DeAnn Reynolds
Biology 475, Neurobiology
This website was created as a project in the course Neurobiology 475 at University of Nevada Reno for Dr. Grant Mastick to summarize the article "A critical role for IGF-II in memory consolidation and enhancement" by Dillion Y. Chen et al. January 2011. Nature: vol. 469. pg 491-497.
Introduction:
Memories, we all have them, and as we get older we tend to lose them, but can we actually keep them as sharp as they are when they are first made? Insulin like growth factor-two (IGF-II) might be that solution to problems with memory.
IGF-II is a naturally occurring chemical with in the body.
Like most experiments preformed in a lab, transgenic mice were used to test this hypothesis that IGF-II can enhance and consolidate memories.
The goal aims to improve the way things are remembered within the subject, and if those memories can be kept longer. IGF-II over expression was investigated for its role in the formation of memories and how it is expressed in a subject. IGF-II (pictured below) is very similar in structure to insulin (see drawing right)
Introduction:
Memories, we all have them, and as we get older we tend to lose them, but can we actually keep them as sharp as they are when they are first made? Insulin like growth factor-two (IGF-II) might be that solution to problems with memory.
IGF-II is a naturally occurring chemical with in the body.
Like most experiments preformed in a lab, transgenic mice were used to test this hypothesis that IGF-II can enhance and consolidate memories.
The goal aims to improve the way things are remembered within the subject, and if those memories can be kept longer. IGF-II over expression was investigated for its role in the formation of memories and how it is expressed in a subject. IGF-II (pictured below) is very similar in structure to insulin (see drawing right)
Currently it is known:
- CREB (cAMP response element-binding protein) is important for the formation of memories.
- In mammals the over expression of Adenylyl Cyclase1 increased the synthesis of CAMP (Cyclic adenosine monophosphate) production in the amygdala.
- CAMP is a second messenger that binds and regulates ion channels that are involved in signal transduction; it is also a transporter molecule within a cell.
- IGF-II is a target gene for C/EBP beta that helps to consolidate, and helps in regrowth and repair.
- It is know that IGF-II can cross the blood brain barrier, which makes it an ideal chemical to use when we are thinking about enhancing memories.
- We know that some events are “burned” in to our memories due to a traumatic or an extremely happy events.
- We know that the amygdala and the hippocampus are primarily responsible for the formation and retention of memories.
- Some research has shown that injections directly in to the hippocampus seem to work best, as well as activating the fear response in the amygdala can improve memory retention.
- It is known that if you retrieve a memory once it is stable in the brain, it becomes easily altered when it is retrieved again.
- We also know that the brain consolidates memories in order to make them stronger and stick.
Experimental System:
Several strategies were used when conducting this experiment. Transgenic mice were used along with special apparatuses.
Terms to know:
Several strategies were used when conducting this experiment. Transgenic mice were used along with special apparatuses.
Terms to know:
Inhibitory Avoidance-
This experimental system is a form of shock therapy, where there are two sides of a box that a rat is placed in. (see drawing to left) This kind of training triggers the amygdala, the fear center of the brain, as well as the hippocampus, which is known to consolidate memories, and help in spatial movement. The box is set up so that one side is very bright and white, while the other is dark and black, and the floor is rigged to give a small voltage shock. The dark and black room is used to simulate a traumatic experience.
Contextual and Auditory Fear Conditioning-
Uses high frequency sounds to trigger a fear response in the subject. This test requires the subject to be placed in a Plexiglas walled box, with a metal floor (see drawing to right). The subjects were allowed to feel out the room the day before any experiments were performed without the sound. On the day of the real test the subjects were placed in the box for 2 minutes without sound then, for 30 seconds, a high-pitched noise was turned on for two 30-second intervals. On the second interval the subject also received shocks to their feet. Freezing levels from the subject’s, defined as lack of movement except for breathing, were recorded as well.
Cannulae Implants and Hippocampal and Amygdala Injections-
Stainless steel tubes were inserted in to both right and left sections of the hippocampus (see drawing below). This was also done with the amygdala but with a smaller gauge stainless steel tube. This allowed direct access to parts of the subject’s brain that they wanted to influence with each solution. This was done so multiple experiments on the same subjects could be performed.
The solutions used were:
The solutions used were:
- Antisense oligonucleotides - a sequence of nucleotide bases that are complementary to genes within a strand of mRNA in the test subject.
- Anisomycin mixed with saline - this solution interferes with protein binding sites, and it can also help in activating signal transduction pathways.
Chromatin Immunoprecipitation (ChIP)-
Chromatin is used to investigate the interactions between the protein and the mRNA in a cell. Immunoprecipitation is used to predict a protein antigen out of a solution; it uses antibodies that bind to the protein the researchers are looking for. It helps the researchers figure out if the proteins are linked to transcription factors, or other types of binding sites.
