Projection-specific deficits in synaptic transmission in adult Sapap3-knockout mice

Increased grooming in Sapap3-KO mice without skin lesions is age independent.

“Increased grooming in Sapap3-KO mice without skin lesions was age independent.”

• Also stated in: § Increased grooming in young adult · hedged

  “Our experiments suggest that excessive grooming occurs in Sapap3-KO mice independent of age.”

• Also stated in: § DISCUSSION · boosted

  “We show that Sapap3-KO mice, a mouse model widely used to study OCD, groomed excessively already in early adulthood, without significant increase of grooming phenotype between 7 weeks and 6 months of age.”

Hypolocomotion, i.e., slowness of movement, is consistently observed in Sapap3-KO mice.

“Hypolocomotion, i.e., slowness of movement, was consistently observed in Sapap3-KO mice.”

• Also stated in: § Increased grooming in young adult · plain

  “We conclude that grooming does not fully account for hypolocomotion, but that Sapap3-KO mice exhibit a general slowness of forward movements.”

Decreased synaptic strength at the input from the primary motor cortex in Sapap3-KO mice is evident by reduction in AMPA receptor function.

“Decreased synaptic strength was evident at the input from the primary motor cortex by reduction in AMPA receptor function.”

• Also stated in: § Increased grooming in young adult · hedged

  “These investigations imply that a reduction of AMPAR currents underlies the reduced A/N ratio at M1/M2-to-DLS synapses, reflecting reduced synaptic strength.”

Synaptic deficits in Sapap3-KO mice involve projections from the motor cortices and cingulate cortex to the dorsolateral and dorsomedial striatum.

“Synaptic deficits persisted in adulthood and involved the projections from the motor cortices and cingulate cortex to the dorsolateral and dorsomedial striatum.”

• Also stated in: § Increased grooming in young adult · plain

  “At synapses from M1/M2 to dorsolateral D1-and D2-MSNs we found significantly decreased A/ N ratios in Sapap3-KO mice (Fig. 3d).”

• Also stated in: § Increased grooming in young adult · plain

  “Decreased A/N ratios were also evident at Cg1/M2-to-DMS synapses, in D1-and D2-MSNs of Sapap3-KO mice (Fig. 3h), but not at synapses of OFC-to-central striatum D1-and D2-MSNs (Fig. 3l).”

Synaptic deficits in Sapap3-KO mice persist in adulthood.

“Synaptic deficits persisted in adulthood and involved the projections from the motor cortices and cingulate cortex to the dorsolateral and dorsomedial striatum.”

• Also stated in: § Increased grooming in young adult · boosted

  “This showed that synaptic deficits of 3 weeks old mice [20] persist to adulthood.”

• cites [20] Wan Y, Feng G, Calakos N. Sapap3 deletion causes mGluR5-dependent silencing of AMPAR synapses. J Neurosci. 2011;31:16685–91. sourceNot checked yet

  Not checked in the free run.

Overall Sapap3-KO mice showed significantly elevated grooming time compared to WT.

“Overall Sapap3-KO showed significantly elevated grooming time (p = 5 × 10 -5 , Mann-Whitney comparison; mean grooming time (within a 5min test period) averaged across tested age range (±SD): 30 s ± 39 for WT and 95 s ± 74 for KO, N = 23 WT and 39 KO).”

mEPSC amplitude did not differ between genotypes in dorsal striatum PV+ cells.

“Neither mEPSC amplitude nor frequency differed between genotypes (Fig. 1i-k).”

There is no correlation between age and time spent grooming for Sapap3-KO mice.

“However, there was no correlation between age and time spent grooming (Fig. 1b) or age and number of grooming bouts (Fig. 1c) for the Sapap3-KO.”

No genotype differences were evident in cortical areas M1 and S1 for PV+ cell counts.

“No genotype differences were evident in the cortical areas M1 and S1 (Fig. 1g).”

Post-hoc analysis of striatal PV+ cell counts was not significant.

“Across striatal areas (Fig. 1f) we found a significant main genotype effect in the ANOVA, however, post-hoc analysis was not significant.”

mEPSC frequency did not differ between genotypes in dorsal striatum PV+ cells.

“Neither mEPSC amplitude nor frequency differed between genotypes (Fig. 1i-k).”

There is no correlation between age and number of grooming bouts for Sapap3-KO mice.

“However, there was no correlation between age and time spent grooming (Fig. 1b) or age and number of grooming bouts (Fig. 1c) for the Sapap3-KO.”

A/N ratios on D1-MSNs were significantly decreased in Sapap3-KO relative to WT littermates.

“A/N ratios on D1-and D2-MSNs were significantly decreased in Sapap3-KO relative to WT littermates (Fig. 2d).”

At synapses from M1/M2 to dorsolateral D1- and D2-MSNs, A/N ratios were significantly decreased in Sapap3-KO mice.

“At synapses from M1/M2 to dorsolateral D1-and D2-MSNs we found significantly decreased A/ N ratios in Sapap3-KO mice (Fig. 3d).”

Decreased A/N ratios were evident at Cg1/M2-to-DMS synapses in D1- and D2-MSNs of Sapap3-KO mice.

