Neuropilin 2 Signaling Mediates Corticostriatal Transmission, Spine Maintenance, and Goal-Directed Learning in Mice

Abstract

The striatum represents the main input structure of the basal ganglia, receiving massive excitatory input from the cortex and the thalamus. The development and maintenance of cortical input to the striatum is crucial for all striatal function including many forms of sensorimotor integration, learning, and action control. The molecular mechanisms regulating the development and maintenance of corticostriatal synaptic transmission are unclear. Here we show that the guidance cue, Semaphorin 3F and its receptor Neuropilin 2 (Nrp2), influence dendritic spine maintenance, corticostriatal short-term plasticity, and learning in adult male and female mice. We found that Nrp2 is enriched in adult layer V pyramidal neurons, corticostriatal terminals, and in developing and adult striatal spiny projection neurons (SPNs). Loss of Nrp2 increases SPN excitability and spine number, reduces short-term facilitation at corticostriatal synapses, and impairs goal-directed learning in an instrumental task. Acute deletion of Nrp2 selectively in adult layer V cortical neurons produces a similar increase in the number of dendritic spines and presynaptic modifications at the corticostriatal synapse in the Nrp2^-/- mouse, but does not affect the intrinsic excitability of SPNs. Furthermore, conditional loss of Nrp2 impairs sensorimotor learning on the accelerating rotarod without affecting goal-directed instrumental learning. Collectively, our results identify Nrp2 signaling as essential for the development and maintenance of the corticostriatal pathway and may shed novel insights on neurodevelopmental disorders linked to the corticostriatal pathway and Semaphorin signaling.SIGNIFICANCE STATEMENT The corticostriatal pathway controls sensorimotor, learning, and action control behaviors and its dysregulation is linked to neurodevelopmental disorders, such as autism spectrum disorder (ASD). Here we demonstrate that Neuropilin 2 (Nrp2), a receptor for the axon guidance cue semaphorin 3F, has important and previously unappreciated functions in the development and adult maintenance of dendritic spines on striatal spiny projection neurons (SPNs), corticostriatal short-term plasticity, intrinsic physiological properties of SPNs, and learning in mice. Our findings, coupled with the association of Nrp2 with ASD in human populations, suggest that Nrp2 may play an important role in ASD pathophysiology. Overall, our work demonstrates Nrp2 to be a key regulator of corticostriatal development, maintenance, and function, and may lead to better understanding of neurodevelopmental disease mechanisms.

Keywords: cortical pyramidal neurons; neurodevelopment; rotarod; semaphorin signaling; short-term plasticity; striatum.

Figure 1.

Nrp2 protein is expressed in cortical neurons and striatal SPNs. A, Schematic of a coronal brain section where representative confocal images were obtained from imaging the cortex (red box) and striatum (blue box). B, Expression of Nrp2 is detected in cortical neurons (black arrowheads), mainly in layer V, and sparsely in layers II/III and VI. C, High magnification of layer V cortical neurons reveals Nrp2 localization in pyramidal neuron soma and apical dendrites (white arrows). D, No expression is observed in adult Nrp2^−/− cortical neurons. E, Adult WT (left column) and Nrp2^−/− (right column) sections triple labeled with anti-Nrp2, anti-VGLUT1, and anti-Darpp-32. Nrp2 expression is detected in VGLUT1⁺ terminals (in merged panel, white arrowheads, boxed inset). F, WT P3, P14, P21, and adult brain sections coimmunolabeled with anti-Nrp2 (red) and anti-Darpp-32 (green). SPNs coexpressing Nrp2 and Darpp-32 (white arrowheads). Scale bars: B, 200 μm; (in C) C, D, 50 μm; E, 20 μm; E, z-plane, 8 μm; F, 20 μm; F, z-plane, 8 μm.

Figure 2.

Changes in SPN intrinsic properties and spine density in Nrp2^−/− mice. A, B, Membrane voltage responses to injected current pulses of an SPN recorded in WT (A) and Nrp2^−/− (B) mice. Note that similar current injection elicits higher firing frequency in Nrp2^−/− mice (B, red) than in WT mice (A, blue). C, Current–voltage curves of SPNs recorded in WT (blue) and Nrp2^−/− (red) mice. D, Graph representing the spike frequency in response to injected current pulses in WT (blue) and Nrp2^−/− mice (red). E, Box plots representing the rheobase current in WT (blue) and Nrp2^−/− (red) mice. F, Representative current-clamp traces showing the rheobase current of an SPN recorded in WT (F1) and Nrp2^−/− mice (F2). Note that the rheobase current is lower in F2 (Nrp2^−/−) than in F1 (WT).

Figure 3.

