1. Role of muscarinic M1 receptors in inhibitory avoidance and contextual fear conditioning Juliana Carlota Kramer Soares, Raquel Vecchio Fornari, Maria Gabriela Menezes Oliveira Presented by Kristi Tschetter

2. Cholinergic System Cholinergic receptors are nicotinic and muscarinic Cholinergic receptors are nicotinic and muscarinic Involved in learning and memory Involved in learning and memory Important role in modulation of aversively motivated tasks Important role in modulation of aversively motivated tasks Contextual fear conditioning Contextual fear conditioning Inhibitory avoidance Inhibitory avoidance

3. Muscarinic Receptors Include M1-M5 Include M1-M5 M1 like include M1, M3, and M5 M1 like include M1, M3, and M5 M2 like include M2 and M4 M2 like include M2 and M4 Activation of M1 like receptors usually produces excitatory responses Activation of M1 like receptors usually produces excitatory responses Activation of M2 like receptors usually produce inhibitory responses Activation of M2 like receptors usually produce inhibitory responses Scopolamine is an antagonist to muscarinic receptors Scopolamine is an antagonist to muscarinic receptors

4. Contextual Fear Conditioning (CFC) An aversive stimulus is presented (ex: footshock) in a determined envoirmental context An aversive stimulus is presented (ex: footshock) in a determined envoirmental context Later exposing the animal to the same context might elicit a conditioned fear response often characterized by somatomotoric immobility (freezing) Later exposing the animal to the same context might elicit a conditioned fear response often characterized by somatomotoric immobility (freezing)

5. Inhibitory Avoidance (IA) Instrumental conditioning Instrumental conditioning The animal is punished for a response The animal is punished for a response If the animal crosses from the light to the dark compartment he receives a footshock If the animal crosses from the light to the dark compartment he receives a footshock The shock depends on the animals response, and after the initial experience the animal has the possibility of avoiding the shock The shock depends on the animals response, and after the initial experience the animal has the possibility of avoiding the shock

6. Scopolamine Antagonist, non-selective for muscarinic receptors Antagonist, non-selective for muscarinic receptors When it is administered before a training session it interferes with acquisition of both CFC and IA When it is administered before a training session it interferes with acquisition of both CFC and IA The cholinergic system has a lack of selectivity of scopolamine, which suggests that the cholinergic system shares a common mechanism with CFC and IA The cholinergic system has a lack of selectivity of scopolamine, which suggests that the cholinergic system shares a common mechanism with CFC and IA

7. M1 Muscarinic Subtype: Function in learning and memory processes Function in learning and memory processes Widely distributed in the hippocampus, cerebral cortex, and the amygdala Widely distributed in the hippocampus, cerebral cortex, and the amygdala Pirenzepine, biperiden, and trihexyphenidyl are selective M1 antagonists. Administration of these selective antagonists impairs the acquisition of IA Pirenzepine, biperiden, and trihexyphenidyl are selective M1 antagonists. Administration of these selective antagonists impairs the acquisition of IA When dicyclomine (antagonist of M1 receptors) is administered before training both CFC and IA are impaired When dicyclomine (antagonist of M1 receptors) is administered before training both CFC and IA are impaired

8. Materials and Methods: Wistar male rats, 3-4 months old Wistar male rats, 3-4 months old Controlled temperature 23 ± 2°C Controlled temperature 23 ± 2°C 12:12h light-dark cycle 12:12h light-dark cycle Food and water ad libitum Food and water ad libitum Drugs Drugs Dicyclomine chloride dissolved in 0.9% saline and injected i.p. in a volume of 1.0 ml/kg Dicyclomine chloride dissolved in 0.9% saline and injected i.p. in a volume of 1.0 ml/kg Doses used 16, 32, and 64 mg/kg Doses used 16, 32, and 64 mg/kg Maintained at 30°C in a water bath Maintained at 30°C in a water bath Control animals received 0.9% saline Control animals received 0.9% saline

