Partial connectomes of labeled dopaminergic circuits reveal non-synaptic communication and axonal remodeling after exposure to cocaine
- Department of Neurobiology, University of Chicago, United States
- Argonne National Laboratory, United States
- Department of Anesthesia & Critical Care, University of Chicago, United States
Figures
Figure 1
Experimental design for dopamine connectomics.
(A) Adeno-associated viruses (AAVs) expressing Cre-dependent Apex2 were bilaterally injected into the ventral tegmental area (VTA) of transgenic mice expressing Cre in dopamine transporter positive …
Figure 2
Dopaminergic (DA) axon varicosities are either empty or filled with different sized vesicles.
(A) Pie chart showing ratio of each kind of DA varicosity across a population of DA axons (n = 409 varicosities scored across 75 axons, 1 mouse). (B) Single 2D images showing representative examples …
Figure 3 with 1 supplement
Monte Carlo simulation of dopaminergic (DA) varicosity types.
(A) Mito-Apex2 DA axons containing six or more varicosities in the field of view are depicted with the linear order of their varicosities. Each varicosity is shown as a colored circle with each …
-
Figure 3—source data 1
Table of p-values for Monte Carlo simulations.
The top row (light blue) represents each data point in the x-axis of Figure 3B that counts the number of axons containing greater than the specified number of varicosities/axon. The left column (light yellow) separates each varicosity type. p-values were calculated by dividing the number of times the simulated axon had a specified varicosity type (yellow column) appear more than the number of specified times (blue row) by the total number of Monte Carlo simulations (100,000).
- https://cdn.elifesciences.org/articles/71981/elife-71981-fig3-data1-v1.pdf
Figure 3—figure supplement 1
Inter-varicosity distances and frequency of mitochondria at varicosity sites in dopaminergic (DA) axons.
(A) Histogram of pair-wise inter-varicosity distances for all DA axon varicosity types (n = 170 varicosities across 29 axons). Average inter-varicosity distance is 5.3 µm, 4.1 µm standard deviation. …
Figure 4 with 3 supplements
Dopaminergic (DA) axons make rare synapses on the soma and shaft of nucleus accumbens (NAc) resident neurons.
Three examples of DA axons making synapses on the soma (left column) and shaft (right column) are depicted. Mito-Apex2 DA axons are shaded in light blue with an asterisk marking an Apex2-positive …
Figure 4—figure supplement 1
Montage of identified dopaminergic (DA) axons making soma synapses.
Three (1–3) putative DA soma synapses shown in Figure 4 are more clearly depicted by showing eight serial electron microscopy (EM) sections spanning the synapse (z1–z8). Red arrows point to the …
Figure 4—figure supplement 2
Montage of identified dopaminergic (DA) axons making shaft synapses.
Three (1–3) putative DA shaft synapses shown in Figure 4 are more clearly depicted by showing eight serial electron microscopy (EM) sections spanning the synapse (z1–z8). Red arrows point to the …
Figure 4—figure supplement 3
Montage of representative chemical synapses.
Three (1–3) chemical synapses identified by a prominent vesicle cloud and post-synaptic density (PSD) in apposition to a post-synaptic neurite were identified in the 6 nm resolution mito-Apex2 …
Figure 5 with 1 supplement
Dopamine contact points are physical interdigitations between dopaminergic (DA) axons and either afferent axons or nucleus accumbens (NAc) dendrites.
(A) Image montage of every other electron microscopy (EM) serial section (Z1–Z9) from a mouse expressing mito-Apex2 in DA neurons. Highlighted in blue is the mito-Apex2 DA axon, and in red is the …
Figure 5—figure supplement 1
Image montage of dopaminergic (DA) axon interdigitations.
(A) Electron microscopy (EM) z-series of the interdigitation between a cyto-Apex2 DA axon (blue) and vesicle-filled chemical synapse (CS) axon (red) that corresponds to the 3D rendering in Figure 5B,…
Figure 6 with 3 supplements
Cocaine increases branching of Apex2 dopamine axons in cocaine sensitized mice.
(A) Two representative reconstructions of dopaminergic (DA) axons each from saline- (left, blue) and cocaine- (right, red) treated mice. Green circles represent contact points (i.e., spinules) where …
Figure 6—figure supplement 1
Cocaine-treated mice have increased locomotor activity.
(A) Cartoon depicting cocaine sensitization protocol: Mice received a single intraperitoneal (IP) injection of cocaine (10 mg/kg) or equivalent volume of saline, placed in a novel cage environment, …
Figure 6—figure supplement 2
Contact points from control and cocaine 20 nm resolution datasets.