“Decreased A/N ratios were also evident at Cg1/M2-to-DMS synapses, in D1-and D2-MSNs of Sapap3-KO mice (Fig. 3h), but not at synapses of OFC-to-central striatum D1-and D2-MSNs (Fig. 3l).”

RI was 1 in D1-MSNs of WT and KO, indicating absence of GluA2-lacking AMPARs.

“The RI was 1 in D1-MSNs of WT and KO, indicating the absence of GluA2-lacking AMPARs (Fig. 2e).”

A/N ratios were not decreased at synapses of OFC-to-central striatum D1- and D2-MSNs in Sapap3-KO mice.

“Decreased A/N ratios were also evident at Cg1/M2-to-DMS synapses, in D1-and D2-MSNs of Sapap3-KO mice (Fig. 3h), but not at synapses of OFC-to-central striatum D1-and D2-MSNs (Fig. 3l).”

There was a small but significant genotype difference in RI in D2-MSNs.

“There was a small but significant genotype difference in D2-MSNs (Fig. 2e), suggesting differences in AMPAR subunit composition at this synapse, with slightly more GluA2-lacking AMRARs in the WT condition.”

A/N ratios on D2-MSNs were significantly decreased in Sapap3-KO relative to WT littermates.

“A/N ratios on D1-and D2-MSNs were significantly decreased in Sapap3-KO relative to WT littermates (Fig. 2d).”

PPR did not differ between genotypes.

“Pairedpulse ratios (PPR), a measure for presynaptic release probability, did not differ between genotypes (Fig. 2f), which is consistent with findings from 3 weeks old mice [20].”

• cites [20] Wan Y, Feng G, Calakos N. Sapap3 deletion causes mGluR5-dependent silencing of AMPAR synapses. J Neurosci. 2011;31:16685–91. sourceNot checked yet

  Not checked in the free run.

There were no genotype differences in PPR at either cortical input.

“There were no genotype differences in RI (Fig. 3e, i, m) and PPR (Fig. 3f, j, n) at either cortical input.”

Frequency of inter-event interval of unitary events was decreased in Sapap3-KO at M1/M2-to-DLS synapses.

“Furthermore, frequency of inter-event interval of unitary events was decreased in Sapap3-KO (Fig. 4d), indicating that lower neurotransmitter release or lower numbers of synapses in the Sapap3-KO mice add to the synaptic weakening from reduced AMPAR currents.”

There were no genotype differences in AMPAR and NMDAR currents at OFC-to-cDS synapses.

“Comparison of absolute amplitudes (Fig. S3) indicated decreased AMPAR currents at Sapap3-KO M1/M2-to-DLS synapses, and no genotype differences in AMPAR and NMDAR currents at OFC-to-cDS synapses.”

M1/M2-to-DLS AMPAR currents were significantly decreased in Sapap3-KO compared to WT littermates.

“Cumulative probability analysis of amplitude revealed that M1/M2-to-DLS AMPAR currents were significantly decreased in Sapap3-KO, compared to WT littermates (Fig. 4c).”

Comparison of absolute amplitudes indicated decreased AMPAR currents at Sapap3-KO M1/M2-to-DLS synapses.

“Comparison of absolute amplitudes (Fig. S3) indicated decreased AMPAR currents at Sapap3-KO M1/M2-to-DLS synapses, and no genotype differences in AMPAR and NMDAR currents at OFC-to-cDS synapses.”

There were no genotype differences in RI at either cortical input.

“There were no genotype differences in RI (Fig. 3e, i, m) and PPR (Fig. 3f, j, n) at either cortical input.”

There were no genotype differences in decay kinetics (Tau w) of NMDAR currents.

“Decay kinetics (weighted tau; Tau w ) of NMDAR currents can give information about relative abundance of the subunits GluN2A and GluN2B, but there were no genotype differences in Tau w (Fig. 4h).”

Velocity of forward movement (not including time immobile) was significantly decreased in Sapap3-KO mice compared to WT littermates.

“Interestingly, velocity of forward movement (not including time immobile) was also significantly decreased in Sapap3-KO mice compared to WT littermates (Fig. 5e), but we found similar numbers of immobility events (Fig. 5f), which suggests that decreased velocity does not arise from an increased drive to stop for grooming.”

Sapap3-KO mice do not have genotype differences in NMDAR function expressed through subunit composition.

“We did not find genotype differences in NMDAR function that are expressed through subunit composition.”

Hypolocomotion did not increase with age in mice tested between 7 and 28 weeks of age.

“Similar to grooming (Fig. 1b, c), hypolocomotion did not increase with age in mice tested between 7 and 28 weeks of age (Fig. S7).”

Sapap3-KO mice spent less time mobile compared to WT littermates.

“Furthermore, Sapap3-KO mice covered less distance during the exploration of a novel arena (Fig. 5c) and spent less time mobile, compared to WT littermates (Fig. 5d), but locomotion and grooming did not correlate (Fig. S6).”

Sapap3-KO mice covered less distance during exploration of a novel arena.

“Furthermore, Sapap3-KO mice covered less distance during the exploration of a novel arena (Fig. 5c) and spent less time mobile, compared to WT littermates (Fig. 5d), but locomotion and grooming did not correlate (Fig. S6).”

