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. 2014 Jul 3;158(1):198-212.
doi: 10.1016/j.cell.2014.04.045.

Autism-associated neuroligin-3 Mutations Commonly Impair Striatal Circuits to Boost Repetitive Behaviors

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Free PMC article

Autism-associated neuroligin-3 Mutations Commonly Impair Striatal Circuits to Boost Repetitive Behaviors

Patrick E Rothwell et al. Cell. .
Free PMC article

Abstract

In humans, neuroligin-3 mutations are associated with autism, whereas in mice, the corresponding mutations produce robust synaptic and behavioral changes. However, different neuroligin-3 mutations cause largely distinct phenotypes in mice, and no causal relationship links a specific synaptic dysfunction to a behavioral change. Using rotarod motor learning as a proxy for acquired repetitive behaviors in mice, we found that different neuroligin-3 mutations uniformly enhanced formation of repetitive motor routines. Surprisingly, neuroligin-3 mutations caused this phenotype not via changes in the cerebellum or dorsal striatum but via a selective synaptic impairment in the nucleus accumbens/ventral striatum. Here, neuroligin-3 mutations increased rotarod learning by specifically impeding synaptic inhibition onto D1-dopamine receptor-expressing but not D2-dopamine receptor-expressing medium spiny neurons. Our data thus suggest that different autism-associated neuroligin-3 mutations cause a common increase in acquired repetitive behaviors by impairing a specific striatal synapse and thereby provide a plausible circuit substrate for autism pathophysiology.

