Roundabout receptor 2 maintains inhibitory control of the adult midbrain
- University of Washington, United States
- Boston University Medical Center, United States
Figures
Figure 1 with 3 supplements
Robo2 VTA mutants have a reduction in the frequency of sIPSCs.
(A) Robo2 is expressed in the VTA, predominantly in dopamine neurons (TH+ cells, arrows), but also non-dopamine neurons (TH- cells, arrowheads). Scale bar = 100 µm (Robo2), 10 µm (merge/zoom). (B) The genomic structure of the Robo2lox/lox allele before and after Cre recombination, adapted from Lu et al. (2007). (C) Schematic of strategy to virally inactivate Robo2 in the VTA (left). Representative image (right) showing expression of Cre-GFP and TH in the VTA. Scale bar = 100 µm. (D) sIPSC representative traces for control and mutant Ih positive cell. (E) sIPSC frequency (left) and cumulative distribution plot (right) for control (n = 13) and mutant (n = 16) Ih positive cells. Unpaired t-test, t(27) = 3.944, ***p<0.001. Two-way repeated measures ANOVA, genotype x time interaction, F(27,100) = 17.30, ****p<0.0001. (F) sIPSC amplitude (left) and cumulative distribution plot (right) for control and mutant Ih positive cells. (G) sIPSC representative trace for control and mutant Ih negative cell. (H) sIPSC frequency (left) and cumulative distribution plot (right) for control (n = 10) and mutant (n = 14) Ih negative cells. Unpaired t-test, t(22) = 3.459, **p<0.01. Two-way repeated measures ANOVA, genotype x time interaction, F(22,100)=11.40, ****p<0.0001. (I) sIPSC amplitude (left) and cumulative distribution plot (right) for control and mutant Ih negative cells. Bars represent mean ± SEM.
Figure 1—figure supplement 1
Robo2 VTA mutants.
(A) In situ hybridization for Robo2 in the rostral, medial, and caudal VTA (image credit: Allen Institute). Scale bar = 500 µm. (B) Traces of the viral transduction of AAV1-Cre-GFP in Robo2lox/lox animals (n = 10) in the rostral, medial, and caudal VTA. Each color represents one animal and corresponds across the three sections and to the cell count quantification in (C). Scale bar = 500 µm. (C) Cell count of AAV1-Cre-GFP positive cells in the rostral, medial, and caudal VTA. (D) Slc6a3 (DAT) mRNA is enriched in midbrain dopamine (n = 3 animals) but not midbrain GABA neurons (n = 3 animals). (E) Slc32a1 (Vgat) mRNA is enriched in midbrain GABA but not midbrain dopamine neurons. (F) Robo2 mRNA is enriched in both midbrain dopamine and midbrain GABA neurons. (H) The distribution of cell types in Robo2lox/lox animals injected with either AAV1-∆Cre-GFP (n = 4) or AAV1-Cre-GFP (n = 4). Bars represent mean ± SEM. (G) Expression of the control virus, AAV1-∆Cre-GFP does not affect expression of Robo2 (arrows in top). AAV1-Cre-GFP causes loss of Robo2 in GFP+ cells of the VTA (arrowheads in bottom), but Robo2 expression is still expressed in GFP- cells. Scale bar = 10 µm.
Figure 1—figure supplement 2
Robo2 VTA mutants have normal sEPSCs.
(A) Representative trace from a recorded Ih positive cell. (B) sEPSC representative traces for control and mutant Ih positive cell. (C) sEPSC frequency (left) and cumulative distribution plot (right) for control (n = 10) and mutant (n = 11) Ih positive cells. (D) sEPSC amplitude (left) and cumulative distribution plot (right) of control and mutant Ih positive cells. (E) Representative trace from a recorded Ih negative cell. (F) sEPSC representative trace for control and mutant Ih negative cell. (G) sEPSC frequency (left) and cumulative distribution plot (right) for control and mutant Ih negative cells. (H) sEPSC amplitude (left) and cumulative distribution plot (right) for control and mutant Ih negative cells. Bars represent mean ± SEM.
