The Future: Circuit-Testing TS &

Accepted Manuscript

Title: Back to the Future: Circuit-Testing TS and OCD

Author: Frank H. Burton

PII: S0165-0270(17)30269-8 DOI: http://dx.doi.org/doi:10.1016/j.jneumeth.2017.07.025 Reference: NSM 7801

To appear in: Journal of Neuroscience Methods

Received date: 25-1-2017 Revised date: 3-7-2017 Accepted date: 25-7-2017

Please cite this article as: Burton Frank H.Back to the Future: Circuit-Testing TS and OCD.Journal of Neuroscience Methods http://dx.doi.org/10.1016/j.jneumeth.2017.07.025

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Back to the Future: Circuit-Testing TS & OCD

Running Title: Brain Circuit-Testing TS & OCD

Frank H. Burtona,b

aDepartment of Pharmacology, University of Minnesota, 6-120 Jackson Hall, 321 Church Street

SE, Minneapolis MN 55455-0217 USA bMinneapolis Medical Research Foundation, Hennepin County Medical Center, 701 Park Ave,

Shapiro S3.111, Minneapolis MN 55415-1623 USA

Author Correspondence: Frank H. Burton, Ph.D., Department of Pharmacology, University of

Minnesota, 6-120 Jackson Hall, 321 Church Street S.E., Minneapolis MN 55455-0217 USA;

Tel/V-mail: +1-612-873-6895; E-mail: [email protected]

1 TS & OCD CTX Circuit-Tests AMY

Chronic Neuropotentiation GLU Photic Neurostimulation FSI

Chemogenetic Neuropotentiation ACH DA Activity-Dependent D2 D1 Chemogenetic Neurostimulation STR STR Chemogenetic Neuroblockade IP DP Photic Neuroinhibition SNc Chemogenetic Neuroinhibition

Activity-Dependent Chemogenetic Neuroinhibition “URGE / MOVE” Highlights

• The first transgenic circuit-test 20 years ago was also the first model of TS & OCD. • This “Ticcy” mouse model has D1+ neuropotentiated corticostriatal glutamate output. • TS- & OCD-like sensorimotor gating & gait disorders were newly shown in these mice. • Chemogenetics & optogenetics are further defining these TS- & OCD-like CSTC circuits. • Activity-dependent chemogenetics may in the next 20 years explain (& treat) TS & OCD.

Abstract

A decade before the rise of optogenetics, the first behavioral “circuit-test” -- transgenically modulating the output of a genetically-specified brain circuit element to examine its effect on behavior -- was performed. The behaviors emulated in those mice were comorbid tics and compulsions, elicited by a gene borrowed from cholera bacteria and tailored to intracellularly neuropotentiate glutamatergic somatosensory cortical and limbic output neurons of cortico/amygdalo-striato-thalamo-cortical (CSTC) loop circuits. Two decades later, cutting-edge chemogenetic and optogenetic methods are again being devoted to further characterize the circuits thought to trigger, mediate, aggravate, or ameliorate TS & OCD symptoms. These tour de force studies support essential roles in tics and compulsions for topographically-parallel corticostriatal and amygdalar glutamatergic output neurons; their target dorsal striatal & ventral striatal (nucleus accumbens) medium spiny neurons (MSNs) of the direct striatothalamic (urge & motor activating) vs. indirect striatopallidal (urge & motor suppressing) output pathways; and their converging modulatory dopaminergic and histaminergic afferents. Going “back to the future” to circuit-map tics and compulsions will give us precision targets for future psychological, drug, medtech, and gene therapies; look for “dopamine bypasses” on your next trip in the DeLorean.

Abbreviations: ACH (acetylcholine); AMY; amygdala; cAMP, 3',5'-cyclic adenosine monophosphate; CSTC, cortico/amygdalo-striato-thalamo-cortical; CT, cholera toxin; CTX, cortex;

D1, dopamine receptor subtype 1; D1+, D1 receptor-expressing; D1CT-7, Dopamine receptor D1 gene (DRD1)-promoter/cholera toxin A1 subunit transgenic sub-strain 7 mouse; D2+, dopamine D2

2 receptor subtype-expressing; DA, dopamine; DP, striatal direct pathway; DREADD, designer receptor exclusively activated by designer drug; E-SARE, enhanced synaptic activity-responsive element; FSIs, striatal fast spiking interneurons; GABA, gamma-amino butyric acid; Gi, inhibitory G- protein; GLU, glutamate; GPC, G-protein coupled; GPCR, G-protein coupled receptor; Gs, Gs/olf,

Gq, stimulatory G-proteins; IP, striatal indirect pathway; MSNs, striatal medium spiny neurons; NPY, neuropeptide Y; NAc, nucleus accumbens; OCD; obsessive-compulsive disorder; OFC (orbitofrontal cortex); PPI (prepulse inhibition); SNc, substantia nigra pars compacta; STN, subthalamic nucleus;

STR, striatum or striatal; TS, Tourette's syndrome; TTM, trichotillomania.

Keywords: Tourette; compulsion; glutamate; transgenic; optogenetic; chemogenetic

1. Introduction

The often comorbid tics and compulsions in Tourette’s syndrome (TS) and Obsessive-

Compulsive Disorder (OCD) may involve overlapping or parallel brain circuits (Robertson, 2000;

American Psychiatric Association, 2013). A role for cortical and amygdalar glutamatergic output subcircuits of CSTC loops in eliciting or mediating neurogenic tics and compulsions was proposed late last century, then elaborated in the intervening two decades (Campbell et al., 1999a,

1999b; McGrath et al., 2000; Carlsson, 2000; Rosenberg et al., 2000; Nordstrom and Burton,

2002; Singer et al., 2010; Milad and Rauch, 2012; Nordstrom et al., 2015). Functional MRI of TS

3 shows primary hyperactivity of excitatory somatosensory, insular and efferent motor output circuits, which elicits premonitory urges and tics (Bohlhalter et al., 2006; Wang et al., 2011), and secondary hypoactivity of motor-suppressing executive-control circuits (Swerdlow and

Sutherland, 2005). The latter may include depletion or deficiency of these regulatory interneuron populations: 1) cortical GABAergic inhibitory interneurons; 2) striatal (STR) cholinergic interneurons that normally excite GABAergic STR “indirect pathway” (IP) medium spiny neurons (MSNs) suppressing tics and compulsions; and 3) STR parvalbumin-positive GABAergic fast-spiking interneurons (FSIs) that normally inhibit GABAergic STR “direct pathway” (DP)

MSNs activating tics and compulsions (Kalanithi et al., 2005; Kataoka et al., 2010; Burguiere et al., 2013; Xu et al., 2015a).

