Impact of Acute Injection of M108 into the Hippocampus on CFC

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Long-Term Synaptic Plasticity Recordings

Before application of long-term induction protocols, a stable baseline of 20 min was recorded. As mentioned above, slices were bathed in aCSF either in control condition, or with the peptide. Picrotoxin (50 μM) was added depending on the protocol. Throughout the recordings for long-term plasticity, the solution was recirculated. We looked at different forms of long-term plasticity: LTP, LTD and an intermediate LTD stage called sub-LTD, which in control conditions is not sufficient to induce an LTD response.
LTP was induced by a high frequency stimulation protocol consisting of 2 pulses at 100 Hz for 1 sec with a 20 sec inter stimulus interval (ISI).
Sub-LTD was induced by low frequency stimulation consisting of 300 pulses at 1 Hz. LTD was induced with a train of 900 pulses of 1 Hz in presence of 50 μM Picrotoxin. After induction, recording continued for an hour to observe the long-term synaptic plasticity changes and compare alterations in response between control conditions and presence of the peptide or in mutant conditions. The first third of the fEPSP slope was calculated for analysis of fEPSPs. The time courses of LTP and LTD were obtained by normalizing each experiment to the average value of all points of the last 20 min stable baseline before induction. In bar graphs, LTP or LTD magnitude was measured during the last 15 min of recording (45–60 min after induction) and calculated as percentage change fEPSP slope from baseline average. Recordings alternated between control and testing conditions throughout the day for acute effects with sAƞ-α or between days for testing the MISEPA2 line.

Short-Term Synaptic Plasticity Recordings

To test for alterations in short-term synaptic plasticity we applied three different protocols to look at different facets of short-term plasticity.

Paired-Pulse Ratio

The paired-pulse ratio (PPR) characterizes alterations for the probability of release at the pre-synapse. The PPR protocol consists of two stimuli delivered ranging from 100 to 400 ms ISI, if not stated otherwise in Results. PPRs were calculated as fEPSP2slope/fEPSP1slope (10 sweeps average per ISI).

Synaptic Fatigue

Synaptic fatigue exploits the fact that if stimulation is occurring at a high enough frequency, the neurotransmitter release will be at a faster rate than the re-uptake cycle ultimately leading to reduced transmitter release, a characteristic of synaptic fatigue. Thus, synaptic fatigue can measure alterations in release probability and neurotransmitter depot under drug the different conditions. Synaptic fatigue was measured via a stimulation protocol consisting of a train of 15 pulses at 40 Hz. Measurements of all fEPSPs were normalized to the first fEPSP of the train for statistical analysis.

Input/ Output

The Input/ Output (I/O) curve protocol measures basal synaptic transmission. We controlled the pre-synaptic input by setting the fiber volley (FV) amplitude and measured the post-synaptic response to this input. The I/O curves were generated by calculating the initial slope of the fEPSP to avoid population spike contamination with a corresponding FV amplitude ranging from 0.1 to 0.4 mV or 0.1 to 0.3 mV in increments of 0.1 mV measuring 10 sweeps average. The protocol was performed twice, first under aCSFI condition and then again after slices were perfused for 20 min in aCSFI with peptide or, serving as within subject control, in aCSFI without peptide. Recordings of control and peptide conditions for all experiments were interleaved within the same day.

Microscopy imaging for injection side in bilateral hippocampal injections

To be sure that injection of sAƞ-α reached target region, injection sites were verified post-mortem after termination of behavioral experiments. Mice were killed by cervical dislocation and immediately their whole brain harvested as described above. Great attention was directed towards harvesting the whole brain without damage of tissue. Brains were stored overnight in 4 % formaldehyde (PFA) solution for tissue fixation. The next day solution was exchanged to 0.1 M Phosphate buffer (PB) solution and brains kept cold in a fridge until further processing.
Brains were precut in a mouse brain mold and sliced at 40 μm with a vibratome (Microm HM600V, Thermo Scientific) in cold 0.1 M PB solution. After mounting brain slices on microscope slides, images were taken with an DMD108 Leica microscope to verify injection site.