Nrp2^−/− SPNs display supernumerary spines, but normal dendritic arborization. A, B, Representative images taken from a WT and Nrp2^−/− SPN back-filled with biocytin. 2D confocal images are reconstructed from complete z-stacks. C, D, Single-plane images of WT and Nrp2^−/− SPNs were taken at higher magnification within the area indicated by the red-dashed boxes in A and B, respectively. E, Quantification of the average number of spines per 50 μm from reconstructed z-stack images of 2D neurons. Error bars are the mean ± SD; unpaired t test, *p = 0.0022. Scale bars: A (for A, B), C (for C, D), 20 μm. F, G, 3D reconstruction of the dendritic arborization of two SPNs recorded and biocytin filled in WT (F; SPN1, SPN2) and Nrp2^−/− mice (G; SPN3, SPN4). Left panels correspond to x–y view, and right panels are rotated 90° around the y-axis. Scale bars, 50 μm. H, I, Box plot representing the number of primary dendrites (H) and the order number of dendritic branching (I) of 3D reconstructed SPNs. J, K, Graphs representing the number of intersections (E) and the dendritic length (F) of 3D reconstructed SPNs. Sholl analysis revealed no effect of genotype on dendritic arborization. WT (blue) and Nrp2^−/− (red) mice; error bars are the mean ± SEM, unpaired t test. H, I, p = 0.419 and p = 0.483, respectively; J, K, p = 0.413 and p = 0.495, respectively.

Figure 4.

Defects in corticostriatal short-term plasticity onto SPNs in Nrp2^−/− mice. A, Schematic illustrating the experimental paradigm where SPNs were recorded in coronal striatal slices. A bipolar stimulating electrode was placed in the corpus callosum to stimulate corticostriatal fibers. B, Example of voltage responses to current steps for injection of an SPN. C, D, Representative examples of EPSPs measured in SPNs after a pair of electrical stimuli. ISIs: 20 ms in C; 100 ms in D. Responses of SPNs recorded in WT are represented in blue, and mutant in red. Note that in WT SPNs, there is a strong paired-pulse facilitation that is impaired in Nrp2^−/−. E, Box plots representing the paired-pulse ratio of the responses at different ISIs (20, 40, 60, 80, 100, 120, and 200 ms) for the two genotypes. Responses are expressed as a ratio of the response to the first stimulus. F, Representative current-clamp traces showing corticostriatal EPSPs elicited by paired stimuli with increasing ISIs in WT mice (F1) and Nrp2^−/− (F2). G, Summary graph of PPRs recorded from medium spiny neurons plotted against ISIs for cortical stimulation in WT (blue) and Nrp2^−/− (red). Box plots represent the minimum, maximum interquartile range, the mean, and the median. Statistical analysis was made using unpaired t test (E) where ***p = 0.0009, 20 ms; ***p = 0.0010, 40 ms; ***p = 0.0002, 60 ms; *p = 0.0117, 80 ms; *p = 0.0391, 100 ms; **p = 0.0071, 120 ms; and *p = 0.0303, 200 ms comparison between WT and Nrp2^−/−, and two-way ANOVA (G).

Figure 5.

Nrp2^−/− mice are impaired in goal-directed instrumental action. A, We trained Nrp2^−/− and WT mice on an instrumental learning task with a selective satiety procedure to devalue the instrumental outcome or an alternative outcome. B, All mice acquired an instrumental response that increased in rate with training; however, the rate of acquisition was reduced in Nrp2^−/− mice. C, When the instrumental outcome was devalued, WT mice significantly reduced their responding compared with when an alternative food was devalued. In contrast, Nrp2^−/− mice responded at similar rates following both devaluation sessions. Paired t test, *p = 0.046; error bars indicate ±1 SEM.

Figure 6.

Specific deletion of Nrp2 in adult layer V pyramidal neurons leads to increased dendritic spine numbers in vivo. A, Nrp2flox mice (F₀) were crossed with Etv1-CreERT2 (F₀) mice to generate heterozygous (F₁) mice, and the F₁ mice were backcrossed with Nrp2flox mice to generate homozygous floxed Nrp2flox mice with or without Etv1-Cre⁺ alleles (F₂). B, F₁ progeny developed in A were crossed with Thy1-GFP⁺ mice to generate triple heterozygous Nrp2⁺/f;Etv1⁺/Cre;Thy1-GFP⁺ mice. Triple heterozygous mice were crossed with Nrp2f/f;Etv1⁺/Cre to generate Nrp2f/f;Etv1⁺/Cre;Thy1-GFP⁺ and littermate controls (F₂). All F₂ in both A and B were treated twice on consecutive days with tamoxifen at 4–6 months. C–K, Schematic of a coronal adult brain section; red box indicates the cortical region of images in D–G immuno-labeled with anti-GFP showing successful Cre recombination in layer V neurons (E, red box enlarged in G) but not in littermate control (D, red box enlarged in F); green box indicates striatal region of image in H–K; no GFP⁺ striatal neurons were detected in H (green box enlarged in J) or in I (green box enlarged in K). Scale bars: E (for D, E), I (for H, I), G (for F, G), K (for J, K), 50 μm. L–N, Representative image of a neuron from a Nrp2+/f;Etv1^+/+;Thy1-GFP+ adult mouse brain. O–Q, Representative image of a neuron from a Nrp2f/f;Etv1⁺/Cre;Thy1-GFP⁺ adult mouse brain. Yellow and white boxes outline middle regions, 45–90 μm from the soma and proximal regions, and 0–45 μm from the soma, respectively. White arrows show dendritic spines in proximal regions M and P, enlarged from yellow box region in L and O, respectively. White arrows show dendritic spines in middle regions N and Q, enlarged from yellow box region in L and O, respectively. R, Quantification of spine density on layer V apical dendrites in proximal and middle regions. A significantly higher number of spines are found on Nrp2f/f;Etv1⁺/Cre;Thy1-GFP⁺ neurons compared with littermate controls for both regions. Error bars are the mean ± SEM; unpaired t test; *p = 0.0157; **p = 0.0027 compared with littermate controls. Scale bars: O (for L, O), Q (for M, N, P, Q), 15 μm.

