Microcircuits for spatial coding in the medial entorhinal cortex.

The hippocampal formation is critically involved in learning and memory and contains a large proportion of neurons encoding aspects of the organism's spatial surroundings. In the medial entorhinal cortex (MEC), this includes grid cells with their distinctive hexagonal firing fields as well as a host of other functionally defined cell types including head direction cells, speed cells, border cells, and object-vector cells. Such spatial coding emerges from the processing of external inputs by local microcircuits. However, it remains unclear exactly how local microcircuits and their dynamics within the MEC contribute to spatial discharge patterns. In this review we focus on recent investigations of intrinsic MEC connectivity, which have started to describe and quantify both excitatory and inhibitory wiring in the superficial layers of the MEC. Although the picture is far from complete, it appears that these layers contain robust recurrent connectivity that could sustain the attractor dynamics posited to underlie grid pattern formation. These findings pave the way to a deeper understanding of the mechanisms underlying spatial navigation and memory.

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.

Mutations in plasticity-related-gene-1 (PRG-1) protein contribute to hippocampal seizure susceptibility and modify epileptic phenotype.

The Phospholipid Phosphatase Related 4 gene (PLPPR4,  *607813) encodes the Plasticity-Related-Gene-1 (PRG-1) protein. This cerebral synaptic transmembrane-protein modulates cortical excitatory transmission on glutamatergic neurons. In mice, homozygous Prg-1 deficiency causes juvenile epilepsy. Its epileptogenic potential in humans was unknown. Thus, we screened 18 patients with infantile epileptic spasms syndrome (IESS) and 98 patients with benign familial neonatal/infantile seizures (BFNS/BFIS) for the presence of PLPPR4 variants. A girl with IESS had inherited a PLPPR4-mutation (c.896C > G, NM_014839; p.T299S) from her father and an SCN1A-mutation from her mother (c.1622A > G, NM_006920; p.N541S). The PLPPR4-mutation was located in the third extracellular lysophosphatidic acid-interacting domain and in-utero electroporation (IUE) of the Prg-1p.T300S construct into neurons of Prg-1 knockout embryos demonstrated its inability to rescue the electrophysiological knockout phenotype. Electrophysiology on the recombinant SCN1Ap.N541S channel revealed partial loss-of-function. Another PLPPR4 variant (c.1034C > G, NM_014839; p.R345T) that was shown to result in a loss-of-function aggravated a BFNS/BFIS phenotype and also failed to suppress glutamatergic neurotransmission after IUE. The aggravating effect of Plppr4-haploinsufficiency on epileptogenesis was further verified using the kainate-model of epilepsy: double heterozygous Plppr4-/+|Scn1awt|p.R1648H mice exhibited higher seizure susceptibility than either wild-type, Plppr4-/+, or Scn1awt|p.R1648H littermates. Our study shows that a heterozygous PLPPR4 loss-of-function mutation may have a modifying effect on BFNS/BFIS and on SCN1A-related epilepsy in mice and humans.

SORCS1 and SORCS3 control energy balance and orexigenic peptide production.

SORCS1 and SORCS3 are two related sorting receptors expressed in neurons of the arcuate nucleus of the hypothalamus. Using mouse models with individual or dual receptor deficiencies, we document a previously unknown function of these receptors in central control of metabolism. Specifically, SORCS1 and SORCS3 act as intracellular trafficking receptors for tropomyosin-related kinase B to attenuate signaling by brain-derived neurotrophic factor, a potent regulator of energy homeostasis. Loss of the joint action of SORCS1 and SORCS3 in mutant mice results in excessive production of the orexigenic neuropeptide agouti-related peptide and in a state of chronic energy excess characterized by enhanced food intake, decreased locomotor activity, diminished usage of lipids as metabolic fuel, and increased adiposity, albeit at overall reduced body weight. Our findings highlight a novel concept in regulation of the melanocortin system and the role played by trafficking receptors SORCS1 and SORCS3 in this process.

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.

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.

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 Ca(2+) 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 cell's position on the superficial-to-deep vertical axis. Since burstiness of spiking correlates with both the cell's location on this axis and its incorporation in intracortical microcircuitry, the neuronal output mode may encode a given cell's involvement in sensory versus associative processing.

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 Ca(2+) 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 Ca(2+) suggest that presynaptic KAR can also reduce transmitter release downstream and therefore independent of Ca(2+) influx.

Parvalbumin Interneurons Are Differentially Connected to Principal Cells in Inhibitory Feedback Microcircuits along the Dorsoventral Axis of the Medial Entorhinal Cortex.

