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||<tablebgcolor="#eeeeee" tablestyle="float:left;font-size:0.85em;margin:0 0 0 0; "> {{attachment:coincidence.jpg||align="left"}}<<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>> ''' Fig 1.''' ''An example of coincidence detection-''<<BR>> ''a neuron receives two excitatory potentials''<<BR>> ''very close in time'' || ||<tablebgcolor="#eeeeee" tablestyle="float:left;font-size:0.85em;margin:0 0 0 0; "> {{attachment:coincidence.jpg||align="left"}}<<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>><<BR>> ''' Fig 1.''' ''An example of coincidence detection-''<<BR>> ''a neuron receives two excitatory potentials''<<BR>> ''very close in time'' ||

Transgenic Tools of Memory and Learning

Memory and learning has long been known to be affected by genetics, but only in the last few decades have we developed ways to directly examine the connection between them. The use of transgenic animals, or animals whose genetic code has been modified by adding foreign genetic material, enables us to show the effect of the presence or lack of certain genes. In the context of learning and memory, we can compare non-transgenic and transgenic animals whose memory-related genes are different, and compare their performance in memory tests. Since these genes are much the same in humans, these experiments can help us understand and even look for a cure for some neurodegenerative diseases, such as Alzheimer’s Disease.


Memory and Learning

Through experience behaviour is altered in humans and other animals, this is commonly known as learning where information about the environment is processed and new behaviour is formed. This information can be instantly acquired through the mechanism of memory. From learning memory is stored and the two types are short term and long-term memory. The former one persists for a shorter time span (minutes to hours) whereas long term persists for a much longer time span (days to years). (Pradeep et al, 2011)

Basics of Memory Formation

coincidence.jpg








































Fig 1. An example of coincidence detection-
a neuron receives two excitatory potentials
very close in time

It is generally accepted that long-term memory is created or degraded through the formation or elimination of certain synapses as well as the synthesis of proteins and mRNA. Alternatively it has been suggested that the basis of short-term memory is determined through the change of function and release of neurotransmitters at certain synapses.

Synaptic activity can be modified by a group of proteins, such as NDMA glutamate-receptors, which functions as a coincidence detector. Coincidence detection (Fig. 1) is the process by which two separate signals (excitatory potential, neurotransmitter release) at the same time are detected, the signal is integrated and produces a potential in the receiving neuron which could not be elicited by each single signal on its own. During the learning process, repetition is key. This means that the same neuronal pathways will be activated repeatedly. Hebb's rule states how this will lead to the formation of memory: repeatedly used synapses will form a stronger connection. (Gerstner, 2010)

Using the NDMA receptor process as an example:

1. The presynaptic neuron fires with high enough frequency (or more than one fire at the same time, see coincidence detection).

2. The postsynaptic cell is strongly depolarised, allowing Mg2+ to be removed from the NDMA receptors (in resting state, the receptors are blocked by Mg2+). At the same time, NDMA receptors bind glutamate (a neurotransmitter).

3. Voltage-dependent NDMA receptors open, allowing Ca2+ to enter the cell.

4. This activates protein kinase mechanicsms which will increase the number and activity of AMPA receptors in the postsynaptic cell.

5. The higher number of receptors means that a smaller signal will elicit a response, and response is increased (short term memory is formed).

6. Repeated stimulation of the protein kinase mechanisms will lead to a cascade resulting in the activation of gene expression and therefore protein synthesis, for example of CREB.

