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/lp/springer-journals/acetylcholinesterase-inhibition-ameliorates-deficits-in-motivational-sN9CSwMhHn
- Publisher
- Springer Journals
- Copyright
- Copyright © 2012 by Martinowich et al; licensee BioMed Central Ltd.
- Subject
- Biomedicine; Neurosciences; Neurology; Behavioral Therapy; Psychiatry
- eISSN
- 1744-9081
- DOI
- 10.1186/1744-9081-8-15
- pmid
- 22433906
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- See Article on Publisher Site
Abstract
Background: Apathy is frequently observed in numerous neurological disorders, including Alzheimer’s and Parkinson’s, as well as neuropsychiatric disorders including schizophrenia. Apathy is defined as a lack of motivation characterized by diminished goal-oriented behavior and self-initiated activity. This study evaluated a chronic restraint stress (CRS) protocol in modeling apathetic behavior, and determined whether administration of an anticholinesterase had utility in attenuating CRS-induced phenotypes. Methods: We assessed behavior as well as regional neuronal activity patterns using FosB immunohistochemistry after exposure to CRS for 6 h/d for a minimum of 21 d. Based on our FosB findings and recent clinical trials, we administered an anticholinesterase to evaluate attenuation of CRS-induced phenotypes. Results: CRS resulted in behaviors that reflect motivational loss and diminished emotional responsiveness. CRS- exposed mice showed differences in FosB accumulation, including changes in the cholinergic basal forebrain system. Facilitating cholinergic signaling ameliorated CRS-induced deficits in initiation and motivational drive and rescued immediate early gene activation in the medial septum and nucleus accumbens. Conclusions: Some CRS protocols may be useful for studying deficits in motivation and apathetic behavior. Amelioration of CRS-induced behaviors with an anticholinesterase supports a role for the cholinergic system in remediation of deficits in motivational drive. Keywords: Apathy, Motivation, Chronic stress, Cholinergic, FosB, c-fos, Nucleus accumbens, Basal forebrain Background symptoms [11-14], and it has been established that ani- Apathy is characterized by severe loss of motivation to mal models of chronic stress cause behavioral changes participate in activities, social withdrawal and emotional similar to symptoms of depression in humans [15]. indifference [1]. Apathy shares some overlapping fea- Exposure to extreme forms of chronic stress, including tures with depression, but can be distinguished by lack time spent in prisoner of war and concentration camps of dysphoric symptoms including sadness, hopelessness as well as survival of the atomic bombing, has been and guilt [2,3]. Apathy is a frequent neuropsychiatric documented to result in an apathetic syndrome[16-19]. syndrome affecting up to 92% of individuals diagnosed For example, prisoners of the Korean War have been with Alzheimer’s disease (AD) [4-6], and up to 70% of described as having a reactive syndrome that included those with Parkinson’s disease (PD) [2,7-10]. Despite its extreme withdrawal of involvement and a paucity of emotion, which could not be explained by depression or prevalence, relatively little is known about the underly- ing neuropathology [10]. Stress exposure is an estab- psychosis, but was best characterized as “apathy” [16]. lished risk factor for development of neuropsychiatric Visual observation of routine animal behavior led us to hypothesize that a 6 hr/d/> 21 d chronic restraint stress (CRS) protocol could be useful for modeling features of * Correspondence: keri.martinowich@libd.org apathy. The objectives of the study were to 1) character- Mood and Anxiety Disorders Program (MAP), National Institute of Mental ize the loss of motivation and initiative in CRS-exposed Health (NIMH), National Institutes of Health (NIH), 35 Convent Drive, Building 35, Room 1C-1012, Bethesda, MD 20892-3711, USA animals, 2) map long-term changes in neuronal activity Full list of author information is available at the end of the article © 2012 Martinowich et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Martinowich et al. Behavioral and Brain Functions 2012, 8:15 Page 2 of 14 http://www.behavioralandbrainfunctions.com/content/8/1/15 in CRS-exposed animals and 3) determine whether facil- Saccharin and quinine preference itating the cholinergic system could ameliorate CRS- 21 d prior to CRS each mouse was given simultaneous induced phenotypes. access to two, dual-ball sipper-top bottles (Ancare, Bell- more, NY, USA): one with purified Milli-Q water and Methods one containing 50 mg/L saccharine (Sigma Aldrich, St. Louis, MO, USA). Bottles were weighed and refilled Animals every 3 d; positions were reversed at each change to We used adult (8 wk) male C57BL/6J mice (Jackson prevent side bias. After 21 d, mice were divided into Laboratories, Bar Harbor, ME, USA) that were double- balanced groups with mice sharing a divider cage placed housed in a standard mouse cage containing a metal divider splitting the cage into two separate compart- in the same experimental group. Individual animals with ments; each mouse retains an individual feeding com- saccharin preference < 65% were excluded from the partment and water bottle. Mice were maintained under study (~3% of total animals used). For the quinine a 12:12 hour light-dark cycle (6:00 AM to 6:00 PM). All experiment, the saccharine solution was replaced with procedures were