1 

Cortical/amygdalar glutamatergic-circuit breakers alleviate 1 

tics in a transgenic Tourette's syndrome model 2 

3 

Eric J. Nordstroma,b, Katie C. Bittnera,1, Michael J. McGratha,2, Clinton R. Parks IIIa,3, 4 

Frank H. Burton§,a,b 5 

6 

aDepartment of Pharmacology, University of Minnesota, 6-120 Jackson Hall, 321 7 

Church Street SE, Minneapolis MN 55455-0217 USA 8 

bMinneapolis Medical Research Foundation, Hennepin County Medical Center, 701 9 

Park Ave, Shapiro S3.111, Minneapolis MN 55415-1623 USA 10 

11 

1Present Address: HHMI Janelia Farm Research Campus, 19700 Helix Dr, Ashburn, VA 12 

20147, USA 13 

2Present Address: Seager, Tufte & Wickem, 1221 Nicollet Avenue, Suite 800, 14 

Minneapolis, MN 55403-2420 USA 15 

3Present Address: SpaceNews, Inc. 1414 Prince Street, Suite 300, Alexandria, VA 22314, 16 

USA 17 

18 

§Corresponding Author: Frank H. Burton, Ph.D., Department of Pharmacology, 19 

University of Minnesota, 6-120 Jackson Hall, 321 Church Street S.E., Minneapolis MN 20 

55455-0217 USA; E-mail: burto006@umn.edu 21 

  2 

22 

Running Title: Cortico/amygdalostriatal circuit breakers alleviate tics 23 

24 

Keywords: Tics, glutamate, transgenic, D1CT-7, mice, ritanserin, prazosin, moxonidine, 25 

bromocriptine 26 

27 

Total number of pages: 57 28 

29 

Number of Figures: 5 30 

31 

  3 

Abbreviations 32 

33 

5-HT, 5-hydroxytryptamine; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; 34 

AMY; amygdala; ANOVA (analysis of variance); cAMP, 3',5'-cyclic adenosine 35 

monophosphate; CGN, "cortical/limbic glutamatergic neuron" hyperactivity model of tics and 36 

compulsions; CSTC, cortico/amygdalo-striato-thalamo-cortical; CT, cholera toxin; CTX, 37 

cortex; D1, dopamine receptor subtype 1; D1+, D1 receptor-containing; D1CT-7, Dopamine 38 

receptor 1 gene (DRD1)-promoter/cholera toxin A1 subunit transgenic sub-strain 7 mouse; 39 

D2, dopamine receptor subtype 2; DA, dopamine; DP, striatal direct pathway; DTM, 40 

dermatillomania; GABA, gamma-amino butyric acid; Glu, glutamate; GS, stimulatory G 41 

protein; I-1, imidazoline-1; IP, striatal indirect pathway; NMDA, N-methyl-D-aspartate; OC; 42 

obsessive-compulsive; OCD; obsessive-compulsive disorder; PCP, phencyclidine; SEM, 43 

standard error of the mean; SNc, substantia nigra pars compacta; STR, striatum; Tg, 44 

transgenic; TS, Tourette's syndrome; TS+OCD, Tourette's syndrome and obsessive-45 

compulsive disorder; TTM, trichotillomania. 46 

  4 

ABSTRACT 47 

48 

The brain circuit that evokes the tic symptoms of Tourette's syndrome (TS) is unknown 49 

but thought to involve hyperactivity of the cortico/amygdalo-striato-thalamo-cortical 50 

(CSTC) circuit loop. We previously engineered a transgenic mouse model of TS by 51 

expressing an artificial neuropotentiating transgene (encoding the cAMP-elevating, 52 

intracellular A1 subunit of cholera toxin) within a cortical-amygdalar subset of dopamine 53 

D1 receptor-expressing neurons whose potentiation excites pyramidal glutamatergic 54 

somatosensory-motor-orbitofrontal cortical and limbic output circuits to the striatum that 55 

are thought to be hyperactive in TS and comorbid obsessive-compulsive (OC) spectrum 56 

disorders. These D1CT-7 ("Ticcy") transgenic mice's tics responded to clonidine, a 57 

therapeutic TS drug, and their corticostriatal circuit was, like TS patients', sensitive to 58 

inhibition by postsynaptic D2 receptor antagonists. To test the hypothesis that 59 

cortical/amygdalar glutamatergic output circuit hyperactivity mediates tics, we've now 60 

examined in these mice the tic-alleviating ability of drugs that counter different elements 61 

of this circuit -- namely, anti-serotonoceptive and anti-noradrenoceptive corticostriatal 62 

pyramidal output blockers, agmatinergic (imidazoline receptor-mediated) anti-63 

GABAergic striatothalamic presynaptic inhibitors, and anti-dopaminergic nigrostriatal 64 

presynaptic inhibitors. Drugs of each class (the serotonin 5-HT2a,c receptor antagonist 65 

ritanserin, the NE alpha-1 receptor antagonist prazosin, the presynaptic 66 

agmatine/imidazoline I1 receptor agonist moxonidine, and the presynaptic dopamine D2 67 

receptor agonist bromocriptine) each fully alleviate tic symptoms in the Ticcy mice. This 68 

  5 

supports a hyperglutamatergic "tic circuit" wherein cortical/amygdalar pyramidal 69 

projection neurons' glutamatergic overexcitation of both striatal effector neurons and 70 

nigral modulatory neurons hyperactivates these target subcircuits and unbalances their 71 

integration to create tics, illuminating novel drug strategies for treating TS. 72 

73 

INTRODUCTION 74 

75 

Touretteʼs syndrome (TS) is characterized by voluntarily suppressible, urge-driven 76 

motor and/or vocal tics and repeated complex movements, which are more prevalent 77 

and severe in males than females, and often arising in childhood (Robertson, 2000). TS 78 

is also frequently (>50%) co-morbid with obsessive-compulsive disorder (OCD), 79 

including its related OC-spectrum hair- and skin- pulling and picking disorders 80 

trichotillomania (TTM) and dermatillomania (DTM) or excoriation disorder (American 81 

Psychiatric Association, 2013), which suggests all these neurological disorders share 82 

overlapping brain circuitry. For example, analogous to motor urges in TS, OCD is 83 

characterized by anxiogenic thought-urges, or “obsessions,” and urge-driven ritualistic 84 

or repeated actions, or “compulsions,” arising either as compensatory behaviors to 85 

reduce anxiety (American Psychiatric Association, 2013) or, more so in childhood-onset 86 

TS+OCD and OCD, as primary compulsions without obsessions (Rapoport et al., 1992). 87 

Both animal and clinical studies support a role for hyperactivity of cortical and 88 

amygdalar forebrain glutamatergic output neurons in causing or mediating tic and OC 89 

disorders (Campbell et al., 1999a; McGrath et al., 2000; Carlsson, 2000; Rosenberg et 90 

  6 

al., 2000; Nordstrom and Burton, 2002; Singer et al., 2010; Milad and Rauch, 2012). TS 91 

and OCD are associated with cortical neuron hyperexcitability and impaired 92 

sensorimotor gating (Swedo et al., 1992; Breiter et al., 1996; Ziemann et al., 1997; 93 

Edgley and Lemon, 1999; Gilbert et al., 2004), possibly from impaired neural inhibition 94 

in some inherited forms, e.g., CNTNAP2 gene deletion (Verkerk et al., 2003), or from 95 

diminished activity of cortical inhibitory (GABAergic) interneurons (Minzer et al., 2004). 96 

Observed reductions in forebrain metabolic activity and blood flow in TS (Swerdlow and 97 

Sutherland, 2005) may be consistent with diminished metabolic activity of cortical 98 

inhibitory interneurons, where the interneuron "brake's" failure permits increased 99 

excitatory glutamate output from the smaller population of corticostriatal pyramidal 100 

projection neurons, as has been postulated to occur in other psychotic-spectrum 101 

disorders and glutamate-modulating psychogenic drug symptoms (Homayoun and 102 

Moghaddam, 2007). Consistent with that interpretation, elevated glutamatergic efflux to 103 

the striatum is observed in TS and OCD (Campbell et al., 1999a; McGrath et al., 2000; 104 

Carlsson, 2000; Rosenberg et al., 2000; Nordstrom and Burton, 2002; Singer et al., 105 

2010). Possibly as a response to corticostriatal glutamatergic hyperexcitation, TS and 106 

OCD patients also display striatal desensitization and volumetric damage (Peterson et 107 

al., 1993; Peterson et al., 1998; Menzies et al., 2008), wherein glutamate may either 108 

trigger cross-talk pharmacodynamic up-regulation (supersensitization) of neuroinhibitory 109 

striatal dopamine D2 receptors (Wolf et al., 1996) or directly up-regulate nigrostriatal 110 

dopamine release (Singer et al., 2010) and conversely desensitize striatal D2 receptors 111 

(Denys et al., 2013), but in either case dopaminergically counter-inhibiting the 112 

  7 

glutamatergically overexcited D2+ striatal neurons (also then explaining why TS 113 

symptoms are sensitive to inhibition by D2 antagonists) (Campbell et al., 1999b). The 114 

apparently distinct mechanisms of action of two early classes of TS drug -- inhibitory 115 

postsynaptic D2 receptor antagonists like haldol or pimozide, and the stress-relieving 116 

inhibitory norepinephrine (NE) alpha-2 receptor agonist clonidine -- also are consistent 117 

with a hyperglutamatergic forebrain circuit model of TS and OCD. Both classes counter 118 

corticostriatal glutamatergic circuit activity and its movement-stimulating effects: D2 119 

antagonists by disinhibiting both cortical GABAergic inhibitory interneurons and striatal 120 

movement-suppressing indirect pathway neurons (Campbell et al., 1999b; Nordstrom 121 

and Burton, 2002; Minzer et al., 2004), and clonidine by possibly blocking noradrenergic 122 

excitation of amygdalostriatal and/or corticostriatal glutamatergic pyramidal output 123 

neurons (Lichter and Jackson, 1996; Nordstrom and Burton, 2002). 124 

The first (and so far only) genetically-engineered model of TS that exhibits tics is 125 

the D1CT-7 "Ticcy" transgenic mouse (Campbell et al., 1999a; Nordstrom and Burton, 126 