Figure 1—figure supplement 3
Robo2 VTA mutants.
(A) Representative examples of asymmetrical and symmetrical synapses in control and mutant mice. Blue shading indicates presynaptic terminal. Pink shading indicates postsynaptic terminal. Scale bar = 500 nm. (B) Average number of synapses of specified type observed from control (n = 16 images from 2 mice) and mutant (n = 14 images from 2 mice) mice. Synapses were identified by the presence of synaptic vesicles juxtaposed to a clearly identifiable postsynaptic site in the presence or absence of a postsynaptic density. Symmetrical synapses, control versus mutant: Unpaired t-test, t(28) = 0.08934, p=0.9295. Asymmetrical synapses, control versus mutant: Unpaired t-test, t(28) = 1.119, p=0.2725. (C) Average waveform of Ih positive cells in control (n = 13) and mutant (n = 16) mice. (D) Quantification of tau value in Ih positive cells in controls (n = 13) and mutants (n = 16). Unpaired t-test, t(27) = 1.619, p=0.1171. (E) Average waveform of Ih negative cell in control (n = 10) and mutant (n = 14) mice. (F) Quantification of tau value in Ih negative cell in controls (n = 10) and mutants (n = 14). Unpaired t-test, t(22) = 0.6904, p=0.4972. Bars represent mean ± SEM.
Figure 2 with 1 supplement
Robo2 VTA mutants show increased burst activity.
(A,B) Firing rate histogram of a representative cell displaying inhibition to quinpirole that is reversed with eticlopride in control (A) and mutant animal (B). (Left inset) Waveform of represented cell on each wire of the tetrode. (Right inset) Cumulative spikes of represented cell across time (min). Scale bar = 0.5 ms. (C) Firing rate of dopamine cells in controls (n = 42) and mutants (n = 50) is unchanged. (D) Burst rate of dopamine cells. (E) Burst duration of dopamine cells is longer in mutant cells. Unpaired t-test, t(90) = 2.17, *p<0.05. (F) Spikes per burst in dopamine cells are increased in mutants. Unpaired t-test, t(90) = 2.33, *p<0.05. (G) Firing rate of non-dopamine cells during in vivo electrophysiological recordings in controls (n = 61) and mutants (n = 71). (H) Total normalized distribution of the inter-spike interval (ISI) in controls and mutants. (I) Skew of the ISI in non-dopamine cells is reduced in Robo2 VTA mutants. Unpaired t-test, t(130) = 2.42, *p<0.05. (J) Coefficient of variation of the ISI in non-dopamine cells is reduced in mutants. Unpaired t-test, t(130) = 2.66, **p<0.01. Bar represent mean ± SEM.
Figure 2—figure supplement 1
in vivo electrophysiology.
(A) Schematic showing tetrode placement in controls and mutants. (B) Representative image showing placement of tetrode in the VTA for in vivo electrophysiological recordings. (C) The distribution of dopamine and non-dopamine cells recorded showed no difference between controls (n = 103) and mutants (n = 121). (D) Cumulative cell count of dopamine and non-dopamine cells in response to quinpirole (right) or eticlopride (top) or both (left) in control animals. (E) Cumulative cell count of dopamine and non-dopamine cells in response to quinpirole (right) or eticlopride (top) or both (left) in mutant animals. (F) The average change in dopamine cells in response to quinpirole (top) or eticlopride (bottom) showed no difference between controls and mutants. Bars represent mean ± SEM.
Figure 3 with 1 supplement
Robo2 VTA mutants have increased phasic dopamine release.
(A) Schematic of experimental design showing stimulation of the PPTg and FSCV recording in the NAc. (B) Representative voltammogram of dopamine recorded in the NAc in control (left) and mutant (right). (C–G) Average dopamine oxidation current recorded in NAc after PPTg stimulation is increased in mutants (n = 6) compared to controls (n = 5). (C) 50 μA: Two-way repeated measures ANOVA, effect of genotype, F(1,9) = 12.75, **p<0.01. (D) 100 μA: Two-way repeated measures ANOVA, effect of genotype, F(1,9) = 5.84, *p<0.05. (E) 150 μA: Two-way repeated measures ANOVA, effect of genotype, F(1,9) = 8.65, *p<0.05. (F) 200 μA. (G) 250 μA. (H) Area under the curve of the dopamine current across all stimulation intensities. Two-way repeated measures ANOVA, effect of genotype, F(1,9) = 5.99, *p<0.05. Bar represent mean ± SEM.
