ERK/MAPK Signaling Is Required for Pathway-Specific Striatal Motor Functions

Abstract

The ERK/MAPK intracellular signaling pathway is hypothesized to be a key regulator of striatal activity via modulation of synaptic plasticity and gene transcription. However, prior investigations into striatal ERK/MAPK functions have yielded conflicting results. Further, these studies have not delineated the cell-type-specific roles of ERK/MAPK signaling due to the reliance on globally administered pharmacological ERK/MAPK inhibitors and the use of genetic models that only partially reduce total ERK/MAPK activity. Here, we generated mouse models in which ERK/MAPK signaling was completely abolished in each of the two distinct classes of medium spiny neurons (MSNs). ERK/MAPK deletion in D1R-MSNs (direct pathway) resulted in decreased locomotor behavior, reduced weight gain, and early postnatal lethality. In contrast, loss of ERK/MAPK signaling in D2R-MSNs (indirect pathway) resulted in a profound hyperlocomotor phenotype. ERK/MAPK-deficient D2R-MSNs exhibited a significant reduction in dendritic spine density, markedly suppressed electrical excitability, and suppression of activity-associated gene expression even after pharmacological stimulation. Our results demonstrate the importance of ERK/MAPK signaling in governing the motor functions of the striatal direct and indirect pathways. Our data further show a critical role for ERK in maintaining the excitability and plasticity of D2R-MSNs.SIGNIFICANCE STATEMENT Alterations in ERK/MAPK activity are associated with drug abuse, as well as neuropsychiatric and movement disorders. However, genetic evidence defining the functions of ERK/MAPK signaling in striatum-related neurophysiology and behavior is lacking. We show that loss of ERK/MAPK signaling leads to pathway-specific alterations in motor function, reduced neuronal excitability, and the inability of medium spiny neurons to regulate activity-induced gene expression. Our results underscore the potential importance of the ERK/MAPK pathway in human movement disorders.

Keywords: MAP kinase; activity-induced gene expression; basal ganglia; dendritic spine; hyperlocomotion; medium spiny neuron.

Figure 1.

ERK-deficient MSNs show proper targeting of axonal projections. A, Quantification of the relative percentage of ERK2-positive D1R-MSNs in control and ERK:D1 mutant mice at P21. ERK2 (green) is coexpressed with D1^tdTomato (red) in D1R-MSNs of littermate control mice, but is lost in virtually all D1R-MSNs of ERK:D1 mutant mice (*p < 0.001; n = 3 mice/genotype, 150 cells/mouse). Scale bar, 50 μm. B, Quantification of the percentage of ERK2-postive D2R-MSNs in littermate control and ERK:D2 mutant mice at P21. ERK2 (green) is expressed in in both D1^tdTomato-positive D1R-MSNs and D1^tdTomato-negative D2R-MSNs of littermate control mice, but is lost in virtually all D1^tdTomato-negative D2R-MSNs of ERK:D2 mutant mice (*p < 0.001; n = 3 mice/genotype, 150 cells/mouse). Scale bar, 50 μm. C, D, Control (ERK^−/−;ERK2^w/w:D1^Cre) and ERK:D1 mutant mice were backcrossed with Cre-dependent fluorescent reporter Ai3 mice to label all D1R-MSN projections with enhanced yellow fluorescent protein (eYFP). Normal D1R axon targeting to the GPi and SNr is observed in control (C) and ERK:D1 mutant (D) mice. Scale bar, 1 mm. E, F, Control (ERK^−/−;ERK2^w/w:D2^Cre) and ERK:D2 mutant mice were backcrossed with Ai3 mice to label D2R-MSN axonal projections with eYFP. Normal D2R axon targeting to the GPe is observed in control (E) and ERK:D2 mutant (F) mice. Insets show magnified images of GPe. Scale bar, 1 mm. G, Quantification of weight gain during the second and third postnatal weeks in ERK:D1 mutant mice compared with ERK^−/−;ERK2 ^Fl/Fl littermate controls. ERK:D1 animals show significant deficits in weight gain beginning at P7 and continuing through P19 (n = 6 animals/genotype, main effect for genotype F_(1,10) = 84.96, *p < 0.0001, Bonferroni post hoc comparison). H, Kaplan–Meier survival curve of ERK:D1 (blue line, n = 80 mice) and ERK:D2 (green line; n = 71 mice) mice compared with ERK1^−/−;ERK2^Fl/Fl controls (black line, n = 57 mice). ERK:D1 mice show a significant reduction in survival (*p < 0.0001, post hoc Gehan–Breslow–Wilcoxon test). All data are presented as mean ± SEM.

