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THE BIOLOGICAL BASIS FOR LEARNING & MEMORY"The patient is a set of biological functions. But I think one has to realize that the whole is more than the sum of the parts. There is something wonderful and special about each person as a unique individual—a unique set of biological functions, if you will. The ultimate aim is to use reductionism, not only to take things apart, but to put them together again. You have to be a reductionist and a holist at the same time." Dr Eric Kandel, September 2000 HHMI Bulletin, Vol. 13, No. 3, pp. 6-8 The major functional difference between plants & animals is
that the latter posses a brain that enables rapid organismic responses to the
environment to promote genetic survival & reproduction. The human mind outshines all other species in terms of its sophisticated cognitive abilities, emotional complexity & the gift of consciousness. However, we share, even with the invertebrates, the same physiological mechanism that permits the brain to learn from experience how to best respond to environmental challenges. Learning is the process of our brain's acquisition of information about the environment. Learning can be considered an evolutionary adaptation to transient order not lasting long enough for a direct evolutionary adaptation, the order being of two types: relations in the world (classical or Pavlovian conditioning) and the consequences of one's own actions in the world (operant or instrumental conditioning) (Brembs & Heisenberg, 2000). Experience induces short & long-term synaptic changes, which we call short & long-term memory. Memory is thus the process of storing acquired information from experience. What sorts of changes does memory produce in the circuits of the brain? How is memory initially stored? How is memory maintained? What are the molecular mechanisms whereby a transient short-term memory is converted into an enduring self-maintained long-term memory? Psychiatrist Eric R. Kandel began using a reductionist approach in the late 1950s to explore the mechanisms of learning & memory storage in molecular terms, to answer these questions. He was awarded the 2000 Nobel Prize in Physiology/Medicine for this research at Columbia University, together with Arvid Carlsson of the University of Göteborg & Paul Greengard of the Rockefeller University. Kandel believed the mechanisms were likely to be evolutionarily conserved, such that a molecular analysis of learning in simple animals like the sea slug Aplysia was likely to reveal mechanisms of general importance. Aplysia has 20,000 nerve cells in its nervous system with 10 ganglia (nerve clusters) each containing 2,000 nerve cells. Each ganglion controls a family of behaviours such that the number of cells committed to a single behavioural act is in the order of about 100 cells. The basic transmission in Aplysia is glutaminergic. There is an NMDA receptor that comes into play in classical conditioning. Serotonin is involved in the short and long term memory process.
These forms of learning shared features of more advanced learning in more complex animals: (1) stages of learning (physiological stages to the laying down of memory) - short term memory (mins), long-term memory (days) & long-term memory (weeks) (2) conversion from short-term memory to long-term memory required repetition (3) long-term memory for each memory differed from short-term memory in that it required the synthesis of new protein. Kandel's lab focused on the implicit memory for sensitisation in Aplysia, a form of learned fear where an animal recognises an aversive stimulus & learns to enhance its reflex response. Sensitisation of the gill-withdrawal reflex by applying a noxious stimulus to another part of the body, such as the tail, enhances the withdrawal reflex of both the siphon and the gill. A single shock will lead to short-term memory lasting minutes. A train of five stimuli will lead to memory lasting days, requiring protein synthesis. More training leads to memory lasting weeks. They examined the neural circuit for this behaviour & found that the number of nerve cells that mediate the behaviour were invariant & they made invariant connections with one another. The 24 sensory neurones that picked up from the siphon's skin made direct connections with 6 motor neurones that innervated the gill. There were interneurons that modulated the firing of the motor neurones. The same was true for the circuits mediating other behaviours (e.g. habituation & classical conditioning). It was determined that (1) all forms of learning led to changes in the strength of synaptic connections in the brain i.e. genes specify precisely how cells connect with each other & environment influences the strength of these pre-existing connections (2) different forms of learning can modulate the strength of the same synaptic connection in opposite ways (sensitisation -> strengthening of connections b/w sensory & motor neurones while habituation -> weakening of the same connections) (3) memory storage is reflected in the duration of the changes. With regard to the short-term process for sensitisation, he found that tactile stimulation activated serotonergic cells & other modulatory cells, which engage a 7-transmembrane spanning receptor that activates adenyl cyclase, responsible for intracellular cAMP synthesis, which activates cAMP-dependent protein kinase enzyme (PKA), which has 2 regulatory spindle-shaped subunits & 2 catalytic subunits. The regulatory subunits normally inhibit the catalytic subunits. When cAMP levels rise, the cAMP binds the regulatory subunits, they undergo a conformational change that frees the catalytic subunit, which permits the enzyme to act in the cytoplasm on ion channels & other machinery to enhance transmitter release. With regard to the long-term process, repeated stimuli activates the serotonergic receptor more & more, increasing the cAMP levels more & more, leading