Strengthened Inputs from Secondary Motor Cortex to Striatum in a Mouse Model of Compulsive Behavior

“…(TS) (Leckman et al., 2010), grooming disorders (e.g., skin-picking, trichotillomania) (Chamberlain et al., 2009; Flessner et al., 2012), and obsessive-compulsive disorder (OCD) (Karno et al., 1988; Ayuso-Mateos, 2006; Menzies et al., 2008), little is known about their underlying neural mechanisms.”

“, 2013), studies demonstrating aberrant activity in corticostriatal circuits in OCD patients (Chamberlain et al., 2008; Menzies et al., 2008; Harrison et al., 2009; Del Casale et al., 2011; Figee et al., 2013; Nakamae et al., 2014) suggest that cortical regions may be useful targets for noninvasive neurostimulation vi…”

“…both hyperactivity in the striatum (caudate head) and increased corticostriatal functional connectivity at baseline and when symptoms are expressed (Rauch et al., 1994, 1997; Saxena et al., 1998; Maia et al., 2008; Menzies et al., 2008; Chamberlain et al., 2009; Harrison et al., 2009; Leckman et…”

“Although striatal deep brain stimulation has reported efficacy in OCD (Greenberg et al., 2010; Figee et al., 2013), studies demonstrating aberrant activity in corticostriatal circuits in OCD patients (Chamberlain et al., 2008; Menzies et al., 2008; Harrison et al., 2009; Del Casale et al., 2011;…”

“This suggests that pathologically enhanced plasticity mechanisms may be present at M2 corticostriatal synapses, which could contribute to abnormal learning phenotypes (Burguière et al., 2013) and persistence of compulsive grooming behavior (Welch et al., 2007) observed in Sapap3-KOs.”

“We first focused on LOFC projections, as this region has been implicated in compulsive behavior (Saxena et al., 1998; Maltby et al., 2005; Chamberlain et al., 2008; Burguière et al., 2013) and related behavioral constructs, including action selection, cognitive flexibility, and reversal learning…”

“Similar to SMA/pre-SMA in humans and primates (Isoda and Hikosaka, 2007), M2 in mice is involved in behavioral planning and movement preparation during behavioral selection (Gremel and Costa, 2013; Guo et al., 2014; Cao et al., 2015; Li et al., 2015; Rothwell et al., 2015; Barthas and Kwan, 2017).”

“Because M2 is associated with planning of sequenced movements (Sul et al., 2011; Cao et al., 2015; Li et al., 2015) and is thought to be homologous to pre-SMA (which is implicated in OCD) (Mantovani et al.”

“…functional connectivity at baseline and when symptoms are expressed (Rauch et al., 1994, 1997; Saxena et al., 1998; Maia et al., 2008; Menzies et al., 2008; Chamberlain et al., 2009; Harrison et al., 2009; Leckman et al., 2010; Del Casale et al., 2011; Denys et al., 2013; Figee et al., 2013).”

“We first focused on LOFC projections, as this region has been implicated in compulsive behavior (Saxena et al., 1998; Maltby et al., 2005; Chamberlain et al., 2008; Burguière et al., 2013) and related behavioral constructs, including action selection, cognitive flexibility, and reversal learning…”

“…functional connectivity at baseline and when symptoms are expressed (Rauch et al., 1994, 1997; Saxena et al., 1998; Maia et al., 2008; Menzies et al., 2008; Chamberlain et al., 2009; Harrison et al., 2009; Leckman et al., 2010; Del Casale et al., 2011; Denys et al., 2013; Figee et al., 2013).”

“In addition, SPN hyperactivity has been described during compulsive grooming as well as at baseline (Burguière et al., 2013).”

“Similar to SMA/pre-SMA in humans and primates (Isoda and Hikosaka, 2007), M2 in mice is involved in behavioral planning and movement preparation during behavioral selection (Gremel and Costa, 2013; Guo et al.”

“In addition, pre-SMA activity has been associated with the “urge” to move in both TS patients and healthy subjects (Bohlhalter et al., 2006; Neuner et al., 2014).”

“…mice (Burguière et al., 2013) reflected differences in excitatory inputs to central striatum, which could augment SPN activity directly or via decreasing feedforward inhibition mediated by FSIs (Parthasarathy and Graybiel, 1997; Mallet et al., 2005; Gittis et al., 2010, 2011; Berke, 2011).”

“Generally, LOFC is known to support functions essential for flexible selection of be- haviors, including reversal learning (Schoenbaum et al., 2002; Bohn et al., 2003; Sul et al., 2010).”

