Embryonic and postnatal neurogenesis produce functionally distinct subclasses of dopaminergic neuron
- Institute of Psychiatry, Psychology and Neuroscience, King’s College London, United Kingdom
- Harvard University, United States
Figures
Figure 1
Two subtypes of DA neuron can be characterised based on size, location and presence of an AIS.
(A) Left: example image of olfactory bulb stained with an anti-TH antibody (blue). Dashed lines indicate subregions of the glomerular layer (GL). The asterisk indicates an AIS-positive DA cell. …
Figure 2
DA neurons that lack an AIS also lack the axonal marker TRIM-46, and all their processes co-stain with the dendritic marker MAP-2.
(A) Example image of a DA cell in a wild-type mouse stained for TH (blue), AnkG (magenta) and the axonal marker TRIM-46 (green). Asterisks indicate soma position; lines indicate the emergence of the …
Figure 3
AIS-positive DA cells have more widely ramified dendritic morphology.
(A) Schematic representation of the experimental strategy adopted to achieve sparse labelling of DA cells: P1-2 neonates or E12 embryos from VGAT-Cre or DAT-Cre lines were injected with floxed …
Figure 4
AIS-positive DA cells are generated exclusively in early embryonic development.
(A) Schematic representation of the experimental strategy for birthdating experiments: pregnant wild-type mice were injected with a single dose of BrdU at different gestational days. Tissue was …
Figure 5
Fully mature adult-born DA neurons are small and never possess an AIS.
(A) Schematic representation of the experimental strategy: pregnant wild-type mice were injected with a single dose of BrdU, and their tissue was collected 4 months later to allow full maturation of …
Figure 6
In neonates there is a preponderance of large DA neurons that can already possess an AIS.
(A) Low-magnification example images of tissue from P0 and P28 wild-type mice stained with an antibody against TH (blue). Arrows indicate cells with a soma area bigger than 100 μm2; ‘GL’ indicates …
Figure 7
AIS-positive cells form the vast majority of embryonically-generated DA neurons that persist throughout adult life.
(A) Schematic representation of the experimental strategy: pregnant wild-type mice were injected with a single dose of BrdU at E12. Tissue was collected from their offspring when they reached 6 …
Figure 8 with 1 supplement
AIS-positive DA neurons have greater intrinsic excitability.
(A) Example image of a fixed, 50 µm OB slice from a P28 DAT-tdT (red) mouse, immunostained with an anti-TH antibody (blue). While most TH-positive neurons exhibit red tdT fluorescence, some are …
Figure 8—figure supplement 1
AIS-positive DA neurons also have greater intrinsic excitability with strict, objective phase plane plot classification.
(A) Example current-clamp traces of phase plane plots of single APs fired by monophasic (AIS-negative, black, top) and biphasic (AIS-positive, magenta, bottom) DAT-tdTomato neurons. Dashed box …
Figure 9
Both OB DA cell subtypes display diverse odour response types.
(A) Schematic representation of the experimental strategy for in vivo recordings: adult DAT-GCaMP6s mice (anaesthetised with ketamine/xylazine) were presented with a panel of eight odours. Resulting …
Figure 10 with 1 supplement
Large putative AIS-positive DA cells respond less strongly to odours and are more broadly tuned.
(A) Example Δf/f GCaMP responses to the eight odours (rows, 3 s stimulus timing is indicated by blue shaded bars) for three big and three small example cells (columns; soma areas are indicated below …
Figure 10—figure supplement 1
Decay kinetics of late-latency responses do not differ between small and big OB DA neurons.
(A) Example mean traces of late-latency excitatory and inhibitory responses that returned to baseline within the imaging timeframe (10.5 s after stimulus onset). Blue box shows stimulus timing; …
Tables
Table 1
Morphological properties of AIS-negative and AIS-positive DA neurons.