Figure 3—figure supplement 1
Similar dopamine levels were recorded after MFB stimulation or microdialysis.
(A) Schematic showing the recording electrode sites in the controls and mutants. (B) Representative image showing recording electrode placement in the NAc. (C) Representative image showing stimulating electrode placement in the PPTg. (D) Representative image showing stimulating electrode placement in the MFB. (E–I) Average dopamine oxidation current recorded in NAc after MFB stimulation shows no difference between controls (n = 4) and mutants (n = 4). (E) 50 μA: Two-way repeated measures ANOVA, effect of genotype, F(1,6) = 2.54, p=0.1620. (F) 100 μA: Two-way repeated measures ANOVA, effect of genotype, F(1,6) = 3.49, p=0.1110. (G) 150 μA: Two-way repeated measures ANOVA, effect of genotype, F(1,6) = 2.08, p=0.1997. (H) 200 μA: Two-way repeated measures ANOVA, effect of genotype, F(1,6) = 0.53, p=0.4958. (I) 250 μA: Two-way repeated measures ANOVA, effect of genotype, F(1,6) = 0.04, p=0.8467. (J) Area under the curve of the dopamine current across all stimulation intensities after MFB stimulation shows no difference between controls and mutants. Two-way repeated measures ANOVA, Genotype X stimulation intensity interaction, F(4,24) = 0.06, p=0.9930. (K,L) Tonic dopamine levels calculated as percent area ratio of dopamine to D4-dopamine across four time points (K) and averaged (L) for controls (n = 5) and mutants (n = 5). Unpaired t-test, t(8) = 0.1853, p=0.8576. Bars represent mean ± SEM.
Figure 4 with 2 supplements
Behavioral characterization of Robo2 VTA mutants show phenotypes in locomotion and cocaine sensitization.
(A) Locomotion measured across three consecutive days and nights showing mutants (n = 31) are hyperactive relative to controls (n = 36). Two-way repeated measures ANOVA, genotype x time interaction, F(252,16380) = 2.53, ****p<0.0001. (B) Total distance traveled across three consecutive nights. Two-way repeated ANOVA, effect of genotype, F(1,65) = 12.49, ***p<0.001, Bonferroni’s multiple comparison test, ***p<0.001, **p<0.01. (C–D) Locomotor response to cocaine (20 mg/kg) in controls (n = 20) and mutants (n = 11) on day 1 (C), Two-way repeated measures ANOVA, genotype x time interaction, F(35,1015) = 7.10, ****p<0.0001 and day 5 (D), Two-way repeated measures ANOVA, genotype x time interaction, F(35,1015) = 8.23, ****p<0.0001. (E) Normalized locomotor response to cocaine by subtracting 60 min pre-cocaine from 60 min post-cocaine. Two-way repeated measures ANOVA, effect of genotype, F (1,29) = 18.26, ***p<0.001, Bonferroni’s multiple comparison test, ****p<0.0001, **p<0.01. (F) Locomotor response to amphetamine (2.5 mg/kg) in controls (n = 19) and mutants (n = 17). Two-way repeated measures ANOVA, genotype x time interaction, F(35,1190) = 6.43, ****p<0.0001. (G) Locomotor response to the DAT blocker, GBR-12909 (10 mg/kg) in controls (n = 19) and mutants (n = 17). Two-way repeated measures ANOVA, genotype x time interaction, F(35,1190) = 2.94, ****p<0.0001. (H) Normalized locomotor response to amphetamine and GBR-12909 by subtracting 60 min pre-drug from 60 min post-drug. AMP: Unpaired t-test, t(34) = 2.968 **p<0.01,. GBR: Unpaired t-test, t(34) = 2.061, *p<0.05. (I) Discrimination between a CSp and CSm is impaired in AAV1-Cre-GFP::Robo2lox/lox animals (n = 16) compared to controls (n = 22). Two-way repeated measures ANOVA, genotype x time interaction, F(6,216) = 2.40, *p<0.05, Bonferroni’s multiple comparison test, **p<0.01 and *p<0.05. Bars represent mean ± SEM.