The frequent comorbidity of TS and OCD suggests their symptoms overlap not only in their interconnecting and parallel CSTC circuitry but also in their pathophysiology: Compulsions are usually preceded by obsessions and anxiety and involve orbitofrontal cortex (OFC) & amygdalar hyperactivity, while tics are preceded by discomfiting premonitory sensations & urges and involve OFC-interconnected somatosensory cortex & amygdalar hyperactivity; also, both show impaired sensorimotor gating, which normally filters out irrelevant inputs (Godar and

Bortolato, 2017). Consequently, both OCD and TS involve hyper-attentiveness to the obsessions and/or urges, which are temporarily alleviated by performing the desired acts and/or motions, likely by feedback inhibition from prefrontal & motor cortex collaterals. Stress also aggravates both disorders, likely from hyperglutamatergic amygdalocortical output (Godar and Bortolato,

2017). Maladaptive habits also develop in both disorders, likely from overlap and parallelism of their CSTC circuitry, including their cortical & amygdalar elements and target dorsal striatum

(STR) & ventral STR (nucleus accumbens, or NAc) elements, which modulate not only motor

4 activity but urges, aversion vs. reward, and habit formation (Bortolato and Pittenger, 2017).

Consistent with their parallel neuropathophysiology, both TS and OCD are associated with hyperactivity of regional (somatosensory or orbitofrontal) cortical output neurons, and deficient cortical inhibitory gating evidenced by PPI (prepulse inhibition) deficits (Swedo et al.,

1992; Breiter et al., 1996; Ziemann et al., 1997; Edgley and Lemon, 1999; Gilbert et al., 2004;

Mantovani et al., 2006; Swerdlow and Sutherland, 2006; Ahmari et al., 2012; Bortolato and

Pittenger, 2017). Transcranial magnetic stimulation confirms regional cortical disinhibition in both disorders (Ziemann et al., 1997; Gilbert et al., 2004; Mantovani et al., 2006), while elevated corticostriatal glutamatergic efflux was confirmed in OCD (Rosenberg et al., 2000). Similarly, both monkey and rat stereotaxic drug studies showed that disinhibiting sensorimotor corticostriatal glutamate (GLU) output was essential to generate tic-like symptoms, while inhibiting it was essential to diminish them (Pogorelov et al., 2015). These findings suggested that corticostriatal GLU can elicit, not just mediate, TS and OCD symptoms.

CSTC loop hyperactivity in TS arises polygenically, most often from diffuse disruptions in synapse formation or function (Huang et al., 2017), but may etiologically arise in some TS cases as impairment either of inhibitory interneurons (Verkerk et al., 2003; Minzer et al., 2004;

Penagarikano et al., 2011) or of inhibitory neurotransmission. Absence of histamine may disinhibit nigrostriatal, striatal, and cortical output in Hdc (histidine decarboxylase) gene deletion- associated hyperdopaminergic forms of human TS or mouse TS-like orofacial and sniffing stereotypies (Ercan-Sencicek et al., 2010; Karagiannidis et al., 2013; Castellan Baldan et al.,

2014; Xu et al., 2015b). Diminished removal or reuptake of dopamine (DA) likewise triggers hyperdopaminergic TS-like and/or OCD-like symptoms in mice (Bortolato et al., 2011; Fox et al.,

2013). But even these hyperdopaminergic etiologies involve STR immediate-early gene induction

5 that still remains dependent on coincident GLU input (Rapanelli et al., 2014; Castellan Baldan et al., 2014), suggesting DA and GLU coinduce STR output to mediate or elicit tics and compulsions. This is consistent with reported OCD-like symptoms in mice with genetic knockouts of Sapap3, a corticostriatal synapse protein that normally inhibits GLU-induced STR

MSN output (Welch et al., 2007; Ting and Feng, 2008; Burguiere et al., 2013; Wan et al., 2014); or of Slitrk5, which normally inhibits OFC hyperactivity (Shmelkov et al., 2010).

Starting with the first transgenic “brain circuit-test” of behavior, I review the ongoing transgenic, optogenetic, and chemogenetic alterations of CSTC circuit element output to understand and treat the abnormal urge- & motor- symptoms of TS and comorbid OCD-spectrum disorders. These studies support the importance of CSTC subcircuit hyperactivation vs. inhibition in inducing vs. suppressing these psychomotor symptoms.

2. Back to the Future I: Transgenic Neuropotentiating Circuit-Test of TS & OCD

The first transgenic behavioral circuit-test (and genetically-engineered mouse model of TS &

OCD) indicated that chronic cortical & amygdalar CSTC hyperactivity causes tic & compulsion- like behavior. D1CT-7 transgenic mice, which exhibit TS-like head & body twitches & OCD-like complex behavioral perseveration (Campbell et al., 1999a, 1999b; Nordstrom and Burton, 2002), were created with a neuropotentiating transgene that instead of directly depolarizing and firing neurons enhanced their responsiveness to their endogenous fast-acting neurotransmitters (Fig. 1).

______A decade before microbial channelrhodopsin-based optogenetics, this was achieved by DA D1 receptor (DRD1) gene promoter-targeted expression of a microbial exon encoding the exclusively

6 intracellular A1 subunit of cholera toxin (CT) (Campbell et al., 1999a). Transgenic CT physiologically engineers cellular hyperactivity -- and potentiates neurons -- by covalently ADP- ribosylating their stimulatory Galpha-s protein (Gs), which chronically activates adenylate cyclase, elevating intracellular levels of the second messenger 3',5'-cyclic adenosine monophosphate

(cAMP) (Burton et al., 1991; Zeiger et al., 1997). Furthermore, in D1CT founder lineage 7, CT expression was regionally restricted to a cortical/limbic subset of D1+ neurons with no expression in striatal D1+ neurons (Campbell et al., 1999a). The potentiated cortical/limbic D1+ neurons induce glutamatergic hyperactivation of the same somatosensory, orbitofrontal, amygdalar, and efferent STR motor output circuits showing primary hyperactivity in human TS and OCD

(Campbell et al., 1999a, 1999b; Bohlhalter et al., 2006; Wang et al., 2011; Church and Shlaggar,

2014). Those circuits’ chronic hyperactivation in the D1CT-7 mice similarly causes OCD-like perseveration of multiple complex behaviors (Campbell et al., 1999a), as well as TS-like twitches, leading to the mouse model’s nickname, “Ticcy mice” (Nordstrom and Burton, 2002). The D1CT-

7 mice’s perseveration of many otherwise normal behaviors is more complex than the stereotyped sniffing & locomotion that can be imposed upon normal (and even upon D1CT-7) mice by broad- acting drugs like cocaine, which are thought to activate a broader set of parallel DA-responsive

CSTC-subcircuits (Campbell et al., 1999c). The D1CT-7 mice’s chronic cortical-limbic GLU output to the STR also unbalances its STR DP vs. IP MSN output pathways in favor of DP MSN output, yet confers behavioral supersensitivity to D2 antagonists, the first drug class shown to be therapeutic for TS & OCD (Campbell et al., 1999b; Nordstrom and Burton, 2002; Nordstrom et al., 2015). The mice also show TS- & OCD- like symptom exacerbation by stress (McGrath et al.,

1999a, 1999b; Godar et al., 2016), TS-like temporary supressibility of twitching, juvenile onset of twitching, more severe & frequent twitching in males (Nordstrom and Burton, 2002), and

7 alleviation of twitching by clonidine, a second pharmacological class of TS therapeutic drug

(Nordstrom and Burton, 2002; Campbell et al., 1999b). These traits are among the greatest behavioral similarities to TS & OCD of animal models reported to date (Burke and Lombroso,

2004; Pittenger et al., 2011). The D1CT-7 mice's potentiated cortical circuitry is glutamatergic:

Although neither normal nor D1CT-7 mice show spontaneous seizures, normal mice show slower- onset & calmer pentylenetetrazole-kindled (cortical glutamatergic) seizures (Campbell et al.,

2000), as well as less-pronounced glutamatergic drug-induced locomotion than do D1CT-7 mice

(McGrath et al., 2000). Based on their specific neuropotentiated somatosensory & limbic CSTC circuit elements (Table 1), D1CT-7 mice comprised a direct test of the hypothesis that cortico/amygdalo-striatal hyperglutamatergic output can cause tic- and compulsion-like behaviors

(Campbell et al., 1999a; Carlsson, 2000; Nordstrom and Burton, 2002).