Two image montages of electron microscopy (EM) serial section (Z1–Z8) from either a saline- (top: 1,2) or cocaine- (bottom: 1,2) treated mice expressing cyto-Apex2 in dopaminergic (DA) neurons, and …
Figure 6—figure supplement 3
Cocaine does not change the proportion of dopamine targets.
Pie charts showing proportion of contact points (i.e., ‘spinules’) that are with chemical synapse (CS) axons (red) and dendrites (green) in saline- (top) and cocaine- (bottom) treated mice (+saline: …
Figure 7
Cocaine results in the formation of large swellings filled with mitochondria in Apex2+ dopaminergic (DA) axons.
(A) Left: 2D electron microscopy (EM) image of Apex2 labeled DA axon bulb in cocaine-treated mouse (square) as compared to a neighboring neuronal soma (circle). Right: zoomed in 2D EM image of Apex2 …
Figure 8
Dopamine axon swellings are surrounded by glia.
(A) 3D reconstruction of a dopamine axon swelling from a cocaine sensitized mouse. Bottom image is rotated 90 degrees relative to top view. White arrows point to swelling. (B) 3D reconstruction of …
Figure 9 with 1 supplement
Cocaine results in increased mitochondrial length in dopaminergic (DA) axons and medium spiny neuron (MSN) dendrites.
(A) Cartoon depicting neurons where mitochondrial length was measured. Left: in the nucleus accumbens (NAc), we measured mitochondria in: (C) Apex2+ DA axons, (D) MSN dendrites, and (E) excitatory …
Figure 9—figure supplement 1
Cocaine does not change the number of mitochondria in dopaminergic (DA) axons.
Scatter plot of the number of mitochondria versus length of axon (μm). (+saline: 0.14 ± 0.02 mitochondria/µm length of axon, n = 96 mitochondria counted across 18 axons, two mice; +cocaine: 0.16 ± …
Author response image 1
Original plot (left) and updated with more data collected (right).
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus) | Slc6a3tm1(cre)Xz/J | Provided by the Xiaoxi Zhuang lab (The University of Chicago), PMID:15763133 | RRID:IMSR_JAX:020080 | Also available at Jackson Laboratories: https://www.jax.org/strain/020080 |
| Chemical compound, drug | 3,3’-Diaminobenzidine | Sigma-Aldrich | D12384 | 50 mg/ml |
| Chemical compound, drug | Sodium cacodylate buffer, pH 7.4 | Electron Microscopy Sciences | 11653 | 0.2 M stock, use at 0.1 M |
| Chemical compound, drug | Sodium hydrosulfite | Sigma-Aldrich | 157953 | 0.8% (w/v) |
| Chemical compound, drug | Sodium bicarbonate | Sigma-Aldrich | S5761 | 0.1 M stock, used at 60% (v/v) |
| Chemical compound, drug | Sodium carbonate | Sigma-Aldrich | S7795 | 0.1 M stock, used at 40% (v/v) |
| Chemical compound, drug | Osimum tetroxide | Electron Microscopy Sciences | 19150 | 4% aqueous stock solution, use at 2% |
| Chemical compound, drug | Paraformaldehyde | Electron Microscopy Sciences | 15710 | 16% aqueous stock solution, use at 2% |
| Chemical compound, drug | Glutaraldehyde | Electron Microscopy Sciences | 16220 | 25% aqueous stock solution, use at 2.5% |
| Chemical compound, drug | Postassium ferrocyanide | Sigma-Aldrich | P3289 | 2.5% |
| Chemical compound, drug | Pyrogallol | Sigma-Aldrich | P0381 | 4% |
| Chemical compound, drug | Uranyl acetate | Electron Microscopy Sciences | 22400–4 | 4% aqueous stock solution, use at 1% |
| Chemical compound, drug | Lead (II) nitrate | Sigma-Aldrich | 228621 | 0.66% |
| Chemical compound, drug | Embed 812 kit | Electron Microscopy Sciences | 14120 | 49% Embed 812, 28% DDSA, 21% NMA, and 2.0% DMP 30 |
| Chemical compound, drug | Cocaine | Obtained through DEA license (Xiaoxi Zhuang, The University of Chicago) | 10 mg/kg |
Author response table 1
| Varicosity type | Average no | SEM |
|---|---|---|
| small | 30.89583333 | 3.2 |
| large | 9.222222222 | 0.8 |
| large/Small:Total | 35.07894737 | 5.5 |
| large/small:large | 6.921052632 | 0.9 |
| large/small:large | 28.15789474 | 4.8 |
Additional files
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/71981/elife-71981-transrepform1-v1.pdf
-
Source code 1
MATLAB code to generate Figure 3B Monte Carlo simulations.
- https://cdn.elifesciences.org/articles/71981/elife-71981-supp1-v1.zip
