Different Roles for M1 and M2 Receptors Within Perirhinal Cortex in Object Recognition and Discrimination ⇑ Susan J

Neurobiology of Learning and Memory 110 (2014) 16–26

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Neurobiology of Learning and Memory

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Different roles for M1 and M2 receptors within perirhinal cortex in object recognition and discrimination ⇑ Susan J. Bartko a,b, , Boyer D. Winters c, Lisa M. Saksida a,b, Timothy J. Bussey a,b a Department of Experimental Psychology, University of Cambridge, Downing St., Cambridge CB2 3EB, UK b MRC and Wellcome Trust Behavioural and Clinical Neuroscience Institute, University of Cambridge, Cambridge CB2 3EB, UK c Department of Psychology and Collaborative Neuroscience Program, University of Guelph, Guelph, Ontario N1G 2W1, Canada article info abstract

Article history: Recognition and discrimination of objects and individuals are critical cognitive faculties in both humans Received 11 September 2013 and non-human animals, and cholinergic transmission has been shown to be essential for both of these Revised 30 December 2013 functions. In the present study we focused on the role of M1 and M2 muscarinic receptors in perirhinal Accepted 6 January 2014 cortex (PRh)-dependent object recognition and discrimination. The selective M antagonists pirenzepine Available online 24 January 2014 1 and the snake toxin MT-7, and a selective M2 antagonist, AF-DX 116, were infused directly into PRh. Pre-sample infusions of both pirenzepine and AF-DX 116 significantly impaired object recognition mem- Keywords: ory in a delay-dependent manner. However, pirenzepine and MT-7, but not AF-DX 116, impaired oddity Medial temporal lobe discrimination performance in a perceptual difficulty-dependent manner. The findings indicate distinct Pirenzepine AF-DX 116 functions for M1 and M2 receptors in object recognition and discrimination. Muscarinic toxin 7 Ó 2014 Elsevier Inc. All rights reserved. Muscarinic receptor Muscarinic subtypes

1. Introduction perirhinal cortex (PRh) (Tang, Mishkin, & Aigner, 1997; Warburton et al., 2003; Winters & Bussey, 2005b; Winters, Saksida, & Bussey, The ability to recognize and discriminate between objects and 2006). A recent study by Wu, Saunders, Mishkin, and Turchi (2012) individuals are important cognitive functions, which can be se- conducted in monkeys revealed significant impairments in visual verely impaired in neurodegenerative diseases such as Alzheimer’s recognition following intra-PRh infusions of the M1 receptor antag- disease (Hajilou & Done, 2007; Irle, Kessler, Markowitsch, & Hof- onist pirenzepine but not the M2 receptor antagonist methoctr- mann, 1987; Laatu, Revonsuo, Jaykka, Portin, & Rinne, 2003; Purdy, amine. However, little is known regarding which specific McMullen, & Freedman, 2002). Consistent with compromised receptor subtypes are involved in recognition memory in the rat cholinergic function in such diseases, cholinergic mechanisms have or what the role is of specific muscarinic receptor subtypes in been shown to be important in both recognition and discrimina- perceptual discrimination. Thus the current series of experiments tion (Aigner & Mishkin, 1986; De Rosa & Hasselmo, 2000; focused on the role of M1 and M2 receptor subtypes in PRh- McGaughy, Koene, Eichenbaum, & Hasselmo, 2005; Robbins dependent object recognition and discrimination in the rat. et al., 1997; Vannucchi, Scali, Kopf, Pepeu, & Casamenti, 1997; PRh-dependent recognition memory was assessed using two Winters, Saksida, & Bussey, 2008). Understanding the more specific delays (0 s and 3 h delay) in a standard spontaneous object recog- mechanisms underlying object recognition and discrimination is nition task. Object discrimination was examined using the simulta- thus essential for understanding normal brain function, how this neous oddity discrimination task (Bartko, Winters, Cowell, Saksida, function is compromised in certain neurodegenerative diseases, & Bussey, 2007a) across three levels of perceptual difficulty (Low, and what therapeutic targets might be useful for ameliorating Medium, and High). Since M1 and M2 receptors have high affinity cognitive decline in such conditions. for pirenzepine (Caulfield & Birdsall, 1998; Ehlert, Roeske, & Non-selective muscarinic receptor antagonists impair object Yamamura, 1995) and AF-DX 116 (Araujo, Lapchak, Regenold, & recognition memory when given systemically (Robbins et al., Quirion, 1989; Hammer, Giraldo, Schiavi, Monferini, & Ladinsky, 1997; Warburton et al., 2003) or when infused directly into the 1986; Quirion, Araujo, Regenold, Araujo, & Quirion, 1989; Rege- nold, Araujo, & Quirion, 1989), respectively, these two antagonists ⇑ were infused directly into PRh to examine M1 and M2 receptor Corresponding author. Address: Cornerstone Research Group, 3228 South function in PRh in object recognition and oddity discrimination Service Road, Suite 204, Burlington, Ontario L7N 3H8, Canada. tasks. M receptor function in PRh was also assessed using a E-mail address: [email protected] (S.J. Bartko). 1

1074-7427/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.nlm.2014.01.002 S.J. Bartko et al. / Neurobiology of Learning and Memory 110 (2014) 16–26 17 muscarinic snake toxin, MT-7 (also called MTX7 and m1-toxin1). Rompun, Dunlops) and placed in a stereotaxic frame (David Kopf

Although pirenzepine is considered an M1 speciﬁc antagonist, Instruments, Tujunga, CA) with the incisor bar set at À3.2 mm. and its use allows comparisons with previous studies using this The scalp was cut and retracted to expose the skull. Holes were compound, the selectivity of pirenzepine for M1 opposed to M4 drilled, and the guide cannulas were implanted according to the receptors is only 4–6 times greater (Madison, Jones, Tom-Moy, & following coordinates, measured relative to the skull at bregma Brown, 1987; Nasman, Jolkkonen, Ammoun, Karlsson, & Akerman, (Paxinos and Watson, 1997): anteroposterior, À5.5 mm; lateral, 2000). MT-7 is one of the most selective ligands available for tar- ±6.6 mm; dorsoventral, À6.5 mm. The cannulas were secured to geting M1 receptor subtypes (Olianas, Adem, Karlsson, & Onali, the skull using four jeweler screws and dental acrylic. Obturators 2004); it has more than 20,000 times higher selectivity for M1 cut to extend 1.1 mm beyond the tip of the guide cannulas and receptors than any other muscarinic receptor subtype (Karlsson, with an outer diameter of 0.36 mm were inserted into the guides Jolkkonen, Mulugeta, Onali, & Adem, 2000). and remained there except during infusions. Animals were allowed

