article 2 Critical role of hippocampal muscarinic acetylcholine receptors on memory reconsolidation in mice

Question 1

abstract

article 2 notes

Answer 1

• Ach (plays an important role in mnemonic phenomena)
• The hippocampus is known to play a role in learning and memory
• Not clear: ach receptors contribute to this brain region’s role during memory rhetorical

ach receptors (two types)
* Ionotropic nicotinic acetylcholine
* Metabotropic muscarinic acetylcholine

2010:
* important role of hippocampal a7 nicotinic
* acetylcholine receptors in memory reconsolidation process of inhibitory avoidance in mice

present work:
* The authors are further investigating the implications of hippocampal muscarinic ach receptors (mAchRs)

• They are administrating agonists and antagonists of the different mAchRs subtypes in the hippocampus

FOUND:
* m1 and m2, but not m3 subtypes involved in memory reconsolidation

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Question 2

introduction

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Answer 2

memory consolidation:
* a process that occurs - - after a learning experience (memory trace and memory formation)

memory rehearsal:
* allows us to omit, incorporate, modify, obliterate
memory is not accurate but rather a dynamic process

after retrieval, memory can be destabilized (reformulated/ modified)
- this process was named memory reconsolidation
reconsolidation is a kinda of similar consolation but not identical
- Reconsolidation is not a recapitulation of memory consolidation

AcH plays an important role in mnemonic phenomena
- lesions of the cholinergic system or administration of anticholinergic drugs can cause memory impairments
- drugs that enhance cholinergic activity = enhance memory

memory reconsolidation can be impaired when ACH synthesis is disrupted by cv administration of HC-3 (inhibitor of HACU)
- HC-3: interfere with the rate limiting step

ach receptors (two types)
- Ionotropic nicotinic acetylcholine
- Metabotropic muscarinic acetylcholine

five different mAchRs are known (M1-M5)
- Can increase or inhibit neural activity
- Located post synaptically
- Promote neural transmission when bound to ACh

M1 and m3
* forebrain, hippocampus, cerebral cortex, corpus striatum, thalamus (m3 less expressed)

M5
* substantia nigra and VTA

M2
* pre- and post-synaptic site, reducing or inhibiting ACh-mediated activity
* Highly abundant in non-cholinergic neurons that project throughout the brain (hippocampus, neocortex

M4
* corpus striatim in CNS, prejunctional nerve terminals in the PNS

• A previous study in 2010: hippocampal α7 nicotinic acetylcholine receptors (α7nAchRs) are able to modulate memory reconsolidation of an inhibitory avoidance response in mice

Present study:
* investigate implications of hippocampus mAchRs
- mice administered agonists and antagonists of mAchRs different times after memory reticle
- Behavior responses were evaluated in retention tests

Results:
* suggested that mAchRs are involved in memory reconsolidation process depending on dose and strength of memory

Also studied:
- role of hippocampal role, m1, m2, m3 on memory reconsolidation process
- Finding that only m1 and m2, but not m3 have specific processes

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Question 3

materials and method

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Answer 3

subjects
- cf-1 male mice, 60-70 days old, 25-30g
- housed in groups of 10 in stainless-steel cages
- 12-hour light-dark cycle, temperature regulated (23-25°c)
- experiments conducted between 08:00-14:00h

drugs
- oxotremorine sesquifumarate (oxo)
- scopolamine hydrochloride (sco)
- pirenzepine
- solifenacine
- all dissolved in sterile saline solution immediately before use

inhibitory avoidance task
- one-trial learning, step-through type situation
- dark compartment (20x20x15 cm) with steel grid floor
- small illuminated platform (5x5 cm) attached to front
- two footshock training conditions:
low footshock (lfs): 0.3 ma, 50 hz, 3 sec
high footshock (hfs): 0.4 ma, 50 hz, 3 sec
- retention test: measure step-through latency (max 300 sec)

intra-dorsal-hippocampal (dhpc) injections:
- surgery under light ether anesthesia using stereotaxic instrument
- coordinates: ap: -1.90 mm, l/r: ±1.50 mm, dv: -1.2 mm
- bilateral infusions via 30-gauge needle
- 0.5 μl volume per injection
- accuracy verified by histological examination

experimental design
- various experiments conducted manipulating:
drug type and dose
timing of drug administration (immediate vs delayed)
presence/absence of memory reactivation
training condition (lfs vs hfs)
- multiple control experiments to verify specificity of effects
- sample sizes generally 8-10 mice per group
- replication of key experiments