“…mice (Burguière et al., 2013) reflected differences in excitatory inputs to central striatum, which could augment SPN activity directly or via decreasing feedforward inhibition mediated by FSIs (Parthasarathy and Graybiel, 1997; Mallet et al., 2005; Gittis et al., 2010, 2011; Berke, 2011).”

“…and increased corticostriatal functional connectivity at baseline and when symptoms are expressed (Rauch et al., 1994, 1997; Saxena et al., 1998; Maia et al., 2008; Menzies et al., 2008; Chamberlain et al., 2009; Harrison et al., 2009; Leckman et al., 2010; Del Casale et al., 2011; Denys et…”

“In Sapap3-KO mice, the shift toward M2 motor control and away from LOFC associative control of central striatum may particularly interfere with flexible switching between habitual and goaldirected behaviors, which has been implicated in OCD symptomatology (Gillan and Robbins, 2014).”

“Consistent with this idea, orbitofrontal cortex (OFC) and presupplementary motor area (pre-SMA), which are hyperactive in OCD patients (Maltby et al., 2005; Yücel et al., 2007; Leckman et al., 2010; Del Casale et al., 2011; de Wit et al., 2012; Grützmann et al., 2016), have been identified as promising targets for rTM…”

“Generally, LOFC is known to support functions essential for flexible selection of be- haviors, including reversal learning (Schoenbaum et al., 2002; Bohn et al., 2003; Sul et al., 2010).”

“…movements (Sul et al., 2011; Cao et al., 2015; Li et al., 2015) and is thought to be homologous to pre-SMA (which is implicated in OCD) (Mantovani et al., 2010; de Wit et al., 2012), we investigated whether M2 was a source of upregulated excitatory drive to central striatum in Sapap3-KOs (Fig.”

“…both hyperactivity in the striatum (caudate head) and increased corticostriatal functional connectivity at baseline and when symptoms are expressed (Rauch et al., 1994, 1997; Saxena et al., 1998; Maia et al., 2008; Menzies et al., 2008; Chamberlain et al., 2009; Harrison et al., 2009; Leckman et…”

“…sequenced movements (Sul et al., 2011; Cao et al., 2015; Li et al., 2015) and is thought to be homologous to pre-SMA (which is implicated in OCD) (Mantovani et al., 2010; de Wit et al., 2012), we investigated whether M2 was a source of upregulated excitatory drive to central striatum in…”

“…1998; Maltby et al., 2005; Chamberlain et al., 2008; Burguière et al., 2013) and related behavioral constructs, including action selection, cognitive flexibility, and reversal learning (Schoenbaum et al., 2002; Takahashi et al., 2009; Sul et al., 2010; Dalton et al., 2016; Gremel et al., 2016).”

“Similar to SMA/pre-SMA in humans and primates (Isoda and Hikosaka, 2007), M2 in mice is involved in behavioral planning and movement preparation during behavioral selection (Gremel and Costa, 2013; Guo et al., 2014; Cao et al., 2015; Li et al., 2015; Rothwell et al., 2015; Barthas and Kwan, 2017).”

“…with this idea, orbitofrontal cortex (OFC) and presupplementary motor area (pre-SMA), which are hyperactive in OCD patients (Maltby et al., 2005; Yücel et al., 2007; Leckman et al., 2010; Del Casale et al., 2011; de Wit et al., 2012; Grützmann et al., 2016), have been identified as promising…”

“Consistent with this, Gremel et al. (2016) reported that activity in LOFC corticostriatal projections is essential for updating action-value associations (Gremel et al., 2016).”

“Because M2 is associated with planning of sequenced movements (Sul et al., 2011; Cao et al., 2015; Li et al., 2015) and is thought to be homologous to pre-SMA (which is implicated in OCD) (Mantovani et al.”

“…(OFC) and presupplementary motor area (pre-SMA), which are hyperactive in OCD patients (Maltby et al., 2005; Yücel et al., 2007; Leckman et al., 2010; Del Casale et al., 2011; de Wit et al., 2012; Grützmann et al., 2016), have been identified as promising targets for rTMS (Berlim et al., 2013).”

“…mice (Burguière et al., 2013) reflected differences in excitatory inputs to central striatum, which could augment SPN activity directly or via decreasing feedforward inhibition mediated by FSIs (Parthasarathy and Graybiel, 1997; Mallet et al., 2005; Gittis et al., 2010, 2011; Berke, 2011).”

“Mutations in the Sapap3 gene, which encodes the synapse-associated protein 90/ postsynaptic density-95-associated protein, have been associated with TS, pathological grooming disorders, and OCD in humans (Bienvenu et al., 2009; Züchner et al., 2009; Crane et al., 2011).”