Dissociation of function between PRh M1 and M2 receptors was to recover for at least 7 days before the beginning of behavioral predicted in this series of experiments because M1 and M2 recep- testing. tors have disparate neurochemical functions. Since M1 receptors are located primarily postsynaptically (Alcantara et al., 2001; Hers- 2.1.3. Histology ch, Gutekunst, Rees, Heilman, & Levey, 1994), antagonism of these After behavioral testing, rats were anesthetized by intraperito- receptors was expected to have effects similar to those of scopol- neal injection of 2 ml of Euthatal (Rhône Mérieux) and perfused amine, disturbing the initial encoding of object information. Be- transcardially with 100 ml of PBS, pH 7.4, followed by 250 ml of cause PRh lesions disrupt object processing in object recognition 4% paraformaldehyde (PFA), pH 7.4. The brains were removed, and oddity discrimination, intra-PRh infusions of M1 antagonists postfixed in 4% PFA at 4 °C for 24 h, and then immersed in 25% su- were predicted to impair both object recognition and oddity dis- crose in PBS until they sank. Coronal sections (60 lm) were cut on crimination. Conversely, M2 receptors are located primarily pre- a freezing microtome through the extent of the lesioned area, and synaptically (Mrzljak, Levey, & Goldman-Rakic, 1993; Packard, every fifth section was mounted on a gelatin coated glass slide and Regenold, Quirion, & White, 1990) and function as autoreceptors stained with cresyl violet. Slides were examined under a light (Galarraga et al., 1999; Quirion, Aubert, Araujo, Hersi, & Gaudreau, microscope to verify the cannulae placements, and approximate 1993); therefore, antagonising M2 receptors might increase cholin- cannula tip locations were mapped onto sections by comparing ergic activity in PRh, possibly facilitating performance in object visually with slides from Paxinos and Watson (1998). recognition and oddity discrimination tasks. The series of experiments examined the effects of intra-PRh infusions of pirenzepine 2.1.4. Infusion procedure (Experiment 1), AF-DX 116 (Experiment 2), and MT-7 (Experiment For all experiments, the basic procedure was as follows. All 3) in object recognition. The effects of pirenzepine (Experiment 4), infusions took place in a preparation room separate from the AF-DX 116 (Experiments 5 and 6), and MT-7 (Experiment 7) were behavioral testing area. Animals were gently restrained by the also examined in oddity discrimination. experimenter throughout the infusion procedure. The obturators were removed, and the 28-gauge infusion cannulae, which were cut to extend 1 mm beyond the tip of the guides, were inserted into 2. Materials and methods the guides. Bilateral infusions were conducted simultaneously using two 1 ll Hamilton syringes, which were connected to the 2.1. General methods infusion cannulae by propylene tubing. The syringes were driven by a Harvard Apparatus (Kent, UK) precision syringe pump, which 2.1.1. Subjects delivered a predetermined amount of ll to each hemisphere The subjects were 69 male Lister hooded rats (Experiment 1, (pirenzepine and AF-DX 116 = 1 ll, MT-7 = 0.5 ll). The infusion n = 9 [original n = 10; one rat died following surgery]; Experiment cannulae were left in place for an addition 1.5 min to allow for dif- 2, n = 10; Experiment 3, n = 14; Experiment 4, n = 10; Experiment fusion of the infusate. The infusion cannulae were then removed 5, n = 10; Experiment 6, n = 9 [original n = 10; one rat was excluded and the obturators replaced, and the rat was placed in a holding from analysis based on cannula placement]; Experiment 7, n =7 cage prior to the object recognition and oddity tasks. Animals were [original n = 10; one rat died during experimentation and two rats placed in the object recognition and oddity apparatus 15 min were excluded based on cannulae placement]; Harlan Olac, Bicest- after the end of the infusion. After each trial, rats were returned er, UK), weighing 270–320 g before surgery and housed in pairs in to home cages. For all experiments, the experimenter was blind a room with a 12 h light/dark cycle (lights on at 7:00 P.M.). All to the drug infusion condition. behavioral testing was conducted during the dark phase of the cy- For all experiments, all rats received two habituation sessions cle when rats are most active. Overhead lights were used during before the beginning of behavioral testing, rats experienced a testing, which is a very small fraction of the animals’ dark cycle ‘‘mock’’ infusion identical in all aspects to the procedure described (i.e., a few minutes at most per testing day). During testing, rats above except that the injection cannulae contained no liquid. This were fed 15 g of laboratory chow after daily behavioral sessions was done to habituate the animals to the general protocol of the to maintain weights at 85–90% of free-feeding body weight. Water infusion procedure, including insertion of the infusion cannulae was available ad libitum throughout the experiment. All experi- and the sound of the pump. mentation was conducted in accordance with the United Kingdom Animals (Scientific Procedures) Act (1986). 2.2. Drug preparation and doses