2002) -- created as an early, pre-millennial foray into "brain circuit-testing" with an 127 

artificial gene to induce neuropotentiation. Preceding by a decade the development of 128 

optogenetic light-activated artificial channel transgenes designed to directly depolarize 129 

and fire neurons, the artificial transgene in Ticcy mice in contrast chronically induces the 130 

intracellular second-messenger, cAMP (3',5'-cyclic adenosine monophosphate), to 131 

potentiate neurons' responsiveness to their own endogenous neurotransmitters 132 

(Campbell et al., 1999a). This was achieved by D1 receptor gene promoter-targeted 133 

expression of an artificial transgene encoding the exclusively intracellular A1 subunit of 134 

  8 

cholera toxin (CT), which, by covalently activating the stimulatory G protein GS, 135 

chronically stimulates adenylate cyclase activity and cAMP production (Burton et al., 136 

1991; Zeiger et al., 1997). Furthermore, D1CT expression in the D1CT-7 ("Ticcy") line 137 

of such transgenic mice is regionally restricted to a cortical/limbic subset of brain D1 138 

receptor-containing (D1+) neurons, with no expression in striatum. These cortical/limbic 139 

D1+ neurons, once potentiated by intracellular CT, induce in D1CT-7 mice voluntarily-140 

suppressible, juvenile-onset tics (Nordstrom and Burton, 2002) and compulsions 141 

(Campbell et al., 1998; Campbell et al., 1999a), with increased tic severity in males 142 

(Nordstrom and Burton, 2002), stress sensitivity (McGrath et al., 1999a,b), and 143 

alleviation by TS drugs of multiple classes (Nordstrom and Burton, 2002; Campbell et 144 

al., 1999b) -- thus showing the greatest behavioral homology to TS+OCD of any animal 145 

model reported so far (Burke and Lombroso, 2004). Based on their cortical circuit's 146 

hyperglutamatergic status (normal mice show slower onset and calmer 147 

pentylenetetrazole-kindled cortical seizures, and less pronounced glutamatergic drug-148 

induced locomotion) (Campbell et al., 2000; McGrath et al., 2000) and associated tic-149 

compulsion phenotype (Campbell et al., 1999a; Nordstrom and Burton, 2002), and on 150 

the known cortical and limbic excitatory output triggered by these transgenically-151 

potentiated D1+ neurons (which comprise cortical somatosensory/insular/piriform D1+ 152 

glutamatergic pyramidal output neurons and amygdalar intercalated nucleus D1+ 153 

GABAergic stellate interneurons, that respectively directly and indirectly trigger 154 

glutamatergic excitation of the striatum from deep-layer somatosensory-motor-155 

orbitofrontal cortical pyramidal and amygdalar pyramidal output neurons) (Campbell et 156 

  9 

al., 1999a,c; Campbell et al., 2000), the Ticcy D1CT-7 transgenic mice comprised a 157 

direct test of the hypothesis that corticostriatal and/or amygdalostriatal glutamatergic 158 

circuit hyperactivity can cause tics and compulsions (Campbell et al., 1999a; Carlsson, 159 

2000; Nordstrom and Burton, 2002). 160 

Studies and reviews of the D1CT-7 mice (Sah and Sallee, 2002; Burke and 161 

Lombroso, 2004; Swerdlow and Sutherland, 2005; Joel, 2006; Ting and Feng, 2008; 162 

Wang et al., 2009; Wu et al., 2012) have so far helped inspire several clinical studies. 163 

These include studies of glutamate's role in tics and compulsions (Chakrabarty et al., 164 

2005; Singer et al., 2010), successful clinical trials of antiglutamatergic drugs for the 165 

OC-spectrum disorders OCD (Lafleur et al., 2006; Grant et al., 2007) and TTM (Grant et 166 

al., 2009); and successful clinical trials of the D1 antagonist, ecopipam, for TS (Gilbert 167 

et al., 2014) and OC-spectrum gambling (impulse-control) disorder (Grant et al., 2014). 168 

Additional clinical study recommendations could be provided by understanding what 169 

pharmacological targets within the Ticcy mice's cortical/amygdalar glutamate-dependent 170 

hyperactive circuitry can act as "circuit breakers" to suppress tics. In this study we have 171 

confirmed that four such postulated circuit breakers -- serotonin 5-HT2a,c receptor 172 

antagonists, NE alpha-1 receptor antagonists, presynaptic agmatine/imidazoline I1 173 

receptor agonists, and presynaptic dopamine D2 receptor agonists -- are capable of 174 

short-circuiting tics elicited by neurogenic hyperactivity of forebrain 175 

cortico/amygdalostriatal and cortico/amygdalonigral glutamatergic outputs, and thus are 176 

prospective sources of new clinical treatments to alleviate neurogenic tics in TS. 177 

178 

  10 

EXPERIMENTAL PROCEDURES 179 

180 

Animal subjects 181 

182 

Studies of drug effects on tic incidence and locomotion used 30 adult Balb/c-inbred (JAX 183 

labs, Bar Harbor, ME, USA) female wild-type control (C) mice and 32 adult Balb/c-184 

inbred female hemizygous D1CT-7 ("Ticcy") transgenic (Tg) sibling mice. Because Tg 185 

females breed and nurse poorly due to Tg-induced anxiogenic fleeing from males and 186 

over-grooming and biting of pups (Campbell et. al., 1999a; Nordstrom and Burton, 187 

2002), Tg males must be used as breeders to maintain the Ticcy mouse colony, while 188 

Tg females are used for drug studies. All animals were naive to behavioral or drug 189 

assays prior to testing, and experiments were carried out with the investigators blinded 190 

as to the animals' transgenic or control genotype status and drug injection status. All 191 

mice were housed in groups of 2-5 in a temperature-controlled room on a 12-hour day-192 

night cycle, allowed unrestricted access to food and water with the exception of testing 193 

times, and drug-treated and videotaped at the same daytime range of hours to control 194 

for the possibility of circadian fluctuation in drug response. Care was taken to ensure 195 

that the animals used in this study received no unnecessary discomfort. All animals 196 

were maintained and procedures were performed in accordance with the Animal 197 

Welfare Act and the NIH Guide for the Care and Use of Laboratory Animals, under the 198 

approval of the University of Minnesota Institutional Animal Care and Use Committee. 199 

  11 

The University of Minnesota Research Animal Resources facility is fully accredited by 200 

the American Association for the Accreditation of Laboratory Animal Care. 201 

202 

Drugs & Injections 203 

204 

Except where indicated, for each drug study mice in both genotype groups were 205 

administered stock solutions of drug or saline vehicle, 24 hours apart, at the injection 206 

volumes and at the acute doses, as well as assayed behaviorally at the post-injection 207 

times, that were previously reported to induce a maximal behavioral effect; and the 208 

behavioral observation of videotapes were performed blinded as to the animals' 209 

transgenic or control genotype status and drug or saline injection status, while 210 

observation counts were confirmed by a repeat observer. 211 

Ritanserin (Research Biochemicals International, Natick, MA, USA) was prepared 212 

as a stock solution (0.1 mg/ml ritanserin, 0.04% Tween-80 in saline) by suspending 10 213 

mg drug in 2 ml of 2% Tween-80 followed by 50-fold dilution in saline vehicle (0.9% 214 

NaCl). All administrations of ritanserin or vehicle were delivered intraperitoneally (i.p.) in 215 

an injection volume of 10 ml/kg body weight. The 1 mg/kg ritanserin dosage was chosen 216 

for this study based on its reported efficacy in alleviating abnormal behaviors triggered 217 

via serotonin 5-HT2a receptors without any concomitant inhibition of spontaneous 218 

locomotor activity (Ninan and Kulkarni, 1998), which we confirmed as described in 219 

Results. Likewise, Tween-80/saline vehicles ranging in Tween-80 concentrations of up 220 

to 32% reportedly have no motor-inhibiting effects (Castro et al., 1995), which we 221 

  12 

confirmed by comparison of both tics and locomotor activity levels in saline-injected 222 

versus 0.04% Tween-80/saline-injected transgenic mice (not shown). 223 

Prazosin hydrochloride was obtained from Research Biochemicals International 224 

(Natick, MA) and dissolved in 0.9% saline. The drug was administered in a volume of 225 

10ml/kg body weight, at a previously reported effective dosage of 3mg/kg (i.p.) 226 

(Wellman and Davies, 1992; Wellman et al., 1997; Cheng and Kuo, 2003). 227 

Moxonidine hydrochloride was obtained from Research Biochemicals 228 

International (Natick, MA) and dissolved in 0.9% saline. The drug was administered i.p. 229 

in a volume of 10ml/kg body weight, at a previously reported effective dosage of 230 

0.5mg/kg (Zhu et al., 1999). 231 

Bromocriptine methanesulfonate was obtained from Research Biochemicals 232 

International (Natick, MA) and dissolved in 0.9% saline. The drug was administered in a 233 

volume of 10ml/kg body weight, at a previously reported effective dosage of 5 mg/kg 234 

(i.p.) (Jackson et al., 1988). At this dosage, bromocriptineʼs D2 agonist action is 235 

reported to be on presynaptic autoreceptors at two hours post-injection (Jackson et al., 236 

1988). 237 

238 

Tic Behavior Quantification 239 

240 

The incidence of tic-like behavior was determined in videotapes of transgenic versus 241 

control non-transgenic littermate female mice. Tics were defined as any very brief (0.05-242 

