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Prefrontal cortical neuregulin-ErbB modulation of inhibitory control in rats
European Journal of Pharmacology 781 (2016) 157–163
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Behavioural pharmacology
Prefrontal cortical neuregulin-ErbB modulation of inhibitory control in rats Maarten Loos a,b, Dustin Schetters c, Myrthe Hoogeland c, Sabine Spijker a, Taco J. de Vries a,c, Tommy Pattij c,n a Department of Molecular and Cellular Neurobiology, Neuroscience Campus Amsterdam, Center for Neurogenomic and Cognitive Research, VU University, Amsterdam, The Netherlands b Sylics (Synaptologics BV), Amsterdam, The Netherlands c Department of Anatomy and Neurosciences, Neuroscience Campus Amsterdam, VU University Medical Center, Amsterdam, The Netherlands
art ic l e i nf o
a b s t r a c t
Article history: Received 4 February 2016 Received in revised form 7 April 2016 Accepted 11 April 2016 Available online 11 April 2016
Impulse control disturbances are key features of various neuropsychiatric and neurological disorders, such as attention-deficit/hyperactivity disorder, drug addiction, Parkinson disease and schizophrenia. Whereas over the last years accumulating evidence has highlighted monoaminergic modulation of the processes underlying impulse control, investigating novel mechanisms beyond monoamines may provide new intervention strategies to ameliorate impulse control disturbances. Recent work has associated the neuregulin (Nrg)-ErbB pathway with several neuropsychiatric diseases, as well as indicated its involvement in murine measures of impulse control. The aim of the present study was to investigate whether this Nrg-ErbB signaling pathway also modulates impulsive action in rats. To this end, a group of rats was trained in the 5-choice serial reaction time task (5-CSRTT), an operant paradigm that provides measures of visuospatial attention and inhibitory control processes. Upon stable baseline performance, the ErbB tyrosine kinase receptor inhibitor JNJ-28871063 (JNJ) was intracranially infused into the medioprefrontal cortex prior to test sessions. Results showed that JNJ dose-dependently improved measures of impulsive action. Importantly, other measures in the 5-CSRTT reflecting visuospatial attention or aspects of motivational behavior were not altered by JNJ. In conclusion, the present data strengthen a role for the Nrg-ErbB4 pathway in the prefrontal cortex in cognitive functioning, and in particular point towards involvement in the processes underlying impulse control. & 2016 Elsevier B.V. All rights reserved.
Keywords: Cognition ErbB4 Impulsivity Prelimbic cortex Neuregulin Rat
1. Introduction ErbB receptors belong to a family of receptor tyrosine kinases that are activated by ligands of the neuregulin family. NeuregulinErbB signaling plays an important role in neural development, including neural circuit assembly, synaptic plasticity and neurotransmission (Mei and Nave, 2014). With regard to the receptor ErbB kinase family, at least four different ErbB receptor kinases have been identified, namely ErbB1, ErbB2, ErbB3 and ErbB4 (Birchmeier, 2009). In recent years, particularly the neuregulinErbB4 signaling pathway has been associated with neuropsychiatric disorders, such as attention-deficit/hyperactivity disorder (ADHD), bipolar disorder, schizophrenia and nicotine dependence in genome wide association studies (Sonuga-Barke et al., 2008; Pan et al., 2011; Mei and Nave, 2014; Turner et al., 2014). Since n
Corresponding author. E-mail address: [email protected] (T. Pattij).
http://dx.doi.org/10.1016/j.ejphar.2016.04.015 0014-2999/& 2016 Elsevier B.V. All rights reserved.
maladaptive impulsivity and more specifically inhibitory control deficits, are shared symptoms in these disorders (Moeller et al., 2001; Gilmour et al., 2013; Ethridge et al., 2014), alterations in neuregulin-ErbB signaling may provide a novel common underlying mechanism. Indeed, in support we recently identified a gene within the neuregulin family, neuregulin 3 (Nrg3) that was causally related to impulsive behavior in mice (Loos et al., 2014). To date, much of our understanding of the neural mechanisms of inhibitory control subserving impulsivity originates from rodent studies. The collective work in these models has strongly implicated various neurotransmitter systems including the monoaminergic, glutamatergic, opioid and more recently GABAergic system in inhibitory control (Pattij and Vanderschuren, 2008; Dalley et al., 2011; Caprioli et al., 2014). In addition, the medioprefrontal cortex (mPFC) and connected ventral striatal brain regions comprise a crucial neuroanatomical circuit, since lesions, reversible inactivation and functional disconnections of mPFC subregions and connected striatum impair inhibitory control
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capacities (Chudasama et al., 2003; Christakou et al., 2004; Feja and Koch, 2014). Of abovementioned brain regions the mPFC contains high densities of ErbB4 receptor kinase, which are mainly expressed in GABA-ergic interneurons and not pyramidal neurons and are conserved in various species ranging from rodents to humans (Fazzari et al., 2010; Neddens et al., 2011). As such, ErbB4 activation has been found to promote GABA release and thereby to modulate functioning of glutamatergic pyramidal neurons and thus N-methyl-D-Aspartate (NMDA) receptor signaling (Woo et al., 2007; Chen et al., 2010; Vullhorst et al., 2015). Several studies have demonstrated glutamatergic modulation of inhibitory control in the mPFC (Mirjana et al., 2004; Murphy et al., 2005; Counotte et al., 2011; Murphy et al., 2012). Hence, this places the ErbB4 receptor kinase in the position to impact on cognitive functions, including inhibitory control. The aim of the present study was to investigate whether, in addition to our previous murine observations (Loos et al., 2014), neuregulin-ErbB signaling in the mPFC could also contribute to inhibitory control across species in rats. For this purpose, rats were trained in the 5-choice serial reaction time task (5-CSRTT), a translational paradigm measuring aspects of visuospatial attention and inhibitory control (Bari et al., 2008). Upon stable baseline performance, intra-mPFC infusions with the ErbB kinase inhibitor JNJ-28871063 (Emanuel et al., 2008) were performed to assess its effects on visuospatial attention and inhibitory control.