9. IA Apparatus: 2 compartments connected by a sliding door 2 compartments connected by a sliding door Safe compartment-walls were white Safe compartment-walls were white Compartment where animals received footshock- walls where black with a visual pattern, 2 squares and 3 squares of white cardboard Compartment where animals received footshock- walls where black with a visual pattern, 2 squares and 3 squares of white cardboard The tops were covered by transparent acrylic The tops were covered by transparent acrylic The floor was a metal grid The floor was a metal grid Footshocks 1mA and 1sec long could be delivered Footshocks 1mA and 1sec long could be delivered

10. Open Field Apparatus: 80cm in diameter 80cm in diameter Walls 30cm high Walls 30cm high Floor was divided into three concentric circles and subdivided by painted black lines into 18 sectors Floor was divided into three concentric circles and subdivided by painted black lines into 18 sectors

11. Tone Fear Conditioning (TFC) A white cylindrical chamber A white cylindrical chamber 35cm in diameter and 30cm high 35cm in diameter and 30cm high Covered with transparent acrylic Covered with transparent acrylic Floor made of white acrylic Floor made of white acrylic Used a 60-dB tone Used a 60-dB tone

12. Inhibitory Avoidance (IA) Task: Performed in 2 sessions (training and test) Performed in 2 sessions (training and test) Training- animals placed individually inside the light (safe) compartment of the avoidance apparatus. 10 seconds later the door was opened as soon as the animal entered the dark compartment with all four paws the door was closed and 1 footshock was delivered (1mA for 1sec). The latency of the animal to enter the dark side was recorded. Immediately after the footshock the animal was removed and returned to homecage. Training- animals placed individually inside the light (safe) compartment of the avoidance apparatus. 10 seconds later the door was opened as soon as the animal entered the dark compartment with all four paws the door was closed and 1 footshock was delivered (1mA for 1sec). The latency of the animal to enter the dark side was recorded. Immediately after the footshock the animal was removed and returned to homecage. Test- 24h after training. Animal placed in light compartment. 10 sec later the door was opened. Latency for the animal to cross (all 4 paws in) to the dark side was recorded. If the animal did not cross after 300 sec it was removed. No footshock was delivered during the test. Test- 24h after training. Animal placed in light compartment. 10 sec later the door was opened. Latency for the animal to cross (all 4 paws in) to the dark side was recorded. If the animal did not cross after 300 sec it was removed. No footshock was delivered during the test.

13. Contextual Fear Conditioning (CFC) Task Carried out during 2 consecutive days. Carried out during 2 consecutive days. Day 1 Training: Animals placed individually in dark compartment with sliding door closed during all CFC procedures. 2 min later, one footshock was delivered (1mA for 1sec). Immediately after the animal was removed and returned to its homecage. Day 1 Training: Animals placed individually in dark compartment with sliding door closed during all CFC procedures. 2 min later, one footshock was delivered (1mA for 1sec). Immediately after the animal was removed and returned to its homecage. Day 2 Test: 24h after training, each animal is placed in the same context, dark side with closed door. Testing occurred for 5min. No footshock was delivered. Freezing time of the animal was measured. Day 2 Test: 24h after training, each animal is placed in the same context, dark side with closed door. Testing occurred for 5min. No footshock was delivered. Freezing time of the animal was measured. Freezing-complete immobility of the animal, with the absence of vibrissae movements and sniffing. Freezing-complete immobility of the animal, with the absence of vibrissae movements and sniffing.

14. Locomotor Activity in Open Field Animals individually placed in the open field for 5 min. Animals individually placed in the open field for 5 min. Total number of sectors that the animal crossed were measured. Total number of sectors that the animal crossed were measured.