performed in accordance with guide- 15 mg/L quinine hydrochloride (Sigma Aldrich, St. lines set forth by the National Institute of Mental Health Louis, MO, USA) solution for 3 d. Animal Care and Use Committee in the Guide for the Care and Use of Laboratory Animals. Figure 1 gives an Restraint stress overview of the experimental design and groups used in Mice were placed in 50 mL plastic conical tubes with the studies. holes cut at the tips to allow for unrestricted breathing Group 1 21d 24d 3d quinine aversion CRS saccharin preference Group 2 21d 21d 6d 3d 2d 2d home cage monitoring (no CRS during 22h of monitoring) saccharin preference CRS Group 3 21d 28d CRS saccharin preference Group 4 21d 21d 6d 1d 1d 1d phenserine tx CRS saccharin preference Figure 1 Experimental design. Group 1 (n = 10 control and n = 14 CRS-exposed for saccharin preference and quinine aversion; body and organ weight analysis performed in n = 8 for control versus CRS). Group 2 (for HCS, n = 8 control and n = 9 CRS-exposed; n = 8 control and n = 8 CRS-exposed for TST, FST and EZM). Group 3 (n = 6 Control and CRS-exposed). Group 4 (n = 12 Control and n = 16 CRS-exposed; CRS group further divided, n = 8 saline and n = 8 phenserine). For cFos immunohistochemistry (IHC) experiment control group further divided into n = 6 water exposure and n = 6 urine exposure; n = 6 saline/CRS/urine exposure and n = 6 phenserine/CRS/urine exposure were analyzed. body weight measurement body weight, organ weight measurements EZM perfuse for FosB IHC TST FST nest-building test strength assessment odor habituation/dishabituation perfuse for cFos IHC Martinowich et al. Behavioral and Brain Functions 2012, 8:15 Page 3 of 14 http://www.behavioralandbrainfunctions.com/content/8/1/15 and gauze was inserted in the remaining space. Mice were dried with a paper towel and returned to their home were restrained for 6 h/d (10:00 AM-4:00 PM). All cage. Water was replaced for each trial. behavioral tests were performed before placement in restrainers (6 AM-10 AM). Elevated zero maze The ring-shaped platform consisted of two walled (white HomeCage scan Plexiglas) sections separated by open sections of equal 24 h after CRS, mice were placed into clean cages con- length. Each mouse was placed such that it was in an taining 100 mL sawdust bedding with food and water. open section, directly facing a walled section. Activity Animals were monitored for 22 h using Sony digital was video-tracked for 5 min and analyzed using Clever cameras and CaptureStar video capturing software with Systems TopScan (Clever Sys Inc., Leesburg, VA, USA). infrared illumination during the dark phase. Automated video analysis of home cage behaviors was performed Drug treatments using HomeCageScan software (Clever Systems, Reston, Phenserine ((-)-N-phenylcarbamoyleseroline) was synthe- VA). Behaviors were detected by utilizing information sized as a water-soluble (L)-tartrate salt (> 99.9% optical about the entire body of the animal, identifying animal and chemical purity)[22]. CRS-exposed mice were admi- body parts such as head, tail, forelimbs, hind limbs, nistered either 0.9% saline or phenserine (1 mg/kg, i.p) in upper/lower back, abdomen, etc., and using sequence the evening after the CRS session and again in the morn- data to automatically recognize and analyze animal ing, 1 h prior to behavioral experiments. Animals within behaviors in durations > 6 frames (30 frames/s). the CRS group were randomly divided into the continued CRS group and the CRS/phenserine group. Odor Habituation/Dishabituation We used a modified version of an odor discrimination Tissue preparation task [20,21] to assess effects of CRS on response to an Animals were anaesthetized under isofluorane and trans- appetitive social stimulus, i.e female estrous urine. Cot- cardially perfused with 50 mL of 4% paraformaldehyde ton-tipped applicators were soaked with water and fas- (PFA) in phosphate buffered saline (PBS). Brains were tened to the roof of each cage such that mice must rear removed from the skull and postfixed overnight at 4°C in up to sniff. Duration of sniffing was measured over a 4% PFA/PBS, then transferred to 30% sucrose/PBS for 3 min presentation. This was repeated 3X, then replaced 72 h for cryopreservation. Brains were mounted on a by 1% imitation vanilla, and finally by urine from an freezing stage (Physitemp Instruments, Inc., Clifton, NJ) estrous-stage female. Contact with the applicator with set to -25°C and coronal sections (50 μm) were cut using an open mouth was considered chewing, and not scored a sliding microtome (Leica, Germany) and collected in as sniffing. PBS containing 0.015 M sodium azide. Nest building Immunohistochemistry Old nesting material was removed, and two unused Every 6th (prefrontal cortex and brainstem) or 12th nestlets (paper-based nesting material compacted into (hypothalamus, hippocampus) section was rinsed free- white squares) [2 g/each] were placed on the cage floor. floating in PBS/0.5% Tween-20. Non-specific binding was After4h, nestletmaterialthathadnotbeeneither blocked with 3% normal goat serum for 30 min. Sections shredded or incorporated into a nest was weighed. were incubated with an anti-fos B antibody directed against the N-terminus, which detects both the full- Tail suspension test length FosB as well as its truncated form, deltaFosB (sc- Mice were suspended by their tails for 6 min using a 48, rabbit IgG, 1:500, Santa Cruz Biotechnology, Santa 15 cm piece of lab tape