Young dentate granule cells mediate pattern separation, whereas old granule cells facilitate pattern completion.

Adult-born granule cells (GCs), a minor population of cells in the hippocampal dentate gyrus, are highly active during the first few weeks after functional integration into the neuronal network, distinguishing them from less active, older adult-born GCs and the major population of dentate GCs generated developmentally. To ascertain whether young and old GCs perform distinct memory functions, we created a transgenic mouse in which output of old GCs was specifically inhibited while leaving a substantial portion of young GCs intact. These mice exhibited enhanced or normal pattern separation between similar contexts, which was reduced following ablation of young GCs. Furthermore, these mutant mice exhibited deficits in rapid pattern completion. Therefore, pattern separation requires adult-born young GCs but not old GCs, and older GCs contribute to the rapid recall by pattern completion. Our data suggest that as adult-born GCs age, their function switches from pattern separation to rapid pattern completion.

Hippocampal GABAergic Inhibitory Interneurons.

In the hippocampus GABAergic local circuit inhibitory interneurons represent only ~10-15% of the total neuronal population; however, their remarkable anatomical and physiological diversity allows them to regulate virtually all aspects of cellular and circuit function. Here we provide an overview of the current state of the field of interneuron research, focusing largely on the hippocampus. We discuss recent advances related to the various cell types, including their development and maturation, expression of subtype-specific voltage- and ligand-gated channels, and their roles in network oscillations. We also discuss recent technological advances and approaches that have permitted high-resolution, subtype-specific examination of their roles in numerous neural circuit disorders and the emerging therapeutic strategies to ameliorate such pathophysiological conditions. The ultimate goal of this review is not only to provide a touchstone for the current state of the field, but to help pave the way for future research by highlighting where gaps in our knowledge exist and how a complete appreciation of their roles will aid in future therapeutic strategies.

GluN2D-Containing N-methyl-d-Aspartate Receptors Mediate Synaptic Transmission in Hippocampal Interneurons and Regulate Interneuron Activity.

N-methyl-d-aspartate receptors (NMDARs) are ionotropic glutamatergic receptors that have been implicated in learning, development, and neuropathological conditions. They are typically composed of GluN1 and GluN2A-D subunits. Whereas a great deal is known about the role of GluN2A- and GluN2B-containing NMDARs, much less is known about GluN2D-containing NMDARs. Here we explore the subunit composition of synaptic NMDARs on hippocampal interneurons. GluN2D mRNA was detected by single-cell PCR and in situ hybridization in diverse interneuron subtypes in the CA1 region of the hippocampus. The GluN2D subunit was detectable by immunoblotting and immunohistochemistry in all subfields of the hippocampus in young and adult mice. In whole-cell patch-clamp recordings from acute hippocampal slices, (+)-CIQ, the active enantiomer of the positive allosteric modulator CIQ, significantly enhanced the amplitude of the NMDAR component of miniature excitatory postsynaptic currents (mEPSCs) in CA1 interneurons but not in pyramidal cells. (+)-CIQ had no effect in slices from Grin2d-/- mice, suggesting that GluN2D-containing NMDARs participate in excitatory synaptic transmission onto hippocampal interneurons. The time course of the NMDAR component of the mEPSC was unaffected by (+)-CIQ potentiation and was not accelerated in slices from Grin2d-/- mice compared with wild-type, suggesting that GluN2D does not detectably slow the NMDAR EPSC time course at this age. (+)-CIQ increased the activity of CA1 interneurons as detected by the rate and net charge transfer of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from CA1 pyramidal cells. These data provide evidence that interneurons contain synaptic NMDARs possessing a GluN2D subunit, which can influence interneuron function and signal processing.

Neto auxiliary protein interactions regulate kainate and NMDA receptor subunit localization at mossy fiber-CA3 pyramidal cell synapses.