Figure 2.

ERK signaling is required for pathway-specific regulation of locomotor behavior. A, Quantification of total distance traveled in a 30 min testing period by ERK:D1 (blue bar) and paired littermate control (gray bar) mice at P21. ERK:D1 animals show a significant reduction in locomotor activity (*p < 0.01, n = 10 mice/genotype). B, Representative recordings of total distance traveled (30 min) in control and ERK:D1 animals. C, Quantification of total distance traveled in a 30 min testing period by ERK:D2 mutant mice (green bar) and paired littermate controls (gray bar). ERK:D2 mutants show significantly more locomotor activity than controls (*p < 0.001, n = 10mice/genotype). D, Representative tracks of cumulative open-field activity for control and ERK:D2 mutant mice. E, Three hour open-field analysis of ERK:D2 mice and controls. Shown is total distance traveled as a function of time. ERK:D2 mutant mice (green trace) showed significantly increased movement throughout the entire 3 h testing period compared with control animals (gray trace), which steadily reduce activity throughout the trial (*p < 0.001; n = 10 mice/genotype). All data are presented as mean ± SEM.

Figure 3.

ERK signaling is required for proper spinogenesis and expression of synaptic plasticity genes. A, Representative images of D1R-MSN and D2R-MSNs labeled with AAV8-CAG-GFP virus (green). D1R-MSNs are identified by their expression of the D1^tdTomato transgene (red) and D2R-MSNs are D1^tdTomato negative. GFP-expressing D2R-MSNs appear green, whereas GFP-expressing D1R-MSNs appear yellow. Scale bar, 20 μm. B, AAV8-CAG-GFP efficiently labels MSN dendritic spines. D2R-MSNs in ERK:D2 mutant mice show a significant reduction in spine density compared with littermate controls. In contrast, there is no difference in spine density on D1R-MSNs between mutant and control animals. Scale bar, 5 μm. C, Quantification of mean spine density shows a reduction in dendritic spines in D2R-MSNs (*p < 0.05; n = 3 mice/genotype, 15 dendrites/mouse), but not D1R-MSNs (p < 0.65; n = 3 mice/genotype, 15 dendrites/mouse), in ERK:D2 mutant mice compared with littermate controls. D, E, Microarray analysis of P17 striatum (n = 3 male mice/genotype) showing dramatic reduction in the expression of activity-induced immediate early genes (D) and genes associated with synaptic plasticity (E). *p < 0.05.

Figure 4.

