Nalfurafine is a G-protein-biased KOR (kappa opioid receptor) agonist that produces analgesia and lacks central nervous system adverse effects. Here, we examined the cardiovascular and renal responses to intravenous and oral nalfurafine alone and in combination with furosemide, hydrochlorothiazide, or amiloride. We hypothesized that nalfurafine, given its distinct mechanism of vasopressin inhibition, would increase urine output to these diuretics and limit electrolyte loss. Following catheterization, conscious Sprague-Dawley rats received an isotonic saline infusion and were then administered an intravenous bolus of nalfurafine, a diuretic, or a combination. Mean arterial pressure, heart rate, and urine output were recorded for 90 minutes. In another study, rats were placed in metabolic cages and administered drug in an oral volume load. Hourly urine samples were then collected for 5 hours. Intravenous and oral nalfurafine produced a marked diuresis, antinatriuresis, antikaliuresis, and a decrease in mean arterial pressure. Compared with diuretic treatment alone, intravenous coadministration with nalfurafine significantly increased urine output to furosemide and hydrochlorothiazide and decreased sodium and potassium excretion. Notably, mean arterial pressure was reduced with nalfurafine/diuretic combination therapy compared to diuretics alone. Similarly, oral coadministration of nalfurafine significantly increased urine output to hydrochlorothiazide and decreased sodium and potassium excretion, whereas combination with furosemide only limited the amount of sodium excreted. Further, both intravenous and oral coadministration of nalfurafine enhanced the diuresis to amiloride and decreased sodium excretion. Together, these findings demonstrate that nalfurafine enhances the diuresis to standard-of-care diuretics without causing an excessive loss of electrolytes, offering a new approach to treat several cardiovascular conditions.

Background and Purpose Partial agonists of the nociceptin opioid peptide (NOP) receptor have potential therapeutic use as antihypertensive and water diuretics (aquaretics). To date, peptide NOP receptor ligands have failed to progress in clinical trials due to poor pharmacokinetics and adverse effects. Nonpeptide, small-molecule NOP receptor ligands may be more suitable as therapeutic agents. This study investigated the cardiovascular and renal responses produced by the novel nonpeptide NOP agonists AT-403, AT-090, AT-127, and AT-039. Experimental Approach Changes in mean arterial pressure (MAP), heart rate (HR), renal excretory function and occurrence of sedation and hyperphagia were determined before and after i.v. bolus injection or infusion of the NOP agonists in conscious Sprague-Dawley rats. Additional studies involving (i) measurement of renal sympathetic nerve activity (RSNA) and (ii) renal denervation were conducted to investigate the role of the renal nerves in the cardiorenal responses to AT-039. Key Results Bolus i.v. injection of AT-403, AT-090, AT-127 and AT-039 produced significant decreases in MAP and HR and a sodium-sparing diuresis. AT-403, AT-090, and AT-127, but not AT-039, induced sedation and hyperphagia at all doses tested. Infusion i.v. of AT-039 produced hypotension and aquaresis without adverse central nervous system effects or change in HR, responses that were also observed in renal denervated rats. Conclusions and Implications Nonpeptide NOP agonists decrease blood pressure and produce aquaresis in conscious rodents. Due to lack of sedation and hyperphagia, AT-039 represents a novel NOP agonist that may be useful for treatment of hypertension and/or volume overload/hyponatraemic states.

Difelikefalin is a peripherally restricted kappa opioid receptor (KOR) agonist that was recently approved by the FDA to treat pruritis in dialysis patients. Here, we investigated the cardiovascular and renal responses to difelikefalin, and using the KOR antagonist norbinaltorphimine (norBNI), examined whether any difelikefalin-induced changes in the renal excretion of water and/or electrolytes were mediated through a central or peripheral KOR pathway. The effects of norBNI pretreatment on nalfurafine, a KOR agonist that crosses the blood-brain barrier, were also examined. We hypothesized that difelikefalin would alter urine output differently than nalfurafine, given that KOR agonists produce diuresis via activating central KORs to inhibit vasopressin release. Following catheterization, conscious Sprague-Dawley rats were infused i.v. with isotonic saline and pretreated with norBNI centrally via an intracerebroventricular (ICV) cannula or peripherally via an intravenous catheter. After stabilization, difelikefalin or nalfurafine was administered i.v. and urine output, heart rate and mean arterial pressure (MAP) were recorded for 90 min. Difelikefalin produced a significant increase in urine output, and significant decrease in urinary sodium and potassium excretion, urine osmolality, and MAP. ICV norBNI pretreatment markedly attenuated the increase in urine output caused by difelikefalin and nalfurafine but did not inhibit the electrolyte effects. However, IV norBNI pretreatment prevented all responses to difelikefalin and nalfurafine. Together, these findings demonstrate that difelikefalin and nalfurafine utilize central KOR pathways to elicit diuresis and a decrease in MAP but enhance renal tubular electrolyte reabsorption through a peripheral KOR pathway, providing important insight into two clinically useful KOR agonists.