2. Materials and methods 2.1. Subjects Sixteen male Wistar rats were obtained from Harlan CPB (Horst, The Netherlands). At the start of the experiments animals weighed approximately 250 g, and were housed two per cage in macrolon cages (42.5 26.6 18.5 cm; length width height) under a reversed 12 h light/dark cycle (lights on at 7.00 P.M.) at controlled room temperature (21 72 °C) and relative humidity of 607 15%. Animals were maintained at approximately 90% of their free-feeding weight, starting one week prior to the beginning of the experiments by restricting the amount of standard rodent food chow. Water was available ad libitum throughout the entire experiment. All experiments were conducted with the approval of the animal ethical committee of the VU University Medical Center and VU University Amsterdam, the Netherlands, and all efforts were made to minimize animal suffering and reduce the number of animals used. 2.2. Apparatus Experiments were conducted in identical rat five hole nose poke operant chambers with stainless steel grid floors (MEDNPW-5L, Med Associates Inc., St. Albans, VT, USA) housed in sound-insulating and ventilated cubicles. Set in the curved wall of each box was an array of five holes. Each nose poke unit was equipped with an infrared detector and a yellow light emitting diode stimulus light. Rodent food pellets (45 mg, Formula P, BioServ, Frenchtown, USA) could be delivered at the opposite wall via a dispenser. In addition, a white house light could illuminate the chamber. A computer equipped with MED-PC version 1.17 (Med Associates Inc.) controlled experimental sessions and recorded data. Animals were tested once daily from Monday until Friday, during the dark phase of the light/dark cycle.
2.3. Behavioral procedure 5-choice serial reaction time task A detailed description of the 5-CSRTT behavioral procedure in our laboratory has been provided previously (Van Gaalen et al., 2006; Wiskerke et al., 2012). In short, rats were trained to detect and respond to a brief visual stimulus in one of 5 nose poke units in order to obtain a food reward. Each session terminated after 100 trials or 30 min, whichever occurred first. Initially the duration of this stimulus was 32 s and was gradually decreased to 1 s over sessions until animals reached stable baseline performance (accuracy 480% correct choice and o20% errors of omission). Responding during stimulus presentation or within the limited hold (LH) period of 2 s was counted as a correct response. Incorrect, premature responses during the fixed 5-s intertrial interval, and errors of omission (no responses or a response after the LH) did not lead to the delivery of a food reward and resulted in a 5-s time-out period during which the houselight was extinguished. Perseverative responses after correct choice, i.e., repeated responding during stimulus presentation into any stimulus unit following correct stimulus detection and before pellet collection, were measured but did not have any programmed consequences. The primary measure visuospatial attention was accurate choice calculated as [number correct trials/(correct þincorrect trials)]*100 and for inhibitory control the primary measure was the number of premature responses. In addition, the following other parameters were measured that reflect behavioral control: omission errors, i.e., the total number of omitted trials during a session; the number of perseverative responses after correct choice, measuring aspects of compulsive behavior (Robbins, 2002); response latency to make a correct choice, i.e., the mean time between stimulus onset and nose poke in the illuminated unit; and feeder latency, i.e., the latency to collect a pellet following correct choice. 2.4. Surgical procedure Upon stable baseline performance in the 5-CSRTT animals were prepared for cannulation surgery by terminating the food restriction and providing free access to food for three days prior to surgery. Placement of indwelling double guide cannulae (model C235G/1.5, Plastics One, Roanoke, VA, USA) occurred under inhalation anaesthesia using a combination of oxygen (0.8 l/min) and isoflurane (1.75–2.5%; Pharmachemie BV, Haarlem, the Netherlands) in a stereotaxic instrument (David Kopf Instruments, Tujunga, CA, USA). Guide cannulae were positioned 1 mm above border of the prelimbic and infralimbic cortex and anchored to the skull with four stainless steel screws and dental acrylic cement. This infusion site was chosen, because our previous murine work on neuregulin-ErbB mechanisms of inhibitory control did not preferentially target a specific mPFC subregion (Loos et al., 2014). The coordinates (in mm, relative to bregma) used for placement of intracranial cannulae were A/Pþ3.2 mm, M/L 70.75 mm, D/V3.6 mm ventral to the skull, calculated from Paxinos and Watson (1998). Rats received 0.5 ml/kg of the analgesic Ketofen (1%; Merial, Amstelveen, the Netherlands) and 0.33 ml/kg of the antibiotic Baytril (2.5%; Bayer, Mijdrecht, the Netherlands) prior to surgery. Following surgery, the animals were housed individually and had ad libitum access to food for a week before retraining in the 5-choice serial reaction time task. 2.5. Intracranial infusion procedure Intracranial infusions were carried out when stable baseline performance was re-established, which took 17 sessions. Initially, during two sham infusion sessions, animals were habituated to insertion of the injectors into the guide cannulae (injectors: 33
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gauge and extending 1 (7 0.04) mm beyond guide cannulae). During the infusion experiments at least two baseline training sessions lapsed in between infusion days during which no infusions were conducted. Rats were bilaterally infused with a total volume of 0.5 μl of the ErbB4 tyrosine kinase receptor inhibitor JNJ-28871063 over a period of 2 min at a rate of 0.25 μl/min using 10 μl Hamilton syringes driven by a syringe infusion pump (Harvard Apparatus, South Natick, MA, USA). Following infusion, the injectors remained in place for an additional 60 s to allow diffusion of the drug, rats were placed in the operant cages and testing commenced 5 min later. Since only few studies have reported on the in vivo effects of JNJ-28871063 (Emanuel et al., 2008; Golani et al., 2014) and no intracranial infusion studies have been conducted as yet, drug doses were not counterbalanced across rats and the order of testing different doses of JNJ-28871063 was saline – 10 μg – 15 μg – 20 μg/side. To assure that results on infusion days were attributable to drug effects and not explained by residual drug-induced changes in baseline performance, repeated measures ANOVAs were performed on the 4 baseline training sessions immediately preceding drug infusion test sessions.
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3. Results 3.1. Histological verification of cannulae placement Following histological analyses, correct placement of the cannulae in the medioprefrontal cortex was verified for all animals. As shown in Fig. 1, infusion sites were located around the border of the prelimbic and infralimbic cortices. Two animals were excluded from all analyses because in both of these rats one of the bilateral cannulae tips was positioned in the midline region and outside the cortical layers. Therefore, in total n¼14 animals were included in all analyses. 3.2. Effects of JNJ-28871063 on performance in the 5-CSRTT Stable baseline performance was achieved after approximately 30 sessions of training and animals were trained for an additional week on the task before surgery. The first intracranial infusion session was carried out after 23 training sessions to re-establish baseline performance, including both sham infusion sessions.