Neto1 and Neto2 auxiliary subunits coassemble with NMDA receptors (NMDARs) and kainate receptors (KARs) to modulate their function. In the hippocampus, Neto1 enhances the amplitude and prolongs the kinetics of KAR-mediated currents at mossy fiber (MF)-CA3 pyramidal cell synapses. However, whether Neto1 trafficks KARs to synapses or simply alters channel properties is unresolved. Therefore, postembedding electron microscopy was performed to investigate the localization of GluK2/3 subunits at MF-CA3 synapses in Neto-null mice. Postsynaptic GluK2/3 Immunogold labeling was substantially reduced in Neto-null mice compared with wild types. Moreover, spontaneous KAR-mediated synaptic currents and metabotropic KAR signaling were absent in CA3 pyramidal cells of Neto-null mice. A similar loss of ionotropic and metabotropic KAR function was observed in Neto1, but not Neto2, single knock-out mice, specifically implicating Neto1 in regulating CA3 pyramidal cell KAR localization and function. Additional controversy pertains to the role of Neto proteins in modulating synaptic NMDARs. While Immunogold labeling for GluN2A at MF-CA3 synapses was comparable between wild-type and Neto-null mice, labeling for postsynaptic GluN2B was robustly increased in Neto-null mice. Accordingly, NMDAR-mediated currents at MF-CA3 synapses exhibited increased sensitivity to a GluN2B-selective antagonist in Neto1 knockouts relative to wild types. Thus, despite preservation of the overall MF-CA3 synaptic NMDAR-mediated current, loss of Neto1 alters NMDAR subunit composition. These results confirm that Neto protein interactions regulate synaptic localization of KAR and NMDAR subunits at MF-CA3 synapses, with implications for both ionotropic and metabotropic glutamatergic recruitment of the CA3 network.

An enhancer-AAV approach selectively targeting dentate granule cells of the mouse hippocampus.

The mammalian brain contains a diverse array of cell types, including dozens of neuronal subtypes with distinct anatomical and functional characteristics. The brain leverages these neuron-type specializations to perform diverse circuit operations and thus execute different behaviors properly. Through the use of Cre lines, access to specific neuron types has improved over past decades. Despite their extraordinary utility, development and cross-breeding of Cre lines is time consuming and expensive, presenting a significant barrier to entry for investigators. Furthermore, cell-based therapeutics developed in Cre mice are not clinically translatable. Recently, several adeno-associated virus (AAV) vectors utilizing neuron-type-specific regulatory transcriptional sequences (enhancer-AAVs) were developed that overcome these limitations. Using a publicly available RNA sequencing (RNA-seq) dataset, we evaluated the potential of several candidate enhancers for neuron-type-specific targeting in the hippocampus. Here, we demonstrate that a previously identified enhancer-AAV selectively targets dentate granule cells over other excitatory neuron types in the hippocampus of wild-type adult mice.

Divergent opioid-mediated suppression of inhibition between hippocampus and neocortex across species and development.

Opioid receptors within the CNS regulate pain sensation and mood and are key targets for drugs of abuse. Within the adult rodent hippocampus (HPC), µ-opioid receptor agonists suppress inhibitory parvalbumin-expressing interneurons (PV-INs), thus disinhibiting the circuit. However, it is uncertain if this disinhibitory motif is conserved in other cortical regions, species, or across development. We observed that PV-IN mediated inhibition is robustly suppressed by opioids in HPC but not neocortex in mice and nonhuman primates, with spontaneous inhibitory tone in resected human tissue also following a consistent dichotomy. This hippocampal disinhibitory motif was established in early development when immature PV-INs and opioids already influence primordial network rhythmogenesis. Acute opioid-mediated modulation was partially occluded with morphine pretreatment, with implications for the effects of opioids on hippocampal network activity during circuit maturation as well as learning and memory. Together, these findings demonstrate that PV-INs exhibit a divergence in opioid sensitivity across brain regions that is remarkably conserved across evolution and highlights the underappreciated role of opioids acting through immature PV-INs in shaping hippocampal development.

A novel enhancer-AAV approach selectively targeting dentate granule cells.

The mammalian brain contains the most diverse array of cell types of any organ, including dozens of neuronal subtypes with distinct anatomical and functional characteristics. The brain leverages these neuron-type-specializations to perform diverse circuit operations and thus execute different behaviors properly. Through the use of Cre lines, access to specific neuron types has steadily improved over past decades. Despite their extraordinary utility, development and cross-breeding of Cre lines is time-consuming and expensive, presenting a significant barrier to entry for many investigators. Furthermore, cell-based therapeutics developed in Cre mice are not clinically translatable. Recently, several AAV vectors utilizing neuron-type-specific regulatory transcriptional sequences (enhancer-AAVs) were developed which overcome these limitations. Using a publicly available RNAseq dataset, we evaluated the potential of several candidate enhancers for neuron-type-specific targeting in the hippocampus. Here we identified a promising enhancer-AAV for targeting dentate granule cells and validated its selectivity in wild-type adult mice.

