminar terminal distribution, PrS and PaS inputs may monosynaptically convey directional information to principal neurons in all layers of MEC through synapses on their extensive dendritic arbors.

Dendritic Compartment and Neuronal Output Mode Determine Pathway-Specific Long-Term Potentiation in the Piriform Cortex

The apical dendrite of layer 2/3 pyramidal cells in the piriform cortex receives two spatially distinct inputs: one projecting onto the distal apical dendrite in sensory layer 1a, the other targeting the proximal apical dendrite in layer 1b. We observe an expression gradient of A-type K+ channels that weakens the backpropagating action potential-mediated depolarization in layer 1a compared with layer 1b. We find that the pairing of presynaptic and postsynaptic firing leads to significantly smaller Ca2+ signals in the distal dendritic spines in layer 1a compared with the proximal spines in layer 1b. The consequence is a selective failure to induce long-term potentiation (LTP) in layer 1a, which can be rescued by pharmacological enhancement of action potential backpropagation. In contrast, LTP induction by pairing presynaptic and postsynaptic firing is possible in layer 1b but requires bursting of the postsynaptic cell. This output mode strongly depends on the balance of excitation and inhibition in the piriform cortex. We show, on the single-spine level, how the plasticity of functionally distinct synapses is gated by the intrinsic electrical properties of piriform cortex layer 2 pyramidal cell dendrites and the cellular output mode.

Anatomical Organization and Spatiotemporal Firing Patterns of Layer 3 Neurons in the Rat Medial Entorhinal Cortex

Layer 3 of the medial entorhinal cortex is a major gateway from the neocortex to the hippocampus. Here we addressed structure–function relationships in medial entorhinal cortex layer 3 by combining anatomical analysis with juxtacellular identification of single neurons in freely behaving rats. Anatomically, layer 3 appears as a relatively homogeneous cell sheet. Dual-retrograde neuronal tracing experiments indicate a large overlap between layer 3 pyramidal populations, which project to ipsilateral hippocampus, and the contralateral medial entorhinal cortex. These cells were intermingled within layer 3, and had similar morphological and intrinsic electrophysiological properties. Dendritic trees of layer 3 neurons largely avoided the calbindin-positive patches in layer 2. Identification of layer 3 neurons during spatial exploration (n = 17) and extracellular recordings (n = 52) pointed to homogeneous spatial discharge patterns. Layer 3 neurons showed only weak spiking theta rhythmicity and sparse head-direction selectivity. A majority of cells (50 of 69) showed no significant spatial modulation. All of the ∼28% of neurons that carried significant amounts of spatial information (19 of 69) discharged in irregular spatial patterns. Thus, layer 3 spatiotemporal firing properties are remarkably different from those of layer 2, where theta rhythmicity is prominent and spatially modulated cells often discharge in grid or border patterns. SIGNIFICANCE STATEMENT Neurons within the superficial layers of the medial entorhinal cortex (MEC) often discharge in border, head-direction, and theta-modulated grid patterns. It is still largely unknown how defined discharge patterns relate to cellular diversity in the superficial layers of the MEC. In the present study, we addressed this issue by combining anatomical analysis with juxtacellular identification of single layer 3 neurons in freely behaving rats. We provide evidence that the anatomical organization and spatiotemporal firing properties of layer 3 neurons are remarkably different from those in layer 2. Specifically, most layer 3 neurons discharged in spatially irregular firing patterns, with weak theta-modulation and head-directional selectivity. This work thus poses constraints on the spatiotemporal patterns reaching downstream targets, like the hippocampus.

