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Activation of type 4 dopaminergic receptors in the prelimbic area of medial prefrontal cortex is necessary for the expression of innate fear behavior
Accepted Manuscript Title: Activation of type 4 dopaminergic receptors in the prelimbic area of medial prefrontal cortex is necessary for the expression of innate fear behavior Authors: Macarena D. Vergara, Victor N. Keller, Jos´e A. Fuentealba, Katia Gysling PII: DOI: Reference:
S0166-4328(16)30858-0 http://dx.doi.org/doi:10.1016/j.bbr.2017.01.050 BBR 10690
To appear in:
Behavioural Brain Research
Received date: Revised date: Accepted date:
25-10-2016 26-1-2017 30-1-2017
Please cite this article as: Vergara Macarena D, Keller Victor N, Fuentealba Jos´e A, Gysling Katia.Activation of type 4 dopaminergic receptors in the prelimbic area of medial prefrontal cortex is necessary for the expression of innate fear behavior.Behavioural Brain Research http://dx.doi.org/10.1016/j.bbr.2017.01.050 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Activation of type 4 dopaminergic receptors in the prelimbic area of medial prefrontal cortex is necessary for the expression of innate fear behavior
Macarena D. Vergara1, Victor N. Keller1,3, José A. Fuentealba2* and Katia Gysling1* 1
Department of Cellular and Molecular Biology, Faculty of Biological Science, Pontificia Universidad Católica de Chile, Santiago, Chile. 2
Department of Pharmacy, Faculty of Chemistry, Pontificia Universidad Católica de Chile, Santiago, Chile. 3
Present address: Department of Psychology, College of Social Science, Michigan State University, East Lansing MI, United States of America. *Co-corresponding authors
Correspondence to: Katia Gysling, PhD Department of Cellular and Molecular Biology Faculty of Biological Science Pontificia Universidad Católica de Chile, Santiago, Chile Phone: 562-23542659 Email: [email protected] or José Fuentealbas, PhD Department of Pharmacy Faculty of Chemistry Pontificia Universidad Católica de Chile, Santiago, Chile Tel: +56-2-2354-5908 E-mail address: [email protected]
Abstract
The prelimbic area (PL) of the medial Prefrontal cortex (mPFC) is involved in the acquisition and expression of conditioned and innate fear. Both types of fear share several neuronal pathways. It has been documented that dopamine (DA) plays an important role in the regulation of aversive memories in the mPFC. The exposure to an aversive stimulus, such as the smell of a predator odor or the exposure to footshock stress is accompanied by an increase in mPFC DA release. Evidence suggests that the type 4 dopaminergic receptor (D4R) is the molecular target through which DA modulates fear expression. In fact, the mPFC is the brain region with the highest expression of D4R; however, the role of D4R in the expression of innate fear has not been fully elucidated. Therefore, the principal objective of this work was to evaluate the participation of mPFC D4R in the expression of innate fear. Rats were exposed to the elevated plus-maze (EPM) and to the cat odor paradigm after the intra PL injection of L-745,870, selective D4R antagonist, to measure the expression of fear-related behaviors. Intra PL injection of L-745,870 increased the time spent in the EPM open arms and decreased freezing behavior in the cat odor paradigm. Our results also showed that D4R is expressed in GABAergic and pyramidal neurons in the PL region of PFC. Thus, D4R antagonism in the PL decreases the expression of innate fearbehavior indicating that the activation of D4R in the PL is necessary for the expression of innate fear-behavior. Keywords: dopamine, prefrontal cortex, innate fear, anxiety, cat odor
1. Introduction The medial Prefrontal cortex (mPFC) receives information from cortical and subcortical structures, and the reciprocal connections with limbic structures allow mPFC to exert a control in emotional memories (McDonald et al., 1996; Dalley et al., 2004). Specifically, PL (prelimibic) and IL (infralimbic) areas of the mPFC have been related with conditioned and innate fear (Quirk et al., 2000; Dielenberg et al., 2001a,b; Lauzon et al., 2009, 2012; Chan et al., 2011). PL has been associated with fear expression and IL has been associated with fear extinction (Sotres-Bayon et al., 2004; Shah et al., 2004; Vidal-Gonzalez et al., 2006; Sotres-Bayon and Quirk, 2010). In conditioned fear the animal associates a neutral stimulus to an aversive stimulus, so the animal learns to fear the neutral stimulus. Instead, innate fear, also named unconditioned fear, does not involve an aversive learning and is expressed when the animal faces risky situations that were recurrent throughout evolutionary history, such as darkness, height, pain, presence of a predator, etc (Jinks and McGregor, 1997; Antoniadis and McDonald, 2001; Dielenberg et al., 2001b; VidalGonzalez et al., 2006). The mPFC receives dopaminergic afferents from the ventral tegmental area (VTA) (Falon 1981; Lammel et al., 2008). DA in the mPFC has been associated with the modulation of aversive memories (Wedzony et al., 1996; Yoshioka et al., 1996; Morrow et al., 2000; Espejo 2003; Giogi et al., 2003; Pezze et al., 2003; Pezze and Feldon, 2004). It has been shown that DA regulates the expression of aversive memories, modulating the connection between mPFC and BLA (Floresco and Tse, 2007). Activation of dopaminergic receptors
has been associated with the modulation of conditioned (Inoue et al., 2000; El-Ghundi et al., 2001; De Oliveira et al., 2006, Hikind and Maroun, 2008) and innate fear (Espejo, 1997; Lauzon et al., 2009). Specifically, the activation of type 4 dopaminergic receptors (D4R) potentiates encoding of emotional memories associated with conditioned fear (Laviolette et al., 2005). D4R is highly expressed in the mPFC (Wedzony et al., 2000; Rivera et al., 2008; De Almedia and Mengod, 2010), particularly in PL (Rivera et al, 2008). However, the specific D4R expression in pyramidal and/or GABAergic neurons has not been fully elucidated. The role of D4R in innate fear has scarcely been studied. In the interesting work of Lauzon et al. (2009) it was shown that intra PL injection of PD-168007, a selective D4R agonist, increases the emotional relevance of a non-aversive stimulus ("footshock" of 0.4 mA) generating fear associated behavior. On the other hand, the injection of PD-168007 attenuates the increase of freezing behavior induced by an aversive stimulus (footshock of 0.8 mA). These results indicate that the activation of PFC D4R modifies the expression of conditioned fear depending on the magnitude of the stimulus presented. Furthermore, D4R activation has been associated with anxiety-like behavior (Shah et al., 2004). These authors showed that the intra PL injection of L-745,870 (1 nmol/0.5 µL), a selective D4R antagonist (Kulagowski et al., 1996; Patel et al., 1997), increases the time spent in the open arms of the elevated plus maze (EPM). This result suggests that D4R activation increases anxiety-like behavior. However, the role of PL D4R in innate fear behavior remains an open question. In the present study we used the cat odor paradigm which allows measuring freezing behavior in rats exposed to an innate aversive stimulus and the EPM re-exposure paradigm to assess aversive emotions. In both behavioral
paradigms, L-745,870 was injected in the PL and the fear-related behaviors were measured. The hypothesis of this work was that the activation of D4R in the PL region of mPFC is necessary for the expression of innate fear. We also studied the specific expression of D4R in PL and IL in GABAergic and/or glutamatergic neuronal phenotypes. 2. Materials and Methods
2.1 Animals All experiments were conducted in accordance with institutional (Catholic University of Chile) and international guidelines (NIH Guide for the Care and Use of Laboratory Animals). Adult male Sprague–Dawley rats (250–300 g) were grown in the animal care facility of Catholic University of Chile under the supervision of a veterinarian. Food and water were available ad libitum. All experiments were conducted during the light hours of the light/dark cycle.
