# data[].attributes.body
1. Sharma, P. et al. Comprehensive review of mechanisms of pathogenesis involved in alzheimer’s disease and potential therapeutic strategies. Progress in Neurobiology 174, 53–89 (2019).

2. van Hoesen, G. W., Hyman, B. T. & Damasio, A. R. Entorhinal Cortex Pathology in alzheimer's disease. Hippocampus 1,1–8 (1991).

3. What are the signs of alzheimer's disease? National Institute on Aging Available at: https://www.nia.nih.gov/health/what-are-signs-alzheimers-disease. (Accessed: 6th December 2022)

4. Bekris, L. M., Yu, C.-E., Bird, T. D. & Tsuang, D. W. Review article: Genetics of Alzheimer Disease. Journal of Geriatric Psychiatry and Neurology 23, 213–227 (2010).

5. Menting, K. W. & Claassen, J. A. β-secretase inhibitor; a promising novel therapeutic drug in Alzheimer's disease. Frontiers in Aging Neuroscience 6, (2014).

6. Luo, Q. et al. High-throughput dual-color precision imaging for brain-wide mapping of the connectome with cytoarchitectonic landmarks at the Cellular Level (conference presentation). Neural Imaging and Sensing (2017). doi:10.1117/12.2251624

7. Ballinger, E. C., Ananth, M., Talmage, D. A. & Role, L. W. Basal Forebrain Cholinergic Circuits and Signaling in Cognition and Cognitive Decline. Neuron 91, 1199–1218 (2016).

8. Shirey, J. K. et al. A selective allosteric potentiator of the M1 muscarinic acetylcholine receptor increases activity of medial prefrontal cortical neurons and restores impairments in reversal learning. J. Neurosci. 29, 14271–14286 (2009).

9. Gulledge, A. T., Bucci, D. J., Zhang, S. S., Matsui, M. & Yeh, H. H. M1 receptors mediate cholinergic modulation of excitability in neocortical pyramidal neurons. J. Neurosci. 29, 9888–9902 (2009).

10. Major, A. J., Vijayraghavan, S. & Everling, S. Muscarinic Attenuation of Mnemonic Rule Representation in Macaque Dorsolateral Prefrontal Cortex during a Pro- and Anti-Saccade Task. J. Neurosci. 35, 16064–16076 (2015).

11. Sun, Q. et al. Acetylcholine deficiency disrupts extratelencephalic projection neurons in the prefrontal cortex in a mouse model of alzheimer’s disease. Nature Communications 13, (2022).

12. Oakley, H. et al. Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial alzheimer's disease mutations: Potential factors in amyloid plaque formation. J. Neuroscience 26, 10129–10140 (2006).

13. Donocoff, R.S., Teteloshvili, N., Chung, H. et al. Optimization of tamoxifen-induced Cre activity and its effect on immune cell populations. Sci Rep 10, 15244 (2020).

14. Principle of cre-dio system. GeneMedi Available at: https://www.genemedi.net/i/principle-of-cre-dio-system. (Accessed: 6th December 2022)

15. Biundo, F., Del Prete, D., Zhang, H., Arancio, O. & D’Adamio, L. A role for tau in learning, memory and synaptic plasticity. Scientific Reports 8, (2018).

16.  Carroll, J. C. et al. Sex differences in β-amyloid accumulation in 3xTg-AD mice: Role of neonatal sex steroid hormone exposure. Brain Research 1366, 233–245 (2010).

17. Tuscher, J. J., Taxier, L. R., Fortress, A. M. & Frick, K. M. Chemogenetic inactivation of the dorsal hippocampus and medial prefrontal cortex, individually and concurrently, impairs object recognition and spatial memory consolidation in female mice. Neurobiology of Learning and Memory 156, 103–116 (2018).