Blue Evans Staining to Verify Correct Placement of Canula Guides and Distribution after Injection

Blue Evans dye was the first method used to verify the correct distribution of M108 in the brain and ventricle via the cannula guides to the designated area, either hippocampal CA1 region or right ventricle (see Figure 25).
Mice were anesthetized with Ketamine/Xylazine i.p. (150 μl/ 20 gr bodyweight: 750 μl Ketamine and 250 μl Xylazine in 4 ml saline) injection as preparation for the following perfusion and connected to the injector. 0.3 μl blue Evans at 0.2 μl/ min was injected. This lower volume compared to M108 injections in the actual experiments is due to experience in our laboratory, as blue Evans tends to distribute quickly.

Perfusion Surgery

Before starting perfusion surgery, we confirmed surgical plane of anesthesia by use of toe pinch-response method. Perfusion was performed under the hood due to the toxic 4 % PFA solution used.
Both the 4 % PFA and 1x phosphate buffered saline (PBS) (PBSx10 Euromedex diluted in H2O) acting as perfusion buffer are stored on ice, the fixative tubing connected to a pump (Drifton) was placed in the perfusion buffer container and carefully filled to avoid air bubbles and a fresh needle (AGANITMNEEDLE 26Gx ½” (0.45x 13 mm), Terumo®) was placed on the outlet and put aside until needed.
Animals were placed on a polyester platform and pinned down with four needles into the limbs, stretching the body out. A small lateral incision just beneath the rib cage cutting through the integument and abdominal wall was made. The diaphragm was cut, and the incision further enlarged by cutting carefully along the rib cage up to the collarbone on both sides to expose the pleural cavity. The sternum was lifted away and fixed next to the head with a needle. The exposed heart was carefully freed from connective tissue. Blunt tweezers were used to position the heart for needle insertion. The needle connected to the fixation tubing was inserted into the posterior end of the left ventricle taking care to not damage the heart (Figure 26). Finally, while keeping the needle stable, an incision to the right atrium using iris scissors was made to create an outlet.

Table of contents :

