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

  1. Gregg Wildenberg
  2. Anastasia Sorokina
  3. Jessica Koranda
  4. Alexis Monical
  5. Chad Heer
  6. Mark Sheffield
  7. Xiaoxi Zhuang
  8. Daniel McGehee
  9. Bobby Kasthuri

  • Curated by eLife

    eLife logo

    Evaluation Summary:

    This study investigates the morphological features of dopaminergic (DA) axons in the Nucleus Accumbens using serial electron microscopy (EM) reconstructions of genetically labelled DA axons. The authors report that DA axons (1) show extremely sparse classical synapses, (2) have varicosities with highly diverse vesicle content, and (3) undergo strong remodelling upon exposing mice to cocaine. The volume EM data set on DA axons presented is valuable. The present findings are suggestive of a mode of dopamine signaling that does not involve classical synapses and are potentially important for understanding the biology of DA neurons, whose dysfunctions have consequences on neurological disorders from Parkinson's disease to schizophrenia.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

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.

Article activity feed

  1. Version published to 10.7554/elife.71981 on eLife
  2. eLife

    Author Response:

    Reviewer #1 (Public Review):

    The reviewer believes that there is a fundamental problem with the approach of the current MS. Dense reconstruction from serial EM images is a powerful tool for revealing the connectivity matrix in many brain area, where the majority of synaptic connections are made by glutamatergic pyramidal and GABAergic interneurons. Many studies have convincingly demonstrated that the site of synaptic communications among these cells is the well-known EM defined synapses with a presynaptic cloud of vesicles, a rigid presynaptic active zone membrane facing a rigid postsynaptic membrane that either has or does not have a pronounced postsynaptic density. We know from many EM localization results that e.g. the active zone contains the essential molecules of the release sites, the presynaptic Ca2+ channels and the PSD contains the appropriate receptors. Thus, with this information, when the connectome is created from serial EM sections, the sites of communication can be defined based on the EM images. To the knowledge of the reviewer, such pre-existing information is lacking for the DA varicosities. The authors argue that almost all varicosities lack synapses. Verification of such a statement would require the molecular characterization of these varicosities, demonstrating that the molecules essential for vesicle docking/priming/release are lacking. However, if these molecules are present in these varicosities without forming an apparent active zone, then the conclusion of the MS is misleading.

    We thank the reviewer for this point and now believe we have addressed it in the discussion.

    The authors demonstrate the clear labeling of DA neuronal processes using the cytoplasmic-targeted Apex2. However, due to the well-known masking effect of DAB precipitate in the cytoplasm, which prevent the unequivocal identification of vesicles, the authors decided to use the mitochondria targeted Apex2 in the first half of the MS. However, for the cocaine part, they turned to the cytoplasmic version for some reasons. They then analyzed the axonal branching structure and the varicosities/contact points. The reviewer cannot see how this later was achieved with densely filled DAB containing structures.

    We apologize for the confusion. There are several reasons we turned to cyto-Apex for the cocaine part of the MS. We have added a broader discussion of this topic and reproduced below.

    "we used cyto-Apex2 for reconstructing axons and their contact points for a several reasons. First, we found that tracing axons in low resolution EM data sets, for both controls and experimental groups, was substantially easier in cytoplasmic Apex2 axons, thus increasing our tracing throughput. In addition, we found that contact points, (e.g., spinules), were also easier to detect. Detecting either a darkly Apex2 labeled cytoplasmic process in an unlabeled structure, or vice versa, was easier because of the stark contrast between Apex2 labeled and unlabeled processes. In Figure 6 and Figure 6-Fig Supplement 2 an example of this difference is shown. While it is possible that cytoplasmic Apex2 expression could potentially obscure the internal contents of varicosities (e.g., synaptic vesicles or endoplasmic reticulum), we found little evidence that cyto-Apex2 obscured the relevant ultra-structural features that were investigated here."

    The evidence for DA varicosities making synapses (Fig4) is not convincing. The presented EM images does not have the quality/resolution to see the opposing rigid pre- and postsynaptic membranes and the widening of extracellular space in the cleft.

    To make the results clearer, we now show an 8-panel montage of each putative DA synapse as Figure 4- Figure Supplement 1 and 2. We hope that showing the serial sections that span the synapse will make details of the pre- and post-synaptic membranes more convincing. All the images in the manuscript are SEM, not TEM.

    Analyzing the structures immediately next to DA varicosities is questionable. If DA is indeed a volume transmitter, how would the authors know how far it can exert its effect. 1 or 5 microns? If 5 microns, there are many structures of all kinds (axon, glia, spine, dendrite) and only their DA receptor content will tell whether they are sensitive or not (and not necessarily their physical distance) to the released DA.

    We agree with the reviewer that this analysis distracts from the main finding of the story and is better investigated with a more accurate model of which types of cellular processes are sensitive to released DA. We have removed these experiments and their discussion from the manuscript.