There were similar numbers of immobility events in Sapap3-KO and WT littermates.

“Interestingly, velocity of forward movement (not including time immobile) was also significantly decreased in Sapap3-KO mice compared to WT littermates (Fig. 5e), but we found similar numbers of immobility events (Fig. 5f), which suggests that decreased velocity does not arise from an increased drive to stop for grooming.”

Locomotion and grooming did not correlate in Sapap3-KO mice.

“Furthermore, Sapap3-KO mice covered less distance during the exploration of a novel arena (Fig. 5c) and spent less time mobile, compared to WT littermates (Fig. 5d), but locomotion and grooming did not correlate (Fig. S6).”

Frequency of inter-event interval of unitary events was decreased in Sapap3-KO at OFC-to-cDS synapses.

“However, frequency of interevent interval of unitary events was decreased in Sapap3-KO at OFC-to-cDS (Fig. S4c), indicating that synaptic components other than the number of functional AMPAR per synapses weaken OFC-to-cDS control.”

Three syllables (#2, 4, 6) were significantly more frequently used in Sapap3-KO than in WT littermates.

“Three syllables (# 2, 4, 6) were significantly more frequently used in Sapap3-KO than in WT littermates.”

Behavior of Sapap3-KO was composed of more of the slower syllables than the behavior of WT animals.

“Consistent with the lower velocity detected with the conventional locomotion analysis, behavior of Sapap3-KO was composed of more of the slower syllables than the behavior of WT animals.”

At M1/M2 corticostriatal connections, AMPAR transmission is reduced in Sapap3-KO compared to WT mice.

“Using optogenetic projection targeting, we found that the M1/M2 corticostriatal connections are a key locus of the Sapap3-KO synaptic pathology, expressed by reduced AMPAR transmission, reducing the efficacy of synaptic transmission compared to WT mice.”

The present study demonstrates the corticostriatal synaptic deficit in adult mice.

“In the present study we extend the characterization by demonstrating the corticostriatal synaptic deficit in adult mice.”

MoSeq grooming syllables correlated well with manual scoring.

“Nevertheless, our results indicate that MoSeq can be used to quantify grooming, as MoSeq grooming syllables correlated well with manual scoring (Fig. S9).”

At M1/M2 corticostriatal connections, synaptic transmission efficacy is reduced in Sapap3-KO compared to WT mice.

“Using optogenetic projection targeting, we found that the M1/M2 corticostriatal connections are a key locus of the Sapap3-KO synaptic pathology, expressed by reduced AMPAR transmission, reducing the efficacy of synaptic transmission compared to WT mice.”

M1/M2 corticostriatal connections are a key locus of the Sapap3-KO synaptic pathology.

“Using optogenetic projection targeting, we found that the M1/M2 corticostriatal connections are a key locus of the Sapap3-KO synaptic pathology, expressed by reduced AMPAR transmission, reducing the efficacy of synaptic transmission compared to WT mice.”

The OFC-to-DS projection was found unaltered in this study.

“Another projection which is implicated in habit formation is the OFC-to-DS projection [47,48] which we found unaltered.”

The expression in the aM2 was clearly weaker than in the OFC.

“However, the expression in the aM2 was clearly weaker than in the OFC and we recorded cells in the medial part of the central striatum (Fig. S2e), where OFC terminals dominate over anterior secondary motor cortex terminals [22].”

A/N ratios were significantly different between WT and KO mice in D1-MSNs and D2-MSNs at the M1/M2 input.

“AMPAR/NMDAR (A/N) ratios were significantly different between WT and KO mice in D1-MSNs (p = 0.009) and D2-MSNs (p = 0.003) at the M1/M2 input.”

Locomotion phenotypes are evident in Sapap3-KO mice by conventional behavioral analysis and quantification of sub-second behavioral syllables.

“Locomotion phenotypes are evident in Sapap3-KO mice by conventional behavioral analysis and quantification of sub-second behavioral syllables.”

A/N ratios were significantly different between WT and KO mice in D1-MSNs and D2-MSNs at the Cg1/M2 input.

“A/N ratios were significantly different between WT and KO mice in D1-MSNs (p = 0.045) and D2-MSNs (p = 0.005) at the Cg1/ M2 input.”

Corticostriatal synaptic deficits are present in mice at 10-20 weeks of age.

“Corticostriatal synaptic deficits in mice at 10-20 weeks of age.”

Paired-pulse ratios did not differ between WT and KO mice in D1-MSNs and D2-MSNs.

“Paired-pulse ratios (PPR) did not differ between WT and KO mice in D1-MSNs (p = 0.43) and D2-MSNs (p = 0.97).”

A/N ratios were not significantly different between WT and KO mice in D1-MSNs and D2-MSNs at the OFC input.

“A/N ratios were not significantly different between WT and KO mice in D1-MSNs (p = 0.86) and D2-MSNs (p = 0.60) at the OFC input.”

Corticostriatal synaptic deficits are input-specific in Sapap3-KO mice.

“Input specificity of corticostriatal synaptic deficits in Sapap3-KO mice.”

RI did not differ between WT and KO in D1-MSNs and D2-MSNs at the OFC input.

