DRNDA and DRN5-HT are electrophysiologically distinct cell-types.

(A) Scheme of the location of the DRN (pink) in a coronal section (top) and at higher magnification together with two patch pipettes (center). Bottom: A representative slice stained post recording for TPH, TH and neurobiotin revealing serotonergic neurons (arrows). The ventricle is indicated with a dashed line. (B) Top: Differential interference contrast (DIC) microscopy image (left) of neurons that were filled with Alexa488 (right) and neurobiotin. Center, bottom: Staining of the same neurons revealing a TPH-positive (DRN5-HT) neuron and a TPH- and TH-negative cell. (C) Top: DIC image of recorded neurons that were filled with Alexa488 and neurobiotin. Center, bottom: Staining of the same neurons revealing tdTomato- and TH-positive (DRNDA) neurons. (D) Representative fluorescent (top), DIC (center) image and overlay (bottom) of a tdTomato-positive neuron in a DAT-tdTomato mouse. (E) Representative recordings depicting postinhibitory hypoexcitability, slowly ramping currents and rebound oscillations in DRNDA neurons. (F) Representative voltage responses to current injections in a TPH-positive (DRN5-HT) and TH-positive (DRNDA) neuron. (G) Ramping current injections reveal action potential (AP) amplitude accommodation. Grey circles indicate the onset and peak of APs. (H) Amplitude and duration of the AP and action potential afterhyperpolarization (AHP) in a DRN5-HT and DRNDA neurons. Grey circles indicate onset, peak, and end of the AP and AHP. (I) Quantification of electrophysiological properties distinguishing DRN5-HT from DRNDA neurons (n= 28-43 cells per group, N = 9; Wilcoxon Rank Sum Test). (J) Principal component analysis (PCA) of five electrophysiological parameters (insert) and hierarchical cluster analysis based on PCA1 and PCA2 (Ward’s method, Euclidean distance). Intrinsic properties were sufficient to separate TPH-positive cells (blue dash) from TH-positive (red dash) cells. Bottom dashes indicate wild type (black) and DAT-tdTomato (gray) mice. Data are shown as mean ± SEM, ***p < 0.001. Scale bars: A, 100 μm; B-D, 10 μm.

DRNDA and DRN5-HT have distinct morphological profiles.

(A) Top: representative digital reconstruction of a DRN5-HT from a wild-type mouse. Bottom: representative digital reconstruction of a DRNDA from a wild-type mouse. Scale bar: 10 μm. (B-F) Morphological parameters describing the soma size and shape of DRN5-HT and DRNDA neurons in Sham-lesion mice (DRN5-HT: n = 20, N = 3; DRNDA: n = 27, N = 3; unpaired t-test). (G-J) Morphological parameters describing the dendritic tree of DRN5-HT and DRNDA neurons in Sham-lesion mice (DRN5-HT: n = 8, N = 3; DRNDA: n = 7, N = 3; unpaired t-test). Data are shown as mean ± SEM, ***p < 0.001.

Lesions targeting primarily nigrostriatal dopamine increase the excitability of DRN5-HT neurons whereas loss of NA affects their action potentials.

(A) Top: Pie charts showing the number of spontaneously active (dark) and silent (pale) DRN5-HT neurons in three conditions: Sham (left), 6-OHDA-injected mice (center) and 6-OHDA-injected mice pre- treated with desipramine (DMI + 6-OHDA, right). Bottom: Representative recordings of spontaneously active DRN5-HT neurons (I = 0 pA). (B) Quantification of the rheobase (left), the firing frequency of spontaneously active cells (center), and the resting membrane potential of silent DRN5-HT neurons (right). (C) Representative action potentials (APs) of DRN5-HT at low (left) and high (right) temporal. Grey circles indicate onset, offset, and peak of the APs as well as the end of the afterhyperpolarization (AHP). (D) Quantification of the amplitude (left) and duration (right) of the APs of DRN5-HT neurons. (E) Same as in (D) for the AHP. (F) Representative responses of DRN5-HT neurons to current steps (I = +75 pA). (G) Quantification of firing frequency / injected current. (H) Quantification of the delay to the first AP when injected with current eliciting 2Hz firing. (I) Quantification of the membrane time constant (tau) of DRN5-HT neurons (Sham: n = 22 - 32, N = 6 - 7; 6-OHDA: n = 11 - 16, N = 7; DMI + 6-OHDA: n = 18 – 21, N = 4; unpaired t-test). Data are shown as mean ± SEM, * p < 0.05, ** p < 0.01.

