Activity-dependent compartmentalization of dendritic mitochondria morphology through local regulation of fusion-fission balance in neurons in vivo.

Neuronal mitochondria play important roles beyond ATP generation, including Ca2+ uptake, and therefore have instructive roles in synaptic function and neuronal response properties. Mitochondrial morphology differs significantly between the axon and dendrites of a given neuronal subtype, but in CA1 pyramidal neurons (PNs) of the hippocampus, mitochondria within the dendritic arbor also display a remarkable degree of subcellular, layer-specific compartmentalization. In the dendrites of these neurons, mitochondria morphology ranges from highly fused and elongated in the apical tuft, to more fragmented in the apical oblique and basal dendritic compartments, and thus occupy a smaller fraction of dendritic volume than in the apical tuft. However, the molecular mechanisms underlying this striking degree of subcellular compartmentalization of mitochondria morphology are unknown, precluding the assessment of its impact on neuronal function. Here, we demonstrate that this compartment-specific morphology of dendritic mitochondria requires activity-dependent, Ca2+ and Camkk2-dependent activation of AMPK and its ability to phosphorylate two direct effectors: the pro-fission Drp1 receptor Mff and the recently identified anti-fusion, Opa1-inhibiting protein, Mtfr1l. Our study uncovers a signaling pathway underlying the subcellular compartmentalization of mitochondrial morphology in dendrites of neurons in vivo through spatially precise and activity-dependent regulation of mitochondria fission/fusion balance.

Functional architecture of intracellular oscillations in hippocampal dendrites.

Fast electrical signaling in dendrites is central to neural computations that support adaptive behaviors. Conventional techniques lack temporal and spatial resolution and the ability to track underlying membrane potential dynamics present across the complex three-dimensional dendritic arbor in vivo. Here, we perform fast two-photon imaging of dendritic and somatic membrane potential dynamics in single pyramidal cells in the CA1 region of the mouse hippocampus during awake behavior. We study the dynamics of subthreshold membrane potential and suprathreshold dendritic events throughout the dendritic arbor in vivo by combining voltage imaging with simultaneous local field potential recording, post hoc morphological reconstruction, and a spatial navigation task. We systematically quantify the modulation of local event rates by locomotion in distinct dendritic regions and report an advancing gradient of dendritic theta phase along the basal-tuft axis, then describe a predominant hyperpolarization of the dendritic arbor during sharp-wave ripples. Finally, we find spatial tuning of dendritic representations dynamically reorganizes following place field formation. Our data reveal how the organization of electrical signaling in dendrites maps onto the anatomy of the dendritic tree across behavior, oscillatory network, and functional cell states.

Activity-dependent subcellular compartmentalization of dendritic mitochondria structure in CA1 pyramidal neurons.

Neuronal mitochondria play important roles beyond ATP generation, including Ca2+ uptake, and therefore have instructive roles in synaptic function and neuronal response properties. Mitochondrial morphology differs significantly in the axon and dendrites of a given neuronal subtype, but in CA1 pyramidal neurons (PNs) of the hippocampus, mitochondria within the dendritic arbor also display a remarkable degree of subcellular, layer-specific compartmentalization. In the dendrites of these neurons, mitochondria morphology ranges from highly fused and elongated in the apical tuft, to more fragmented in the apical oblique and basal dendritic compartments, and thus occupy a smaller fraction of dendritic volume than in the apical tuft. However, the molecular mechanisms underlying this striking degree of subcellular compartmentalization of mitochondria morphology are unknown, precluding the assessment of its impact on neuronal function. Here, we demonstrate that this compartment-specific morphology of dendritic mitochondria requires activity-dependent, Camkk2-dependent activation of AMPK and its ability to phosphorylate two direct effectors: the pro-fission Drp1 receptor Mff and the recently identified anti-fusion, Opa1-inhibiting protein, Mtfr1l. Our study uncovers a new activity-dependent molecular mechanism underlying the extreme subcellular compartmentalization of mitochondrial morphology in dendrites of neurons in vivo through spatially precise regulation of mitochondria fission/fusion balance.

Compartment-specific tuning of dendritic feature selectivity by intracellular Ca2+ release.