Figure 7.

SPNs recorded in Nrp2 CKO mice have normal intrinsic properties. A, B, Membrane voltage responses to injected current pulses of a SPN recorded in a WT (A) and Nrp2 CKO (B) mice. C, Current–voltage curves of SPNs recorded in WT (blue line) and Nrp2 CKO mice (red line). D, Graph representing the spike frequency in response to injected current pulses in WT (blue line) and Nrp2 CKO mice (red line). E, Box plots representing the input resistance measured at rest (WT: blue; Nrp2 CKO: red). F, Box plots representing the resting membrane potential (WT: blue; Nrp2 CKO: red). G, H, Representative current-clamp traces showing the rheobase current of an SPN recorded in WT (G) and Nrp2 CKO (H) mice. I, Box plots representing rheobase current (WT: blue; Nrp2 CKO: red). Box plots represent the minimum, maximum interquartile range, the mean, and the median. Statistical analysis was performed using unpaired t test (F) and two-way ANOVA (G).

Figure 8.

Altered corticostriatal short-term plasticity in Nrp2 CKO mice. A, Schematic illustrating the experimental paradigm where SPNs were recorded in coronal striatal slices. A bipolar stimulating electrode was placed in the corpus callosum to stimulate corticostriatal fibers. B, C, Representative examples of EPSPs measured in SPNs after a pair of electrical stimuli. ISIs: 20 ms in K; 100 ms in L (K1, L1: WT; K2 and L2: Nrp2 CKO mice). Note that in WT SPNs there is a strong paired-pulse facilitation that is impaired in Nrp2 CKO. D, E, Representative current-clamp traces showing corticostriatal EPSPs elicited by paired stimuli with increasing ISIs in WT (M) and Nrp2 CKO (N) mice. F, Box plots representing the paired-pulse ratio of the responses at different ISIs (20, 40, 60, 80, 100, 120, and 200 ms) for the two genotypes and unpaired t test was performed (***p = 0.0003, 20 ms; ****p < 0.0001, 40 ms; ***p = 0.0002, 60 ms; ****p < 0.0001, 80 ms; **p = 0.0016, 100 ms; **p = 0.0043, 120 ms; ****p < 0.0001, 200 ms comparison between WT and Nrp2^−/−). Responses are expressed as the amplitude of the second EPSP divided by the amplitude of the first one. G, Summary graph of PPRs recorded from medium spiny neurons plotted against ISIs for cortical stimulation in WT (blue) and Nrp2 CKO (red).

Figure 9.

The effects of selective Nrp2 deletion in adult layer V cortical neurons on sensorimotor and instrumental learning. A, B, No significant difference was detected between WT and Nrp2 CKO mice for distance or time spent in the center of the open field test. C, No significant difference between WT and Nrp2 CKO mice for time spent in the open arm of the elevated zero maze. D, CKO mice showed a significant impairment in rotarod performance. Their overall latency to fall was significantly reduced, and post hoc comparisons revealed significantly reduced latency on trials 6–9. E, CKO and control mice did not differ on acquisition of an instrumental response and performance across sessions with increasing response requirements. Following devaluation of the instrumental outcome, both groups of mice showed a significant reduction in response rate compared with when the outcome is still valued. *p = 0.004; **p = 0.001. Error bars represent ±1 SEM.

Figure 10.

Schematic diagram illustrating the distinct roles of Nrp2 in corticostriatal pathway development, maintenance, and function. Loss of Nrp2 in Nrp2^−/− global KO mice increases spine number on apical dendrites of layer V cortical neurons as well as on striatal SPNs. CKO mice with adult loss of Nrp2 specifically from layer V cortical neurons (Nrp2f/f;Etv1-CreER) also show an increased number of cortical dendritic spines. Both Nrp2 KO and CKO mice show reduced paired-pulse facilitation relative to WT mice at corticostriatal synapses, indicating a modification in presynaptic neurotransmitter release. Nrp2 KO increased SPN intrinsic excitability relative to WT mice, consistent with the increased number of spines on SPN dendrites, whereas CKO mice showed no change in SPN excitability. Nrp2 KO animals were impaired in the rotarod and in an instrumental goal-directed task, whereas CKO mice showed only impaired rotarod performance.