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Question 4

Experimental Groups

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Answer 4

Oxotremorine (OXO) and Scopolamine (SCO) Effects:
* 8 groups of 5 mice each (replicated for 10/group)
* Half trained with low footshock (LFS), half with high footshock (HFS)
* 48 hours post-training: First retention test (T1)
* Immediately after T1: Bilateral dHPC infusion of saline or OXO (1.00-10.00 ng/hippocampus)
* 24 hours later: Second retention test (T2)
* Similar experiment with SCO (1.00-10.00 μg/hippocampus)

Control Experiments:
* No Reactivation: 6 groups (3 LFS, 3 HFS), 5 mice each
* 48 hours post-training: Saline, OXO (3.0 or 10.0 ng), or SCO (10.0 μg) without T1
* 24 hours later: Retention test (T2)
* Delayed Infusion: Same as main experiment, but drug infusions 3 hours after T1

OXO and SCO Interaction:
* 7 groups of 5 mice each (replicated for 10/group), HFS training
* T1 48 hours post-training
* Immediately after T1: Saline, b) SCO (10.0 μg/hippocampus), c) SCO (10.0 μg) + OXO (0.3-30.0 ng) cocktail
* 24 hours later: T2
* Similar experiment with OXO (3.0 ng) + SCO (3-30 μg) cocktail

Specific mAChRs Antagonists:
* Pirenzepine (M1 antagonist), AF-DX116 (M2 antagonist), Solifenacine (M3 antagonist)
* 4 groups of 10 mice each for each antagonist, HFS training
* T1 48 hours post-training
* Immediately after T1: Saline or antagonist at various doses
* 24 hours later: T2
* For Pirenzepine and AF-DX116: Additional test 21 days post-T1 (T3)

LFS Condition for Pirenzepine and AF-DX116:
* Similar to HFS experiments, but with LFS training

Control Experiments for Pirenzepine and AF-DX116:
* No Reactivation: 6 groups (3 LFS, 3 HFS), 5 mice each (replicated for 10/group)
* 48 hours post-training: Saline, Pirenzepine (10.0 μg), or AF-DX116 (3.5 μg) without T1
* 24 hours later: T2
* Delayed Infusion: Same as main experiment, but drug infusions 3 hours after T1

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Question 5

results

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Answer 5

Oxotremorine (OXO) Effects:
* Low Footshock (LFS): Enhanced retention performance at 10.0 ng/hippocampus
* High Footshock (HFS): Impaired retention performance at 3.0 ng/hippocampus
* Dose-dependent effects in both cases

Scopolamine (SCO) Effects:
* Impaired retention performance in both LFS and HFS conditions
* Effective dose: 10.0 μg/hippocampus in both conditions
* Dose-dependent effects

Specificity to Reconsolidation:
* No effect when OXO or SCO administered without memory reactivation
* No effect when administered 3 hours after reactivation
* Indicates effects are specific to reconsolidation time window

OXO and SCO Interaction:
* SCO effects reversed by increasing doses of OXO
* OXO effects reversed by increasing doses of SCO
* Suggests interaction at mAChR level

Specific mAChR Antagonists (HFS condition):
* Pirenzepine (M1 antagonist): Impaired retention performance dose-dependently
* AF-DX116 (M2 antagonist): Impaired retention performance dose-dependently
* Solifenacine (M3 antagonist): No significant effect
* Effects of Pirenzepine and AF-DX116 persisted for at least 21 days

Specific mAChR Antagonists (LFS condition):
* Pirenzepine: Impaired retention performance dose-dependently
* AF-DX116: Enhanced retention performance dose-dependently (inverted U-shape curve)

Control Experiments for Pirenzepine and AF-DX116:
* No effect when administered without memory reactivation
* No effect when administered 3 hours after reactivation
* Confirms specificity to reconsolidation processes

Anatomical Control:
* No effect when OXO or SCO injected into primary somatosensory cortex
* Confirms site-specific effect in hippocampus

Key Findings:
* Hippocampal mAChRs play a critical role in memory reconsolidation
* Effects depend on training conditions (LFS vs HFS)
* M1 and M2 subtypes, but not M3, are involved in reconsolidation
* Effects are specific to the reconsolidation time window and to the hippocampus
* mAChR agonists and antagonists can bidirectionally modulate reconsolidation, depending on dosage and training conditions