2.1.2. Surgery Physiological saline (0.9% sodium chloride, pH 7.0; Aquapharm;

The surgical protocol was identical to the protocol used previ- Animalcare, York, UK) and the M1 antagonist pirenzepine dihydro- ously for rat PRh cannulations (Winters & Bussey, 2005a). For each chloride (47 mM in physiological saline; Sigma–Aldrich, UK) were experiment, all rats were implanted bilaterally with 22-gauge prepared for Experiments 1 and 2. For Experiments 3, 4, and 5, indwelling guide cannulas. Before surgery, all animals were deeply phosphate buffered saline (0.01 M PBS) and the M2 antagonist anesthetized by ketamine hydrochloride (100 mg/kg intramuscular AF-DX 116 (Tocris, UK) were prepared. AF-DX 116 was first dis- (i.m.), Ketaset, Dunlops, Dumfries, UK) and xylazine (9 mg/kg i.p., solved in hydrochloric acid (HCl; 1 M) and then brought to a PH 18 S.J. Bartko et al. / Neurobiology of Learning and Memory 110 (2014) 16–26 of 7–8 with sodium hydroxide (NAOH; 1 M). The solution was then order of exposure to object pairs as well as the designated sample diluted in 0.01 M PBS to the final concentrations. In Experiments 3 and novel objects for each pair were counterbalanced within and and 5, three different concentrations were used (.25 mM, 2.5 mM, across groups. The time spent exploring objects was assessed from and 25 mM AF-DX 116) in addition to the PBS condition (0.01 M video recordings of the sample and choice phases. Data were col- PBS). In Experiment 4 only the 2.5 mM concentration of AF-DX lected by scoring exploratory bouts using a personal computer run- 116 and PBS were used. In Experiments 6 and 7, physiological sal- ning a program written in Visual Basic 6.0 (Microsoft, Redmond, ine (0.9% sodium chloride, pH 7.0; Aquapharm; Animalcare, York, WA). All trials were run with the experimenter blind to the drug UK) and MT-7 (4 lg/ll) were prepared. The MT-7 dose was chosen treatment conditions. based on a previous study that used comparable doses to examine behavioral effects following intracranial infusions (McCool, Patel, 2.3.4. Object recognition test Talati, & Ragozzino, 2008). Each test session consisted of two phases. In the sample phase, two identical objects (A1 and A2) were placed in the Y-shaped 2.3. Spontaneous object recognition apparatus on the second sliding door platform (door one was not needed for experiments and therefore was removed from the appa- 2.3.1. Apparatus ratus for the duration of all experimental testing). The rat was Spontaneous object recognition was conducted in a Y-shaped placed in the start box with the guillotine door lowered. The apparatus constructed in such a way as to minimize the influence guillotine door was then raised to allow the rat into the exploration of spatial or contextual factors and the presence of stimuli in the area of the maze. When the rat exited the start box, the guillotine testing room external to the object exploration area (Forwood, door was lowered to prevent re-entry, and the sample phase began. Winters, & Bussey, 2005; Winters, Forwood, Cowell, Saksida, & The time spent exploring the two objects was scored by an exper- Bussey, 2004). The apparatus had high, homogenous white walls imenter viewing the rat on a video screen. The cumulative duration constructed from Perspex (Lucite International, Southampton, of exploratory bouts, the beginning and end of which were indi- UK) to prevent the rat from looking out into the room and thereby cated by pressing a given key on the computer keyboard, was cal- maximizing attention to the stimuli. All walls were 40 cm high and culated by the computer program. Exploration of an object was each arm was 27 cm in length and 10 cm wide. The start arm con- defined as directing the nose to the object at a distance of tained a guillotine door 18 cm from the rear of the arm. This pro- <2.0 cm and/or touching it with the nose. Turning around or sitting vided a start box area within which the rat could be confined at on the object was not considered exploratory behavior. The sample the start of a given trial. Within each arm of the Y-apparatus were phase ended when the rat had explored the identical objects for a two sliding doors. The first sliding door was located 9 cm from the total of 25 s or when 5 min had passed. start box guillotine door (located at 18 cm from the rear of the start At the end of the sample phase, the rat was removed from the Y- box) and the second sliding door was located 18 cm from the first shaped apparatus for the duration of the 3 h retention delay. At the sliding door. Each sliding door was 9.8 cm wide and 40 cm high, at- start of the delay, rats were transferred to holding cages in a room tached to the bottom of the sliding door was a platform adjacent to the testing room for the duration of the delay. After the (8 Â 9.8 cm) where the stimuli could be placed. The floor and walls delay, the rat was placed back in the start box of the Y-shaped were wiped down with a dry paper towel between trials but other- apparatus and released into the exploration area for the choice wise were not cleaned during the experiment. A lamp illuminated phase. The Y-shaped apparatus now contained an identical copy the apparatus and a white shelf 50 cm from the top of the appara- of the sample (familiar) object (A3) in one arm and a new object tus created a ceiling on which a video camera was mounted to re- (B) in the other. The exploration arms in which the choice objects cord trials. were placed were counterbalanced between rats and across sessions. The rat was allowed to explore the objects for 2 min, at the 2.3.2. Objects end of which it was removed and returned to its home cage. The Triplicate copies were obtained for the objects, which were time spent exploring the novel and familiar objects was recorded made of plastic, metal, or glass. For any given trial, the objects in for all 2 min of the choice phase, but attention was focused on a pair were composed of the same material so that it was unlikely the first minute, during which object discrimination is typically they could readily be distinguished by olfactory cues. The height of greatest (Dix & Aggleton, 1999). A discrimination ratio was calcu- the objects ranged from 5 to 20 cm, and the objects were secured lated, which was the proportion of total exploration time spent to the platform of the doors (for sample objects) and to the appa- exploring the novel object (i.e., the difference in time spent explor- ratus (for choice objects) with Blu-Tack (Bostik, Stafford, UK). As far ing the novel and familiar objects divided by the total time spent as could be determined, the objects had no natural significance for exploring the objects), for the first minute of the choice phase on the rats, and they had never been associated with a reinforcer. each object recognition trial. This measure takes into account individual differences in the total amount of exploration time. 2.3.3. General procedure Rats were tested in two delay conditions: zero-delay and 3-h All rats were habituated in two consecutive daily sessions in delay. The procedure for the zero-delay object recognition condi- which they were allowed to explore the empty Y-apparatus for tion was identical to the procedure described above. However, 5 min. Before being placed in the apparatus for the habituation ses- when the sample phase ended (when the rat explored the identical sion, rats experienced a mock infusion procedure, as described objects for 25 s or when 5 min had passed), the sliding door (where above. After the mock infusion, the rats were taken from the prep- the objects were affixed) between the sample and choice objects aration area to the testing room and placed in the start box; the was removed, thereby immediately presenting the choice objects guillotine door was opened to allow the rat to explore the main to the rat. area of the apparatus. The guillotine door was lowered when the rat exited the start box to prevent re-entry into this area of the 2.4. Simultaneous oddity discrimination task apparatus. The experimenter did not begin timing the trial until the rat exited the start box. Testing began 24 h after the second 2.4.1. Apparatus habituation session. Rats were given a series of test trials (one The oddity apparatus has been described previously (Bartko per day) with a minimum interval of 24 h between trials. A differ- et al., 2007a; Bartko, Winters, Cowell, Saksida, & Bussey, 2007b). ent object pair was used for each trial for a given animal, and the The exploration area was triangular in shape. The oddity apparatus S.J. Bartko et al. / Neurobiology of Learning and Memory 110 (2014) 16–26 19 had high, homogenous white walls constructed from Perspex (Lu- given animal, and the order of exposure to object pairs, the desig- cite International) to prevent the rat from looking out into the nated repeated objects and odd object for each pair, and the odd room. All walls were 40 cm high and the start box contained a guil- object location were counterbalanced within and across groups. lotine door 25 cm from the rear of the box, providing a start box The time spent exploring objects was assessed from video record- area within which the rat could be confined at the start of a trial. ings of the trials. Data were collected by scoring exploratory bouts The back wall (bottom part of the triangle) was positioned using a personal computer running a program written in Visual Ba- 19.5 cm from the guillotine door. The back wall was 35 cm long sic 6.0 (Microsoft). with four 3 cm wide dividers positioned between the three 10 cm spaces (where the objects were placed) along the back wall. 2.4.4. Oddity test All object sets used in a given trial were placed in the apparatus 2.4.2. Lego objects before the rat was placed in the start box. The guillotine door was Three different perceptual conditions were examined. These then raised to allow the rat into the exploration area of the appa- were the low, medium, and high condition stimulus pairs (see ratus. When the rat exited the start box, the guillotine door was Fig. 1 for example stimuli) that were assessed in a visual discrim- lowered to prevent re-entry, and testing began. The time spent ination experiment for perceptual similarity in Bartko et al. (2007a). exploring the three objects during a testing phase was scored by an experimenter viewing the rat on a video screen. The cumulative 2.4.3. General procedure duration of exploratory bouts, the beginning and end of which All rats were habituated in two consecutive daily sessions in were indicated by pressing a given key on the computer keyboard, which they were allowed to explore the empty oddity apparatus was calculated by the computer program. for 5 min. For these habituation sessions, the rats were placed in Exploration of an object was defined as directing the nose to the the start box and the guillotine door was opened to allow the rat object at a distance of <2 cm and/or touching it with the nose. to explore the main area of the apparatus. The guillotine door Exploration of two identical and one odd object was recorded for was lowered when the rat exited the start box to prevent re-entry 5 min. We calculated an oddity preference percentage score, the into this area of the apparatus. The experimenter did not begin exploration of the odd object divided by the total exploration of timing the trial until the rat exited the start box. Testing began the odd and identical objects. Using this score, an oddity prefer- 24 h after the second habituation session. Rats were given a series ence score of 33.0% would indicate chance performance (the rat of test trials (one per day) with a minimum interval of 24 h explored all objects equally). An oddity preference score of between trials. A different object trio was used for each trial for a 100.0% would indicate maximum preference for the odd object; it is not, however, a realistic score because it could only be obtained if the rat explores only the odd object. In reality, the rat must explore all objects before preference for the odd object can be expressed. Therefore, an oddity preference score significantly > 33.3% (chance performance) would represent a meaning- ful score on this task.