wrapped around the tip of the Cruz, CA) or with an anti-c-fos antibody (PC38, 1:1000, tail. Sessions were videotaped and later scored by an Calbiochem) for 24 h at 4°C. Sections were rinsed in observer blinded to groups. Any significant movement PBS/0.5% Tween-20, incubated for 2 h at room tempera- of the body or the limbs was considered as mobility. ture with biotinylated goat anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA). Endogenous per- Forced swim test oxidase activity was blocked using 0.3% hydrogen perox- Transparent plexiglass cylinders, 25 cm tall × 12 cm dia- ide for 30 min. The HRP-DAB reaction was carried out meter were filled with 30°C water to ~21 cm so mice were using an avidin/biotin peroxidase complex (VectaStain not able to touch the floor or escape. Mice were placed in ABC Kit, Vector Laboratories). Sections were incubated the water for 6 min, videotaped and later analyzed with in ABC for 1 hr and DAB-cobalt (Sigma, St. Louis MO) Clever Systems Forced Swim Test Scan (Clever Sys Inc., for 3 min. They were then mounted on SuperFrost-Plus Leesburg,VA, USA).At the endofeachsession, mice treated slides (Fisher Scientific, Pittsburgh, PA), air-dried, Martinowich et al. Behavioral and Brain Functions 2012, 8:15 Page 4 of 14 http://www.behavioralandbrainfunctions.com/content/8/1/15 Nissl stained, dehydrated with alcohol rinses, cleared [F = 1.452, Student’s t-test P = 0.0001] (Figure 2f) 7,7 with CitriSolv, and coverslipped with Permount. weight in CRS-exposed animals. The decrease in initiative to both approach an appetitive Image analysis stimulus and avoid an aversive stimulus indicates a poten- Section images were captured using a Leica DMRB light tial deficit in motivation. Chronic stress models have fre- microscope equipped with a CoolSNAP digital camera quently been used to model symptoms of anhedonia, and and IPLab software. Cell density in the hippocampal den- these models consistently reveal related deficits in other tate gyrus, nucleus accumbens, and cortical areas were depressive- and anxiety-like behaviors. We tested CRS- analyzed using ImageJ. Cells in thalamic, hypothalamic, exposed animals (n = 8) as compared to control animals and septal nuclei were manually counted by a blinded (n = 8) in two measures of behavioral despair, the tail sus- individual. Anatomical boundaries were determined by pension test (TST) and the forced swim test (FST). We using the Franklin and Paxinos mouse brain atlas [23]. observed no difference in immobility times in the TST [F = 1.385, Student’s t-test P = 0.8802] (Figure 3a), but 7,7 Statistical analysis saw a significant decrease in immobility times in the FST Data are presented as group means ± standard error of [F = 5.994, Student’s t-test P < 0.0001] (Figure 3b). This 7,7 the mean (SEM). As appropriate, Student’s t-test, one- result is likely confounded by decreased body-weights in way ANOVA with Newman Keul’s post hoc or two-way CRS-exposed animals (Figure 2c). We also tested CRS- ANOVA with Bonferroni post hoc were performed using exposed animals in the elevated zero maze (EZM) where GraphPad Prism 5. Statistical significance was defined as an increase in time spent in the open portion of the maze p < 0.05. indicates decreased anxiety-like behavior. Control and CRS-exposed animals spent similar amounts of times in Results the open portions of the maze [F = 3.854, Student’s 7,7 Motivational deficits following chronic stress t-test P = 0.1749] (Figure 3c). Animals exposed to chronic stress consistently show deficits in the sucrose and/or saccharin preference test, Changes in home cage behavior following chronic stress findings which have contributed to establishing these To better understand the full range of effects of CRS on tests as analogs of anhedonia [24-26]. A significant normal home cage activity, we utilized automated beha- decrease in saccharin preference was found in CRS- vioral recognition software, HomeCage Scan (CleverSys exposed animals (n = 14) when compared to control Inc., Reston, VA, USA), to monitor home cage activity animals (n = 10) beginning ~17 d after CRS initiation for 22 h (Figure 1, Group2). We began recording 24 h [ANOVA F = 17.33, P < 0.0001 for CRS treatment; following CRS, and analyzed the following behaviors: rear 1,311 F =5.59, P < 0.0001 for time; F =2.63, P = up,hangcuddled,drink,eat,groom,sleep,chew,twitch, 6,311 6,311 0.0166 for time-CRS interaction; Bonferroni post-test, sniff, remain low and walk slowly (software definitions in P > 0.05 at d5, d10, d17, d25 and d31, P <0.05atd38 Table 1). Prior to experimentation, accuracy of the soft- and P < 0.01 at d44] (Figure 2a). Directly following the ware in analyzing all behaviors was verified and calibrated last measurement for saccharin preference we changed to experienced hand-scorers. to a 2-bottle preference test between water and mildly Our analysis revealed significant differences between bitter quinine solution (Figure 1, Group 1). As expected, control and CRS-exposed mice in total incidence as well control mice avoided the quinine, while CRS-exposed as in diurnal patterns of numerous behaviors. CRS- animals drank at levels close to chance [Student’s t-test, exposed mice spent significantly less total time rearing, P = 0.0012) (Figure 2b). To validate physiological mar- hanging cuddled, sniffing and walking slowly, but showed kers of chronic stress we measured total body weight as no difference in total time spent grooming drinking, eat- well as weights