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Question 6

discussion

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Answer 6

Memory Reconsolidation Process:
* Allows for updating content, strength, and expectations of stored memories
* Involves memory destabilization and re-stabilization
* Dependent on factors like training strength, memory age, and mismatch between expectation and realitY

Role of Acetylcholine (ACh):
* Essential for various mnemonic phenomena
* Cholinergic system lesions or anticholinergic drugs impair memory
* Enhancing cholinergic activity can improve memoRY

Muscarinic Acetylcholine Receptors (mAChRs):
* Five subtypes (M1-M5) with different locations and functions
* M1, M3, M5 are postsynaptic and promote neural transmission
* M2, M4 are presynaptic and postsynaptic, acting as auto-receptors to regulate synaptic activitY

Main Findings:
* Oxotremorine (OXO, mAChR agonist) enhanced or impaired reconsolidation depending on training conditions
* Scopolamine (SCO, mAChR antagonist) impaired reconsolidation regardless of training conditions
* Effects were specific to the reconsolidation time window and dependent on memory reactivation

Specific mAChR Subtypes:
* M1 and M2, but not M3, were involved in reconsolidation
* Pirenzepine (M1 antagonist) impaired reconsolidation in both LFS and HFS conditions
* AF-DX116 (M2 antagonist) had bidirectional effects depending on training conditions

Interpretation of Results:
* OXO effects similar to those observed in consolidation studies (Gold & Van Buskirk, 1976)
* Suggests post-retrieval treatments modulate memory processes similar to, but not identical to, those after learning
* Inverted U-shape curve for AF-DX116 in LFS condition typical of neuromodulatory effects

Clinical Implications:
* Findings relevant to understanding cognitive impairments in schizophrenia and other disorders
* Potential for developing new therapeutic approaches targeting mAChRs

Limitations and Future Directions:
* Study focused on acute drug administration
* Need for further investigation of mechanisms underlying mAChR involvement in reconsolidation
* Potential for exploring therapeutic applications in neurodegenerative and psychiatric diseases

Conclusion:
* Hippocampal M1 and M2 mAChRs play a critical role in memory reconsolidation
* Effects depend on training conditions and specific receptor subtypes
* Findings open new avenues for understanding memory processes and developing potential treatments for memory-related disorders

article 2 notes

Question 7

What is the main focus of this study?

Answer 7

The critical role of hippocampal muscarinic acetylcholine receptors (mAChRs) in memory reconsolidation in mice.

Question 8

What are the two main types of acetylcholine receptors?

Answer 8

Nicotinic receptors (nAChRs) - ligand-gated ion channels

Muscarinic receptors (mAChRs) - seven-transmembrane G-protein-coupled receptors

Question 9

What behavioral task was used to assess memory in this study?

Answer 9

Inhibitory avoidance (IA) task, a one-trial learning, step-through type situation

Question 10

How did Oxotremorine (OXO) affect memory reconsolidation in mice trained with low footshock (LFS)

Answer 10

OXO enhanced retention performance at 10.0 ng/hippocampus in mice trained with LFS.

Question 11

What is the clinical relevance of this study’s findings

Answer 11

The results may have implications for developing new therapeutic approaches for cognitive impairments in neurodegenerative and psychiatric diseases, particularly those involving muscarinic receptors.

Question 12

What acetylcholine receptor subtypes were investigated in this study?

Answer 12

The study investigated muscarinic acetylcholine receptors (mAChRs), specifically M1, M2, and M3 subtypes.

Question 13

What was the main finding regarding M1 and M2 receptors?

Answer 13

M1 and M2, but not M3 subtypes, were found to be involved in memory reconsolidation processes in mice.

Question 14

What is memory reconsolidation?

Answer 14

A process where memories become labile after retrieval and can be modified before being re-stabilized.

Question 15

How many different mAChR subtypes are known?

Answer 15

Five different mAChRs are known (M1-M5).

Question 16

Where are M1-like mAChRs (M1, M3, M5) primarily located?

Answer 16

They are located postsynaptically, promoting neural transmission when bound to ACh.

Question 17

What strain and sex of mice were used in the study?

Answer 17

CF-1 male mice.

Question 18

What were the two footshock training conditions used?

Answer 18

Low footshock (LFS) of 0.3 mA and high footshock (HFS) of 0.4 mA.

Question 19

How were drugs administered to the mice?

Answer 19

Through bilateral intra-dorsal-hippocampal (dHPC) injections.