to the regulatory subunits staying off the catalytic subunits sufficiently long so that the catalytic subunit moves into the nucleus, recruiting the MAP kinase in the process, so that they both translocate to the nucleus to activate genes. Therefore, the experience activates genes, a transcriptional activator, including CREB (cAMP response element binding protein), which binds to an element in DNA (cAMP response element) that is a regulatory element that controls gene expression. The activator is under an inhibitory constraint by a repressor, CREB2, that normally prevents the action of CREB1. Therefore, to lay an experience in long-term memory, you not only need to activate the activator but you need to get rid of the repressor. There is a family of inhibitory restraints preventing long-term memory storage that ensure a high threshold. There are allelic variations so that some people have weak forms of the CREB2 repressor, allowing them to put events into long-term memory more easily. There are signalling mechanisms whereby you can remove that restraint, to put an experience into long-term memory very easily, known as "flashbulb" memory. This process of memory storage, which by-passes the usual circuits described through the loss or bypassing of the usual high threshold of multiple repressors of long-term memory storage, may be the molecular mechanism whereby the memory disorder of PTSD occurs. Kandel suggests there it is likely the neurotransmitter modulators can turn on & block memories in situations where people are exposed to frightening situations. When CREB2 is removed & CREB1 is activated, a family of genes, including ubiquitin hydrolase, joins the proteosome, which clips the regulatory subunits (inhibitory constraints on the catalytic subunits), getting rid of a second inhibitory constraint, freeing the catalytic subunit to activate the same substrates activated in the short-term process but for a prolonged period and not requiring serotonin or cAMP signalling. This is the simplest long-term memory -- the co-opting of the same signalling system occurring in short-term memory, but with no signalling required to maintain it, with the catalytic subunits acting as free-canons. This carries the memory for 10-12 hours. What gives memory its enduring quality is the activation of additional genes that give rise to the growth of new synaptic connections, which is the stable, self-maintaining form of memory storage in the brain. Following sensitisation, there was a doubling of the number of synaptic connections, from 1,200 -> 2,800 in Aplysia. There is a retraction of these synaptic connections in the brain with forgetting & following continued habituation (1,200 -> 800 synaptic connections). The homunculus is a representational map in the brains of monkeys & humans, discovered by Wade, Marshall & Penfield, which was initially thought to be stable over time. Mike Muznik discovered that the finger representation varied amongst monkeys & that the internal representation representation of their hand areas expanded when used more, in his experiment, to press a lever to provide food. Tal conducted a study of violinists & found that the right hand had representations like the rest of us (their bowing hand) but their left hands had substantially larger representations. The left hand representation is larger if the violinists started before puberty. Those who started later are better than "wild type" (those who never played) but never catch up to the former group. Therefore, the earlier the experience, the profounder the effect on internal representation (brain structure). These results provide an insight into how a long-term memory is established & the structural changes that maintain it. We know that short-term changes are synapse-specific & not associated with protein synthesis & synapse growth. But the long-term process, by turning on transcription, turns on the whole cell. Kandel showed that transcriptionally-dependent structurally-associated long-term changes can also be synapse-specific, despite each neurone having as many as 1,000 synapses. How does this occur? Kandel tested the model that proteins are sent to all the synapses, when transcription is activated, but only those that had been "marked" by the short-term process can utilise those proteins productively. A single pulse (the short-term process) was found to serve as a marking signal. Therefore, the short-term process has two functions: (1) a mechanism for short-term memory storage & (2) acting to mark the synapse for the long-term process to act at that point to grow new synapses. Dendritic spines contain within them the machinery for local protein synthesis (as well as nucleus). Kandel showed that this is a critical component for stabilising the "mark". Signal transmitters can therefore mediate four major actions:
The above process related to implicit memory storage in Aplysia (procedural memory storage or non-conscious recall of skills & habits involving simple memory such as sensitisation, habituation, classical & operant conditioning). In parallel is a more complicated form of memory storage known as explicit (or declarative) memory storage -- the conscious recall of facts and events, of information about people, places and objects. This memory requires the medial temporal lobe & hippocampus in the mammalian brain. Kandel has examined the synaptic mechanisms contributing to memory for space in mice, a complex form of explicit memory storage. Mice are good at learning things about objects and places, not people, requiring the hippocampus & MTL. Peranderson had characterised the three major pathways of the hippocampus, that synaptic strengthening could be induced by a brief train called long-term potentiation (similar to facilitation in Aplysia) lasting several hours not requiring new protein synthesis but covalent modification like Aplysia but a different 2nd messenger system. In the hippocampus, the modulatory