2.2 Immunohistochemistry of D4R Rats were deeply anesthetized with ketamine-xylazine (50-5mg/kg, respectively) and were intracardially perfused with 50 ml of saline, followed by 500 ml of 4% paraformaldehyde (PFA) in phosphate buffer. Then, the animals were guillotined and the brain was post-fixed with 4% PFA for 2 hours and left in 20% sucrose during 2 days. The brains were then cut into 30 µm slices using a Leyca cryostat and slices were placed in 1x PBS. Brain slices were washed with 1x PBS at room temperature (3x10 minutes) and incubated during 1 hour with a blocking solution (1% BSA, 0.2% Triton X-100 and 0.02% azide). Thereafter, the
slices were incubated 48 hours at 4ºC with 1:500 anti-D4R rabbit polyclonal antibody (Calbiochem, Cat. N° 324405). Slices were washed with 1x PBS (3x15 minutes) and the tissue sections were placed for 1 hour in rabbit-biotinylated anti-rabbit IgG (Vector Laboratories, CA) diluted 1:1000 in blocking solution. After a rinse in 1x PBS, the slices were placed in an avidin-biotin complex reagent (Vectastain ABC Kit) in 1x PBS for 1 hour and then rinsed again in 1x PBS. The peroxidase contained in the ABC reagent was visualized by placing the sections in a solution of DAB (1 mg/mL, Sigma), Tris-buffer pH 7.6 (50 mM Tris acid, 150 mM NaCl) and 0.5% H2O2 for 10 minutes. Tissue slices were washed in 1x PBS and mounted in glass slides with 0.1% gelatin, dried for 2 hours and coverslipped. The sections were observed under a light microscope (Olympus) and microphotographs were captured with a digital camera.
2.3 Immunofluorescence of D4R and GABAergic markers To perform the dual immunofluorescence for D4R and markers of GABAergic neurons (parvalbumin (PV), GAD 65/67 or GAD 67), slices were washed with 1x PBS at room temperature (3x10 min). The slices were then incubated during 1 hour with a blocking solution (1% BSA, 0.2% Triton X-100 and 0.02% azide). Thereafter, slices were incubated overnight with 1:200 anti-D4R rabbit polyclonal antibody and 1:200 anti PV (Sigma, p3088), GAD 65/67 goat polyclonal (Santa Cruz, sc-7513) or GAD 67 mouse polyclonal (Millipore, MAB5406). Slices were washed with 1x PBS (3x15 minutes), mounted in glass slides with 0.1% gelatin, allowed to dry for 2 hours and then incubated with the secondary fluorescent antibody (1:1000, Invitrogen) for 2 hours. Finally, the slices were washed with 1x PBS (3x15 minutes) and coverslipped with Dako (Invitrogen). A light microscope
(Olympus), equipped with fluorescent lamp and digital camera, was used to analyze the sections and to capture the microphotographs.
2.4 Animal surgery The animals were injected with ketamine-xylazine (50-5mg/kg, respectively) and placed in a stereotaxic apparatus (Stoelting, Wood Dale, IL, USA), and an incision was made to expose the skull. Stainless steel guide cannulae (plastic One, Roanoke, VA, USA) were bilaterally implanted, targeting the PL cortex (+2.9 mm, AP ±1.9 ML, -1.9 DV; all cannulae angled at 15º towards the midline, Fig. 1). The cannulae were secured to the skull with two screws and cranioplastic cement. After the surgery, rats were injected with 150 µL of 1% ketoprofen and 200 µL of 5 % enrofloxacin, and were returned to their home cages after waking from anesthesia.
2.5 Intracranial infusion procedure Bilateral intra PL infusions were performed with an infusion pump (Harvad Apparatus) at a rate of 0.5 µL/min (0.5 µL final volume). The cannulae were left in place for 1 minute after the end of the infusion. The selective D4R antagonist L-745,870 trihydrochloride (5 nmol/0.5 µL; Tocris, Ellisville, MO, USA) was dissolved in saline. 2.6 Elevated plus maze test The testing of EPM (EPM1) started 7 days after surgery. Rats were handled for 5 minutes in each of 4 consecutive days prior to testing. The test began 3 min after the end of the
infusion procedure. The maze was made of a wooden, plus-shaped apparatus with a height of 70 cm. It consisted of two 50x10 cm open arms and two 50x10x50 enclosed arms. Testing was conducted in a quiet and dimly illuminated room. All testing occurred between 11.00 and 16.00 hours, and behavior was recorded for 5 minutes. For the EPM re-exposure paradigm (EPM2), animals were re-exposed to the maze 24 hours after EPM1. The maze was cleaned with 50% ethanol after each rat was tested. The videos were analyzed using XPlo-Rat (Garcia et al., 2005). An entry was defined when the rat has all four paws in an arm of the maze. Time in open arms (OA) and in closed arm entries was measured.
2.7 Cat odor paradigm Rats were exposed to cat odor 7 days after surgery. The test apparatus consisted in an open rectangular arena (60x26x36) with Plexiglass walls. The cat collar was worn by a domestic cat for a period of 3 weeks. After this time, the collar was collected from the cat and stored at 4ºC until use. A collar that was not worn by a cat was used as control. To minimize responses to the novel environment, all rats were given 4 consecutive habituation days in which they were placed in the chamber test for 20 minutes without collar presence. After each habituation session, rats were returned to their home cages. On the test day, the collar that was worn by a cat or the control collar were placed in the test chamber before the test. The rats were placed in the test chamber 3 min after the end of the saline or antagonist infusion procedure. Testing was conducted in a quiet and dimly illuminated room. All testing occurred between 11.00 and 16.00 hours, and behavior was recorded for 20 minutes. Protocol 1: Rats were exposed once to either the odor or the control collar for 20 minutes, 3 minutes after intra PL injection of saline or L-745,870 and the behavior was videotaped.
Protocol 2: Rats were re-exposed three times to the odor or to the control collar, separated by 24 hours each. The intra PL injection of L-745,870 was made in the second test day. The total time of freezing during the test in all groups was measured.
2.8 Histology After the test, rats were immediately sacrificed by decapitation and the brain was stored in 4% PFA for at least 48 hours. The brains were cut into 30 µm slices, stained with cresyl violet and mounted onto glass slides.
2.9 Statistical Analysis All statistical analyses were performed using Prism 5 Graphpad software. Data were analyzed by one-way ANOVA, two-way ANOVA, and t-student test. Bonferroni and Tukey post hoc test were used. All data are reported as mean ± SEM.
3. Results 3.1 D4R are located in pyramidal-type and GABAergic neurons of the PL and IL areas of mPFC A specific antibody against D4R was used to perform immunohistochemistry in coronal slices containing rat PFC. Immunoreactivity for D4R was widely distributed in the PFC, including the PL and IL sub-areas. (Fig. 2). Different types of neuronal morphologies were observed in both PL and IL. The main phenotypes expressed in the PFC are pyramidal glutamatergic neurons (Kubota et al., 1994; Fremeau et al., 2004; Geisler et al., 2007; Geisler and Wise, 2008) which project to different areas of the brain, and GABAergic neurons, which mostly correspond to interneurons (Gabbot et al., 1997). In figure 2 it is possible to observe positive D4R labeling in PL neurons with classical pyramidal morphology (arrows) characteristic of cortical glutamatergic neurons and in small neuronal cell bodies of potentially GABAergic phenotype. In order to assess the phenotype of the small neurons, we performed immunofluorescence using markers of GABAergic neurons such as GAD 65/67 and PV. Figure 3 shows the co-localization of D4R with GAD 65/67, GAD 67 and PV, indicating that D4R is also expressed in PFC GABAergic neurons. Positive D4R neurons were also observed in IL neurons of pyramidal morphology (Fig. 2).