Summary
Index of Figures
Index of Tables
Abbreviations
1. Introduction
Alzheimer’s Disease and Amyloid Precursor Protein processing
1.1.1 The Amyloid Precursor Protein (APP)
1.1.2 Amyloidogenic Pathway
1.1.2.1 ß-Secretase
1.1.2.2 Amyloid ß
1.1.3 Non-Amyloidogenic Pathway
1.1.3.1 α -Secretase
1.1.3.2 P3
1.1.4 γ-Secretase
1.1.5 η-Secretase Pathway
1.1.5.1 η -Secretase
1.1.5.2 Amyloid η-α and Aη-β
1.1.6 Soluble APP
1.1.6.1 sAPP-α
1.1.6.2 sAPPβ
1.1.6.3 sAPP-η
1.1.7 Carboxyl-Terminal Fragment
1.1.7.1 AICD
1.1.8 Other APP Processing Pathways
The Hippocampus
1.2.1 Anatomy of the Hippocampus
1.2.2 Functional Role of the Hippocampus
1.2.3 Hippocampus and Memory
1.2.3.1 Definition of Memory
1.2.3.2 Memory formation
1.2.3.3 Working memory
1.2.4 Spatial Memory
1.2.5 Anxiety
1.2.6 An Object to Study in Neurobiology
Methods to Study Physio-Pathological Role of Proteins
1.3.1 Electrophysiology
1.3.1.1 The Synapse
1.3.1.1.1 Glutamate
1.3.1.1.1.1 mGluRs
1.3.1.1.1.2 iGluR
1.3.1.1.2 AMPAR
1.3.1.1.3 NMDAR
1.3.1.2 Long-Term Synaptic Plasticity
1.3.1.2.1 LTP
1.3.1.2.1.1 Mechanism of LTP
1.3.1.2.2 LTD
1.3.1.2.2.1 Mechanisms of LTD
1.3.1.3 Short-Term Synaptic Plasticity
1.3.1.3.1 Facilitation
1.3.1.3.2 Depression
1.3.1.4 Synaptic plasticity reflects behavioral outcome
1.3.2 Behavioral Studies
1.3.2.1 Experimental Design
1.3.2.1.1 Husbandry
1.3.2.2 Behavior
1.3.2.2.1 Spatial Navigation Tasks
1.3.2.2.2 Aversive Learning
1.3.2.2.3 Recognition Memory
1.3.2.2.4 Anxiety
2. Objectives
3. Material and Methods
Animal Model
3.1.1 Acute Effects of Aη on Synaptic Plasticity in Electrophysiology Field Recordings
3.1.2 Acute Effects of Synthetic Aη-α Injections into the CA1 Hippocampal Region or Lateral Ventricle
3.1.3 MISEPA2: A Transgenic Mouse Line Overexpressing Aη-α in the Brain
3.1.4 MISEPA4: A Transgenic Mouse Line Overexpressing Aη-α in the Brain with an Elevated Expression Level
Compared to MISEPA2 Line
3.1.5 APPΔEta: A Mouse Model Without η-Secretase Processing of APP due to Deletion the Enzymatic Recognition
Site on APP
3.1.1 Immunofluorescence and Western Blot Verify Expression of Aη-α in MISEPA2 Mice and Absence of Aη-α in
APPΔEta Mice
3.1.2 Housing Conditions
3.1.3 Genotyping
3.1.3.1 Lysis
3.1.4 Polymerase-Chain Reaction Protocol for MISEPA2 and MISEPA4 Mouse Line
3.1.5 Polymerase-Chain Reaction Protocol for APPΔEta Mouse Line
3.1.6 Gel Electrophoresis
Electrophysiology
3.2.1 Peptides
3.2.2 Solutions
3.2.3 Harvesting and Slicing of Mice Hippocampi
3.2.4 Rig Set-Up
3.2.5 Field Recordings
3.2.5.1 Long-Term Synaptic Plasticity Recordings
3.2.5.2 Short-Term Synaptic Plasticity Recordings
3.2.5.2.1 Paired-Pulse Ratio
3.2.5.2.2 Synaptic Fatigue
3.2.5.2.3 Input/ Output
Acute M108 Injection
3.3.1 Surgery
3.3.2 Injection Volume and Concentration
3.3.3 Verification of Injection Site and Distribution of M108
3.3.3.1 Microscopy imaging for injection side in bilateral hippocampal injections
3.3.3.2 Blue Evans Staining to Verify Correct Placement of Canula Guides and Distribution after Injection
3.3.3.2.1 Perfusion Surgery
3.3.3.2.2 Perfusion
3.3.3.3 Western Blot
3.3.3.3.1 Brain Harvesting and Storage
3.3.3.3.2 RIPA Extraction Protocol
3.3.3.3.3 Immunoblotting
Behavioral Testing
3.4.1 Experimental Design for MISEPA2 and MISEPA4 Lines
3.4.2 Experimental Design of M108 Injected Mice Submitted to Behavioral Tasks
3.4.3 Experimental Design for APPΔEta Mice
3.4.4 Morris Water Maze
3.4.5 Novel Object Recognition
3.4.6 Contextual Fear Conditioning
3.4.6.1 CFC Protocol for the M108 Mice
3.4.6.2 CFC Protocol for APPΔEta Mice
3.4.7 T-Maze
3.4.7.1 Injection of M108 During T-Maze Testing
3.4.7.2 Familiar versus New Arm
3.4.7.2.1 Alterations of the T-Maze Task for Testing APPΔEta Mice
3.4.7.3 Forced Alternation
3.4.8 Open Field
3.4.9 Light-Dark Box
3.4.10 3-Chambers Social Interaction Task
3.4.11 Actimeter
Statistical Analysis
3.5.1 Electrophysiology
3.5.2 Behavioral Testing
4. Results
Consequences of Elevated Aƞ Levels on Synaptic Plasticity and Behavior
4.1.1 Effect of Acute Increase of Aη-α Levels on Synaptic Function and Behavior
4.1.1.1 Impact of Acutely Elevated Aƞ-α Levels on Synaptic Plasticity