2.6. Assessment of cannulae placement Following completion of the behavioral procedures, animals were deeply anaesthetized using sodium pentobarbital (Ceva Sante Animale BV, Maassluis, the Netherlands; 60 mg/ml, i.p.). Subsequently, animals were perfused transcardially with 100 ml 0.9% NaCl, followed by 500 ml 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS, pH 7.2). Brains were removed rapidly and post-fixed for 1 h in the same fixative at room temperature, then stored in 5% sucrose in 0.1 M PBS at 4 °C. Coronal sections of 40 mm were cut on a cryostat and subsequently stained with thionine for the determination of the infusion sites. Only animals with correct cannulae placements were included in the analyses. 2.7. Drugs JNJ-28871063 hydrochloride (Santa Cruz Biotechnology, Dallas, TX, USA) was dissolved in 100% DMSO (Sigma-Aldrich, St. Louis, MO, USA). Drug doses were freshly prepared on each test day and were intracranially infused as described above. 2.8. Statistical analyses Data were analyzed using IBM SPSS Statistics version 20 (IBM Corporation, Armonk, NY, USA) and were subjected to repeated measures analysis of variance (ANOVA) with days (baseline training) as within subjects variable, or drug dose as within subjects variable. The homogeneity of variance across groups was determined using Mauchly's tests for equal variances and in case of violation of homogeneity, Huynh-Feldt epsilon (ε) adjusted degrees of freedom and resulting more conservative probability values were depicted and used for subsequent analyses. As neuregulin-ErbB signaling was hypothesized to modulate inhibitory control, in case of statistically significant main dose effects planned comparisons were conducted using a general linear model simple contrast that compares the mean of each dose to the mean of vehicle infusion. Two-tailed Pearson's correlation coefficients were also calculated in order to explore the relationship between the magnitude of JNJ-28871063 effects, i.e., change in premature responding, and baseline premature responding under vehicle condition. The level of probability for statistically significant effects was set at 0.05. Graphs were produced using GraphPad Prism version 5.02 for Windows (GraphPad Software, San Diego, CA, USA).
Fig. 1. Schematic drawing of coronal sections depicting cannulae placement into the medioprefrontal cortex at a level of 3.70 mm, 3.20 mm and 2.70 mm rostral to bregma. Drawings are adapted from Paxinos and Watson (1998).
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Fig. 2. Behavioral performance during baseline training sessions immediately preceding drug infusion test days. Accurate choice (left y-axis, closed circles) and number of omission errors (right y-axis, open circles) (A); Number of premature responses (left y-axis, closed circles) and perseverative responses (right y-axis, open circles) (B). Latency to make a correct stimulus detection (left y-axis, closed circles) and latency to collect food reward after correct choice (right y-axis, open circles) (C). In total n ¼14 rats were included in the analyses and data are depicted as mean þ S.E.M.
Analysis of the four baseline training sessions immediately preceding drug infusion sessions indicated that for all task parameters behavioral performance was stable [Fig. 2A, accurate choice: F(3,39) ¼2.12, P¼0.11 and omission errors: F(3,39) ¼0.50, P¼0.68; Fig. 2B, premature responses: F(3,39) ¼0.084, P¼0.97 and perseverative responses: F(3,39) ¼0.92, P¼0.44; Fig. 2C, response latencies: F(3,39) ¼ 0.16, P¼0.92 and feeder latencies: F(3,39) ¼ 1.60, ε ¼0.42, P¼0.23]. Thus, baseline performance did not shift either by drug infusions or repeated exposure to the DMSO vehicle in which JNJ-2881063 was dissolved. As indicated in Fig. 3, intracranial infusion of JNJ-2881063 improved inhibitory control by reducing the number of premature responses (vehicle: 20.8 73.8 responses; 10 mg/side: 13.1 72.6 responses; 15 mg/side: 10.4 72.2 responses; 10 mg/side: 11.1 72.3 responses) [Fig. 3A, F(3,39) ¼ 3.53, P ¼0.022]. Further planned comparisons revealed that the intermediate and high dose of JNJ2881063 significantly decreased premature responding compared to vehicle infusion [F(1,13) ¼7.44, P¼ 0.017 and F(1,13) ¼6.80, P¼0.022, respectively]. In contrast, none of the other behavioral parameters were affected by JNJ-2881063 [Fig. 3B, accurate choice: F(3,39) ¼ 0.46, P¼0.71; Fig. 3C, omission errors: F(3,39) ¼0.70, P¼0.56; Fig. 3D, perseverative responses: F(3,39) ¼1.78, P¼ 0.17;
Fig. 3E, response latencies: F(3,39) ¼ 2.24, ε ¼0.77, P¼0.12; Fig. 3F, feeder latencies: F(3,39) ¼1.48, ε ¼0.59, P¼0.25]. In order to explore whether JNJ-2881063 differentially affected premature responding depending on baseline inhibitory control under vehicle condition, Pearson correlation analyses were performed. As indicated in Fig. 4, these analyses revealed that the effect size of JNJ-2881063 on premature responding correlated negatively with baseline inhibitory control for all doses [10 μg/ side: r ¼ 0.83, Po0.001; Fig. 4 A, 15 μg/side: r ¼ 0.84, Po0.001; Fig. 4B; 20 μg/side: r ¼ 0.82, Po0.001].
4. Discussion The present data reveal an ErbB-dependent mechanism in modulating inhibitory control in the mPFC. We found that intramPFC infusion of the ErbB kinase inhibitor JNJ-2881063 exerted specific behavioral effects by dose-dependently reducing the number of premature responses in the 5-CSRTT and thus resulting in improved inhibitory control. In contrast, other parameters in the task reflecting aspects of visuospatial attention and behavioral control were not affected by JNJ-2881063. Furthermore, these
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Fig. 3. Effects of bilateral intra-mPFC infusion of the ErbB-specific inhibitor JNJ-2881063 (JNJ) on behavioral performance in the 5-choice serial reaction time task, namely the number of premature responses (A), accurate choice (B), number of omissions (C), number of perseverative responses (D), latency to correct stimulus detection (E) and latency to collect food reward after correct choice (F). In total n ¼14 rats were included in the analyses and data are depicted as mean þS.E.M. *p o 0.05 planned comparisons versus vehicle infusion.