Early awareness of the D1CT-7 hyperglutamatergic CSTC model (Sah and Sallee, 2002;

Burke and Lombroso, 2004; Joel, 2006; Ting and Feng, 2008; Wang et al., 2009; Pittenger et al.,

2011; Wu et al., 2012; Ahmari and Dougherty, 2015) helped spur clinical studies of GLU’s role in

TS and OCD (Chakrabarty et al., 2005; Singer et al., 2010) and new drug trials for TS, OCD, and

OCD-spectrum disorders. These included successful trials of antiglutamatergics for OCD and trichotillomania (TTM) (Lafleur et al., 2006; Grant et al., 2007; Grant et al., 2009). A D1 antagonist, potentially able to inhibit both D1+ cortical-limbic neurons’ induced GLU output and

D1+ STR DP MSN output, proved effective for TS (Gilbert et al., 2014) as well as for OCD- spectrum gambling disorder (Grant et al., 2014). And a neurosteroid inhibitor of D1+ corticostriatal subcircuits known to activate STR DP MSN output alleviated TS (Bortolato et al.,

2007; Bortolato et al., 2008; Muroni et al., 2011; Frau et al., 2013; Frau et al., 2016). D1CT-7

8 mice have since been used to identify new classes of prospective CSTC “circuit-breaker” drugs able to block TS-like symptoms (Nordstrom et al., 2015).

Most recently, D1CT-7 mice were shown to exhibit stress-induction of multiple TS- &

OCD- like symptoms, and to exhibit a sensorimotor gating deficit -- impaired prepulse inhibition

(PPI) -- that increases its face validity as a model of the impaired sensorimotor gating in TS

(Godar et al., 2016; Godar and Bortolato, 2017; Bortolato and Pittenger, 2017) Moreover, new methods for characterizing TS-like motor symptoms -- microsecond-resolution force-plate actometer tests of mouse gait, and sticky-tape removal tests of sensorimotor integration -- revealed stress-aggravated hyper-vigorous gait and sensorimotor integration deficits in D1CT-7 mice that match those reported for TS (Fowler et al., 2017) (Table 1). These D1CT-7 mouse findings will help define how topographically overlapping and parallel CSTC loop abnormalities manifest urge- or obsession-driven psychomotor, sensorimotor, locomotor, and compulsive symptoms in TS & OCD-spectrum disorders. In turn this will also shed light on other CSTC loop- regulated psychotic-spectrum & psychomotor disorders in which such symptoms also arise -- including autism; schizophrenia; DA agonists’ psychotic, dyskinesic, hypermotivating, & compulsive effects; and, conversely, DA antagonists’ antipsychotic & anti-tic, bradykinesic

(pseudoparkinsonian), demotivating, & anti-compulsive effects.

However, the D1CT-7 mouse model circuit test is experimentally limited in that its chronic cortical-limbic D1+ neuropotentiation can’t be deactivated or further spatially or temporally restricted (Fig. 1). Although the chronic nature of this mouse’s potentiated forebrain

GLU output likely contributes to its marked symptomatic resemblance to the similarly chronic disorders TS & OCD (Ahmari et al., 2013), it can’t provide the precise behavioral circuit-testing

9 of each CSTC circuit element required to query these individual elements’ roles in these disorders. Consequently, since the first use in the ‘90s of neuropotentiating transgenes chronically expressed either globally in all neurons (Burton et al., 1998) or selectively in cortical-limbic D1+ neurons (Campbell et al., 1999a), newer circuit-tests have used chemical- or photic- regulated

“chemogenetic” or “optogenetic” transgenic methods (Fig. 1). These methods have been used to either stimulate (Table 1) or inhibit (Table 2) different CSTC circuit elements putatively involved in TS & OCD, in a spatially, temporally, and/or synaptic activity-dependent manner.

3. Back to the Future II: Chemogenetic and Optogenetic TS & OCD Circuit-Tests

3.1. Chemogenetic Neuroblockade

Doxycycline-reversible chronic (2-week) blockade of mouse STR & NAc D1+ DP vs. D2+ IP

MSNs by transgenic tetanus toxin (Fig. 1 & Table 2) revealed that blocking urge- & motor- activating DP output inhibits not only basal & acute DA drug-induced locomotion but also inhibits OCD-like DA-drug sensitization & reward-seeking/learning. Conversely, blocking urge-

& motor-suppressive IP output increases not only basal locomotion but also TS- & OCD-like lack of aversive self-control (Hikida et a., 2010). However, unlike later-performed optogenetic and chemogenetic acute IP inhibition, chronic IP blockade prevented acute DA drug-induced locomotion and retarded DA-drug sensitization, rather than causing a predicted TS- & OCD-like increase in such behaviors. Thus, either residual IP activity may be required to permit DA- induced behaviors; or chronic IP loss may cross-desensitize D1 receptors in DP MSNs to rebalance lost IP output with minimal DP output, allowing basal locomotion but preventing DA- dependent hyperkinesias (and thus possibly also tics & dyskinesias).

10 3.2. Optogenetic Channels

Scientists contributing to the first transgenic brain circuit-test using modified bacterial neuropotentiating transgenes (Campbell et al., 1999a) later contributed to the first optogenetic brain circuit-tests using modified bacterial and algal photoactivatable or photoinhibitable channelrhodopsin, halorhodopsin, or archaerhodopsin neurostimulating or neuroinhibiting transgenes (Adamantidis et al., 2007; Tsai et al., 2009). Rather than chronically potentiating genetically-specified neurons, optogenes (usually also stereotaxically-injected into different mouse brain regions on viral vectors) directly depolarize (fire) or hyperpolarize (silence) genetically-specified neurons in response to spatially- and temporally- restricted photon excitation from cranial fiber-optic leads or light-emitting diode (LED) implants. Optogenetic neurostimulatory (Table 1) or neuroinhibitory (Table 2) brain circuit-tests of the CSTC circuit elements presumed to mediate TS- & OCD-like urge- & motor- symptoms in mice are diagrammed in Fig 1. These studies have refined the topographically-overlapping behavioral effects of corticostriatal GLU output neurons; their target STR & NAc DP vs. IP output MSNs that activate vs. suppress TS-like motor & motor-initiation urges and OCD-like repetitive & perseverant behaviors and habits; and their adjacent STR FSI or ACH+ regulatory interneurons:

