Presynaptic mitochondrial morphology in monkey prefrontal cortex correlates with working memory and is improved with estrogen treatment.

Humans and nonhuman primates are vulnerable to age- and menopause-related decline in working memory, a cognitive function reliant on the energy-demanding recurrent excitation of neurons within Brodmann's Area 46 of the dorsolateral prefrontal cortex (dlPFC). Here, we tested the hypothesis that the number and morphology (straight, curved, or donut-shaped) of mitochondria in dlPFC presynaptic boutons are altered with aging and menopause in rhesus monkeys (Macaca mulatta) and that these metrics correlate with delayed response (DR) accuracy, a well-characterized measure of dlPFC-dependent working memory. Although presynaptic bouton density or size was not significantly different across groups distinguished by age or menses status, DR accuracy correlated positively with the number of total and straight mitochondria per dlPFC bouton. In contrast, DR accuracy correlated inversely with the frequency of boutons containing donut-shaped mitochondria, which exhibited smaller active zone areas and fewer docked synaptic vesicles than those with straight or curved mitochondria. We then examined the effects of estrogen administration to test whether a treatment known to improve working memory influences mitochondrial morphology. Aged ovariectomized monkeys treated with vehicle displayed significant working memory impairment and a concomitant 44% increase in presynaptic donut-shaped mitochondria, both of which were reversed with cyclic estradiol treatment. Together, our data suggest that hormone replacement therapy may benefit cognitive aging, in part by promoting mitochondrial and synaptic health in the dlPFC.

The aging mouse brain: cognition, connectivity and calcium.

Aging is a complex process that differentially impacts multiple cognitive, sensory, neuronal and molecular processes. Technological innovations now allow for parallel investigation of neuronal circuit function, structure and molecular composition in the brain of awake behaving adult mice. Thus, mice have become a critical tool to better understand how aging impacts the brain. However, a more granular systems-based approach, which considers the impact of age on key features relating to neural processing, is required. Here, we review evidence probing the impact of age on the mouse brain. We focus on a range of processes relating to neuronal function, including cognitive abilities, sensory systems, synaptic plasticity and calcium regulation. Across many systems, we find evidence for prominent age-related dysregulation even before 12 months of age, suggesting that emerging age-related alterations can manifest by late adulthood. However, we also find reports suggesting that some processes are remarkably resilient to aging. The evidence suggests that aging does not drive a parallel, linear dysregulation of all systems, but instead impacts some processes earlier, and more severely, than others. We propose that capturing the more fine-scale emerging features of age-related vulnerability and resilience may provide better opportunities for the rejuvenation of the aged brain.

DeepBouton: Automated Identification of Single-Neuron Axonal Boutons at the Brain-Wide Scale.

Fine morphological reconstruction of individual neurons across the entire brain is essential for mapping brain circuits. Inference of presynaptic axonal boutons, as a key part of single-neuron fine reconstruction, is critical for interpreting the patterns of neural circuit wiring schemes. However, automated bouton identification remains challenging for current neuron reconstruction tools, as they focus mainly on neurite skeleton drawing and have difficulties accurately quantifying bouton morphology. Here, we developed an automated method for recognizing single-neuron axonal boutons in whole-brain fluorescence microscopy datasets. The method is based on deep convolutional neural networks and density-peak clustering. High-dimensional feature representations of bouton morphology can be learned adaptively through convolutional networks and used for bouton recognition and subtype classification. We demonstrate that the approach is effective for detecting single-neuron boutons at the brain-wide scale for both long-range pyramidal projection neurons and local interneurons.

Astrocytic Coverage of Dendritic Spines, Dendritic Shafts, and Axonal Boutons in Hippocampal Neuropil.

Distal astrocytic processes have a complex morphology, reminiscent of branchlets and leaflets. Astrocytic branchlets are rod-like processes containing mitochondria and endoplasmic reticulum, capable of generating inositol-3-phosphate (IP3)-dependent Ca2+ signals. Leaflets are small and flat processes that protrude from branchlets and fill the space between synapses. Here we use three-dimensional (3D) reconstructions from serial section electron microscopy (EM) of rat CA1 hippocampal neuropil to determine the astrocytic coverage of dendritic spines, shafts and axonal boutons. The distance to the maximum of the astrocyte volume fraction (VF) correlated with the size of the spine when calculated from the center of mass of the postsynaptic density (PSD) or from the edge of the PSD, but not from the spine surface. This suggests that the astrocytic coverage of small and larger spines is similar in hippocampal neuropil. Diffusion simulations showed that such synaptic microenvironment favors glutamate spillover and extrasynaptic receptor activation at smaller spines. We used complexity and entropy measures to characterize astrocytic branchlets and leaflets. The 2D projections of astrocytic branchlets had smaller spatial complexity and entropy than leaflets, consistent with the higher structural complexity and less organized distribution of leaflets. The VF of astrocytic leaflets was highest around dendritic spines, lower around axonal boutons and lowest around dendritic shafts. In contrast, the VF of astrocytic branchlets was similarly low around these three neuronal compartments. Taken together, these results suggest that astrocytic leaflets preferentially contact synapses as opposed to the dendritic shaft, an arrangement that might favor neurotransmitter spillover and extrasynaptic receptor activation along dendritic shafts.