Figures

Figure 1
Figure 1. ASD-associated NL3 mutations enhance repetitive and stereotyped behaviors
(A) Summary of behavioral heterogeneity in ASD mouse models produced by NL3 mutations. (B) Illustration of a mouse on a rotarod (left), and diagram of the rotarod testing protocol (right). The effects of motor coordination and learning are illustrated below the protocol. (C-F) Performance of littermate wild-type (WT, n=22) and NL3-KO mice (n=23) on the accelerating rotarod. Time to fall off is presented at 4 to 40 rpm (C) and 8 to 80 rpm (D); the terminal speed of rotation (E) was used to calculate initial coordination and learning rate (F). Inset in C shows percentage of mice reaching maximum performance time (300 s) plotted as a function of trial. (G-J) Same as C-F, but comparing WT (n=19) and NL3-R451C mutant mice (n=16). (K-N) Quantitative video analysis of acquisition rates of repetitive motor routines during rotarod training (n=8 WT and 9 NL3-KO mice). K illustrates the analyzed parameters: step location, step length, and step timing. L-N depicts the standard deviation (SD) of step location (L), step length (M), and time between steps (N) calculated on trials 7 and 12 during rotarod training to measure variability in the motor routine (left panels), as well as the correlation with time to fall off the rotarod (right panels). (O & P) Behavior of WT and NL3-KO mice in an open field test, showing time course of activity across the entire session (O), as well as total distance travelled and the number of ambulatory episodes (P). Insets depict movement path of individual mice 10-20 min after the test begins. (Q & R) Open field activity for WT and NL3-R451C mice. (S-U) Analysis of stereotyped behaviors by measurements of the force plate variance as percentage of body weight (%BW) during low-mobility bouts (LMB, S), hind limb jumps (T), and rotational bias during locomotion (U) in WT (n=10) and NL3-KO mice (n=12). Data are means + SEM; *significant difference between groups (ANOVA). Also see Table S1 and Figure S1.
Figure 2
Figure 2. Conditional removal of NL3 in cerebellar Purkinje cells does not affect rotarod performance but causes hyperactivity
(A) Strategy for generation of NL3 conditional knockout (NL3-cKO) mice. Top, structure of the 5′ end of the wild-type mouse Nlgn3 gene; middle, structure of the cKO Nlgn3 gene in which loxP511 sites flank exons 2 and 3 (“flox”); and bottom, structure of the KO gene after Cre-recombinase mediated deletion of exons 2 and 3. (B) Levels of NL1, NL2, and NL3 mRNAs in brains of NL3-cKO mice without (n=4) or with nestin-Cre (Nes-Cre, n=4). (C) NL3 immunoblot of whole brain protein from NL3-cKO mice with and without Nes-Cre. (D & E) Analysis of littermate NL3-cKO mice without (n=16) or with Nes-Cre (n=16) in the open field test. Data show the time course of activity across the entire session (D) as well as the total distance traveled and the number of ambulatory episodes (E). (F) Illustration of genetic cross to selectively delete NL3 from cerebellar Purkinje cells. (G-J) Rotarod performance of NL3-cKO mice without (n=11) or with L7-cre expression in cerebellar Purkinje cells (n=11). Time to fall off is presented at 4 to 40 rpm (G) and 8 to 80 rpm (H); the terminal speed of rotation (I) was used to calculate initial coordination and learning rate (J). (K & L) Behavior in a test of open field activity after Purkinje cell deletion of NL3. Data are means ± SEM; *significant difference between groups (ANOVA). Also see Figure S2.
Figure 3
Figure 3. Enhanced acquisition of repetitive motor routines requires NL3 expression in striatal D1-MSNs but not D2-MSNs
(A) Illustration of genetic cross used to specifically delete NL3 from D1-MSNs. (B-E) Rotarod performance of NL3-cKO mice without (n=16) and with D1-Cre (n=12). Time to fall off is presented at 4 to 40 rpm (B) and 8 to 80 rpm (C); the terminal speed of rotation (D) was used to calculate initial coordination and learning rate (E). (F & G) Behavior of the same mice in a test of open field activity, showing time course of activity across the entire session (F) as well as total distance travelled and number of ambulatory episodes (G). (H-N) Behavior of NL3-cKO mice without (n=8) and with A2a-Cre (n=10), which directs Cre expression exclusively to D2-MSNs. (O) Illustration of cytosol aspiration from individual D1- and D2-MSNs of the NAc (top), and quantitative RT-PCR results from individual cells showing mRNA expression of cell type-specific markers (bottom). (P-R) Relative mRNA expression quantified as the ratio between D1- and D2-MSNs, showing cell type-specific markers in the NAc (P) as well as neuroligins in the NAc (Q) and dorsal striatum (R). Data are means ± SEM; *significant difference between groups (ANOVA). Also see Figures S3 & S4.
Figure 4
Figure 4. Conditional deletion of NL3 in the NAc but not the dorsal striatum causes enhanced rotarod learning