Loss of Grin2a causes a transient delay in the electrophysiological maturation of hippocampal parvalbumin interneurons.

N-methyl-D-aspartate receptors (NMDARs) are ligand-gated ionotropic glutamate receptors that mediate a calcium-permeable component to fast excitatory neurotransmission. NMDARs are heterotetrameric assemblies of two obligate GluN1 subunits (GRIN1) and two GluN2 subunits (GRIN2A-GRIN2D). Sequencing data shows that 43% (297/679) of all currently known NMDAR disease-associated genetic variants are within the GRIN2A gene, which encodes the GluN2A subunit. Here, we show that unlike missense GRIN2A variants, individuals affected with disease-associated null GRIN2A variants demonstrate a transient period of seizure susceptibility that begins during infancy and diminishes near adolescence. We show increased circuit excitability and CA1 pyramidal cell output in juvenile mice of both Grin2a+/- and Grin2a-/- mice. These alterations in somatic spiking are not due to global upregulation of most Grin genes (including Grin2b). Deeper evaluation of the developing CA1 circuit led us to uncover age- and Grin2a gene dosing-dependent transient delays in the electrophysiological maturation programs of parvalbumin (PV) interneurons. We report that Grin2a+/+ mice reach PV cell electrophysiological maturation between the neonatal and juvenile neurodevelopmental timepoints, with Grin2a+/- mice not reaching PV cell electrophysiological maturation until preadolescence, and Grin2a-/- mice not reaching PV cell electrophysiological maturation until adulthood. Overall, these data may represent a molecular mechanism describing the transient nature of seizure susceptibility in disease-associated null GRIN2A patients.

Evolutionary conservation of hippocampal mossy fiber synapse properties.

Various specialized structural/functional properties are considered essential for contextual memory encoding by hippocampal mossy fiber (MF) synapses. Although investigated to exquisite detail in model organisms, synapses, including MFs, have undergone minimal functional interrogation in humans. To determine the translational relevance of rodent findings, we evaluated MF properties within human tissue resected to treat epilepsy. Human MFs exhibit remarkably similar hallmark features to rodents, including AMPA receptor-dominated synapses with small contributions from NMDA and kainate receptors, large dynamic range with strong frequency facilitation, NMDA receptor-independent presynaptic long-term potentiation, and strong cyclic AMP (cAMP) sensitivity of release. Array tomography confirmed the evolutionary conservation of MF ultrastructure. The astonishing congruence of rodent and human MF core features argues that the basic MF properties delineated in animal models remain critical to human MF function. Finally, a selective deficit in GABAergic inhibitory tone onto human MF postsynaptic targets suggests that unrestrained detonator excitatory drive contributes to epileptic circuit hyperexcitability.

A blueprint for the spatiotemporal origins of mouse hippocampal interneuron diversity.

Although vastly outnumbered, inhibitory interneurons critically pace and synchronize excitatory principal cell populations to coordinate cortical information processing. Precision in this control relies upon a remarkable diversity of interneurons primarily determined during embryogenesis by genetic restriction of neuronal potential at the progenitor stage. Like their neocortical counterparts, hippocampal interneurons arise from medial and caudal ganglionic eminence (MGE and CGE) precursors. However, while studies of the early specification of neocortical interneurons are rapidly advancing, similar lineage analyses of hippocampal interneurons have lagged. A "hippocampocentric" investigation is necessary as several hippocampal interneuron subtypes remain poorly represented in the neocortical literature. Thus, we investigated the spatiotemporal origins of hippocampal interneurons using transgenic mice that specifically report MGE- and CGE-derived interneurons either constitutively or inducibly. We found that hippocampal interneurons are produced in two neurogenic waves between E9-E12 and E12-E16 from MGE and CGE, respectively, and invade the hippocampus by E14. In the mature hippocampus, CGE-derived interneurons primarily localize to superficial layers in strata lacunosum moleculare and deep radiatum, while MGE-derived interneurons readily populate all layers with preference for strata pyramidale and oriens. Combined molecular, anatomical, and electrophysiological interrogation of MGE/CGE-derived interneurons revealed that MGE produces parvalbumin-, somatostatin-, and nitric oxide synthase-expressing interneurons including fast-spiking basket, bistratified, axo-axonic, oriens-lacunosum moleculare, neurogliaform, and ivy cells. In contrast, CGE-derived interneurons contain cholecystokinin, calretinin, vasoactive intestinal peptide, and reelin including non-fast-spiking basket, Schaffer collateral-associated, mossy fiber-associated, trilaminar, and additional neurogliaform cells. Our findings provide a basic blueprint of the developmental origins of hippocampal interneuron diversity.

