Partial connectomes of labeled dopaminergic circuits reveal non-synaptic communication and axonal remodeling after exposure to cocaine

Dopaminergic (DA) neurons exert profound influences on behavior including addiction. However, how DA axons communicate with target neurons and how those communications change with drug exposure remains poorly understood. We leverage cell type-specific labeling with large volume serial electron microscopy to detail DA connections in the nucleus accumbens (NAc) of the mouse (Mus musculus) before and after exposure to cocaine. We find that individual DA axons contain different varicosity types based on their vesicle contents. Spatially ordering along individual axons further suggests that varicosity types are non-randomly organized. DA axon varicosities rarely make specific synapses (<2%, 6/410), but instead are more likely to form spinule-like structures (15%, 61/410) with neighboring neurons. Days after a brief exposure to cocaine, DA axons were extensively branched relative to controls, formed blind-ended ‘bulbs’ filled with mitochondria, and were surrounded by elaborated glia. Finally, mitochondrial lengths increased by ~2.2 times relative to control only in DA axons and NAc spiny dendrites after cocaine exposure. We conclude that DA axonal transmission is unlikely to be mediated via classical synapses in the NAc and that the major locus of anatomical plasticity of DA circuits after exposure to cocaine are large-scale axonal re-arrangements with correlated changes in mitochondria.

Oligodendrocyte precursor cells prune axons in the mouse neocortex

Neurons in the developing brain undergo extensive structural refinement as nascent circuits adopt their mature form1. This transformation is facilitated by the engulfment and degradation of excess axonal branches and inappropriate synapses by surrounding glial cells, including microglia and astrocytes2,3. However, the small size of phagocytic organelles and the complex, highly ramified morphology of glia has made it difficult to determine the contribution of these and other glial cell types to this process. Here, we used large scale, serial electron microscopy (ssEM) with computational volume segmentation to reconstruct the complete 3D morphologies of distinct glial types in the mouse visual cortex. Unexpectedly, we discovered that the fine processes of oligodendrocyte precursor cells (OPCs), a population of abundant, highly dynamic glial progenitors4, frequently surrounded terminal axon branches and included numerous phagolysosomes (PLs) containing fragments of axons and presynaptic terminals. Single- nucleus RNA sequencing indicated that cortical OPCs express key phagocytic genes, as well as neuronal transcripts, consistent with active axonal engulfment. PLs were ten times more abundant in OPCs than in microglia in P36 mice, and declined with age and lineage progression, suggesting that OPCs contribute very substantially to refinement of neuronal circuits during later phases of cortical development.

Desmosomal connectomics of all somatic muscles in an annelid larva

Cells form networks in animal tissues through synaptic, chemical and adhesive links. In muscle tissue, cells often connect through desmosomes, adhesive structures anchored by intermediate filaments. To study desmosomal networks, we reconstructed 852 muscle cells and their desmosomal partners by serial electron microscopy in an entire larva of the annelid Platynereis. Muscle cells adhere to each other, to epithelial, glial, ciliated, and bristle-producing cells and to the basal lamina, forming a desmosomal connectome of over 2,000 cells. The aciculae - chitin rods that form an endoskeleton in the segmental parapodia - are highly connected hubs in this network. This agrees with the many degrees of freedom of their movement, as revealed by video microscopy. Mapping motoneuron synapses to the desmosomal connectome highlighted the extent of tissue influenced by individual motoneuron classes. Our work shows how cellular-level maps of synaptic and adherent force networks can elucidate body mechanics.

Candelabrum cells are molecularly distinct, ubiquitous interneurons of the cerebellar cortex with specialized circuit properties

To understand how the cerebellar cortex transforms mossy fiber (MF) inputs into Purkinje cell (PC) outputs, it is vital to delineate the elements of this circuit. Candelabrum cells (CCs) are enigmatic interneurons of the cerebellar cortex that have been identified based on their morphology, but their electrophysiological properties, synaptic connections, and function remain unknown. Here we clarify these properties using electrophysiology, snRNA sequencing, in situ hybridization, and serial electron microscopy. We find that CCs are the most abundant PC layer interneuron. They are GABAergic, molecularly distinct, and present in all cerebellar lobules. Their high resistance renders CC firing highly sensitive to synaptic inputs. CCs are excited by MFs and granule cells, and strongly inhibited by PCs. CCs in turn inhibit molecular layer interneurons, which leads to PC disinhibition. Thus, inputs, outputs and local signals all converge onto CCs to allow them to assume a unique role in controlling cerebellar output.

Cocaine causes rapid remodeling of mesoaccumbens dopaminergic axons, synapses, and mitochondria

Detailing the ways drugs of abuse physically alter dopaminergic circuits would provide new mechanisms for explaining addictive behaviors, future targets for therapeutic intervention, and insights into the nature of synaptic plasticity. We combine recent advances in genetic labeling with large volume serial electron microscopy to detail how normal dopaminergic (DA) axons interact with putative targets in the Nucleus Accumbens (NAc) and how those interactions change in mice briefly exposed to cocaine. We find that while most DA axonal boutons are devoid of obvious signs of synapses (i.e. synaptic vesicles or synaptic densities), many DA boutons physically interdigitate with dendrites or excitatory and inhibitory axons. A brief exposure to cocaine results in large-scale remodeling: extensive DA axonal branching and frequent occurrences of axonal blind-ended “bulbs”, filled with mitochondria and reminiscent of axonal retraction in the developing and damaged brain. The number of physical interdigitations and vesicle filled boutons in DA axons scales linearly with the length of axon in both controls and cocaine exposed animals and the size or the type of interaction (i.e. axo-axonic or axo-dendritic) do not change. Finally, we find in cocaine exposed animals, mitochondrial lengths are increased ~2.5 times relative to control. Mitochondrial elongation is cell type specific: primarily in DA neurons and downstream spiny dendrites, and localized to DA axons and not DA soma or dendrites. We show for the first time the effects of cocaine on remodeling of dopamine axon morphology and mitochondria and reveal new details on how dopamine neurons physically associate with downstream targets.

