Role of muscarinic Role of muscarinic M1 receptors in M1 receptors in

inhibitory inhibitory avoidance and avoidance and contextual fear contextual fear conditioningconditioning

Juliana Carlota Kramer Soares, Raquel Juliana Carlota Kramer Soares, Raquel Vecchio Fornari, Maria Gabriela Vecchio Fornari, Maria Gabriela

Menezes OliveiraMenezes Oliveira

Presented by Kristi TschetterPresented by Kristi Tschetter

Cholinergic SystemCholinergic System

Cholinergic receptors are nicotinic Cholinergic receptors are nicotinic and muscarinicand muscarinic

Involved in learning and memoryInvolved in learning and memory Important role in modulation of Important role in modulation of

aversively motivated tasksaversively motivated tasks Contextual fear conditioningContextual fear conditioning Inhibitory avoidanceInhibitory avoidance

Muscarinic ReceptorsMuscarinic Receptors

Include M1-M5Include M1-M5 M1 like include M1, M3, and M5M1 like include M1, M3, and M5 M2 like include M2 and M4M2 like include M2 and M4 Activation of M1 like receptors usually Activation of M1 like receptors usually

produces excitatory responses produces excitatory responses Activation of M2 like receptors usually Activation of M2 like receptors usually

produce inhibitory responsesproduce inhibitory responses Scopolamine is an antagonist to Scopolamine is an antagonist to

muscarinic receptorsmuscarinic receptors

Contextual Fear Contextual Fear Conditioning (CFC)Conditioning (CFC)

An aversive stimulus is presented (ex: An aversive stimulus is presented (ex: footshock) in a determined envoirmental footshock) in a determined envoirmental contextcontext

Later exposing the animal to the same Later exposing the animal to the same context might elicit a conditioned fear context might elicit a conditioned fear response often characterized by response often characterized by somatomotoric immobility (freezing)somatomotoric immobility (freezing)

Inhibitory Avoidance (IA)Inhibitory Avoidance (IA)

Instrumental conditioningInstrumental conditioning The animal is punished for a responseThe animal is punished for a response If the animal crosses from the light to If the animal crosses from the light to

the dark compartment he receives a the dark compartment he receives a footshockfootshock

The shock depends on the animals The shock depends on the animals response, and after the initial response, and after the initial experience the animal has the experience the animal has the possibility of avoiding the shockpossibility of avoiding the shock

ScopolamineScopolamine

Antagonist, non-selective for Antagonist, non-selective for muscarinic receptorsmuscarinic receptors

When it is administered before a When it is administered before a training session it interferes with training session it interferes with acquisition of both CFC and IAacquisition of both CFC and IA

The cholinergic system has a lack of The cholinergic system has a lack of selectivity of scopolamine, which selectivity of scopolamine, which suggests that the cholinergic system suggests that the cholinergic system shares a common mechanism with shares a common mechanism with CFC and IACFC and IA

M1 Muscarinic Subtype:M1 Muscarinic Subtype: Function in learning and memory processesFunction in learning and memory processes Widely distributed in the hippocampus, Widely distributed in the hippocampus,

cerebral cortex, and the amygdalacerebral cortex, and the amygdala Pirenzepine, biperiden, and trihexyphenidyl Pirenzepine, biperiden, and trihexyphenidyl

are selective M1 antagonists. are selective M1 antagonists. Administration of these selective Administration of these selective antagonists impairs the acquisition of IAantagonists impairs the acquisition of IA

When dicyclomine (antagonist of M1 When dicyclomine (antagonist of M1 receptors) is administered before training receptors) is administered before training both CFC and IA are impairedboth CFC and IA are impaired

Materials and Methods:Materials and Methods:

Wistar male rats, 3-4 months oldWistar male rats, 3-4 months old Controlled temperature 23 Controlled temperature 23 ± 2°C± 2°C 12:12h light-dark cycle12:12h light-dark cycle Food and water ad libitumFood and water ad libitum

DrugsDrugs Dicyclomine chloride dissolved in 0.9% saline Dicyclomine chloride dissolved in 0.9% saline

and injected i.p. in a volume of 1.0 ml/kgand injected i.p. in a volume of 1.0 ml/kg Doses used 16, 32, and 64 mg/kgDoses used 16, 32, and 64 mg/kg Maintained at 30Maintained at 30°C in a water bath°C in a water bath Control animals received 0.9% salineControl animals received 0.9% saline