Markedly reduced excitability of ERK-deleted D2R-MSNs. A, Image of pipette recording from a D2R-MSN negative for D1^tdTomato (red). B, Representative mEPSC recordings of D2R-MSNs from control (gray) and ERK:D2 mutant (black) mice. C, Summary of mEPSC frequencies and amplitudes from control (gray; n = 3 mice, n = 10 neurons) and ERK:D2 mutant (black; n = 3 mice, n = 8 neurons) D2R-MSNs. mEPSC frequency is significantly reduced in ERK:D2 mutant D2R-MSNs (*p < 0.01), whereas mEPSC amplitude is unchanged. D, Representative mIPSC recordings of D2R-MSNs from control (gray) and ERK:D2 mutant (black) mice. E, Summary of mIPSC frequencies and amplitudes from control (gray; n = 3 mice; n = 10 neurons) and ERK:D2 mutant (black; n = 3 mice; n = 7 neurons) D2R-MSNs. Both mIPSC frequency and amplitude are reduced significantly in ERK:D2 mutant D2-MSNs (*p < 0.01). F, Representative image of recording pipette in a D1^tdTomato-positive (red) D1R-MSN. G, Representative mEPSC recordings of D1R-MSNs from control (red) and ERK:D2 mutant (black) mice. H, Summary of mEPSC frequencies and amplitudes from control (red; n = 3 mice; n = 8 neurons) and ERK:D2 mutant (black; n = 3 mice; n = 7 neurons) D1R-MSNs. There is no significant change in mEPSC frequency or amplitude in ERK:D2 mutant D1R-MSNs compared with control. I, Representative mIPSC recordings of D1R-MSNs from control (red) and ERK:D2 mutant (black) mice. J, Summary of mIPSC frequencies and amplitudes from control (red; n = 3 mice; n = 8 neurons) and ERK:D2 mutant (black; n = 3 mice; n = 7 neurons) D1R-MSNs. There is no significant difference between ERK:D2 mutant D1R-MSNs and control MSNs. K, Representative traces of whole-cell patch-clamp recordings from D2R-MSNs in control (gray) and ERK:D2 mutant (black) mice. L, Relationship between elicited action potential responses and somatic current injection in D2R-MSNs of control (gray; n = 3 mice; n = 9 neurons) and ERK:D2 mutant (black; n = 3 mice; n = 10 neurons) mice. D2R-MSNs from ERK:D2 mutant mice have a significantly reduced capacity to elicit action potentials (*p < 0.001). M, Representative traces of whole-cell patch-clamp recordings from D1R-MSNs in control (red) and ERK:D2 mutant (black) mice. N, Relationship between elicited action potential responses and somatic current injection in D1R-MSNs of control (red; n = 3 mice; n = 9 neurons) and ERK:D2 mutant (black; n = 3 mice; n = 9 neurons) mice. Intrinsic excitability in D1R-MSNs of ERK:D2 mutant mice was slightly, but significantly, reduced compared with controls (*p < 0.01).

Figure 5.

Activity-regulated gene expression is strongly suppressed in ERK-deleted D2R-MSNs. A, Representative image of cataleptic response to haloperidol using horizontal bar test. B, Quantification of cataleptic response (freezing) to haloperidol (1 mg/kg) or vehicle in littermate control and ERK:D2 mutant mice. Control mice exhibit a robust cataleptic response to haloperidol compared with vehicle-treated controls (*p = 0.001, n = 7 mice/genotype/condition). The cataleptic response is effectively abolished in the mutant mice (*p = 0.001, haloperidol-treated ERK:D2 vs haloperidol treated control; n = 7 mice/condition). Vehicle-treated mice show no cataleptic response (n = 7 mice per genotype per treatment). C, D, c-FOS (green) expression in control and ERK:D2 mutant striatum 1 h after haloperidol administration. CTIP2 (blue) identifies all MSNs; D1^tdTomato (red) identifies the D1R-MSN subpopulation. All D2R-MSNs are CTIP2(+);tdTomato(−). Insets are magnified images showing all three labels (top) and c-FOS only (bottom) demonstrating that c-FOS is strongly upregulated in D2R-MSNs (yellow arrows) in control (C) but not ERK:D2 animals (D). E, Quantification of MSN-specific c-FOS expression after haloperidol (1 mg/kg) or vehicle administration (*p < 0.001; n = 3 mice/genotype; 200–250 cells/mouse). F, No changes in c-FOS expression are observed in D1R-MSNs in either control or ERK:D2 mutant animals. (n = 3 animals/genotype, 200–250 cells/animal). G, H, ARC (green) expression in control and ERK:D2 mutant striatum 1 h after haloperidol administration. CTIP2 (blue) identifies all MSNs; D1^tdTomato (red) identifies the D1R-MSN subpopulation. All D2R-MSNs are CTIP2(+);tdTomato(−). Insets are magnified images demonstrating that ARC is upregulated in D2R-MSNs (yellow arrows) in control (G) but not ERK:D2 animals (H). I, Quantification of D2R-MSN-specific ARC expression after haloperidol (1 mg/kg) or vehicle administration (*p < 0.001; n = 3 mice/genotype; 200–250 cells/mouse). J, D1R-MSNs in ERK:D2 mutant mice express significantly more ARC compared with controls regardless of treatment (#p < 0.05, *p < 0.001; n = 3 mice/genotype; 200–250 cells/mouse). All data are presented as mean ± SEM. Scale bar, 50 μm; inset, 10 μm.