Upstream CSTC circuit elements optogenetically circuit-tested in mice include photostimulated GLU output from M1 and M2 motor CTX, OFC, and lateral OFC (Table 1); and photoinhibited GLU output from infralimbic CTX (Table 2). Chronic (5-day) optogenetic neurostimulation of M2 motor CTX and OFC pyramidal projection neurons’ GLU outputs to the

STR respectively reproduced two TS- & OCD-like symptoms earlier seen in D1CT-7 mice – increased locomotion and perseverative grooming (Ahmari et. al., 2013). In contrast, acute optogenetic neurostimulation of lateral OFC neurons that selectively excited not STR MSNs but

11 mutant, functionally-deficient STR FSIs restored those FSIs’ failed inhibition of OCD-like grooming in a gene knockout mouse model of OCD (Burguiere et al., 2013). This interestingly suggests that corticostriatal GLU can excite not only STR MSN but also MSN-inhibiting STR interneurons, identifying a potential corticostriatal subcircuit for executive control (voluntary suppression) of tics and compulsions. Acute optogenetic stimulation of OFC output also confers

OCD-like increased value to devalued acts (Gremel and Costa, 2013), while acute optogenetic inhibition of infralimbic CTX GLU output neurons cancels new acquired habits & prevents their acquisition (Smith et al., 2012; Smith and Graybiel, 2013).

Downstream CSTC circuit elements optogenetically circuit-tested in mice include photostimulated dorsomedial STR D1+ DP MSNs and STR D2+ IP MSNs, NAc D2+ IP MSNs, and further-downstream STN afferent fiber inputs (Table 1); and photoinhibited NAc ACH+ interneurons (Table 2). Optogenetic stimulation of dorsomedial STR D1+ DP MSN vs. STR D2+

IP MSN output neurons respectively triggered vs. inhibited TS- & OCD-like increases in locomotion & locomotor initiations (Kravitz et al., 2010). More ventrally-located STR optogenetic stimulation of NAc D2+ IP MSN output inhibited OCD-like DA-entrained

“compulsive” drug seeking/requesting (Bock et al., 2013). Downstream of STR IP outputs, differential frequencies of optogenetic stimulation of STN neurons’ GLU-responsive afferent fiber inputs reproduced both the motor activating vs. inhibiting effects of frequency-dependent deep-brain-stimulation (DBS) implants, and the effect of M1 motor CTX hyperdirect pathway

GLU input to the STN (Gradinaru et al., 2009). Optogenetic photoinhibition of another STR interneuron population, NAc cholinergic (ACH+) interneurons, indirectly increased NAc MSN output in a manner that specifically inhibited DA-drug reward-seeking/learning, while photostimulating these interneurons had no effect (Witten et al., 2010).

3.3. Chemogenetic Designer Receptors

Other new brain circuit-testing techniques include stereotaxic viral vector-delivered transgenes encoding G-protein-coupled (stimulatory Gs-, Gs/olf-, or Gq-, and inhibitory Gi-coupled)

“Designer Receptor Exclusively Activated by Designer Drugs” (“DREADDs”) for drug-based

“remote control” of receptor-coupled second messenger signaling (Rogan and Roth, 2011) (Fig.

1). Such G-protein-coupled DREADD circuit-tests in rodents are confirming predicted roles for cortical and striatal neurons in mediating TS- & OCD-like symptoms, and for the first time are functionally interrogating the subsets of these neurons that are synaptically-activated by replicating actual etiological causes of human TS & OCD (Table 1 & 2):

First, in mice, acute Gi-DREADD neuroinhibition of OFC GLU output, as predicted, disrupts any valuation of goal-directed acts, opposite to the OCD-like overvaluation of even useless acts that is conferred by optogenetic neurostimulation of OFC GLU output (Gremel and

Costa, 2013). Similarly, Gs-DREADD neurostimulation of STR & NAc IP MSNs reduces basal

& novelty-induced locomotion and prevents its acute DA-drug sensitization (Farrell et al., 2013), while Gi-DREADD neuroinhibition of NAc IP MSNs induces OCD-like “compulsive”

(unrewarded and hypermotivated) drug-seeking/requesting (Bock et al., 2013).

In rats, Gi-DREADD neuroinhibition of dorsal STR DP vs. IP MSNs similarly inhibits vs. causes OCD-like increases in DA-drug sensitization/retention of locomotion (Ferguson et al.,

2011). Also, Gi-DREADD neuroinhibition vs. Gs-DREADD neurostimulation of dorsomedial

STR DP MSNs inhibits vs. causes an OCD-like increase in retention of rewarding feeding strategies (Ferguson et al., 2013). This latter study comparing Gi- vs. Gs-DREADD effects on the same neurons specifically implies that the level of cAMP-dependent neuropotentiation within

13 dorsomedial STR DP MSNs mediates retaining reward-specific information needed to improve later performance -- a process that when aberrantly excessive could engender compulsions.

3.4. Activity-Dependent Chemogenetic Designer Receptors

A new derivation of chemogenetic DREADD methods is now being used to further specify genetically-defined neurons by their prior synaptic-activation in response to drugs, electrical stimuli, or neurotransmitters -- including neurotransmitter changes elicited by disease states. Expression of such “Activity-Dependent” DREADDs is achieved by using a synthetic

“Enhanced Synaptic Activity-Responsive [DNA] Element” (E-SARE) to drive neuronal activity- dependent gene expression of Cre-loxP recombinase to permanently assemble the DREADD transgene (Kawashima et al., 2013). This approach has been elegantly used to reveal the roles in manifesting mouse TS- & OCD-like symptoms played only by those CTX or STR neurons that can be synaptically-activated by an actual known etiological cause of TS & OCD, loss of histamine neurotransmission (Fig. 1; Table 1 & 2). These studies functionally examined the medial prefrontal CTX (mPFC) neurons and dorsal STR neurons selectively supersensitized or synaptically activated by experimentally-reproduced genetic CNS histamine deficiency (Rapanelli et al., 2017a), or by temporary Gi-DREADD-induced loss of hypothalamocortical & hypothalamostriatal histamine inputs (Rapanelli et al. 2017b). Gs-DREADD neurostimulation of such histamine-depletion-activated mPFC neurons initiated “long-range” (ambulatory) locomotion, while that of histamine-depletion-activated dorsal STR neurons caused TS- & OCD- like stereotypic “short-range” locomotion, excess grooming, and head shakes (Table 1 & 2).

Moreover, Gi-DREADD inhibition of these same neurons reverses the mice’s histamine- deficiency dependent TS- & OCD-like behaviors (Table 2). Hence these hyperactivated CSTC

14 circuit elements mediate not only twitches & urge-driven behaviors in mice, but likely also human tics & comorbid compulsions in at least one established etiological form of TS & OCD, which has polygenic etiologies (Huang et al., 2017).

