Cellular And Synaptic Physiology Of Hippocampal Interneurons

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Although cortical and hippocampal GABAergic inhibitory interneurons represent only 20% of the total cortical cell population their anatomical diversity is unparalleled in the mammalian central nervous system; for example there are currently upwards of 20 acknowledged distinct members within the CA1 hippocampal formation alone. Their anatomical diversity is rich, with the morphologies of many cell types remaining local to a particular subfield, while other cell types extend wide arbor dendrites and axons that cross numerous cortical and hippocampal layers and subfields. Inhibitory interneurons often demonstrate exquisite targeting of their axons to differential postsynaptic structures. For example, axons can target selective subcellular domains (e.g. the perisomatic, axon initial segment or specific dendritic domains) to compartmentalize or time electrical activity in either a positive or negative manner. Alternatively, axons can make projections several millimeters in length, to innervate thousands of postsynaptic targets to co-ordinate the activity of both homogeneous and distributed neuronal ensembles. Moreover, each cortical interneuron subtype is unique in its proliferative history, migration during corticogenesis as well as postnatal integration into cortical circuitry. Indeed several developmentally regulated neural circuit disorders such as epilepsy, schizophrenia and autism are likely associated with deficits in the numbers and function of distinct interneuron cohorts. For all of these reasons inhibitory interneurons have recently become the intense focus of investigators drawn from a wide variety of backgrounds. Over the last year we have have continued our study of glutamatergic and GABAergic synaptic transmission made onto both principal neurons and inhibitory interneurons and their downstream targets within the hippocampal formation. We are also using genetic approaches to examine the role of inhibitory interneurons in the onset and progression of circuit degradation in a variety of mouse models. We use a multi-parametric approach of cortical and hippocampal development and circuit function, which has been an extremely fruitful research avenue and is a perfect example of a research strategy well suited to the intramural environment. Evolutionary conservation of Mossy fiber synapses: Computational models based primarily on rodent data predict that mossy fiber (MF) synaptic connections between hippocampal dentate gyrus granule cells (DGGCs) and CA3 pyramidal cells (PCs) are essential for encoding contextual memories. MF encoding of memory is considered to relate directly to a host of peculiar specialized structural/functional synaptic properties including sparse innervation by large multirelease site terminals supporting a remarkable frequency-dependent dynamic range of transmission onto the most proximal dendrites of CA3 PCs. Though investigated to exquisite detail in model organisms, synapses, including MFs, have undergone relatively minimal functional interrogation in the human brain. To determine the translational relevance of rodent MF findings to the human brain Ken Pelkey in collaboration with neurosurgeon Dr Kareem Zaghloul (NINDS) and Katalin Toth (University of Ottowa) evaluated the basic synaptic properties of MF connections within human hippocampal tissue resected for treatment of pharmacoresistant epilepsy. Human MF transmission exhibits 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 presynaptically expressed long-term potentiation, and strong cAMP sensitivity of presynaptic release. Moreover, serial array tomography electron microscopy confirmed evolutionary conservation of MF synapse ultrastructure. The astonishing congruence of the core features shared between rodent and human MF synapses argues that the basic properties of MF transmission reported in experimental animal models are also critical to human MF function. However, of interest from the disease perspective, we observed a dramatic selective deficit in GABAergic inhibitory tone onto human MF postsynaptic targets, suggesting that unrestrained detonator excitatory drive contributes to circuit hyperexcitability in epilepsy. Loss of the NMDA receptor subunit Grin2a Causes a Transient Delay in the Maturation of Hippocampal Parvalbumin Interneurons: N-methyl-D-aspartate receptors (NMDARs) comprise a family of ligand-gated ionotropic glutamate receptors that mediate a calcium-permeable component to fast excitatory neurotransmission. NMDARs are heterotetrameric assemblies of two obligate GluN1 subunits (encoded by the GRIN1 gene) and two GluN2 subunits (encoded by the GRIN2A-GRIN2D genes). 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. To explore this new clinical finding at that circuit and cellular level, we conducted studies using Grin2a+/- and Grin2a-/- mice at various stages during neurodevelopment. 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 other GRIN genes (including Grin2b) nor can they be attributed to perturbations in the intrinsic excitability or action-potential firing properties of CA1 pyramidal cells. 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 PV interneurons. Overall, we report that Grin2a+/+ mice reach electrophysiological maturation between the neonatal and juvenile neurodevelopmental timepoints, with Grin2a+/- mice not reaching electrophysiological maturation until preadolescence, and Grin2a-/- not reaching electrophysiological maturation until adulthood. Overall, these data may represent a molecular mechanism describing the transient nature of seizure burden in disease-associated null GRIN2A patients. Development of tools for interrogation of cellular and circuit evolutionary conservation Over the last few years we have begun to explore evolutionary conservation of interneuron function from rodents to humans. This has necessitated the generation of tools that allow unequivocal identification of INs in species from which few studies exist. In two ongoing collaborations with the labs of Dr. Gord Fishell (MIT/Broad) and Matt Rowan (Emory) we have developed and characterized several AAV vectors utilizing neuron-type-specific regulatory transcriptional sequences (enhancer-AAVs), which have allowed us to overcome the limitations set by using mouse Cre-lines. 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. We achieved this in compact vectors by integrating structural improvements of AAV vectors with innovative molecular tools. In a series of manuscripts, we have illustrate