(A) Diagram of AAV constructs and stereotaxic injection of AAVs into the dorsal striatum (DS) or the NAc (left), and representative images showing GFP from viral injection localized to DS or NAc (right). (B-E) Rotarod performance of NL3-cKO mice after injection of ΔCre (Control, n=15) or Cre into the DS (n = 11). Time to fall off is presented at 4 to 40 rpm (B) and 8 to 80 rpm (C); the terminal speed of rotation (D) was used to calculate initial coordination and learning rate (E). (F & G) Behavior of the same mice in a test of open field activity, showing time course of activity across the entire session (F), as well as the total distance travelled and the number of ambulatory episodes (G). (H-M) Behavior of control mice (n=26) and NL3-cKO mice receiving injection of Cre into NAc (n=9). For this comparison, the control group includes NL3-cKO mice receiving injection of ΔCre as well as injection of Cre into DS. Data are means ± SEM; *significant difference between groups (ANOVA). Also see Figure S3.
Figure 5
Figure 5. Behavioral phenotypes caused by NL3 loss-of-function are rescued by targeted expression of NL3 in D1-MSNs of the NAc
(A) Schematic of Cre-dependent NL3 rescue construct (DIO-NL3), with illustration of rescue in D1-MSNs of the NAc (lower left) and image of Venus expression in the NAc (lower right) as well as co-localization of rescue construct with D1 -tomato (inset). (B-E) Rotarod performance of mice with DIO-NL3 injection in NAc, including a control group lacking Cre (n=10) and those carrying D1-Cre (n=9). Time to fall off is presented at 4 to 40 rpm (B) and 8 to 80 rpm (C); the terminal speed of rotation (D) was used to calculate initial coordination and learning rate (E). (F and G) Behavior of the same mice in a test of open field activity, showing time course of activity across the entire session (F) as well as total distance travelled and number of ambulatory episodes (G). Data are means ± SEM.
Figure 6
Figure 6. Subregion- and cell type-specific behavioral functions of D1- and D2-MSNs
(A) Schematic of Cre-dependent Kir2.1 construct (DIO-Kir), and confocal images from mice carrying D1-tomato showing co-localized expression in D1-Cre (lower left) and mutually exclusive expression in A2a-Cre (lower right). (B and C) Examples of current-clamp recordings from uninfected (B, left) and infected (B, right) D2-MSNs in a double transgenic mouse carrying D1-tomato and A2a-Cre, and plot of the average number of spikes fired in response to a constant depolarizing current (C, left), as well as summary graphs of the average input resistance (IR) (C, upper right) and rheobase (C, lower right). (D-F) Rotarod performance of WT (n=15) and D1-Cre mice (n=16) with DIO-Kir2.1 injections in the NAc, showing time to fall off at 4 to 40 rpm (D) and 8 to 80 rpm (E), as well as the terminal speed of rotation (F). (G) Time course of open field activity in the same mice as D-F. (H-K) Rotarod performance and open field activity of WT (n=12) and A2a-Cre mice (n=13) with DIO-Kir2.1 injections in the NAc. (L-O) Rotarod performance and open field activity of WT (n=9) and D1-Cre mice (n=15) with DIO-Kir2.1 injection in the dorsal striatum (DS). (P-S) Rotarod performance and open field activity of WT (n=7) and A2a-Cre mice (n=12) with DIO-Kir2.1 injection in the DS. (T-V) Summary graphs comparing the effects of DIO-Kir2.1 in different striatal cell types and subregions. Data are presented as percentage of WT level on the first rotarod trial (T), last rotarod trial (U), and open field activity (V). Data are means ± SEM; *significant difference between groups (ANOVA).
Figure 7
Figure 7. NL3 deletion decreases inhibitory synaptic transmission only in D1-MSNs but not D2-MSNs of the NAc
(A and B) Representative traces of mEPSCs in WT and NL3-KO cells (top), cumulative distribution of mEPSC inter-event intervals (lower left; inset shows average mEPSC frequency) and cumulative distribution of mEPSC amplitudes (lower right; inset shows average mEPSC amplitude) in WT (n=23) or NL3-KO (n=23) D1-MSNs (A), and WT (n=15) or NL3-KO (n=17) D2-MSNs (B). (C and D) EPSC changes upon bath application of the group I mGluR agonist DHPG (100 μM), recorded in WT (n=7) or NL3-KO (n=6) D1-MSNs (C), and in WT (n=5) or NL3-KO (n=9) D2-MSNs (D). (E and F) Representative traces, frequency, and amplitude of mIPSCs recorded in WT (n=17) or NL3-KO (n=17) D1-MSNs (E), and WT (n=12) or NL3-KO (n=15) D2-MSNs (F). (G and H) Evoked IPSC amplitudes during bath application of the endocannabinoid receptor CB1 antagonist AM251 (2.5 μM) in WT (n=5) or NL3-KO (n=3) D1-MSNs (G), or during bath application of the CB1 agonist WIN55, 212 (1 μM) in WT (n=7) or NL3-KO (n=8) D1-MSNs (H). (I) Illustration of the protocol used to measure the inhibition/excitation ratio. (J and K) Representative traces of evoked AMPAR- and GABAR-mediated currents in WT or NL3-KO cells (top), amplitudes of AMPAR and GABAR currents (lower left), and average inhibition/excitation ratio (lower right) from WT (n=11) or NL3-KO (n=9) D1-MSNs (J), and WT (n=8) or NL3-KO (n=7) D2-MSNs (K). Data are means ± SEM; *significant difference between groups (ANOVA). Also see Figures S5, S6 & S7.

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