4. Back to the Future III: New Directions in TS & OCD Circuit Testing & Therapy

4.1. Future Circuit-Tests

Today’s transgenic, optogenetic, and chemogenetic arsenal is ripe for fine-tuned CSTC circuit- testing of not only feedforward cortical, amygdalar, STR, or STN circuit elements, but also feedback thalamocortical, thalamolimbic, and thalamostriatal circuit elements and their modulatory neurotransmitter inputs (Ellender et al., 2011; Halassa et al., 2014; Rapanelli et al.,

2017a, 2017b). STR neurons also include not only the currently investigated major DP & IP MSN output subpopulations and FSI or ACH+ interneuron subpopulations, but also STR NPY+ low- threshold-spiking interneurons and STR interneurons defined by selective responsiveness to thalamic GLU instead of cortical/amygdalar GLU (Gerfen and Surmeier, 2011). Circuit-testing these CSTC loop STR subpopulations will refine our understanding of the role of CSTC loop dysregulation in tics and compulsions. Also, new TS & OCD circuit-testing methods are in the offing, including not just neuromodulatory G-protein-coupled DREADDs but depolarizing- or hyperpolarizing-“channel-coupled” DREADDs, or “Pharmacologically Selective Actuator

Molecules activated by Pharmacologically Selective Effector Molecules” (PSAMs-PSEMs)

(Sternson and Roth, 2014). Other new methods include neuromodulatory optogenes -- optogenetic photon-activated G-protein-coupled receptors, or “optoXRs” (Airan et al., 2009). Also, to bypass the potential issue of pharmacodynamic desensitization of optogenetic channels’ or DREADD receptors’ transmembrane domains, bacterial optogenetic adenylate cyclases have been developed

15 (Ryu et al., 2010; Stierl et al., 2011). These work like a photic-regulated version of the original circuit-testing neuropotentiation method, bacterial intracellular cholera toxin (Burton et al., 1991).

4.2. Future Circuit-Therapies

Optogenetic channels’ computer-programmed imposition of precise firing patterns on neurons is an optimal technique for brain-behavior circuit-testing. However, as a prospective therapeutic tool, it may risk reprogramming not only psychomotor urges but also normal behaviors unless combined with computerized monitoring-&-control implants analogous to next-generation DBS devices. As one alternative, neuropotentiating Gs-DREADDs would have less risk of robotizing behavior because they would only enhance targeted neurons’ responsiveness to their own endogenous signals. Adeno-Associated Virus- (AAV-) delivery of Gs-DREADDs to somatosensory CTX or OFC D2+ inhibitory GABAergic “executive control” interneurons, or to

STR IP D2+ “urge- & motor- suppressing” GABAergic output MSNs, could “bypass” the inhibitory effect of basal DA upon them. Their consequent designer-drug-neuropotentiation would then increase their ability to inhibit the intractable urges, premonitory sensations, obsessions, tics, movements, and compulsive habits they otherwise routinely fail to inhibit.

To illustrate the unprecedented utility of the newest chemogenetic methods, how might such a future “dopamine-bypass” gene therapy be constructed to work effectively in human patients requiring neurosurgical intervention? The enkephalin ENK gene promoter, which can abundantly express AAV-delivered DREADD transgenes in STR IP D2+ MSN GABAergic output neurons (Table 2), isn’t as abundantly expressed in the CTX D2+/parvalbumin+

GABAergic interneurons known to locally inhibit pyramidal GLU output neurons. But newly developed AAV-delivered DREADD transgenes using mouse or human Distal-less homeobox

16 (hDlx) enhancers specifically confer, in the CTX as well as other telencephalon regions of multiple species, GABAergic neuron-specific DREADD expression (Dimidschstein et al., 2016).

In mouse somatosensory CTX, AAV-hDlx-GFP-expressed Green Fluorescent Protein marker is coexpressed in all its parvalbumin+ GABAergic inhibitory interneurons, and designer-drug-

(CNO-) dependent AAV-hDlx-Gq-DREADD neurostimulation is confirmed to inhibit adjacent

CTX pyramidal neuron GLU output. This suggests two potentially feasible future gene therapies for otherwise intractable & severe TS &/or OCD. The first strategy would be local somatosensory

CTX or OFC (or, alternatively, dorsomedial STR) injection of an AAV-hDlx-Gs-DREADD (or, in dorsomedial STR, AAV-ENK-Gs-DREADD) vector alone. A second strategy, likely more specific and thus safer, would be similar local co-injection of both the synaptic-activity- & tamoxifen- dependent transient Cre recombinase expression vector, AAV-E-SARE-ERT2CreERT2-PEST (Table

1 & 2), and a Cre-reconstructible version of the DREADD vector, AAV-hDlx(or ENK)-DIO-Gs-

DREADD. In this second strategy, regional co-injection of both vectors would be followed 2 weeks later (Rapanelli et al., 2017a, 2017b) by brief treatment of the TS & OCD patients with 4-

OH-tamoxifen while asking them to voluntarily suppress their tics or compulsions. This should trigger transient Cre recombinase expression and its permanent reconstruction of the CNO-drug- responsive Gs-DREADD transgene only in the patients’ most relevant “executive control” neurons – the subset of their AAV-vector-infected inhibitory GABAergic neurons that are synaptically-activated while the patients actively suppress their tics or compulsions.

Such “DA-bypass” therapies, along with other possible transgenic circuit-therapy methods, may eventually serve as 21st century cures for the most severe presentations of TS &

OCD. Moreover, such methodologies will also be applicable to quell the symptoms of other intransigent behavioral disorders likewise mediated by topographically overlapping or parallel

17 CSTC circuits. But regardless of what transgenic therapies may in two decades complement our therapeutic arsenal for intractable TS & OCD and their related psychomotor & psychotic- spectrum disorders, our past two decades of using designer genes to CSTC circuit-test TS & OCD allows us to envision such future treatments today.

Conflict of Interest

The author declares no actual or potential conflict of interest.

Acknowledgements

This work was supported by a grant from the University of Minnesota Foundation to FHB.

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35 Figure Legend & Tables

Fig. 1. Schematic of transgenic behavioral circuit-tests of the CSTC circuit elements mediating rodent TS- & OCD-like behaviors. Cortico/amygdalo-striato-thalamo-cortical

(CSTC) circuit elements tested by neurostimulatory methods are summarized in Table 1, while those tested by neuroinhibitory methods are summarized in Table 2. Triangles: Cortex & amygdala (CTX AMY) cortico/amygdalo-striatal (or, left arrow, cortico-subthalamic nuclear hyperdirect pathway) excitatory glutamatergic (green GLU) pyramidal projection output neuron circuit elements. Large circles: Dorsal or ventral (nucleus accumbens) striatum (STR) dopamine

(DA) excitable, D1-receptor-expressing (green D1) striatonigral direct pathway (DP) and DA- inhibitable, D2-receptor-expressing (red D2) striatopallidal indirect pathway (IP) medium spiny output neuron circuit elements. Small circles: STR fast-spiking inhibitory (FSI) or cholinergic

(ACH) regulatory interneuron subcircuit elements. Ovoid: Substantia nigra pars compacta (SNc) nigrostriatal dopaminergic (DA) modulatory neuron subcircuit element. Rectangle: Integrated urge- & motor- output from DP vs. IP efferent (globus pallidus/subthalamic nucleus/substantia nigra pars reticulata/thalamus) circuit elements. Pointing vs. blocking arrows respectively signify excitatory vs. inhibitory outputs on either efferent circuit elements or, for the bottom rectangular

“urge/move” box, integrated thalamocortical GLU output underlying urges & movements.