of the adrenals, thymus and testes. Body ing, sleeping, twitching, chewing and remaining low weights of non-stressed animals (n = 8) significantly (Table 2). In the first 41/2 hours prior to dark cycle onset, increased over the experiment while body weights of CRS-exposed mice spent less time rearing up (Figure 4a), CRS-exposed animals (n = 8) did not [ANOVA F = hanging cuddled (Figure 4b), sniffing (Figure 4c), remain- 1,28 28.79, P < 0.0001 for CRS treatment; F =30.79, P < ing low (Figure 4k) and walking slowly (Figure 4d). How- 1,28 0.0001 for time; F = 30.54, P < 0.0001 for time-CRS ever, they spent more time drinking (Figure 4f) and resting 1,28 interaction; Bonferroni post-test, P < 0.001 for Control (Figure 4h). It should be noted that differences in behavior versus CRS after CRS exposure] (Figure 2c). As between the control and CRS-exposed groups at experi- expected, there was a significant increase in adrenal mentonsetcouldreflectdifferences in novelty response weight [F = 3.097, Student’s t-test P = 0.0022] (Figure since the cage was changed before beginning the record- 7,6 1d) and significant decreases in both thymus [F = ing. As expected there was a spike in most active behaviors 7,7 (Figure 4a-d, f, g, j, k) at dark cycle initiation and a sharp 2.186, Student’s t-test P < 0.0001] (Figure 2e) and testes Martinowich et al. Behavioral and Brain Functions 2012, 8:15 Page 5 of 14 http://www.behavioralandbrainfunctions.com/content/8/1/15 ** Control 10 CRS CRS Control CRS 0 5 10 15 20 25 30 35 40 45 50 Day cd *** ** 0.03 *** 0.02 0.01 0.00 before after *** 0.15 *** 1.0 0.8 0.10 0.6 0.05 0.4 0.2 0.00 0.0 Figure 2 CRS-exposed mice exhibit a decreased appetitive response, and fail to avoid a mildly aversive stimulus.(a) Preference ratios for saccharin versus water in control and CRS-exposed mice in Group 1 (see Figure 1). Shaded area denotes CRS exposure. (b) Preference ratio for quinine versus water in control and CRS-exposed mice. (c) Control animals gain significantly more weight than CRS-exposed animals over the course of the experiment, while CRS animals do not gain a significant amount of weight and weigh significantly less than controls after CRS- exposure. (d) Increased adrenal weight in CRS-exposed animals; presented as % of total body weight. CRS exposure leads to decreased thymus weight. (f) CRS exposure leads to a decreased testes weight. Results here and in subsequent figures are reported as mean ± SEM; * = p < 0.05, ** = p < 0.01 and *** = p < 0.001. saccharine preference (%) body weight (g) Thymus (% of Body Weight) Adrenals (% of Body Weight) Testes (% of Body Weight) Quinine Preference (%) Martinowich et al. Behavioral and Brain Functions 2012, 8:15 Page 6 of 14 http://www.behavioralandbrainfunctions.com/content/8/1/15 ab c 250 100 Control 100 40 CRS *** TST FST EZM Figure 3 CRS-exposure does not affect depressive- and anxiety- like behavior.(a) Control and CRS-exposed animals spend a similar amount of time immobile in the tail suspension test. (b) CRS-exposed animals spend significantly less time immobile in the FST. (c) CRS-exposed animals do not differ in the amount of time spent in the open portion of an elevated zero maze. drop in inactive behaviors (Figure 4h, i) in both groups. At chewing (Figure 4j), walking slowly (Figure 4d), sniffing this time, control and CRS-exposed animals were similar (Figure 4c) and remaining low (Figure 4k). in sniffing (Figure 4c), walking slowly (Figure 4d) and To examine overall trends in behavior, we created a remaining low (Figure 4k). However, the CRS-exposed behavior-array analysis grid displaying fold increases and group showed lower levels of rearing (Figure 4a) and decreases between control and CRS-exposed mice (Fig- hanging behavior (Figure 4b), but higher levels of drinking ure 5). Each box represents an individual behavior and (Figure 4f), eating (Figure 4g) and chewing (Figure 4j) dur- time point within the experiment. The intensity of color ing the first ~4 hours of the dark phase. During the shift for each box represents the magnitude of behavioral from dark to light (hours 15-18) CRS-exposed mice spent change between control and CRS-exposed mice. Overall, significantly more time resting than control mice (Figure CRS-exposed mice displayed significant decreases in 4h). As opposed to control mice, whose active behaviors exploratory behaviors such as rear-up and sniff, as well showed a short, final peak right at the dark to light transi- as in locomotor behaviors including walk slowly, remain tion, CRS-exposed animalsshowednopeakinrearing low, and hang cuddled for several hours directly prior to (Figure 4a), hanging (Figure 4b), eating (Figure 4g), the onset of the dark cycle and before the onset of the Table 1 Software definitions for scoring HomeCage Scan behaviors Behavior Software Definition Rear Up Begins with the mouse lifting its front paws off the ground and standing on its hind legs. Rearing ends when the mouse comes back down and places one front paw back on the ground. Rearing may also include “partially reared” in which the mouse hunches its back and its front paws are off the ground, and is about halfway from being in the fully stretched, completely reared position. Hang Both forelimbs and hindlegs are above the midpoint between the floor and the top of the cage. Cuddled Drink Behavior starts when mouth is at level with the drinking spout. Behavior ends when mouth withdraws from the drinking spout. Sniffing behaviors directly