2.5. Data analysis

2.5.1. Object recognition task Group means were taken from object recognition testing (duration of sample phase, object exploration in the first minute of the choice phase, and the discrimination ratio from the first minute of the choice phase). Planned comparison t-tests were used when hypotheses predicted an effect of drug in one delay condition but not the other. In Experiment 1, means were submitted to two- way ANOVA with repeated measures for drug and delay. In Exper- iment 3, means were submitted to one-way ANOVA with repeated measures for drug. Planned comparison t-tests were used when hypotheses predicted an effect of group in one trial condition but not the other and paired-samples t-tests were used for post hoc analyses of within-subject effects. In Experiment 7, means were submitted to two-way ANOVA with repeated measures for the within-subjects factor of delay and the between-subjects factor of group. Independent-samples t-tests were used for post hoc analyses of between-subjects effects. All object recognition statistical analyses were conducted with a significance level of a = 0.05.

2.5.2. Oddity task Total object exploration during the oddity task (total exploration of the odd and identical objects) was analyzed because a difference in exploration could affect oddity preference. Preference for the odd object (exploration of the odd object divided by total exploration) was also analyzed. The odd object preference score for each drug condition was analyzed for the second minute of exploration during the oddity task, because consistent with a pre- Fig. 1. Examples of Lego stimuli used in the (A) Low, (B) Medium, and (C) High conditions. All three stimulus conditions were used in Experiments 4, 5, and 7. For vious oddity experiment (Bartko et al., 2007b), this was the first Experiment 6, only the high stimulus condition was used. time point at which the control animals showed a significant pref- 20 S.J. Bartko et al. / Neurobiology of Learning and Memory 110 (2014) 16–26 erence for the odd object in all conditions. Means from each of 3 days (Liang et al., 2001). Animals were placed into the Y appara- these measures were submitted to two-way ANOVA with repeated tus 60 min after the end of the infusion (disengagement of the measures for drug and condition in Experiments 4–6. Planned pump) for the first object recognition trial. The second trial oc- comparison t-tests were used when hypotheses predicted an effect curred 24 h after the first trial and the third trial occurred 24 h of drug in the higher perceptual conditions but not the low condi- after the second trial. Twenty-four hours after the third zero-delay tion. Paired-samples t tests were used for post hoc analysis of with- object recognition trial, rats were given a second infusion (the rats in-subject effects. In Experiment 7, means from each of these were given the same drug they received on the first trial) and run measures were submitted to a two-way ANOVA with repeated on the three-hour delay condition for 3 trials. Experiment 4 exam- measures for the within-subjects factor of condition and the be- ined the effects of pirenzepine and saline in the simultaneous odd- tween-subjects factor of group. Independent-samples t-tests were ity discrimination task. All rats were run for four trials on each used for post hoc analyses of within-subjects effects. Tests of sig- condition (Low, Medium, and High), receiving two saline trials nificance in all oddity experiments were performed at a = 0.05. and two pirenzepine trials per condition, for a total of 12 trials. For each condition, drug conditions were counterbalanced be- 3. Experimental details tween rats. Experiment 5 examined the effects of 2.5 mM AF- DX116 and PBS in the oddity task. All rats were run for four trials See Table 1 for an overview of Experiments 1–7. Experiment 1 on each condition (Low, Medium, and High), receiving two saline assessed the effects of pre-sample intra-PRh infusions of physio- trials and two AF-DX 116 trials per condition, for a total of 12 trials. logical saline and the M1 antagonist pirenzepine dihydrochloride For each condition, drug conditions were counterbalanced be- in object recognition, using a 0 s and 3 h retention delay between tween rats. Experiment 6 examined the effects of the different con- the sample and choice phases. All rats were run for four trials on centrations of AF-DX 116 (0.25 mM, 2.5 mM, and 25 mM) and PBS the 3 h delay condition, receiving saline infusions for half of trials in only the High condition of the oddity task. Experiment 7 exam- and pirenzepine infusions on the other half. These same rats were ined the effects of MT-7 and saline in the simultaneous oddity dis- then run for four trials on the zero-delay condition, receiving saline crimination task. Using a between-subjects design of drug (MT-7 infusions for half of the trials and pirenzepine infusions for the and saline), a new cohort of seven rats (three rats received saline other half. For each delay condition, drug conditions were counter- and four rats received MT-7) was used in the oddity task. The drug balanced between rats. Experiment 2 examined the effects of concentrations used and the amount of drug delivered was identi- pre-sample intra-PRh infusions of phosphate-buffered saline and cal to Experiment 3. All rats were run for three trials on each con- the three doses (0.25 mM, 2.5 mM, and 25 mM) of M2 antagonist dition (Low, Medium, and High), for a total of 9 trials. Twenty-four AF-DX 116 in object recognition, using a 3 h delay between the hours after the third oddity trial, rats were given a second infusion sample and choice phase. All rats were tested for two trials for each (the rats were given the same drug they received on the first trial) drug condition, for a total of eight trials, on the 3 h delay condition. and run for three trials, in a similar manner to the first three trials. Drug condition was counterbalanced between rats. In Experiment Twenty-four hours after the sixth trial, rats were given a third infu- 3 the effects of intra-PRh infusions of physiological saline and sions (the rats were given the same drug they received on the first MT-7 were examined in object recognition using two retention de- and second trial) and run for three final trials. The three perceptual lays (0 s and 3 h) between sample and choice phase. A between- conditions were counterbalanced within and between rats. subjects design of drug (saline or MT-7). Fourteen rats received infusions before the sample phase; seven rats received saline and 4. Results seven rats received MT-7. Each rat received 0.5 ll of drug to each hemisphere over 2 min. Rats were first tested in the zero-delay 4.1. Histology condition. Each rat received an infusion on the first day of each delay condition (0 s and 3 h) since the efficacy of MT-7 has been All rats included for analyses had guide cannulae located bilat- examined previously in the rat striatum (Liang, Gutierrez-Ford, & erally with injection needle tips terminating in PRh near the border Potter, 2001), which showed that MT-7 blockade was immediate between areas 35 and 36 within cortical layers 2–5 (Burwell, and the halftime for the turnover of M1 receptors was roughly 2001). These placements were located in the approximate midsec-

Table 1 Overview of Experiments1–7. Abbreviations: OR = Object Recognition, PBS = Phosphate-Buffered Saline, KEY = ÃN is the number of animals included in the ﬁnal analysis.