4.1.1.1.1 Aƞ-α, the secreted APP fragment processed by ƞ- and α-secretases, acutely modulates post-synaptic plasticity mechanisms shifting the balance towards depression of synaptic st
4.1.1.2 Impact of Acute in vivo Injection of M108 into the Brain
4.1.1.2.1 Optimization of Protocol for in vivo Delivery of M108 Peptide
4.1.1.2.2 Presence of M108 in the Hippocampus Post-Injection Confirmed via Blue Evans Dye and Western Blot
4.1.1.2.3 Impact of Acute Injection of M108 into the Hippocampus on CFC
4.1.1.2.3.1 Acute Injection of M108 Prior to Conditioning Session in CFC Does Not Impact Memory Formation but Increases Memory Extinction During Secondary Downstream Retrieval
4.1.1.2.3.2 Contextual Memory Formation Is Not Impacted by Acute in vivo M108 Injection Irrespective of Time-point of Injection
4.1.1.2.4 Effect of Acute Injection of M108 into the Right Lateral Ventricle on Performance in T-Maze
4.1.1.2.4.1 Times of Injections Rather Than Delay of Retrieval Leads to Performance Impairment in M108 Injected Mice in a Familiar versus New Arm T-maze Task
4.1.1.2.4.2 Performance in M108 Injected Mice Is Not Impaired in a Forced Alternation T-Maze
4.1.1.3 Discussion
4.1.2 Effect of Chronic Enrichment of Aη-α Levels on Synaptic Function and Behavior
4.1.2.1 Impact of Chronically Elevated Aƞ-α Levels in a MISEPA2 Mouse Line on Synaptic Plasticity and Behavior
4.1.2.1.1 Influence of Chronically Elevated Aƞ-α Levels in a MISEPA2 Mouse Line on Synaptic Plasticity
4.1.2.1.1.1 Impaired LTP in MISEPA2 Mice
4.1.2.1.1.2 Short-Term Synaptic Plasticity and Basal Transmission Unaltered in MISEPA2 Mice
4.1.2.1.2 Influence of Chronically Elevated Aη-α Levels in a MISEPA2 Mouse Line on Behavior
4.1.2.1.2.1 No Impairment of Performance in a Spatial Memory Dependent MWM Task for MISEPA2 Mice
4.1.2.1.2.2 Indication of External Factors Influencing Contextual Memory of MISEPA2 MICE in CFC
4.1.2.1.2.3 Unaltered Diurnal Activity in MISEPA2 Mice
4.1.2.2 Impact of Chronically Elevated Aƞ-α Levels in a MISEPA4 Mouse Line on Synaptic Plasticity and Behavior
4.1.2.2.1 Synaptic Plasticity in MISEPA4 Mouse Line
4.1.2.2.1.1 LTP is Normal in MISEPA4 Mice
4.1.2.2.1.2 Impaired Basal Transmission in MISEPA4 Mice but No Alterations in Short-Term Synaptic Plasticity
4.1.2.2.2 Impact of Chronically Elevated Aη-α Levels in a MISEPA4 Mouse Line on Behavior
4.1.2.2.2.1 No Effect on Recognition Memory in NOR for MISEPA4 Mice
4.1.2.2.2.2 No Impairment of Performance in a Spatial Memory Dependent MWM Task for MISEPA4 Mice
4.1.2.2.2.3 Insufficient CFC Task Set-Up to Examine an Effect on Contextual Learning for MISEPA4 Mice
4.1.2.2.2.4 Normal Diurnal Activity in MISEPA4 Mice
4.1.2.3 Discussion
Consequences of Inhibition of the APP Processing η-Secretase Pathway on Synaptic Plasticity and Behavior
4.2.1 Impact of Deficiency in η-Secretase Processed APP in an APPΔEta Mouse Line on Synaptic Plasticity
4.2.1.1 No Alterations of LTP in APPΔEta Mice
4.2.1.2 Deficiency in ƞ-Secretase Processed APP Prevents LTD, a Phenotype Rescued by Acute Application of M108
4.2.1.3 Short-Term Synaptic Plasticity and Basal Transmission are Normal APPΔEta Mice
4.2.2 Impact of Loss of η-Secretase-dependent Cleavage of APP on Behavior
4.2.2.1 Indication of Reduced Anxiety for HOMO in Open Field
4.2.2.2 Indication of Reduced Anxiety for APPΔEta Mice in the Light-Dark Box
4.2.2.3 Regular Social Interaction Observed for APPΔEta Mice in the 3-Chambers Social Interaction Task
4.2.2.4 Impaired Spatial Memory in HOMO in T-Maze Task
4.2.2.5 APPΔEta Mice Display Loss of Spatial Memory in the MWM
4.2.2.6 APPΔEta Mice Display Normal Contextual Fear Memory
4.2.2.7 Diurnal Activity is Altered in HOMO Mice (Preliminary Data)
4.2.3 Discussion
Synaptic Plasticity, Under Acute and Chronic elevated Aη-α Conditions
Behavioral Studies, Under Acute and Chronic elevated Aη-α Conditions
Comparing the Acute and Chronic elevated Aη-α Conditions
Depletion of Aη-α levels
APPΔEta and Alternatives: A Comparison
Comparing the Outcomes of Aη-α Level Modification
Probing the η-Secretase Pathway: Prospective Approaches
6. Conclusion
Bibliography
Supplementary ..