Synaptic plasticity at the dentate gyrus granule cell to somatostatin-expressing interneuron synapses supports object location memory.

Somatostatin-expressing interneurons (SOMIs) in the mouse dentate gyrus (DG) receive feedforward excitation from granule cell (GC) mossy fiber (MF) synapses and provide feedback lateral inhibition onto GC dendrites to support environment representation in the DG network. Although this microcircuitry has been implicated in memory formation, little is known about activity-dependent plastic changes at MF-SOMI synapses and their influence on behavior. Here, we report that the metabotropic glutamate receptor 1α (mGluR1α) is required for the induction of associative long-term potentiation (LTP) at MF-SOMI synapses. Pharmacological block of mGluR1α, but not mGluR5, prevented synaptic weight changes. LTP at MF-SOMI synapses was postsynaptically induced, required increased intracellular Ca2+, involved G-protein-mediated and Ca2+-dependent (extracellular signal-regulated kinase) ERK1/2 pathways, and the activation of NMDA receptors. Specific knockdown of mGluR1α in DG-SOMIs by small hairpin RNA expression prevented MF-SOMI LTP, reduced SOMI recruitment, and impaired object location memory. Thus, postsynaptic mGluR1α-mediated MF-plasticity at SOMI input synapses critically supports DG-dependent mnemonic functions.

Integration of the CA2 region in the hippocampal network during epileptogenesis.

The CA2 pyramidal cells are mostly resistant to cell death in mesial temporal lobe epilepsy (MTLE) with hippocampal sclerosis, but they are aberrantly integrated into the epileptic hippocampal network via mossy fiber sprouting. Furthermore, they show increased excitability in vitro in hippocampal slices obtained from human MTLE specimens or animal epilepsy models. Although these changes promote CA2 to contribute to epileptic activity (EA) in vivo, the role of CA2 in the epileptic network within and beyond the sclerotic hippocampus is still unclear. We used the intrahippocampal kainate mouse model for MTLE, which recapitulates most features of the human disease including pharmacoresistant epileptic seizures and hippocampal sclerosis, with preservation of dentate gyrus (DG) granule cells and CA2 pyramidal cells. In vivo recordings with electrodes in CA2 and the DG showed that EA occurs at high coincidence between the ipsilateral DG and CA2 and current source density analysis of silicon probe recordings in dorsal ipsilateral CA2 revealed CA2 as a local source of EA. Cell-specific viral tracing in Amigo2-icreERT2 mice confirmed the preservation of the axonal projection from ipsilateral CA2 pyramidal cells to contralateral CA2 under epileptic conditions and indeed, EA propagated from ipsi- to contralateral CA2 with increasing likelihood with time after KA injection, but always at lower intensity than within the ipsilateral hippocampus. Furthermore, we show that CA2 presents with local theta oscillations and like the DG, shows a pathological reduction of theta frequency already from 2 days after KA onward. The early changes in activity might be facilitated by the loss of glutamic acid decarboxylase 67 (Gad67) mRNA-expressing interneurons directly after the initial status epilepticus in ipsi- but not contralateral CA2. Together, our data highlight CA2 as an active player in the epileptic network and with its contralateral connections as one possible router of aberrant activity.

Multifunctional Fibers as Tools for Neuroscience and Neuroengineering.