0.1 sec, as determined by a duration of 1.5 to 3 frames in 30 fps videotape recordings) 243 

  13 

isolated head and/or body jerk or shake, other than those associated with acoustic 244 

startle or obvious shedding of litter visible on the coat. By this definition normal mice 245 

exhibit tic-like twitches only infrequently, compared to 3- to 5-fold more frequent tic-like 246 

twitches in D1CT-7 transgenic mice (Nordstrom and Burton, 2002). The effect on tic 247 

incidence of vehicle versus drug treatment was determined as the mean number of 248 

tics/15 min in transgenic or control mice observed over a 15 minute period beginning 30 249 

minutes after either vehicle or drug injection and 15 minutes after introduction of the 250 

mice into a new cage (i.e., after a 15 minute cage habituation period), with these 251 

exceptions: 1) In the ritanserin study, 15 minute cage habituation was omitted to avoid 252 

confounding a reported anxiolytic effect of ritanserin (Gao and Cutler, 1993) with any 253 

potential anxiolytic effect of cage habituation, while tic counts were instead observed 254 

and analyzed over a 30 minute period beginning 30 minutes after either vehicle or drug 255 

injection -- however, because post-hoc analysis confirmed there was no significant 256 

effect of 15 min cage habituation upon control or transgenic tic incidence in the 257 

presence or absence of ritanserin treatment (not shown), the tics/30min data were 258 

adjusted for figure display to the standard tics/15 min; 2) In the bromocriptine study, the 259 

post-injection observations commenced not 30 minutes but two hours after injection of 260 

bromocriptine, in keeping with the reported maximal presynaptic agonist action of the 261 

drug in mice (Jackson et al., 1998), while pre-injection observational data was obtained 262 

15 minutes prior to bromocriptine injection with no saline vehicle injection, to match 263 

Jackson et al.'s prior reported drug design. No significant difference was observed in 264 

Ticcy mouse tic counts between the bromocriptine study's "15 min no-drug pre-injection 265 

  14 

vs. 2 hr post-injection drug" design and the remaining drug studies' "24 hr-separated 266 

vehicle- vs. drug- injection" design. Videotapes and/or drug & vehicle samples were 267 

coded to blind observers to the mice's genotype and drug-injection status, and logged 268 

data were confirmed by at least one additional independent observer. 269 

270 

Locomotion Behavior Quantification 271 

272 

In all studies, to measure spontaneous locomotor activity levels during the observation 273 

periods the same videotapes as described above were analyzed for the number of 274 

cage-midline crossings (the observed number of locomotion-dependent cage midline 275 

incursions, which reproduces an automated beam-break design, as described by 276 

Nordstrom and Burton, 2002). Data are displayed as the number of midline crossings/15 277 

min. At least two observers blinded to subject genotype and treatment independently 278 

scored the numbers of midline crossings from the original videotapes, confirming 279 

excellent interrater reliability (Intraclass Correlation Coefficient [ICC] > 0.8). Additionally, 280 

for the bromocriptine study, because this dopaminergic motor output-inhibiting drug 281 

(unlike ritanserin, prazosin and moxonidine) is reported to also diminish mouse motor 282 

activity (Jackson et al., 1998), a more comprehensive behavioral analysis of this drug's 283 

locomotor suppressing effects was also performed, as described in the next section. 284 

285 

Waveform display analysis of bromocriptine treated mice 286 

287 

  15 

Waveform display analysis was performed as previously described (Campbell et al., 288 

1998). Briefly, the above-described videotapes of drug-naive D1CT-7 or control females 289 

littermates were continuously observed for 15 minutes prior to bromocriptine injection 290 

and 2 hours post-injection, in each case after 15 minutes of habituation to a new cage. 291 

EthoMac (v1.10, © The University of Minnesota) software was used for behavioral state 292 

entry, and for calculation and tabulation of behavioral state event timing, number, and 293 

duration. Scored behaviors included: 1) climbing/leaping (animal standing on its hind 294 

paws in the corner of the cage moving at least three limbs); 2) still (remaining in one 295 

position with an occasional head movement); 3) rear; 4) gnaw (gnawing against the side 296 

of the Plexiglas cage); 5) (horizontal) locomotion; 6) dig (into the sawdust bedding); 7) 297 

groom; 8) hang (from the wire bar cage lid); 9) eat (bedding picked from the cage 298 

bottom and put into the mouth); 10) sniff; 11) other (any activity that does not fit into the 299 

previous categories). The total observer-scored number of locomotion events for each 300 

mouse under each drug condition, as tabulated by the EthoMac logs, was confirmed by 301 

at least one independent, genotype- and treatment- blinded observer from original 302 

coded videotapes, confirmed to have ICC > 0.8 interrater reliability, then statistically 303 

compared for the extent of bromocriptine and genotype effects and displayed as the 304 

mean number of locomotion events/15 min. 305 

306 

Statistical Analyses 307 

308 

Overall statistical significance of a ritanserin, prazosin, moxonidine, or bromocriptine 309 

  16 

drug (within-groups) effect, Ticcy transgenic (Tg) vs. control (C) wild-type genotype 310 

(between-groups) effect, or a drug by genotype interaction, was determined in Statview 311 

4.5 (Abacus Corp., Berkeley CA, USA) by initial repeated-measures analysis of variance 312 

(repeated-measures ANOVA) on both tic and locomotion behavioral measures, followed 313 

by individual between-group unpaired, two-tailed Studentʼs t-test comparisons of 314 

genotype effect and within-group paired, two-tailed Studentʼs t-test comparisons of drug 315 

effect, with significance assumed at P < 0.05, for parametrically-distributed locomotion 316 

data; or individual between-group Mann-Whitney U-tests of genotype effect and within-317 

group Wilcoxon Signed Rank tests of drug effect, with significance assumed at tied P < 318 

0.05, for the non-parametrically-distributed tic incidence data. Because elevated tic 319 

counts in the Ticcy genotype population routinely sort into a non-parametric biphasic 320 

distribution caused by the presence within the Ticcy group of epigenetically-variable but 321 

individually-consistent "super (6-fold) ticcers" and "elevated (3-fold) ticcers," the use of a 322 

repeated-measures drug design, as performed by Nordstrom and Burton (2002) and 323 

herein, where each subjects' behavior is tested both with and without drug injection, 324 

permits drug effects to be tested reliably on such populations even though the mean 325 

elevation and standard error of tic incidence may vary from one drug study to another 326 

depending on each study population's random percentage of "super-ticcers." All data 327 

were expressed as the mean plus standard error of the mean (S.E.M.) of the number of 328 

tics per 15 minutes, cage midline crossings per 15 minutes, or locomotion events per 15 329 

minutes, occurring during the videotaped windows of observation. 330 

331 

  17 

RESULTS 332 

333 

Deep-layer cortical pyramidal glutamatergic output neurons are known to express 334 

excitatory serotonin 5-HT2a,c receptors (Sheldon and Aghajanian, 1991; Nestler, 1997; 335 

Jakab and Goldman-Rakic, 1998; Marek and Aghajanian, 1998; Marek and Aghajanian, 336 

1999; Aghajanian and Marek, 1999), suggesting a potential therapeutic role in TS for 5-337 

HT2a,c antagonists like ritanserin or ketanserin (shown effective in a small trial of six TS 338 

patients by Bonnier et al., 1999). Ritanserin also has anxiolytic activities both clinically 339 

and in rodents (Ceulemans et al., 1985; Danjou et al., 1992; Gao and Cutler, 1993), 340 

putatively due to its similar inhibition of amygdalar 5-HT2c receptors (Gibson et al., 341 

1994) -- which may also trigger reduced excitatory amygdalar glutamatergic output to 342 

the limbic cortex, orbitofrontal cortex, and motor striatal circuits. The ability of this pure 343 

5-HT2a,c antagonist, ritanserin (Ceulemans et al., 1985), to suppress tics at a 344 

concentration not inhibitory to mouse locomotor activity was tested in Fig. 1. While the 345 

Ticcy D1CT-7 transgenic (Tg) mice show multiple times the number of TS-like twitches 346 

compared to control non-transgenic control (C) mice, their tics are restored to control 347 

levels by acute ritanserin (1 mg/kg, i.p.) treatment (Fig.1, Panel A, black bars). 348 

Ritanserin treatment did not significantly decrease the control mice's normal, baseline 349 

level of infrequent twitching (Fig. 1, Panel A, white bars). Nor did ritanserin treatment 350 

significantly reduce in either Ticcy or control mice the level of general locomotor activity 351 

(Fig. 2, Panel B), which is displayed as the number of cage midline crossings/15 min. 352 

This is consistent with previous reports that this 1mg/kg ritanserin dose in rodents, 353 

  18 

although psychoactive in reducing anxiety, does not inhibit spontaneous locomotion 354 

(Ninan and Kulkarni, 1998). These data indicate that acute ritanserin treatment 355 

selectively suppresses abnormal ticcing without inhibiting normal, baseline spontaneous 356 

locomotor activities. 357 

A drug thought to decrease corticostriatal glutamatergic output is the alpha-1 358 

antagonist, prazosin (Fig. 2), whose alpha-1 NE receptors were shown to be co-359 

expressed, with the 5-HT2a,c receptor targets of ritanserin, on deep-layer cortical 360 

pyramidal glutamatergic output neurons (Marek and Aghajanian, 1999). Consequently, 361 

the ability of prazosin to suppress corticostriatal glutamatergic tics, at a concentration 362 

reportedly not inhibitory to mouse locomotor activity but capable of psychoactively 363 

countering dopamine-dependent anorexia (Wellman and Davies, 1992; Wellman et al., 364 