Real-Time Quantitative RT-PCR-
Polymerase chain reaction (PCR) is used to amplify the target or sample DNA. PCR labels the DNA or RNA with florescent dye that reacts with any double stranded form of DNA or RNA. Researchers are looking for a specific sequence on the sample DNA or RNA. DNA or RNA is detected as the reaction progresses in real-time.
Synaptoneurosomal Preparation and Western Blot Analysis -
Synaptoneurosomal preparation involves using purified synapse that contains both a pre-synaptic and post-synaptic terminus. The western blot analysis is used to detect protein in the tissue that has been homogenated by a centrifuge. Brain tissue was collected from the dorsal hippocampus mixed with buffer and centrifuged. Western blot analysis use electrophoresis to separate out the proteins on to a gel by size from the centrifuged mixture. An example of the output can be seen here. (See drawing left)
Experiments and Results:
C/EBP-beta-dependent IGF-II Expression is Regulated by Training-
- C/EBP-beta is a transcription factor and CCAAT enhancer binding protein. It is used to see if C/EBP-beta regulates IGF-II expression in a target gene.
- Inhibitory avoidance training was used. The subject was placed in the white box portion of the apparatus with its tail facing the door. After ten seconds the subject was allowed to enter the black room of the box where it received shocks on its feet for an hour. The control mice were exposed to the box’s without the foot shock or euthanized them right after.
- The trained subjects, the ones who received shocks to their feet in the black room, had no change in their hippocampus after 6 hours of IGF-II injection treatment. After twenty to thirty-six hours, it was shown that the IGF-II mRNA expression had increased in the hippocampus among animals trained with inhibitory avoidance compared to those who did not receive shocks. However, it was observed that expression of memory retention declined after seventy-two to ninety-six hours had gone by (see chart 1 below). Quantitative real-time PCR was used to confirm the western blot, confirming there was an increase in IGF-II mRNA twenty hours after training.
- Part two of this experiment involved an injection of C/EBP-beta mixed with oligodeoxynucleotide into both sides of the hippocampus. The subjects under went five hours of inhibitory avoidance training. It was found that the oligodeoxynucleotide blocks C/EBP-beta completely, and disrupts the subject’s ability to form and retain memories (see charts 2 and 3 below).
- Chromatin immunoprecipitation confirmed that C/EBP-beta does bind to a promoter region on an exon portion of IGF-II receptor region. C/EBP-beta can be increased in the hippocampus.
Limited Temporal Requirement of IGF-II During Memory Consolidation-
- IGF-II expression was knocked down in the dorsal hippocampus. The subjects were given an injection of IGF-II oligodeoxynucleotide antisense to inhibit IGF-II from binding to receptor sites in the hippocampus. Using inhibitory avoidance to hinder the subject’s memory retention. In some subjects injections were given eight hours after training as well as at the beginning.
- Subjects that were injected at both times had developed amnesia and did not remember any of their training. IGF-II is extremely important for memory consolidation after inhibitory avoidance training in order to process memories from learning or testing. Blocking IGF-II only hinders the brains ability to process things that were previously learned (see chart 4 on right).
IGF-II Significantly Enhances Memory and Prevents Forgetting-
- IGF-II was injected directly into the hippocampus after inhibitory avoidance training and auditory fear conditioning, to try to prevent the subjects from forgetting the training they learned. This was done to find out if direct injections in the hippocampus can enhance memory?
- Injections increased memory retention between twenty-four hours and seven days later in auditory fear conditioning, but it did not seem to have any effect when inhibitory avoidance training was performed forty-eight hours after auditory fear conditioning (see chart 5 on right).
- Subjects were re-tested three weeks later to see how or if the memory of training was retained.
- Subjects memories were retained three weeks after initial test (see chart 6 on left)
- A second experiment was conducted to test the amygdala. Direct injections to the amygdala showed no change in the subject’s memory. The amygdala plays a small or little role in the retention of memories.
IGF-II- Mediated Memory Enhancement- Effect on Reconsolidation-
- Would using IGF-II help keep memories after they have been retrieved?
- Bilateral injections in to the hippocampus were used. Testing on the amygdala was left out because the previous experiment showed that IGF-II did not affect it.
- Memories are unstable after retrieval. Twenty-four hours after inhibitory avoidance training IGF-II direct injections were given to the subjects. Forty-eight hours after the injection the subject’s memories were tested; this didn’t seem to have an effect on the retention of memory. Why?
- Revision of the test - subjects were injected twenty-four hours after training but only after there was a retrieval of a memory; this showed to enhance the retention of the subject’s memory (see chart 7 on left).