3.2 D4R in PL area of mPFC regulates the expression of anxiety and fear related behaviors To evaluate the role of PL D4R in innate fear, we used two experimental paradigms: reexposure to EPM and to the cat odor paradigm.
3.2.1 Antagonism of PL D4R decreases anxiety and fear related behaviors in the EPM: The EPM is the most popular and validated anxiety test (Pellow et al., 1985). In addition, re-exposure to a second EPM test has been associated with aversive emotions (Treit et al., 1993; Rodgers et al., 1996). The antagonist used in this work, L-745,870, is highly selective for D4R (Kulagowski et al., 1996; Patel et al., 1997). We infused 5 nmol/0.5 µl of L-745,870 bilaterally in PL. This dose was decided based in previous studies (Bernaerts and Tirelli, 2003; Shah et al., 2004; Zhang et al., 2004). Bilateral infusion of L-745,870 in the PL, before the re-exposure to a second EPM test (EPM2), was used to assess whether activation of D4R is involved in the expression of fear-related behavior. Intra PL infusion of L-745,870, 3 min before EPM1, increased the time spent in the open arm, indicating an anxiolytic effect for the D4R antagonism (Fig. 4A) (saline: 31.58±7.833 s vs L-745,870: 131.5±46.81 s), as previously shown by Shah et al (2004). As shown in figure 4A and 4C, control saline animals showed a significant decrease in time spent in open arms after EPM re-exposure (EPM2) (4A: EPM1 saline: 31.58±7.83 sec vs EPM2 saline: 9.04±21.16 sec; 4C: EPM1: 45.76±8.28 sec vs EPM2 saline: 22.14±6.669 sec). Interestingly, the decrease in time spent in open arms after re-exposure was not observed after intra PL infusion of L-745,870, 3 min before EPM2 (EPM1: 64.99±10.93 s vs EPM2: 60.37±14.61), indicating that D4R antagonism attenuates the expression of fearrelated behavior (Fig. 4C). The change of the animal behavior in both exposures to the EPM was not due to a change in locomotor activity, because there was no significant difference in locomotor activity between treatment and control groups (Fig. 4B and 4D). (Fig 4B: EPM1: saline 1076±99.27 cm vs L-745,870 713.8±152.3 cm; EPM 2: saline
1162±136 cm vs 976±107.9 cm. 4D: EPM 1: saline 1345±125.7 cm vs L-745,870 1450±243.9 cm; EPM 2: saline 747±115 cm vs L-745,870 885.3±134.2 cm). 3.2.2 Antagonism of D4R in the PL decreases fear related behavior in the cat odor test: As it has been well documented (Dielenberg et al., 2001a; Staples et al., 2006; Collins, 2011), rodents exposed to a cat odor collar show increased freezing compared to rodents exposed to a control collar. Figure 5 shows the effect of the intra PL infusion of L-745,870 on total time in freezing behavior during a single 20-min exposure session. A One Way ANOVA revealed a significant effect of treatment (F(4,14)= 10.19, p=0.0004). A Tukey's Multiple Comparison Test showed a significant increase in freezing behavior in control rats exposed to the cat odor collar (control: 2.4±0.2 min vs odor: 5.6±0.7 min; p< 0.05), as well as in rats with intra PL saline infusion (control saline: 1.79±0.59 vs odor saline: 5.8±0.7; p<0.01). After intra PL infusion of L-745,870, the expression of freezing in animals exposed to the cat odor was not significantly different than the control levels (control saline: 1.79±0.59; odor antagonist: 1.9±0.5), but was significantly different from odor saline (odor saline: 5.8±0.7 vs odor antagonist: 1.9±0.5; p<0.01) indicating that D4R antagonism in PL significantly attenuates the expression of behavior associated with innate fear. The latency of the freezing behavior was not significantly different between the groups (Fig. 6) indicating that the observed decrease in freezing behavior, in the presence of the D4R antagonist, was not due to an impairment in the access to the evocation of innate fear.
To evaluate the behavior after repeated exposures to the cat odor, the rats were exposed to three consecutive tests separated by 24 hours each. The exposure to the cat odor increased significantly the freezing behavior in the first test (Fig.7A) (two-way ANOVA, control saline: 1.41±0.5 vs odor saline: 4.88±0.17; Bonferroni post-test: ***p<0.001). However, a significant decrease in freezing behavior following the cat odor was observed in the second test (Fig 7B; odor saline day 1: 4.88±0.17 vs odor saline day 2: 2.07±0.28 **p<0.01). Surprisingly, a tendency to an increase in freezing behavior was observed in the third reexposure to the odor test, observing a “u-shape” in the levels of freezing behavior in the odor saline groups (Fig. 7B; odor saline day 2: 2.07±0.28 vs odor saline day 3: 3.22±0.85). To study the effect of D4R antagonism on freezing behavior during cat odor re-exposure, L-745,870 was infused 3 min prior to the second test. Intra PL infusion of L-745,870 did not change the level of freezing behavior compared to odor saline rats in the second day (odor saline: 2.07±0.28 vs odor antagonist: 2.01±0.63). Rats injected with L-745,870, 3 min before the second exposure, maintained low levels of freezing during the third re-exposure day. Instead, odor saline rats showed a significant increase in freezing behavior, compared with odor antagonist rats in the third test (odor saline: 3.22±0.85 vs odor antagonist: 0.91±0.21, p<0.01).
4. Discussion In the present study we tested the hypothesis that the activation of D4R in the PL region of mPFC is involved in the expression of innate fear. Our results show that intra PL infusion of L-745,870 significantly decreased fear-related behavior in both the cat odor test and the EPM re-exposure, indicating that the activation of D4R in PL favors the expression of innate fear. In addition, our results show that D4R is expressed in mPFC pyramidal and GABAergic neurons. Pyramidal neurons of the PL project to other areas of the brain, such as the amygdala, important brain region for fear behaviors (Aggleton et al. 1980; Ottersen, 1982; Russchen, 1982; Cassell et al. 1989). The deeper layers of the mPFC also receive projections from brain regions associated with fear responses, including the amygdala (Porrino et al., 1981; Amaral and Price, 1984; Bacon et al., 1996). Thus, the observed localization of D4R in PL pyramidal neurons could be an anatomical substrate for the regulation of fear behavior (Peters et al., 2009). Previous studies have described the expression of D4R in the PFC, but not in specific sub-regions of this brain area (Ariano et al, 1997; Defagot et al., 1997; Wedzony et al. 2000; Noaín et al., 2006; Rivera et al., 2008). These studies described the expression of D4R in PL, but its expression in IL remained an open question. Our results confirm the expression of D4R in PL and they also show D4R expression in IL. D4R was also observed in GABAergic neurons of the PL and IL, identified by the expression of GAD 65/67 or the calcium binding protein parvalbumin (PV). The wide expression of D4R in PL and IL, suggests a modulatory role of D4R in pathways that has been proposed to facilitate and attenuate fear expression, respectively (Shah et al., 2004; Sotres-Bayon et al., 2004; Vidal-Gonzalez et al., 2006; Sotres-Bayon and Quirk, 2010).