Multifunctional devices for modulation and probing of neuronal activity during free behavior facilitate studies of functions and pathologies of the nervous system. Probes composed of stiff materials, such as metals and semiconductors, exhibit elastic and chemical mismatch with the neural tissue, which is hypothesized to contribute to sustained tissue damage and gliosis. Dense glial scars have been found to encapsulate implanted devices, corrode their surfaces, and often yield poor recording quality in long-term experiments. Motivated by the hypothesis that reducing the mechanical stiffness of implanted probes may improve their long-term reliability, a variety of probes based on soft materials have been developed. In addition to enabling electrical neural recording, these probes have been engineered to take advantage of genetic tools for optical neuromodulation. With the emergence of optogenetics, it became possible to optically excite or inhibit genetically identifiable cell types via expression of light-sensitive opsins. Optogenetics experiments often demand implantable multifunctional devices to optically stimulate, deliver viral vectors and drugs, and simultaneously record electrophysiological signals from the specified cells within the nervous system. Recent advances in microcontact printing and microfabrication techniques have equipped flexible probes with microscale light-emitting diodes (µLEDs), waveguides, and microfluidic channels. Complementary to these approaches, fiber drawing has emerged as a scalable route to integration of multiple functional features within miniature and flexible neural probes. The thermal drawing process relies on the fabrication of macroscale models containing the materials of interest, which are then drawn into microstructured fibers with predefined cross-sectional geometries. We have recently applied this approach to produce fibers integrating conductive electrodes for extracellular recording of single- and multineuron potentials, low-loss optical waveguides for optogenetic neuromodulation, and microfluidic channels for drug and viral vector delivery. These devices allowed dynamic investigation of the time course of opsin expression across multiple brain regions and enabled pairing of optical stimulation with local pharmacological intervention in behaving animals. Neural probes designed to interface with the spinal cord, a viscoelastic tissue undergoing repeated strain during normal movement, rely on the integration of soft and flexible materials to avoid injury and device failure. Employing soft substrates, such as parylene C and poly-(dimethylsiloxane), for electrode and µLED arrays permitted stimulation and recording of neural activity on the surface of the spinal cord. Similarly, thermally drawn flexible and stretchable optoelectronic fibers that resemble the fibrous structure of the spinal cord were implanted without any significant inflammatory reaction in the vicinity of the probes. These fibers enabled simultaneous recording and optogenetic stimulation of neural activity in the spinal cord. In this Account, we review the applications of multifunctional fibers and other integrated devices for optoelectronic probing of neural circuits and discuss engineering directions that may facilitate future studies of nerve repair and accelerate the development of bioelectronic medical devices.

Theta frequency decreases throughout the hippocampal formation in a focal epilepsy model.

Mesial temporal lobe epilepsy is characterized by focal, recurrent spontaneous seizures, sclerosis and granule cell dispersion (GCD) in the hippocampal formation. Changes in theta rhythm properties have been correlated with the severity of hippocampal restructuring and were suggested as a cause of memory deficits accompanying epilepsy. For severe sclerosis, it has even been questioned whether theta band oscillations persist. We asked how theta oscillations change with graded restructuring along the longitudinal hippocampal axis and whether these changes correlate with the overall severity of temporal lobe epilepsy. We recorded local field potentials in the medial entorhinal cortex and along the septo-temporal axis of the dentate gyrus at sites with different degrees of GCD in freely behaving epileptic mice. Theta frequency was decreased at all recording positions throughout the dentate gyrus and in the medial entorhinal cortex, irrespective of the extent of GCD or the rate of severe epileptic events. The frequency reduction by up to 1.7 Hz, corresponding to 1/3 octaves within the theta range, was present during rest, exploration and running. Despite the frequency reduction, theta oscillations remained coherent across the hippocampal formation and were modulated by running speed as in controls. The reduction in theta frequency thus is likely not a consequence of the local restructuring but rather a global phenomenon affecting the hippocampal formation as a whole.

Synaptic Remodeling of Entorhinal Input Contributes to an Aberrant Hippocampal Network in Temporal Lobe Epilepsy.

The hippocampus is reciprocally connected with the entorhinal cortex. Although several studies emphasized a role for the entorhinal cortex in mesial temporal lobe epilepsy (MTLE), it remains uncertain whether its synaptic connections with the hippocampus are altered. To address this question, we traced hippocampo-entorhinal and entorhino-hippocampal projections, assessed their connectivity with the respective target cells and examined functional alterations in a mouse model for MTLE. We show that hippocampal afferents to the dorsal entorhinal cortex are lost in the epileptic hippocampus. Conversely, entorhino-dentate projections via the medial perforant path (MPP) are preserved, but appear substantially altered on the synaptic level. Confocal imaging and 3D-reconstruction revealed that new putative contacts are established between MPP fibers and dentate granule cells (DGCs). Immunohistochemical identification of pre- and postsynaptic elements indicated that these contacts are functionally mature synapses. On the ultrastructural level, pre- and postsynaptic compartments of MPP synapses were strongly enlarged. The length and complexity of postsynaptic densities were also increased pointing to long-term potentiation-related morphogenesis. Finally, whole-cell recordings of DGCs revealed an enhancement of evoked excitatory postsynaptic currents. In conclusion, the synaptic rearrangement of excitatory inputs to DGCs from the medial entorhinal cortex may contribute to the epileptogenic circuitry in MTLE.