- Results - memory enhancement with IGF-II only occurs after inhibitory avoidance training in a small window of time after memories were retrieved.
IGF-II Facilitates Long-Term potentiation (LPT)-
- Long-term potentiation means that there is a long lasting enhancement in the signal transduction between two neurons.
- Does IGF-II affect the synapses plasticity by changing how it sends signals within the hippocampus? Would this help in effectively making a drug treatment for enhancing and retaining memories in the long term?
- Brains of the subjects were removed and placed in an artificial cerebral fluid to keep them alive in order to perform multiple experiments on them. Small slices of each brain were prepared.
- Shaffer collateral pathway- This pathway is a small branch of axons that are given off by pyramidal cells, neurons that have the shape of a pyramid with long tree like dendrites, within the hippocampus. They are important when memories and emotions are being formed.
- Brains of the subjects were removed and placed in an artificial cerebral fluid to keep them alive in order to perform multiple experiments on them. Small slices of each brain were prepared.
- First a baseline transmission was formed to give a normal non action potential state. They observed a synaptic transmission that slowly returned to the baseline state after 100 minutes. They then performed a similar test but with IGF-II injections on the sliced sections of the hippocampus and a small stimulus were given to start an action potential (see chart 8 below).
- IGF-II created a stable long-term potentiation that increased from the baseline by a little more than six and a half percent and only slightly effected the basal synaptic transmission.
- Slices that were prepared with IGF-II and the IGF-II Receptor antibody and given the same small amount of stimulus showed there were no retention and the graph looked like the control. It did not show any response on the basal synaptic transmission or the long-term potentiation. By blocking IGF-II with the IGF-II Receptor antibody, it doesn’t allow the hippocampus to keep or retain memories (see chart 8 above).
Conclusion:
- In rats they proved that injecting the hippocampus with IGF-II could improve memories.
- Memory can be enhanced, but that there is a limited time frame when it is optimal to inject the hippocampus with IGF-II.
- Retrieval of memories can also enhance memories when given an IGF-II injection twenty-four hours after memories were retrieved.
- When they tested the subjects two weeks later they had retained their memories, it was conclusive that you can enhance and keep memories once retrieved after IGF-II treatment.
- Inhibitory avoidance training allows the IGF-II to increase and the C/EBP-beta helps in memory retention. This experiment showed how important C/EBP-beta is in regulating the increase of IGF-II downstream in the hippocampus. They found that using an oligodeoxynucleotide will block the activity of C/EBP-beta and disrupt the subject’s memory.
- During memory consolidation experiments, when they doubly knocked down IGF-II expression in the hippocampus, the subjects showed to have amnesia after training. This told the researchers that the IGF-II present in the hippocampus is very important for memory consolidation after inhibitory avoidance training within a time frame that only lasts between one day and less than four days.
- The hippocampus and not the amygdala is the source of where memories are enhanced the most. IGF-II in the hippocampus showed to be very good at enhancing memories and helped with forgetting. The dose of IGF-II was also important in memory retention giving 2.5ng seemed to be the best amount to administer to see an enhanced memory retention after twenty-four hours later
- Long-term potentiation, a long lasting signal transmission between two neurons, was promoted during the administration of growth factor IGF-II it in turn regulated the subjects ability to remember things. This experiment showed that IGF-II is an important chemical when it comes to enhancing and re-consolidating memories in the hippocampus.
- IGF-II might not recruit the activation of new cells, but uses cells that have been transcriptionally ‘marked’ with antibodies. This is done during training which target synaptic mechanisms within the hippocampus, and may mark those at activated synapses as well. They also went on to say that they are not exactly sure how this works and that IGF-II may recruit new cells for activation. Further work needs to be done to investigate why this is.
Significance/ Future Directions:
As neurons and synapses degenerate and memories are lost we currently don’t know how to fix them once they are gone. Understanding how neurons are lost or degenerated in mice could lead to rebuilding them or enhancing their abilities. Eventually this research may cross over in to Alzheimer’s, and dementia research, or enhance how fast a subject can learn. The more we understand about how our memories are made and stored, the better we can understand how to enhance and retain them.
There are many unanswered questions from this research that could lead in many future directions. Questions like, why is there such a small time window when memories can be reconsolidated? I think there should be further research done to find out what enzyme in the synapses breaks down naturally occurring IGF-II and find a way to suppress it in animals that may over express it. What if the amygdala does play a larger role in memory retention, instead of being enhanced by IGF-II? What if it releases chemical signals or hormones to help the hippocampus maintain and keep memories? I think it would be worthwhile to further research the roles of the amygdala with memory retention.