Size-Dependent Axonal Bouton Dynamics following Visual Deprivation In Vivo.

Persistent synapses are thought to underpin the storage of sensory experience, yet little is known about their structural plasticity in vivo. We investigated how persistent presynaptic structures respond to the loss of primary sensory input. Using in vivo two-photon (2P) imaging, we measured fluctuations in the size of excitatory axonal boutons in L2/3 of adult mouse visual cortex after monocular enucleation. The average size of boutons did not change after deprivation, but the range of bouton sizes was reduced. Large boutons decreased, and small boutons increased. Reduced bouton variance was accompanied by a reduced range of correlated calcium-mediated neural activity in L2/3 of awake animals. Network simulations predicted that size-dependent plasticity may promote conditions of greater bidirectional plasticity. These predictions were supported by electrophysiological measures of short- and long-term plasticity. We propose size-dependent dynamics facilitate cortical reorganization by maximizing the potential for bidirectional plasticity.

Long-term stability of axonal boutons in the mouse barrel cortex.

Many lines of evidence indicate that postsynaptic dendritic spines are plastic during development and largely stable in adulthood. It remains unclear to what degree presynaptic axonal terminals undergo changes in the developing and mature cortex. In this study, we examined the formation and elimination of fluorescently-labeled axonal boutons in the living mouse barrel cortex with transcranial two-photon microscopy. We found that the turnover of axonal boutons was significantly higher in 3-week-old young mice than in adult mice (older than 3 months). There was a slight but significant net loss of axonal boutons in mice from 1 to 2 months of age. In both young and adult barrel cortex, axonal boutons existed for at least 1 week were less likely to be eliminated than those recently-formed boutons. In adulthood, 80% of axonal boutons persisted over 12 months and enriched sensory experience caused a slight but not significant increase in the turnover of axonal boutons over 2-4 weeks. Thus, similar to postsynaptic dendritic spines, presynaptic axonal boutons show remarkable stability after development ends. This long-term stability of synaptic connections is likely important for reliable sensory processing in the mature somatosensory cortex.

Long-range orbitofrontal and amygdala axons show divergent patterns of maturation in the frontal cortex across adolescence.

The adolescent transition from juvenile to adult is marked by anatomical and functional remodeling of brain networks. Currently, the cellular and synaptic level changes underlying the adolescent transition are only coarsely understood. Here, we use two-photon imaging to make time-lapse observations of long-range axons that innervate the frontal cortex in the living brain. We labeled cells in the orbitofrontal cortex (OFC) and basolateral amygdala (BLA) and imaged their axonal afferents to the dorsomedial prefrontal cortex (dmPFC). We also imaged the apical dendrites of dmPFC pyramidal neurons. Images were taken daily in separate cohorts of juvenile (P24-P28) and young adult mice (P64-P68), ages where we have previously discovered differences in dmPFC dependent decision-making. Dendritic spines were pruned across this peri-adolescent period, while BLA and OFC afferents followed alternate developmental trajectories. OFC boutons showed no decrease in density, but did show a decrease in daily bouton gain and loss with age. BLA axons showed an increase in both bouton density and daily bouton gain at the later age, suggesting a delayed window of enhanced plasticity. Our findings reveal projection specific maturation of synaptic structures within a single frontal region and suggest that stabilization is a more general characteristic of maturation than pruning.

Optical imaging of structural and functional synaptic plasticity in vivo.

The adult brain has long been viewed as a collection of neuronal networks that maintain a fixed configuration of synaptic connections. Brain plasticity and learning was thought to depend exclusively on changes in the gain and offset of these connections. Over the last 50 years, molecular and cellular studies of neuroplasticity have altered this view. Brain plasticity is now viewed as a continuum of structural changes that could vary from long-range axon growth to the twitching of dendritic spines and synaptic receptor composition dynamics. Plasticity proteins similar to those that drive neuronal development may underpin brain plasticity, and consequently could regulate adaptations to new experiences and learning. In vivo imaging has confirmed that neuronal plasticity in the adult brain involves subtle structural changes at synaptic connections, including synapse formation and pruning. Synaptic structural changes are associated with experience-dependent plasticity, learning, brain traumas and neurodegeneration. Owing to the expanding toolbox of in vivo imaging we have come to the brink of understanding the causal relationship between structural synaptic network dynamics and functional brain plasticity. This review summarizes the technical developments in the imaging of laboratory animals' brains in vivo and the insights they have provided into the mechanisms of brain plasticity and learning.