The difference is thus that short-term memory does not require protein synthesis but derive from the alterations in synaptic strength. Long-term memory however generally requires transcription and translation of new proteins that enhance already active synapses through increased strength or a rise in number of active synapses. (Pradeep et al, 2011)








Associated Genes

DLG3: Discs, large homolog 3 (DLG3). It encodes synapse-associated protein 102 (SAP102). This protein affects the NDMA receptor complex. It is found in the postsynaptic density of excitatory synapses during development of the brain. It is important in long-term memory formation. (Cold Spring Harbor Laboratory)

DLG4: Discs, large homolog 4 (DLG4). Encodes DLG4 protein in humans. Rats and mice have Psd95 proteins which are almost identical (99%), and which have an important role in spatial learning. Lack of them may negatively affect the long-term potentiation of the brain. The reason for this is likely that the synaptic NMDA receptors, which are able to bind these proteins, will no longer aid in the neuroplasticity and learning pathways. (Cold Spring Harbor Laboratory)

CREB1: Activates cAMP response element-binding (CREB) proteins- transcription factors which bind to sequences of DNA called cAMP response elements. It can either switch on or off specific genes. CREB is activated when receptors on cells bind a signal, initiating the cAMP production. The cAMP activates a protein kinase which activates CREB. It is involved in long-term memory formation. CREB proteins in neurons are involved in the formation of long-term memories and are involved in long-term potentiation. (Cold Spring Harbor Laboratory, Pradeep et al, 2011)

CREB2: Activating Transcription Factor 2 (ATF2). Opposite effect to CREB1 (inhibits CREB). (Cold Spring Harbor Laboratory, Pradeep et al, 2011)

BDNF: Encodes Brain-derived neurotrophic factor protein (BDNF). Has a double role in the Central and Peripheral Nervous System- it supports existing neurons and aids in the formation of new neurons and synapses (important for long-term memory). (Pradeep et al, 2011)

MARCKS: the gene encodes the MARCKS protein which is a substrate of protein kinase C and Ca2+/Calmodulin that is expressed highly in the hippocampal granule cells and mossy fiber axons, a reduction in MARCKS causes an increase in infrapyramidal mossy fiber (IP-MF) cells and the expression of PKCɛ which plays a role in spatial learning (McNamara et al 1998)

PSEN1: Encodes Presenilin-1, part of a protease complex that cleaves amyloid precursor protein into beta-amyloid (mutation in the PSEN-1 gene can lead to accumulation of beta-amyloid, a sign of Alzheimer's Disease) (Sy et al, 2011)

APP: Amyloid Precursor Protein. As well as PSEN-1 (see above), mutations in the APP gene can result in Alzheimer's disease. (Sy et al, 2011)


Basics of the Transgenic Method

  • lentivirus.png |
    Fig 2.
    Lentiviral (Retroviral) Method of Creating Transgenic Animals

Transgenic tools refer to the techniques and processes of introducing foreign DNA into another animal of the same species or even DNA between different species. In this way specific genetic traits can be investigated in another one. The most common host for this investigatory technique is rodents due to the high amount of techniques currently in use to create transgenic mice. Other species however include plants, insects, worms and other vertebrates such as fish. The cell or transgene is incorporated in the host animal at a very early stage, usually at gamete or embryonic level. In this way the specific gene used for investigation is spread in the entire genome of the animal in question. Genes can be "knocked-in", which means inserting an active form, or "knocked-out" (inhibited). Transgenetics have been developed for reasons from investigating human genetic diseases to genetically engineering agricultural crops. The usefulness and medical importance of these techniques in understanding human disease cannot be understated; however there is a great ethical debate regarding the production of enhanced genetic material as for instance genetically perfected crops and the possible impact on the environment.

Microinjection

One of the most common tools used to create transgenic mice, where a gene is microinjected into the nucleus of a cell and randomly incorporated into the host genome. The gene is injected into a fertilized egg in vitro, which is then returned to the mother. A successful microinjection means that the desired gene now resides within the offspring.

Retrovirus mediated gene transfer

Retroviruses replicate by entering a host cell and using reverse transcription to make DNA from their RNA, which the cell then takes into its own genome and can thus express it. RNA from a lentivirus (type of retrovirus, used because of its unique ability to infect also non-dividing cells) instead of DNA is used in this method as a vector creating a chimera (an organism with several sets of genetic material- not all cells will contain the introduced DNA) (Fig. 2). After several breeding cycles, homozygous offspring is produced that carry the desired genetic material.