Mean values ± SEM of morphological properties for sparsely labelled AIS-negative (n = 14) and AIS-positive (n = 9) DA cells. Statistical differences between groups (AIS-negative vs AIS-positive) …
| Morphological properties | |||
|---|---|---|---|
| AIS-negative (mean ± sem) | AIS-positive (mean ± sem) | Test type, p-value | |
| Soma area (μm2) | 70.49 ± 2.71 | 139.00 ± 17.16 | tW, 0.003 |
| Distance of soma from nerve layer (μm) | 75.07 ± 12.79 | 141.60 ± 31.38 | MW, 0.02 |
| Number of primary dendrites | 3.14 ± 0.23 | 3.44 ± 0.29 | t, 0.43 |
| Length of primary dendrites (μm) | 12.14 ± 2.97 | 26.13 ± 5.23 | MW, 0.01 |
| Area under Sholl curve (μm) | 338.60 ± 42.58 | 871.10 ± 167.20 | tW, 0.01 |
| Furthest intersection (μm) | 77.86 ± 8.23 | 148.30 ± 13.94 | t, 0.0001 |
| Maximum no. of intersections | 8.36 ± 0.68 | 12.56 ± 1.68 | tW, 0.04 |
| Radius for maximum no. of intersections (μm) | 36.16 ± 4.45 | 80.00 ± 13.97 | tW, 0.02 |
Table 2
Intrinsic electrophysiological properties of DAT-tdTomato neurons.
Mean values ± SEM of passive, action potential and repetitive firing properties for monophasic (putative AIS-negative, n = 15) and biphasic (putative AIS-positive, n = 11) DAT-tdTomato cells. …
| Intrinsic electrophysiological properties | |||
|---|---|---|---|
| Monophasic (mean ± sem) | Biphasic (mean ± sem) | Test type, p-value | |
| Passive properties | |||
| Soma area (μm2) | 57 ± 4.8 | 89 ± 6.8 | t,<0.01 |
| Membrane capacitance (pF) | 19 ± 2 | 22 ± 2 | t, 0.39 |
| Resting membrane potential (mV) | −78 ± 1.9 | −74 ± 2.9 | MW, 0.31 |
| Input Resistance (MΩ) | 960 ± 272 | 572 ± 115 | MW, 0.13 |
| Action potential properties | |||
| Threshold (pA/pF) | 7.5 ± 1.0 | 4.6 ± 0.4 | t, 0.02 |
| Threshold (mV) | −30 ± 1.0 | −33 ± 1.0 | t, 0.13 |
| Max voltage reached (mV) | 19 ± 2.4 | 18 ± 4.0 | t, 0.80 |
| Peak amplitude (mV) | 49 ± 2.2 | 50 ± 4.4 | t, 0.79 |
| Width at half-height (ms) | 0.55 ± 0.03 | 0.50 ± 0.04 | t, 0.37 |
| Rate of rise (max dV/dt) (mV/ms) | 230 ± 16 | 251 ± 31 | t, 0.53 |
| Onset rapidness (1/ms) | 3.95 ± 0.28 | 8.22 ± 1.66 | t, 0.03 |
| After hyper polarization AHP (mV) | −54 ± 1.4 | −55 ± 1.5 | t, 0.72 |
| AHP relative to threshold (mV) | 25 ± 1.3 | 24 ± 1.3 | t, 0.95 |
| Repetitive firing properties | |||
| Rheobase (pA/pF) | 3.6 ± 1.0 | 1.7 ± 0.8 | MW, 0.28 |
| Max number of action potentials | 10 ± 2 | 21 ± 4 | t, 0.01 |
| First action potential delay (ms) | 168 ± 38 | 273 ± 45 | t, 0.08 |
| Inter-spike interval CV | 0.28 ± 0.04 | 0.24 ± 0.03 | t, 0.46 |
| Slope of input/output curve (Hz/(pA/pF)) | 1.85 ± 0.54 | 3.53 ± 0.48 | t, 0.04 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers |
|---|---|---|---|
| Strain, strain background (M. musculus) | C57BL/6J mice | Charles River | Strain code 027 |
| Strain, strain background (M. musculus) | DAT-Cre, B6.SJL-Slc6a3tm1.1(cre)Bkmn/J | The Jackson Laboratory | Jax stock 006660 |
| Strain, strain background (M. musculus) | VGAT-Cre, Slc32a1tm2(cre)Lowl/J | The Jackson Laboratory | Jax stock 016962 |
| Strain, strain background (M. musculus) | flex-tdTomato, B6.Cg–Gt(ROSA)26Sortm9(CAG-tdTomato)Hze, | The Jackson Laboratory | Jax stock 007909 |
| Strain, strain background (M. musculus) | flex-GCaMP6s animals, Ai96; B6;129S6-Gt(ROSA)26Sortm96(CAG-GCaMP6s)Hze/J | The Jackson Laboratory | Jax stock 024106 |