The adverse effects of mu opioid agonists have spurred a renewed interest in using kappa opioid receptor (KOR) agonists as analgesics. KOR agonists also have potential for development as diuretics for the treatment of edema and hypertension. Here, we evaluated the discriminative stimulus, antinociceptive, and diuretic effects of the kappa agonist (±)-trans-U-50488 and its stereoisomers (-)-(1S,2S)-U-50488 or (+)-(1R,2R)-U-50488) alone and in combination with the cannabinoid agonist (-)-CP 55,940. To establish (±)-U-50488 as a discriminative stimulus, rats (n = 12) were trained to discriminate intraperitoneal (i.p.) administration of 5.6 mg/kg of (±)-trans-U-50488 from saline under a fixed-ratio 20 (FR-20) schedule of food reinforcement. Then, antinociception was assessed using two procedures: warm water tail withdrawal and von Frey paw withdrawal. Diuretic effects were assessed in separate rats (n = 6/group). Doses of (±)-U-50488 and (-)-U-50488 that served as discriminative stimuli produced significant increases in urine output, but at lower doses than those that produced antinociception. In contrast, (+)-U-50488 alone had no discriminative stimulus or diuretic effects at the doses tested, but did produce antinociception in the von Frey assay. When three cannabinoids and morphine were tested in the (±)-U-50488 discrimination procedure to determine the similarity of these drugs' discriminative stimulus effects to those for (±)-U-50488, the rank order similarity was (-)-CP 55,940 > (-)-trans-THC > (+)-WIN 55,212-2 ≥ morphine. (-)-CP 55,940 alone (0.056 mg/kg) partially substituted for the discriminative stimulus effects of (±)-U-50488 and produced significant diuretic and antinociceptive effects. (-)-CP 55,940 in combination with (±)-U-50488 also produced a two-fold leftward shift in the discriminative stimulus curve for (±)-U-50488, and near-additive antinociception with (±)-U-50488 and (+)-U-50488. Further, the diuretic effect of (-)-CP 55,940 was enhanced by a dose of (+)-U50488, which itself did not alter urine output. These data together indicate that a combination of cannabinoid and kappa opioid agonists can enhance diuresis, but may have limited potential for serving as opioid-sparing pharmacotherapeutics for treatment of pain.

Kappa opioid receptor (KOR) agonists produce a variety of beneficial effects, including a water diuresis, but their translation into the clinic has been hindered by psychotomimetic adverse effects. Nalfurafine is a novel, G protein biased KOR agonist that has been shown to produce several desired effects of KOR agonists, while avoiding central adverse effects. To more fully explore the clinical potential of this drug, this study examined the cardiovascular and renal responses to i.v. nalfurafine alone or in combination with the clinically used diuretics: furosemide, hydrochlorothiazide (HCTZ), and amiloride. Following chronic instrumentation, conscious Sprague-Dawley rats were continuously infused i.v. with isotonic saline; after stabilization, rats were administered i.v. bolus nalfurafine, diuretics, diuretics combined with nalfurafine, or vehicle, and mean arterial pressure (MAP), heart rate (HR), and urine output were recorded for 90 min. IV nalfurafine produced a marked diuresis, antinatriuresis, antikaliuresis, and decrease in MAP without eliciting a change in HR. As compared to diuretic treatment alone, co-administration of nalfurafine notably increased the total urine output to furosemide and HCTZ while reducing the amount of sodium and potassium excreted. When combined with amiloride, nalfurafine also increased the diuresis and decreased the amount of sodium excreted. In contrast to these diuretics administered alone, MAP was reduced with nalfurafine combination therapy. Together, these findings demonstrate that nalfurafine has a clinically important action to augment the diuresis to classical diuretics without causing excessive loss of electrolytes characteristic of these drugs. Combination therapy of nalfurafine with loop/thiazide diuretics may offer a new approach to treat several cardiovascular conditions such as hypertension, volume overloaded states, and electrolyte abnormalities.