“RI did not differ between WT and KO in D1-MSNs (p = 0.56) and D2-MSNs (p = 0.70) at the OFC input.”

The extent of grooming phenotype remains constant across adulthood.

“In conclusion, we report here behavioral phenotypes that coexist with projection-specific corticostriatal deficits in young adult Sapap3-KO mice and showed that the extent of grooming phenotype remains constant across adulthood.”

RI did not differ between WT and KO in D1-MSNs and D2-MSNs at the Cg1/M2 input.

“RI did not differ between WT and KO in D1-MSNs (p = 0.78) and D2-MSNs (p = 0.84) at the Cg1/M2 input.”

RI did not differ between WT and KO in D1-MSNs and D2-MSNs at the M1/M2 input.

“Rectification index (RI) did not differ between WT and KO in D1-MSNs (p = 0.63) and D2-MSNs (p = 0.38) at the M1/M2 input.”

PPR did not differ between WT and KO mice in D1-MSNs and D2-MSNs at the M1/M2 input.

“Paired-pulse ratios (PPR) did not differ between WT and KO mice in D1-MSNs (p = 0.053) and D2-MSNs (p = 0.67) at the M1/M2 input.”

PPR did not differ between WT and KO mice in D1-MSNs and D2-MSNs at the OFC input.

“PPR did not differ between WT and KO mice in D1-MSNs (p = 0.80) and D2-MSNs (p = 0.084) at the OFC input.”

Behavioral phenotypes coexist with projection-specific corticostriatal deficits in young adult Sapap3-KO mice.

“In conclusion, we report here behavioral phenotypes that coexist with projection-specific corticostriatal deficits in young adult Sapap3-KO mice and showed that the extent of grooming phenotype remains constant across adulthood.”

Excessive grooming is present across age in Sapap3-KO mice.

“Excessive grooming across age in Sapap3-KO mice, but no major genotype effect on PV + cells in the striatum.”

PPR did not differ between WT and KO mice in D1-MSNs and D2-MSNs at the Cg1/M2 input.

“PPR did not differ between WT and KO mice in D1-MSNs (p = 0.14) and D2-MSNs (p = 0.66) at the Cg1/M2 input.”

AMPAR/NMDAR ratios were significantly different between WT and KO mice in D1-MSNs and D2-MSNs.

“AMPAR/NMDAR (A/N) ratios were significantly different between WT and KO mice in D1-MSNs (p = 0.015) and D2-MSNs (p = 0.003).”

There is no major genotype effect on PV+ cells in the striatum.

“Excessive grooming across age in Sapap3-KO mice, but no major genotype effect on PV + cells in the striatum.”

93.8% of all syllable blocks had a duration of less than 1 s.

“In total, 93.8% of all blocks had a duration of less than 1 s.”

Three syllables were significantly used more by Sapap3-KO compared to WT mice.

“Three syllables were significantly used more by Sapap3-KO, compared to WT mice.”

Deficits in corticostriatal control provide a link to the pathophysiology of OCD.

“Deficits in corticostriatal control provide a link to the pathophysiology of OCD.”

• cites [1] Gillan CM, Robbins TW. Goal-directed learning and obsessive-compulsive disorder. Philos Trans R Soc Lond B Biol Sci. 2014;369:1655. sourceNot checked yet

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• cites [2] Graybiel AM, Rauch SL. Toward a neurobiology of obsessive-compulsive disorder. Neuron. 2000;28:343–7. sourceNot checked yet

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Construct validity for the Sapap3-KO OCD model is supported by more frequent SAPAP3 mutations in OCD patients than in healthy controls.

“Construct validity for this disease model is supported by more frequent SAPAP3 mutations in OCD patients than in healthy controls [7].”

• cites [7] Zuchner S, Wendland JR, Ashley-Koch AE, Collins AL, Tran-Viet KN, Quinn K, et al. Multiple rare SAPAP3 missense variants in trichotillomania and OCD. Mol Psychiatry. 2009;14:6–9. sourceNot checked yet

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Sapap3-KO mice exhibit excessive grooming.

“Such phenotypes are excessive grooming [9][10][11], anxiety-like behavior [9,12], behavioral inflexibility [13,14], and deficits in the balance of habitual and goal-directed behavior [15,16].”

• cites [9] Welch JM, Lu J, Rodriguiz RM, Trotta NC, Peca J, Ding JD, et al. Cortico-striatal synaptic defects and OCD-like behaviours in Sapap3-mutant mice. Nature.2007;448:894–900. sourceNot checked yet

  Not checked in the free run.

• cites [10] van den Boom BJG, Pavlidi P, Wolf CJH, Mooij AH, Willuhn I. Automated classification of self-grooming in mice using open-source software. J Neurosci Methods. 2017;289:48–56. sourceNot checked yet

  Not checked in the free run.

• cites [11] Burguiere E, Monteiro P, Feng G, Graybiel AM. Optogenetic stimulation of lateral orbitofronto-striatal pathway suppresses compulsive behaviors. Science 2013;340:1243–6. sourceNot checked yet

  Not checked in the free run.

Compulsive-like motor phenotypes of Sapap3-KO mice are well-suited to preclinically investigate OCD.