1997; Cheng and Kuo, 2003), was tested (Fig. 2). Like ritanserin, acute prazosin 365 

treatment (3 mg/kg, i.p.) treatment of the Ticcy D1CT-7 transgenic (Tg) mice restored 366 

their elevated tic counts to the level of control (C) littermates (Fig. 2, Panel A, black 367 

bars). Prazosin treatment, also like ritanserin, didn't significantly decrease the control 368 

mice's normal, baseline level of infrequent twitches (Fig. 2, Panel A, white bars), nor 369 

significantly alter locomotion in either the Ticcy transgenic mice or control mice (Fig. 2, 370 

Panel B). These data indicate that acute treatment with prazosin, whose alpha 1 NE 371 

receptors are known to co-localize with ritanserin-targeted serotonin 5-HT2a,c receptors 372 

on cortical glutamatergic output neurons, selectively suppresses, as does ritanserin, 373 

abnormal ticcing without inhibiting normal, baseline spontaneous locomotor activities. 374 

The agmatine/imidazoline-1 agonist moxonidine (Fairbanks and Wilcox, 1999; 375 

  19 

Zhu et al., 1999; Taksande et al., 2010; Dixit et al., 2014), a less-sedating, less-376 

hypotensive and less alpha-2 NE receptor-specific relative of the TS-drug clonidine, 377 

may exert distinct central nervous system actions due to its imidazoline-1 (I-1) receptor 378 

specificity. For example, whereas clonidine's presynaptic agonist action on alpha-2 NE 379 

receptors is thought to decrease NE stimulation of anxiogenic amygdalar glutamatergic 380 

output to the limbic cortex and striatum (Lichter and Jackson, 1996; Nordstrom and 381 

Burton, 2002), moxonidine, by selectively acting as a presynaptic I-1 receptor agonist to 382 

inhibit the striatal GABAergic direct- and indirect- pathway neurons targeted by 383 

glutamate (Tanabe et al., 2006) and to reduce glutamate-triggered neurotoxicity 384 

(Bakuridze et al., 2009), may suppress the more distal, striatal-output element within the 385 

cortico/amygdalo-striato-thalamo-cortical (CSTC) tic-circuit. Consequently, we tested 386 

the ability of acute i.p. moxonidine to block hyperglutamatergic-mediated tics at a 0.5 387 

mg/kg dose sufficient for peripheral reduction of blood pressure (Zhu et al., 1999), as 388 

well as for CNS reduction of drug withdrawal-induced anxiety and endogenous anxiety-389 

dependent compulsive (marble-burying) behavior in rodents (Taksande et al., 2010; 390 

Dixit et al., 2014) (Fig. 3). Like acute ritanserin and prazosin treatments, acute 391 

moxonidine treatment of the Ticcy D1CT-7 transgenic (Tg) mice restored their elevated 392 

tic counts to the level of control (C) littermates (Fig. 3, Panel A, black bars) without 393 

decreasing the control mice's normal, baseline level of infrequent twitches (Fig. 3, Panel 394 

A, white bars) or altering locomotion in either the transgenic or control mice (Fig. 3, 395 

Panel B). These data suggest that acute treatment with moxonidine, whose imidazoline 396 

I-1 receptors are known to localize presynaptically on and inhibit striatal GABAergic 397 

  20 

neurons, can suppress cortico/amygdalostriatal glutamate-induced abnormal ticcing 398 

without inhibiting normal, baseline spontaneous locomotor activities. 399 

The final candidate "tic circuit breaker" drug we examined was the D2 receptor 400 

agonist, bromocriptine (Fig. 4), which at low doses is selectively presynaptic in its action 401 

on striatonigral dopaminergic axon terminals (Ceccherini-Nelli and Guazzelli, 1994) -- 402 

thus reducing nigral dopaminergic efflux onto motion-activating, DA receptor-expressing 403 

striatal neurons (Campbell et al., 1999b), which should counter the tic-inducing effect of 404 

coincident hyperglutamatergic stimulation of the striatal D1+ direct pathway neurons. 405 

Bromocriptine, however, is known to also reduce general locomotor activity as a 406 

consequence of its retardation of DA input to the striatum (Jackson et al., 1988), as do 407 

more classical postsynaptic D2 antagonist drugs (such as haloperidol) for tic and OC-408 

spectrum disorders (Cohen et al., 1992). We consequently examined bromocriptine's 409 

ability to diminish tics and, in this case, total locomotion events, modulated by 410 

dopaminergic inputs to the same GABAergic striatal neurons that stimulate motion and 411 

urges through the direct pathway and suppress them through the indirect pathway, and 412 

that in the Ticcy mice are co-excited by cortical/amygdalar glutamatergic inputs (Fig. 4). 413 

At 2 hours post-injection, when in rodents bromocriptine is documented to exert its 414 

highest presynaptic D2 agonist effect to inhibit nigrostriatal axonal DA release (Jackson 415 

et al., 1988), the elevated tics in the Ticcy D1CT-7 transgenic (Tg) mice were reduced to 416 

the level of untreated control (C) littermates (Fig. 4, Panel A, black bars). But unlike 417 

acute ritanserin, prazosin, or moxonidine, the acute bromocriptine treatment also 418 

significantly decreased control mice's normal, baseline level of infrequent twitches (Fig. 419 

  21 

4, Panel A, white bars) as well as their locomotion events (Fig. 4, Panel B, white bars), 420 

indicating that bromocriptine likely exerts some of its tic-suppressing effects by more 421 

generalized inhibition of baseline levels of locomotor initiation being mediated by the 422 

striatal subcircuit targeted by the convergent cortical/amygdalar glutamate and nigral DA 423 

inputs. This is consistent with the presumed role of presynaptic striatal dopamine D2 424 

receptors to reduce various dopamine-dependent motor activities and increase 425 

catalepsy in mice (Jackson et al., 1988). It is also consistent with DA's known role as a 426 

modulatory inducer of locomotion, both normally and in DA-replacement therapies for 427 

parkinsonism, and with mild parkinsonian effects being within the known clinical side-428 

effect profile of presynaptic D2 agonist therapeutic drugs (including pergolide and 429 

aripiprazole). The diminishment of locomotion events induced by bromocriptine in 430 

control mice was not evident in Ticcy mice (Fig. 4, Panel B), although their incidence of 431 

vertical motor events (climbing-leaping) was diminished (not shown), suggesting that 432 

bromocriptine indeed exerts an indiscriminate motor-circuit inhibiting influence on both 433 

Ticcy and control mice. The incidence of locomotion events in all untreated or 434 

bromocriptine treated mice (Fig. 4, Panel B) was roughly twice their incidence of cage 435 

midline crossings (not shown), consistent with prior reports that locomotor initiations in 436 

both control and Ticcy mice represent initiations of extended locomotor sequences 437 

(Campbell et al., 1999a; Nordstrom and Burton, 2002). 438 

439 

DISCUSSION 440 

441 

  22 

The D1CT-7 transgenic "Ticcy mouse" model of tics and compulsions was the first 442 

performed brain "circuit test" of a complex psychiatric or psychomotor disease -- the first 443 

symptomatic model to be created by transgenic neuropotentiation of a molecularly-specified 444 

and regionally-restricted circuit element in the brain (Campbell et al., 1999a; Nordstrom and 445 

Burton, 2002). The particular circuit element potentiated in the brains of Ticcy mice is D1+ 446 

somatosensory cortical and limbic neurons that trigger deep-layer cortical and amygdalar 447 

glutamatergic excitation of efferent striatothalamic GABAergic circuits and of efferent 448 

nigrostriatal dopaminergic circuits, and that are thought to be hyperactive in human TS and 449 

OCD. Hence the Ticcy mice permit examination of the biological role in tics of coincident 450 

glutamatergic and dopaminergic action upon striatal neurons, as well as permit examination 451 

of new classes of drugs that may therapeutically block the circuitry eliciting or mediating 452 

Tourette's like tic symptoms. 453 

What is the role in tics of coincident glutamate and dopamine action upon striatal 454 

neurons, and what is its circuitry? In isolation, glutamate- and excitatory DA D1- receptor 455 

coexpressing striatal direct-pathway GABAergic neurons would respond to either glutamate 456 

or dopamine by triggering tics, while glutamate- and inhibitory DA D2- receptor 457 

coexpressing striatal indirect-pathway GABAergic neurons would respond to glutamate by 458 

suppressing tics or to DA by triggering tics. (Locomotor induction by psychoactive NMDA 459 

glutamate receptor antagonists may be due to these drugs either primarily inhibiting the 460 

indirect-pathway striatal neurons the suppress motion and urges, or primarily inhibiting 461 

cortical GABAergic interneurons that suppress corticostriatal glutamate output to both 462 

striatal NMDA and AMPA glutamate receptors (Homayoun and Moghaddam, 2007)). 463 

  23 

But what happens if hyperglutamatergic output to both the striatum and substantia 464 

nigra is initiated from a hyperactive cortex and/or amygdala (whether it be by transgenic 465 

potentiation in the Ticcy mice of D1+ intermediate-cortical layer glutamatergic neurons and 466 

amygdalar neurons that activate these deep-layer cortical and amygdalar glutamatergic 467 

output neurons, or by cortical GABAergic interneurons' inhibition by psychoactive drugs like 468 

PCP, or by various mutations or epigenetic changes of cortical/amygdalar glutamate output 469 

in subtypes of neurogenic TS)? If such "cortical/limbic glutamatergic neuron hyperactivity" 470 

occurs, the convergence of its hyperglutamatergic input to striatal circuits with its parallel 471 

hyperglutamatergic-triggered nigral dopamine input to those same striatal circuits is 472 

predicted to elicit chronic tics by chronically unbalancing those striatal circuits, in favor of tic 473 

induction by the striatal direct pathway, as detailed below: 474 

Our current model of the "tic circuit," with the sites and mechanisms of action of the 475 

four tested "circuit breakers," is diagrammed in Fig. 5. This tic circuit is based on ours and 476 

others' prior "cortical/limbic glutamatergic neuron (CGN) hyperactivity" circuit model of tics 477 

and compulsions (Campbell et al., 1999a; Carlsson, 2000; Rosenberg et al., 2000; 478 