Complementary Sensory and Associative Microcircuitry in Primary Olfactory Cortex

The three-layered primary olfactory (piriform) cortex is the largest component of the olfactory cortex. Sensory and intracortical inputs converge on principal cells in the anterior piriform cortex (aPC). We characterize organization principles of the sensory and intracortical microcircuitry of layer II and III principal cells in acute slices of rat aPC using laser-scanning photostimulation and fast two-photon population Ca2+ imaging. Layer II and III principal cells are set up on a superficial-to-deep vertical axis. We found that the position on this axis correlates with input resistance and bursting behavior. These parameters scale with distinct patterns of incorporation into sensory and associative microcircuits, resulting in a converse gradient of sensory and intracortical inputs. In layer II, sensory circuits dominate superficial cells, whereas incorporation in intracortical circuits increases with depth. Layer III pyramidal cells receive more intracortical inputs than layer II pyramidal cells, but with an asymmetric dorsal offset. This microcircuit organization results in a diverse hybrid feedforward/recurrent network of neurons integrating varying ratios of intracortical and sensory input depending on a cells position on the superficial-to-deep vertical axis. Since burstiness of spiking correlates with both the cells location on this axis and its incorporation in intracortical microcircuitry, the neuronal output mode may encode a given cells involvement in sensory versus associative processing.

A novel control software that improves the experimental workflow of scanning photostimulation experiments

Optical uncaging of caged compounds is a well-established method to study the functional anatomy of a brain region on the circuit level. We present an alternative approach to existing experimental setups. Using a low-magnification objective we acquire images for planning the spatial patterns of stimulation. Then high-magnification objectives are used during laser stimulation providing a laser spot between 2 microm and 20 microm size. The core of this system is a video-based control software that monitors and controls the connected devices, allows for planning of the experiment, coordinates the stimulation process and manages automatic data storage. This combines a high-resolution analysis of neuronal circuits with flexible and efficient online planning and execution of a grid of spatial stimulation patterns on a larger scale. The software offers special optical features that enable the system to achieve a maximum degree of spatial reliability. The hardware is mainly built upon standard laboratory devices and thus ideally suited to cost-effectively complement existing electrophysiological setups with a minimal amount of additional equipment. Finally, we demonstrate the performance of the system by mapping the excitatory and inhibitory connections of entorhinal cortex layer II stellate neurons and present an approach for the analysis of photo-induced synaptic responses in high spontaneous activity.

Excitatory Microcircuits within Superficial Layers of the Medial Entorhinal Cortex

The distinctive firing pattern of grid cells in the medial entorhinal cortex (MEC) supports its role in the representation of space. It is widely believed that the hexagonal firing field of grid cells emerges from neural dynamics that depend on the local microcircuitry. However, local networks within the MEC are still not sufficiently characterized. Here, applying up to eight simultaneous whole-cell recordings in acute brain slices, we demonstrate the existence of unitary excitatory connections between principal neurons in the superficial layers of the MEC. In particular, we find prevalent feed-forward excitation from pyramidal neurons in layer III and layer II onto stellate cells in layer II, which might contribute to the generation or the inheritance of grid cell patterns.

GluK1 inhibits calcium dependent and independent transmitter release at associational/commissural synapses in area CA3 of the hippocampus

CA3 pyramidal cells receive three main excitatory inputs: the first one is the mossy fiber input, synapsing mainly on the proximal apical dendrites. Second, entorhinal cortex cells form excitatory connections with CA3 pyramidal cells via the perforant path in the stratum lacunosum moleculare. The third input involves the ipsi‐and contralateral connections, termed the associational/commissural (A/C) pathway terminating in the stratum radiatum of CA3, thus forming a feedback loop within this region. Since this excitatory recurrent synapse makes the CA3 region extremely prone to seizure development, understanding the regulation of synaptic strength of this connection is of crucial interest. Several studies suggest that kainate receptors (KAR) play a role in the regulation of synaptic strength. Our aim was to characterize the influence of KAR on A/C synaptic transmission: application of ATPA, a selective agonist of the GluK1 KAR, depressed the amplitude fEPSP without affecting the size of the fiber volley. Blockade of GABA receptors had no influence on this effect, arguing against the influence of interneuronal KARs. Pharmacological and genetic deletion studies could show that this effect was selectively due to GluK1 receptor activation. Several lines of evidence, such as PPF changes, coefficient of variance–analysis and glutamate uncaging experiments strongly argue for a presynaptic locus of suppression. This is accompanied by an ATPA‐mediated reduction in Ca2+ influx at excitatory synaptic terminals, which is most likely mediated by a G‐Protein dependent mechanism, as suggested by application of pertussis toxin. Finally, analysis of miniature EPSCs in the presence and absence of extracellular Ca2+ suggest that presynaptic KAR can also reduce transmitter release downstream and therefore independent of Ca2+ influx.