Mossy fiber sprouting and pyramidal cell dispersion in the hippocampal CA2 region in a mouse model of temporal lobe epilepsy.

Dentate granule cells and the hippocampal CA2 region are resistant to cell loss associated with mesial temporal lobe epilepsy (MTLE). It is known that granule cells undergo mossy fiber sprouting in the dentate gyrus which contributes to a recurrent, proepileptogenic circuitry in the hippocampus. Here it is shown that mossy fiber sprouting also targets CA2 pyramidal cell somata and that the CA2 region undergoes prominent structural reorganization under epileptic conditions. Using the intrahippocampal kainate mouse model for MTLE and the CA2-specific markers Purkinje cell protein 4 (PCP4) and regulator of G-Protein signaling 14 (RGS14), it was found that during epileptogenesis CA2 neurons survive and disperse in direction of CA3 and CA1 resulting in a significantly elongated CA2 region. Using transgenic mice that express enhanced green fluorescent protein (eGFP) in granule cells and mossy fibers, we show that the recently described mossy fiber projection to CA2 undergoes sprouting resulting in aberrant large, synaptoporin-expressing mossy fiber boutons which surround the CA2 pyramidal cell somata. This opens up the potential for altered synaptic transmission that might contribute to epileptic activity in CA2. Indeed, intrahippocampal recordings in freely moving mice revealed that epileptic activity occurs concomitantly in the dentate gyrus and in CA2. Altogether, the results call attention to CA2 as a region affected by MTLE-associated pathological restructuring.

Altered θ coupling between medial entorhinal cortex and dentate gyrus in temporal lobe epilepsy.

PURPOSE: Temporal lobe epilepsy is often accompanied by neuron loss and rewiring in the hippocampus. We hypothesized that the interaction of subnetworks of the entorhinal-hippocampal loop between epileptic events should show significant signatures of these pathologic changes. METHODS: We combined simultaneous recording of local field potentials in entorhinal cortex (EC) and dentate gyrus (DG) in freely behaving kainate-injected mice with histologic analyses and computational modeling. KEY FINDINGS: In healthy mice, theta band activity was synchronized between EC and DG. In contrast, in epileptic mice, theta activity in the EC was delayed with respect to the DG. A computational neural mass model suggests that hippocampal cell loss imbalances the coupling of subnetworks, introducing the shift. SIGNIFICANCE: We show that pathologic dynamics in epilepsy encompass ongoing activity in the entorhinal-hippocampal loop beyond acute epileptiform activity. This predominantly affects theta band activity, which links this shift in entorhinal-hippocampal interaction to behavioral aspects in epilepsy.

Reelin Is Required for Maintenance of Granule Cell Lamination in the Healthy and Epileptic Hippocampus.

One characteristic feature of mesial temporal lobe epilepsy is granule cell dispersion (GCD), a pathological widening of the granule cell layer in the dentate gyrus. The loss of the extracellular matrix protein Reelin, an important positional cue for neurons, correlates with GCD formation in MTLE patients and in rodent epilepsy models. Here, we used organotypic hippocampal slice cultures (OHSC) from transgenic mice expressing enhanced green fluorescent protein (eGFP) in differentiated granule cells (GCs) to monitor GCD formation dynamically by live cell video microscopy and to investigate the role of Reelin in this process. We present evidence that following treatment with the glutamate receptor agonist kainate (KA), eGFP-positive GCs migrated mainly toward the hilar region. In the hilus, Reelin-producing neurons were rapidly lost following KA treatment as shown in a detailed time series. Addition of recombinant Reelin fragments to the medium effectively prevented the KA-triggered movement of eGFP-positive GCs. Placement of Reelin-coated beads into the hilus of KA-treated cultures stopped the migration of GCs in a distance-dependent manner. In addition, quantitative Western blot analysis revealed that KA treatment affects the Reelin signal transduction pathway by increasing intracellular adaptor protein Disabled-1 synthesis and reducing the phosphorylation of cofilin, a downstream target of the Reelin pathway. Both events were normalized by addition of recombinant Reelin fragments. Finally, following neutralization of Reelin in healthy OHSC by incubation with the function-blocking CR-50 Reelin antibody, GCs started to migrate without any direction preference. Together, our findings demonstrate that normotopic position of Reelin is essential for the maintenance of GC lamination in the dentate gyrus and that GCD is the result of a local Reelin deficiency.