“…mice (Burguière et al., 2013) reflected differences in excitatory inputs to central striatum, which could augment SPN activity directly or via decreasing feedforward inhibition mediated by FSIs (Parthasarathy and Graybiel, 1997; Mallet et al., 2005; Gittis et al., 2010, 2011; Berke, 2011).”

“…prominent, disabling, and notoriously treatment-resistant, symptoms in multiple severe neuropsychiatric disorders, including Tourette syndrome (TS) (Leckman et al., 2010), grooming disorders (e.g., skin-picking, trichotillomania) (Chamberlain et al., 2009; Flessner et al., 2012), and…”

“Mutations in the Sapap3 gene, which encodes the synapse-associated protein 90/ postsynaptic density-95-associated protein, have been associated with TS, pathological grooming disorders, and OCD in humans (Bienvenu et al., 2009; Züchner et al., 2009; Crane et al., 2011).”

“In addition, pre-SMA activity has been associated with the “urge” to move in both TS patients and healthy subjects (Bohlhalter et al., 2006; Neuner et al., 2014).”

“…1998; Maltby et al., 2005; Chamberlain et al., 2008; Burguière et al., 2013) and related behavioral constructs, including action selection, cognitive flexibility, and reversal learning (Schoenbaum et al., 2002; Takahashi et al., 2009; Sul et al., 2010; Dalton et al., 2016; Gremel et al., 2016).”

“For instance, it has been shown that KOs have higher levels of the NR2B subunit in the striatum relative to WTs (Welch et al., 2007), and increased NR2B expression in hippocampus has been associated with enhanced LTP and heightened learning (Cao et al., 2007; Xu et al., 2009).”

“…functional connectivity at baseline and when symptoms are expressed (Rauch et al., 1994, 1997; Saxena et al., 1998; Maia et al., 2008; Menzies et al., 2008; Chamberlain et al., 2009; Harrison et al., 2009; Leckman et al., 2010; Del Casale et al., 2011; Denys et al., 2013; Figee et al., 2013).”

“Although we did not observe a strong laminar pattern, labeled cells were present in layer V, as anticipated based on past work (Levesque et al., 1996).”

“…mice (Burguière et al., 2013) reflected differences in excitatory inputs to central striatum, which could augment SPN activity directly or via decreasing feedforward inhibition mediated by FSIs (Parthasarathy and Graybiel, 1997; Mallet et al., 2005; Gittis et al., 2010, 2011; Berke, 2011).”

“Similar to SMA/pre-SMA in humans and primates (Isoda and Hikosaka, 2007), M2 in mice is involved in behavioral planning and movement preparation during behavioral selection (Gremel and Costa, 2013; Guo et al., 2014; Cao et al., 2015; Li et al., 2015; Rothwell et al., 2015; Barthas and Kwan, 2017).”

“…mice (Burguière et al., 2013) reflected differences in excitatory inputs to central striatum, which could augment SPN activity directly or via decreasing feedforward inhibition mediated by FSIs (Parthasarathy and Graybiel, 1997; Mallet et al., 2005; Gittis et al., 2010, 2011; Berke, 2011).”

“Of particular interest, M2-striatal projections have been shown to be critical for initiation of behavioral sequences (Rothwell et al., 2015).”

“Because FSIs make up only 1% of the striatal cell population (Luk and Sadikot, 2001; Berke, 2011), we used a transgenic fluorescent strategy to target these neurons using acute slice electrophysiology.”

“…neuropsychiatric disorders, including Tourette syndrome (TS) (Leckman et al., 2010), grooming disorders (e.g., skin-picking, trichotillomania) (Chamberlain et al., 2009; Flessner et al., 2012), and obsessive-compulsive disorder (OCD) (Karno et al., 1988; Ayuso-Mateos, 2006; Menzies et al.,…”

“Sapap3-KOs demonstrate ex vivo abnormalities in striatal spiny projection neurons (SPNs), including both increased NMDAreceptor mediated and reduced AMPA-receptor mediated transmission in dorsal striatum (Welch et al., 2007; Wan et al., 2011, 2014; Ade et al., 2016).”

“For instance, it has been shown that KOs have higher levels of the NR2B subunit in the striatum relative to WTs (Welch et al., 2007), and increased NR2B expression in hippocampus has been associated with enhanced LTP and heightened learning (Cao et al., 2007; Xu et al., 2009).”

“…been shown to have alterations in NMDA signaling that can impact synaptic strength, including differential expression of NMDA receptor subunits, increased NMDAmediated currents in dorsolateral striatum, and reduced AMPA/ NMDA ratios in dorsolateral striatum (Welch et al., 2007; Wan et al., 2011).”