beneficial effects of JNJ-2881063 on impulsive action were strongly correlated to baseline levels of premature responding. Whereas most evidence regarding our understanding of neuregulin-ErbB signaling in the mPFC is derived from in vitro studies or electrophysiological recordings in brain slices (e.g., Woo et al., 2007; Chen et al., 2010; Vullhorst et al., 2015), to the best of our knowledge the current study is the first to demonstrate an in vivo behavioral correlate in the rat brain. In addition to neuropsychiatric disorders such as ADHD, bipolar disorder and nicotine dependence, the neuregulin-ErbB pathway has been particularly associated with schizophrenia. A large body of genetic linkage and genome-wide association studies have pinpointed neuregulins and ErbB4 as candidate genes contributing to schizophrenia (for recent review, see Mei and Nave (2014)). Post-mortem studies have further corroborated this and revealed alterations in gene and protein expression levels of neuregulin and ErbB4 in the brains of schizophrenia patients (for review, see Pan et al. (2011)). Interestingly, it has been shown that chronic antipsychotic treatment in rats reduces neuregulin-ErbB4 signaling in
mice (Hahn et al., 2006) and, more recently, that the same treatment decreases ErbB4 receptor expression in the rat prefrontal cortex (Deng et al., 2015). This suggests that antipsychotic treatment-induced changes in neuregulin-ErbB4 signaling may contribute to its therapeutic effects in schizophrenia. Whereas the current study did not employ an animal model of schizophrenia, it is of interest that disturbances in various executive functions are important endophenotypes in schizophrenia, including attention/ vigilance, cognitive flexibility and executive control (Gilmour et al., 2013; Swerdlow et al., 2015). With regard to the latter, disturbed executive or inhibitory control is also a prominent feature of other neuropsychiatric disorders mentioned earlier. As such, the current findings together with our recent observations in mice (Loos et al., 2014) highlight the neuregulin-ErbB pathway as a putative candidate mechanism underlying inhibitory control deficits. Whereas in our previous study targeted deletion of neuregulin 3 and overexpression of neuregulin 3 in the mPFC indicated that chronic stimulation of mPFC neuregulin-ErbB signaling changed inhibitory control in the murine 5-CSRTT, the present findings extend those
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Fig. 4. Baseline-dependent effects of intracranial JNJ infusion at 15 μg/side (A) and 20 μg/side (B) on improving impulsive action in the 5-CSRTT. In total n ¼ 14 rats were included in the analyses.
showing that acute inhibition of mPFC neuregulin-ErbB signaling by JNJ-2881063 also affects inhibitory control. These data indicate that neuregulin-ErbB signaling pathway is actively engaged in inhibitory control during execution of a cognitive task such as the 5-CSRTT. Functionally, in the mPFC ErbB4 activation has been shown to inhibit NMDA receptor signaling by promoting release of GABA from interneurons (Woo et al., 2007; Chen et al., 2010; Vullhorst et al., 2015; for review, see Mei and Nave (2014)). Thus, in the present study intra-mPFC infusion of the ErbB kinase inhibitor JNJ2881063 is likely to have opposite effects and facilitate glutamate transmission. In support of the notion that this may improve inhibitory control, positive allosteric mGlu receptor modulation with the compound ADX47273 has been found to reduce premature responding in the 5-CSRTT (Isherwood et al., 2015). Nonetheless, it should be mentioned that in this latter study the compound was systemically administered potentially providing different results from intra-mPFC administration. Conversely, in line with the NMDA receptor hypofunction hypothesis of schizophrenia (Gilmour et al., 2012), antagonism of NMDA receptors in the mPFC has been shown to increase premature responding in the 5-CSRTT (Mirjana et al., 2004; Murphy et al., 2005, 2012). Taken together, ErbB4-mediated modulation of glutamate transmission in the mPFC could be a possible explanation by which JNJ-2881063 improves inhibitory control processes. A limitation of the current study is the effect of JNJ-2881063 on inhibition of other ErbB kinases. Whereas this compound has the lowest IC50 value for inhibition of ErbB4 kinase, the in vitro IC50 for other ErbB kinases including ErbB1 and ErbB2 is in the same concentration range (o2 fold difference in IC50; Emanuel et al., 2008). As a result, not only ErbB4 kinase inhibition, but also ErbB1, ErbB2 or ErbB3 kinase inhibition could play a role in the effects of JNJ-2881063 on inhibitory control. Neuroanatomically, ErbB1, ErbB2 and ErbB3 are abundantly expressed in the midbrain particularly during early development and expression levels of these kinases decline during adulthood (Abe et al., 2009). However, during adulthood residual levels of ErbB1 are still co-expressed in ErbB4-positive GABAergic interneurons in the cortex (Fox and Kornblum, 2005). Furthermore, in the adult forebrain ErbB3 is primarily expressed in glial cells (Gerecke et al., 2001) and
although ErbB3 kinase activity itself is weak, it signals by heterodimerizing with other ErbBs (Pinkas-Kramarski et al., 1998). The fact that in the current study JNJ-2881063 was infused into the mPFC, containing higher ErbB4 expression compared to ErbB1 expression in GABA-ergic interneurons (Fazzari et al., 2010; Neddens et al., 2011), favors an ErbB4-mediated mechanism, yet we cannot exclude other ErbB-mediated mechanisms. Also in the present study, JNJ-2881063 was infused in the mPFC at a level bordering the infralimbic and prelimbic cortices, because our previous murine study indicated expression of Nrg3 across different subregions of the mPFC without preferential expression within specific mPFC subregions (Loos et al., 2014). Previous lesion work in rats has demonstrated intact functioning of the infralimbic cortex to be crucial to inhibitory control in the 5-CSRTT (Chudasama et al., 2003). In this regard, the current study does not allow to delineate whether neuregulin-ErbB involvement in inhibitory control in this task is restricted to the infralimbic cortex. The majority of infusion sites were located in the ventral parts of the prelimbic cortex, although these JNJ-2881063 infusion effects may also be partly mediated via infralimbic mechanisms due to drug diffusion. Future work is warranted to unravel subregion-specificity of neuregulin-ErbB mechanisms in the mPFC. Taken together, this study is the first to demonstrate the role of ErbB-dependent signaling in impulsive action in rats. As such, the current data expand on our earlier findings in mice (Loos et al., 2014) and provide further insight into the importance of neuregulin-ErbB signaling in executive cognitive functions across species. This is highly relevant given the association of alterations in this signaling pathway with various psychiatric disorders including schizophrenia, ADHD, nicotine dependence and bipolar disorder.