Nordstrom and Burton, 2002), with a recent refinement adding corticonigral glutamate 479 

excitation of nigrostriatal DA efflux (Singer et al., 2010). 480 

In this hyperglutamatergic circuit model of neurogenic tics, balanced circuit 481 

element outputs control motor activities and urges in normal individuals (Fig. 5, Panel 482 

A). In contrast, excess cortical/amygdalar glutamate output to the striatum and 483 

substantia nigra is proposed to initiate tics in the Ticcy mice and in some forms of 484 

neurogenic TS (Fig. 5, Panel B), by exciting the GABAergic striatal "direct pathway" 485 

  24 

neurons that co-express excitatory dopamine D1 receptors, and by simultaneously 486 

exciting nigral neurons to release reinforcing DA onto those same striatal neurons' D1 487 

receptors. Meanwhile, the simultaneous cortico/amygdalostriatal glutamatergic 488 

excitation of tic-suppressing GABAergic striatal "indirect pathway" neurons, which co-489 

express inhibitory dopamine D2 receptors, would be counteracted. Either of two 490 

mechanisms (one pharmacodynamic, the other circuit-based) would block the ability of 491 

the indirect pathway, once stimulated by forebrain glutamate output, to suppress tics: 1) 492 

Forebrain glutamatergic hyperexcitation of these D2+ striatal neurons could trigger their 493 

inhibitory D2 receptors to pharmacodynamically cross-supersensitize (Wolf et al.,1996), 494 

countering the over-excitation by glutamate; or 2) The forebrain's parallel 495 

cortico/amygdalonigral hyperglutamatergic excitation of the substantia nigra would 496 

trigger excessive nigrostriatal DA release to those same striatal indirect pathway 497 

neurons' inhibitory D2 receptors (Singer et al., 2010), again countering the neurons' 498 

over-excitation by forebrain glutamate. Either glutamate-induced mechanism still would 499 

cause tics to be chronically expressed, by unbalancing the tic-stimulating direct pathway 500 

and tic-inhibiting indirect pathway in favor of chronic direct pathway activity (Fig. 5, 501 

Panel A vs. B). 502 

Given this neurogenic hyperglutamatergic "tic circuit" (Fig. 5, Panel B), where 503 

glutamate and dopamine synergize with each other at the striatal direct pathway to elicit 504 

tics, but antagonize each other at the indirect pathway to fail in suppressing tics, the 505 

mechanisms by which the four tested "circuit-breaker" drugs would most likely then act 506 

on the tic circuit to alleviate or suppress tics are also diagrammed (Fig. 5, Panel B vs. 507 

  25 

Panels C-F): 508 

First, ritanserin and prazosin both act as antagonists of different, but co-509 

expressed, excitatory receptors located on the deep-layer cortical pyramidal output 510 

neurons that glutamatergically excite the striatum, while prazosin may furthermore 511 

inhibit amygdalar glutamate output to limbic cortex and directly to the striatum (Fig. 5, 512 

Panels C and D). Blocking cortico/amygdalostriatal hyperglutamatergic output would 513 

then "short-circuit" these neurons' chronic excitation of their target striatal and nigral 514 

neurons, alleviating tics. Moxonidine would be proposed to act more distally, on the 515 

striatal GABAergic neurons themselves, by presynaptically inhibiting their 516 

hyperactivated striatothalamic output that excites tics (Fig. 5, Panel E). And 517 

bromocriptine would act on input nigrostriatal dopaminergic neurons' D2 autoreceptors 518 

to presynaptically inhibit their dopamine efflux both to the excitatory D1 receptors on the 519 

glutamatergically co-excited striatal direct pathway GABAergic neurons that stimulate 520 

tics, and to the inhibitory D2 receptors on the glutamatergically excited but 521 

dopaminergically cross-inhibited indirect pathway GABAergic neurons that suppress tics 522 

(Fig. 5, Panel F). The effectiveness of these drugs in either selectively alleviating tics 523 

(i.e., ritanserin, prazosin, moxonidine) or, as in humans with TS, less selectively 524 

suppressing both tics and locomotion (i.e., bromocriptine) undergirds the potential 525 

validity of this forebrain hyperglutamatergic circuit model of neurogenic tics and 526 

topographically parallel circuit-triggered OC- and psychotic- spectrum disorders. 527 

The reported effects of drugs that should conversely "overload" rather than 528 

"circuit-break" this tic-circuit also bolster its validity. For example, a PCP-like drug that 529 

  26 

aggravated, rather than diminished, abnormal motor-urge symptoms in the Ticcy mice 530 

(McGrath et al., 2000) has since been established to aggravate, rather than diminish, 531 

corticostriatal glutamatergic output (Homayoun and Moghaddam, 2007). 532 

533 

Clinical Implications for Tourette's syndrome drug discovery 534 

What is the therapeutic potential of these four "tic circuit-breakers" for the treatment of 535 

human TS? The TS-like tic behavior of these mice, and thus perhaps of some 536 

neurogenic forms of TS, is initiated by increased cortical/limbic glutamatergic output 537 

from serotonoceptive plus noradrenoceptive pyramidal output neurons, which should be 538 

amenable to inhibition by at least some 5-HT2 serotonin receptor as well as alpha-1 NE 539 

receptor antagonists. Hence tics might respond not only to weak-to-strong-D2/strong-5-540 

HT2 antagonists, such as the atypical neuroleptics risperidone (Bruun and Budman, 541 

1996) and ziprasidone (Sallee et al., 2000), which showed effectiveness in TS pilot 542 

studies, but may also respond to "pure" 5-HT2 antagonists like ritanserin, which is now 543 

used in humans for other psychological disorders including anxiety (Ceulemans et al., 544 

1985; Barone et al., 1986; Danjou et al., 1992), putatively reflecting the drug's 5-HT2c-545 

antagonist-mediated suppression of amygdalar and limbic-cortex glutamatergic output 546 

(Gibson et al., 1994). Furthermore, ketanserin, a ritanserin-related, non-anxiolytic, 547 

hypotensive antagonist of both 5-HT2a and alpha-1 NE receptors (Hosie, et al., 1987), 548 

has proven partially effective in a small clinical trial of childhood-onset TS (Bonnier, et 549 

al., 1999), which suggests both ritanserin, as a 5-HT2a,c antagonist, and prazosin, as 550 

an alpha-1 NE antagonist, should be clinically studied individually as separate 551 

  27 

prospective human TS treatments. To our knowledge neither ritanserin nor prazosin 552 

have yet been tested clinically for their efficacy in alleviating TS. 553 

Could ritanserin be superior to ketanserin for TS, given these drugs' distinct 554 

receptor specificities and affinities? The receptor binding profiles of ritanserin vs. 555 

ketanserin suggests that ritanserin could prove therapeutically more effective. For 556 

example, the glutamatergic pyramidal cortical output neurons our data suggest may be 557 

hyperactive in TS and its comorbid OC-spectrum disorders carry both 5-HT2a and 5-558 

HT2c receptors, as well as alpha-1 NE receptors (Sheldon and Aghajanian, 1991; 559 

Marek and Aghajanian, 1998; Marek and Aghajanian, 1999). Ketanserin, as a 5-HT2a 560 

antagonist but only weak alpha-1 NE antagonist (Brogden and Sorkin, 1990), would 561 

thus be predicted to more weakly block cortical glutamate output from these neurons 562 

than ritanserin, which efficiently inhibits both 5-HT2a and 5-HT2c receptors. Moreover, 563 

ritanserin, as a 5-HT2c antagonist, unlike ketanserin, should also reduce tic severity by 564 

reducing amygdalar glutamate output and consequent anxiety (Gibson et al., 1994). In 565 

TS, tic severity correlates with the level of anxiety, and other anxiolytic drugs can lessen 566 

tics (Goetz, 1992). Finally, ritanserin counters drug-dependent tic- and 567 

compulsion/craving- like symptoms, associated with its ability to block 5-HT2a,c 568 

receptor stimulation of the glutamatergic cortical output neurons whose hyperactivity 569 

underlies tics in the Ticcy mice and, we propose, in some forms of neurogenic TS 570 

(Sheldon and Aghajanian, 1991; Willins and Meltzer, 1997; Marek and Aghajanian, 571 

1998; Marek and Aghajanian, 1999; Ciccocioppo et al., 1999; Campbell et al., 1999a; 572 

Nordstrom and Burton, 2002). 573 

  28 

The alpha-1 NE antagonist, prazosin, should also prove worthy to test for human 574 

TS. As an central alpha-1 adrenergic (but more NE selective) receptor antagonist, 575 

prazosin reduces the excitatory influence of NE on its limbic and cortical targets and, 576 

like ritanserin (which antagonizes the same neurons but through 5-HT2a,c receptors), 577 

thus reduces subsequent cortical/amygdalar excitatory glutamatergic output to the 578 

striatum and substantia nigra. Prazosin clinically has served as an antihypertensive 579 

drug, but more recently was found to alleviate alcohol craving (Simpson et al., 2009), 580 

which is thought to involve striatal circuits shared with impulse control disorder, an OC-581 

spectrum disorder (Grant et al., 2014). Given that TS is an urge-driven twitch disorder 582 

often comorbid with OCD (Frankel et al., 1988), and given that target receptors for both 583 

ritanserin and prazosin are co-expressed in the tic-circuit glutamatergic neurons, 584 

prazosin too is a good candidate to vet for the ability to alleviate tics in human TS. 585 

