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.

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.

An enhancer-AAV toolbox to target and manipulate distinct interneuron subtypes.

In recent years, we and others have identified a number of enhancers that, when incorporated into rAAV vectors, can restrict the transgene expression to particular neuronal populations. Yet, viral tools to access and manipulate fine neuronal subtypes are still limited. Here, we performed systematic analysis of single cell genomic data to identify enhancer candidates for each of the cortical interneuron subtypes. We established a set of enhancer-AAV tools that are highly specific for distinct cortical interneuron populations and striatal cholinergic neurons. These enhancers, when used in the context of different effectors, can target (fluorescent proteins), observe activity (GCaMP) and manipulate (opto- or chemo-genetics) specific neuronal subtypes. We also validated our enhancer-AAV tools across species. Thus, we provide the field with a powerful set of tools to study neural circuits and functions and to develop precise and targeted therapy.

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 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.

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.

Activity-dependent tuning of intrinsic excitability in mouse and human neurogliaform cells.

The ability to modulate the efficacy of synaptic communication between neurons constitutes an essential property critical for normal brain function. Animal models have proved invaluable in revealing a wealth of diverse cellular mechanisms underlying varied plasticity modes. However, to what extent these processes are mirrored in humans is largely uncharted thus questioning their relevance in human circuit function. In this study, we focus on neurogliaform cells, that possess specialized physiological features enabling them to impart a widespread inhibitory influence on neural activity. We demonstrate that this prominent neuronal subtype, embedded in both mouse and human neural circuits, undergo remarkably similar activity-dependent modulation manifesting as epochs of enhanced intrinsic excitability. In principle, these evolutionary conserved plasticity routes likely tune the extent of neurogliaform cell mediated inhibition thus constituting canonical circuit mechanisms underlying human cognitive processing and behavior.

Paradoxical network excitation by glutamate release from VGluT3+ GABAergic interneurons.

In violation of Dale's principle several neuronal subtypes utilize more than one classical neurotransmitter. Molecular identification of vesicular glutamate transporter three and cholecystokinin expressing cortical interneurons (CCK+VGluT3+INTs) has prompted speculation of GABA/glutamate corelease from these cells for almost two decades despite a lack of direct evidence. We unequivocally demonstrate CCK+VGluT3+INT-mediated GABA/glutamate cotransmission onto principal cells in adult mice using paired recording and optogenetic approaches. Although under normal conditions, GABAergic inhibition dominates CCK+VGluT3+INT signaling, glutamatergic signaling becomes predominant when glutamate decarboxylase (GAD) function is compromised. CCK+VGluT3+INTs exhibit surprising anatomical diversity comprising subsets of all known dendrite targeting CCK+ interneurons in addition to the expected basket cells, and their extensive circuit innervation profoundly dampens circuit excitability under normal conditions. However, in contexts where the glutamatergic phenotype of CCK+VGluT3+INTs is amplified, they promote paradoxical network hyperexcitability which may be relevant to disorders involving GAD dysfunction such as schizophrenia or vitamin B6 deficiency.

Viral manipulation of functionally distinct interneurons in mice, non-human primates and humans.

Recent success in identifying gene-regulatory elements in the context of recombinant adeno-associated virus vectors has enabled cell-type-restricted gene expression. However, within the cerebral cortex these tools are largely limited to broad classes of neurons. To overcome this limitation, we developed a strategy that led to the identification of multiple new enhancers to target functionally distinct neuronal subtypes. By investigating the regulatory landscape of the disease gene Scn1a, we discovered enhancers selective for parvalbumin (PV) and vasoactive intestinal peptide-expressing interneurons. Demonstrating the functional utility of these elements, we show that the PV-specific enhancer allowed for the selective targeting and manipulation of these neurons across vertebrate species, including humans. Finally, we demonstrate that our selection method is generalizable and characterizes additional PV-specific enhancers with exquisite specificity within distinct brain regions. Altogether, these viral tools can be used for cell-type-specific circuit manipulation and hold considerable promise for use in therapeutic interventions.

Satellite NG2 progenitor cells share common glutamatergic inputs with associated interneurons in the mouse dentate gyrus.

Several studies have provided evidence that NG2-expressing (NG2(+)) progenitor cells are anatomically associated to neurons in gray matter areas. By analyzing the spatial distribution of NG2(+) cells in the hilus of the mouse dentate gyrus, we demonstrate that NG2(+) cells are indeed closely associated to interneurons. To define whether this anatomical proximity reflected a specific physiological interaction, we performed patch-clamp recordings on hilar NG2(+) cells and interneurons between 3 and 21 postnatal days. We first observed that hilar NG2(+) cells exhibit spontaneous glutamatergic EPSCs (sEPSCs) whose frequency and amplitude increase during the first 3 postnatal weeks. At the same time, the rise time and decay time of sEPSCs significantly decreased, suggesting that glutamatergic synapses in NG2(+) cells undergo a maturation process that is reminiscent of what has been reported in neurons during the same time period. We also observed that hilar interneurons and associated NG2(+) cells are similarly integrated into the local network, receiving excitatory inputs from both granule cells and CA3 pyramidal neurons. By performing pair recordings, we found that bursts of activity induced by GABAergic antagonists were strongly synchronized between both cell types and that the amplitude of these bursts was positively correlated. Finally, by applying carbachol to increase EPSC activity, we observed that closely apposed cells were more likely to exhibit synchronized EPSCs than cells separated by >200 microm. The finding that NG2(+) cells are sensing patterns of activity arising in closely associated neurons suggests that NG2(+) cell function is finely regulated by the local network.