| Transfected construct (Adeno-associated virus) | AAV9.EF1a.ChR2-YFP lox/lox virus | Penn Vector Core, USA | AV-9-PV1522, |
| Transfected construct (retrovirus) | floxed rv::dio-GFPlox/lox | Oscar Marin | Ciceri et al., 2013 Nat Neuroscience 16(9):1199–210 |
| Antibody | polyclonal Anti-Tyrosine Hydroxylase, raised in Rabbit; use 1:500 | Millipore | catalogue number AB152; RRID:AB_390204 |
| Antibody | monoclonal Anti-Tyrosine Hydroxylase, raised in mouse; use 1:500 | Millipore | clone (LNC1) - catalog number MAB318; RRID: AB_2313764 |
| Antibody | polyclonal Anti-Tyrosine Hydroxylase, raised in chicken; use 1:250 | Abcam | catalog number ab76442; RRID:AB_1524535 |
| Antibody | monoclonal anti-Ankyrin-G IgG2a, raised in mouse; use 1:500 | Neuromab | clone (106/36) - catalog number75–146; RRID: AB_10673030 |
| Antibody | monoclonal anti-Ankyrin-G IgG2b, raised in mouse; use 1:500 | Neuromab | clone (106/65) - catalog number75–147; RRID: AB_10675130 |
| Antibody | monoclonal anti-Ankyrin-G IgG1, raised in mouse; use 1:500 | Neuromab | clone (106/20) - catalog number75–187; RRID:AB_10674433 |
| Antibody | monoclonal Phospho-IκBα (Ser32) (14D4), raised in rabbit; use 1:1000 | Cell Signaling Technology | catalog number 2859; RRID:AB_561111 |
| Antibody | polyclonal Anti-TRIM46, raised in rabbit; use 1:500 | Gift from Casper Hoogenraad | van Beuningen et al. (2015) Neuron. 88:1208–1226 |
| Antibody | monoclonal anti-MAP-2, raised in mouse; use 1:500 | Gift from Phillip Gordon-Weeks | |
| Antibody | monoclonal anti-chemical BrdU, raised in Rat; use 1:200 | Serotec | clone BU1/75 (ICR1)- catalog number OBT0030; RRID:AB_609568 |
| Antibody | polyclonal anti-GFP, raised in chicken; use 1:2000 | Abcam | catalog number ab13970; RRID:AB_300798 |
| Antibody | polyclonal anti-GFP, raised in guinea pig; use 1:500 | Synaptic Systems | catalog 132 005; RRID:AB_11042617 |
| Chemical compound, drug | Heparin | Alfa Aesar | CAS A16198 |
| C | |||
| Chemical compound, drug | PIPES | Sigma | CAS P6757 |
| Chemical compound, drug | 5-Bromo-2′-deoxyuridine | Sigma | CAS 59143 |
| Chemical compound, drug | Alexa 488 | Thermo Fisher Scientific | A10436 |
| Chemical compound, drug | Methyl Propionate | Sigma | CAS 81988 |
| Chemical compound, drug | Methyl Butyrate | Sigma | CAS 246093 |
| Chemical compound, drug | Ethyl Valerate | Sigma | CAS 290866 |
| Chemical compound, drug | Hexanal | Sigma | CAS 115606 |
| Chemical compound, drug | Methyl Tiglate | Penta | CAS 13–73400 |
| Chemical compound, drug | Valeraldehyde | Sigma | CAS 110132 |
| Chemical compound, drug | Propyl Acetate | Tokyo Chemical Industry | CAS A0044 |
| Chemical compound, drug | Pentyl Acetate | Sigma | CAS 109549 |
| Chemical compound, drug | Diethyl Phthalate | Sigma | CAS 84662 |
| Software, algorithm | ImageJ software (Fiji) | NIH; Schneider et al. (2012) | RRID:SCR_003070 |
| Software, algorithm | ClampFit 10.4 | pClamp | Molecular Devices; RRID:SCR_011323 |
| Software, algorithm | Prism 5.3 | GraphPad | RRID:SCR_002798 |
| Software, algorithm | Matlab | Mathworks | RRID:SCR_001622 |
| Software, algorithm | Vaa3D | Allen Institute for Brain Science | RRID:SCR_002609 |
| Software, algorithm | IBM SPSS Statistics | IBM | RRID:SCR_002865 |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.32373.017