Nalfurafine, a G protein-biased kappa opioid receptor (KOR) agonist that produces analgesia and is devoid of CNS adverse effects, is used in Japan to treat pruritis in dialysis patients. Our lab has shown that in rats, IV nalfurafine produces a marked diuresis, antinatriuresis, antikaliuresis, and decrease in blood pressure. Here, we examined the cardiovascular and renal responses to IV and oral nalfurafine in combination with furosemide or hydrochlorothiazide (HCTZ). We hypothesized that combining nalfurafine with these diuretics would increase urine output, given its distinct mechanism of vasopressin inhibition, and limit electrolyte loss. Following chronic catheterization, conscious Sprague-Dawley rats received an isotonic saline infusion, and after stabilization, were administered an IV bolus diuretic alone or in combination with nalfurafine. Mean arterial pressure (MAP), heart rate (HR), and urine output were recorded for 90-min. In a separate study, rats were placed in metabolic cages, and following 2-hour acclimation, were administered drug in a volume load (20 cc/kg) via oral gavage. Hourly urine samples were then collected for five hours. When compared to diuretic treatment alone, IV co-administration with nalfurafine significantly increased total urine output to furosemide and HCTZ while reducing the amount of sodium and potassium excreted (Table 1). Notably, MAP was reduced with nalfurafine/diuretic combination therapy compared to diuretics alone. Similarly, oral co-administration of nalfurafine significantly increased the urine output to HCTZ and reduced the amount of sodium and potassium excreted, whereas combination with furosemide only limited the amount of sodium excreted (Table 1). Together, these findings demonstrate that nalfurafine enhances the diuresis to standard-of-care diuretics without causing an excessive loss of electrolytes, which may offer a new approach to treat several cardiovascular conditions.

Phenotyping mouse model systems of human disease has proven to be a difficult task, with frequent poor inter- and intra-laboratory replicability, particularly in behavioral domains such as social and cognitive function. However, establishing robust animal model systems with strong construct validity is of fundamental importance as they are central tools for understanding disease pathophysiology and developing therapeutics. To complete our studies of mouse model systems relevant to autism spectrum disorder (ASD), we present a replication of the main findings from our two published studies of five genetic mouse model systems of ASD. To assess the intra-laboratory robustness of previous results, we chose the two model systems that showed the greatest phenotypic differences, the Shank3/F and Cntnap2, and repeated assessments of general health, activity and social behavior. We additionally explored all five model systems in the same framework, comparing all results obtained in this three-yearlong effort using informatics techniques to assess commonalities and differences. Our results showed high intra-laboratory replicability of results, even for those with effect sizes that were not particularly large, suggesting that discrepancies in the literature may be dependent on subtle but pivotal differences in testing conditions, housing enrichment, or background strains and less so on the variability of the behavioral phenotypes. The overall informatics analysis suggests that in our behavioral assays we can separate the set of tested mouse model system into two main classes that in some aspects lie on opposite ends of the behavioral spectrum, supporting the view that autism is not a unitary concept.

Sympathoexcitation and increased inflammation are both involved in mediating hypertension, and decreasing sympathetic nerve activity is vital for reducing inflammation in this disease. However, the mechanism(s) by which the nervous system communicates with the immune system in hypertension is still largely unknown. To examine this interaction in this study, twenty‐week‐old spontaneously hypertensive rats (SHR) underwent renal denervation (RDN) via radiofrequency ablation of the renal artery. After two months, kidneys were harvested and separated into cortex and medulla, and lymphocytes were detected by flow cytometry. RDN significantly decreased CD4+ T cells, CD8+ T cells, and B cells in the renal medulla but had no effect on these immune cells in the cortex. In a separate group of intact SHR, Thy‐1, a reported activator of T cells, was measured in leukocytes isolated from the renal cortex and medulla. Thy‐1 mRNA expression was significantly increased in T cells, non‐T cells, CD45+ cells, and CD45− cells from the leukocytes isolated from the cortex compared to those isolated from the medulla. These findings suggest that Thy‐1 in cortical leukocytes might activate the immune system in the renal cortex independent of the renal nerves. Downregulation of Thy‐1 in the renal cortex could increase the benefit of RDN and decrease renal inflammation in hypertension.Support or Funding InformationNIH NIGMS P30 GM106392