Dendritic calcium signaling is central to neural plasticity mechanisms that allow animals to adapt to the environment. Intracellular calcium release (ICR) from the endoplasmic reticulum has long been thought to shape these mechanisms. However, ICR has not been investigated in mammalian neurons in vivo. We combined electroporation of single CA1 pyramidal neurons, simultaneous imaging of dendritic and somatic activity during spatial navigation, optogenetic place field induction, and acute genetic augmentation of ICR cytosolic impact to reveal that ICR supports the establishment of dendritic feature selectivity and shapes integrative properties determining output-level receptive fields. This role for ICR was more prominent in apical than in basal dendrites. Thus, ICR cooperates with circuit-level architecture in vivo to promote the emergence of behaviorally relevant plasticity in a compartment-specific manner.

Local feedback inhibition tightly controls rapid formation of hippocampal place fields.

Hippocampal place cells underlie spatial navigation and memory. Remarkably, CA1 pyramidal neurons can form new place fields within a single trial by undergoing rapid plasticity. However, local feedback circuits likely restrict the rapid recruitment of individual neurons into ensemble representations. This interaction between circuit dynamics and rapid feature coding remains unexplored. Here, we developed "all-optical" approaches combining novel optogenetic induction of rapidly forming place fields with 2-photon activity imaging during spatial navigation in mice. We find that induction efficacy depends strongly on the density of co-activated neurons. Place fields can be reliably induced in single cells, but induction fails during co-activation of larger subpopulations due to local circuit constraints imposed by recurrent inhibition. Temporary relief of local inhibition permits the simultaneous induction of place fields in larger ensembles. We demonstrate the behavioral implications of these dynamics, showing that our ensemble place field induction protocol can enhance subsequent spatial association learning.

Dendritic Excitability and Synaptic Plasticity In Vitro and In Vivo.

Much of our understanding of dendritic and synaptic physiology comes from in vitro experimentation, where the afforded mechanical stability and convenience of applying drugs allowed patch-clamping based recording techniques to investigate ion channel distributions, their gating kinetics, and to uncover dendritic integrative and synaptic plasticity rules. However, with current efforts to study these questions in vivo, there is a great need to translate existing knowledge between in vitro and in vivo experimental conditions. In this review, we identify discrepancies between in vitro and in vivo ionic composition of extracellular media and discuss how changes in ionic composition alter dendritic excitability and plasticity induction. Here, we argue that under physiological in vivo ionic conditions, dendrites are expected to be more excitable and the threshold for synaptic plasticity induction to be lowered. Consequently, the plasticity rules described in vitro vary significantly from those implemented in vivo.

Early cis-regulatory events in the formation of retinal horizontal cells.

During retinal development, multipotent and restricted progenitor cells generate all of the neuronal cells of the retina. Among these are horizontal cells, which are interneurons that modulate the light-induced signal from photoreceptors. This study utilizes the identification of novel cis-regulatory elements as a method to examine the gene regulatory networks that direct the development of horizontal cells. Here we describe a screen for cis-regulatory elements, or enhancers, for the horizontal cell-associated genes PTF1A, ONECUT1 (OC1), TFAP2A (AP2A), and LHX1. The OC1ECR22 and Tfap2aACR5 elements were shown to be potential enhancers for OC1 and TFAP2A, respectively, and to be specifically active in developing horizontal cells. The OC1ECR22 element is activated by PTF1A and RBPJ, which translates to regulation of OC1 expression and suggests that PTF1A is a direct activator of OC1 expression in developing horizontal cells. The region within the Tfap2aACR5 element that is responsible for its activation was determined to be a 100 bp sequence named Motif 4. Both OC1ECR22 and Tfap2aACR5 are negatively regulated by the nuclear receptors THRB and RXRG, as is the expression of OC1 and AP2A, suggesting that nuclear receptors may have a role in the negative regulation of horizontal cell development.

OTX2 represses sister cell fate choices in the developing retina to promote photoreceptor specification.

During vertebrate retinal development, subsets of progenitor cells generate progeny in a non-stochastic manner, suggesting that these decisions are tightly regulated. However, the gene-regulatory network components that are functionally important in these progenitor cells are largely unknown. Here we identify a functional role for the OTX2 transcription factor in this process. CRISPR/Cas9 gene editing was used to produce somatic mutations of OTX2 in the chick retina and identified similar phenotypes to those observed in human patients. Single cell RNA sequencing was used to determine the functional consequences OTX2 gene editing on the population of cells derived from OTX2-expressing retinal progenitor cells. This confirmed that OTX2 is required for the generation of photoreceptors, but also for repression of specific retinal fates and alternative gene regulatory networks. These include specific subtypes of retinal ganglion and horizontal cells, suggesting that in this context, OTX2 functions to repress sister cell fate choices.