Figure 4—figure supplement 1
Robo2 VTA mutants have altered gait.
(A) Hindleg stride length in controls (n = 8 animals) and mutants (n = 8 animals). (B) Foreleg stride length. (C) Hindleg base. (D) Foreleg base, unpaired t-test, t(14) = 2.469, *p<0.05. (E) Print separation, unpaired t-test, t(14) = 2.992, **p<0.01. (F) Representative footprints from control and mutant showing forelimbs (green) and hindlimbs (red). Scale bar = 1 cm. Bars represent mean ± SEM.
Figure 4—figure supplement 2
Additional pharmacological studies on Robo2 VTA mutant mice.
(A) Conditioned place preference (CPP) to cocaine (15 mg/kg) in controls (n = 11) and mutants (n = 10) showed no difference between genotypes. (B) Locomotor response to saline in controls (n = 28) and mutants (n = 28), Two-way repeated ANOVA, effect of genotype, F(1, 54) = 5.19, *p<0.05. (C) Locomotor response to fluoxetine (20 mg/kg) in controls (n = 7) and mutants (n = 8). Two-way repeated ANOVA, genotype x time interaction, F(35, 455) = 2.17, ***p<0.001. (D) Locomotor response to nisoxetine (10 mg/kg) in controls (n = 13) and mutants (n = 11). (E) Normalized locomotor response to saline, fluoxetine, and nisoxetine by subtracting 90 min pre-injection from 90 min post-injection. (Nisoxetine) unpaired t-test, t(13) = 2.780, *p<0.05. (F) Robo2 VTA mutants have no significant difference in head entries during Pavlovian conditioning between control and mutant mice. (G) Acoustic pre-pulse inhibition at different pre-pulse intensities is not different between control (n = 5) and mutant mice (n = 5). (H) Response to NMDA receptor antagonist MK-801 (0.2 mg/kg) shows a difference in mutant mice (n = 8) compared to control mice (n = 7), Two-way repeated measures ANOVA, genotype x time interaction, F(35,455) = 1.47, *p<0.05. Bars represent mean ± SEM.
Figure 5 with 1 supplement
Adult or embryonic inactivation of Robo2 in dopamine neurons does not recapitulate the Robo2 VTA mutant phenotypes.
(A) Schematic showing expression of tamoxifen-inducible Cre specifically in adult dopamine neurons. Representative images from Slc6a3-iCreERT2 crossed to a Cre-dependent reporter line, Gt(ROSA)26Sortm(CAG-tdTomato)Hze, shows induction of the tdTomato reporter is specific to animals receiving tamoxifen injections. Scale bar = 100 µm. (B) Locomotion measured across three consecutive days and nights showing mutants (n = 12) are normal compared to control animals (n = 12). (C) Discrimination between CSp and CSm (left) is normal in Slc6a3-iCreERT2::Robo2lox/lox animals (n = 12) compared to controls (n = 12). (D) Schematic showing expression of Cre in dopamine neurons starting in embryonic development and continuing into adulthood. (E) Locomotion measured across three consecutive days and nights showing mutants (n = 12) are normal compared to control animals (n = 11). (F) Discrimination between CSp and CSm (left) is normal in Slc6a3-Cre::Robo2∆/lox animals (n = 11) compared to controls (n = 12). (G) Schematic of strategy for injecting AAV1-Cre-GFP into the midbrain of Slc6a3-Cre; Robo2∆/lox animals. (H) Locomotion measured across three consecutive days and nights showing mutants (n = 13) are hyperactive relative to controls (n = 15). Two-way repeated measures ANOVA, genotype x time interaction, F(252,6552) = 3.17, ****p<0.0001. (I) Discrimination between a CSp and CSm is impaired in AAV1-CreGFP::Slc6a3-Cre::Robo2∆/lox animals (n = 6) compared to controls (n = 8). Two-way repeated measures ANOVA, genotype x time interaction, F(6,72) = 2.68, *p<0.05, Bonferroni’s multiple comparison test, **p<0.01. (J, K) Locomotor response to cocaine (20 mg/kg) in controls (n = 15) and mutants (n = 13) on day 1 (d), Two-way repeated measures ANOVA, genotype x time interaction, F (35,910)=6.57, ****p<0.0001 and day 5 (e), Two-way repeated measures ANOVA, genotype x time interaction, F (35,910)= 17.03, ****p<0.0001. (L) Normalized locomotor response to cocaine by subtracting 60 min pre-cocaine from 60 min post-cocaine on day 1 and day 5, Two-way repeated measures ANOVA, genotype x time interaction, F (1,26)= 10.97, **p<0.01, Bonferroni’s multiple comparison test, ****p<0.0001, **p<0.01. Bars represent mean ± SEM.