Disclosure/Conflict of Interest Part of the work described in the study was carried out with the financial support of Grant ERAB EA 1135 awarded to TJV.
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Behavioural pharmacology
Prefrontal cortical neuregulin-ErbB modulation of inhibitory control in rats Maarten Loos a,b, Dustin Schetters c, Myrthe Hoogeland c, Sabine Spijker a, Taco J. de Vries a,c, Tommy Pattij c,n a Department of Molecular and Cellular Neurobiology, Neuroscience Campus Amsterdam, Center for Neurogenomic and Cognitive Research, VU University, Amsterdam, The Netherlands b Sylics (Synaptologics BV), Amsterdam, The Netherlands c Department of Anatomy and Neurosciences, Neuroscience Campus Amsterdam, VU University Medical Center, Amsterdam, The Netherlands
art ic l e i nf o
a b s t r a c t
Article history: Received 4 February 2016 Received in revised form 7 April 2016 Accepted 11 April 2016 Available online 11 April 2016
Impulse control disturbances are key features of various neuropsychiatric and neurological disorders, such as attention-deficit/hyperactivity disorder, drug addiction, Parkinson disease and schizophrenia. Whereas over the last years accumulating evidence has highlighted monoaminergic modulation of the processes underlying impulse control, investigating novel mechanisms beyond monoamines may provide new intervention strategies to ameliorate impulse control disturbances. Recent work has associated the neuregulin (Nrg)-ErbB pathway with several neuropsychiatric diseases, as well as indicated its involvement in murine measures of impulse control. The aim of the present study was to investigate whether this Nrg-ErbB signaling pathway also modulates impulsive action in rats. To this end, a group of rats was trained in the 5-choice serial reaction time task (5-CSRTT), an operant paradigm that provides measures of visuospatial attention and inhibitory control processes. Upon stable baseline performance, the ErbB tyrosine kinase receptor inhibitor JNJ-28871063 (JNJ) was intracranially infused into the medioprefrontal cortex prior to test sessions. Results showed that JNJ dose-dependently improved measures of impulsive action. Importantly, other measures in the 5-CSRTT reflecting visuospatial attention or aspects of motivational behavior were not altered by JNJ. In conclusion, the present data strengthen a role for the Nrg-ErbB4 pathway in the prefrontal cortex in cognitive functioning, and in particular point towards involvement in the processes underlying impulse control. & 2016 Elsevier B.V. All rights reserved.
Keywords: Cognition ErbB4 Impulsivity Prelimbic cortex Neuregulin Rat
1. Introduction ErbB receptors belong to a family of receptor tyrosine kinases that are activated by ligands of the neuregulin family. NeuregulinErbB signaling plays an important role in neural development, including neural circuit assembly, synaptic plasticity and neurotransmission (Mei and Nave, 2014). With regard to the receptor ErbB kinase family, at least four different ErbB receptor kinases have been identified, namely ErbB1, ErbB2, ErbB3 and ErbB4 (Birchmeier, 2009). In recent years, particularly the neuregulinErbB4 signaling pathway has been associated with neuropsychiatric disorders, such as attention-deficit/hyperactivity disorder (ADHD), bipolar disorder, schizophrenia and nicotine dependence in genome wide association studies (Sonuga-Barke et al., 2008; Pan et al., 2011; Mei and Nave, 2014; Turner et al., 2014). Since n
Corresponding author. E-mail address: [email protected] (T. Pattij).
http://dx.doi.org/10.1016/j.ejphar.2016.04.015 0014-2999/& 2016 Elsevier B.V. All rights reserved.
maladaptive impulsivity and more specifically inhibitory control deficits, are shared symptoms in these disorders (Moeller et al., 2001; Gilmour et al., 2013; Ethridge et al., 2014), alterations in neuregulin-ErbB signaling may provide a novel common underlying mechanism. Indeed, in support we recently identified a gene within the neuregulin family, neuregulin 3 (Nrg3) that was causally related to impulsive behavior in mice (Loos et al., 2014). To date, much of our understanding of the neural mechanisms of inhibitory control subserving impulsivity originates from rodent studies. The collective work in these models has strongly implicated various neurotransmitter systems including the monoaminergic, glutamatergic, opioid and more recently GABAergic system in inhibitory control (Pattij and Vanderschuren, 2008; Dalley et al., 2011; Caprioli et al., 2014). In addition, the medioprefrontal cortex (mPFC) and connected ventral striatal brain regions comprise a crucial neuroanatomical circuit, since lesions, reversible inactivation and functional disconnections of mPFC subregions and connected striatum impair inhibitory control
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capacities (Chudasama et al., 2003; Christakou et al., 2004; Feja and Koch, 2014). Of abovementioned brain regions the mPFC contains high densities of ErbB4 receptor kinase, which are mainly expressed in GABA-ergic interneurons and not pyramidal neurons and are conserved in various species ranging from rodents to humans (Fazzari et al., 2010; Neddens et al., 2011). As such, ErbB4 activation has been found to promote GABA release and thereby to modulate functioning of glutamatergic pyramidal neurons and thus N-methyl-D-Aspartate (NMDA) receptor signaling (Woo et al., 2007; Chen et al., 2010; Vullhorst et al., 2015). Several studies have demonstrated glutamatergic modulation of inhibitory control in the mPFC (Mirjana et al., 2004; Murphy et al., 2005; Counotte et al., 2011; Murphy et al., 2012). Hence, this places the ErbB4 receptor kinase in the position to impact on cognitive functions, including inhibitory control. The aim of the present study was to investigate whether, in addition to our previous murine observations (Loos et al., 2014), neuregulin-ErbB signaling in the mPFC could also contribute to inhibitory control across species in rats. For this purpose, rats were trained in the 5-choice serial reaction time task (5-CSRTT), a translational paradigm measuring aspects of visuospatial attention and inhibitory control (Bari et al., 2008). Upon stable baseline performance, intra-mPFC infusions with the ErbB kinase inhibitor JNJ-28871063 (Emanuel et al., 2008) were performed to assess its effects on visuospatial attention and inhibitory control.