Experiment 1 2 3 4 5 6 7 NÃ 91014101097 Task OR OR OR Oddity Oddity Oddity Oddity Delay or condition 0 s and 3 h 3 h 0 s and 3 h Low, medium, high Low, High Low, medium, high medium, high Drugs/doses Pirenzepine AF-DX 116 MT-7 Pirenzepine AF-DX 116 AF-DX 116 MT-7 47 mM 0.25 mM 4 lg/ll 47 mM 2.5 mM 0.25 mM 4 lg/ll 2.5 mM 2.5 mM Saline 25 mM Saline Saline PBS 25 mM Saline PBS PBS Result Impairment during Impairment with Impairment Impairment in the High No effect of No effect of Impairment in the the 3 h delay but the 2.5 mM AF-DX during the 3 h condition but not the Low 2.5 mM AF- any dose of Medium and High not the 0 s delay 116 dose but not delay but not the and Medium perceptual DX 116 dose AF-DX 116 in perceptual following intra- the 0.25 mM and 0 s delay conditions following in any the High conditions PRh pirenzepine 25 mM AF-DX 116 following intra- intra-PRh pirenzepine perceptual perceptual following intra- infusion doses PRh MT-7 infusion condition condition PRh MT-7 infusion infusion S.J. Bartko et al. / Neurobiology of Learning and Memory 110 (2014) 16–26 21

Fig. 2. (A) Photomicrograph illustrating typical cannula tracks in PRh of a representative brain section from Experiment 1. Rats from Experiments 2–7 had similar PRh cannula placements. (B) Schematic representation of the infusion needle tip placements from Experiment 1. Cannulae were consistently located between 5.80 and 6.30 mm posterior to bregma. Some needle tips overlap in the figure. tion of the rostral–caudal extent of PRh, between 5.80 and 4.2.1.3. Recognition during the choice phase. Intra-PRh infusions of 6.30 mm posterior to bregma (see Fig. 2). Histological analysis of pirenzepine did not affect performance in object recognition in the placements also did not indicate that the frequency of the the zero-delay condition; however, infusions of pirenzepine se- MT-7 infused resulted in any gross damage. verely disrupted object recognition when the retention delay between the sample and choice phase was three hours (see 4.2. Experiment 1 Fig. 3A). A two-way ANOVA with repeated measures conducted on the drug and delay revealed a main effect of drug (F(1,8) = 6.98, Experiment 1 examined the effects of pre-sample intra-PRh p = 0.03). However, the delay (F(1,8) = 1.89, p > 0.05) and the drug by delay interaction (F(1,8) = 2.0, p > 0.05) were not significant. As infusions of physiological saline and the M1 antagonist pirenzepine dihydrochloride in object recognition, using a 0 s and 3 h retention we predicted an effect in the 3 h delay condition, but not the delay between the sample and choice phases. We predicted a de- zero-delay condition due to the fact that memory of the objects in PRh is required in the delay condition but not when objects lay-dependent effect based on our previous findings in M1 knockout mice (M1RÀ/À) which showed impairments in M1RÀ/À mice are presented nearly simultaneously in the 0 s delay condition, on an object recognition task at a 3 h delay, but not when the delay planned comparisons were also conducted. Planned comparison was very short (Bartko et al., 2011). t-tests revealed no significant effect of drug in the zero-delay condition (t(8) = 0.66, p > 0.05) but a highly significant effect in the 3 h delay condition (t(8) = 3.54, p = 0.008). 4.2.1. Spontaneous object recognition 4.2.1.1. Duration of the sample phase. The total time required to 4.3. Experiment 2 complete 25 s of exploration in the sample phase was analyzed because significant group differences at this stage of a trial might lead Experiment 2 examined the effects of pre-sample intra-PRh to differences in subsequent recognition performance. This analy- infusions of PBS and the M2 antagonist AF-DX 116 in object recog- sis revealed no significant difference of drug (F < 1), delay (F < 1), nition, using a 3 h delay between the sample and choice phase. and the interaction of drug by delay was also not significant Three different concentrations were used (0.25 mM, 2.5 mM, and (F < 1). The mean sample phase duration (±SEM) for each drug in 25 mM AF-DX 116) in addition to the PBS condition (0.01 M PBS). each delay were as follows: Zero-delay: Saline = 98.81 ± 17.0 s and Pirenzepine = 99.45 ± 20.16 s; 3 h delay: Saline = 103.20 ± 4.3.1. Spontaneous object recognition 23.26 s and Pirenzepine = 100.68 ± 23.64 s. 4.3.1.1. Duration of the sample phase. Analysis of the total time required to complete 25 s of exploration in the sample phase re- 4.2.1.2. Object exploration during choice phase. Analysis of the first vealed no significant effect of drug (F < 1). The mean sample minute of object exploration during the choice phase revealed no phase duration (±SEM) for each drug were as follows: significant effect of drug (F < 1). However, analysis of the choice PBS = 73.08 ± 22.14 s, 0.25 mM AF-DX 116 = 71.09 ± 19.53 s; exploration revealed a significant effect of delay (F(1,8) = 29.42, 2.5 mM AF-DX 116 = 76.1 ± 22.16 s, and 25 mM AF-DX p = 0.001). Importantly, however, the interaction of drug by delay 116 = 78.49 ± 25.42 s. was not significant (F < 1). The mean exploration of the objects in the choice phase (±SEM) for each drug in each delay were as 4.3.1.2. Object exploration during choice phase. Analysis of the total follows: Zero-delay: Saline = 16.47 ± 2.38 s and Pirenzepine = mean object exploration during the first minute of the choice 16.41 ± 3.55 s; 3 h delay: Saline = 22.06 ± 0.95 s and Pirenzepine = phase revealed no significant effect of drug (F < 1). The mean 20.67 ± 0.89 s. (±SEM) for each drug is as follows: BS = 18.85 ± 2.08 s; 0.25 mM 22 S.J. Bartko et al. / Neurobiology of Learning and Memory 110 (2014) 16–26

a non-competitive antagonist of the M1 receptors by binding stably to an allosteric site and is currently the most selective ligand avail-

able for targeting M1 receptor subtypes (Olianas et al., 2004). MT-7 has more than 20,000 times higher selectivity for M1 receptors than any other muscarinic receptor subtype (Karlsson et al., 2000). The effects of intra-PRh infusions of physiological saline and MT-7 (4 lg/ll) were examined in object recognition using two retention delays (0 s and 3 h) between sample and choice phase using a between-subjects design.

4.4.1. Spontaneous object recognition 4.4.1.1. Duration of the sample phase. Analysis of the total time required to complete 25 s of exploration in the sample phase revealed no signiﬁcant difference of drug (F < 1), delay

(F(1,12) = 2.12), and the interaction of drug by delay was also not signiﬁcant (F < 1). The mean sample phase duration (±SEM) for each drug in each delay were as follows: Zero-delay: Control 75.16 ± 15.90 s and MT-7 = 83.88 ± 24.39 s; 3 h delay: Control = 86.55 ± 20.25 s and Pirenzepine = 87.32 ± 22.55 s.