“Sapap3-KOs demonstrate ex vivo abnormalities in striatal spiny projection neurons (SPNs), including both increased NMDAreceptor mediated and reduced AMPA-receptor mediated transmission in dorsal striatum (Welch et al., 2007; Wan et al., 2011, 2014; Ade et al., 2016).”

“…reduced activity in LOFC corticostriatal terminals is necessary for habit expression, and habit expression is dependent on endocannabinoid-mediated LTD (Gremel et al., 2016); because both phenomena are enhanced in Sapap3-KOs (Chen et al., 2011), KOs could display enhanced habit expression.”

“…(OFC) and presupplementary motor area (pre-SMA), which are hyperactive in OCD patients (Maltby et al., 2005; Yücel et al., 2007; Leckman et al., 2010; Del Casale et al., 2011; de Wit et al., 2012; Grützmann et al., 2016), have been identified as promising targets for rTMS (Berlim et al., 2013).”

“While FSIs are canonically thought to strongly inhibit SPN firing (Owen et al., 2018), other reports show that FSI firing can potentiate activity in a subset of SPNs (O’Hare et al., 2017).”

“Similar to SMA/pre-SMA in humans and primates (Isoda and Hikosaka, 2007), M2 in mice is involved in behavioral planning and movement preparation during behavioral selection (Gremel and Costa, 2013; Guo et al., 2014; Cao et al., 2015; Li et al., 2015; Rothwell et al., 2015; Barthas and Kwan, 2017).”

“Mutations in the Sapap3 gene, which encodes the synapse-associated protein 90/ postsynaptic density-95-associated protein, have been associated with TS, pathological grooming disorders, and OCD in humans (Bienvenu et al., 2009; Züchner et al., 2009; Crane et al., 2011).”

“Generally, LOFC is known to support functions essential for flexible selection of be- haviors, including reversal learning (Schoenbaum et al., 2002; Bohn et al., 2003; Sul et al., 2010).”

“…including Tourette syndrome (TS) (Leckman et al., 2010), grooming disorders (e.g., skin-picking, trichotillomania) (Chamberlain et al., 2009; Flessner et al., 2012), and obsessive-compulsive disorder (OCD) (Karno et al., 1988; Ayuso-Mateos, 2006; Menzies et al., 2008), little is known about…”

“, 2013), studies demonstrating aberrant activity in corticostriatal circuits in OCD patients (Chamberlain et al., 2008; Menzies et al., 2008; Harrison et al., 2009; Del Casale et al., 2011; Figee et al., 2013; Nakamae et al., 2014) suggest that cortical regions may be useful targets for noninvasive neurostimulation vi…”

“They found that in the SAPAP3 mutant, lOFC input to striatal SPNs was reduced in strength, while M2 input to both SPNs and fast-spiking interneurons (FSIs) in striatum was increased 6-fold, suggesting that it is M2, and not lOFC inputs, that drive compulsive grooming (Corbit et al., 2019).”

“The OFC and M2 can differentially regulate the activity of neurons in specific DS regions in vitro (Corbit et al., 2019), with”

“In another study, striatal medium spiny neurons showed an increased response to the in-vitro optogenetic activation of M2 terminals in the striatum in the Sapap3-KO mice, which suggests a dysregulation of activity in the cortico-striatal circuit [24].”

“Thus, reconsidering cortico-striatal circuitry as a substrate for pathological RBs and taking into account specific indications of an implication of the sensorimotor cortico-striatal loops also in the Sapap3 mice [28, 40], we here raised the question whether compulsive-like self-grooming in Sapap3 mice might be comple…”

“We have shown that M2 connectivity to both central striatal SPNs and fast-spiking interneurons (FSIs), whichstrongly inhibitnearby SPNs 79 – 81 , issigni fi cantlyincreased in KO mice relative to WTs 37 .”

“Past studies 27,47–49 assessed EPSCs in mice at ages when overt behavioral symptoms do not occur.”

“Sap90/postsynaptic density protein-95–associated protein 3 (Sapap3) null mice show compulsive-like grooming and abnormalities in glutamatergic neurotransmission and function (Welch et al., 2007; Burguière et al., 2013; Wan et al., 2014; Corbit et al., 2019).”

“…grooming in the Sapap3 -KO mice is also supported by recent studies describing abnormal synaptic properties of striatal PVI in this animal model 40 as well as by observations in other mouse models suffering from pathological repetitive behaviors where abnormally low PVI density in striatal…”

“Furthermore, in OCD-relevant mouse model, M2 postsynaptic responses in the central striatum were significantly increased, which suggested that strengthened M2-striatal inputs might contribute in striatal hyperactivity and compulsive behaviors, where M2 is homologous to preSMA/SMA in human (63).”