2. Materials and methods 2.1. Subjects Sixteen male Wistar rats were obtained from Harlan CPB (Horst, The Netherlands). At the start of the experiments animals weighed approximately 250 g, and were housed two per cage in macrolon cages (42.5 26.6 18.5 cm; length width height) under a reversed 12 h light/dark cycle (lights on at 7.00 P.M.) at controlled room temperature (21 72 °C) and relative humidity of 607 15%. Animals were maintained at approximately 90% of their free-feeding weight, starting one week prior to the beginning of the experiments by restricting the amount of standard rodent food chow. Water was available ad libitum throughout the entire experiment. All experiments were conducted with the approval of the animal ethical committee of the VU University Medical Center and VU University Amsterdam, the Netherlands, and all efforts were made to minimize animal suffering and reduce the number of animals used. 2.2. Apparatus Experiments were conducted in identical rat five hole nose poke operant chambers with stainless steel grid floors (MEDNPW-5L, Med Associates Inc., St. Albans, VT, USA) housed in sound-insulating and ventilated cubicles. Set in the curved wall of each box was an array of five holes. Each nose poke unit was equipped with an infrared detector and a yellow light emitting diode stimulus light. Rodent food pellets (45 mg, Formula P, BioServ, Frenchtown, USA) could be delivered at the opposite wall via a dispenser. In addition, a white house light could illuminate the chamber. A computer equipped with MED-PC version 1.17 (Med Associates Inc.) controlled experimental sessions and recorded data. Animals were tested once daily from Monday until Friday, during the dark phase of the light/dark cycle.
2.3. Behavioral procedure 5-choice serial reaction time task A detailed description of the 5-CSRTT behavioral procedure in our laboratory has been provided previously (Van Gaalen et al., 2006; Wiskerke et al., 2012). In short, rats were trained to detect and respond to a brief visual stimulus in one of 5 nose poke units in order to obtain a food reward. Each session terminated after 100 trials or 30 min, whichever occurred first. Initially the duration of this stimulus was 32 s and was gradually decreased to 1 s over sessions until animals reached stable baseline performance (accuracy 480% correct choice and o20% errors of omission). Responding during stimulus presentation or within the limited hold (LH) period of 2 s was counted as a correct response. Incorrect, premature responses during the fixed 5-s intertrial interval, and errors of omission (no responses or a response after the LH) did not lead to the delivery of a food reward and resulted in a 5-s time-out period during which the houselight was extinguished. Perseverative responses after correct choice, i.e., repeated responding during stimulus presentation into any stimulus unit following correct stimulus detection and before pellet collection, were measured but did not have any programmed consequences. The primary measure visuospatial attention was accurate choice calculated as [number correct trials/(correct þincorrect trials)]*100 and for inhibitory control the primary measure was the number of premature responses. In addition, the following other parameters were measured that reflect behavioral control: omission errors, i.e., the total number of omitted trials during a session; the number of perseverative responses after correct choice, measuring aspects of compulsive behavior (Robbins, 2002); response latency to make a correct choice, i.e., the mean time between stimulus onset and nose poke in the illuminated unit; and feeder latency, i.e., the latency to collect a pellet following correct choice. 2.4. Surgical procedure Upon stable baseline performance in the 5-CSRTT animals were prepared for cannulation surgery by terminating the food restriction and providing free access to food for three days prior to surgery. Placement of indwelling double guide cannulae (model C235G/1.5, Plastics One, Roanoke, VA, USA) occurred under inhalation anaesthesia using a combination of oxygen (0.8 l/min) and isoflurane (1.75–2.5%; Pharmachemie BV, Haarlem, the Netherlands) in a stereotaxic instrument (David Kopf Instruments, Tujunga, CA, USA). Guide cannulae were positioned 1 mm above border of the prelimbic and infralimbic cortex and anchored to the skull with four stainless steel screws and dental acrylic cement. This infusion site was chosen, because our previous murine work on neuregulin-ErbB mechanisms of inhibitory control did not preferentially target a specific mPFC subregion (Loos et al., 2014). The coordinates (in mm, relative to bregma) used for placement of intracranial cannulae were A/Pþ3.2 mm, M/L 70.75 mm, D/V3.6 mm ventral to the skull, calculated from Paxinos and Watson (1998). Rats received 0.5 ml/kg of the analgesic Ketofen (1%; Merial, Amstelveen, the Netherlands) and 0.33 ml/kg of the antibiotic Baytril (2.5%; Bayer, Mijdrecht, the Netherlands) prior to surgery. Following surgery, the animals were housed individually and had ad libitum access to food for a week before retraining in the 5-choice serial reaction time task. 2.5. Intracranial infusion procedure Intracranial infusions were carried out when stable baseline performance was re-established, which took 17 sessions. Initially, during two sham infusion sessions, animals were habituated to insertion of the injectors into the guide cannulae (injectors: 33
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gauge and extending 1 (7 0.04) mm beyond guide cannulae). During the infusion experiments at least two baseline training sessions lapsed in between infusion days during which no infusions were conducted. Rats were bilaterally infused with a total volume of 0.5 μl of the ErbB4 tyrosine kinase receptor inhibitor JNJ-28871063 over a period of 2 min at a rate of 0.25 μl/min using 10 μl Hamilton syringes driven by a syringe infusion pump (Harvard Apparatus, South Natick, MA, USA). Following infusion, the injectors remained in place for an additional 60 s to allow diffusion of the drug, rats were placed in the operant cages and testing commenced 5 min later. Since only few studies have reported on the in vivo effects of JNJ-28871063 (Emanuel et al., 2008; Golani et al., 2014) and no intracranial infusion studies have been conducted as yet, drug doses were not counterbalanced across rats and the order of testing different doses of JNJ-28871063 was saline – 10 μg – 15 μg – 20 μg/side. To assure that results on infusion days were attributable to drug effects and not explained by residual drug-induced changes in baseline performance, repeated measures ANOVAs were performed on the 4 baseline training sessions immediately preceding drug infusion test sessions.
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3. Results 3.1. Histological verification of cannulae placement Following histological analyses, correct placement of the cannulae in the medioprefrontal cortex was verified for all animals. As shown in Fig. 1, infusion sites were located around the border of the prelimbic and infralimbic cortices. Two animals were excluded from all analyses because in both of these rats one of the bilateral cannulae tips was positioned in the midline region and outside the cortical layers. Therefore, in total n¼14 animals were included in all analyses. 3.2. Effects of JNJ-28871063 on performance in the 5-CSRTT Stable baseline performance was achieved after approximately 30 sessions of training and animals were trained for an additional week on the task before surgery. The first intracranial infusion session was carried out after 23 training sessions to re-establish baseline performance, including both sham infusion sessions.