4.4.1.2. Object exploration during choice phase. Analysis of the total mean object exploration during the choice phase revealed no sig- niﬁcant effect of drug (F < 1) or condition (F < 1). The interaction of drug by condition (F < 1) was also not signiﬁcant. The mean (±SEM) for each drug in each delay were as follows: Zero-delay: Saline = 12.79 ± 5.16 s and MT-7 = 12.85 ± 3.19 s; 3 h delay: Saline = 11.53 ± 1.38 and MT-7 = 12.02 ± 0.92 s.

4.4.1.3. Recognition during the choice phase. The MT-7 group performed significantly worse than the Saline group in the 3 h delay condition but not in the zero-delay condition (see Fig. 3C), replicating the pirenzepine-induced object recognition deficit in Fig. 3. (A) Blockade of M1 receptors following pre-sample intra-PRh infusions of Experiment 1. A two-way ANOVA with repeated measures con- pirenzepine impaired object recognition memory with a 3 h retention delay ducted on the discrimination ratio revealed a main effect of drug between sample and choice phases in Experiment 1. Data are presented as average discrimination ratio ± SEM, ÃÃ p = 0.008. (B) Pre-sample, intra-PRh infusions of (F(1,12) = 6.53, p = 0.02. There was no significant effect of delay 2.5 mM AF-DX 116 impaired recognition memory with a 3 h retention delay (F < 1) and the interaction of drug by delay was also not significant between sample and choice phases in Experiment 2. Data are presented as average (F < 1). Since a significant difference was predicted in the 3 h delay discrimination ratio ± SEM, ÃÃ p = 0.03. (C) Infusion of the snake toxin MT-7 impaired condition but not the zero-delay condition, planned comparisons object recognition memory with a 3 h retention delay between sample and choice t-tests were used to examine group performance separately in each phases (Experiment 3). Data are presented as average discrimination ratio ± SEM. ÃÃ p = 0.05. delay condition. There was no significant difference between drug in the zero-delay condition (t(12) = 0.67, p > 0.05) but a significant effect in the 3 h delay condition (t(12) = 2.18, p = 0.05). Thus, AF-DX 116 = 18.51 ± 6.05 s; 2.5 mM AF-DX 116 = 16.91 ± 3.74 s; infusions of MT-7 resulted in impaired performance in object rec- and 25 mM AF-DX 116 = 17.07 ± 5.35 s. ognition only when a three-hour retention delay occurred between the sample and choice phase. 4.3.1.3. Recognition during the choice phase. Intra-PRh infusions of the medium concentration of AF-DX 116 (2.5 mM) disrupted object 4.5. Experiment 4 recognition (see Fig. 3B). A one-way ANOVA with repeated measures conducted on the drug concentration revealed a main effect Experiment 4 examined the effects of pirenzepine and saline in of drug (F(3,27) = 3.50, p = 0.02). Paired samples t-tests revealed a the simultaneous oddity discrimination task. significant difference between PBS and 2.5 mM AF-DX 116

(t(9) = 2.5, p = 0.03). However, paired samples t-tests revealed no 4.5.1. Simultaneous oddity discrimination signiﬁcant differences between the following drug conditions: 4.5.1.1. Object exploration during the oddity task. Total exploration

PBS and 0.25 mM AF-DX 116 (t(9) = 0.19, p > 0.05), PBS and of the odd and the identical objects during the oddity task was 25 mM AF-DX 116 (t(9) = 0.78, p > 0.05, 0.25 mM and 2.5 mM not affected by pirenzepine. Analysis of variance revealed no AF-DX 116 (t(9) = 2.7, p > 0.05), 0.25 mM and 25 mM AF-DX 116 significant effect of drug (F < 1) or condition (F < 1). The interaction (t(9) = 0.89, p > 0.05, and 2.5 mM and 25 mM AF-DX 116 of drug by condition was also not significant (F < 1). The mean (t(9) = 1.90, p > 0.05). exploration during the oddity discrimination task (±SEM) for each drug condition in each perceptual difficulty condition were as fol- 4.4. Experiment 3 lows: Low: Saline = 28.71 ± 7.32 s and Pirenzepine = 29.82 ± 9.06 s; Medium: Saline = 28.51 ± 7.98 s and Pirenzepine = 26.33 ± 8.41 s; Experiment 3 was designed to examine more specific blockade and High: Saline = 25.84 ± 6.56 s and Pirenzepine = 26.71 ± 7.79 s. of M1 receptors by infusing snake toxin MT-7 into PRh. Although pirenzepine is considered an M1-specific antagonist, the selectivity 4.5.1.2. Preference for the odd object. Analysis of the percent prefer- of pirenzepine for M1 opposed to M4 receptors is only 4–6 times ence for the odd object at 2 min revealed a significant main effect greater (Madison et al., 1987; Nasman et al., 2000). MT-7 acts as of drug (F(1,9) = 6.38, p = 0.03) (see Fig. 4A). However, there was no S.J. Bartko et al. / Neurobiology of Learning and Memory 110 (2014) 16–26 23

Fig. 4. (A) Pre-trial intra-PRh infusion of pirenzepine impaired oddity discrimination in Experiment 4. Data are presented as 2 min cumulative average oddity preference scores (the exploration of the odd object divided by the total exploration of the odd and identical objects combined) ± SEM, ÃÃ p = 0.04. (B) Pre-trial intra-PRh infusions of 2.5 mM AF-DX 116 did not result in oddity discrimination performance that is signiﬁcantly different from PBS infusions in Experiment 5. Data are presented as 2 min cumulative average oddity preference scores ± SEM. (C) Pre-trial intra-PRh infusions of 25, 2.5, and 0.25 mM AF-DX 116 did not result in oddity discrimination performance in the High condition that was signiﬁcantly different from PBS infusions in Experiment 6. Data are presented as 2 min cumulative average oddity preference scores ± SEM. (D) Pre-trial intra-PRh infusion of MT-7 impaired oddity discrimination in Experiment 7. Data are presented as 2 min cumulative average oddity preference scores ± SEM. ÃÃ p = 0.03, ÃÃÃ p = 0.001.

significant effect of condition (F(2,18) = 1.22, p > 0.5) and the inter- 4.6.1.2. Preference for the odd object. Analysis of the percent prefer- action of drug by condition was also not significant (F < 1). As we ence for the odd object at 2 min revealed no significant effect of predicted impairments in the higher perceptual conditions but drug (F(1,9) = 3.12, p > 0.05), condition (F < 1), or the interaction of not the low condition, planned-comparison t-tests revealed no sig- drug by condition (F < 1) (see Fig. 4B). No significant effect of drug nificant drug difference in the Low condition (t(9) = 0.06, p > 0.5) or was demonstrated in minutes three through five. the Medium condition (t(9) = 0.97, p > 0.05). However, the Piren- zepine performance in the High condition was significantly lower 4.7. Experiment 6 than Saline performance (t(9) = 2.32, p = 0.04). When pirenzepine was infused pre-trial, the rats did not show preference for the Experiment 6 examined the effects of the different concentra- odd object when the stimuli were perceptually similar in the High tions of AF-DX 116 (0.25 mM, 2.5 mM, and 25 mM) and PBS in only condition. However, when the novel object was not perceptually the High condition of the oddity task. similar to the familiar object in the choice phase, in the Low condition, pirenzepine infusions did not affect performance in the task. 4.7.1. Simultaneous oddity discrimination This pattern of impairment was demonstrated across minutes 4.7.1.1. Object exploration during the oddity task. Total exploration three through five. of the odd and the identical objects during the oddity task was

not affected by AF-DX 116 (F(3,24) = 1.61, p > 0.05). The mean explo- 4.6. Experiment 5 ration during the oddity discrimination task (±SEM) for each drug condition in the High condition were as follows: PBS = 27.24 ± Experiment 5 examined the effects of 2.5 mM AF-DX 116 and 5.69 s, 0.25 mM AF-DX 116 = 26.43 ± 5.17 s, 2.5 mM AF-DX 116 = PBS in the oddity task. 27.30 ± 7.42 s and 25 mM AF-DX 116 = 22.38 ± 3.32 s.