Neto1 is an auxiliary subunit of native synaptic kainate receptors.

Ionotropic glutamate receptors of AMPA, NMDA, and kainate receptor (KAR) subtypes mediate fast excitatory synaptic transmission in the vertebrate CNS. Auxiliary proteins have been identified for AMPA and NMDA receptor complexes, but little is known about KAR complex proteins. We previously identified the CUB (complement C1r/C1s, Uegf, Bmpl) domain protein, Neto1, as an NMDA receptor-associated polypeptide. Here, we show that Neto1 is also an auxiliary subunit for endogenous synaptic KARs. We found that Neto1 and KARs coimmunoprecipitated from brain lysates, from postsynaptic densities (PSDs) and, in a manner dependent on Neto1 CUB domains, when coexpressed in heterologous cells. In Neto1-null mice, there was an ∼50% reduction in the abundance of GluK2-KARs in hippocampal PSDs. Neto1 strongly localized to CA3 stratum lucidum, and loss of Neto1 resulted in a selective deficit in KAR-mediated neurotransmission at mossy fiber-CA3 pyramidal cell (MF-CA3) synapses: KAR-mediated EPSCs in Neto1-null mice were reduced in amplitude and decayed more rapidly than did those in wild-type mice. In contrast, the loss of Neto2, which also localizes to stratum lucidum and interacts with KARs, had no effect on KAR synaptic abundance or MF-CA3 transmission. Indeed, MF-CA3 KAR deficits in Neto1/Neto2-double-null mutant mice were indistinguishable from Neto1 single-null mice. Thus, our findings establish Neto1 as an auxiliary protein required for synaptic function of KARs. The ability of Neto1 to regulate both NMDARs and KARs reveals a unique dual role in controlling synaptic transmission by serving as an auxiliary protein for these two classes of ionotropic glutamate receptors in a synapse-specific fashion.

An update on cholinergic regulation of cholecystokinin-expressing basket cells.

Information processing and transfer within cortical circuits requires precise spatiotemporal coordination of excitatory principal cell activity by a relatively small population of inhibitory interneurons that exhibit remarkable anatomical, molecular and electrophysiological diversity. One subtype of interneuron, the cholecystokinin-expressing basket cell (CCKBC), is particularly well suited to integrate and impart emotional features of an animal's physiological state to principal cell entrainment through the inhibitory network as CCKBCs are highly susceptible to neuromodulation by local and subcortically generated signals commonly associated with 'mood' such as cannabinoids, serotonin and acetylcholine. Here we briefly review recent studies that have elucidated the cellular mechanisms underlying cholinergic regulation of CCKBCs.

Resilient Hippocampal Gamma Rhythmogenesis and Parvalbumin-Expressing Interneuron Function Before and After Plaque Burden in 5xFAD Alzheimer's Disease Model.