The medial entorhinal cortex (mEC) shows a high degree of spatial tuning, predominantly grid cell activity, which is reliant on robust, dynamic inhibition provided by local interneurons (INs). In fact, feedback inhibitory microcircuits involving fast-spiking parvalbumin (PV) basket cells (BCs) are believed to contribute dominantly to the emergence of grid cell firing in principal cells (PrCs). However, the strength of PV BC-mediated inhibition onto PrCs is not uniform in this region, but high in the dorsal and weak in the ventral mEC. This is in good correlation with divergent grid field sizes, but the underlying morphologic and physiological mechanisms remain unknown. In this study, we examined PV BCs in layer (L)2/3 of the mEC characterizing their intrinsic physiology, morphology and synaptic connectivity in the juvenile rat. We show that while intrinsic physiology and morphology are broadly similar over the dorsoventral axis, PV BCs form more connections onto local PrCs in the dorsal mEC, independent of target cell type. In turn, the major PrC subtypes, pyramidal cell (PC) and stellate cell (SC), form connections onto PV BCs with lower, but equal probability. These data thus identify inhibitory connectivity as source of the gradient of inhibition, plausibly explaining divergent grid field formation along this dorsoventral axis of the mEC.

Somatostatin interneurons activated by 5-HT2A receptor suppress slow oscillations in medial entorhinal cortex.

Serotonin (5-HT) is one of the major neuromodulators present in the mammalian brain and has been shown to play a role in multiple physiological processes. The mechanisms by which 5-HT modulates cortical network activity, however, are not yet fully understood. We investigated the effects of 5-HT on slow oscillations (SOs), a synchronized cortical network activity universally present across species. SOs are observed during anesthesia and are considered to be the default cortical activity pattern. We discovered that (±)3,4-methylenedioxymethamphetamine (MDMA) and fenfluramine, two potent 5-HT releasers, inhibit SOs within the entorhinal cortex (EC) in anesthetized mice. Combining opto- and pharmacogenetic manipulations with in vitro electrophysiological recordings, we uncovered that somatostatin-expressing (Sst) interneurons activated by the 5-HT2A receptor (5-HT2AR) play an important role in the suppression of SOs. Since 5-HT2AR signaling is involved in the etiology of different psychiatric disorders and mediates the psychological effects of many psychoactive serotonergic drugs, we propose that the newly discovered link between Sst interneurons and 5-HT will contribute to our understanding of these complex topics.

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 Ca(2+) 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.

Up and Down States and Memory Consolidation Across Somatosensory, Entorhinal, and Hippocampal Cortices.

In the course of a day, brain states fluctuate, from conscious awake information-acquiring states to sleep states, during which previously acquired information is further processed and stored as memories. One hypothesis is that memories are consolidated and stored during "offline" states such as sleep, a process thought to involve transfer of information from the hippocampus to other cortical areas. Up and Down states (UDS), patterns of activity that occur under anesthesia and sleep states, are likely to play a role in this process, although the nature of this role remains unclear. Here we review what is currently known about these mechanisms in three anatomically distinct but interconnected cortical areas: somatosensory cortex, entorhinal cortex, and the hippocampus. In doing so, we consider the role of this activity in the coordination of "replay" during sleep states, particularly during hippocampal sharp-wave ripples. We conclude that understanding the generation and propagation of UDS may provide key insights into the cortico-hippocampal dialogue linking archi- and neocortical areas during memory formation.

Publisher Correction: Propagation of hippocampal ripples to the neocortex by way of a subiculum-retrosplenial pathway.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Layer 3 Pyramidal Cells in the Medial Entorhinal Cortex Orchestrate Up-Down States and Entrain the Deep Layers Differentially.

Up-down states (UDS) are synchronous cortical events of neuronal activity during non-REM sleep. The medial entorhinal cortex (MEC) exhibits robust UDS during natural sleep and under anesthesia. However, little is known about the generation and propagation of UDS-related activity in the MEC. Here, we dissect the circuitry underlying UDS generation and propagation across layers in the MEC using both in vivo and in vitro approaches. We provide evidence that layer 3 (L3) MEC is crucial in the generation and maintenance of UDS in the MEC. Furthermore, we find that the two sublayers of the L5 MEC participate differentially during UDS. Our findings show that L5b, which receives hippocampal output, is strongly innervated by UDS activity originating in L3 MEC. Our data suggest that L5b acts as a coincidence detector during information transfer between the hippocampus and the cortex and thereby plays an important role in memory encoding and consolidation.

Propagation of hippocampal ripples to the neocortex by way of a subiculum-retrosplenial pathway.