Stem Cells

Embryonic stem cells can also be introduced and grown in mice, these ES cells are then recovered and injected into mice embryos. Through inbreeding the result after some generations is an offspring with the desired alteration.

Other techniques, or a combination of different techniques, can be used depending on what is being investigated. Relating to the study of memory and learning the focus of studying can be either to investigate a specific gene or protein and its consequence relating for example to degenerative disease or the lack of the same gene or protein and the reversed conclusion. (Buy, 1997)


Examples of Transgenic Method in Memory and Learning

1. To investigate the effects of neural stem cells on Alzheimer’s Disease (AD), Blurton-Jones et al used triple-transgenic mice (3xTg-AD) as a model for AD. They knocked in PSEN-1 into single-celled embryos, then injected two human transgenes: APP with the Swedish mutation and MAPT with the P30IL mutation. These mice developed signs of AD. They then injected neuronal stem cells (NSC) to try to counteract the signs of AD. They had 4 groups of mice: Tg with injected NSC, non-Tg with injected NSC, Tg with a control injection, and non-Tg with a control injection. Their memory was tested in a Morris Water Maze and, as expected, the Tg mice performed worse than non-Tg mice, and the Tg-mice injected with NSC performed better than the ones with control injection. (Blurton-Jones et al, 2009)

2. An aspect of learning and memory which has not been much studied on a genetic level is vocal learning (as shown by songbirds), because of a lack of transgenic songbirds. This is due to the failure in songbirds of methods used on chickens and quails. Agate et al succesfully created the first transgenic zebra finches, using the lentiviral method, which uses a type of retrovirus as a vessel for genes, to insert GFP (Green Fluorescent Protein, widely used in transgenics as an indicator that the inserted gene is successfully expressed). While this did not investigate learning and memory itself, it opens the door for future transgenic experiments about vocal learning. (Agate et al, 2009)

3. MARCKS(reduced myristoylated alanine-rich C kinase) is a substrate of brain protein kinase C (PKC), which is expressed highly in the granule cells and their axons (known as mossy fibres) of the hippocampus. Heterozygous MARCKS mice were used to study the effects of a 50% reduction in MARCKS. The result was that hippocamal PCK expression was increased, spatial learning became more proficient and interpyrimidal mossy fibre length increased. Homozygous MARCKS mice were also studied, the absence of MARCKS showed many brain abnormality especially in the neocortex and hippocampus. The insertion of transgenic human MARCKS produced mice that didn’t show brain abnormality, suggesting that lack of MARCKS alone was responsible of the brain abnormality's. (McNamara et al 1998)

4. Rubinstein-Taybi Syndrome has an association with mutations of the CREB binding protein gene, the syndrome causes retarded growth and impaired mental function, to examine this mice were produced that expressed a truncated version of the protein in the fore-brain interneurons. These mice suffered from defects in long term memory and spatial learning (both of these tasks are hippocampal dependent). This suggests that CREB binding protein is involved in hippocampal synaptic plasticity and hippocampal dependent long term memory formation. (Wood et al 2005)

5. Until recently, investigations of learning and memory in the entorhinal hippocampal circuit have been hindered by the sensitive nature of conducting studies in brain functions. The entorhinal hippocampal circuit or entorhinal cortex is an area located in the medial temporal lobe with widespread functions on memory. Memory formation, optimization during sleep and memory consolidation takes place here. Investigations on these functions have previously been limited to observational studies on awake animals. With development of pharmacogenetic and optogenic transgenes the limitations on this approach have changed. Pharmacogenetic transgenes are designed neurotransmitter receptors that can be externally activated and controlled. Optogenic transgenes use light to control the membrane potential and allow immediate determination of a response. These tools together enable researchers to elicit a controlled action in specific cells while monitoring the response in related brain tissue and so broaden the understanding of memory formation. (Lykken and Kentros, 2014)