“OCD also manifests with intrusive, distressing thoughts which obviously escape investigation in mice, but compulsive-like motor phenotypes of Sapap3-KO mice are well-suited to preclinically investigate OCD.”

Pathological body care such as nail biting seems to be associated with SAPAP3 mutations.

“Particularly pathological body care such as nail biting seems to be associated with SAPAP3 mutations [8].”

• cites [8] Bienvenu OJ, Wang Y, Shugart YY, Welch JM, Grados MA, Fyer AJ, et al. Sapap3 and pathological grooming in humans: results from the OCD collaborative genetics study. Am J Med Genet B Neuropsychiatr Genet. 2009;150B:710–20. sourceNot checked yet

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Sapap3-KO mice exhibit anxiety-like behavior.

“Such phenotypes are excessive grooming [9][10][11], anxiety-like behavior [9,12], behavioral inflexibility [13,14], and deficits in the balance of habitual and goal-directed behavior [15,16].”

• cites [9] Welch JM, Lu J, Rodriguiz RM, Trotta NC, Peca J, Ding JD, et al. Cortico-striatal synaptic defects and OCD-like behaviours in Sapap3-mutant mice. Nature.2007;448:894–900. sourceNot checked yet

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• cites [12] Ade KK, Wan Y, Hamann HC, O’Hare JK, Guo W, Quian A, et al. Increased metabotropic glutamate receptor 5 signaling underlies obsessive-compulsive disorder-like behavioral and striatal circuit abnormalities in mice. Biol Psychiatry. 2016;80:522–33. sourceNot checked yet

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Sapap3-KO mice exhibit deficits in the balance of habitual and goal-directed behavior.

“Such phenotypes are excessive grooming [9][10][11], anxiety-like behavior [9,12], behavioral inflexibility [13,14], and deficits in the balance of habitual and goal-directed behavior [15,16].”

• cites [15] Hadjas LC, Luscher C, Simmler LD. Aberrant habit formation in the Sapap3-knockout mouse model of obsessive-compulsive disorder. Sci Rep. 2019;9:12061. sourceNot checked yet

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• cites [16] Ehmer I, Feenstra M, Willuhn I, Denys D. Instrumental learning in a mouse model for obsessive-compulsive disorder: impaired habit formation in Sapap3 mutants. Neurobiol Learn Mem. 2020;168:107162. sourceNot checked yet

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Studies that have used Sapap3-KO mice to test treatment approaches for OCD remain limited.

“Despite construct, face and predictive validity of the Sapap3-KO mouse model, studies that have used Sapap3-KO mice to test treatment approaches for OCD remain limited [17,18].”

• cites [17] Pinhal CM, van den Boom BJG, Santana-Kragelund F, Fellinger L, Bech P, Hamelink R, et al. Differential effects of deep brain stimulation of the internal capsule and the striatum on excessive grooming in Sapap3 mutant mice. Biol Psychiatry. 2018;84:917–25. sourceNot checked yet

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• cites [18] Todorov G, Mayilvahanan K, Ashurov D, Cunha C. Amelioration of obsessive-compulsive disorder in three mouse models treated with one epigenetic drug: unraveling the underlying mechanism. Sci Rep. 2019;9:8741. sourceNot checked yet

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Sapap3-KO mice exhibit behavioral inflexibility.

“Such phenotypes are excessive grooming [9][10][11], anxiety-like behavior [9,12], behavioral inflexibility [13,14], and deficits in the balance of habitual and goal-directed behavior [15,16].”

• cites [13] Manning EE, Dombrovski AY, Torregrossa MM, Ahmari SE. Impaired instrumental reversal learning is associated with increased medial prefrontal cortex activity in Sapap3 knockout mouse model of compulsive behavior. Neuropsychopharmacology. 2019;44:1494–504. sourceNot checked yet

  Not checked in the free run.

• cites [14] van den Boom BJG, Mooij AH, Miseviciute I, Denys D, Willuhn I. Behavioral flexibility in a mouse model for obsessive-compulsive disorder: Impaired Pavlovian reversal learning in SAPAP3 mutants. Genes Brain Behav. 2019;18:e12557. sourceNot checked yet

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OCD-like behavioral phenotypes in Sapap3-KO mice have mostly been studied in mice of about 6 months or older, shortly before the onset of lesions.

“Skin lesions arising from excessive grooming typically appear in Sapap3-KO mice at advanced age, therefore, OCD-like behavioral phenotypes have mostly been studied in mice of about 6 months or older, shortly before the onset of lesions [9,10,13,14,17].”

• cites [9] Welch JM, Lu J, Rodriguiz RM, Trotta NC, Peca J, Ding JD, et al. Cortico-striatal synaptic defects and OCD-like behaviours in Sapap3-mutant mice. Nature.2007;448:894–900. sourceNot checked yet

  Not checked in the free run.

• cites [10] van den Boom BJG, Pavlidi P, Wolf CJH, Mooij AH, Willuhn I. Automated classification of self-grooming in mice using open-source software. J Neurosci Methods. 2017;289:48–56. sourceNot checked yet

  Not checked in the free run.