Currently, more than half of the drugs in clinical use are chiral compounds. In many instances the enantiomers of chiral drugs demonstrate pronounced differences in biological activity, toxicology, pharmacokinetics, and metabolism. Administration of racemic U‐50,488, a highly selective chiral κ‐opioid receptor agonist, produces a marked sodium and potassium sparing diuresis (i.e. aquaresis), dysphoria, and analgesia in conscious rats. Though U‐50,488 enantiomers have been the subject of various behavioral and analgesic studies, there is a distinct lack of data pertaining to isomers and kappa induced aquaresis. Therefore, the aim of the present study was to pharmacologically characterize the renal excretory effects produced by the isolated (−)‐(1S,2S)‐U‐50,488 versus (+)‐(1R,2R)‐U50,488 enantiomers. Methods Male Sprague‐Dawley rats with an indwelling intracerebroventricular (ICV) cannula were surgically implanted with an arterial, venous, and bladder catheter and continuously infused intravenously (i.v.) with iso‐saline. After stabilization, mean arterial pressure (MAP), heart rate (HR), and urine flow rate (V) were measured in conscious rats for 20‐min before (control) and 90‐min after (experimental) ICV bolus injection (1 ug) of (−)‐(1S,2S)‐U‐50,488, (+)‐(1R,2R)‐U50,488, racemic (+/−)‐U‐50,488, or vehicle (n=6/group). Urine samples were analyzed for urinary sodium excretion (UNaV) and urinary potassium excretion (UKV). Results ICV injection of racemic U‐50,488 (1 ug) produced a significant diuretic (control (C), 56±6, 30‐min, 152±7 ul/min), antinatriuretic (C, 8.7±2.2, 30 min, 3.7±2 ueq/min), and antikaluretic (C, 1.1±0.5; 0.4±0.4 ueq/min) response. At the same dose, both the (−)‐(1S,2S)‐U‐50,488 and (+)‐(1R,2R)‐U50,488 isomers produced a significant sodium and potassium sparing diuresis without changing MAP or HR. At 1 ug, (−)‐(1S,2S)‐U‐50,488 significantly increased peak urine flow rate (C, 58±6 ul/min; 20 min 196±29 ul/min) and decreased urinary sodium (C, 8.9±2.3 μeq/min; 30 min 3.9±0.8 μeq/min) and potassium (C, 0.8±0.2 μeq/min, 30 min, 0.2±0.1 μeq/min) excretion. In contrast, 1 ug of (+)‐(1R,2R)‐U50,488 increased urine flow rate to a lessor magnitude (C, 57±6 ul/min; 10 min 119±26 ul/min) while decreasing urinary sodium excretion (C, 8.0±0.7 μeq/min, 30 min 4.3±1.5 μeq/min) but not urinary potassium excretion. ICV pretreatment (1ug, 10 min) with the kappa antagonist nor‐BNI abolished the kappa‐mediated diuresis by both enantiomers and racemic U‐50,488.ConclusionsThese results indicate (−)‐(1S,2S)‐U‐50,488 is the predominant isomer responsible for producing the centrally mediated aquaresis observed with racemic U‐50,488. Blockade of the aquaresis by ICV nor‐BNI indicates that both enantiomers mediate their renal excretory effects through activation of central kappa receptors. Further renal excretory studies are needed to fully elucidate the pharmacological variances between these two compounds and determine whether a selective entaniomer of U‐50,488 may be used therapeutically as a water diuretic.Support or Funding InformationNIH P30GM106392 (DRK)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