The EPM has been used to measure anxiety behavior in rodents (Pellow et al., 1985), sustained by the effect of anxiolytic drugs. However, during EPM re-exposure (a second exposure to the test) animals display an increased avoidance of the open arms and a corresponding preference for the enclosed arms of the apparatus, compared to a first exposure (Rodgers et al., 1996). It has also been widely described in the literature that anxiolytic drugs do not affect the behavior of rats exposed to a second EPM test, if they were not administered anxiolytic drugs in the first test. This effect is known as "one-trial tolerance" (File and Zangrossi, 1993a). It has been postulated that an emotional change, from a state of anxiety to one of fear, occurs in the animals when they are exposed to the second EPM test (Treit et al., 1993; Rodgers et al., 1996). D4R antagonism in the PL significantly increased the time spent in the open arm in the first test showing an anxiolytic effect of L-745,870 and confirming that the activation of D4R in PL facilitates anxiety behavior (Shah et al., 2004). However, the role of D4R in a second exposure to EPM has not been previously assessed. Administration of the D4R antagonist before the second exposure to EPM increased open arm time. As mentioned above, it has been proposed that when the animals are re-exposed to the EPM, a change occurs from an anxiety state in the EPM1 to a fear state in the EPM2 (Treit et al., 1993; Rodgers et al., 1996), indicating an aversive component associated with fear in the EPM2. Taken together, our results indicate that D4R antagonism in PL increases the time spent in the open arms during both exposures to the EPM, showing that the anxiolytic effect of the L-745,870 does not present the “one trial” tolerance effect. In conclusion, the activity of D4R in the PL is necessary for the expression of anxiety and fear-related behaviors.
The cat odor paradigm is one of the most used tests to measure behavioral responses associated with innate fear. It has been widely reported that exposing rats to cat odor, either through a collar worn by a cat or a urine sample, increases freezing behavior (Dielenberg et al., 2001a,b; Collins, 2011). Our results show that intra PL injection of a D4R antagonist decreases the freezing response of animals exposed to cat odor, indicating that activation of D4R is necessary for the expression of behaviors associated with innate fear. A decrease in the expression of freezing in animals with intra PL infusion of the D4R antagonist was not accompanied by changes in freezing latency. Cannulation of the animal or infusion of the vehicle solution did not affect the animal’s behavior. Thus, D4R in the PL allows for the expression of freezing behavior upon encountering an aversive stimulus. These results are in line with previous evidence (Shah and Treit, 2003) showing that intra mPFC infusion of L-745,870 decreases burying in the shock-probe test. Although contradictory results were observed in a study that uses the “knock out” mice lacking D4R (Falzone et al., 2002). It is possible as Shah at al. (2004) argued, that this discrepancy could be related to different experimental approaches. To further assess the role of PL D4R in innate fear we retested the response of the animals to the aversive cat odor. Several studies have described that when animals are re-exposed to the cat odor test animals continue showing high levels of fear-related behaviors during several days (Staples et al., 2005; File et al., 1993b; Zangrossi and File, 1994). However, our results show a significant decrease in the levels of freezing behavior in the second test. Interestingly, in a third test, the animals showed a tendency to increase the levels of freezing behavior compared to the second exposure. Further studies should better evaluate the possible changes after repeated exposures. It is tempting to suggest that changes in memory strength could occurred. While fear elicited by the cat odor is innate, the exposure
to a novel environment with the cat odor leads to a new contextual memory formation (Dielenberg et al., 2001a). The memory formation starts with short-term memory and if the animal is re-exposed several times to the stimulus, the memory becomes a long-term memory. During this process of memory formation, the strength of the memory changes in a time dependent manner (McGaugh, 2000). Short and long term memories show greater strength compared to the transition period, a mnemonic process that could underlie the behavior observed in figure 7B. In this same experimental protocol, another group of animals was re-exposed to the cat odor after PL infusion of L-745,870. In this case, D4R antagonism in PL did not affect freezing behavior in the second exposure, but the decrease was still present in the third test. This behavior was not observed in the group with odor and administration of saline. The infusion of the D4R antagonist in PL prevented the ushape in the freezing behavior, maintaining low levels of freezing in the third test. It is tempting to suggest that the activation of D4R in PL is important to the change of the neuronal pathway involved in the fear behavior from PL-amygdala to PL-paraventricular nucleus of thalamus (Do-Monte et al., 2015). In conclusion, the present study shows that the activation of D4R in PL is involved in both anxiety and innate fear behavior. The anatomical evidence shows that D4R are expressed in both PL GABAergic and non-GABAergic neurons. Further studies should address which of these phenotypes are involved in anxiety and innate fear behavior. Acknowledgements This study was funded by FONDECYT grant N° 1150244. MDV was recipient of a CONICYT graduate fellowship.
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6. Figures legends
Figure 1: Representative position of guide cannulae. A: shows the representation of the bilateral position of guide cannulae in the PL cortex used for the injection of L-745,870. B: Image of the scar left by the cannula in one of the hemispheres. Figure 2: D4R is located in PL and IL. Immunohistochemistry shows the expression of D4R in PL (A and B) and IL (C and D). In both areas the receptor is more expressed in different types of neuronal cell bodies in the inner layers of the cortex. Arrows show D4R positive pyramidal type-neurons and arrow heads show D4R positive smaller neurons, possible GABAergic neuron. Bars: A and B: 50 µm, C and D: 20 µm. Figure 3: D4R is expressed in GABAergic neurons of the PL. Immunofluorescence of D4R and PV, GAD 65/67 and GAD 67 (GABAergic markers) in the PL. A: D4R colocalizes with PV-positive neuronal cell bodies in PL cortex. B: D4R co-localizes with GAD 65/67-positive neuronal cell bodies in the PL. C: D4R co-localizes with GAD 67positive neuronal cell bodies in the PL. Bars: 20 µm. Figure 4: Antagonism of D4R in the PL increases the exploration of the open arms in the EPM. A: Time spent in the open arms in EPM1 and EPM2 of animals treated with vehicle (saline) or L-745,870 (D4R antagonist), 3 min before the first test (EPM1). The administration of L-745,870 (5 nmol) before EPM1 increases the exploration of the open arms, indicating a decrease of the anxiety in this animal group. The effect of L-745,870 does not persist in the second test. C: Time spent in the open arms in EPM1 and EPM2 of animals treated with vehicle (saline) or L-745,870, 3 min before the second test (EPM2).
The administration of L-745,870 before EPM2 increases the exploration of the open arms, indicating a decrease of fear in this animal group. B and D: Locomotor activity of the two groups of animals used in EPM1 and EPM2. *p <0.05; according to t-test analysis. Figure 5: D4R antagonism in the PL decreases freezing behavior in the cat odor test. Control: non-cannulated animals exposed to a collar without cat odor. Control saline: cannulated animals exposed to a collar without cat odor and administration of saline in the PL. Odor: non-cannulated animals exposed to a collar with cat odor. Odor saline: cannulated animals exposed to a collar with cat odor and administration of saline in the PL. Odor antagonist: cannulated animals exposed to a collar with cat odor and administration of L-745,870 (5 nmol) in the PL. **p= 0.0004; according to one-way ANOVA followed by Tukey post-test. Figure 6: D4R antagonism in the PL does not change the latency of the freezing behavior in the cat odor test. Control: non-cannulated animals exposed to a collar without cat odor. Control saline: cannulated animals exposed to a collar without cat odor and administration of saline in the PL. Odor: non-cannulated animals exposed to a collar with cat odor. Odor saline: cannulated animals exposed to a collar with cat odor and administration of saline in the PL. Odor antagonist: cannulated animals exposed to a collar with cat odor and administration of L-745,870 (5 nmol) in the PL. Figure 7: D4R antagonism in the PL maintains attenuated innate fear behavior. Control saline: cannulated animals exposed to a collar without cat odor and administration of saline in the PL. Odor saline: cannulated animals exposed to a collar with cat odor and administration of saline in the PL. Odor antagonist: cannulated animals exposed to a collar with cat odor and administration of L-745,870 (5 nmol) in the PL. *p<0.05, **p<0.005,