4.6.1. Simultaneous oddity discrimination 4.7.1.2. Preference for the odd object. Analysis of the percent prefer- 4.6.1.1. Object exploration during the oddity task. Infusions of ence for the odd object at 2 min revealed no significant effect of 2.5 mM AF-DX 116 and PBS did not affect total exploration of the drug (F < 1) (see Fig. 4C). The odd object preference score for each odd and the identical objects during the oddity task. Analysis of drug condition was analyzed for all five minutes of the oddity task variance revealed no significant effect of drug (F < 1), condition to examine if there was an effect of drug during any minutes of the task. These analyses revealed no significant effect of drug at any (F(2,18) = 2.97, p > 0.05), and the interaction of drug by condition was also not significant (F < 1). The mean exploration during the minute of the task (all minutes, F < 1). oddity discrimination task (±SEM) for each drug condition in each perceptual condition were as follows: Low: PBS = 32.72 ± 7.73 s 4.8. Experiment 7 and 2.5 mM AF-DX 116 = 30.55 ± 12.16 s; Medium: PBS = 23.26 ± 7.85 s and 2.5 mM AF-DX 116 = 27.82 ± 5.59 s; and Experiment 7 examined the effects of MT-7 and saline in the High: PBS = 27.91 ± 8.45 s and 2.5 mM AF-DX 116 = 27.96 ± 3.10 s. simultaneous oddity discrimination task. 24 S.J. Bartko et al. / Neurobiology of Learning and Memory 110 (2014) 16–26

4.8.1. Simultaneous oddity discrimination paired object recognition, the dose that had the largest effect on 4.8.1.1. Object exploration during the oddity task. Analysis of vari- memory had no effect on challenging perceptual discriminations; ance of the total exploration of the odd and identical objects during indeed the performance of the animals infused with AF-DX 116 the oddity ask revealed no significant effect of drug (F(1,5) = 1.05, was numerically (but not significantly) higher than that of controls p > 0.05). However, there was a significant effect of condition (discussed below).

(F(2,10) = 5.14, p = 0.02). Importantly, the interaction of group by In the present study, pre-sample infusions of pirenzepine and condition was not significant (F(2,10) = 1.92, p > 0.05). The mean MT-7 significantly impaired object recognition memory (in a exploration during the oddity discrimination task (±SEM) for each delay-dependent manner) and perceptual discrimination. This drug in each condition was as follows: Low: Saline = 37.47 ± finding is consistent with previous studies that have shown that

11.08 s and MT-7 = 32.15 ± 3.37 s; Medium: Saline = 28.54 ± 3.05 s M1 receptor antagonism can impair learning and memory (Aura, and MT-7 = 21.24 ± 2.71 s; and High: Saline = 25.16 ± 1.88 s and Sirvio, & Riekkinen, 1997; Herrera-Morales, Mar, Serrano, & MT-7 = 29.65 ± 8.30 s. Bermudez-Rattoni, 2007; McCool et al., 2008; Tzavos, Jih, & Ragozzino, 2004; Wu et al., 2012). For example, in an object recognition procedure for mice analogous to that used here, Bartko et al. 4.8.1.2. Preference for the odd object. Analysis of the percent prefer- (2011) reported substantial impairments in M receptor knockout ence for the odd object at 2 min revealed significant effects of drug 1 mice (M1RÀ/À), consistent with the findings of the present study. (F(1,5) = 7.90, p = 0.03) and condition (F(2,10) = 4.11, p = 0.05) (see Another finding by Bartko et al. (2011), however, appears inconsis- Fig. 4D). Although the drug by condition interaction was not signif- tent with the present study, namely that M1RÀ/À mice were unim- icant (F < 1), planned-comparison t-tests revealed a significant paired in the acquisition of difficult visual discrimination tasks in a group effect in the Medium condition (t(5) = 6.67, p = 0.001) and touchscreen apparatus. However, in these tasks, animals are the High condition (t (5) = 2.80, p = 0.03), but not in the Easy condi- exposed to the stimuli over a large number of trials, whereas the tion (t(5) = 1.01, p > 0.05). Despite the small sample size, there was oddity discrimination in the present study proceeds during a single a significant impairment in the more perceptually challenging con- exposure to the testing stimuli. It may be that such a task, which ditions, suggesting a highly robust effect. This pattern of impair- limits the time over which animals can form a robust representa- ment was also demonstrated in minutes three through five. The tion of the stimuli, is particularly sensitive to perturbations which performance deficit in the High condition following MT-7 repli- lead to impoverished stimulus representations. However, it is cates the oddity performance deficits produced following intra- worth noting that in the rat, intra-PRh infusion of scopolamine PRh infusions of pirenzepine in Experiment 2. (or muscimol, or AP-5), can impair acquisition of a similar touchscreen task 5. Discussion (Winters, Bartko, Saksida, & Bussey, 2010). Thus an alternative explanation for the preserved perceptual discrimination learning In the present study we sought to understand in more detail the in M1RÀ/À mice may be that compensatory mechanisms can devel- involvement of muscarinic receptor subtypes in PRh-dependent ob- op in such constitutive models, which may mask impairments and ject recognition and discrimination. These experiments revealed a which are less likely to accompany acute pharmacological functional dissociation between M1 and M2 receptors: activity at interventions. both M1 and M2 receptors in PRh is important for object recognition The object recognition impairment in the 3 h delay condition fol- memory across a 3-h delay, but only M1 receptor activity was nec- lowing pre-sample intra-PRh infusions of M1 antagonists pirenzep- essary for object discrimination in a perceptual oddity task. Indeed, ine and MT-7 but not infusions of the M2 antagonist AF-DX 116 is if anything, antagonism of M2 receptors in PRh improved object also consistent with a recent study conducted by Wu et al. (2012). discrimination. Neither M1 nor M2 antagonism resulted in general In this study, intra-PRh infusions of pirenzepine impaired visual perceptual impairments or deficits in the ability to respond nor- recognition memory; however, infusions of the M2 antagonist mally toward novel and familiar objects, as shown by normal per- methoctramine into PRh did not result in recognition memory def- formance in the perceptually easy conditions and at short delays; icits. The memory impairment caused by local administration of the these impairments were revealed only when the loads on percep- non-selective muscarinic receptor antagonist scopolamine resulted tual discrimination and retention across a delay were increased. in similar deficits caused by the same dose of pirenzepine. This find- Previous studies have shown that cortical acetylcholine (ACh) ing suggests that recognition memory impairments observed in can enhance perceptual processing in visual tasks (Furey et al., previous studies (Tang et al., 1997; Warburton et al., 2003; Winters