• cites [13] Manning EE, Dombrovski AY, Torregrossa MM, Ahmari SE. Impaired instrumental reversal learning is associated with increased medial prefrontal cortex activity in Sapap3 knockout mouse model of compulsive behavior. Neuropsychopharmacology. 2019;44:1494–504. sourceNot checked yet

  Not checked in the free run.

• cites [14] van den Boom BJG, Mooij AH, Miseviciute I, Denys D, Willuhn I. Behavioral flexibility in a mouse model for obsessive-compulsive disorder: Impaired Pavlovian reversal learning in SAPAP3 mutants. Genes Brain Behav. 2019;18:e12557. sourceNot checked yet

  Not checked in the free run.

• cites [17] Pinhal CM, van den Boom BJG, Santana-Kragelund F, Fellinger L, Bech P, Hamelink R, et al. Differential effects of deep brain stimulation of the internal capsule and the striatum on excessive grooming in Sapap3 mutant mice. Biol Psychiatry. 2018;84:917–25. sourceNot checked yet

  Not checked in the free run.

Skin lesions arising from excessive grooming typically appear in Sapap3-KO mice at advanced age.

“Skin lesions arising from excessive grooming typically appear in Sapap3-KO mice at advanced age, therefore, OCD-like behavioral phenotypes have mostly been studied in mice of about 6 months or older, shortly before the onset of lesions [9,10,13,14,17].”

• cites [9] Welch JM, Lu J, Rodriguiz RM, Trotta NC, Peca J, Ding JD, et al. Cortico-striatal synaptic defects and OCD-like behaviours in Sapap3-mutant mice. Nature.2007;448:894–900. sourceNot checked yet

  Not checked in the free run.

• cites [10] van den Boom BJG, Pavlidi P, Wolf CJH, Mooij AH, Willuhn I. Automated classification of self-grooming in mice using open-source software. J Neurosci Methods. 2017;289:48–56. sourceNot checked yet

  Not checked in the free run.

• cites [13] Manning EE, Dombrovski AY, Torregrossa MM, Ahmari SE. Impaired instrumental reversal learning is associated with increased medial prefrontal cortex activity in Sapap3 knockout mouse model of compulsive behavior. Neuropsychopharmacology. 2019;44:1494–504. sourceNot checked yet

  Not checked in the free run.

• cites [14] van den Boom BJG, Mooij AH, Miseviciute I, Denys D, Willuhn I. Behavioral flexibility in a mouse model for obsessive-compulsive disorder: Impaired Pavlovian reversal learning in SAPAP3 mutants. Genes Brain Behav. 2019;18:e12557. sourceNot checked yet

  Not checked in the free run.

• cites [17] Pinhal CM, van den Boom BJG, Santana-Kragelund F, Fellinger L, Bech P, Hamelink R, et al. Differential effects of deep brain stimulation of the internal capsule and the striatum on excessive grooming in Sapap3 mutant mice. Biol Psychiatry. 2018;84:917–25. sourceNot checked yet

  Not checked in the free run.

The trend in striatal PV+ cell counts is consistent with reduced striatal PV+ cell abundance previously reported in 3-10 months old Sapap3-KO mice.

“This trend is consistent with reduced striatal PV + cell abundance previously reported in 3-10 months old Sapap3-KO mice [11].”

• cites [11] Burguiere E, Monteiro P, Feng G, Graybiel AM. Optogenetic stimulation of lateral orbitofronto-striatal pathway suppresses compulsive behaviors. Science 2013;340:1243–6. sourceNot checked yet

  Not checked in the free run.

Variable expression of Sapap3 during postnatal synapse maturation has been reported.

“Clearly, the age mismatch for behavioral and neuronal phenotypes in the Sapap3-KO model needs to be addressed, particularly given that variable expression of Sapap3 during postnatal synapse maturation has been reported [24].”

• cites [24] Kindler S, Rehbein M, Classen B, Richter D, Bockers TM. Distinct spatiotemporal expression of SAPAP transcripts in the developing rat brain: a novel dendritically localized mRNA. Brain Res Mol Brain Res. 2004;126:14–21. sourceNot checked yet

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Excessive grooming occurs in Sapap3-KO mice independent of age.

“Our experiments suggest that excessive grooming occurs in Sapap3-KO mice independent of age.”

There are inputs (i.e., thalamic) that seem unaffected by the KO of Sapap3.

“Furthermore, for a thorough understanding of the pathologies in the Sapap3-KO mice, refinement of circuit specificity of synaptic dysfunction is necessary, as there are inputs (i.e., thalamic) that seem unaffected by the KO of Sapap3 [21].”

• cites [21] Wan Y, Ade KK, Caffall Z, Ilcim Ozlu M, Eroglu C, Feng G, et al. Circuit-selective striatal synaptic dysfunction in the Sapap3 knockout mouse model of obsessive-compulsive disorder. Biol Psychiatry. 2014;75:623–30. sourceNot checked yet

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Corticostriatal synaptic deficits in Sapap3-KO mice have been assessed in mice aged 3-8 weeks.

“In contrast, corticostriatal synaptic deficits have been assessed in mice aged 3-8 weeks [19][20][21][22][23].”