***p<0.0005; according to two-way ANOVA with repeated measure followed by Bonferroni post-test.
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S0166-4328(16)30858-0 http://dx.doi.org/doi:10.1016/j.bbr.2017.01.050 BBR 10690
To appear in:
Behavioural Brain Research
Received date: Revised date: Accepted date:
25-10-2016 26-1-2017 30-1-2017
Please cite this article as: Vergara Macarena D, Keller Victor N, Fuentealba Jos´e A, Gysling Katia.Activation of type 4 dopaminergic receptors in the prelimbic area of medial prefrontal cortex is necessary for the expression of innate fear behavior.Behavioural Brain Research http://dx.doi.org/10.1016/j.bbr.2017.01.050 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Activation of type 4 dopaminergic receptors in the prelimbic area of medial prefrontal cortex is necessary for the expression of innate fear behavior
Macarena D. Vergara1, Victor N. Keller1,3, José A. Fuentealba2* and Katia Gysling1* 1
Department of Cellular and Molecular Biology, Faculty of Biological Science, Pontificia Universidad Católica de Chile, Santiago, Chile. 2
Department of Pharmacy, Faculty of Chemistry, Pontificia Universidad Católica de Chile, Santiago, Chile. 3
Present address: Department of Psychology, College of Social Science, Michigan State University, East Lansing MI, United States of America. *Co-corresponding authors
Correspondence to: Katia Gysling, PhD Department of Cellular and Molecular Biology Faculty of Biological Science Pontificia Universidad Católica de Chile, Santiago, Chile Phone: 562-23542659 Email: [email protected] or José Fuentealbas, PhD Department of Pharmacy Faculty of Chemistry Pontificia Universidad Católica de Chile, Santiago, Chile Tel: +56-2-2354-5908 E-mail address: [email protected]
Abstract
The prelimbic area (PL) of the medial Prefrontal cortex (mPFC) is involved in the acquisition and expression of conditioned and innate fear. Both types of fear share several neuronal pathways. It has been documented that dopamine (DA) plays an important role in the regulation of aversive memories in the mPFC. The exposure to an aversive stimulus, such as the smell of a predator odor or the exposure to footshock stress is accompanied by an increase in mPFC DA release. Evidence suggests that the type 4 dopaminergic receptor (D4R) is the molecular target through which DA modulates fear expression. In fact, the mPFC is the brain region with the highest expression of D4R; however, the role of D4R in the expression of innate fear has not been fully elucidated. Therefore, the principal objective of this work was to evaluate the participation of mPFC D4R in the expression of innate fear. Rats were exposed to the elevated plus-maze (EPM) and to the cat odor paradigm after the intra PL injection of L-745,870, selective D4R antagonist, to measure the expression of fear-related behaviors. Intra PL injection of L-745,870 increased the time spent in the EPM open arms and decreased freezing behavior in the cat odor paradigm. Our results also showed that D4R is expressed in GABAergic and pyramidal neurons in the PL region of PFC. Thus, D4R antagonism in the PL decreases the expression of innate fearbehavior indicating that the activation of D4R in the PL is necessary for the expression of innate fear-behavior. Keywords: dopamine, prefrontal cortex, innate fear, anxiety, cat odor
1. Introduction The medial Prefrontal cortex (mPFC) receives information from cortical and subcortical structures, and the reciprocal connections with limbic structures allow mPFC to exert a control in emotional memories (McDonald et al., 1996; Dalley et al., 2004). Specifically, PL (prelimibic) and IL (infralimbic) areas of the mPFC have been related with conditioned and innate fear (Quirk et al., 2000; Dielenberg et al., 2001a,b; Lauzon et al., 2009, 2012; Chan et al., 2011). PL has been associated with fear expression and IL has been associated with fear extinction (Sotres-Bayon et al., 2004; Shah et al., 2004; Vidal-Gonzalez et al., 2006; Sotres-Bayon and Quirk, 2010). In conditioned fear the animal associates a neutral stimulus to an aversive stimulus, so the animal learns to fear the neutral stimulus. Instead, innate fear, also named unconditioned fear, does not involve an aversive learning and is expressed when the animal faces risky situations that were recurrent throughout evolutionary history, such as darkness, height, pain, presence of a predator, etc (Jinks and McGregor, 1997; Antoniadis and McDonald, 2001; Dielenberg et al., 2001b; VidalGonzalez et al., 2006). The mPFC receives dopaminergic afferents from the ventral tegmental area (VTA) (Falon 1981; Lammel et al., 2008). DA in the mPFC has been associated with the modulation of aversive memories (Wedzony et al., 1996; Yoshioka et al., 1996; Morrow et al., 2000; Espejo 2003; Giogi et al., 2003; Pezze et al., 2003; Pezze and Feldon, 2004). It has been shown that DA regulates the expression of aversive memories, modulating the connection between mPFC and BLA (Floresco and Tse, 2007). Activation of dopaminergic receptors
has been associated with the modulation of conditioned (Inoue et al., 2000; El-Ghundi et al., 2001; De Oliveira et al., 2006, Hikind and Maroun, 2008) and innate fear (Espejo, 1997; Lauzon et al., 2009). Specifically, the activation of type 4 dopaminergic receptors (D4R) potentiates encoding of emotional memories associated with conditioned fear (Laviolette et al., 2005). D4R is highly expressed in the mPFC (Wedzony et al., 2000; Rivera et al., 2008; De Almedia and Mengod, 2010), particularly in PL (Rivera et al, 2008). However, the specific D4R expression in pyramidal and/or GABAergic neurons has not been fully elucidated. The role of D4R in innate fear has scarcely been studied. In the interesting work of Lauzon et al. (2009) it was shown that intra PL injection of PD-168007, a selective D4R agonist, increases the emotional relevance of a non-aversive stimulus ("footshock" of 0.4 mA) generating fear associated behavior. On the other hand, the injection of PD-168007 attenuates the increase of freezing behavior induced by an aversive stimulus (footshock of 0.8 mA). These results indicate that the activation of PFC D4R modifies the expression of conditioned fear depending on the magnitude of the stimulus presented. Furthermore, D4R activation has been associated with anxiety-like behavior (Shah et al., 2004). These authors showed that the intra PL injection of L-745,870 (1 nmol/0.5 µL), a selective D4R antagonist (Kulagowski et al., 1996; Patel et al., 1997), increases the time spent in the open arms of the elevated plus maze (EPM). This result suggests that D4R activation increases anxiety-like behavior. However, the role of PL D4R in innate fear behavior remains an open question. In the present study we used the cat odor paradigm which allows measuring freezing behavior in rats exposed to an innate aversive stimulus and the EPM re-exposure paradigm to assess aversive emotions. In both behavioral
paradigms, L-745,870 was injected in the PL and the fear-related behaviors were measured. The hypothesis of this work was that the activation of D4R in the PL region of mPFC is necessary for the expression of innate fear. We also studied the specific expression of D4R in PL and IL in GABAergic and/or glutamatergic neuronal phenotypes. 2. Materials and Methods
2.1 Animals All experiments were conducted in accordance with institutional (Catholic University of Chile) and international guidelines (NIH Guide for the Care and Use of Laboratory Animals). Adult male Sprague–Dawley rats (250–300 g) were grown in the animal care facility of Catholic University of Chile under the supervision of a veterinarian. Food and water were available ad libitum. All experiments were conducted during the light hours of the light/dark cycle.