18. Retrograde tracing technique. Encyclopedia of Pain 2126–2126 doi:10.1007/978-3-540-29805-2_3825

# data[].attributes.body
Cholinergic neurons degenerate in AD neurons

Cholinergic neurons connect to ET neurons and their degeneration results in ET neuronal dysfunctions

ET dysfunctions causes recognition memory impairment in expression.

Sun et al. designed a novel study to map a pathway involved in memory impairments in AD mice models, which could be targeted in therapeutic studies working to improve AD symptoms.

# data[].attributes.headline
Strengths | Double-flexed inverted open reading frame CRE system

# data[].attributes.subject
Strengths | Double-flexed inverted open reading frame CRE system

# data[].attributes.body
Look at whether ET-cholinergic pathway still stands in male 5xFAD mice, by repeating similar experiments.

Could repeat the experiments using wireless devices to elicit activation of ChR2 and NpHR, because a fiber optic line has potential to overheat and be toxic toward the mice, and act as a confounding variable. It also allows less tethering of mice models and allows them to explore the area without anxiety, which may factor into their responses in the NOR test.

# data[].attributes.body
Authors should measure effects of inhibition and activation of ChR2 and NpHR in the cholinergic neurons on activity levels in the dorsal hippocampus and how it translates to NOR.

They could use retrograde tracing methods to see whether dorsal hippocampus has direct connections to the ET neurons. In retrograde tracing, the tracer is inserted into a region of interest of neurons and it jumps backward from the dendrite of neuron it is injected in to the axon of the neuron connecting to it and hence maps out the connections to that neuron (18).

Expected results: Cholinergic neuronal upregulation increases dorsal hippocampus activation in 5xFAD mice models during the habituation and training phase of NOR. This would suggest that dorsal hippocampus is necessary for ORM memory encoding. Dorsal hippocampus would then also show connections to and send signals directly to the ET neurons, which is why it would be indirectly involved in memory expression. Thus making the ORM pathway in AD be, cholinergic-hippocampus-ET neurons.

# data[].attributes.body
Did not measure whether tau protein differences in mice model affected ORM

In the beginning of the experiment, it was measured that increased Aβ burden showed a weak but negative correlation with ORM. Therefore, it could act as a weak confound behind the experimental results and this much shift was accounted for in their results. However, the same was not done with phosphorylated tau proteins, which also characterizes AD pathology and is involved in memory (15).

Overexpression of amyloid precursor protein (APP) in 5xFAD is artificial

This level of APP buildup requires time in humans. Which is why whether sudden increase in APP is the real reason behind significant cholinergic neuron and ET neuron degeneration or if it is normally the case cannot be separated, thus decreasing external validity (11).

Only studied female 5xFAD mice, who have a more severe amyloid-beta accumulation than male mice (16).

This suggests that the experiments have a low generalisation outside of the lab.

Only looked at ORM expression and not consolidation and encoding, which is done by dorsal hippocampus (17).

An important established brain structure involved in memory consolidation is the dorsal hippocampus. This study only focused on cholinergic neuronal connections with the ET neurons and not with hippocampus. Which is why, the suggested pathway may be incomplete with hippocampus coming before ET neurons, as it sends its axons to mPFC, as well.

However, an argument against this is that the authors' significant result differences showing better ORM by increasing cholinergic or ET neuronal activity were only found in the testing phase, and not habituation or training phase, which is when memory is consolidated and hippocampus is involved.

But role of hippocampus in the ET-cholinergic pathway would still need to confirmed with future studies.

# data[].attributes.body
Optogenetics allowed control of channels down to milliseconds due to usage of light. This is important, as previous data on lesion studies in PFC and its links with memory suggests that timing is critical between surgery and NOR measurements, with greater drop in NOR scores as time passes (11).

Optogenetics is reversible and the inserted channels can be inhibited at different phases of the NOR test. This allowed the authors to look at whether differences in memory in AD patients under the ET-cholinergic pathway were only during the expression stage or encoding and consolidation, as well (11).

Use of tamoxifen allowed authors more control opportunities, negating the effects of stereotaxic surgery for fiber optic insertion, as well as confounds caused by AAV virus vehicle insertion (13).

