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.

Mammalian neurons operate at length scales spanning six orders of magnitude; they project millimeters to centimeters across brain regions, are composed of micrometer-scale-diameter myelinated axons, and ultimately form nanometer scale synapses. Capturing these anatomical features across that breadth of scale has required imaging samples with multiple independent imaging modalities. 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 micrometer-scale synchrotron x-ray tomography ( μ CT), 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.

Mammalian 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, 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.

Despite the prevalence and high heritability of Attention-Deficit/Hyperactivity Disorder (ADHD), genetic etiology remains elusive. Clinical evidence points in part to reduced function of the striatum, but which specific genes are differentially expressed and how they sculpt striatal physiology to predispose ADHD are not well understood. As an exploratory tool, a polygenic mouse model of ADHD was recently developed through selective breeding for high home cage activity. Relative to the Control line, the High-Active line displays hyperactivity and motor impulsivity which are ameliorated with amphetamine. This study compared gene expression in the striatum between Control and High-Active mice to develop a coherent hypothesis for how genes might affect striatal physiology and predispose ADHD-like symptoms. To this end, striatal transcriptomes of High-Active and Control mice were analyzed after mice were treated with saline or amphetamines. The pseudogene Gm6180 for n-cofilin (Cfl1) displayed 20-fold higher expression in High-Active mice corresponding with reduced Cfl1 expression suggesting synaptic actin dysregulation. Latrophilin 3 (Lphn3), which is associated with ADHD in human populations and is involved in synapse structure, and its ligand fibronectin leucine rich transmembrane protein 3 (Flrt3), were downregulated in High-Active mice. Multiple genes were altered in High-Active mice in a manner predicted to downregulate the canonical Wnt pathway. A smaller and different set of genes including glyoxalase (Glo1) were differentially regulated in High-Active as compared to Control in response to amphetamine. Together, results suggest genes involved in excitatory synapse regulation and maintenance are downregulated in ADHD-like mice. Consistent with the molecular prediction, stereological analysis of the striatum from a separate set of mice processed for imunohistochemical detection of synaptophysin revealed approximately a 46% reduction in synaptophysin immunoreactivity in High-Active relative to Control. Results provide a new set of molecular targets related to synapse maintenance for the next generation of ADHD medicines.

SummaryDetailing the ways drugs of abuse physically change 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 and how those interactions change in mice briefly exposed to cocaine. We find that while most DA boutons are devoid of obvious signs of synapses (i.e. synaptic vesicles or synaptic densities) many DA boutons physically interdigitate with both dendrites and excitatory and inhibitory axons. After a brief exposure to cocaine, we find evidence of large-scale structural remodeling: extensive axonal branching and frequent occurrences of axonal blind-ended “bulbs”, filled with mitochondria, reminiscent of axonal retraction in the developing and damaged brain. The number of physical interdigitations and vesicle filled boutons between DA axons and targets scales linearly with the length of axon whether in controls or cocaine exposed animals and the size or the type of interaction (i.e. axo-axonic or axo-dendritic) does not change. Finally, we find significant cell-type and sub-cellular specific increases in mitochondrial length in response to cocaine. Specifically, mitochondria in dopamine axons and local Nucleus Accumbens (NAc) dendrites are ~3.5 times and 2 times longer, respectively, in cocaine treated mice than controls. These results show for the first time the effects of cocaine on remodeling of dopamine circuitry and reveal new details on how dopamine neurons physical associate with downstream targets.

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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.