“central striatum [103]  Inhibition of striatal cells increases grooming behavior; in‐ hibition of striatal indirect pathway neurons decreases grooming [105] Striatum  ═ mEPSC amplitude/frequency (PV+ cells), ═ PPR,”

“These distinctions are even apparent for cortical sub-regions, which display profound heterogeneity in anatomical and functional connectivity to striatum, as well as divergent synaptic phenotypes in response to loss of the postsynaptic scaffolding molecule SAPAP3 (Corbit et al., 2019).”

“By assessing functional synaptic inputs in the central striatum, a knockout mouse study also revealed weakened inputs from the lateral orbitofrontal cortex and strengthened inputs from the secondary motor area in mice with OCD-like behaviors, thus suggesting a model of imbalanced cortical inputs to the striatum [67].”

“Unlike the central “associative” striatum, where deletion of SAPAP3 has no effect on SPN somatic excitability, 28 deletion of SAPAP3 significantly enhanced SPN excitability in the dorsolateral striatum (Figure 1).”

“While hypoactivation of OFC characterizes deficits in 278 reversal learning in animal models of OCD (108), recent data suggest that synaptic 279 strengthening and weakening of secondary motor cortex and OFC striatal inputs, respectively, 280 contribute to compulsive behavior in an OCD model (109).”

“Furthermore, striatal MSNs showed an increased response to the in vitro optogenetic activation of secondary motor area (M2) terminals in the striatum of the Sapap3 KO mice, supporting a potential role for M2-striatal circuit may contribute to compulsive behaviors [65].”

“For example, striatal input from the secondary motor cortex, which is strengthened in SAPAP3 (Corbit et al., 2019), has not yet been tested in SA.”

“The anterior lateral motor cortex was selected because those pyramidal neurons project to the striatum, and the spike trains were collected during a learning task known to involve the striatum [41,42].”

“Rodent models of repetitive, stereotyped, and compulsive behavior demonstrate hyperactivity in the striatal interneurons and their cortical projections [55, 56].”

“Given that axonal calibre is positively related to synaptic strength [38,39], the here‐observed structural phenotype of a decrease of axon calibre in the associative striatum is in line with and corroborates a previously reported decrease in cortico‐striatal synaptic strength in the associative striatum [30,64].”

“…to findings implicating the striatum [67–82], the glutamatergic system [83–87], the monoaminergic system [88], the orbitofrontal cortex [89–91], the motor cortex [73], the medial prefrontal cortex [92], and dopamine receptors [93] to obsessive-compulsive symptoms and aberrant grooming behaviors.”

“32, 46 While there been reports of alterations to premotor circuits in In the present findings, impaired sensitivity to recent action information was causally tied to M2-DMS activity, replicating prior findings.”

“Deletion in Dlgap3 has been shown to result in altered synaptic strength and circuit dynamics in the striatum (Burguière et al. 2013; Corbit et al. 2019 ; Hadjas et al. 2020; Ramírez-Armenta et al. 2022; Welch et al. 2007 ), especially imbalance between the dynamics of striatal subcircuits due to…”

“…human studies (Breiter and Rauch, 1996; Fettes et al., 2017; Menzies et al., 2008; Olver et al., 2009) and in animal studies (Ahmari et al., 2013; Corbit et al., 2019; Mondrag ´ on-Gonz ´ alez et al., 2024) striatal abnormalities were tied to OCD, with particular focus on striatal DA…”

“Local interneurons inhibit MSNs (5) and suppress glutamate release from M2 (6) and non-M2 (7) terminals.”

“In addition, in previous ex vivo slice electrophysiology work in WTs and Sapap3-KOs, 17 we showed that ALM projects monosynaptically to CS, contacting both SPNs and fast-spiking interneurons (FSIs).”

“…striatum and its cortical inputs ( Nordstrom & Burton, 2002 ; McCairn et al., 2009 ; Worbe et al., 2012 ; Ahmari et al., 2013 ; Worbe et al., 2015 ; Corbit et al., 2019 ) altering their interactions with other brain regions and associated structures ( O'Connell & Hofmann, 2012 ; Albin, 2018 ) and…”

“Unlike the central “associative” striatum of SAPAP3 KO mice (which also displays corticostriatal synaptic impairments) (Corbit et al., 2019), we found that somatic excitability is elevated in both direct and indirect pathway SPNs in the dorsolateral striatum.”