Came up with a causal mechanism, rather than just correlational study due to manipulation of independent variables (activity of optogenetic channels to induce or inhibit action potential in ET or cholinergic neurons).

Authors use a double-flexed open reading frame CRE system, which allows the inverted gene to stay inverted, as it takes out one of the complemetary lox sites after one inversion due to binding of Cre, which means Cre cannot bind again and reverse the expression of ChR2 and NpHR, allowing for stable and similar expression of these channels in different cells (14).

# data[].attributes.body
This a novel study in which the causational mechanism of ET-cholinergic pathway has been studied and linked with memory loss in AD.

# data[].attributes.body
The greater the age of the mice, the more impaired their novel object recognition test scores. This is because AD is an age-dependent progressive condition. Both a study by Sun et al. (11) and Oakley et al. (12) suggests that 6-month-old 5xFAD mice show the greatest ORM impairment when compared to 2 or 4-month-old mice, which was their rationale for using 6-month-old 5xFAD mice for their studies.

# data[].attributes.body
Sun et al. used immunochemical staining to look at (11):

Neuronal activity:

GCaMPs — a green fluorescent protein (GFP) bound to calmodulin, whose high expression suggests greater calcium cation intake, and hence activity in the cell

Expression of channels in specified neurons:

mCherry — red, paired with channelrhodopsin (ChR2) or halorhodopsin (NpHR) to look at expression of these channels in ET or cholinergic neurons

To validate optogenetic channel expression, they stained for specific markers of ET neurons (Fezf2) and cholinergic neurons (ChAT) and merged images

Used immunohistochemistry: antibody 1 binds to the antigen, antibody 2 binds to antibody 1, and gives off signal

As indicators of activity or neurotransmitter concentration:

C-fos (violet) for all activated neurons

Ach2.0 (green) for measurement of acetylcholine levels in cells.

# data[].attributes.headline
 Materials and Methods | Experimental Design

# data[].attributes.subject
 Materials and Methods | Experimental Design

# data[].attributes.body
Data suggests that there is a direct contact between cholinergic neurons and ET neurons in the pyramidal V neurons of PFC. ET neurons come second in the pathway to the cholinergic neurons as upregulation of ET neurons does not increase acetylcholine, whereas increase in cholinergic activity does increase activity in ET neurons, and thus increases ORM.

In their studies, it was also shown that activation of cholinergic pathway improved locomotion. However, those increases were not paired with activation of ET neurons. This complements the hypothesis that AD is a progressive disease with multiple pathway and that ET neurons are only involved in memory.

# data[].attributes.body
Talha Anwar Chaudhry undertook the introduction and materials and methods section of the presentation and poster. He helped in writing up discussion section from points chosen by Jackson Leslie, and gathering information from the lecture to highlight advantages of optogenetics appraoch. He helped with visualization of the padlet, as well as curation of references and explanation of axis in figures. The future direction and conclusion section was written up by Talha, as well.

Chanwoo Kim took on data curation, summary, and captions of results section. He also proofread and provided input on which figures and results we should be focusing on, as well as set up the draft version of padlet. He was also the one responsible for mentioning general issues with optogenetics that are mentioned in the discussion section's weaknesses.

Jackson Leslie helped in data summary to address the hypothesis. He was responsible for the general discussion sections (significance, analysis of findings) and critiques of the paper.

Each section included input from other section heads, and final decisions were made when an unanimous decision was reached.

# data[].attributes.body
Sun et al. used 5xFAD mice of Fezf2-CreER and ChaT-
CreER driver line

CreER (paired with Fezf2 and ChaT) utilizes the CRE recombinase system in presence of tamoxifen to express genes in ET neurons

Tamoxifen allows the CRE to move from the cytoplasm of cells into the nucleus and allows the lox system to work. Without tamoxifen, this specially designed CRE system will not allow CRE-lox system to work. This allows the authors more control over their experiment and another set of controls (13).