Figure 5—figure supplement 1
Embryonic or adult inactivation of Robo2 does not recapitulate the Robo2 VTA mutant phenotypes.
(A, B) Locomotor response to cocaine (20 mg/kg) in controls (n = 12) and mutants (n = 12) on day 1 (A) and day 5 (B). (C) Normalized locomotor response to cocaine by subtracting 60 min pre-cocaine from 60 min post-cocaine. (D) There was no difference in head entries during Pavlovian conditioning between controls (n = 12) and mutants (n = 12). (E) Schematic of AAV1-FLEX-Rpl22HA injection into the VTA of Slc6a3-Cre::Robo2△/lox and control mice (right). Inactivation of Robo2 in dopamine neurons resulted in a significant de-enrichment of ribosomal-associated Robo2 mRNA, **p<0.01. (F, G) Locomotor response to cocaine (20 mg/kg) in controls (n = 11) and mutants (n = 12) on day 1 (F) and day 5 (G). (H) Normalized locomotor response to cocaine by subtracting 60 min pre-cocaine from 60 min post-cocaine. (I) There was no difference in head entries during Pavlovian conditioning between controls (n = 11) and mutants (n = 12). (J) Total distance traveled across three consecutive nights. Two-way repeated measures ANOVA, effect of genotype, F(1,26) = 50.42, ****p<0.0001, Bonferroni’s multiple comparison test, ****p<0.0001. (K) There was no difference in head entries during Pavlovian conditioning between controls (n = 6) and mutants (n = 8). Bars represent mean ± SEM.
Figure 6
Midbrain GABA neuron inactivation recapitulates Robo2 VTA mutant phenotypes.
(A) Schematic of strategy for injecting AAV1-GFP-TetX into the midbrain of Vgat-Cre animals (left). Schematic of Cre-dependent AAV1-GFP-TetX (top). Representative image (bottom) showing viral transduction is specific to non-dopamine neurons (TH- neurons). Scale bar = 100 µm (25 µm in zoom). (B) Locomotion measured across three consecutive days and nights showing mutants (n = 9) are hyperactive relative to controls (n = 6). Two-way repeated measures ANOVA, genotype x time interaction, F(252,3276) = 1.35, ***p<0.001. (C,D) Locomotor response to cocaine (20 mg/kg) in controls (n = 6) and mutants (n = 9) on day 1 (C), Two-way repeated measures ANOVA, genotype x time interaction, F(35, 455) = 1.34, p=0.0987, and on day 5 (D), Two-way repeated measures ANOVA, genotype x time interaction, F(35, 455) = 2.86, p<0.0001. (E) Normalized locomotor response to cocaine by subtracting 60 min pre-cocaine from 60 min post-cocaine on day 1 and day 5, Two-way repeated measures ANOVA, genotype x time interaction, F(1,13) = 5.35, *p<0.05. Bars represent mean ± SEM.
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Roundabout receptor 2 maintains inhibitory control of the adult midbrain
eLife 6:e23858.
https://doi.org/10.7554/eLife.23858