OBJECTIVES/SPECIFIC AIMS: We have reported that radiofrequency renal denervation (RF-RDN) in SHR at 20-weeks of age, decreased blood pressure (BP) and fibrosis in kidney cortex and medulla when rats were sacrificed at 6 months. However, whether RF-RDN can have similar benefits in older rats remains unknown. This study examined whether performing RF-RDN in older rats also has a beneficial effect on BP and renal fibrosis. METHODS/STUDY POPULATION: Baseline systolic and diastolic BP (SBP/DPB) was measured (telemetry) in nine-month-old SHR and Wistar Kyoto rats (WKY). Groups of rats then received bilateral RF-RDN or Sham-RDN (SHR-RDN, n=9; SHR-Sham, n=10; WKY-RDN, n=5; WKY-Sham, n=8). Rats were then sacrificed at 12-months of age. Kidneys were harvested, sectioned, and assessed for fibrosis by Masson’s trichrome stain. A pathologist, who was blinded to treatment groups, evaluated each kidney section for fibrosis. RESULTS/ANTICIPATED RESULTS: Compared to SHR with Sham-RDN, RF-RDN prevented a further increase in systolic and diastolic BP from baseline (9-month) in SHR as they aged to 12-months (SHR-Sham mmHg: 9-month 193±4/127±4; 12-month 207±3/142±5; SHR-RDN mmHg: 9-month 197±3/132±2; 12-month 197±4/132±3). RF-RDN did not alter SBP or DBP in aged WKY. One-year-old SHR with prior Sham-RDN showed extensive renal fibrosis in kidney cortex and medulla. In contrast, RF-RDN significantly decreased renal fibrosis in the medulla, but not cortex. There was no fibrosis in kidneys of age matched WKY. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings suggest that RF-RDN may be a potential therapy for halting progression of hypertension and decreasing medullary fibrosis in the aged population.

Kappa (κ) opioid receptor agonists can produce prominent analgesic and diuretic effects. However, these agonists also produce dysphoria, which markedly reduces their clinical utility. The purpose of the present experiment was to determine if the optical isomers of the κ agonist U50,488 ((±)‐trans‐3,4‐dichloro‐N‐methyl‐N‐[2‐(1‐pyrrolidinyl)‐cyclohexyl]benzeneacetamide) might be differentially effective in producing these κ‐mediated effects. In addition, the effects of specific cannabinoids were tested alone and in combination with the U50,488 racemate on several measures of κ activity. To establish some of U50,488's subjective effects as a discriminate stimulus, a group (n=12) of rats were trained to discriminate 5.6 mg/kg from saline under a fixed‐ratio 20 (FR‐20) schedule of food reinforcement. Then, following sessions in which the enantiomers or the cannabinoids were administered, antinociception was assessed in these rats using both a warm‐water tail‐withdrawal procedure and a paw‐withdrawal (Von Frey) procedure. In separate groups of rats (n=6/group), U50,488 and its enantiomers, (−)‐(1S,2S)‐U50,488 and (+)‐(1R,2R)‐U50,488, were administered intracerebroventricularly (ICV) to determine their capacity for producing an aquaresis. With respect to the discrimination procedure, both (−)‐(1S,2S)‐U50,488 and delta‐9‐tetrahydrocannabinol (Δ9‐THC) produced dose‐dependent increases in U50,488‐lever responding, but only (−)‐(1S,2S)‐U50,488 produced full (>80%) substitution. In contrast, both (+)‐(1R,2R)‐U50,488 and the mu opioid receptor agonist morphine (negative control) engendered less than 20% U50,488‐lever responding up to doses that significantly decreased overall response rate. Doses of U50,488 and (−)‐(1S,2S)‐U50,488 that produced U50,488‐appropriate responding also produced antinociception by dose‐dependently increasing both tail‐ and paw‐withdrawal latencies, whereas (+)‐(1R,2R)‐U50,488 was inactive in both assays. Finally, a synergistic interaction between the cannabinoids and κ opioids occurred when a dose of 0.056 mg/kg of CP‐55,940, a non‐selective CB1/CB2 receptor agonist, was administered prior to ineffective doses of U50,448. This dose of CP‐55,940 produced a small effect alone and potentiated the effects of low doses of U50,488 in the paw‐withdrawal assay (i.e., there was an upward shift in the dose‐effect curve). While all of the κ agonists tested produced an aquaresis, as measured by an increase in urine output, an equivalent dose of each produced a rank order of (−)‐(1S,2S)‐U50,488 > U50,488 > (+)‐(1R,2R)‐U50,488. These increases in urine output were also blocked by a pretreatment with the κ antagonist norbinaltorphimine (nor‐BNI), indicating these effects were mediated by κ opioid receptors. Taken together, these data indicate that the (−)‐(1S,2S)‐U50,488 enantiomer produces discriminative, antinociceptive, and diuretic effects that are most similar to those of the racemate, and provide evidence for a synergistic interaction between κ opioids and cannabinoid receptors. Further studies are needed in order to reevaluate the clinical potential of κ opioid receptor agonists as therapeutic targets as either analgesics or diuretics.Support or Funding InformationNIH P30GM106392 (DRK)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