The proposed shared neuroanatomical basis of tics in the Ticcy mice and in (at 586 

least some forms of) human TS suggests that both ritanserin and prazosin will be 587 

capable of alleviating tics, by suppression of cortical (and also amygdalar, for ritanserin) 588 

glutamate output. This prediction was supported by our present study, where both drugs 589 

were able to completely normalize tics in the Ticcy D1CT-7 transgenic mice. But what of 590 

drugs, like moxonidine and bromocriptine, that work downstream of the primary cortical 591 

and amygdalar sites that glutamatergically elicited TS-like tic behavior in these mice? 592 

The Ticcy mice's tics are not initiated, but are subsequently mediated, by 593 

increased striatothalamic GABAergic output from glutamate-excited striatal neurons of 594 

the direct pathway -- neurons which should be amenable to inhibition by presynaptic 595 

  29 

imidazoline I-1 receptor agonists like moxonidine. In humans, moxonidine has so far 596 

been approved only as a centrally acting antihypertensive treatment, but is contra-597 

indicated for use in parkinsonian patients -- suggesting it may reduce motor initiation 598 

events, which would include tics. In mice, moxonidine is not only antihypertensive but 599 

inhibits anxiogenic and also anxiety-dependent compulsive behaviors (Taksande et al., 600 

2010; Dixit et al., 2014), suggesting moxonidine is a good clinical candidate to treat TS. 601 

Finally, because striatal dopamine D2 receptors are not only postsynaptic but 602 

also presynaptic, existing as inhibitory autoreceptors on dopamine-releasing 603 

nigrostriatal terminals, at low doses bromocriptine acts selectively as a presynaptic D2 604 

dopamine autoreceptor agonist (Jackson et al., 1988), similarly to pergolide, a 605 

presynaptic dopamine D1/D2 receptor agonist recently shown to reduce human TS 606 

symptoms (Gilbert et al., 2000). Only at higher doses does bromocriptine act as a 607 

postsynaptic D2 receptor agonist, mimicking dopamineʼs effects on the indirect striatal 608 

pathway (Jackson et al., 1988). Low-dose bromocriptine has previously been shown to 609 

reduce symptoms of OCD (Ceccherini-Nelli and Guazzelli, 1994), hence it could be 610 

tested for TS, although it might have no better clinical effect than current presynaptic DA 611 

receptor agonists like pergolide and the novel atypical neuroleptic, aripiprazole (which 612 

has a mild postsynaptic D2 antagonist and presynaptic D2 agonist action). 613 

Nevertheless, the Ticcy mice's responsiveness to bromocriptine as well as postsynaptic 614 

D2 antagonist TS drugs like pimozide (Campbell et al., 1999b) or the presynaptic alpha-615 

2 NE receptor agonist clonidine (Nordstrom and Burton, 2002) supports the predictive 616 

validity of this tic-circuit for candidate TS drug selection. 617 

  30 

618 

Clinical Implications for OC-spectrum disorders 619 

How effective would these drugs be on compulsions in humans? Although our findings 620 

predict effectiveness of these drugs in tic alleviation in TS and TS+OCD, one limitation of 621 

this study is that it has no implications for these drugs' treatment of OCD. Of these four 622 

acute drug administration studies, we only observed one, bromocriptine, to depress a 623 

compulsive behavior in Ticcy mice (data not shown). But bromocriptine was also the one 624 

drug that also suppressed normal mouse locomotion, and the compulsive behavior it 625 

suppressed, climbing-leaping, is a (vertical) locomotion-dependent behavior, meaning it 626 

could have been attenuated due to bromocriptine's broader attenuation of locomotion 627 

(Jackson et al., 1998). Broader locomotor suppression may likewise be the mechanism by 628 

which anti-dopaminergic drugs alleviate human compulsions and tics, which might be 629 

triggered by cortical/limbic glutamate hyperexcitation but still require co-stimulation of 630 

striatal dopamine receptors -- explaining why drugs that attenuate the nigrostriatal release 631 

of dopamine confer therapeutic benefits but also parkinsonian adverse effects (Ceccherini-632 

Nelli and Guazzelli, 1994; Gilbert et al., 2000). Interestingly, no bromocriptine-elicited 633 

decrease was seen in horizontal locomotion in the Ticcy mice. One possible explanation is 634 

that the drug depressed the mice's vertical climbing/leaping motor behaviors to a more 635 

horizontal locomotion behavior. An alternative possibility is that there may be some 636 

selectivity of action of dopamine receptor blockade upon different subsets of the 637 

topographically parallel striatal motor circuits that, when excessively stimulated by 638 

cortical/limbic glutamate, trigger distinct motor symptoms. 639 

  31 

Another possible reason why we didn't observe pronounced suppressive effects on 640 

Ticcy mouse compulsions at drug doses that don't suppress locomotion is that, unlike 641 

tics, human compulsions usually don't respond to single acute drug administrations, but 642 

only to long-term repeated drug administration -- a design we rejected due to the 643 

deleterious effects on the anxiogenic Ticcy mice of repeated physical injections or 644 

surgical pump implantation procedures. We conjecture that compulsions might respond 645 

slower than tics because tics may originate solely from a narrow psychogenic (e.g., 646 

hyperglutamatergic somatosensory cortical) subcircuit, while compulsions may originate 647 

integratively from a broad convergence of parallel psychogenic (e.g., 648 

hyperglutamatergic orbitofrontal cortical) plus anxiogenic (e.g., hyperglutamatergic 649 

amygdalar) subcircuits. Hence drugs other than direct cortical/amygdalar anti-650 

glutamatergics must act longer, or exert broader (e.g., combined antipsychotic-651 

anxiolytic) effects to additionally counter compulsions. 652 

Nevertheless, some data is available on the therapeutic effect of these drugs 653 

exclusively on compulsion-like behaviors in normal mice: Acute moxonidine reportedly 654 

reduces endogenous anxiety-dependent compulsive (marble-burying) behavior in mice 655 

(Dixit et al., 2014) -- an effect we could not study in Ticcy mice due to their ignoring all 656 

marble-burying behavior in lieu of more immediate locomotion-dependent compulsions 657 

(not shown). However, the ability of moxonidine to suppress both compulsive marble-658 

burying in normal mice, and, in this study, tics in Ticcy mice, offers convergent evidence 659 

to support a therapeutic trial of moxonidine for not just TS but OC-spectrum disorders. 660 

Acute ritanserin has been reported to variously inhibit (Bruins Slot et al., 2008), activate 661 

  32 

(Njung'e and Handley, 1991), or have no effect (Ichimaru et al., 1995; Gaikwad et al., 662 

2010) on normal mice's marble-burying, although it has anxiolytic, and thus potentially 663 

anti-compulsive, effects in both rodents and humans (Ceulemans et al., 1985; Danjou et 664 

al., 1992; Gao and Cutler, 1993). We speculate that it may be fruitful to examine the 665 

action of all four of our tested drugs on not only human TS but comorbid TS+OCD, OC-666 

spectrum and impulse control disorders, psychomotor side effects of therapeutic drugs 667 

and drugs of abuse, and psychotic-spectrum disorders -- all of which we believe may 668 

involve hyperactivation of glutamatergic circuit output from topographically-parallel 669 

CSTC circuit loops. 670 

671 

CONCLUSION 672 

673 

Human tic disorders may be induced, as in the Ticcy transgenic mouse model, by 674 

abnormally high levels of cortical/amygdalar glutamate deposition and consequent 675 

coincident dopamine deposition onto target striatal direct and indirect pathway circuits. 676 

Interestingly, our model merges prior glutamate, dopamine, serotonin, norepinephrine, 677 

and agmatine/imidazoline models of tic and related psychotic-spectrum, drug-abuse, 678 

and OC/impulse control-spectrum disorders. In this "five neurotransmitter" hypothesis 679 

(Fig. 5), symptoms like tics, obsessions, compulsions, impulses, cravings, and 680 

hallucinations could be triggered initially by excessive forebrain glutamatergic excitation 681 

of the striatum and of the substantia nigra -- the latter triggering consequent 682 

dopaminergic unbalancing of the glutamate-excited striatal neurons' motion/urge-683 

  33 

activating (direct) vs. motion/urge-suppressing (indirect) striatothalamic outputs, 684 

chronically favoring activated motion/urge symptoms. These symptoms should then 685 

accordingly be counteracted not only by drugs that directly block forebrain glutamatergic 686 

neurons' output, but by antagonists of these neurons' co-expressed excitatory forebrain 687 

serotonin (ritanserin) and norepinephrine (prazosin) receptors; and by presynaptic 688 

agmatinergic or dopaminergic drugs that, respectively, would block the downstream 689 

glutamate-triggered target striatothalamic neurons' GABA output (moxonidine), or the 690 

downstream glutamate-triggered target nigrostriatal neurons' co-modulatory dopamine 691 

output (bromocriptine). Hence our observation that the Ticcy transgenic mice's tics are 692 

fully alleviated, albeit with differing specificity, by acute treatment with all four drugs 693 

confirms the drugs may be "short-circuiting" these mice's initial hyperactive 694 

cortico/amygdalostriatal and cortico/amygdalonigral glutamate output; their target striatal 695 

neurons' consequent glutamate-triggered hyperactive striatothalamic GABAergic output; 696 

and their target nigral neurons' consequent glutamate-triggered hyperactive nigrostriatal 697 

DA output. Our findings suggest that the "cortical/limbic glutamatergic neuron (CGN) 698 

hyperactivity" model of neurogenic tics is a valid tic-circuit model for designing future 699 

interventional therapies for human TS, and suggest new drugs that should be useful to 700 

test in clinical trials. 701 

702 

CONFLICT OF INTEREST 703 

704 

The authors declare no actual or potential conflict of interest. 705 

706 

  34 

ACKNOWLEDGEMENTS 707 

708 

This work was supported by NIH training grant T32DA07097 to MJM; and by NIH 709 

research grant R03MH53553, the Jeff Sutton Memorial Young Investigator Award from 710 

the National Alliance for Research on Schizophrenia and Depression and the Rochester 711 