NG2 cell response in the CNP-EGFP mouse after contusive spinal cord injury.

NG2(+) cells in the adult CNS are a heterogeneous population. The extent to which the subpopulation of NG2(+) cells that function as oligodendrocyte progenitor cells (OPCs) respond to spinal cord injury (SCI) and recapitulate their normal developmental progression remains unclear. We used the CNP-EGFP mouse, in which oligodendrocyte lineage cells express EGFP, to study NG2(+) cells in the normal and injured spinal cord. In white matter of uninjured mice, bipolar EGFP(+)NG2(+) cells and multipolar EGFP(neg)NG2(+) cells were identified. After SCI, EGFP(+)NG2(+) cell proliferation in residual white matter peaked at 3 days post injury (DPI) rostral to the epicenter, while EGFP(neg)NG2(+) cell proliferation peaked at 7 DPI at the epicenter. The expression of transcription factors, Olig2, Sox10, and Sox17, and the basic electrophysiological membrane parameters and potassium current phenotype of the EGFP(+)NG2(+) population after injury were consistent with those of proliferative OPCs during development. EGFP(neg)NG2(+) cells did not express transcription factors involved in oligodendrogenesis. EGFP(+)CC1(+) oligodendrocytes at 6 weeks included cells that incorporated BrdU during the peak of EGFP(+)NG2(+) cell proliferation. EGFP(neg)CC1(+) oligodendrocytes were never observed. Treatment with glial growth factor 2 and fibroblast growth factor 2 enhanced oligodendrogenesis and increased the number of EGFP(neg)NG2(+) cells. Therefore, based on EGFP and transcription factor expression, spatiotemporal proliferation patterns, and response to growth factors, two populations of NG2(+) cells can be identified that react to SCI. The EGFP(+)NG2(+) cells undergo cellular and physiological changes in response to SCI that are similar to those that occur in early postnatal NG2(+) cells during developmental oligodendrogenesis.

NG2-expressing cells in the subventricular zone are type C-like cells and contribute to interneuron generation in the postnatal hippocampus.

The subventricular zone (SVZ) is a source of neural progenitors throughout brain development. The identification and purification of these progenitors and the analysis of their lineage potential are fundamental issues for future brain repair therapies. We demonstrate that early postnatal NG2-expressing (NG2+) progenitor cells located in the SVZ self-renew in vitro and display phenotypic features of transit-amplifier type C-like multipotent cells. NG2+ cells in the SVZ are highly proliferative and express the epidermal growth factor receptor, the transcription factors Dlx, Mash1, and Olig2, and the Lewis X (LeX) antigen. We show that grafted early postnatal NG2+ cells generate hippocampal GABAergic interneurons that propagate action potentials and receive functional glutamatergic synaptic inputs. Our work identifies Dlx+/Mash1+/LeX+/NG2+/GFAP-negative cells of the SVZ as a new class of postnatal multipotent progenitor cells that may represent a specific cellular reservoir for renewal of postnatal and adult inhibitory interneurons in the hippocampus.

Postnatal NG2 proteoglycan-expressing progenitor cells are intrinsically multipotent and generate functional neurons.

Neurogenesis is known to persist in the adult mammalian central nervous system (CNS). The identity of the cells that generate new neurons in the postnatal CNS has become a crucial but elusive issue. Using a transgenic mouse, we show that NG2 proteoglycan-positive progenitor cells that express the 2',3'-cyclic nucleotide 3'-phosphodiesterase gene display a multipotent phenotype in vitro and generate electrically excitable neurons, as well as astrocytes and oligodendrocytes. The fast kinetics and the high rate of multipotent fate of these NG2+ progenitors in vitro reflect an intrinsic property, rather than reprogramming. We demonstrate in the hippocampus in vivo that a sizeable fraction of postnatal NG2+ progenitor cells are proliferative precursors whose progeny appears to differentiate into GABAergic neurons capable of propagating action potentials and displaying functional synaptic inputs. These data show that at least a subpopulation of postnatal NG2-expressing cells are CNS multipotent precursors that may underlie adult hippocampal neurogenesis.

Exploring the Interneuron Canopy Atop the 'Impenetrable Jungle'.