  3. eLife

    Evaluation Summary:

    This study investigates the morphological features of dopaminergic (DA) axons in the Nucleus Accumbens using serial electron microscopy (EM) reconstructions of genetically labelled DA axons. The authors report that DA axons (1) show extremely sparse classical synapses, (2) have varicosities with highly diverse vesicle content, and (3) undergo strong remodelling upon exposing mice to cocaine. The volume EM data set on DA axons presented is valuable. The present findings are suggestive of a mode of dopamine signaling that does not involve classical synapses and are potentially important for understanding the biology of DA neurons, whose dysfunctions have consequences on neurological disorders from Parkinson's disease to schizophrenia.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    The reviewer believes that there is a fundamental problem with the approach of the current MS. Dense reconstruction from serial EM images is a powerful tool for revealing the connectivity matrix in many brain area, where the majority of synaptic connections are made by glutamatergic pyramidal and GABAergic interneurons. Many studies have convincingly demonstrated that the site of synaptic communications among these cells is the well-known EM defined synapses with a presynaptic cloud of vesicles, a rigid presynaptic active zone membrane facing a rigid postsynaptic membrane that either has or does not have a pronounced postsynaptic density. We know from many EM localization results that e.g. the active zone contains the essential molecules of the release sites, the presynaptic Ca2+ channels and the PSD contains the appropriate receptors. Thus, with this information, when the connectome is created from serial EM sections, the sites of communication can be defined based on the EM images. To the knowledge of the reviewer, such pre-existing information is lacking for the DA varicosities. The authors argue that almost all varicosities lack synapses. Verification of such a statement would require the molecular characterization of these varicosities, demonstrating that the molecules essential for vesicle docking/priming/release are lacking. However, if these molecules are present in these varicosities without forming an apparent active zone, then the conclusion of the MS is misleading.
    The authors demonstrate the clear labeling of DA neuronal processes using the cytoplasmic-targeted Apex2. However, due to the well-known masking effect of DAB precipitate in the cytoplasm, which prevent the unequivocal identification of vesicles, the authors decided to use the mitochondria targeted Apex2 in the first half of the MS. However, for the cocaine part, they turned to the cytoplasmic version for some reasons. They then analyzed the axonal branching structure and the varicosities/contact points. The reviewer cannot see how this later was achieved with densely filled DAB containing structures.
    The evidence for DA varicosities making synapses (Fig4) is not convincing. The presented EM images does not have the quality/resolution to see the opposing rigid pre- and postsynaptic membranes and the widening of extracellular space in the cleft.
    Analyzing the structures immediately next to DA varicosities is questionable. If DA is indeed a volume transmitter, how would the authors know how far it can exert its effect. 1 or 5 microns? If 5 microns, there are many structures of all kinds (axon, glia, spine, dendrite) and only their DA receptor content will tell whether they are sensitive or not (and not necessarily their physical distance) to the released DA.

  5. eLife

    Reviewer #2 (Public Review):

    This manuscript provides large-scale EM reconstructions of dopaminergic axons in the mouse striatum. These reconstructions are performed in five mice. One mouse had mitochondrial DAB in dopamine axons and was used for large scale, high-resolution reconstructions. Four mice had cytosolic DAB in dopamine axons, and they were used mostly for lower resolution analyses of effects of cocaine (2 mice) vs control (2 mice). Overall, the data are rigorously acquired and well presented. The paper is easy to follow.

    Key findings of this study are that:
    (1) only a very small subset of dopamine varicosities, 2%, forms synaptic contacts
    (2) there is considerable heterogeneity in vesicle content with varicosities without vesicles, with small vesicles, with large vesicles, or with small and large vesicles
    (3) cocaine induces strong changes in dopamine axon structure with extensive branching and large bulbs

    The findings are very important for understanding dopamine axon biology and dopamine transmission. In particular, the non-synaptic nature of dopamine transmission has been debated for years. While the field mostly agrees that dopamine transmission is unlikely to rely on classical synaptic structure, it was not possible before to assess the actual number of synaptic contacts compared of non-synaptic varicosities, because previous EM studies did not reconstruct dopamine axons but instead relied on single sections. This is an important finding. Similarly, the finding of heterogeneity of vesicle size is important, and may be related to multiple release and signaling modes that have been proposed for dopamine neurons.

    Altogether, the conclusions are supported by the data. Recommendations for improvement include a more detailed discussion of some of the technical limitations that are inherent to the method that is used and suggestions related to data presentation. One important point is that the morphological features used to distinguish synaptic and non-synaptic varicosities have to be described better, and non-DA axons should be assessed in the same dataset by a blind experimenter using the same morphological parameters for comparison.

  6. eLife

    Reviewer #3 (Public Review):

    Fundamental questions about the connectivity of neuromodulatory circuits remain to be solved. The present study focuses on the dopaminergic (DA) system that controls a variety of brain functions including learning, decision making, reward assessment, and social behaviors, and whose dysfunctions being associated with neurological and neuropsychiatric diseases such as Parkinsons's disease, attention-deficit/hyperactivity disorder, and schizophrenia. Specifically, the authors have sought to address the basis by which DA axons might communicate with their target(s) and identify features of structural plasticity of DA axons upon drug exposure. To this end, the authors capitalized on a genetic method to label DA axons that is amenable to electron microscopy (EM), and 3D reconstructions of high-resolution EM images obtained by automated sequential sampling of contiguous sections. Several key findings are presented. Most importantly, the authors present a striking observation that majority of DA axons have varicosities that are largely devoid of synaptic vesicle-like structures and do not form typical morphologically identifiable synapses with target (contacting) cells, which are important in shedding light on the mode by which dopaminergic signaling is implemented. As the manuscript stands, some of the analyses are incomplete to fully support the conclusions.

  7. Version published to 10.1101/2020.09.29.318881v3 on bioRxiv
  8. Version published to 10.1101/2020.09.29.318881v2 on bioRxiv
  9. Version published to 10.1101/2020.09.29.318881v1 on bioRxiv