2.6. Assessment of cannulae placement Following completion of the behavioral procedures, animals were deeply anaesthetized using sodium pentobarbital (Ceva Sante Animale BV, Maassluis, the Netherlands; 60 mg/ml, i.p.). Subsequently, animals were perfused transcardially with 100 ml 0.9% NaCl, followed by 500 ml 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS, pH 7.2). Brains were removed rapidly and post-fixed for 1 h in the same fixative at room temperature, then stored in 5% sucrose in 0.1 M PBS at 4 °C. Coronal sections of 40 mm were cut on a cryostat and subsequently stained with thionine for the determination of the infusion sites. Only animals with correct cannulae placements were included in the analyses. 2.7. Drugs JNJ-28871063 hydrochloride (Santa Cruz Biotechnology, Dallas, TX, USA) was dissolved in 100% DMSO (Sigma-Aldrich, St. Louis, MO, USA). Drug doses were freshly prepared on each test day and were intracranially infused as described above. 2.8. Statistical analyses Data were analyzed using IBM SPSS Statistics version 20 (IBM Corporation, Armonk, NY, USA) and were subjected to repeated measures analysis of variance (ANOVA) with days (baseline training) as within subjects variable, or drug dose as within subjects variable. The homogeneity of variance across groups was determined using Mauchly's tests for equal variances and in case of violation of homogeneity, Huynh-Feldt epsilon (ε) adjusted degrees of freedom and resulting more conservative probability values were depicted and used for subsequent analyses. As neuregulin-ErbB signaling was hypothesized to modulate inhibitory control, in case of statistically significant main dose effects planned comparisons were conducted using a general linear model simple contrast that compares the mean of each dose to the mean of vehicle infusion. Two-tailed Pearson's correlation coefficients were also calculated in order to explore the relationship between the magnitude of JNJ-28871063 effects, i.e., change in premature responding, and baseline premature responding under vehicle condition. The level of probability for statistically significant effects was set at 0.05. Graphs were produced using GraphPad Prism version 5.02 for Windows (GraphPad Software, San Diego, CA, USA).
Fig. 1. Schematic drawing of coronal sections depicting cannulae placement into the medioprefrontal cortex at a level of 3.70 mm, 3.20 mm and 2.70 mm rostral to bregma. Drawings are adapted from Paxinos and Watson (1998).
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Fig. 2. Behavioral performance during baseline training sessions immediately preceding drug infusion test days. Accurate choice (left y-axis, closed circles) and number of omission errors (right y-axis, open circles) (A); Number of premature responses (left y-axis, closed circles) and perseverative responses (right y-axis, open circles) (B). Latency to make a correct stimulus detection (left y-axis, closed circles) and latency to collect food reward after correct choice (right y-axis, open circles) (C). In total n ¼14 rats were included in the analyses and data are depicted as mean þ S.E.M.
Analysis of the four baseline training sessions immediately preceding drug infusion sessions indicated that for all task parameters behavioral performance was stable [Fig. 2A, accurate choice: F(3,39) ¼2.12, P¼0.11 and omission errors: F(3,39) ¼0.50, P¼0.68; Fig. 2B, premature responses: F(3,39) ¼0.084, P¼0.97 and perseverative responses: F(3,39) ¼0.92, P¼0.44; Fig. 2C, response latencies: F(3,39) ¼ 0.16, P¼0.92 and feeder latencies: F(3,39) ¼ 1.60, ε ¼0.42, P¼0.23]. Thus, baseline performance did not shift either by drug infusions or repeated exposure to the DMSO vehicle in which JNJ-2881063 was dissolved. As indicated in Fig. 3, intracranial infusion of JNJ-2881063 improved inhibitory control by reducing the number of premature responses (vehicle: 20.8 73.8 responses; 10 mg/side: 13.1 72.6 responses; 15 mg/side: 10.4 72.2 responses; 10 mg/side: 11.1 72.3 responses) [Fig. 3A, F(3,39) ¼ 3.53, P ¼0.022]. Further planned comparisons revealed that the intermediate and high dose of JNJ2881063 significantly decreased premature responding compared to vehicle infusion [F(1,13) ¼7.44, P¼ 0.017 and F(1,13) ¼6.80, P¼0.022, respectively]. In contrast, none of the other behavioral parameters were affected by JNJ-2881063 [Fig. 3B, accurate choice: F(3,39) ¼ 0.46, P¼0.71; Fig. 3C, omission errors: F(3,39) ¼0.70, P¼0.56; Fig. 3D, perseverative responses: F(3,39) ¼1.78, P¼ 0.17;
Fig. 3E, response latencies: F(3,39) ¼ 2.24, ε ¼0.77, P¼0.12; Fig. 3F, feeder latencies: F(3,39) ¼1.48, ε ¼0.59, P¼0.25]. In order to explore whether JNJ-2881063 differentially affected premature responding depending on baseline inhibitory control under vehicle condition, Pearson correlation analyses were performed. As indicated in Fig. 4, these analyses revealed that the effect size of JNJ-2881063 on premature responding correlated negatively with baseline inhibitory control for all doses [10 μg/ side: r ¼ 0.83, Po0.001; Fig. 4 A, 15 μg/side: r ¼ 0.84, Po0.001; Fig. 4B; 20 μg/side: r ¼ 0.82, Po0.001].
4. Discussion The present data reveal an ErbB-dependent mechanism in modulating inhibitory control in the mPFC. We found that intramPFC infusion of the ErbB kinase inhibitor JNJ-2881063 exerted specific behavioral effects by dose-dependently reducing the number of premature responses in the 5-CSRTT and thus resulting in improved inhibitory control. In contrast, other parameters in the task reflecting aspects of visuospatial attention and behavioral control were not affected by JNJ-2881063. Furthermore, these
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Fig. 3. Effects of bilateral intra-mPFC infusion of the ErbB-specific inhibitor JNJ-2881063 (JNJ) on behavioral performance in the 5-choice serial reaction time task, namely the number of premature responses (A), accurate choice (B), number of omissions (C), number of perseverative responses (D), latency to correct stimulus detection (E) and latency to collect food reward after correct choice (F). In total n ¼14 rats were included in the analyses and data are depicted as mean þS.E.M. *p o 0.05 planned comparisons versus vehicle infusion.