Area Alliance for the Mentally Ill, and grants from the Tourette Syndrome Association 712 

and the University of Minnesota Foundation to FHB. 713 

714 

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716 

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FIGURE LEGENDS 1040 

1041 

Fig 1. Ritanserin alleviates tics in a transgenic model of Tourette's syndrome. 1042 

Panel A. Ritanserin (1 mg/kg, i.p.) normalizes tics in D1CT-7 "Ticcy" transgenic mice. 1043 

Data are shown as a bar graph of the mean number (+ S.E.M.) of head or body twitches 1044 

occurring over 15 min of videotaped observation. Overall significance of genotype effect 1045 

[F(1,17) = 8.771; P = 0.0087, n = 8 Tg, 11 C], drug effect [F(1,17) = 14.113; P = 0.0016, 1046 

n = 8 Tg, 11 C], and genotype x drug interaction [F(1,17) = 8.487; P = 0.0097, n = 8 Tg, 1047 

11 C] was established by repeated measures ANOVA, followed by individual between-1048 

group Mann-Whitney U-test of genotype effect and within-group Wilcoxon Signed Rank 1049 

test of drug effect, with significance established at tied P < 0.05, which revealed both 1050 

elevated tics in transgenic mice and reduction of their tics by ritanserin treatment. 1051 

Panel B. Tic reduction by ritanserin is not associated with reduced locomotion. Data are 1052 

shown as a bar graph of the mean number (+S.E.M.) of cage midline crossings, an 1053 

assay of locomotion, occurring over 15 min of videotaped observation. Non-significance 1054 

of all effects and interactions was established by repeated measures ANOVA, which 1055 

revealed that 1 mg/kg i.p. ritanserin did not alter locomotor activity, indicating that the 1056 

tic-suppressing effect of ritanserin in Ticcy mice occurs in the absence of general 1057 

locomotor inhibition or sedation. 1058 

  50 

Statistics: Initial repeated measures ANOVA (n = 8 transgenic, 11 control non-1059 

transgenic mice) was performed to establish overall significance on tics or locomotion of 1060 

genotype effect, drug effect, or genotype x drug interaction, after which individual 1061 

comparisons of the non-parametrically distributed tic data (see Methods) or the 1062 

parametrically distributed locomotion data were performed by between-group non-1063 

parametric Mann-Whitney U-tests or parametric unpaired 2-tailed Student's t-tests of 1064 

genotype effects, and within-group non-parametric Wilcoxon Signed Rank tests or 1065 

parametric paired 2-tailed Student's t-tests of drug effects, with significance of effect on 1066 

non-parametrically distributed tic counts assumed at tied P < 0.05 and on 1067 

parametrically-distributed locomotor event counts assumed at P < 0.05. 1068 

Abbreviations: Tg (D1CT- 7 "Ticcy" transgenic female mice); C (non-transgenic control 1069 

female mice); Veh (saline vehicle i.p. injection); Rit (1 mg/kg i.p. ritanserin injection); **P 1070 

< 0.01 for between-group (Tg vs C, Veh) comparison (unpaired Mann-Whitney U-test), 1071 

+P < 0.05 for within-group, between-treatment (Tg, Veh vs Rit) comparison (paired 1072 

Wilcoxon Signed Rank test), n = 8 Tg, 11 C mice. 1073 

1074 

Fig 2. Prazosin alleviates tics in a transgenic model of Tourette's syndrome. 1075 

Panel A. Prazosin (3 mg/kg, i.p.) normalizes tics in D1CT-7 "Ticcy" transgenic mice. 1076 

Data are shown as a bar graph of the mean number (+ S.E.M.) of head or body twitches 1077 

occurring over 15 min of videotaped observation. Overall significance of genotype effect 1078 

[F(1,11) = 10.259; P = 0.0084, n = 7 Tg, 6 C], drug effect [F(1,11) = 21.495; P = 0.0007, 1079 

n = 7 Tg, 6 C], and genotype x drug interaction [F(1,11) = 18.424; P = 0.0013, n = 7 Tg, 1080 

  51 

6 C] was established by repeated measures ANOVA, followed by individual between-1081 

group Mann-Whitney U-test of genotype effect and within-group Wilcoxon Signed Rank 1082 

test of drug effect, with significance established at tied P < 0.05, which revealed both 1083 

elevated tics in transgenic mice and reduction of their tics by prazosin treatment. 1084 

Panel B. Tic reduction by prazosin is not associated with reduced locomotion. Data are 1085 

shown as a bar graph of the mean number (+ S.E.M.) of cage midline crossings, an 1086 

assay of locomotion, occurring over 15 min of videotaped observation. Non-significance 1087 

of all effects and interactions was established by repeated measures ANOVA, which 1088 

revealed that 3 mg/kg i.p. prazosin did not alter locomotor activity, indicating that the tic-1089 

suppressing effect of prazosin in Ticcy mice occurs in the absence of general locomotor 1090 

inhibition or sedation. 1091 

Statistics: Initial repeated measures ANOVA (n = 7 transgenic, 6 control non-transgenic 1092 

mice) was performed to establish overall significance on tics or locomotion of genotype 1093 

effect, drug effect, or genotype x drug interaction, after which individual comparisons of 1094 

the non-parametrically distributed tic data (see Methods) or the parametrically 1095 

distributed locomotion data were performed by between-group non-parametric Mann-1096 

Whitney U-tests or parametric unpaired 2-tailed Student's t-tests of genotype effects, 1097 

and within-group non-parametric Wilcoxon Signed Rank tests or parametric paired 2-1098 

tailed Student's t-tests of drug effects, with significance of effect on non-parametrically 1099 

distributed tic counts assumed at tied P < 0.05 and on parametrically-distributed 1100 

locomotor event counts assumed at P < 0.05. 1101 

Abbreviations: Tg (D1CT- 7 "Ticcy" transgenic female mice); C (non-transgenic control 1102 

  52 

female mice); Veh (saline vehicle i.p. injection); Praz (3 mg/kg prazosin i.p. injection); 1103 

**P < 0.01 for between-group (Tg vs C, Veh) comparison (unpaired Mann-Whitney U-1104 

test), +P < 0.05 for within-group, between-treatment (Tg, Veh vs Praz) comparison 1105 

(paired Wilcoxon Signed Rank test), n = 7 Tg, 6 C mice. 1106 

1107 

Fig 3. Moxonidine alleviates tics in a transgenic model of Tourette's syndrome. 1108 

Panel A. Moxonidine (0.5 mg/kg, i.p.) normalizes tics in D1CT-7 "Ticcy" transgenic mice. 1109 

Data are shown as a bar graph of the mean number (+ S.E.M.) of head or body twitches 1110 

occurring over 15 min of videotaped observation. Overall significance of genotype effect 1111 

[F(1,12) = 8.753; P = 0.012, n = 9 Tg, 5 C], drug effect [F(1,12) = 39.656; P < 0.0001, n 1112 

= 9 Tg, 5 C], and genotype x drug interaction [F(1,12) = 18.344; P = 0.0011, n = 9 Tg, 5 1113 

C] was established by repeated measures ANOVA, followed by individual between-1114 

group Mann-Whitney U-test of genotype effect and within-group Wilcoxon Signed Rank 1115 

test of drug effect, with significance established at tied P < 0.05, which revealed both 1116 

elevated tics in transgenic mice and reduction of their tics by moxonidine treatment. 1117 

Panel B. Tic reduction by moxonidine is not associated with reduced locomotion. Data 1118 

are shown as a bar graph of the mean number (+ S.E.M.) of cage midline crossings, an 1119 

assay of locomotion, occurring over 15 min of videotaped observation. Non-significance 1120 

of all effects and interactions was established by repeated measures ANOVA, which 1121 

revealed that 0.5 mg/kg i.p. moxonidine did not alter locomotor activity, indicating that 1122 

the tic-suppressing effect of moxonidine in Ticcy mice occurs in the absence of general 1123 

locomotor inhibition or sedation. 1124 

  53 

Statistics: Initial repeated measures ANOVA (n = 9 transgenic, 5 control non-transgenic 1125 

mice) was performed to establish overall significance on tics or locomotion of genotype 1126 

effect, drug effect, or genotype x drug interaction, after which individual comparisons of 1127 

the non-parametrically distributed tic data (see Methods) or the parametrically 1128 

distributed locomotion data were performed by between-group non-parametric Mann-1129 

Whitney U-tests or parametric unpaired 2-tailed Student's t-tests of genotype effects, 1130 

and within-group non-parametric Wilcoxon Signed Rank tests or parametric paired 2-1131 

tailed Student's t-tests of drug effects, with significance of effect on non-parametrically 1132 

distributed tic counts assumed at tied P < 0.05 and on parametrically-distributed 1133 

locomotor event counts assumed at P < 0.05. 1134 

Abbreviations: Tg (D1CT- 7 "Ticcy" transgenic female mice); C (non-transgenic control 1135 

female mice); Veh (saline vehicle i.p. injection); Mox (0.5 mg/kg moxonidine i.p. 1136 

injection); **P < 0.01 for between-group (Tg vs C, Veh) comparison (unpaired Mann-1137 

Whitney U-test), ++P < 0.01 for within-group, between-treatment (Tg, Veh vs Mox) 1138 

comparison (paired Wilcoxon Signed Rank test), n = 9 Tg, 5 C mice. 1139 

1140 

Fig 4. Bromocriptine alleviates tics in a transgenic model of Tourette's 1141 

syndrome. 1142 

Panel A. Bromocriptine (5 mg/kg, i.p.) normalizes tics in D1CT-7 "Ticcy" transgenic 1143 

mice. Data are shown as a bar graph of the mean number (+ S.E.M.) of head or body 1144 

twitches occurring over 15 min of videotaped observation beginning 15 minutes before 1145 