Figure 6.

Basal and haloperidol-induced behavioral and gene expression changes in ERK:A2a mice. A, B, Open-field locomotor testing of ERK:A2a and control mice (n = 7 animals/genotype). A, There is no significant difference in cumulative distance traveled (1 h test) between ERK:A2a mutant and control littermates. B, Total distance traveled as a function of time. No differences are observed between mutant and control animals. C, Cataleptic response 1 h after haloperidol administration (1 mg/kg). ERK:A2a mutant mice have a significantly reduced cataleptic response compared with littermate controls (*p < 0.001; n = 6 mice/genotype). D, E, Immunohistochemical labeling of activity-induced gene expression 1 h after haloperidol administration. All MSNs are labeled with Ctip2 (blue). D1R-MSNs are distinguished from D2R-MSNs by their expression of D1^tdTomato(red). c-FOS (green) is upregulated in D2R-MSNs of control (D) but not ERK:A2a mutant (E) mice. Insets show higher magnification of all three labels (top) and c-FOS only (bottom). D2R-MSNs are indicated by yellow arrows. F, Quantitative analysis showing a significant reduction in the percentage of D2R-MSNs expressing c-FOS in ERK:A2a mutant animals compared with control animals (*p < 0.001; n = 3 mice/genotype; 200–250 cells/mouse). G, H, ARC (green) is upregulated in D2R-MSNs (yellow arrows) in control (G) but not ERK:A2a mutant (H) animals. I, Quantitative analysis demonstrating a significant decrease in the percentage of D2R-MSNs that upregulate ARC in ERK:A2a mutant mice (*p < 0.05; n = 3 mice/genotype; 200–250 cells/mouse). All data are presented as mean ± SEM. Scale bars, 50 μm; inset, 20 μm.

Figure 7.

Delayed loss of ERK activity in ERK:A2a mice. A, Representative image of P21 ERK:A2a striatum. CTIP2 (blue) labels all MSNs, whereas D2R-MSNs are identified as negative for D1^tdTomato expression (red). A subpopulation of D2R-MSNs in ERK:A2a mutants maintain ERK2 expression (green, white arrows). ERK2-deficient D2R-MSNs are indicated with a white asterisk. Scale bar, 20 μm. B, Quantitative analysis of ERK2 expression in P21 and P28 ERK:A2a striatum. Approximately half of all D2R-MSNs maintain ERK2 expression at P21. By 28, ∼15% of D2R-MSNs continue to express ERK2. (n = 3 animals/genotype; 500–600cells/animal). C, Colocalization of D2^GFP (green) and A2a^Cre:Ai9 (red) at P14. Scale bar, 50 μm. D, Quantification of D2^GFP and A2a^Cre:Ai9 colocalization at P14, P21, and P28. All data are presented as a percentage of total D2R-MSNs counted. Note that the percentage of D2R-MSNs that express both GFP and Ai9 is low at P14. The percentage increases over time, but complete recombination in D2R-MSNs is not observed, even by P28 (n = 3 animals/time point; 500–600 cells/animal). All data are presented as mean ± SEM.