The multifaceted functions of the brain are borne through seemingly infinite spatiotemporal interactions between its resident neural elements. Using a combinatorial approach, Schuman and colleagues (J. Neurosci. 2018;39:125-139) recently identify four layer 1 cortical interneuron subtypes, including a hitherto uncharacterized neuron they term the 'canopy' cell. Properties unique to each of the subtypes likely endow them with distinct roles in top-down processing.

Differential synaptic integration of interneurons in the outer and inner molecular layers of the developing dentate gyrus.

The dentate gyrus (DG) undergoes continued reorganization and lamination during early postnatal development. Interneurons with anatomically identified synaptic contacts migrate from the outer to the inner regions of the molecular layer (ML) of the DG. By using the 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP)-enhanced green fluorescent protein transgenic mouse, we were able to target and physiologically characterize Dlx2(+) developing ML interneurons. We investigated whether synapses on migrating ML interneurons were functional and defined properties of synaptic inputs onto interneurons that were located in the outer ML (OML) or inner ML (IML). Consistent with ongoing maturation, IML interneurons displayed lower input resistances and more hyperpolarized resting membrane potentials than OML interneurons. Both OML and IML interneurons received a direct excitatory monosynaptic input from the entorhinal cortex via the perforant paths, but this input was differentially sensitive to activation of presynaptic group II and III metabotropic glutamate receptors. Furthermore, only IML interneurons also received significant synaptic input from the CA3/hilar region, especially under conditions of experimentally induced disinhibition. These changes are attributed to a significant reorganization of dendritic fields. GABA(A) receptor-mediated innervation of OML and IML interneurons also displayed significant differences in miniature IPSC amplitude, frequency, and decay kinetics. Finally, cell-attached recordings indicated that GABA(A) receptor activation was depolarizing in OML interneurons but predominantly shunting in IML interneurons. Our data provide evidence that developing ML interneurons receive functional glutamatergic and GABAergic inputs and undergo significant changes in synaptic integration during migration from the OML to the IML.

Neto Auxiliary Subunits Regulate Interneuron Somatodendritic and Presynaptic Kainate Receptors to Control Network Inhibition.

Although Netos are considered auxiliary subunits critical for kainate receptor (KAR) function, direct evidence for their regulation of native KARs is limited. Because Neto KAR regulation is GluK subunit/Neto isoform specific, such regulation must be determined in cell-type-specific contexts. We demonstrate Neto1/2 expression in somatostatin (SOM)-, cholecystokinin/cannabinoid receptor 1 (CCK/CB1)-, and parvalbumin (PV)-containing interneurons. KAR-mediated excitation of these interneurons is contingent upon Neto1 because kainate yields comparable effects in Neto2 knockouts and wild-types but fails to excite interneurons or recruit inhibition in Neto1 knockouts. In contrast, presynaptic KARs in CCK/CB1 interneurons are dually regulated by both Neto1 and Neto2. Neto association promotes tonic presynaptic KAR activation, dampening CCK/CB1 interneuron output, and loss of this brake in Neto mutants profoundly increases CCK/CB1 interneuron-mediated inhibition. Our results confirm that Neto1 regulates endogenous somatodendritic KARs in diverse interneurons and demonstrate Neto regulation of presynaptic KARs in mature inhibitory presynaptic terminals.

Downregulation of platelet-derived growth factor-alpha receptor-mediated tyrosine kinase activity as a cellular mechanism for K+-channel regulation during oligodendrocyte development in situ.

Oligodendrocyte maturation has been defined based on expression of developmentally regulated antigens. However, transitions at early stages of the lineage have not been functionally characterized fully in situ. Combining 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP)-promoter driven enhanced green fluorescent protein expression and whole-cell capacitance measurements permitted a reliable distinction between subcortical white matter NG2+ oligodendrocyte progenitors (OPs) and O4+ preoligodendrocytes (pre-OLs) in situ. We focused on K+ channels because their expression has been associated previously with the proliferation and differentiation potential of OPs. Using whole-cell patch clamp, we observed a downregulation of the delayed outward-rectifying current (IKDR) between the NG2+ and O4+ stages but no significant changes in transient K+-channel current (IKA) amplitude. Tyrosine kinase inhibition in NG2+ cells reduced IKDR amplitude with no effect on IKA, which mimicked the endogenous changes observed between OPs and pre-OLs. Tyrosine kinase inhibition also reduced the proliferative capacity of NG2+ OPs in slice cultures. Conversely, acute platelet-derived growth factor receptor-alpha (PDGFR-alpha) activation caused an increase of IKDR in NG2+ but not in O4+ cells. Consistent with this finding, PDGFR-alpha immunoreactivity was confined to NG2+ cells with undetectable levels in O4+ cells, suggesting that PDGFR-alpha signaling is absent in pre-OLs in situ. Importantly, the PDGF-induced increase of IKDR in NG2+ cells was prevented by tyrosine kinase inhibition. Together, these data indicate that PDGFR-alpha and tyrosine kinase activity act via a common pathway that influences functional expression of K+ channels and proliferative capacity of OPs in situ.