before drinking are scored as drinking. Eat Snout is in the plane of the food compartment with minimal body and head movements. “Sniff” and “eat” are differentiated by the total time the snout remains in the food bin, with “sniff” being significantly less duration in the compartment. However, if the mouse sniffs directly before eating, the sniff behavior is scored as eating. Groom Mouse uses front paws to clean itself by rubbing over body and face in circular movements. Repetitive paw movements over a certain period of time are scored as groom. Sleep A minimum of 30 s of no significant movement of the mouse while it is in a non-rearing and non-hanging position is scored as sleep. Twitch Any movement occurring during sleep Sniff Body of mouse is stationary, but snout moves in a bobbing fashion. Scored as an exploratory behavior. Remain Low Prolonged inactivity of the mouse that is not scored by the software as any other behavior. Walk Slowly Mouse is moving across the cage and at least three legs are propelling it forward Time Immobile (sec) Time Immobile (sec) Time in Open Arm (sec) Martinowich et al. Behavioral and Brain Functions 2012, 8:15 Page 7 of 14 http://www.behavioralandbrainfunctions.com/content/8/1/15 Table 2 Home-cage behavior analysis array of control this area undergoes significant changes in gene tran- versus CRS-exposed mice scription and neuronal activity after CRS. This data, Control CRS coupled with reports that treatment with acetylcholines- terase inhibitors (AChE-I) leads to remediation of Behavior Mean (s) SEM Mean (s) SEM F value P value apathetic behavior[10,29], led us to ask whether the Rear Up 81.08 9.21 28.99 4.26 3.12 < 0.0001 CRS-induced behavioral deficits could be rescued by Hang Cuddled 268.7 18.13 118.7 25.23 2.9 0.01 facilitating cholinergic signaling. Sniff 2783 230.2 1717 221.1 1.38 0.01 We administered phenserine, a centrally active and Walk Slowly 1918 143.4 1414 137.1 1.37 0.03 potent AChE-I, 21 d after initiating CRS. Phenserine Groom 8820 771 10050 858.4 1.86 0.34 was administered 2x/d/6 d in accord with its anticholi- Drink 185.6 24.18 334.1 69.94 12.55 0.12 nesterase half-life of 8.25 h before beginning behavioral Eat 4451 503 4773 976.5 5.65 0.8 analysis (Figure 1, Group 4) [30]. Preference ratios for Sleep 27560 883 31670 1799 6.23 0.1 Control animals were 74.5 ± 7.2 and preference ratios for CRS-exposed animals were 50.4 ± 4.7 prior to the Twitch 667.2 75.62 776.3 58.71 1.11 0.27 phenserine study. Saline-injected CRS-exposed animals Chew 930.4 139.9 1330 184.1 2.6 0.14 showed no improvement in saccharin preference, but Remain Low 12120 810 11120 598.6 1.22 0.33 CRS-exposed animals administered phenserine increased preference by ~13% over stress-induced depressed levels [ANOVA F = 6.505, P = 0.007; Newman-Keuls Mul- light cycle (Figure 5), suggesting a decrease in initiation 2,21 tiple Comparisons for Control vs CRS P > 0.05, for Con- of non-essential activities. However, directly prior to dark cycle onset, inactive behaviors such as twitch and trol vs CRS/Phenserine P <0.05and forCRS versus rest increased in the CRS mice. CRS/Phenserine P < 0.01] (Figure 7a). We next looked at the effect of phenserine on the motivation levels of Neuronal activity mapping CRS-exposed animals in a nest-building paradigm. Exist- We used FosB immunohistochemistry to analyze neuronal ing nests and nesting materials were removed, and ani- activity patterns in control and CRS-exposed animals. Ani- mals were provided new nesting material. After 4 h, mals were killed 24 h following the last session of CRS and unused nesting material was measured. CRS-exposed brains processed for FosB immunoreactivity (Figure 1, animals showed significantly decreased motivation in Group 3). The antibody used detects both full-length FosB nest building as determined by incorporation of less as well as truncated deltaFosB, which gradually accumu- nestlet material, but phenserine treatment led to a lates due to its high stability and long half-life. A recent significant improvement [ANOVA F =11.00, P = 2,21 0.0001; Newman-Keuls Multiple Comparisons for Con- report showed that in CRS-exposed rats (1 h/d/10 d), del- trol vs CRS P < 0.001, for Control vs CRS/Phenserine taFosB is the predominant Fos family protein induced and P < 0.05 and for CRS versus CRS/Phenserine P <0.05] that the 35-37 kDa deltaFosB isoform is the only Fos (Figure7b).Weruled out the possibility that the family protein that remains elevated 24 h after the final decreased nest-building could be a result of weakness in stress exposure [27]. In agreement with a previous study the CRS-exposed animals by performing a wire hang [27], CRS-exposed mice (6 hr/d/28 d) showed significant test to check for muscle strength [ANOVA F = increases in FosB immunoreactivity in the medial septum 2,21 1.172, P = 0.3291] (Figure 7c). (MS)/nucleus of the vertical limb of the diagonal band To determine whether loss of motivational drive in the (vDB) and the lateral septum (LS) (Table 3, Figure 6). saccharin preference test transfers to an alternative appeti- Differing from this study [27], we observed significant tive stimulus, we measured the time spent sniffing on increases in FosB immunoreactivity in hypothalamic regions including the arcuate nuclei and the paraventricu- urine from a female estrous mouse in a modified odor lar nucleus (PVN), but no differences in the prelimbic cor- habituation/dishabituation test. We first measured time tex (PrL), the