• cites [19] Chen M, Wan Y, Ade K, Ting J, Feng G, Calakos N. Sapap3 deletion anomalously activates short-term endocannabinoid-mediated synaptic plasticity. J Neurosci. 2011;31:9563–73. sourceNot checked yet

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• cites [20] Wan Y, Feng G, Calakos N. Sapap3 deletion causes mGluR5-dependent silencing of AMPAR synapses. J Neurosci. 2011;31:16685–91. sourceNot checked yet

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• cites [21] Wan Y, Ade KK, Caffall Z, Ilcim Ozlu M, Eroglu C, Feng G, et al. Circuit-selective striatal synaptic dysfunction in the Sapap3 knockout mouse model of obsessive-compulsive disorder. Biol Psychiatry. 2014;75:623–30. sourceNot checked yet

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Synaptic deficits of 3 weeks old mice persist to adulthood.

“This showed that synaptic deficits of 3 weeks old mice [20] persist to adulthood.”

• cites [20] Wan Y, Feng G, Calakos N. Sapap3 deletion causes mGluR5-dependent silencing of AMPAR synapses. J Neurosci. 2011;31:16685–91. sourceNot checked yet

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Decreased A/N ratios of M1/M2-to-DLS synapses indicate decreased synaptic strength.

“The decreased A/N ratios of M1/M2-to-DLS synapses indicate decreased synaptic strength, since typically it is the number of functional AMPAR, not NMDAR, that changes in the synaptic composition when synapses modify their strength [23].”

• cites [23] Luscher C, Malenka RC. NMDA receptor-dependent long-term potentiation and long-term depression (LTP/LTD). Cold Spring Harb Perspect Biol. 2012;4:a005710. sourceNot checked yet

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Interpretations from absolute currents have to be made with caution since the experimental design did not control for ChR2 infection and stimulation light intensity.

“However, interpretations from absolute currents have to be made with caution since our experimental design did not control for ChR2 infection and stimulation light intensity.”

Lower neurotransmitter release or lower numbers of synapses in Sapap3-KO mice add to the synaptic weakening from reduced AMPAR currents.

“Furthermore, frequency of inter-event interval of unitary events was decreased in Sapap3-KO (Fig. 4d), indicating that lower neurotransmitter release or lower numbers of synapses in the Sapap3-KO mice add to the synaptic weakening from reduced AMPAR currents.”

Young adult Sapap3-KO mice have lower locomotor activity in an open-field arena.

“Previously we made the observation that young adult Sapap3-KO mice have lower locomotor activity in an open-field arena [15].”

• cites [15] Hadjas LC, Luscher C, Simmler LD. Aberrant habit formation in the Sapap3-knockout mouse model of obsessive-compulsive disorder. Sci Rep. 2019;9:12061. sourceNot checked yet

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Decreased velocity does not arise from an increased drive to stop for grooming.

“Interestingly, velocity of forward movement (not including time immobile) was also significantly decreased in Sapap3-KO mice compared to WT littermates (Fig. 5e), but we found similar numbers of immobility events (Fig. 5f), which suggests that decreased velocity does not arise from an increased drive to stop for grooming.”

Synaptic components other than the number of functional AMPAR per synapse weaken OFC-to-cDS control in Sapap3-KO.

“However, frequency of interevent interval of unitary events was decreased in Sapap3-KO at OFC-to-cDS (Fig. S4c), indicating that synaptic components other than the number of functional AMPAR per synapses weaken OFC-to-cDS control.”

Excessive grooming in Sapap3-KO mice has been consistently observed across experimental conditions, including testing during light cycle and during the dark period.

“In contrast, excessive grooming in Sapap3-KO mice has been consistently observed across experimental conditions, including testing during light cycle [9,12,13] and during the dark period [9,10,17,33], which shows the robustness of the phenotype.”

• cites [9] Welch JM, Lu J, Rodriguiz RM, Trotta NC, Peca J, Ding JD, et al. Cortico-striatal synaptic defects and OCD-like behaviours in Sapap3-mutant mice. Nature.2007;448:894–900. sourceNot checked yet

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• cites [12] Ade KK, Wan Y, Hamann HC, O’Hare JK, Guo W, Quian A, et al. Increased metabotropic glutamate receptor 5 signaling underlies obsessive-compulsive disorder-like behavioral and striatal circuit abnormalities in mice. Biol Psychiatry. 2016;80:522–33. sourceNot checked yet

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• cites [13] Manning EE, Dombrovski AY, Torregrossa MM, Ahmari SE. Impaired instrumental reversal learning is associated with increased medial prefrontal cortex activity in Sapap3 knockout mouse model of compulsive behavior. Neuropsychopharmacology. 2019;44:1494–504. sourceNot checked yet

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• cites [10] van den Boom BJG, Pavlidi P, Wolf CJH, Mooij AH, Willuhn I. Automated classification of self-grooming in mice using open-source software. J Neurosci Methods. 2017;289:48–56. sourceNot checked yet

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• cites [17] Pinhal CM, van den Boom BJG, Santana-Kragelund F, Fellinger L, Bech P, Hamelink R, et al. Differential effects of deep brain stimulation of the internal capsule and the striatum on excessive grooming in Sapap3 mutant mice. Biol Psychiatry. 2018;84:917–25. sourceNot checked yet

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• cites [33] Ehmer I, Crown L, van Leeuwen W, Feenstra M, Willuhn I, Denys D. Evidence for distinct forms of compulsivity in the SAPAP3 mutant-mouse model for obsessive-compulsive disorder. eNeuro. 2020;7:ENEURO.0245-19.2020. sourceNot checked yet

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Constitutive KO of Sapap3 causes synaptic deficits already in preadolescence.