36 TS & OCD CTX Circuit-Tests AMY

Chronic Neuropotentiation GLU Photic Neurostimulation FSI Chemogenetic Neuropotentiation ACH DA Activity-Dependent D2 D1 Chemogenetic Neurostimulation STR STR Chemogenetic Neuroblockade IP DP

Photic Neuroinhibition SNc Chemogenetic Neuroinhibition

Activity-Dependent Chemogenetic Neuroinhibition “URGE / MOVE”

Fig. 1. Schematic of transgenic behavioral circuit-tests of the CSTC circuit elements mediating rodent TS- & OCD-like behaviors. Cortico/amygdalo-striato-thalamo-cortical (CSTC) circuit elements tested by neurostimulatory methods are summarized in Table 1, while those tested by neuroinhibitory methods are summarized in Table 2. Triangles: Cortex & amygdala (CTX AMY) cortico/amygdalo-striatal (or, left arrow, cortico-subthalamic nuclear hyperdirect pathway) excitatory glutamatergic (green GLU) pyramidal projection output neuron circuit elements. Large circles: Dorsal or ventral (nucleus accumbens) striatum (STR) dopamine (DA) excitable, D1-receptor-expressing (green D1) striatonigral direct pathway (DP) and DA-inhibitable, D2-receptor-expressing (red D2) striatopallidal indirect pathway (IP) medium spiny output neuron circuit elements. Small circles: STR fast-spiking inhibitory (FSI) or cholinergic (ACH) regulatory interneuron subcircuit elements. Ovoid: Substantia nigra pars compacta (SNc) nigrostriatal dopaminergic (DA) modulatory neuron subcircuit element. Rectangle: Integrated urge- & motor- output from DP vs. IP efferent (globus pallidus/subthalamic nucleus/substantia nigra pars reticulata/thalamus) circuit elements. Pointing vs. blocking arrows respectively signify excitatory vs. inhibitory outputs on either efferent circuit elements or, for the bottom rectangular “urge/move” box, integrated thalamocortical GLU output underlying urges & movements.

37 Table 1. Neurostimulatory CSTC circuit-tests of TS- & OCD-like symptoms

Method Transgene(s) Circuit element(s) Behavioral effect(s) Reference(s)

Chronic OCD-like generalized perseveration of multiple complex behaviors (e.g., D1CT-7 (Ticcy) mice Neuropotentiation D1+ layer II-III digging, eating, climbing, commensal grooming) and locomotor hyperactivity; Campbell et al.,

(Intracellular Cholera somatosensory-insular- OCD/TTM-like perseverative self-grooming; Locomotor insensitivity to D1- 1999a, 1999b,

Toxin A1 Subunit) piriform CTX pyramidal antagonists & supersensitivity to therapeutic D2-antagonists [compensatory 1999c; McGrath et

GLU output neurons, & D1+ imbalance between STR DP vs. IP MSNs favoring DP hyperactivity]; Increased al., 2000

AMY intercalated nucleus sensitivity to glutamatergic drug effects

GABA output interneurons,

which trigger GLU output TS-like head & body twitches (juvenile-onset, gender-dimorphic [greater in Nordstrom and

from somatosensory, motor, males], temporarily suppressed during more attention-dependent activities [e.g., Burton, 2002;

& orbitofrontal CTX & AMY bar-hanging, digging, eating, drinking, grooming], ameliorated by TS- drug Nordstrom et al.,

to STR clonidine & by CSTC-“circuit breaker” drugs) 2015

McGrath et al., OCD- & TS-like symptoms aggravated or induced by olfactory or physical 1999a, 1999b; stressors; TS-like impaired PPI (sensorimotor gating) Godar et al., 2016

TS-like hyper-vigorous gait & sensorimotor integration deficits Fowler et al., 2017

Photic M2 motor CTX vs. OFC glia AAV-Ef1a-DIO-ChR2-EYFP OCD-like perseverative grooming (after 5 days chronic OFC neurostimulation); Neurostimulation & GLU output neurons to Ahmari et al., 2013 vector  Emx-Cre mice CTX increased locomotion (upon acute M2 motor CTX neurostimulation) (Optogenetics) VM STR Lateral OFC GLU neuron AAV-CaMKII-ChR2-EYFP vector Reversal of OCD-like perseverative grooming in Sapap3KO mutant model of Burguiere et al., outputs to STR inhibitory  Sapap3KO mice CTX defective FSI-inhibition of STR DP MSNs 2013 FSIs OCD-like increased locomotor initiations & locomotor activity, reduced AAV-Ef1a-DIO-ChR2-EYFP D1+ DP vs. D2+ IP DM STR freezing, and rescued 6-OHDA-induced parkinsonism (DP), vs. parkinsonism- vector  D1-Cre vs. D2-Cre mice Kravitz et al., 2010 MSNs like reduced locomotor initiations & locomotor activity, and increased freezing STR (IP) OCD-like increased valuation of devalued goal-directed acts (increased lever- AAV-CaMKII-ChR2-EYFP vector Gremel and Costa, OFC GLU output neurons press counts when satiated, but not when hungry or when already entrained to  mice CTX 2013 press habitually) OCD-like high-freq-stim of STN input GLU fibers (or of M1 motor CTX GLU M1 motor CTX GLU neuron neuron hyperdirect pathway output to STN) reduced locomotion-suppressive Thy1-ChR2-EYFP-18 mice with 6- Gradinaru et al., outputs to STN, and STN STN GLU neuron output, reversing hemi-parkinsonism; low-freq-stim of STN OHDA hemi-parkinsonism 2009 afferent input GLU fibers input GLU fibers increased STN GLU neuron output, worsening hemi- parkinsonism [matches DBS effects in PD]. AAV-EF1a-DIO-ChR2-EYFP Reduced entrained “compulsive” drug-seeking [cocaine request extinction D2+ IP NAc MSNs Bock et al., 2013 vector  Adora2a-Cre mice NAc without reward; less-motivated, non-increasing requests with reward] Chemogenetic Reduced spontaneous & novelty-induced locomotion; prevented amphetamine- Neuropotentiation Adora2a-rM3Ds-mCherry mice  D2+ IP STR-&-NAc MSNs [reward-] sensitization of locomotion; reduced relative expression of co- Farrell et al., 2013 (Gs-coupled CNO excitatory D2+ NAc NMDA-subtype GLU receptors DREADDs) HSV-DYN-HA-rM3Ds vector  Ferguson et al., D1+ DP DM STR MSNs OCD-like increased retention of more-rewarding strategies [lever choices] rat STR  CNO 2013 AAV9-hSyn-DIO-HA-KORD(Gi)- Activity-Dependent IRES-mCitrine [1] (Table 2), Chemogenetic AAV2-E-SARE-ERT2CreERT2- Neuropotentiation or mPFC & dorsal STR neurons Increased TS- & OCD-like grooming & stereotypic locomotion (dorsal STR) & PEST [2] + AAV5-hSyn-DIO- Neurostimulation previously activated by TS- ambulatory locomotion (mPFC) by DREADD-stimulation of dorsal STR or Rapanelli et al., hM3Dq-mCherry [3] vectors  (E-SARE-Cre- & OCD-like depletion of mPFC neurons previously activated by DREADD-inhibition of hypothalamic 2017b Hdc-Cre mice TMN [1] (Table 2), assembled Gs- or Gq- histamine input (Table 2) [TMN] histamine input (Table 2) mPFC + dorsal STR [2,3]  SalB coupled DREAADs) [1] (Table 2) + 4-OH-TMX [2];