15. Tone Fear Conditioning (TFC) Task 2 day conditioning procedure 2 day conditioning procedure Day 1: Training-animals placed individually in black compartment with sliding door closed (during all TFC procedures). 2 min later a tone (60-dB, CS) sounded for 30sec and in the last sec a footshock (1mA for 1sec, US) was delivered. The animal was removed and returned to homecage. Day 1: Training-animals placed individually in black compartment with sliding door closed (during all TFC procedures). 2 min later a tone (60-dB, CS) sounded for 30sec and in the last sec a footshock (1mA for 1sec, US) was delivered. The animal was removed and returned to homecage. Day 2: Test-animal individually placed in the cylindrical chamber (new context) for 5min. At the end of the 3 rd min of exposure to the apparatus, one tone (60-dB for 30sec) was presented. No footshock was administered. Freezing was measured before and after tone presentation. Day 2: Test-animal individually placed in the cylindrical chamber (new context) for 5min. At the end of the 3 rd min of exposure to the apparatus, one tone (60-dB for 30sec) was presented. No footshock was administered. Freezing was measured before and after tone presentation.

16. Experiment 1: Effects of pre-training of dicyclomine on the acquisition of IA and CFC Saline or dicyclomine (16 or 32 mg/kg) was administered 30 min before training on one of the tasks Saline or dicyclomine (16 or 32 mg/kg) was administered 30 min before training on one of the tasks n=13-15 per group for each procedure n=13-15 per group for each procedure

17. Figure 1

18. Experiment 2: Effects of pre-training/pre-test administration of dicyclomine on IA and CFC Four groups of rats (n=9-11) Four groups of rats (n=9-11) Examine if state-dependent learning occurs in the presence of dicyclomine Examine if state-dependent learning occurs in the presence of dicyclomine Sal/sal group: received saline before both training and test Sal/sal group: received saline before both training and test Dic/sal group: received dicyclomine before training and saline before the test Dic/sal group: received dicyclomine before training and saline before the test Sal/dic group: received saline before training and dicyclomine before the test Sal/dic group: received saline before training and dicyclomine before the test Dic/dic group: received dicyclomine before both training and test Dic/dic group: received dicyclomine before both training and test All injections were administered 30 min before training or test All injections were administered 30 min before training or test

19. Figure 2

20. Experiment 3: Effects of administration of dicyclomine on locomotor activity and Tone fear conditioning Used the same treatment as experiment 2 with different animals to evaluate whether dicyclomine interferes with locomotion Used the same treatment as experiment 2 with different animals to evaluate whether dicyclomine interferes with locomotion Dose (saline or 32 mg/kg dicyclomine) Dose (saline or 32 mg/kg dicyclomine) Day 1: animals were returned to homecage after injection Day 1: animals were returned to homecage after injection Day 2: 30 min after injection each rat was placed in the open field and locomotor activity was recorded for 5 min Day 2: 30 min after injection each rat was placed in the open field and locomotor activity was recorded for 5 min

21. Experiment 3 Continued: Used same treatment as experiment 2 with different animals to evaluate whether dicyclomine interferes with freezing Used same treatment as experiment 2 with different animals to evaluate whether dicyclomine interferes with freezing Dose (saline or 32 mg/kg dicyclomine) Dose (saline or 32 mg/kg dicyclomine) Day 1: all animals received saline and after 30 min each was placed into the TFC for training Day 1: all animals received saline and after 30 min each was placed into the TFC for training Day 2: each rat received saline or dicyclomine and after 30 min was placed in the TFC for testing Day 2: each rat received saline or dicyclomine and after 30 min was placed in the TFC for testing

22. Experiment 4: Effects of post-training administration of dicyclomine on consolidation of IA and CFC Administered saline or dicyclomine (16, 32 mg/kg) after training to evaluate whether dicyclomine impairs the consolidation of IA and CFC Administered saline or dicyclomine (16, 32 mg/kg) after training to evaluate whether dicyclomine impairs the consolidation of IA and CFC Rats were tested 24h later Rats were tested 24h later

23. Figure 3

24. Discussion 1. Both IA and CFC were impaired by administering dicyclomine (16 and 32 mg/kg) 2. Pre-training/pre-test treatment with dicyclomine affected both IA and CFC 3. Pre-test treatment without pre-training treatment with dicyclomine induced a dissociation between the two tasks, affecting the conditioned freezing response but not the avoidance response 4. Post-training administration of dicyclomine did not affect the tasks