IA Apparatus:IA Apparatus: 2 compartments connected 2 compartments connected

by a sliding doorby a sliding door Safe compartment-walls Safe compartment-walls

were whitewere white Compartment where Compartment where

animals received animals received footshock- walls where footshock- walls where black with a visual pattern, black with a visual pattern, 2 squares and 3 squares of 2 squares and 3 squares of white cardboardwhite cardboard

The tops were covered by The tops were covered by transparent acrylictransparent acrylic

The floor was a metal gridThe floor was a metal grid Footshocks 1mA and 1sec Footshocks 1mA and 1sec

long could be deliveredlong could be delivered

Open Field Apparatus:Open Field Apparatus:

80cm in diameter80cm in diameter Walls 30cm highWalls 30cm high Floor was divided Floor was divided

into three into three concentric circles concentric circles and subdivided by and subdivided by painted black lines painted black lines into 18 sectorsinto 18 sectors

Tone Fear Conditioning Tone Fear Conditioning (TFC)(TFC)

A white cylindrical A white cylindrical chamberchamber

35cm in diameter 35cm in diameter and 30cm highand 30cm high

Covered with Covered with transparent acrylictransparent acrylic

Floor made of Floor made of white acrylicwhite acrylic

Used a 60-dB toneUsed a 60-dB tone

Inhibitory Avoidance (IA) Inhibitory Avoidance (IA) Task:Task:

Performed in 2 sessions (training and test)Performed in 2 sessions (training and test) Training- animals placed individually inside the light Training- animals placed individually inside the light

(safe) compartment of the avoidance apparatus. 10 (safe) compartment of the avoidance apparatus. 10 seconds later the door was opened as soon as the seconds later the door was opened as soon as the animal entered the dark compartment with all four animal entered the dark compartment with all four paws the door was closed and 1 footshock was paws the door was closed and 1 footshock was delivered (1mA for 1sec). The latency of the animal delivered (1mA for 1sec). The latency of the animal to enter the dark side was recorded. Immediately to enter the dark side was recorded. Immediately after the footshock the animal was removed and after the footshock the animal was removed and returned to homecage.returned to homecage.

Test- 24h after training. Animal placed in light Test- 24h after training. Animal placed in light compartment. 10 sec later the door was opened. compartment. 10 sec later the door was opened. Latency for the animal to cross (all 4 paws in) to the Latency for the animal to cross (all 4 paws in) to the dark side was recorded. If the animal did not cross dark side was recorded. If the animal did not cross after 300 sec it was removed. No footshock was after 300 sec it was removed. No footshock was delivered during the test.delivered during the test.

Contextual Fear Contextual Fear Conditioning (CFC) TaskConditioning (CFC) Task

Carried out during 2 consecutive days.Carried out during 2 consecutive days. Day 1 Training: Animals placed individually in dark Day 1 Training: Animals placed individually in dark

compartment with sliding door closed during all compartment with sliding door closed during all CFC procedures. 2 min later, one footshock was CFC procedures. 2 min later, one footshock was delivered (1mA for 1sec). Immediately after the delivered (1mA for 1sec). Immediately after the animal was removed and returned to its homecage. animal was removed and returned to its homecage.

Day 2 Test: 24h after training, each animal is placed Day 2 Test: 24h after training, each animal is placed in the same context, dark side with closed door. in the same context, dark side with closed door. Testing occurred for 5min. No footshock was Testing occurred for 5min. No footshock was delivered. Freezing time of the animal was delivered. Freezing time of the animal was measured.measured.

Freezing-complete immobility of the animal, with Freezing-complete immobility of the animal, with the absence of vibrissae movements and sniffing.the absence of vibrissae movements and sniffing.

Locomotor Activity in Open Locomotor Activity in Open FieldField

Animals individually placed in the Animals individually placed in the open field for 5 min.open field for 5 min.

Total number of sectors that the Total number of sectors that the animal crossed were measured.animal crossed were measured.

Tone Fear Conditioning Tone Fear Conditioning (TFC) Task(TFC) Task

2 day conditioning procedure2 day conditioning procedure Day 1: Training-animals placed individually in Day 1: Training-animals placed individually in

black compartment with sliding door closed black compartment with sliding door closed (during all TFC procedures). 2 min later a tone (60-(during all TFC procedures). 2 min later a tone (60-dB, CS) sounded for 30sec and in the last sec a dB, CS) sounded for 30sec and in the last sec a footshock (1mA for 1sec, US) was delivered. The footshock (1mA for 1sec, US) was delivered. The animal was removed and returned to homecage.animal was removed and returned to homecage.