BACKGROUND Angiotensin II (Ang II) activates central Angiotensin II type 1 receptors to increase blood pressure via multiple pathways. However, whether central G alpha proteins contribute to Ang II-induced hypertension remains unknown. We hypothesized that Angiotensin II type 1 receptors couple with G alpha 12 and/or G alpha q to produce sympatho-excitation and increase blood pressure and downregulation of these G alpha-subunit proteins will attenuate Ang II-dependent hypertension. METHODS AND RESULTS After chronic infusion of Ang II (s.c. 350 ng/kg/min) or vehicle for 2 weeks, Ang II evoked an increase in G alpha 12 expression, but not G alpha q in the rostral ventrolateral medulla of Sprague-Dawley rats. In other studies, rats that received Ang II or vehicle infusion s.c. were simultaneously infused i.c.v. with a scrambled (SCR) or G alpha 12 oligodeoxynucleotide (ODN; 50 mu g/day). Central G alpha 12 ODN infusion lowered mean blood pressure in Ang II infused rats compared with SCR ODN infusion (14-day peak; 133 12 vs. 176 +/- 11 mm Hg). Compared to the SCR ODN group, Ang II infused rats that received i.c.v. G alpha 12 ODN showed a greater increase in heart rate to atropine, an attenuated reduction in blood pressure to chlorisondamine, and an improved baroreflex sensitivity. In addition, central G alpha 12 and G alpha q ODN pretreatment blunted the pressor response to an acute i.c.v. injection of Ang II (i.c.v., 200 ng). CONCLUSIONS These findings suggest that central G alpha 12 protein signaling pathways play an important role in the development of chronic Ang II-dependent hypertension in rats.

To expand, analyze and extend published behavioral phenotypes relevant to autism spectrum disorder (ASD), we present a study of three ASD genetic mouse models: Feng’s Shank3 tm2Gfng model, hereafter Shank3/F , Jiang’s Shank3 tm1Yhj model, hereafter Shank3/J , and the Cacna1c deletion model. The Shank3/F and Shank3/J models mimick gene mutations associated with Phelan-Mcdermid syndrome and the Cacna1c model recapitulates the deletion underlying Timothy syndrome. The current study utilizes both standard and novel, computer-vision based behavioral tests, the same methdology used in our previously published companion report on the Cntnap2 null and 16p11.2 deletion models. Overall, some but not all behaviors replicated published findings. Those that replicated, such as social behavior and overgrooming in Shank3 models, also tended to be milder than previous reports. The Shank3/F model, and to a much lesser extent, the Shank3/J and Cacna1c models, showed hypoactivity and a general anxiety-like behavior triggered by external stimuli which pervaded social interactions. We did not detect deficits in a cognitive procedural learning test nor did we observe perseverative behavior in these models. We did, however, find differences in exploratory patterns of Cacna1c mutant mice suggestive of a behavioral effect in a social setting. In addition, Shank3/F but not Shank3/J KO or Cacna1c HET showed differences in sensory-gating. Discrepancies in our current results from previous reports may be dependent on subtle differences in testing conditions, housing enrichment, or background strain. Both positive and negative results from this study will be useful in identifying the most robust and replicable behavioral signatures within and across mouse models of autism. Understanding these phenotypes may shed light of which features to study when screening compounds for potential therapeutic interventions.