2.2 Immunohistochemistry of D4R Rats were deeply anesthetized with ketamine-xylazine (50-5mg/kg, respectively) and were intracardially perfused with 50 ml of saline, followed by 500 ml of 4% paraformaldehyde (PFA) in phosphate buffer. Then, the animals were guillotined and the brain was post-fixed with 4% PFA for 2 hours and left in 20% sucrose during 2 days. The brains were then cut into 30 µm slices using a Leyca cryostat and slices were placed in 1x PBS. Brain slices were washed with 1x PBS at room temperature (3x10 minutes) and incubated during 1 hour with a blocking solution (1% BSA, 0.2% Triton X-100 and 0.02% azide). Thereafter, the
slices were incubated 48 hours at 4ºC with 1:500 anti-D4R rabbit polyclonal antibody (Calbiochem, Cat. N° 324405). Slices were washed with 1x PBS (3x15 minutes) and the tissue sections were placed for 1 hour in rabbit-biotinylated anti-rabbit IgG (Vector Laboratories, CA) diluted 1:1000 in blocking solution. After a rinse in 1x PBS, the slices were placed in an avidin-biotin complex reagent (Vectastain ABC Kit) in 1x PBS for 1 hour and then rinsed again in 1x PBS. The peroxidase contained in the ABC reagent was visualized by placing the sections in a solution of DAB (1 mg/mL, Sigma), Tris-buffer pH 7.6 (50 mM Tris acid, 150 mM NaCl) and 0.5% H2O2 for 10 minutes. Tissue slices were washed in 1x PBS and mounted in glass slides with 0.1% gelatin, dried for 2 hours and coverslipped. The sections were observed under a light microscope (Olympus) and microphotographs were captured with a digital camera.
2.3 Immunofluorescence of D4R and GABAergic markers To perform the dual immunofluorescence for D4R and markers of GABAergic neurons (parvalbumin (PV), GAD 65/67 or GAD 67), slices were washed with 1x PBS at room temperature (3x10 min). The slices were then incubated during 1 hour with a blocking solution (1% BSA, 0.2% Triton X-100 and 0.02% azide). Thereafter, slices were incubated overnight with 1:200 anti-D4R rabbit polyclonal antibody and 1:200 anti PV (Sigma, p3088), GAD 65/67 goat polyclonal (Santa Cruz, sc-7513) or GAD 67 mouse polyclonal (Millipore, MAB5406). Slices were washed with 1x PBS (3x15 minutes), mounted in glass slides with 0.1% gelatin, allowed to dry for 2 hours and then incubated with the secondary fluorescent antibody (1:1000, Invitrogen) for 2 hours. Finally, the slices were washed with 1x PBS (3x15 minutes) and coverslipped with Dako (Invitrogen). A light microscope
(Olympus), equipped with fluorescent lamp and digital camera, was used to analyze the sections and to capture the microphotographs.
2.4 Animal surgery The animals were injected with ketamine-xylazine (50-5mg/kg, respectively) and placed in a stereotaxic apparatus (Stoelting, Wood Dale, IL, USA), and an incision was made to expose the skull. Stainless steel guide cannulae (plastic One, Roanoke, VA, USA) were bilaterally implanted, targeting the PL cortex (+2.9 mm, AP ±1.9 ML, -1.9 DV; all cannulae angled at 15º towards the midline, Fig. 1). The cannulae were secured to the skull with two screws and cranioplastic cement. After the surgery, rats were injected with 150 µL of 1% ketoprofen and 200 µL of 5 % enrofloxacin, and were returned to their home cages after waking from anesthesia.
2.5 Intracranial infusion procedure Bilateral intra PL infusions were performed with an infusion pump (Harvad Apparatus) at a rate of 0.5 µL/min (0.5 µL final volume). The cannulae were left in place for 1 minute after the end of the infusion. The selective D4R antagonist L-745,870 trihydrochloride (5 nmol/0.5 µL; Tocris, Ellisville, MO, USA) was dissolved in saline. 2.6 Elevated plus maze test The testing of EPM (EPM1) started 7 days after surgery. Rats were handled for 5 minutes in each of 4 consecutive days prior to testing. The test began 3 min after the end of the
infusion procedure. The maze was made of a wooden, plus-shaped apparatus with a height of 70 cm. It consisted of two 50x10 cm open arms and two 50x10x50 enclosed arms. Testing was conducted in a quiet and dimly illuminated room. All testing occurred between 11.00 and 16.00 hours, and behavior was recorded for 5 minutes. For the EPM re-exposure paradigm (EPM2), animals were re-exposed to the maze 24 hours after EPM1. The maze was cleaned with 50% ethanol after each rat was tested. The videos were analyzed using XPlo-Rat (Garcia et al., 2005). An entry was defined when the rat has all four paws in an arm of the maze. Time in open arms (OA) and in closed arm entries was measured.
2.7 Cat odor paradigm Rats were exposed to cat odor 7 days after surgery. The test apparatus consisted in an open rectangular arena (60x26x36) with Plexiglass walls. The cat collar was worn by a domestic cat for a period of 3 weeks. After this time, the collar was collected from the cat and stored at 4ºC until use. A collar that was not worn by a cat was used as control. To minimize responses to the novel environment, all rats were given 4 consecutive habituation days in which they were placed in the chamber test for 20 minutes without collar presence. After each habituation session, rats were returned to their home cages. On the test day, the collar that was worn by a cat or the control collar were placed in the test chamber before the test. The rats were placed in the test chamber 3 min after the end of the saline or antagonist infusion procedure. Testing was conducted in a quiet and dimly illuminated room. All testing occurred between 11.00 and 16.00 hours, and behavior was recorded for 20 minutes. Protocol 1: Rats were exposed once to either the odor or the control collar for 20 minutes, 3 minutes after intra PL injection of saline or L-745,870 and the behavior was videotaped.
Protocol 2: Rats were re-exposed three times to the odor or to the control collar, separated by 24 hours each. The intra PL injection of L-745,870 was made in the second test day. The total time of freezing during the test in all groups was measured.
2.8 Histology After the test, rats were immediately sacrificed by decapitation and the brain was stored in 4% PFA for at least 48 hours. The brains were cut into 30 µm slices, stained with cresyl violet and mounted onto glass slides.
2.9 Statistical Analysis All statistical analyses were performed using Prism 5 Graphpad software. Data were analyzed by one-way ANOVA, two-way ANOVA, and t-student test. Bonferroni and Tukey post hoc test were used. All data are reported as mean ± SEM.
3. Results 3.1 D4R are located in pyramidal-type and GABAergic neurons of the PL and IL areas of mPFC A specific antibody against D4R was used to perform immunohistochemistry in coronal slices containing rat PFC. Immunoreactivity for D4R was widely distributed in the PFC, including the PL and IL sub-areas. (Fig. 2). Different types of neuronal morphologies were observed in both PL and IL. The main phenotypes expressed in the PFC are pyramidal glutamatergic neurons (Kubota et al., 1994; Fremeau et al., 2004; Geisler et al., 2007; Geisler and Wise, 2008) which project to different areas of the brain, and GABAergic neurons, which mostly correspond to interneurons (Gabbot et al., 1997). In figure 2 it is possible to observe positive D4R labeling in PL neurons with classical pyramidal morphology (arrows) characteristic of cortical glutamatergic neurons and in small neuronal cell bodies of potentially GABAergic phenotype. In order to assess the phenotype of the small neurons, we performed immunofluorescence using markers of GABAergic neurons such as GAD 65/67 and PV. Figure 3 shows the co-localization of D4R with GAD 65/67, GAD 67 and PV, indicating that D4R is also expressed in PFC GABAergic neurons. Positive D4R neurons were also observed in IL neurons of pyramidal morphology (Fig. 2).