# data[].attributes.body
Sun et al. used 5xFAD mice of Fezf2-CreER and ChaT-
CreER driver line

5xFAD mice carry presenilin-1 mutation, most common mutation in hereditary AD patients (4)

Makes Sun et al.'s results more generalized to the familial cases of AD

Fezf2 is used as an ET neuronal marker; ChaT is used as a cholinergic neuronal marker (11)

Sun et al. stain the cells by using immunohistochemical staining and antibodies for these markers.

# data[].attributes.body
Previously, underactivation and overactivation of M1 receptors showed impaired ORM in AD mice models. ET neuronal dysfunction in mice also showed ORM impairments. However, there was no direct pathway behind this impairment. Given the past research and to figure out what mechanism caused impairment, and therefore should be focus for AD treatments, Sun et al. hypothesized:

Acetylcholine upregulates ET neuron activation in the PFC to increase ORM (11)

# data[].attributes.headline
Study goals | Previous Research on ET neurons in AD

# data[].attributes.subject
Study goals | Previous Research on ET neurons in AD

# data[].attributes.body
Previous research suggests loss of dendritic spine density of extra-telencephalic (ET) projections in prefrontal cortex (PFC) in AD patients (6)

ET neurons are present in the cortical layer V pyramidal neurons of PFC (6)

Correlation of ET dysfunction and short-term memory deficits in AD measured by object recognition memory (ORM)

# data[].attributes.attachment_caption
Subramaniyam, N. P. Lab talk. Sapien Labs | Neuroscience | Human Brain Diversity Project (2022). Available at: https://sapienlabs.org/lab-talk/optical-stimulati

# data[].attributes.body
Under the Cre-lox system/AAV, authors induced ChR2 (excitatory channel) and NpHR (inhibitory channel) by adeno-associated virus in ET neurons of ADf and WTf mice and 5xFADChaT-CreER (ADc) and Wild typeChaT-CreER(WTc) mice (11)

ChR2 is activated by shining blue (473nm) lazer through fiber optic wire inserted in mice brain using stereotaxic surgery (11):

Once activated, it allows positive ions to pump into neurons, causing depolarization and action potential. In cholinergic neurons, it allows release of neurotransmitter acetylcholine

NpHR is activated by yellow (570nm) lazer through fiber optic wire inserted in mice brain using stereotaxic surgery (11):

Once activated, it allows chlorine anions ions to pump into neurons, causing hyperpolarization and inactivity

# data[].attributes.body
Novel objection recognition (NOR) test to measure ORM (11).

NOR involves three phases

Habituation: Two identical objects placed in front of rodents

Training: Switch one object with a novel object

Test: Measure time spent of mice with novel object

Less time mice spent with novel objects indicates impaired ORM (11)

# data[].attributes.headline
Activation of cholinergic terminals extending to ET neurons increases ORM

# data[].attributes.subject
Activation of cholinergic terminals extending to ET neurons increases ORM

# data[].attributes.body
A) Experimental strategy for measuring effects of artificial upregulation of acetylcholine release at ET neuron terminal using ChR2 on ORM.

B) Bar plot of delta object recognition index (i.e. time spent with novel objects - time spent with familiar objects) in ADc mice expressing ChR2 in cholinergic terminals at ET neurons against on and off blue light. Mice spent a statistically significantly (p=0.028) higher time with novel objects in blue light than no blue light (n=5).

C) Bar plot of delta object recognition index in ADc mice expressing GFP in cholinergic terminals at ET neurons against on and off blue light. Mice spent a statistically significantly (p=0.017) higher time with familiar objects in blue light than no blue light (n=5).

# data[].attributes.headline
Lower cholinergic response in ET neurons in AD mice model

# data[].attributes.subject
Lower cholinergic response in ET neurons in AD mice model

# data[].attributes.body
A) Experimental strategy for measuring acetylcholine’s functional response in ET neuron during NOR.