Striatal injection of 6-OHDA induced a hypotrophic phenotype in the DRN5-HT, which is prevented by pre- treatment with DMI.

(A) Representative digital reconstructions of a DRN5-HT neuron in three different conditions: Sham (left), 6-OHDA-injected mice (center) and 6-OHDA-injected mice pre-treated with desipramine (right). Scale bar: 10 μm (B) Representative confocal pictures of soma from DRN5-HT neurons in Sham (top), 6-OHDA-injected mice (center), and 6-OHDA-injected mice pre-treated with desipramine (bottom). Scale bar: 10 μm. (C-G) Morphological descriptors of the soma size and shape in DRN5-HT neurons (Sham: n = 20, N = 4; 6-OHDA: n = 19, N = 4; DMI + 6-OHDA: n = 17, N = 3; unpaired t-test).(H-K) Morphological descriptors of the dendritic tree in DRN5-HT neurons (Sham: n = 8, N = 3, 6-OHDA: n = 6, N = 3: DMI + 6-OHDA: n = 6, N = 2; unpaired t-test). Data are shown as mean ± SEM, ***p<0.001, **p<0.01. Scale bar: 10 µm.

Lesions targeting primarily SN dopamine depolarize DRNDA neurons whereas concomitant loss of NA does not affect their action potentials.

(A) Top: Pie charts showing the proportion of spontaneously active (dark) and silent (pale) DRNDA neurons in three conditions: Sham (left), 6OHDA-injected mice (center) and 6-OHDA-injected mice pre- treated desipramine (DMI, right). Bottom: Representative recordings of spontaneously active DRNDA (I = 0pA). (B) Quantification of the rheobase (left), the firing frequency of spontaneously active (center), and the resting membrane potential of silent DRNDA neurons (right). (C) Representative action potentials (APs) of DRNDA at low (left) and high (right) temporal resolution. Grey circles indicate onset, offset, and peak of APs and the end of the afterhyperpolarization (AHP). (D) Quantification of the amplitude (left) and duration (right) of the APs of DRNDA neurons. (E) Same as in (D) for the AHP. (F) Representative responses of DRNDA neurons to current steps (I = 75pA). Grey circles indicate the delay to the first AP. (G-I) Quantification of firing frequency / injected current (G), the delay to the first AP when injected with current eliciting 2 Hz firing (H), and the membrane time constant (tau, I) of DRNDA neurons recorded (Sham: n = 43, N = 8; 6-OHDA: n = 29, N = 6; DMI + 6-OHDA: n = 40, N = 6; unpaired t-test). Data are shown as mean ± SEM, * p < 0.05.

Striatal injection of 6-OHDA increased soma complexity and branching in DRNDA.

(A) Representative digital reconstructions of a DRNDA neuron in three different conditions: Sham (left), 6-OHDA-injected mice (center) and 6- OHDA-injected mice pre-treated with desipramine (right). (B) Representative confocal pictures of soma from DRNDA neurons in Sham (top), 6-OHDA-injected mice (center), and 6-OHDA-injected mice pre-treated with DMI (bottom). (C- G) Morphological descriptors of the soma size and shape in DRNDA neurons (Sham: n = 27, N = 7, 6-OHDA: n = 16, N = 4; DMI + 6-OHDA: n = 31, N = 5; unpaired t-test). (H-K) Morphological descriptors of the dendritic tree in DRNDA neurons. (Sham: n = 7, N = 3; 6-OHDA: n = 11, N = 4; DMI + 6-OHDA: n = 7, N = 3; unpaired t-test). Data are shown as mean ± SEM, *p<0.05. Scale bar: 10 µm.