Day 2: Test-animal individually placed in the Day 2: Test-animal individually placed in the cylindrical chamber (new context) for 5min. At the cylindrical chamber (new context) for 5min. At the end of the 3end of the 3rdrd min of exposure to the apparatus, min of exposure to the apparatus, one tone (60-dB for 30sec) was presented. No one tone (60-dB for 30sec) was presented. No footshock was administered. Freezing was footshock was administered. Freezing was measured before and after tone presentation.measured before and after tone presentation.

Experiment 1: Effects of pre-training of Experiment 1: Effects of pre-training of dicyclomine on the acquisition of IA dicyclomine on the acquisition of IA

and CFCand CFC

Saline or dicyclomine (16 or 32 Saline or dicyclomine (16 or 32 mg/kg) was administered 30 min mg/kg) was administered 30 min before training on one of the tasksbefore training on one of the tasks

n=13-15 per group for each n=13-15 per group for each procedureprocedure

Figure 1Figure 1

Experiment 2: Effects of pre-Experiment 2: Effects of pre-training/pre-test administration of training/pre-test administration of

dicyclomine on IA and CFCdicyclomine 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 Examine if state-dependent learning occurs in

the presence of dicyclominethe presence of dicyclomine Sal/sal group: received saline before both Sal/sal group: received saline before both

training and testtraining and test Dic/sal group: received dicyclomine before Dic/sal group: received dicyclomine before

training and saline before the testtraining and saline before the test Sal/dic group: received saline before training and Sal/dic group: received saline before training and

dicyclomine before the testdicyclomine before the test Dic/dic group: received dicyclomine before both Dic/dic group: received dicyclomine before both

training and testtraining and test All injections were administered 30 min before All injections were administered 30 min before

training or test training or test

Figure 2Figure 2

Experiment 3: Effects of Experiment 3: Effects of administration of dicyclomine on administration of dicyclomine on locomotor activity and Tone fear locomotor activity and Tone fear

conditioningconditioning Used the same treatment as experiment Used the same treatment as experiment

2 with different animals to evaluate 2 with different animals to evaluate whether dicyclomine interferes with whether dicyclomine interferes with locomotionlocomotion Dose (saline or 32 mg/kg dicyclomine)Dose (saline or 32 mg/kg dicyclomine) Day 1: animals were returned to homecage Day 1: animals were returned to homecage

after injectionafter injection Day 2: 30 min after injection each rat was Day 2: 30 min after injection each rat was

placed in the open field and locomotor placed in the open field and locomotor activity was recorded for 5 minactivity was recorded for 5 min

Experiment 3 Continued:Experiment 3 Continued:

Used same treatment as experiment 2 Used same treatment as experiment 2 with different animals to evaluate with different animals to evaluate whether dicyclomine interferes with whether dicyclomine interferes with freezingfreezing Dose (saline or 32 mg/kg dicyclomine)Dose (saline or 32 mg/kg dicyclomine) Day 1: all animals received saline and after Day 1: all animals received saline and after

30 min each was placed into the TFC for 30 min each was placed into the TFC for trainingtraining

Day 2: each rat received saline or Day 2: each rat received saline or dicyclomine and after 30 min was placed in dicyclomine and after 30 min was placed in the TFC for testingthe TFC for testing

Experiment 4: Effects of post-Experiment 4: Effects of post-training administration of training administration of

dicyclomine on consolidation of IA dicyclomine on consolidation of IA and CFCand CFC

Administered saline or dicyclomine Administered saline or dicyclomine (16, 32 mg/kg) after training to (16, 32 mg/kg) after training to evaluate whether dicyclomine evaluate whether dicyclomine impairs the consolidation of IA and impairs the consolidation of IA and CFCCFC

Rats were tested 24h laterRats were tested 24h later

Figure 3Figure 3

DiscussionDiscussion

1.1. Both IA and CFC were impaired by Both IA and CFC were impaired by administering dicyclomine (16 and 32 administering dicyclomine (16 and 32 mg/kg)mg/kg)

2.2. Pre-training/pre-test treatment with Pre-training/pre-test treatment with dicyclomine affected both IA and CFCdicyclomine affected both IA and CFC

3.3. Pre-test treatment without pre-training Pre-test treatment without pre-training treatment with dicyclomine induced a treatment with dicyclomine induced a dissociation between the two tasks, dissociation between the two tasks, affecting the conditioned freezing affecting the conditioned freezing response but not the avoidance responseresponse but not the avoidance response

4.4. Post-training administration of Post-training administration of dicyclomine did not affect the tasksdicyclomine did not affect the tasks