B) Fluctuation in ACh2.0 fluorescence levels levels in WTf (n=12) and ADf mice (n=9), with higher ACh2.0 expression percentage change in WTf mice.

C) Area under curve (AUC) from the plot of WTf (n=12) and ADf (n=9) mice, with statistically significantly (p=0.0120) higher AUC in WTf mice than ADf mice.

*deltaF/F(%) represents percentage change in fluorescence of ACh2.0 per change in time

# data[].attributes.headline
Lower ET neuronal activity in AD mice model is observed

# data[].attributes.subject
Lower ET neuronal activity in AD mice model is observed

# data[].attributes.body
A) Experimental strategy for measuring ET neuronal activity using GCaMP in ADf and WTf mice.

B) Bar plot quantification of neurons expressing both GCaMP and c-fos in WTf and ADf mice. There is statistically significant (p=0.0008) data suggesting greater colocalization in WTf mice than ADf mice (n=4).

C) Visualization of c-fos and GCaMP staining. Greater number of neurons co-express GCaMP and c-fos in WTf mice than ADf mice.

*AAV: adeno-associated virus; TM: tamoxifen.

# data[].attributes.headline
Inhibition of ET neuronal activity impairs ORM

# data[].attributes.subject
Inhibition of ET neuronal activity impairs ORM

# data[].attributes.body
A) Experimental strategy for measuring effects of artificial downregulation of ET neuron activity using NpHR on ORM.

B) Bar plot of delta object recognition index (i.e. time spent with novel objects - time spent with familiar objects) in ADf mice expressing NpHR in ET neurons of PFC against on and off blue light. Mice spent a statistically significantly (p=0.048) lower time with novel objects than familiar objects in blue light (n=8).

# data[].attributes.headline
Activation of ET neuronal activity rescues ORM

# data[].attributes.subject
Activation of ET neuronal activity rescues ORM

# data[].attributes.body
A) Experimental strategy for measuring effects of artificial upregulation of ET neuron activity using ChR2 on ORM.

B) Bar plot of delta object recognition index (i.e. time spent with novel objects - time spent with familiar objects) in ADf mice expressing ChR2 in ET neurons of PFC against on and off blue light. Mice spent a statistically significantly (p=0.048) higher time with novel objects than familiar objects in blue light (n=8).

C) Bar plot of delta object recognition index in ADf mice expressing GFP in ET neurons of PFC against on and off blue light. Mice spent a similar amount of time with novel objects and familiar objects in blue light than no light (n=5).

# data[].attributes.body
Alzheimer’s disease’s (AD) is the most common form of dementia (1). It is a progressive neurodegenerative disorder, which effects the entorhinal cortex (2) and hippocampus (1).

The symptoms range from memory loss, attention deficits, cognitive dissonance, spatial disorientation, mood disorders, and communication defects (3).

AD is primarily characterized by the neuronal shrinkage due to aggregation of extracellular β-amyloid (Aβ) plaques and intracellular neurofibrillary tangles (NFTs) (1).

Only 5% of AD cases are early-onset, familial AD (FAD) linked to genetics with presenilin (PS) 1 mutations accounting for most of known FAD cases (4). PS1 is a part of the γ-secretase complex, whose hampered proteolytic activity helps to produce Aβ peptides and Aβ intracellular domain (AICD) (4,5). AICD is degraded, however, Aβ causes plaque formation in mutated PS1 mutations.

# data[].attributes.headline
Study goals | Previous Research Cholinergic Neurons in AD

# data[].attributes.subject
Study goals | Previous Research Cholinergic Neurons in AD

# data[].attributes.body
Research suggests role of cholinergic pathway in AD:

Cholinergic neurons that innervate the PFC degenerate in AD patients (7)

ORM increases in AD patients given M1 cholinergic receptor agonists (8)

M1 receptors increase activation of layer V pyramidal neurons (9)

However, in other studies, M1 receptor overactivation also causes impaired short-term memory (10)