SalB [1] or CNO [3]

Transgenic Components: D1CT-7 (Dopamine [DA] receptor DRD1 gene promoter-Cholera Toxin Intracellular A1 Subunit fusion gene transgenic mice, founder-lineage 7 [expressed in D1+ neurons of CTX & AMY, not STR]); AAV (Adeno-Associated Virus vector); Ef1a (Elongation-Factor-1a gene strong constitutive promoter); DIO (Double-floxed Inverted Open-reading-frame [paired loxP sites surrounding inactive antisense-oriented transgene cassette for Cre-loxP recombinase to invert it to active sense-orientation]); ChR2 (Channelrhodopsin-2- photoactivated excitatory cation channel gene cassette); EYFP (Enhanced Yellow Fluorescent Protein yellow marker fusion-gene cassette); Emx (Emx1 cortical homeobox gene promoter); Cre (Cre-loxP recombinase transgene); CaMKII (Calcium Calmodulin Kinase-II alpha gene cortical pyramidal neuron-specific promoter); Sapap3KO (Sapap3 inhibitory STR interneuron scaffolding gene knockout mouse); D1-Cre or D2-Cre (Bacterial Artificial Chromosome transgenic mice containing DA receptor DRD1 or DRD2 gene promoter-regulated Cre-loxP recombinase gene); Thy1-ChR2-EYFP-18 (Thy1 gene projection-neuron- specific promoter-ChR2-EYFP fusion gene transgenic mice, line 18 [STN afferent fiber-specific]); Adora2a-Cre (Bacterial Artificial Chromosome [BAC] transgenic mice containing Adenosine A2a receptor Adora2a D2+ MSN-specific gene promoter-regulated Cre-loxP recombinase gene); rM3Ds (gene for neuropotentiating Gs/Golf-DREADD receptor); mCherry (red fluorescent protein marker fusion-gene cassette); HSV (Herpes Simplex Virus Amplicon vector); DYN (Dynorphin gene [D1+ DP STR MSN-specific] promoter]; HA (Hemagglutinin tag fusion-gene cassette); KORD(Gi) (gene for neuroinhibiting Gi-coupled Kappa Opioid Receptor-derived DREADD receptor); IRES (internal ribosome entry site for expressing downstream gene cassettes); mCitrine (yellow fluorescent protein marker fusion-gene cassette); E-SARE (Enhanced Synaptic Activity Responsive Element [neuroactivation-dependent] transgene promoter); ERT2CreERT2 (fusion-gene cassette encoding Cre- loxP recombinase fusion protein activated by 4-OH-TMX via fused estrogen receptor domains); PEST (fusion-gene cassette for proline-, glutamic acid-, serine-, & threonine-rich peptide sequence conferring rapid protein degradation); hM3Dq (gene for neuropotentiating Gq-DREADD receptor); Hdc-Cre (histaminergic neuron-specific Histidine decarboxylase gene promoter-expressed Cre-loxP recombinase transgenic mice). Neuropharmacological Terms: D1+ (D1 receptor-expressing); CTX (cortex/cortical); GLU (glutamatergic); AMY (amygdala/amygdalar); STR (striatum/striatal); GABA (gamma-amino-butyric acid); OCD (obsessive-compulsive disorder); TTM (trichotillomania); D1+ (DA D1 receptor-expressing); D2+ (DA D2 receptor-expressing); DP (Direct Pathway [striatonigral D1+, motor-urge exciting]); IP (Indirect Pathway [striatopallidal D2+, motor-urge inhibiting]); MSNs (Medium Spiny Neurons [GABAergic STR output neurons]); TS (Tourette’s Syndrome); D2+ (D2 receptor- expressing); PPI (prepulse inhibition); OFC (orbitofrontal CTX); VM (ventromedial); FSIs (inhibitory parvalbumin-positive Fast Spiking Interneurons); DM (dorsomedial); 6-OHDA (6- hydroxydopamine); STN (Subthalamic nucleus); DBS (Deep Brain Stimulation); PD (Parkinson’s Disease); NAc (nucleus accumbens); CNO (clozapine-N-oxide, drug agonist of neuropotentiating Gs/Golf-DREADD receptor rM3Ds, neuroinhibiting Gi-DREADD receptor hM4Di, and neurostimulating Gq-DREADD receptor hM3Dq); TMN (hypothalamic tuberomammillary nucleus [supplying hypothalamocortical & hypothalamostriatal histamine]); mPFC (medial prefrontal cortex); 4-OH-TMX (4-hydroxy-tamoxifen, drug activator of recombinant ERT2CreERT2 tamoxifen-dependent Cre- loxP recombinase); SalB (Salvinorin B, drug agonist of neuroinhibiting Gi-DREADD receptor KORD(Gi)).

38 Table 2. Neuroinhibitory CSTC circuit-tests of TS- & OCD-like symptoms

Circuit Reference Method Transgene(s) Behavioral effect(s) element(s) (s)

Chemogenetic Decreased spontaneous rotational locomotion (hemilateral DP) vs. increased Neuroblockade spontaneous rotational locomotion (hemilateral IP); abrogated acute DA AAV-SP(vs.ENK)-tTA vector TRE- (Intracellular Tetanus D1+ DP vs. D2+ IP [methamphetamine- or cocaine-] induced locomotion (DP or IP); Inhibited OCD-like Hikida et al., CMV-GFP-TN mice STR & NAc  Toxin STR & NAc MSNs cocaine-sensitized locomotion & reward-learning [conditioned place preference] 2010 DOX+/- Light Chain) (DP); TS- & OCD-like inhibited aversive self-control behavior [one-trial shock place- aversion] (IP) Photic Increased NAc MSN output & disrupted DA-dependent reward-seeking (cocaine- Neuroinhibition conditioned place preference); no behavioral effects in absence of cocaine; No AAV-Ef1a-DIO-eNpHR3.0-EYFP vector NAc ACH+ interneuron Witten et al., (Optogenetics) impairment of fear conditioning. No reward-seeking or enhancement of DA-  ChAT-Cre mice NAc subpopulation 2010 dependent reward-seeking from conversely increasing NAc ACH+ interneuron output (AAV-Ef1a-DIO-ChR2-EYFP) Smith et al., Disrupted new habits while restoring older ones [implies neuron output greenlights AAV-CaMKII eNpHR3.0-EYFP vector infralimbic CTX GLU 2012; Smith latest habit]; Prevented OCD-like acquisition of new habits by over-training; No  mice CTX output neurons and Graybiel, effect on motor activity, reward, or aversion memory 2013 Chemogenetic AAV-hSyn-DIO-hM4Di-mCherry + Neuroinhibition OFC GLU output Disrupted OCD-required conferral of value on goal-directed acts (prevented different Gremel and AAV-CaMKII-GFP-Cre vectors  mice (Gi-coupled neurons lever-pressing counts when hungry vs. satiated) Costa, 2013 CTX  CNO DREADDs) HSV-DYN(vs.ENK)-HA-hM4Di vector D1+ DP vs. D2+ IP Inhibited retention of amphetamine-sensitization of locomotion (DP) vs. OCD-like Ferguson et