Intracortical probe arrays with silicon backbone and microelectrodes on thin polyimide wings enable long-term stable recordingsin vivo.

Objective.Recording and stimulating neuronal activity across different brain regions requires interfacing at multiple sites using dedicated tools while tissue reactions at the recording sites often prevent their successful long-term application. This implies the technological challenge of developing complex probe geometries while keeping the overall footprint minimal, and of selecting materials compatible with neural tissue. While the potential of soft materials in reducing tissue response is uncontested, the implantation of these materials is often limited to reliably target neuronal structures across large brain volumes.Approach.We report on the development of a new multi-electrode array exploiting the advantages of soft and stiff materials by combining 7-µm-thin polyimide wings carrying platinum electrodes with a silicon backbone enabling a safe probe implantation. The probe fabrication applies microsystems technologies in combination with a temporal wafer fixation method for rear side processing, i.e. grinding and deep reactive ion etching, of slender probe shanks and electrode wings. The wing-type neural probes are chronically implanted into the entorhinal-hippocampal formation in the mouse forin vivorecordings of freely behaving animals.Main results.Probes comprising the novel wing-type electrodes have been realized and characterized in view of their electrical performance and insertion capability. Chronic electrophysiologicalin vivorecordings of the entorhinal-hippocampal network in the mouse of up to 104 days demonstrated a stable yield of channels containing identifiable multi-unit and single-unit activity outperforming probes with electrodes residing on a Si backbone.Significance.The innovative fabrication process using a process compatible, temporary wafer bonding allowed to realize new Michigan-style probe arrays. The wing-type probe design enables a precise probe insertion into brain tissue and long-term stable recordings of unit activity due to the application of a stable backbone and 7-µm-thin probe wings provoking locally a minimal tissue response and protruding from the glial scare of the backbone.

Bursts with High and Low Load of Epileptiform Spikes Show Context-Dependent Correlations in Epileptic Mice.

Hypersynchronous network activity is the defining hallmark of epilepsy and manifests in a wide spectrum of phenomena, of which electrographic activity during seizures is only one extreme. The aim of this study was to differentiate between different types of epileptiform activity (EA) patterns and investigate their temporal succession and interactions. We analyzed local field potentials (LFPs) from freely behaving male mice that had received an intrahippocampal kainate injection to model mesial temporal lobe epilepsy (MTLE). Epileptiform spikes occurred in distinct bursts. Using machine learning, we derived a scale reflecting the spike load of bursts and three main burst categories that we labeled high-load, medium-load, and low-load bursts. We found that bursts of these categories were non-randomly distributed in time. High-load bursts formed clusters and were typically surrounded by transition phases with increased rates of medium-load and low-load bursts. In apparent contradiction to this, increased rates of low-load bursts were also associated with longer background phases, i.e., periods lacking high-load bursting. Furthermore, the rate of low-load bursts was more strongly correlated with the duration of background phases than the overall rate of epileptiform spikes. Our findings are consistent with the hypothesis that low-level EA could promote network stability but could also participate in transitions towards major epileptiform events, depending on the current state of the network.

Active Control of Dye Release for Neuronal Tracing Using PEDOT-PSS Coated Electrodes.

Penetrating neural probes comprising arrays of microelectrodes are commonly used to monitor local field potentials and multi-unit activity in animal brain over time frames of weeks. To correlate these recorded signals to specific tissue areas, histological analysis is performed after the experimental endpoint. Even if the lesion of the penetrating probe shaft can be observed, a precise reconstruction of the exact electrode positions is still challenging. To overcome these experimental difficulties, we developed a new concept, whereupon recording electrodes are coated with a poly (3, 4-ethylenedioxythiophene/ polystyrenesulfonate) (PEDOT/PSS)-based film. The conducting polymer acts as dye reservoir over several weeks and afterwards provides controlled delivery of neurotracers. This paper presents a recording electrode based on a PEDOT/PSS bilayer optimized for dye delivery and with reduced impedance. Controlled exchange of neurotracer dye is successfully demonstrated in vitro using spectrofluorometry and in neuroblastoma cell cultures. A second PEDOT/PSS capping layer on top of the dye reservoir lowers the passive leakage of dye by a factor of 6.4 and prevents a direct contact of the dye filled layer with the cells. Stability tests over four weeks demonstrate the electrochemical stability of the PEDOT coating, as well as retained functionality of the dye delivery system.