Recent studies have implicated impaired Parvalbumin Fast-Spiking Interneuron (PVIN) function as a precipitating factor underlying abnormalities in network synchrony, oscillatory rhythms, and cognition associated with Alzheimer's disease (AD). However, a complete developmental investigation of potential gamma deficits, induced by commonly used carbachol or kainate in ex vivo slice preparations, within AD model mice is lacking. We examined gamma oscillations using field recordings in acute hippocampal slices from 5xFAD and control mice, through the period of developing pathology, starting at 3 months of age, when there is minimal plaque presence in the hippocampus, through to 12+ months of age, when plaque burden is high. In addition, we examined PVIN participation in gamma rhythms using targeted cell-attached recordings of genetically-reported PVINs, in both wild type and mutant mice. In parallel, a developmental immunohistochemical characterisation probing the PVIN-associated expression of PV and perineuronal nets (PNNs) was compared between control and 5xFAD mice. Remarkably, this comprehensive longitudinal evaluation failed to reveal any obvious correlations between PVIN deficits (electrical and molecular), circuit rhythmogenesis (gamma frequency and power), and Aß deposits/plaque formation. By 6-12 months, 5xFAD animals have extensive plaque formation throughout the hippocampus. However, a deficit in gamma oscillatory power was only evident in the oldest 5xFAD animals (12+ months), and only when using kainate, and not carbachol, to induce the oscillations. We found no difference in PV firing or phase preference during kainate-induced oscillations in younger or older 5xFAD mice compared to control, and a reduction of PV and PNNs only in the oldest 5xFAD mice. The lack of a clear relationship between PVIN function, network rhythmicity, and plaque formation in our study highlights an unexpected resilience in PVIN function in the face of extensive plaque pathology associated with this model, calling into question the presumptive link between PVIN pathology and Alzheimer's progression.

A versatile viral toolkit for functional discovery in the nervous system.

The ability to precisely control transgene expression is essential for basic research and clinical applications. Adeno-associated viruses (AAVs) are non-pathogenic and can be used to drive stable expression in virtually any tissue, cell type, or species, but their limited genomic payload results in a trade-off between the transgenes that can be incorporated and the complexity of the regulatory elements controlling their expression. Resolving these competing imperatives in complex experiments inevitably results in compromises. Here, we assemble an optimized viral toolkit (VTK) that addresses these limitations and allows for efficient combinatorial targeting of cell types. Moreover, their modular design explicitly enables further refinements. We achieve this in compact vectors by integrating structural improvements of AAV vectors with innovative molecular tools. We illustrate the potential of this approach through a systematic demonstration of their utility for targeting cell types and querying their biology using a wide array of genetically encoded tools.

Presynaptic kainate receptor activation preserves asynchronous GABA release despite the reduction in synchronous release from hippocampal cholecystokinin interneurons.

Inhibitory synaptic transmission in the hippocampus in mediated by a wide variety of different interneuron classes which are assumed to play different roles in network activity. Activation of presynaptic kainate receptors (KARs) has been shown to reduce inhibitory transmission but the interneuron class(es) at which they act is only recently beginning to emerge. Using paired recordings we show that KAR activation causes a decrease in presynaptic release from cholecystokinin (CCK)- but not parvalbumin-containing interneurons and that this decrease is observed when pyramidal cells, but not interneurons, are the postsynaptic target. We also show that although the synchronous release component is reduced, the barrage of asynchronous GABA release from CCK interneurons during sustained firing is unaffected by KAR activation. This indicates that presynaptic KARs preserve and act in concert with asynchronous release to switch CCK interneurons from a phasic inhibition mode to produce prolonged inhibition during periods of intense activity.

Common origins of hippocampal Ivy and nitric oxide synthase expressing neurogliaform cells.

GABAergic interneurons critically regulate cortical computation through exquisite spatiotemporal control over excitatory networks. Precision of this inhibitory control requires a remarkable diversity within interneuron populations that is largely specified during embryogenesis. Although interneurons expressing the neuronal isoform of nitric oxide synthase (nNOS) constitute the largest hippocampal interneuron cohort their origin and specification remain unknown. Thus, as neurogliaform cells (NGC) and Ivy cells (IvC) represent the main nNOS(+) interneurons, we investigated their developmental origins. Although considered distinct interneuron subtypes, NGCs and IvCs exhibited similar neurochemical and electrophysiological signatures, including NPY expression and late spiking. Moreover, lineage analyses, including loss-of-function experiments and inducible fate-mapping, indicated that nNOS(+) IvCs and NGCs are both derived from medial ganglionic eminence (MGE) progenitors under control of the transcription factor Nkx2-1. Surprisingly, a subset of NGCs lacking nNOS arises from caudal ganglionic eminence (CGE) progenitors. Thus, while nNOS(+) NGCs and IvCs arise from MGE progenitors, a CGE origin distinguishes a discrete population of nNOS(-) NGCs.