Question 20

How did Scopolamine (SCOP) affect memory reconsolidation?

Answer 20

SCOP impaired retention performance regardless of training conditions (LFS or HFS).

Question 21

What effect did Pirenzepine (M1 antagonist) have on memory reconsolidation?

Answer 21

Pirenzepine impaired retention performance in both LFS and HFS conditions.

Question 22

How did AF-DX116 (M2 antagonist) affect mice trained with LFS?

Answer 22

AF-DX116 enhanced retention performance in a dose-related manner, showing an inverted U-shape curve.

Question 23

What does the inverted U-shape curve typically indicate in neuropharmacological studies?

Answer 23

It typically indicates neuromodulatory effects, where moderate doses have optimal effects while higher doses may be less effective or even detrimental.

Question 24

How might these findings be relevant to schizophrenia research?

Answer 24

Post-mortem studies in schizophrenia patients have shown lower expression of M1 and M4 receptors in brain regions linked to cognitive function.

Question 25

What is a key limitation of this study mentioned in the discussion?

Answer 25

The study focused on acute drug administration, and further investigation of mechanisms underlying mAChR involvement in reconsolidation is needed.

Question 26

What potential clinical applications are suggested by this research?

Answer 26

The findings could lead to new therapeutic approaches for cognitive impairments in neurodegenerative and psychiatric diseases, particularly those involving muscarinic receptors.

Question 27

Describe the main findings regarding the roles of M1 and M2 muscarinic receptor subtypes in memory reconsolidation processes.

Answer 27

Main findings regarding M1 and M2 muscarinic receptor subtypes in memory reconsolidation: - M1 antagonist (Pirenzepine) impaired retention performance in both LFS and HFS conditions. - M2 antagonist (AF-DX116) had different effects depending on training conditions: In LFS: enhanced retention performance in a dose-dependent manner In HFS: impaired retention performance - Both M1 and M2, but not M3, were found to be involved in memory reconsolidation processes.

Question 28

Explain the experimental design used to test whether the effects of Oxotremorine and Scopolamine were specific to memory reactivation processes.

Answer 28

Experimental design to test specificity of Oxotremorine and Scopolamine effects: - Control experiments were conducted where drugs were administered: a) In the absence of memory reactivation (48 hours after training, without T1 session) b) 3 hours after memory reactivation (outside the reconsolidation time window) - If effects were only observed when drugs were given immediately after reactivation, this would indicate specificity to reconsolidation processes.

Question 30

How did the effects of AF-DX116 (M2 antagonist) differ between mice trained with low footshock (LFS) and high footshock (HFS) conditions?

Answer 30

Differences in AF-DX116 effects between LFS and HFS conditions: LFS: AF-DX116 enhanced retention performance in a dose-dependent manner, showing an inverted U-shape curve. HFS: AF-DX116 impaired retention performance.

Question 31

What evidence from the study suggests that the effects of Pirenzepine and AF-DX116 on memory reconsolidation were time-dependent?

Answer 31

Evidence for time-dependent effects of Pirenzepine and AF-DX116: - When administered 3 hours after memory reactivation, neither drug affected retention performance. - Effects were only observed when drugs were given immediately after reactivation, suggesting a specific time window for reconsolidation processes.

Question 32

Compare and contrast the effects of Oxotremorine on memory reconsolidation in mice trained with low footshock versus high footshock conditions.

Answer 32

- LFS: Oxotremorine enhanced retention performance at 10.0 ng/hippocampus. - HFS: Oxotremorine impaired retention performance at 3.0 ng/hippocampus. - This demonstrates that the drug's effects depend on the strength of the original memory.

Question 33

Key findings regarding the M3 muscarinic receptor subtype:

Answer 33

The M3 antagonist Solifenacine did not affect retention performance at any dose tested. This suggests that M3 receptors are not critically involved in memory reconsolidation processes in this paradigm.

Question 34

Implications for understanding and treating cognitive impairments in schizophrenia:

Answer 34

- Post-mortem studies in schizophrenia patients have shown lower expression of M1 and M4 receptors in brain regions linked to cognitive function. - This research provides insight into how M1 and M2 receptors modulate memory processes. - Understanding these mechanisms could lead to new therapeutic approaches for cognitive symptoms in schizophrenia. - The study suggests that targeting specific mAChR subtypes might be a promising avenue for developing treatments for cognitive impairments in schizophrenia and other disorders.