 rat STR  CNO Dorsal STR MSNs increased sensitization to & retention of amphetamine-dependent locomotion (IP) al., 2011 HSV-DYN-HA-hM4Di vector  rat D1+ DP DM STR Ferguson et Inhibited retention of more-rewarding strategies [lever choices] STR  CNO MSNs al., 2013 AAV-hSyn-DIO-hM4Di-mCherry vector Induced OCD-like “compulsive” drug-seeking [cocaine requests continue without Bock et al., D2+ IP NAc MSNs  Adora2a-Cre mice NAc  CNO reward; hyper-motivated, increasing requests with reward] 2013 Activity-Dependent AAV5-hSyn-DIO-hM4Di-mCherry [1], Chemogenetic mPFC & dorsal STR AAV2-E-SARE-ERT2CreERT2-PEST [2] DREADD-inhibiting hypothalamic [TMN] histamine input to mPFC & dorsal STR Neuroinhibition neurons previously + AAV9-hSyn-DIO-HA-KORD(Gi)- increased TS- & OCD-like grooming & stereotypic locomotion; TS- & OCD-like (E-SARE-Cre- activated by Rapanelli et IRES-mCitrine [3] vectors  Hdc-Cre effects reversed by either re-infusing histamine [dorsal STR only] or by DREADD- assembled TS- & OCD-like al., 2017b mice TMN [1], mPFC + dorsal STR inhibiting dorsal STR neurons previously activated by DREADD-inhibition of Gi-coupled depletion of histamine [2,3]  CNO [1] + 4-OH-TMX [2]; hypothalamic histamine input. DREADDs) input CNO [1] +/- SalB [3]

AAV2-E-SARE-ERT2CreERT2-PEST [1] H3+ dorsal STR Inhibited, in a histamine-negative HdcKO mouse TS & OCD model, + AAV9-hSyn-DIO-HA-KORD(Gi)- neurons supersensitized TS- & OCD-like stereotypic locomotion and TS-like stereotypic Rapanelli et IRES-mCitrine [2] vectors  HdcKO by chronic loss of licking/sniffing/shaking induced by prior drug [RAMH]-activation of basally- and/or al., 2017a mice dorsal STR  RAMH + 4-OH- histaminergic input DA- supersensitized histamine H3+ dorsal STR neurons TMX [1]; RAMH +/- SalB [2])

Transgenic Components: AAV (Adeno-Associated Virus vector); SP (Substance P gene [D1+ DP STR MSN-specific] promoter]; ENK (Enkephalin gene [D2+ IP STR MSN-specific] promoter]; tTA (flag-tagged Tetracycline [Doxycycline]-repressed Transactivator fusion-gene cassette); TRE-CMV-GFP-TN (Tetracycline Response Element-Cytomegalovirus promoter-Green Fluorescent Protein-Tetanus Toxin Light Chain fusion-gene transgenic mice); Ef1a (Elongation-Factor-1a gene strong constitutive promoter); DIO (Double-floxed Inverted Open-reading-frame [paired loxP sites surrounding inactive antisense-oriented transgene cassette for Cre-loxP recombinase to invert it to active sense-orientation]); eNpHR (Enhanced Halorhodopsin photoactivated inhibitory chloride channel gene cassette); EYFP (Enhanced Yellow Fluorescent Protein yellow marker fusion-gene cassette); ChAT-Cre (BAC transgenic mice containing choline acetyltransferase gene promoter regulated Cre-loxP recombinase gene); ChR2 (Channelrhodopsin-2- photoactivated excitatory cation channel gene cassette); CaMKII (Calcium Calmodulin Kinase-II alpha gene cortical pyramidal neuron-specific promoter); hSyn (human Synapsin gene promoter); hM4Di (gene cassette for neuroinhibiting Gi-DREADD receptor); GFP (Green Fluorescent Protein fusion-gene cassette); mCherry (red fluorescent protein marker fusion-gene cassette); Cre (Cre-loxP recombinase transgene); HSV (Herpes Simplex Virus Amplicon vector); DYN (Dynorphin gene [D1+ DP STR MSN-specific] promoter]; HA (Hemagglutinin tag fusion-gene cassette); Adora2a-Cre (Bacterial Artificial Chromosome [BAC] transgenic mice containing Adenosine A2a receptor Adora2a D2+ MSN-specific gene promoter-regulated Cre-loxP recombinase gene); E-SARE (Enhanced Synaptic Activity Responsive Element [neuroactivation-dependent] transgene promoter); ERT2CreERT2 (fusion-gene cassette encoding Cre-loxP recombinase fusion protein activated by 4-OH-TMX via fused estrogen receptor domains); PEST (fusion-gene cassette for proline-, glutamic acid-, serine-, & threonine-rich peptide sequence conferring rapid protein degradation); KORD(Gi) (gene for neuroinhibiting Gi-coupled Kappa Opioid Receptor-derived DREADD receptor); IRES (internal ribosome entry site for expressing downstream gene cassettes); mCitrine (yellow fluorescent protein marker fusion-gene cassette); Hdc-Cre (histamine-synthesizing neuron-specific Histidine decarboxylase gene promoter-expressed Cre-loxP recombinase transgenic mice); HdcKO (Histidine decarboxylase gene knockout mouse model of histamine-deficient comorbid TS & OCD). Neuropharmacological Terms: STR (striatum/striatal); NAc (nucleus accumbens); DOX+/- (experimental addition or cessation of neuroblockade-reversing doxycycline, a drug repressor of tTA and its consequent TRE-CMV-GFP-TN expressed Tetanus Toxin light chain); D1+ (DA [dopamine] D1 receptor expressing); DP (Direct Pathway [striatonigral D1+, motor-urge exciting]); D2+ (DA [dopamine] D2 receptor-expressing); IP (Indirect Pathway [striatopallidal D2+, motor-urge inhibiting]); MSNs (Medium Spiny Neurons [GABAergic STR output neurons]); ACH+ ([acetyl]cholinergic); DA (dopamine); CTX (cortex/cortical); GLU (glutamatergic); CNO (clozapine-N-oxide, drug agonist of neuropotentiating Gs/Golf-DREADD receptor rM3Ds and of neuroinhibiting Gi-DREADD receptor hM4Di); OFC (orbitofrontal CTX); DM (dorsomedial); TMN (hypothalamic tuberomammillary nucleus [supplying hypothalamocortical & hypothalamostriatal histamine]); mPFC (medial prefrontal cortex); 4-OH-TMX (4-hydroxy-tamoxifen, drug activator of recombinant ERT2CreERT2 tamoxifen-dependent Cre-loxP recombinase); SalB (Salvinorin B, drug agonist of neuroinhibiting Gi-DREADD receptor KORD(Gi)); H3+ (histamine H3 receptor-expressing neurons); RAMH (R-aminomethylhistamine, drug agonist of H3 receptors).