NMDARs Drive the Expression of Neuropsychiatric Disorder Risk Genes Within GABAergic Interneuron Subtypes in the Juvenile Brain.

Medial ganglionic eminence (MGE)-derived parvalbumin (PV)+, somatostatin (SST)+and Neurogliaform (NGFC)-type cortical and hippocampal interneurons, have distinct molecular, anatomical, and physiological properties. However, the molecular mechanisms regulating their maturation remain poorly understood. Here, via single-cell transcriptomics, we show that the obligate NMDA-type glutamate receptor (NMDAR) subunit gene Grin1 mediates transcriptional regulation of gene expression in specific subtypes of MGE-derived interneurons, leading to altered subtype abundances. Notably, MGE-specific early developmental Grin1 loss results in a broad downregulation of diverse transcriptional, synaptogenic and membrane excitability regulatory programs in the juvenile brain. These widespread gene expression abnormalities mirror aberrations that are typically associated with neurodevelopmental disorders. Our study hence provides a road map for the systematic examination of NMDAR signaling in interneuron subtypes, revealing potential MGE-specific genetic targets that could instruct future therapies of psychiatric disorders.

A biomarker-authenticated model of schizophrenia implicating NPTX2 loss of function.

Schizophrenia is a polygenetic disorder whose clinical onset is often associated with behavioral stress. Here, we present a model of disease pathogenesis that builds on our observation that the synaptic immediate early gene NPTX2 is reduced in cerebrospinal fluid of individuals with recent onset schizophrenia. NPTX2 plays an essential role in maintaining excitatory homeostasis by adaptively enhancing circuit inhibition. NPTX2 function requires activity-dependent exocytosis and dynamic shedding at synapses and is coupled to circadian behavior. Behavior-linked NPTX2 trafficking is abolished by mutations that disrupt select activity-dependent plasticity mechanisms of excitatory neurons. Modeling NPTX2 loss of function results in failure of parvalbumin interneurons in their adaptive contribution to behavioral stress, and animals exhibit multiple neuropsychiatric domains. Because the genetics of schizophrenia encompasses diverse proteins that contribute to excitatory synapse plasticity, the identified vulnerability of NPTX2 function can provide a framework for assessing the impact of genetics and the intersection with stress.

Compartmentalized Ca(2+) channel regulation at divergent mossy-fiber release sites underlies target cell-dependent plasticity.

Hippocampal mossy fibers (MFs) innervate CA3 targets via anatomically distinct presynaptic elements: MF boutons (MFBs) innervate pyramidal cells (PYRs), whereas filopodial extensions (Fils) of MFBs innervate st. lucidum interneurons (SLINs). Surprisingly, the same high-frequency stimulation (HFS) protocol induces presynaptically expressed LTP and LTD at PYR and SLIN inputs, respectively. This differential distribution of plasticity indicates that neighboring, functionally divergent presynaptic elements along the same axon serve as autonomous computational elements capable of modifying release independently. Indeed we report that HFS persistently depresses voltage-gated calcium channel (VGCC) function in Fil terminals, leaving MFB VGCCs unchanged despite similar contributions of N- and P/Q-type VGCCs to transmission at each terminal. Selective Fil VGCC depression results from HFS-induced mGluR7 activation leading to persistent P/Q-type VGCC inhibition. Thus, mGluR7 localization to MF-SLIN terminals and not MFBs allows for MF-SLIN LTD expression via depressed presynaptic VGCC function, whereas MF-PYR plasticity proceeds independently of VGCC alterations.

Transient incorporation of native GluR2-lacking AMPA receptors during hippocampal long-term potentiation.

Postnatal glutamatergic principal neuron synapses are typically presumed to express only calcium-impermeable (CI), GluR2-containing AMPARs under physiological conditions. Here, however, we demonstrate that long-term potentiation (LTP) in CA1 hippocampal pyramidal neurons causes rapid incorporation of GluR2-lacking calcium-permeable (CP)-AMPARs: CP-AMPARs are present transiently, being replaced by GluR2-containing AMPARs approximately 25 min after LTP induction. Thus, CP-AMPARs are physiologically expressed at CA1 pyramidal cell synapses during LTP, and may be required for LTP consolidation.