(-Bromo) vs. two hours after (+Bromo) drug injection. Repeated measures ANOVA 1146 

  54 

showed both a significant overall effect on ticcing incidence of bromocriptine treatment 1147 

[F(1,14) = 42.215; P < 0.0001, n = 8 Tg, 8 C], and a significant genotype x 1148 

bromocriptine interaction [F(1,14) = 5.385; P = 0.0359, n = 8 Tg, 8 C ], justifying 1149 

individual comparisons of the non-parametrically distributed tic count data by between-1150 

group Mann-Whitney U-test of genotype effect and within-group Wilcoxon Signed Rank 1151 

test of drug effect with significance established at tied P < 0.05, which revealed elevated 1152 

tics in transgenic mice and reduction of their tics by bromocriptine treatment, as well as 1153 

reduction of control mice's baseline twitch count by bromocriptine treatment. 1154 

Panel B. Bromocriptine is associated with reduced locomotion in control mice. Data are 1155 

shown as a bar graph of the mean number (+ S.E.M.) of locomotion events occurring 1156 

over 15 min of videotaped observation beginning 15 minutes before (-Bromo) vs. two 1157 

hours after (+Bromo) drug injection. Repeated measures ANOVA showed a significant 1158 

overall effect on locomotion events of drug treatment [F(1,14) = 12.427; P = 0.0034; n = 1159 

8 Tg, 8 C], justifying individual comparison of bromocriptine's effects on the 1160 

parametrically distributed locomotion event count data by within-group, paired 2-tailed 1161 

Studentʼs t-test, which revealed that 5 mg/kg i.p. bromocriptine suppressed locomotion 1162 

events in control mice, indicating that the tic-suppressing effect of bromocriptine in Ticcy 1163 

and control mice occurs in conjunction with a general locomotor inhibiting or sedating 1164 

effect evident in control mice. 1165 

Statistics: Initial repeated measures ANOVA (n = 8 transgenic, 8 control non-transgenic 1166 

mice) was performed to establish overall significance on tics or locomotion of genotype 1167 

effect, drug effect, or genotype x drug interaction, after which individual comparisons of 1168 

  55 

the non-parametrically distributed tic data (see Methods) or the parametrically 1169 

distributed locomotion data were performed by between-group non-parametric Mann-1170 

Whitney U-tests or parametric unpaired 2-tailed Student's t-tests of genotype effects, 1171 

and within-group non-parametric Wilcoxon Signed Rank tests or parametric paired 2-1172 

tailed Student's t-tests of drug effects, with significance of effect on non-parametrically 1173 

distributed tic counts assumed at tied P < 0.05 and on parametrically-distributed 1174 

locomotion event counts assumed at P < 0.05. 1175 

Abbreviations: Tg (D1CT- 7 "Ticcy" transgenic female mice); C (non-transgenic control 1176 

female mice); -Bromo (15 mins pre-injection); +Bromo (5 mg/kg i.p. bromocriptine, 2 hrs 1177 

post-injection); *P < 0.05 for between-group (Tg vs C, -Bromo) comparison of genotype 1178 

effect on non-parametrically distributed tic counts (unpaired Mann-Whitney U-test), +P < 1179 

0.05 for within-group, between-treatment (-Bromo vs +Bromo) comparisons of drug 1180 

effect on non-parametrically distributed Tg and C tic counts (paired Wilcoxon Signed 1181 

Rank tests), ++P < 0.01 for within-group, between-treatment (C, -Bromo vs +Bromo) 1182 

comparison of drug effect on parametrically-distributed locomotion event counts (paired 1183 

2-tailed Students' t-test), n = 8 Tg, 8 C mice. 1184 

1185 

Figure 5. Predicted hyperglutamatergic tic circuit and circuit-breaker drugs' 1186 

actions. 1187 

Panel A. Normal circuit controlling motion and urges. 1188 

Panel B. Abnormal cortical/amygdalar hyperglutamatergic circuit triggers tics. 1189 

Panel C. Ritanserin breaks tic circuit as a cortical/amygdalar 5-HT2a,c antagonist. 1190 

  56 

Panel D. Prazosin breaks tic circuit as a cortical/amygdalar alpha-1 NE antagonist. 1191 

Panel E. Moxonidine breaks tic circuit as a striatothalamic I-1 presynaptic agonist. 1192 

Panel F. Bromocriptine counters tic circuit as a nigrostriatal D2 DA presynaptic agonist. 1193 

Symbols: Triangles, excitatory glutamatergic pyramidal cortical/amygdalar output 1194 

neurons; Filled triangles, hyperactivated glutamatergic output neurons (due to D1CT-7 1195 

transgene-potentiated excitatory afferents in Ticcy mice or genetic/epigenetic alterations 1196 

in neurogenic TS; Circles, target striatal GABAergic neurons; Squares, modulatory 1197 

substantia nigra dopaminergic neurons; , move-urge-exciting or striatal neuron-1198 

exciting neurotransmission; ---|, move-urge-inhibiting or striatal-neuron inhibiting 1199 

neurotransmission; Thicker arrows, increased neurotransmission; Thicker "move-urge" 1200 

box, move-urge excitation (e.g., tics, obsessions, compulsions, impulses, cravings, or 1201 

hallucinations, depending on topographic parallelism of the circuit). 1202 

Abbreviations: GLU, glutamate (excitatory); DA, dopamine (modulatory); CTX, cortex; 1203 

AMY, amygdala; STR, striatum; SNc, substantia nigra pars compacta; D1, dopamine D1 1204 

excitatory postsynaptic receptors; D2, dopamine D2 inhibitory postsynaptic (left) or 1205 

presynaptic (middle) receptors; DP, striatal direct pathway (D1 receptor excited, 1206 

motor/urge-activating); IP, striatal indirect pathway (D2 receptor inhibited, motion/urge-1207 

suppressing); 5-HT2a,c, serotonin 5-HT2a,c excitatory postsynaptic receptors; alpha-1, 1208 

norepinephrine alpha-1adrenergic excitatory postsynaptic receptors; I-1, imidazoline-1 1209 

(agmatine) inhibitory presynaptic receptors; RIT, ritanserin; PRAZ, prazosin; MOX, 1210 

moxonidine; BROMO, bromocriptine. 1211 

1212 

  57 

1213 

1214 

1215 

1216 

1217 

1218 

1219 

1220 

1221 

1222 

1223 

1224 

1225 

Fig 1. Ritanserin alleviates tics in transgenic model of Tourette's syndrome. 1226 

1227 

1228 

0

5

10

15

Veh Rit0

20

40

60

80

Veh Rit

Nu

mb

er

of T

ics

Num

ber

of M

idlin

e C

rossin

gs

A B

+

**

Tg

C

Tg

C

+

  58 

1229 

1230 

1231  1232 

1233 

1234 

1235 

1236 

1237 

1238 

1239 

1240 

1241 

1242 

Fig 2. Prazosin alleviates tics in transgenic model of Tourette's syndrome. 1243 

1244 

1245 

0

5

10

15

Veh Praz0

20

40

60

80

Veh Praz

Nu

mb

er

of T

ics

Num

ber

of M

idlin

e C

rossin

gs

A B

+

**Tg

C

Tg

C

  59 

1246 

1247 

1248 

1249 

1250 

1251 

1252 

1253 

1254 

1255 

1256 

1257 

1258 

1259 

Fig 3. Moxonidine alleviates tics in transgenic model of Tourette's syndrome. 1260 

1261 

1262 

0

5

10

15

Veh Mox0

20

40

60

80

Veh Mox

Nu

mb

er

of T

ics

Num

ber

of M

idlin

e C

rossin

gs

A B

++

**Tg

C

Tg

C

  60 

1263 

1264 

1265 

1266 

1267 

1268 

1269 

1270 

1271 

1272 

1273 

1274 

1275 

1276 

Fig 4. Bromocriptine alleviates tics in transgenic model of Tourette's syndrome. 1277 

1278 

1279 

0

2.5

5

7.5

10

-Bromo +Bromo0

25

50

75

100

125

-Bromo +Bromo

Nu

mb

er

of T

ics

Num

ber

of Locom

otion E

vents

A B

Tg

C

Tg

C*

+

+

++

  61 

1280 

1281 

1282 

1283 

Fig 5. Predicted hyperglutamatergic tic circuit and circuit-breaker drugs' actions. 1284 

1285 

1286 

MOVE-URGE

D2 D1

CTX/AMY

SNc

GLU

DA

5-HT2a,c

alpha-1

I-1 I-1

D2 STR

DP STR

IP

MOVE-URGE

SNc

GLU

D2 D1 DA

CTX/AMY 5-HT2a,c

alpha-1

I-1 I-1

D2 STR

DP STR

IP

MOVE-URGE

D2 D1

CTX/AMY

SNc

GLU

DA

5-HT2a,c RIT

alpha-1

I-1 I-1

D2 STR

DP STR

IP

MOVE-URGE

D2 D1

CTX/AMY

SNc

GLU

DA

5-HT2a,c PRAZ alpha-1

I-1 I-1

D2 STR

DP STR

IP

SNc

GLU

D2 D1 DA

CTX/AMY 5-HT2a,c

alpha-1

I-1 I-1 MOX

MOVE-URGE

D2

MOX

STR

DP STR

IP

SNc

GLU

D2 D1 DA

CTX/AMY 5-HT2a,c

alpha-1

I-1 I-1

D2 BROMO

STR

DP STR

IP

MOVE-URGE

A B C!

!

!

!

!

!

!

!

!

D E F!