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.

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.

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.

Vesicle Pools of Memory at Mossy Fiber Synapses.

In this issue of Neuron, Vandael et al. (2020) reveal that post-tetanic potentiation at dentate gyrus mossy fiber synapses is induced by natural activity patterns. This plasticity is mediated by an increase in readily releasable vesicle pool size and is extended in the absence of activity, forming a "pool engram."

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.

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.

The Hyperpolarization-Activated Cation Current Ih: The Missing Link Connecting Cannabinoids to Cognition.

In this issue of Neuron, Maroso et al. (2016) describe a novel link between cannabinoids and cognition. They show that CB1Rs bidirectionally modulate HCN-mediated Ih in a subset of CA1 pyramidal neurons to influence both short- and long-term circuit dynamics and alter spatial working memory in behaving mice.

Pentraxins coordinate excitatory synapse maturation and circuit integration of parvalbumin interneurons.

Circuit computation requires precision in the timing, extent, and synchrony of principal cell (PC) firing that is largely enforced by parvalbumin-expressing, fast-spiking interneurons (PVFSIs). To reliably coordinate network activity, PVFSIs exhibit specialized synaptic and membrane properties that promote efficient afferent recruitment such as expression of high-conductance, rapidly gating, GluA4-containing AMPA receptors (AMPARs). We found that PVFSIs upregulate GluA4 during the second postnatal week coincident with increases in the AMPAR clustering proteins NPTX2 and NPTXR. Moreover, GluA4 is dramatically reduced in NPTX2(-/-)/NPTXR(-/-) mice with consequent reductions in PVFSI AMPAR function. Early postnatal NPTX2(-/-)/NPTXR(-/-) mice exhibit delayed circuit maturation with a prolonged critical period permissive for giant depolarizing potentials. Juvenile NPTX2(-/-)/NPTXR(-/-) mice display reduced feedforward inhibition yielding a circuit deficient in rhythmogenesis and prone to epileptiform discharges. Our findings demonstrate an essential role for NPTXs in controlling network dynamics highlighting potential therapeutic targets for disorders with inhibition/excitation imbalances such as schizophrenia.