1997; Schon et al., 2005). ACh in the visual cortex enhances the sig- et al., 2006) were due primarily to blockade of M1 receptors. nal-to-noise ratio in visual information processing (Gu, 2003), and Although behavioral tasks examining M1 and M2 receptors have ACh has been shown to increase the strength of afferent input rel- often revealed contradictory results (Anagnostaras et al., 2003; e.g., ative to feedback in certain brain regions (Hasselmo, 2006; Has- Gerber et al., 2001; Miyakawa, Yamada, Duttaroy, & Wess, 2001), selmo & Bower, 1993; Hasselmo & McGaughy, 2004). Similarly it the literature on the roles of these receptors in synaptic plasticity has been suggested that ACh can ‘tune’ perceptual representations has been more consistent. Hippocampal long-term potentiation À/À (Murphy & Sillito, 1991; Sillito & Kemp, 1983). Disruption of such (LTP) is abnormal in M1 receptor knockout (M1R ) mice (Anag- processes may produce an impoverished representation that is nostaras et al., 2003). Furthermore, activating cholinergic receptors insufficiently robust to survive interference or decay, resulting in in hippocampal slices with carbachol enhances LTP of excitatory impaired subsequent memory performance (Winters, Bartko, Saks- synaptic transmission in mouse hippocampal slices but this effect ida, & Bussey, 2007; Winters et al., 2006). Likewise, such represen- is abolished in M1RÀ/À mice (Shinoe, Matsui, Taketo, & Manabe, tations could be insufficient for discrimination between similar 2005). Atropine, a non-specific muscarinic antagonist, suppresses representations, resulting in impairments in perceptual discrimi- the induction of LTP in vivo in the dentate gyrus, and pirenzepine nations or memory retention across a delay, when assessed under and AF-DX 116 suppress the amplitude of LTP (Luo et al., 2008). challenging conditions. Such an account may explain the effects of Cholinergic receptors have been specifically implicated in plas-

M1 antagonism on perceptual discrimination and object discrimi- ticity in PRh. Activation of muscarinic receptors in vitro with carba- nation shown in the present study. This explanation cannot, how- chol induces LTD in PRh, and scopolamine and pirenzepine reverse ever, be applied to the effects of M2 receptor antagonism, because this activation (Massey, Bhabra, Cho, Brown, & Bashir, 2001). The whereas intra-PRh infusions of the M2 antagonist AF-DX 116 im- role of ACh in PRh synaptic plasticity is likely related to its broader S.J. Bartko et al. / Neurobiology of Learning and Memory 110 (2014) 16–26 25 involvement in enhancing cortical information processing, which acetylcholine M1 muscarinic receptor mutant mice. Nature Neuroscience, could influence PRh-mediated object information acquisition. 6(1), 51–58. Araujo, D. M., Lapchak, P. A., Regenold, W., & Quirion, R. (1989). Long-term depression and LTP are likely involved in certain forms Characterization of [3H]AF-DX 116 binding sites in the rat brain: of PRh-dependent long-term object recognition memory (Barker Evidence for heterogeneity of muscarinic-M2 receptor sites. Synapse (New et al., 2006; Massey et al., 2001; Winters & Bussey, 2005a). York, N.Y.), 4(2), 106–114. Aura, J., Sirvio, J., & Riekkinen, P. Jr., (1997). Methoctramine moderately improves However, oddity discrimination, which does not tax long-term memory but pirenzepine disrupts performance in delayed non-matching to memory, might not be expected to require such plasticity mecha- position test. European Journal of Pharmacology, 333(2–3), 129–134. Bainbridge, N. K., Koselke, L. R., Jeon, J., Bailey, K. R., Wess, J., Crawley, J. N., et al. nisms. Because M2 receptors are thought to be presynaptic (2008). Learning and memory impairments in a congenic C57BL/6 strain of mice autoreceptors in at least some brain regions, blocking these recep- that lacks the M2 muscarinic acetylcholine receptor subtype. Behavioural Brain tors in these regions can increase the release of ACh. Therefore, if Research, 190(1), 50–58. Barker, G. R., Warburton, E. C., Koder, T., Dolman, N. P., More, J. C., Aggleton, J. ACh is important for a particular task, M2 receptor antagonism P., et al. (2006). The different effects on recognition memory of perirhinal might facilitate performance on that task. Somewhat paradoxi- kainate and NMDA glutamate receptor antagonism: Implications for cally, however, M2 receptor knockout mice display disrupted underlying plasticity mechanisms. Journal of Neuroscience, 26(13), hippocampal LTP and long-term memory performance, but these 3561–3566. deficits may be related to altered cholinergic–GABA-ergic interac- Bartko, S. J., Romberg, C., White, B., Wess, J., Bussey, T. J., & Saksida, L. M. (2011). Intact attentional processing but abnormal responding in M1 muscarinic tions (Bainbridge et al., 2008; Seeger et al., 2004). Thus, differential receptor-deficient mice using an automated touchscreen method. circuit dynamics underlying long-tem memory might necessitate Neuropharmacology, 61(8), 1366–1378. M receptor activation in some brain regions, but in cases where Bartko, S. J., Winters, B. D., Cowell, R. A., Saksida, L. M., & Bussey, T. J. (2007a). 2 Perceptual functions of perirhinal cortex in rats: Zero-delay object recognition LTP is not required, M2 blockade might be expected to have no ef- and simultaneous oddity discriminations. Journal of Neuroscience, 27(10), fect, or even a facilitatory effect due to the resulting increase of 2548–2559. ACh in the synapse. Accordingly, it is plausible that AF-DX 116 pro- Bartko, S. J., Winters, B. D., Cowell, R. A., Saksida, L. M., & Bussey, T. J. (2007b). Perirhinal cortex resolves feature ambiguity in configural object recognition duced impairments in object memory only when a long retention and perceptual oddity tasks. Learning & Memory, 14(12), 821–832. delay was used because LTP was required. Furthermore, because Burwell, R. D. (2001). Borders and cytoarchitecture of the perirhinal and postrhinal the oddity discrimination task probably does not require LTP, in- cortices in the rat. Journal of Comparative Neurology, 437(1), 17–41. Bussey, T. J., & Saksida, L. M. (2007). Memory, perception, and the ventral visual- tra-PRh infusions of AF-DX 116 would not necessarily be expected perirhinal-hippocampal stream: thinking outside of the boxes. Hippocampus, to disrupt performance in this task. Indeed, consistent with this ac- 17(9), 898–908. count, AF-DX 116 tended if anything to improve oddity discrimina- Caulfield, M. P., & Birdsall, N. J. (1998). International Union of Pharmacology. XVII. 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