6. Alzheimer’s disease is a degenerative disease that leads to dementia and memory loss. With the help of transgenic mice expressing the amyloid precursor protein (APP) the relationship between memory function and Amyloid-ß (Aß) can be investigated. Aß protein is the centre of an hypothesis suggesting it causes plaques which is neurotoxic, the result is neurodegradation and dementia. The evidence suggest that soluble Aß accumulates with age causing degenerative effects on the brain and memory. Further studies are however needed to determine the impact on this variable in relationship with others. Without the help of transgenic mice this and further studies would however not be possible, therefore this is an essential tool for investigating degenerative effects on memory. (Ashe, 2001)

Conclusion

There are many ways to study learning and memory, ranging from simple observation to molecular biology. Recently the development of transgenics has opened up a wide area of knowledge. Scientists are now able to get to the core processes of memory formation, which lies in the expression of genes to form proteins. Alongside finding the general processes in which humans and animals learn and remember, we are able to identify which gene specifically affects what part of the memory process. In the field of medicine this gives us an opportunity to research neurodegenerative diseases, learning disabilities, and other genetic disorders which may have negative effects on learning and memory. Certain mutations in specific genes may cause diseases such as Alzheimer's disease, which means that a DNA test can be used as a diagnostic tool in cases where these diseases are suspected, or where there is a family history of them. In the future, a cure could be found using these methods.

References

Agate R. J., Scott B. B., Haripal B., Lois C., Nottebohm F (2009): Transgenic songbirds offer an opportunity to develop a genetic model for vocal learning Proceedings of the National Academy of Sciences 106:(42) 17963-17967

Ashe K. H. (2001): Learning and Memory in transgenic Mice Modeling Alzheimer´s Disease. Learning and Memory 8: 301-308, Cold Spring Harbor Laboratory Press

Blurton-Jones M., Kitazawa M., Martinez-Coria H., Castello N. A., Müller F., Loring J. F., Yamasaki T. R., Poon W. W., Green K. M., LaFerla F. M. (2009): Neural stem cells improve cognition via BDNF in a transgenic model of Alzheimer disease. Proceedings of the National Academy of Sciences 106:(32) 13594-13599

Buy. M. (1997): Transgenic Animals. Canadian Council on Animal Care Resource Supplement

Cold Spring Harbor Laboratory, Genes for Learning and Memory, accessed 19.10.2014

Gerstner, W. (2012): Biological Learning: Synaptic Plasticity, Hebb Rule and Spike TimingDependent Plasticity. Encyclopedia of Machine Learning 111-132. Springer US

Lykken C., Kentros C.G (2014): Beyond The Bolus: transgenic tools for investigating the neurophysiology of learning and memory. Learning and Memory 15;21(10):506-18, Cold Spring Harbour Laboratory Press

McNamara R. K, Stumpo D. J., Morel L. M., Lewis M. H., Wakeland E. W., Blackshear P. J., and Lenox R. H., (1998) Effect of reduced myristoylated alanine-rich C kinase substrate expression on hippocampal mossy fiber development and spatial learning in mutant mice: Transgenic rescue and interactions with gene background Proceedings of the National Academy of Sciences 95:(24) 14517-14522

Pradeep F. S., Pradeep B. V., Palaniswamy M. (2011) Biology of Learning and Memory Genes. Journal of Neurology and Neurophysiology 120:(2) 1-13

Sy M., Kitazawa M., LaFerla F. M. (2011): The 3xTg-AD Mouse Model: Reproducing and Modulating Plaque and Tangle Pathology. Animal Methods of Dementia 48: 469-482

Wood M. A., Kaplan M. P., Park A., Blanchard E. J., Oliveira A. M. M., Lombardi T. L., Abel T., (2005) Transgenic mice expressing a truncated form of CREB-binding protein (CBP) exhibit deficits in hippocampal synaptic plasticity and memory storage Learning and Memory 12: 111-119 Cold Spring Harbour Laborotory Press

Figures

Fig. 1. Theredman047. Uploaded to Wikimedia Commons Dec. 2007

Fig. 2. Dan Cojocari, University of Toronto. Uploaded to Wikimedia Commons Dec. 2009

TransgeneLearning (last edited 2014-12-01 20:39:07 by 2635E)