Bouts of high frequency activity known as sharp wave ripples (SPW-Rs) facilitate communication between the hippocampus and neocortex. However, the paths and mechanisms by which SPW-Rs broadcast their content are not well understood. Due to its anatomical positioning, the granular retrosplenial cortex (gRSC) may be a bridge for this hippocampo-cortical dialogue. Using silicon probe recordings in awake, head-fixed mice, we show the existence of SPW-R analogues in gRSC and demonstrate their coupling to hippocampal SPW-Rs. gRSC neurons reliably distinguished different subclasses of hippocampal SPW-Rs according to ensemble activity patterns in CA1. We demonstrate that this coupling is brain state-dependent, and delineate a topographically-organized anatomical pathway via VGlut2-expressing, bursty neurons in the subiculum. Optogenetic stimulation or inhibition of bursty subicular cells induced or reduced responses in superficial gRSC, respectively. These results identify a specific path and underlying mechanisms by which the hippocampus can convey neuronal content to the neocortex during SPW-Rs.

Species-specific differences in synaptic transmission and plasticity.

Synaptic transmission and plasticity in the hippocampus are integral factors in learning and memory. While there has been intense investigation of these critical mechanisms in the brain of rodents, we lack a broader understanding of the generality of these processes across species. We investigated one of the smallest animals with conserved hippocampal macroanatomy-the Etruscan shrew, and found that while synaptic properties and plasticity in CA1 Schaffer collateral synapses were similar to mice, CA3 mossy fiber synapses showed striking differences in synaptic plasticity between shrews and mice. Shrew mossy fibers have lower long term plasticity compared to mice. Short term plasticity and the expression of a key protein involved in it, synaptotagmin 7 were also markedly lower at the mossy fibers in shrews than in mice. We also observed similar lower expression of synaptotagmin 7 in the mossy fibers of bats that are evolutionarily closer to shrews than mice. Species specific differences in synaptic plasticity and the key molecules regulating it, highlight the evolutionary divergence of neuronal circuit functions.

Estrus-Cycle Regulation of Cortical Inhibition.

Female mammals experience cyclical changes in sexual receptivity known as the estrus cycle. Little is known about how estrus affects the cortex, although alterations in sensation, cognition and the cyclical occurrence of epilepsy suggest brain-wide processing changes. We performed in vivo juxtacellular and whole-cell recordings in somatosensory cortex of female rats and found that the estrus cycle potently altered cortical inhibition. Fast-spiking interneurons were strongly activated with social facial touch and varied their ongoing activity with the estrus cycle and estradiol in ovariectomized females, while regular-spiking excitatory neurons did not change. In situ hybridization for estrogen receptor ß (Esr2) showed co-localization with parvalbumin-positive (PV+) interneurons in deep cortical layers, mirroring the laminar distribution of our physiological findings. The fraction of neurons positive for estrogen receptor ß (Esr2) and PV co-localization (Esr2+PV+) in cortical layer V was increased in proestrus. In vivo and in vitro experiments confirmed that estrogen acts locally to increase fast-spiking interneuron excitability through an estrogen-receptor-ß-dependent mechanism.

RIM-BP2 primes synaptic vesicles via recruitment of Munc13-1 at hippocampal mossy fiber synapses.

All synapses require fusion-competent vesicles and coordinated Ca2+-secretion coupling for neurotransmission, yet functional and anatomical properties are diverse across different synapse types. We show that the presynaptic protein RIM-BP2 has diversified functions in neurotransmitter release at different central murine synapses and thus contributes to synaptic diversity. At hippocampal pyramidal CA3-CA1 synapses, RIM-BP2 loss has a mild effect on neurotransmitter release, by only regulating Ca2+-secretion coupling. However, at hippocampal mossy fiber synapses, RIM-BP2 has a substantial impact on neurotransmitter release by promoting vesicle docking/priming and vesicular release probability via stabilization of Munc13-1 at the active zone. We suggest that differences in the active zone organization may dictate the role a protein plays in synaptic transmission and that differences in active zone architecture is a major determinant factor in the functional diversity of synapses.

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.

VGLUT2 Functions as a Differential Marker for Hippocampal Output Neurons.

The subiculum is the gatekeeper between the hippocampus and cortical areas. Yet, the lack of a pyramidal cell-specific marker gene has made the analysis of the subicular area very difficult. Here we report that the vesicular-glutamate transporter 2 (VGLUT2) functions as a specific marker gene for subicular burst-firing neurons, and demonstrate that VGLUT2-Cre mice allow for Channelrhodopsin-2 (ChR2)-assisted connectivity analysis.