infra-limbic cortex (IL) or the nucleus spent sniffing a Q-tip dipped in water upon its initial pre- accumbens (NAcc). The differences between our results sentation and then habituation to the smell on the 2nd and those in previous studies may be explained by species and 3rd presentations. Next, we determined whether the and strain differences or from differences in CRS duration animal showed normal dishabituation in response to pre- and protocol [27,28]. sentation of a novel vanilla scent and then habituation to this scentonthe 2ndand 3rdpresentations.Lastly,we Effects of cholinergic facilitation on motivation and introduced female estrous urine and measured dishabitua- apathetic behavior tion and then habituation. Both control and CRS-exposed DeltaFosB accumulation in the MS/vDB, one of the mice showed normal habituation and dishabituation major basal forebrain cholinergic nuclei, suggests that curves in response to the 3 stimuli (Figure 7d). Animals Walk Slowly (s) Groom (s) Drink (s) Eat (s) Sleep (s) Twitch (s) Rear Up (s) Hang Cuddled (s) Sniff (s) Martinowich et al. Behavioral and Brain Functions 2012, 8:15 Page 8 of 14 http://www.behavioralandbrainfunctions.com/content/8/1/15 Figure 4 Animals exposed to CRS show differences in the diurnal patterns of engaging in numerous home cage activities.Data show 22 h behavior plots for (a) rearing up, (b) hanging-cuddled, (c) sniffing, (d) walking slowly, (e) grooming, (f) drinking, (g) eating, (h) sleeping, (i) twitching, (j) chewing, and (k) remaining low. exposed to CRS spent slightly less overall time sniffing on To better understand the brain regions phenserine may water [ANOVA F = 4.101, P = 0.0314; Newman-Keuls act on to mediate its behavioral effects in CRS-exposed 2,21 Multiple Comparisons for Control vs CRS P < 0.05, for animals, we used c-fos immunohistochemistry to examine Control vs CRS/Phenserine P >0.05 and for CRS versus neuronal activation patterns in response to presentation of CRS/Phenserine P > 0.05] (Figure 7e), no difference in estrous urine. For this experiment, animals were divided time spent sniffing on vanilla [ANOVA F = 5.46, P = into 4 groups: saline-injected/non-CRS exposed animals 2,21 0.0149; Newman-Keuls Multiple Comparisons for Control that sniffedonaQ-tipdippedin water, saline-injected/ vs CRS P > 0.05, for Control vs CRS/Phenserine P <0.05 non-CRS exposed animals that sniffed on a Q-tip dipped and for CRS versus CRS/Phenserine P > 0.05] (Figure 7f), in estrous urine, saline-injected/CRS exposed animals that and significantly less time sniffing on estrous urine sniffed on a Q-tip dipped in estrous urine and phenserine- [ANOVA F = 24.03, P < 0.0001; Newman-Keuls Multi- injected/CRS-exposed animals that sniffed on Q-tip 2,21 ple Comparisons for Control vs CRS P < 0.001, for Con- dipped in estrous urine. Animals were allowed to sniff on trol vs CRS/Phenserine P < 0.01 and for CRS versus CRS/ the Q-tip for 3 min and killed 2 h later for c-fos immuno- Phenserine P < 0.01] (Figure 7g). histochemistry. In non-stressed, saline-injected animals 250 1200 200 960 150 720 100 480 50 240 0 0 0 5 10 15 20 25 0 5 10 15 20 25 300 70 240 56 180 42 120 28 60 14 0 0 0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25 800 4000 175 640 3200 140 480 2400 105 320 1600 70 160 800 35 0 0 0 0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25 20 50 400 Control CRS 16 40 320 12 30 240 20 160 4 10 80 0 0 0 0 5 10 15 20 25 0 5 10 15 20 25 0 5 10 15 20 25 Chew (s) Remain Low (s) Martinowich et al. Behavioral and Brain Functions 2012, 8:15 Page 9 of 14 http://www.behavioralandbrainfunctions.com/content/8/1/15 Figure 5 Differences in specific home cage behaviors at discrete periods within the daily cycle. Behavior-array analysis grid displaying fold increases and decreases between control and CRS-exposed mice. Each box represents an individual behavior and time point within the experiment. The intensity of color for each box represents the magnitude of behavioral change between control and CRS-exposed mice. (n = 6), sniffing on estrous urine (hatched bars) as exposed animals lost their preference for saccharin, but opposed to water (white bars) increases c-fos immunor- also failed to avoid a bitter quinine solution (Figure 2a, b). eactivity in numerous brain regions including the medial A similar phenomenon has been reported in rhesus mon- preoptic area (MPO), LS, MS, NAcc and PrL (Figure 8a, b, keys following maternal deprivation [31]. This study pro- c, d and 8e). CRS-exposed animals that were injected with posed that in addition to producing anhedonia, some saline (n = 6) (black bars) did not show the increase in c- chronic stress paradigms may decrease motivation for appetitive stimuli in general [31]. Deficits in the sucrose fos immunoreactivity in the MPO, LS, MS, NAcc and PrL (Figure 8a, b, c, d and 8e) after sniffing on estrous urine. and saccharin preference tests have been reliably used as However, phenserine administration in CRS-exposed ani- measures of anhedonia [24-26], which is defined as the mals (n = 6) was capable of fully rescuing the response to inability to experience pleasure in previously enjoyable estrous in the NAcc (Figure 8d) and partially rescuing the activities such as eating, exercising, socializing and sex response in the MS (Figure 8c). ANOVA and post-hoc sta- [32-34]. The saccharin preference deficit coupled with the tistics for Figure 8 are provided in the accompanying lack of quinine aversion may also indicate apathy, a