beneficial effects of JNJ-2881063 on impulsive action were strongly correlated to baseline levels of premature responding. Whereas most evidence regarding our understanding of neuregulin-ErbB signaling in the mPFC is derived from in vitro studies or electrophysiological recordings in brain slices (e.g., Woo et al., 2007; Chen et al., 2010; Vullhorst et al., 2015), to the best of our knowledge the current study is the first to demonstrate an in vivo behavioral correlate in the rat brain. In addition to neuropsychiatric disorders such as ADHD, bipolar disorder and nicotine dependence, the neuregulin-ErbB pathway has been particularly associated with schizophrenia. A large body of genetic linkage and genome-wide association studies have pinpointed neuregulins and ErbB4 as candidate genes contributing to schizophrenia (for recent review, see Mei and Nave (2014)). Post-mortem studies have further corroborated this and revealed alterations in gene and protein expression levels of neuregulin and ErbB4 in the brains of schizophrenia patients (for review, see Pan et al. (2011)). Interestingly, it has been shown that chronic antipsychotic treatment in rats reduces neuregulin-ErbB4 signaling in
mice (Hahn et al., 2006) and, more recently, that the same treatment decreases ErbB4 receptor expression in the rat prefrontal cortex (Deng et al., 2015). This suggests that antipsychotic treatment-induced changes in neuregulin-ErbB4 signaling may contribute to its therapeutic effects in schizophrenia. Whereas the current study did not employ an animal model of schizophrenia, it is of interest that disturbances in various executive functions are important endophenotypes in schizophrenia, including attention/ vigilance, cognitive flexibility and executive control (Gilmour et al., 2013; Swerdlow et al., 2015). With regard to the latter, disturbed executive or inhibitory control is also a prominent feature of other neuropsychiatric disorders mentioned earlier. As such, the current findings together with our recent observations in mice (Loos et al., 2014) highlight the neuregulin-ErbB pathway as a putative candidate mechanism underlying inhibitory control deficits. Whereas in our previous study targeted deletion of neuregulin 3 and overexpression of neuregulin 3 in the mPFC indicated that chronic stimulation of mPFC neuregulin-ErbB signaling changed inhibitory control in the murine 5-CSRTT, the present findings extend those
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Fig. 4. Baseline-dependent effects of intracranial JNJ infusion at 15 μg/side (A) and 20 μg/side (B) on improving impulsive action in the 5-CSRTT. In total n ¼ 14 rats were included in the analyses.
showing that acute inhibition of mPFC neuregulin-ErbB signaling by JNJ-2881063 also affects inhibitory control. These data indicate that neuregulin-ErbB signaling pathway is actively engaged in inhibitory control during execution of a cognitive task such as the 5-CSRTT. Functionally, in the mPFC ErbB4 activation has been shown to inhibit NMDA receptor signaling by promoting release of GABA from interneurons (Woo et al., 2007; Chen et al., 2010; Vullhorst et al., 2015; for review, see Mei and Nave (2014)). Thus, in the present study intra-mPFC infusion of the ErbB kinase inhibitor JNJ2881063 is likely to have opposite effects and facilitate glutamate transmission. In support of the notion that this may improve inhibitory control, positive allosteric mGlu receptor modulation with the compound ADX47273 has been found to reduce premature responding in the 5-CSRTT (Isherwood et al., 2015). Nonetheless, it should be mentioned that in this latter study the compound was systemically administered potentially providing different results from intra-mPFC administration. Conversely, in line with the NMDA receptor hypofunction hypothesis of schizophrenia (Gilmour et al., 2012), antagonism of NMDA receptors in the mPFC has been shown to increase premature responding in the 5-CSRTT (Mirjana et al., 2004; Murphy et al., 2005, 2012). Taken together, ErbB4-mediated modulation of glutamate transmission in the mPFC could be a possible explanation by which JNJ-2881063 improves inhibitory control processes. A limitation of the current study is the effect of JNJ-2881063 on inhibition of other ErbB kinases. Whereas this compound has the lowest IC50 value for inhibition of ErbB4 kinase, the in vitro IC50 for other ErbB kinases including ErbB1 and ErbB2 is in the same concentration range (o2 fold difference in IC50; Emanuel et al., 2008). As a result, not only ErbB4 kinase inhibition, but also ErbB1, ErbB2 or ErbB3 kinase inhibition could play a role in the effects of JNJ-2881063 on inhibitory control. Neuroanatomically, ErbB1, ErbB2 and ErbB3 are abundantly expressed in the midbrain particularly during early development and expression levels of these kinases decline during adulthood (Abe et al., 2009). However, during adulthood residual levels of ErbB1 are still co-expressed in ErbB4-positive GABAergic interneurons in the cortex (Fox and Kornblum, 2005). Furthermore, in the adult forebrain ErbB3 is primarily expressed in glial cells (Gerecke et al., 2001) and
although ErbB3 kinase activity itself is weak, it signals by heterodimerizing with other ErbBs (Pinkas-Kramarski et al., 1998). The fact that in the current study JNJ-2881063 was infused into the mPFC, containing higher ErbB4 expression compared to ErbB1 expression in GABA-ergic interneurons (Fazzari et al., 2010; Neddens et al., 2011), favors an ErbB4-mediated mechanism, yet we cannot exclude other ErbB-mediated mechanisms. Also in the present study, JNJ-2881063 was infused in the mPFC at a level bordering the infralimbic and prelimbic cortices, because our previous murine study indicated expression of Nrg3 across different subregions of the mPFC without preferential expression within specific mPFC subregions (Loos et al., 2014). Previous lesion work in rats has demonstrated intact functioning of the infralimbic cortex to be crucial to inhibitory control in the 5-CSRTT (Chudasama et al., 2003). In this regard, the current study does not allow to delineate whether neuregulin-ErbB involvement in inhibitory control in this task is restricted to the infralimbic cortex. The majority of infusion sites were located in the ventral parts of the prelimbic cortex, although these JNJ-2881063 infusion effects may also be partly mediated via infralimbic mechanisms due to drug diffusion. Future work is warranted to unravel subregion-specificity of neuregulin-ErbB mechanisms in the mPFC. Taken together, this study is the first to demonstrate the role of ErbB-dependent signaling in impulsive action in rats. As such, the current data expand on our earlier findings in mice (Loos et al., 2014) and provide further insight into the importance of neuregulin-ErbB signaling in executive cognitive functions across species. This is highly relevant given the association of alterations in this signaling pathway with various psychiatric disorders including schizophrenia, ADHD, nicotine dependence and bipolar disorder.
Disclosure/Conflict of Interest Part of the work described in the study was carried out with the financial support of Grant ERAB EA 1135 awarded to TJV.
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