3.2 D4R in PL area of mPFC regulates the expression of anxiety and fear related behaviors To evaluate the role of PL D4R in innate fear, we used two experimental paradigms: reexposure to EPM and to the cat odor paradigm.
3.2.1 Antagonism of PL D4R decreases anxiety and fear related behaviors in the EPM: The EPM is the most popular and validated anxiety test (Pellow et al., 1985). In addition, re-exposure to a second EPM test has been associated with aversive emotions (Treit et al., 1993; Rodgers et al., 1996). The antagonist used in this work, L-745,870, is highly selective for D4R (Kulagowski et al., 1996; Patel et al., 1997). We infused 5 nmol/0.5 µl of L-745,870 bilaterally in PL. This dose was decided based in previous studies (Bernaerts and Tirelli, 2003; Shah et al., 2004; Zhang et al., 2004). Bilateral infusion of L-745,870 in the PL, before the re-exposure to a second EPM test (EPM2), was used to assess whether activation of D4R is involved in the expression of fear-related behavior. Intra PL infusion of L-745,870, 3 min before EPM1, increased the time spent in the open arm, indicating an anxiolytic effect for the D4R antagonism (Fig. 4A) (saline: 31.58±7.833 s vs L-745,870: 131.5±46.81 s), as previously shown by Shah et al (2004). As shown in figure 4A and 4C, control saline animals showed a significant decrease in time spent in open arms after EPM re-exposure (EPM2) (4A: EPM1 saline: 31.58±7.83 sec vs EPM2 saline: 9.04±21.16 sec; 4C: EPM1: 45.76±8.28 sec vs EPM2 saline: 22.14±6.669 sec). Interestingly, the decrease in time spent in open arms after re-exposure was not observed after intra PL infusion of L-745,870, 3 min before EPM2 (EPM1: 64.99±10.93 s vs EPM2: 60.37±14.61), indicating that D4R antagonism attenuates the expression of fearrelated behavior (Fig. 4C). The change of the animal behavior in both exposures to the EPM was not due to a change in locomotor activity, because there was no significant difference in locomotor activity between treatment and control groups (Fig. 4B and 4D). (Fig 4B: EPM1: saline 1076±99.27 cm vs L-745,870 713.8±152.3 cm; EPM 2: saline
1162±136 cm vs 976±107.9 cm. 4D: EPM 1: saline 1345±125.7 cm vs L-745,870 1450±243.9 cm; EPM 2: saline 747±115 cm vs L-745,870 885.3±134.2 cm). 3.2.2 Antagonism of D4R in the PL decreases fear related behavior in the cat odor test: As it has been well documented (Dielenberg et al., 2001a; Staples et al., 2006; Collins, 2011), rodents exposed to a cat odor collar show increased freezing compared to rodents exposed to a control collar. Figure 5 shows the effect of the intra PL infusion of L-745,870 on total time in freezing behavior during a single 20-min exposure session. A One Way ANOVA revealed a significant effect of treatment (F(4,14)= 10.19, p=0.0004). A Tukey's Multiple Comparison Test showed a significant increase in freezing behavior in control rats exposed to the cat odor collar (control: 2.4±0.2 min vs odor: 5.6±0.7 min; p< 0.05), as well as in rats with intra PL saline infusion (control saline: 1.79±0.59 vs odor saline: 5.8±0.7; p<0.01). After intra PL infusion of L-745,870, the expression of freezing in animals exposed to the cat odor was not significantly different than the control levels (control saline: 1.79±0.59; odor antagonist: 1.9±0.5), but was significantly different from odor saline (odor saline: 5.8±0.7 vs odor antagonist: 1.9±0.5; p<0.01) indicating that D4R antagonism in PL significantly attenuates the expression of behavior associated with innate fear. The latency of the freezing behavior was not significantly different between the groups (Fig. 6) indicating that the observed decrease in freezing behavior, in the presence of the D4R antagonist, was not due to an impairment in the access to the evocation of innate fear.
To evaluate the behavior after repeated exposures to the cat odor, the rats were exposed to three consecutive tests separated by 24 hours each. The exposure to the cat odor increased significantly the freezing behavior in the first test (Fig.7A) (two-way ANOVA, control saline: 1.41±0.5 vs odor saline: 4.88±0.17; Bonferroni post-test: ***p<0.001). However, a significant decrease in freezing behavior following the cat odor was observed in the second test (Fig 7B; odor saline day 1: 4.88±0.17 vs odor saline day 2: 2.07±0.28 **p<0.01). Surprisingly, a tendency to an increase in freezing behavior was observed in the third reexposure to the odor test, observing a “u-shape” in the levels of freezing behavior in the odor saline groups (Fig. 7B; odor saline day 2: 2.07±0.28 vs odor saline day 3: 3.22±0.85). To study the effect of D4R antagonism on freezing behavior during cat odor re-exposure, L-745,870 was infused 3 min prior to the second test. Intra PL infusion of L-745,870 did not change the level of freezing behavior compared to odor saline rats in the second day (odor saline: 2.07±0.28 vs odor antagonist: 2.01±0.63). Rats injected with L-745,870, 3 min before the second exposure, maintained low levels of freezing during the third re-exposure day. Instead, odor saline rats showed a significant increase in freezing behavior, compared with odor antagonist rats in the third test (odor saline: 3.22±0.85 vs odor antagonist: 0.91±0.21, p<0.01).
4. Discussion In the present study we tested the hypothesis that the activation of D4R in the PL region of mPFC is involved in the expression of innate fear. Our results show that intra PL infusion of L-745,870 significantly decreased fear-related behavior in both the cat odor test and the EPM re-exposure, indicating that the activation of D4R in PL favors the expression of innate fear. In addition, our results show that D4R is expressed in mPFC pyramidal and GABAergic neurons. Pyramidal neurons of the PL project to other areas of the brain, such as the amygdala, important brain region for fear behaviors (Aggleton et al. 1980; Ottersen, 1982; Russchen, 1982; Cassell et al. 1989). The deeper layers of the mPFC also receive projections from brain regions associated with fear responses, including the amygdala (Porrino et al., 1981; Amaral and Price, 1984; Bacon et al., 1996). Thus, the observed localization of D4R in PL pyramidal neurons could be an anatomical substrate for the regulation of fear behavior (Peters et al., 2009). Previous studies have described the expression of D4R in the PFC, but not in specific sub-regions of this brain area (Ariano et al, 1997; Defagot et al., 1997; Wedzony et al. 2000; Noaín et al., 2006; Rivera et al., 2008). These studies described the expression of D4R in PL, but its expression in IL remained an open question. Our results confirm the expression of D4R in PL and they also show D4R expression in IL. D4R was also observed in GABAergic neurons of the PL and IL, identified by the expression of GAD 65/67 or the calcium binding protein parvalbumin (PV). The wide expression of D4R in PL and IL, suggests a modulatory role of D4R in pathways that has been proposed to facilitate and attenuate fear expression, respectively (Shah et al., 2004; Sotres-Bayon et al., 2004; Vidal-Gonzalez et al., 2006; Sotres-Bayon and Quirk, 2010).