transmitter serotonin does not participate in the short-term process to a significant degree, but is important in the conversion of short to long-term memory. There is a representation for each stage of memory storage, just as in Aplysia. Kandel discovered that when four trains were given, there was a new phase requiring new protein synthesis that required cAMP & cAMP-dependent protein kinase (PKA). This suggested that although the logic for explicit memory is different to implicit storage, the mechanism for converting short to long-term memory might be conserved. They focused on the Schaffer-collateral pathway in the hippocampus, one of its three pathways, because a lesion here in humans (as Larry Squires discovered in patient RB) produces a profound memory deficit. Repeatedly stimulating a glutaminergic synapse activates glutamate receptors & with a train you activate the NMDA receptor, which allows calcium into the neurone, which activates calcium calmodulin dependent kinase 2, which phosphorylates an ion channel giving rise to the short-term change. With repeated stimuli, the calcium influx is adequate to activate an adenylate cyclase, which activates the cAMP dependent protein kinase, MAP kinase, translocates to the nucleus & activates CREB1; there is a CREB2 repressor here too. Genes are turned on which lead to the growth of new synaptic connections. The cAMP process is critical for conversion or STM to LTM, just like in Aplysia. Kandel studied the high-level abstract cognitive module of internal representation of space in the hippocampus of mice that had a single genetic defect (REV-AB, a mutated form of the regulatory subunit that doesn't recognise cAMP in the forebrain). His team determined that there is a normal early phase of LTP (one train leads to a normal STM & early LTM), but a selective defect in the late phase (four trains does not lead to normal LTP). The animal was tested in memory tasks with good temporal resolution. It was placed in a Skinner Box, which was then electrified whilst playing a tone, leading to the animal "freezing" if put back in the box at a later time (Fanslow). The mice have a selective defect for long-term memory, just like mice given an inhibitor of protein synthesis. This has been repeated for mice put in a Barnes maze. Why does the mouse lose its long-term memory for space due to interference with cAMP signalling? The hippocampus has a cognitive map or internal representation of the spatial environment in which it lives in its brain. Different cells encode different positions in space. Different cells fire when the animal assumes different positions in space. All the cells then provide a map of the space. Putting the animal in a new space leads to formation of a new map. In wild type mice, once the map forms over a few minutes it is then maintained. Mutant mice have a good map at one hour but when they are put back in 24 hours later, the cells have rotated, so the map has shifted. Therefore, interfering with the REV-AB gene leads to loss of stabilisation of the map long-term, its internal representation of space. Therefore, explicit & implicit memory use different mechanism for short term storage but a conserved mechanism for long-term storage. This suggests that a common biology underlies memory storage in a number of different contexts. New insights into the nature of modulatory transmitters were gained through the study of memory. These are important both in the initiating of the transition from short to long term memory but also the marking of synaptic connections with local protein synthesis so that long term memory can be stabilised in a synapse specific fashion. Limited understanding has been gained in terms of the systems properties of mechanisms -- how the hippocampus communicates with other brain regions like the neocortex to interact with working memory. Benign senescent forgetfulness is a normal age-related memory loss that occurs in humans > 60 years with onset in 40's unrelated to Alzheimer's Disease. It also occurs as a natural phenomenon in mice. As they age to 12 months , they do less well in the Barnes Maze & at 2 years most mice are quite impaired. There is a selective compromise in the late phase of LTP in these animals, similar to the REV-AB mice. Anything that boosts cAMP signalling will reverse the deficit, such as inhibiting the cAMP-dependent phosphodiesterase that breaks down cAMP or giving more modulatory signal -- dopamine. As the brain ages, there is a reduction particularly of dopamine. D1 D5 agonists (e.g. rolapram) given to mice could restore the late LTP deficit. It causes a partial dose-dependent reversal of their behavioural deficits (Kandel, 2001).
Citation suggestion: Dr Gary Galambos, Cell Biology of the Mind, Evolutionary Psychiatry Interest Group Sydney Australia (http://www.ep.org.au/models/biol_mind.htm) [date accessed]The materials provided on this website may be freely cited but reposting on other websites, publishing or other reproductions, whole or in part, are subject to the written permission of Gary Galambos. Images may be reproduced provided the source is properly acknowledged.Site Copyright (C) 1999-2004 Dr Gary Galambos M.B.B.S. F.R.A.N.Z.C.P.Page last updated: 05 September 2006
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Kandel
(1) delineated a behaviour capable of being modified by
learning (2) defined in cellular detail the neural circuit that was being
modified by learning (3) located within the neural circuit the sites of change
critical for the learning event that carried memory storage over time & used
the techniques of molecular biology to analyse the learning process. They found that
applying a tactile stimulus to the external respiratory organ called the gill
(covered by the mantle shelf ending in the fleshy spout called the siphon)
caused a reflex withdrawal response that could be modified by four types
of learning: (1) habituation (2) sensitisation (3) 