Multi-modal imaging of a single mouse brain over five orders of magnitude of resolution

ABSTRACTMammalian neurons operate at length scales spanning five orders of magnitude; micron-scale-diameter myelinated axons project millimeters across brain regions, ultimately forming nanometer scale synapses on individual post-synaptic neurons. Capturing these anatomical features across that breadth of scale has required imaging samples with multiple independent imaging modalities (e.g. MRI, electron microscopy, etc.). Translating between the different modalities, however, requires imaging the same brain with each. Here, we imaged the same postmortem mouse brain over five orders of spatial resolution using MRI, whole brain micron-scale synchrotron x-ray tomography (μCT), and large volume automated serial electron microscopy. Using this pipeline, we can track individual myelinated axons previously relegated to axon bundles in diffusion tensor MRI or arbitrarily trace neurons and their processes brain-wide and identify individual synapses on them. This pipeline provides both an unprecedented look across a single brain’s multi-scaled organization as well as a vehicle for studying the brain’s multi-scale pathologies.

Cocaine causes rapid remodeling of mesoaccumbens dopaminergic axons, synapses, and mitochondria

SummaryDetailing the ways drugs of abuse physically alter dopaminergic circuits would provide new mechanisms for explaining addictive behaviors, future targets for therapeutic intervention, and insights into the nature of synaptic plasticity. We combine recent advances in genetic labeling with large volume serial electron microscopy to detail how normal dopaminergic (DA) axons interact with putative targets in the Nucleus Accumbens (NAc) and how those interactions change in mice briefly exposed to cocaine. We find that while most DA axonal boutons are devoid of obvious signs of synapses (i.e. synaptic vesicles or synaptic densities), many DA boutons physically interdigitate with dendrites or excitatory and inhibitory axons. A brief exposure to cocaine results in large-scale remodeling: extensive DA axonal branching and frequent occurrences of axonal blind-ended “bulbs”, filled with mitochondria and reminiscent of axonal retraction in the developing and damaged brain. The number of physical interdigitations and vesicle filled boutons in DA axons scales linearly with the length of axon in both controls and cocaine exposed animals and the size or the type of interaction (i.e. axo-axonic or axo-dendritic) do not change. Finally, we find in cocaine exposed animals, mitochondrial lengths are increased ~2.5 times relative to control. Mitochondrial elongation is cell type specific: primarily in DA neurons and downstream spiny dendrites, and localized to DA axons and not DA soma or dendrites. We show for the first time the effects of cocaine on remodeling of dopamine axon morphology and mitochondria and reveal new details on how dopamine neurons physically associate with downstream targets.

The rich get richer: synaptic remodeling between climbing fibers and Purkinje cells in the developing cerebellum begins with positive feedback addition of synapses

SUMMARYDuring postnatal development, cerebellar climbing fibers strongly innervate a subset of their original Purkinje cell targets and eliminate their connections from the rest. In the adult, each climbing fiber innervates a small number of Purkinje cells and each Purkinje cell is innervated by a single climbing fiber. To get insight about the processes responsible for this remapping, we reconstructed serial electron microscopy datasets from mice during the first postnatal week. In contrast to adult connectivity, individual neonatal climbing fibers innervate many nearby Purkinje cells, and multiple climbing fibers innervate each Purkinje cell. Between postnatal days 3 and 7, Purkinje cells retract long dendrites and grow many proximal dendritic processes. On this changing landscape, individual climbing fibers selectively add many synapses to a subset of Purkinje cell targets in a positive-feedback manner, without pruning synapses from other Purkinje cells. The active zone sizes of synapses associated with powerful versus weak inputs are indistinguishable. These results show that changes in synapse number rather than synapse size are the predominant form of early developmental plasticity. Finally, although multiple climbing fibers innervate each Purkinje cell in early postnatal development, the number of climbing fibers and Purkinje cells in a local cerebellar region nearly match. Thus, initial over-innervation of Purkinje cells by climbing fibers is economical, in that the number of axons entering a region is enough to assure that each axon ends up with a postsynaptic target, and that none branched there in vain.HIGHLIGHTSDeveloping climbing fibers establish synapses on many neighboring Purkinje cells unlike the sparse pattern of innervation in later lifeClimbing fibers add many synapses onto a few of their Purkinje targets before the pruning stage in a rich-get-richer type processThe synapse sizes of strengthened and weakened climbing fiber inputs are indistinguishable.Exuberant branching of climbing fiber axons in early postnatal life appears to be economical because the numbers of axons and Purkinje cells in a local region match, ensuring that each axon can establish a long-lasting connection thereBLURBHigh-resolution serial electron microscopy reconstructions reveal that climbing fiber-Purkinje cell synaptic refinement in the developing cerebellum begins with significant synapse addition. Climbing fibers focus their synapses onto a smaller number of Purkinje cells by selectively adding synapses onto some target cells. All axons that project to a region in development play a role in the final connectivity.