Optogenetic entrainment of neural oscillations with hybrid fiber probes.

OBJECTIVE: Optogenetic modulation of neural activity is a ubiquitous tool for basic investigation of brain circuits. While the majority of optogenetic paradigms rely on short light pulses to evoke synchronized activity of optically sensitized cells, many neurobiological processes are associated with slow local field potential (LFP) oscillations. Therefore, we developed a hybrid fiber probe capable of simultaneous electrophysiological recording and optical stimulation and used it to investigate the utility of sinusoidal light stimulation for evoking oscillatory neural activity in vivo across a broad frequency range. APPROACH: We fabricated hybrid fiber probes comprising a hollow cylindrical array of 9 electrodes and a flexible optical waveguide integrated within the core. We implanted these probes in the hippocampus of transgenic Thy1-ChR2-YFP mice that broadly express the blue-light sensitive cation channel channelrhodopsin 2 (ChR2) in excitatory neurons across the brain. The effects of the sinusoidal light stimulation were characterized and contrasted with those corresponding to pulsed stimulation in the frequency range of physiological LFP rhythms (3-128 Hz). MAIN RESULTS: Within hybrid probes, metal electrode surfaces were vertically aligned with the waveguide tip, which minimized optical stimulation artifacts in neurophysiological recordings. Sinusoidal stimulation resulted in reliable and coherent entrainment of LFP oscillations up to 70 Hz, the cutoff frequency of ChR2, with response amplitudes inversely scaling with the stimulation frequencies. Effectiveness of the stimulation was maintained for two months following implantation. SIGNIFICANCE: Alternative stimulation patterns complementing existing pulsed protocols, in particular sinusoidal light stimulation, are a prerequisite for investigating the physiological mechanisms underlying brain rhythms. So far, studies applying sinusoidal stimulation in vivo were limited to single stimulation frequencies. We show the feasibility of sinusoidal stimulation in vivo to induce coherent LFP oscillations across the entire frequency spectrum supported by the gating dynamics of ChR2 and introduce a hybrid fiber probe tailored to continuous light stimulation.

Seizure-Induced Motility of Differentiated Dentate Granule Cells Is Prevented by the Central Reelin Fragment.

Granule cell dispersion (GCD) represents a pathological widening of the granule cell layer in the dentate gyrus and it is frequently observed in patients with mesial temporal lobe epilepsy (MTLE). Recent studies in human MTLE specimens and in animal epilepsy models have shown that a decreased expression and functional inactivation of the extracellular matrix protein Reelin correlates with GCD formation, but causal evidence is still lacking. Here, we used unilateral kainate (KA) injection into the mouse hippocampus, an established MTLE animal model, to precisely map the loss of reelin mRNA-synthesizing neurons in relation to GCD along the septotemporal axis of the epileptic hippocampus. We show that reelin mRNA-producing neurons are mainly lost in the hilus and that this loss precisely correlates with the occurrence of GCD. To monitor GCD formation in real time, we used organotypic hippocampal slice cultures (OHSCs) prepared from mice which express enhanced green fluorescent protein (eGFP) primarily in differentiated dentate granule cells. Using life cell microscopy we observed that increasing doses of KA resulted in an enhanced motility of eGFP-positive granule cells. Moreover, KA treatment of OHSC resulted in a rapid loss of Reelin-producing interneurons mainly in the hilus, as observed in vivo. A detailed analysis of the migration behavior of individual eGFP-positive granule cells revealed that the majority of these neurons actively migrate toward the hilar region, where Reelin-producing neurons are lost. Treatment with KA and subsequent addition of the recombinant R3-6 Reelin fragment significantly prevented the movement of eGFP-positive granule cells. Together, these findings suggest that GCD formation is indeed triggered by a loss of Reelin in hilar interneurons.