Huntington’s Disease (HD) is a progressive neurodegenerative disorder that causes motor, cognitive, and psychiatric symptoms. In these experiments, we tested if operant training at an early age affected adult cognitive deficits in the zQ175 KI Het (zQ175) mouse model of HD. In Experiment 1 we trained zQ175 mice in a fixed-ratio/progressive ratio (FR/PR) task to assay learning and motivational deficits. We found pronounced deficits in response rates and task engagement in naïve adult zQ175 mice (32-33 weeks age), while deficits in zQ175 mice trained from 6-7 weeks age were either absent or less severe. When those mice were re-tested as adults, FR/PR performance deficits were absent or otherwise less severe than deficits observed in naïve adult zQ175 relative to wild type (WT) mice. In Experiment 2, we used a Go/No-go operant task to assess the effects of early cognitive testing on response inhibition deficits in zQ175 mice. We found that zQ175 mice that began testing at 7-8 weeks did not exhibit deficits in Go/No-go testing, but when re-tested at 28-29 weeks age exhibited an initial impairment that diminished with training. These transient deficits were nonetheless mild relative to deficits observed among adult zQ175 mice without prior testing experience. In Experiment 3 we trained mice in a two-choice visual discrimination test to evaluate cognitive flexibility. As in prior experiments, we found performance deficits were mild or absent in mice that started training at 6-9 weeks of age, while deficits in naive mice exposed to training at 28-29 weeks were severe. Re-testing mice at 28-29 weeks age, were previously trained starting at 6-9 weeks, revealed that deficits in learning and cognitive flexibility were absent or reduced relative to effects observed in naive adults. In Experiment 4, we tested working memory deficits with a delayed non-match to position (DNMTP) test. Mice with prior experience exhibited mild working memory deficits, with males zQ175 exhibiting no deficits, and females performing significantly worse than WT mice at a single delay interval, whereas naive zQ175 exhibited severe delay-dependent deficits at all intervals exceeding 1 s. In sum, these experiments indicate that CAG-dependent impairments in motivation, motor control,

Patricia Kabitzke, Daniela Brunner, Dansha He, Pamela A. Fazio, Kimberly Cox, Jane Sutphen, Lucinda Thiede, Emily Sabath, Taleen Hanania, Vadim Alexandrov, Randall Rasmusson, Will Spooren, Anirvan Ghosh, Pamela Feliciano, Barbara Biemans, Marta Benedetti, and Alice Luo Clayton. PsychoGenics, Inc., Tarrytown, NY, USA Department of Psychiatry, Columbia University, New York, NY, USA Department of Physiology and Biophysics, SUNY Buffalo School of Medicine and Biomedical Sciences, Buffalo, NY, USA Roche Pharma Research and Early Development, NORD, Roche Innovation Center, Basel, Switzerland Simons Foundation Autism Research Initiative, New York, NY, USA

Timing is a ubiquitous process that underlies a great variety of human activities and depends on highly conserved neuronal circuitry, the cortico-striatal loops. The peak interval (PI) task is an operant task that conditions subjects to initiate and terminate behavioral responses bracketing a fixed interval associated with reinforcement. Performance in this task depends on the efficacy of temporal control processes that coordinate interval encoding and decoding, instrumental response innitiation, cessation and maintenance, and motor control. Here, we used the PI procedure to characterize temporal control in zQ175 knockin (KI) and BAC HD transgenic (Tg) mice generated to model Huntington's Disease (HD), and contrast the result with previously published R6/2 Tg PI data. HD is a progressive neurodegenerative disorder that involves degeneration of the same neural circuits underlying temporal information processing and control of motor output. Our results indicate that temporal control is disrupted in R6/2 Tg and zQ175 KI mice but intact in BAC HD Tg mice. Trial-by-trial analysis of break-run patterns in response rates indicated that shifts in zQ175 KI response curves were driven by significant delays in response initiation and cessation. Similar temporal control deficits were previously reported in HD patients and R6/2 transgenic HD mice. These findings support the use of zQ175 mice in preclinical studies of HD-related cognitive deficits. They provide evidence of a strong homology between the human and rodent neural bases of temporal information processing, temporal response control, and their pathology in neurodegeneration.