“However, since constitutive KO of Sapap3 causes synaptic deficits already in preadolescence [9,[19][20][21], we hypothesized that excessive grooming behavior must manifest early in life, which we confirmed here.”

• cites [9] Welch JM, Lu J, Rodriguiz RM, Trotta NC, Peca J, Ding JD, et al. Cortico-striatal synaptic defects and OCD-like behaviours in Sapap3-mutant mice. Nature.2007;448:894–900. sourceNot checked yet

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• cites [19] Chen M, Wan Y, Ade K, Ting J, Feng G, Calakos N. Sapap3 deletion anomalously activates short-term endocannabinoid-mediated synaptic plasticity. J Neurosci. 2011;31:9563–73. sourceNot checked yet

  Not checked in the free run.

• cites [20] Wan Y, Feng G, Calakos N. Sapap3 deletion causes mGluR5-dependent silencing of AMPAR synapses. J Neurosci. 2011;31:16685–91. sourceNot checked yet

  Not checked in the free run.

Skin lesions might be a consequence of life-long excessive grooming, rather than of a sudden onset or worsening of the grooming phenotype at advanced age.

“Skin lesions might thus be a consequence of life-long excessive grooming, rather than of a sudden onset or worsening of the grooming phenotype at advanced age.”

Grooming in WT mice is highly sensitive to experimental conditions including stressful situations such as a novel environment or exposure to bright light.

“Grooming in WT mice is highly sensitive to experimental conditions including stressful situations such as a novel environment or exposure to bright light [32].”

• cites [32] Kalueff AV, Stewart AM, Song C, Berridge KC, Graybiel AM, Fentress JC. Neurobiology of rodent self-grooming and its value for translational neuroscience. Nat Rev Neurosci. 2016;17:45–59. sourceNot checked yet

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Behavioral findings revealed movement abnormalities caused by the absence of SAPAP3.

“Overall, our behavioral findings revealed movement abnormalities caused by the absence of SAPAP3, which might be caused by a disturbance in motor circuits, evident here by decreased strength at M1/M2to-DLS synapses.”

No alterations in striatal DA concentrations in Sapap3-KO mice have been observed in tissue homogenates.

“Striatal DA levels affect vigor of movements [34], but no alterations in striatal DA concentrations in Sapap3-KO mice have been observed in tissue homogenates [35].”

• cites [35] Wood J, LaPalombara Z, Ahmari SE. Monoamine abnormalities in the SAPAP3 knockout model of obsessive-compulsive disorder-related behaviour. Philos Trans R Soc Lond B Biol Sci. 2018;373:20170023. sourceNot checked yet

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The study did not establish a direct causal link between synaptic deficits and behavioral phenotypes.

“A limitation of our study is that we did not establish a direct causal link between synaptic deficits and behavioral phenotypes (Supplementary text; Fig. S10).”

Neurological soft signs are more prevalent in OCD patients than in healthy controls.

“Primary dysfunctions are indicated by neurological soft signs (deviations from average performance in tests assessing coordination, sensory integration and involuntary movements) that are more prevalent in OCD patients than in healthy controls [37][38][39][40].”

• cites [37][38][39][40]Not checked yet

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Altered synaptic properties at the Cg1/M2-to-DMS input could underlie OCD-like behavioral inflexibility leading to aberrant habit formation.

“Altered synaptic properties at this input could underlie OCD-like behavioral inflexibility leading to aberrant habit formation [15] since lesioning the M2 impairs goal-directed behavior [46].”

• cites [15] Hadjas LC, Luscher C, Simmler LD. Aberrant habit formation in the Sapap3-knockout mouse model of obsessive-compulsive disorder. Sci Rep. 2019;9:12061. sourceNot checked yet

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• cites [46] Gremel CM, Costa RM. Premotor cortex is critical for goal-directed actions. Front Comput Neurosci. 2013;7:110. sourceNot checked yet

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The significance of the comparison of absolute amplitudes is limited by the lack of control for the number of stimulated synapses.

“The significance of this comparison is limited by the lack of control for the number of stimulated synapses.”

In the medial part of the central striatum, OFC terminals dominate over anterior secondary motor cortex terminals.

“However, the expression in the aM2 was clearly weaker than in the OFC and we recorded cells in the medial part of the central striatum (Fig. S2e), where OFC terminals dominate over anterior secondary motor cortex terminals [22].”

• cites [22] Corbit VL, Manning EE, Gittis AH, Ahmari SE. Strengthened inputs from secondary motor cortex to striatum in a mouse model of compulsive behavior. J Neurosci. 2019;39:2965–75. sourceNot checked yet

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Such a confound cannot be fully excluded in our study.

“Such a confound cannot be fully excluded in our study.”

Opposing roles of mouse medial/ventral and lateral OFC have been shown.

“Furthermore, opposing roles of mouse medial/ventral and lateral OFC have been shown.”