The EPM has been used to measure anxiety behavior in rodents (Pellow et al., 1985), sustained by the effect of anxiolytic drugs. However, during EPM re-exposure (a second exposure to the test) animals display an increased avoidance of the open arms and a corresponding preference for the enclosed arms of the apparatus, compared to a first exposure (Rodgers et al., 1996). It has also been widely described in the literature that anxiolytic drugs do not affect the behavior of rats exposed to a second EPM test, if they were not administered anxiolytic drugs in the first test. This effect is known as "one-trial tolerance" (File and Zangrossi, 1993a). It has been postulated that an emotional change, from a state of anxiety to one of fear, occurs in the animals when they are exposed to the second EPM test (Treit et al., 1993; Rodgers et al., 1996). D4R antagonism in the PL significantly increased the time spent in the open arm in the first test showing an anxiolytic effect of L-745,870 and confirming that the activation of D4R in PL facilitates anxiety behavior (Shah et al., 2004). However, the role of D4R in a second exposure to EPM has not been previously assessed. Administration of the D4R antagonist before the second exposure to EPM increased open arm time. As mentioned above, it has been proposed that when the animals are re-exposed to the EPM, a change occurs from an anxiety state in the EPM1 to a fear state in the EPM2 (Treit et al., 1993; Rodgers et al., 1996), indicating an aversive component associated with fear in the EPM2. Taken together, our results indicate that D4R antagonism in PL increases the time spent in the open arms during both exposures to the EPM, showing that the anxiolytic effect of the L-745,870 does not present the “one trial” tolerance effect. In conclusion, the activity of D4R in the PL is necessary for the expression of anxiety and fear-related behaviors.
The cat odor paradigm is one of the most used tests to measure behavioral responses associated with innate fear. It has been widely reported that exposing rats to cat odor, either through a collar worn by a cat or a urine sample, increases freezing behavior (Dielenberg et al., 2001a,b; Collins, 2011). Our results show that intra PL injection of a D4R antagonist decreases the freezing response of animals exposed to cat odor, indicating that activation of D4R is necessary for the expression of behaviors associated with innate fear. A decrease in the expression of freezing in animals with intra PL infusion of the D4R antagonist was not accompanied by changes in freezing latency. Cannulation of the animal or infusion of the vehicle solution did not affect the animal’s behavior. Thus, D4R in the PL allows for the expression of freezing behavior upon encountering an aversive stimulus. These results are in line with previous evidence (Shah and Treit, 2003) showing that intra mPFC infusion of L-745,870 decreases burying in the shock-probe test. Although contradictory results were observed in a study that uses the “knock out” mice lacking D4R (Falzone et al., 2002). It is possible as Shah at al. (2004) argued, that this discrepancy could be related to different experimental approaches. To further assess the role of PL D4R in innate fear we retested the response of the animals to the aversive cat odor. Several studies have described that when animals are re-exposed to the cat odor test animals continue showing high levels of fear-related behaviors during several days (Staples et al., 2005; File et al., 1993b; Zangrossi and File, 1994). However, our results show a significant decrease in the levels of freezing behavior in the second test. Interestingly, in a third test, the animals showed a tendency to increase the levels of freezing behavior compared to the second exposure. Further studies should better evaluate the possible changes after repeated exposures. It is tempting to suggest that changes in memory strength could occurred. While fear elicited by the cat odor is innate, the exposure
to a novel environment with the cat odor leads to a new contextual memory formation (Dielenberg et al., 2001a). The memory formation starts with short-term memory and if the animal is re-exposed several times to the stimulus, the memory becomes a long-term memory. During this process of memory formation, the strength of the memory changes in a time dependent manner (McGaugh, 2000). Short and long term memories show greater strength compared to the transition period, a mnemonic process that could underlie the behavior observed in figure 7B. In this same experimental protocol, another group of animals was re-exposed to the cat odor after PL infusion of L-745,870. In this case, D4R antagonism in PL did not affect freezing behavior in the second exposure, but the decrease was still present in the third test. This behavior was not observed in the group with odor and administration of saline. The infusion of the D4R antagonist in PL prevented the ushape in the freezing behavior, maintaining low levels of freezing in the third test. It is tempting to suggest that the activation of D4R in PL is important to the change of the neuronal pathway involved in the fear behavior from PL-amygdala to PL-paraventricular nucleus of thalamus (Do-Monte et al., 2015). In conclusion, the present study shows that the activation of D4R in PL is involved in both anxiety and innate fear behavior. The anatomical evidence shows that D4R are expressed in both PL GABAergic and non-GABAergic neurons. Further studies should address which of these phenotypes are involved in anxiety and innate fear behavior. Acknowledgements This study was funded by FONDECYT grant N° 1150244. MDV was recipient of a CONICYT graduate fellowship.
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6. Figures legends
Figure 1: Representative position of guide cannulae. A: shows the representation of the bilateral position of guide cannulae in the PL cortex used for the injection of L-745,870. B: Image of the scar left by the cannula in one of the hemispheres. Figure 2: D4R is located in PL and IL. Immunohistochemistry shows the expression of D4R in PL (A and B) and IL (C and D). In both areas the receptor is more expressed in different types of neuronal cell bodies in the inner layers of the cortex. Arrows show D4R positive pyramidal type-neurons and arrow heads show D4R positive smaller neurons, possible GABAergic neuron. Bars: A and B: 50 µm, C and D: 20 µm. Figure 3: D4R is expressed in GABAergic neurons of the PL. Immunofluorescence of D4R and PV, GAD 65/67 and GAD 67 (GABAergic markers) in the PL. A: D4R colocalizes with PV-positive neuronal cell bodies in PL cortex. B: D4R co-localizes with GAD 65/67-positive neuronal cell bodies in the PL. C: D4R co-localizes with GAD 67positive neuronal cell bodies in the PL. Bars: 20 µm. Figure 4: Antagonism of D4R in the PL increases the exploration of the open arms in the EPM. A: Time spent in the open arms in EPM1 and EPM2 of animals treated with vehicle (saline) or L-745,870 (D4R antagonist), 3 min before the first test (EPM1). The administration of L-745,870 (5 nmol) before EPM1 increases the exploration of the open arms, indicating a decrease of the anxiety in this animal group. The effect of L-745,870 does not persist in the second test. C: Time spent in the open arms in EPM1 and EPM2 of animals treated with vehicle (saline) or L-745,870, 3 min before the second test (EPM2).
The administration of L-745,870 before EPM2 increases the exploration of the open arms, indicating a decrease of fear in this animal group. B and D: Locomotor activity of the two groups of animals used in EPM1 and EPM2. *p <0.05; according to t-test analysis. Figure 5: D4R antagonism in the PL decreases freezing behavior in the cat odor test. Control: non-cannulated animals exposed to a collar without cat odor. Control saline: cannulated animals exposed to a collar without cat odor and administration of saline in the PL. Odor: non-cannulated animals exposed to a collar with cat odor. Odor saline: cannulated animals exposed to a collar with cat odor and administration of saline in the PL. Odor antagonist: cannulated animals exposed to a collar with cat odor and administration of L-745,870 (5 nmol) in the PL. **p= 0.0004; according to one-way ANOVA followed by Tukey post-test. Figure 6: D4R antagonism in the PL does not change the latency of the freezing behavior in the cat odor test. Control: non-cannulated animals exposed to a collar without cat odor. Control saline: cannulated animals exposed to a collar without cat odor and administration of saline in the PL. Odor: non-cannulated animals exposed to a collar with cat odor. Odor saline: cannulated animals exposed to a collar with cat odor and administration of saline in the PL. Odor antagonist: cannulated animals exposed to a collar with cat odor and administration of L-745,870 (5 nmol) in the PL. Figure 7: D4R antagonism in the PL maintains attenuated innate fear behavior. Control saline: cannulated animals exposed to a collar without cat odor and administration of saline in the PL. Odor saline: cannulated animals exposed to a collar with cat odor and administration of saline in the PL. Odor antagonist: cannulated animals exposed to a collar with cat odor and administration of L-745,870 (5 nmol) in the PL. *p<0.05, **p<0.005,
***p<0.0005; according to two-way ANOVA with repeated measure followed by Bonferroni post-test.
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