lack of Table 4. interest in surroundings, social withdrawal and loss of motivation and initiative [35,36]. Apathy, on its own, or Discussion when co-morbid with depression poses a challenge to clin- A major objective of this study was to characterize CRS- icians due to their overlapping symptomatology and fre- induced deficits in motivational drive. As expected CRS- quent co-occurrence [3]. Identifying apathy requires Table 3 deltaFosB immunoreactivity in control versus CRS-exposed mice Brain Region Control CRS Fold Difference Mean (Cells/pixel) Mean (Cells/pixel) CRS vs Control F Value P Value Dentate Gyrus 0 0 1.2891 1.533 0.11 Nucleus Accumbens 0 0 1.4205 2.061 0.07 Prelimbic Cortex 0 0 1.4308 8.433 0.05 Infralimbic Cortex 0 0 1.0169 1.328 0.93 Cingulate Cortex 0 0 1.0033 2.134 0.99 Visual Cortex 0 0 1.1538 1.295 0.25 Mean (cells counted) Mean (cells counted) Medial Septum/vDB 25.67 52.83 2.0580 1.794 0.01 Lateral Septum 568.7 1564 2.7501 2.510 < 0.0001 PVA 164.8 243 1.4745 2.718 0.22 PVN 34.17 125.3 3.6670 15.590 0 Arcuate 10 52.17 5.2170 12.710 0 SCN 5.83 2.5 0.4286 3.628 0.26 Martinowich et al. Behavioral and Brain Functions 2012, 8:15 Page 10 of 14 http://www.behavioralandbrainfunctions.com/content/8/1/15 Figure 6 Changes in deltaFosB immunoreactivity following CRS exposure. Representative image showing increased deltaFosB immunoreactivity in the medial septum (MS) and lateral septum (LS) after exposure to CRS. differentiation between loss of initiation versus loss of abil- estrous urine (Figure 7g), suggesting that these animals ity and emotional indifference versus a primary mood dis- exhibit deficits in motivational drive. CRS-exposed animals turbance [7]. Modeling apathy is important since it does also show lack of motivation in a nest-building paradigm not respond similarly to treatment options for anhedonia [35,36] and decreases in home-cage exploratory behaviors. [37,38]. Accordingly, despite their overlapping symptoma- For example, we saw significant differences in total time tology, there is accumulating evidence that apathy and spent rearing up, hanging cuddled and sniffing (Table 2), anhedonia may have different underlying alterations in and in patterns of diurnal activity. Since alterations in brain circuits [3,39]. sleep and circadian rhythms play a critical role in the We show that loss of motivational drive in CRS-exposed pathophysiology of numerous neuropsychiatric disorders animals in the saccharin preference test can transfer to a [33,40,41], the ability to model circadian alterations is a decrease in motivation for an alternative appetitive stimu- useful experimental tool. lus. First, we showed that CRS-exposed animals showed It has been suggested that apathy may reflect an inter- normal habituation and dishabituation to three different action between cholinergic deficiency and subsequent odors, confirming intact olfactory senses (Figure 7d). neurological changes in limbic regions [42]. Thus, we However, we observed a significant decrease in interest for asked whether deltaFosB accumulation in the MS/vDB, Martinowich et al. Behavioral and Brain Functions 2012, 8:15 Page 11 of 14 http://www.behavioralandbrainfunctions.com/content/8/1/15 ab c ** 4 150 Control CRS 5 2 CRS/Phenserine -5 ef g Control 16 100 CRS *** CRS/Phenserine ** 0 0 1 2 3 1 2 3 1 2 3 Estrous Water Vanilla Urine Figure 7 Phenserine treatment results in partial recovery of CRS-induced deficits in motivation.(a) Phenserine improves the CRS-induced saccharin preference deficit. (b) Phenserine improves the CRS-induced deficit in nesting behavior. (c) No change in muscle strength in CRS- exposed animals as determined by time the animal is able to hang from a wire rod. (d) Motivational drive to approach an appetitive stimulus is decreased by CRS, but partially rescued by phenserine administration. Habituation to the smell of a water-dipped Q-tip was measured over the course of three 3 min presentations by measuring time spent sniffing. Dishabituation and then habituation to a novel scent was assessed by measuring the time spent sniffing an imitation-vanilla dipped Q-tip for three consecutive 3 minute presentations and, lastly, an estrous urine dipped Q-tip. All groups showed normal habituation and dishabituation curves in response to the 3 stimuli. (e) Total time sniffing on the water smell is slightly decreased by CRS exposure. (f) Total time sniffing on the vanilla smell is slightly decreased in CRS-exposed animals administered phenserine. (g) Total time sniffing on estrous urine is robustly decreased by CRS exposure, but significantly improved by phenserine administration. which constitutes the major cholinergic projection to exposed animals and there are no appreciable changes the hippocampal formation, cingulate cortex and the in regional volume between control and CRS-exposed hypothalamus [29] could be influencing cholinergic sig- animals (KM and RJS, unpublished observations). naling. AChE-I treatment reduces incidence of apathy It is also possible that the behavioral effects of phen- and improves functioning in patients who present with serine in our model result from activation of cholinergic cholinergic disturbances in limbic and paralimbic cor- interneurons in areas implicated in motivation and tices[10,29],and restorationoffunctioninthese brain reward. For example, it has been shown that the AChE- regions may underlie the behavioral response to AChE- Is galantamine and donepezil lead to increased dopa- Is [9,43]. In AD, functional loss is thought to be a con- mine release in NAcc [45,46]. Control animals show a sequence of neuronal loss in cholinergic nuclei, and it robust increase in immediate early gene activation in has previously been reported that CRS can result in hip- the NAcc after being exposed to a motivating stimulus, pocampal atrophy [44]. However, it appears that choli- i.e. estrous urine (Figure 8d), but this increase is lost in nergic function in our model may be altered via changes CRS-exposed animals. However, phenserine administra- resulting from alterations in plasticity as opposed to tion rescued this deficit, suggesting that cholinergic neuronal loss because levels of the cholinergic cell mar- facilitation may restore dopaminergic function in the NTR ker p75 are unchanged between control and CRS- CRS-exposed NAcc. This restorative function could Sniffing (s) saccharin preference (% change) Sniffing on water (s) Nesting material (g) Sniffing on vanilla (s) Hanging (s) Sniffing on estrous urine (s) Martinowich et al. Behavioral and Brain Functions 2012, 8:15 Page 12 of 14 http://www.behavioralandbrainfunctions.com/content/8/1/15 a b ** ** saline/no stress/water saline/no stress/urine saline/CRS/urine phenserine/CRS/urine MPO LS ns d ns 15 20 ** ** ** ** 0 0 PrL MS NAcc Figure 8 Treatment with the AChE-I, phenserine provides a partial recovery for CRS-induced deficits in immediate early gene activation following exposure to a motivational stimulus.(a) Control animals (no CRS), injected with saline and exposed to a Q-tip dipped in estrous urine (hatched bars) show significantly increased numbers of c-fos positive nuclei in the medial preoptic area (MPO) as compared to those exposed to a Q-tip dipped in water (white bars). CRS animals administered either saline (black bars) or phenserine (grey bars) do not show an increase in c-fos positive nuclei after sniffing on estrous urine. (b) Control animals (no CRS), injected with saline and exposed to a Q-tip dipped in estrous urine (hatched bars) show significantly increased numbers of c-fos positive nuclei in the LS as compared to those exposed to a Q-tip dipped in water (white bars). CRS-exposed animals administered either saline (black bars) or phenserine (grey bars) do not show an increase in c-fos positive nuclei after sniffing on estrous urine. (c) Control animals (no CRS), injected with saline and exposed to a Q-tip dipped in estrous urine (hatched bars) show significantly increased numbers of c-fos positive nuclei in the MS as compared to those exposed to a Q-tip dipped in water (white bars). CRS-exposed animals administered saline (black bars) do not show an increase in c-fos positive nuclei after sniffing on estrous urine. CRS-exposed animals administered phenserine (grey bars) partially recover the induction in c-fos positive nuclei following estrous urine exposure. (d) Control animals (no CRS), injected with saline and exposed to a Q-tip dipped in estrous urine (hatched bars) show significantly increased numbers of c-fos positive nuclei in the NAcc as compared to those exposed to a Q-tip dipped in water (white bars). CRS animals administered saline (black bars) do not show an increase in c-fos positive nuclei after sniffing on estrous urine. CRS-exposed animals administered phenserine (grey bars) fully recover the induction in c-fos positive nuclei following estrous urine exposure. (e) Control animals (no CRS), injected with saline and exposed to a Q-tip dipped in estrous urine (hatched bars) show significantly increased numbers of c-fos positive nuclei in the PrL as compared to those exposed to a Q-tip dipped in water (white bars). CRS animals administered either saline (black bars) or phenserine (grey bars) do not show an increase in c-fos positive nuclei after sniffing on estrous urine. contribute to phenserine’s role in behavioral rescue of reversing selected CRS-induced phenotypes rather than motivational drive in CRS-exposed animals (Figure 7a, determining the effect of the drug in a naïve population. b, d). However, it remains a caveat of our studies that our The focus of the experiments with phenserine was to study did not include a control group to look at the determine whether an anticholinesterase had utility in effects of phenserine in a non-CRS exposed population. # c-fos positive cells # c-fos positive cells #c -fos positive cells # c-fos positive cells # c-fos positive cells Martinowich et al. Behavioral and Brain Functions 2012, 8:15 Page 13 of 14 http://www.behavioralandbrainfunctions.com/content/8/1/15 Table 4 ANOVA table for statistics in Figure 8 Newman-Keul’s post hoc Brain Region F value P value white vs hatched white vs black white vs gray hatched vs black hatched vs gray black vs hatched MPO 5.03 P < 0.05 P > 0.05 P > 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05 MPO 5.03 P < 0.05 P > 0.05 P > 0.05 P < 0.05 P < 0.05 P < 0.05 P < 0.05 LS 8.76 P < 0.01 P > 0.05 P > 0.05 P < 0.01 P < 0.01 P < 0.001 P < 0.01 MS 4.82 P < 0.05 P > 0.05 P > 0.05 P < 0.01 P < 0.01 P > 0.05 P < 0.01 NAcc 6.8 P < 0.05 P > 0.05 P < 0.05 P < 0.01 P < 0.01 P > 0.05 P < 0.01 Received: 20 June 2011 Accepted: 20 March 2012 Thus, it is possible that the effects of phenserine may Published: 20 March 2012 not be limited to animals exposed to CRS, but may also have similar effects on a control population. References 1. Marin RS: Apathy: a neuropsychiatric syndrome. 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Published: Mar 20, 2012