Another avenue that could be looked at is the C/EBP-beta dependent IGF-II expression; why did the C/EBP-beta top out at twenty hours of retention and not last longer? Does it degrade after a certain amount of time? Or would applying another injection just before the twenty-hour mark extend the memory retention a little longer? We all know that in young animals the plasticity of the brain is greater than that of adult animals, so could it be possible for researchers to use IGF-II in adult test subjects to create plasticity within the brain to keep it malleable like in babies or week-old test subjects?
There are many unanswered questions from this research that could lead in many future directions. Questions like, why is there such a small time window when memories can be reconsolidated? I think there should be further research done to find out what enzyme in the synapses breaks down naturally occurring IGF-II and find a way to suppress it in animals that may over express it. What if the amygdala does play a larger role in memory retention, instead of being enhanced by IGF-II? What if it releases chemical signals or hormones to help the hippocampus maintain and keep memories? I think it would be worthwhile to further research the roles of the amygdala with memory retention.
Another avenue that could be looked at is the C/EBP-beta dependent IGF-II expression; why did the C/EBP-beta top out at twenty hours of retention and not last longer? Does it degrade after a certain amount of time? Or would applying another injection just before the twenty-hour mark extend the memory retention a little longer? We all know that in young animals the plasticity of the brain is greater than that of adult animals, so could it be possible for researchers to use IGF-II in adult test subjects to create plasticity within the brain to keep it malleable like in babies or week-old test subjects?
References:
Article- nature- jan 27 2011. Vol 469. P491-499
"A critical role for IGF-II in memory consolidation and enhancement" -Dillion Y. Chen, Sarah A. Stern, Ana Garcia-Osta, et al.
http://www.nature.com/nature/journal/v469/n7331/abs/nature09667.html
Article – Science- vol. 331 march 4, 2011. 1207-1210
"Enhancement of Consolidated Long-Term Memory by Overexpression of Protein Kinase Mk in the Neocortex" -Reut Shema, Sharon Haramati, Shiri Ron, Shoshi Hazvi, et al.
http://0-www.sciencemag.org.innopac.library.unr.edu/content/331/6021/1207.full
Article- Cellular/Molecular 5688 • The Journal of Neuroscience, April 18, 2012 • 32(16):5688 –5703
"IkB Kinase/Nuclear Factor kB-Dependent Insulin-Like Growth Factor 2 (Igf2) Expression Regulates Synapse Formation and Spine Maturation via Igf2 Receptor Signaling" -Michael J. Schmeisser, Bernd Baumann, Svenja Johannsen, et al.
http://0-www.jneurosci.org.innopac.library.unr.edu/content/32/16/5688
Review- Cell Press may 2012 vol. 35, No.5. 274-283
"Memory enhancement: consolidation, reconsolidation and insulin-like growth factor 2" -Cristina M. Alberini and Dillion Y. Chen
http://0-www.sciencedirect.com.innopac.library.unr.edu/science/article/pii/S0166223611002153
** All charts re-created from the article "A critical role for IGF-II in memory consolidation and enhancement". Chen et all.
*All drawings created by DeAnn Reynolds
"A critical role for IGF-II in memory consolidation and enhancement" -Dillion Y. Chen, Sarah A. Stern, Ana Garcia-Osta, et al.
http://www.nature.com/nature/journal/v469/n7331/abs/nature09667.html
Article – Science- vol. 331 march 4, 2011. 1207-1210
"Enhancement of Consolidated Long-Term Memory by Overexpression of Protein Kinase Mk in the Neocortex" -Reut Shema, Sharon Haramati, Shiri Ron, Shoshi Hazvi, et al.
http://0-www.sciencemag.org.innopac.library.unr.edu/content/331/6021/1207.full
Article- Cellular/Molecular 5688 • The Journal of Neuroscience, April 18, 2012 • 32(16):5688 –5703
"IkB Kinase/Nuclear Factor kB-Dependent Insulin-Like Growth Factor 2 (Igf2) Expression Regulates Synapse Formation and Spine Maturation via Igf2 Receptor Signaling" -Michael J. Schmeisser, Bernd Baumann, Svenja Johannsen, et al.
http://0-www.jneurosci.org.innopac.library.unr.edu/content/32/16/5688
Review- Cell Press may 2012 vol. 35, No.5. 274-283
"Memory enhancement: consolidation, reconsolidation and insulin-like growth factor 2" -Cristina M. Alberini and Dillion Y. Chen
http://0-www.sciencedirect.com.innopac.library.unr.edu/science/article/pii/S0166223611002153
** All charts re-created from the article "A critical role for IGF-II in memory consolidation and enhancement". Chen et all.
*All drawings created by DeAnn Reynolds