Neuroscience's Holy Grail has long been the engram - a neuron or set of neurons which physically hold a memory. To a large extent, that search has ended, thanks to scientists like Nobel prizewinner Eric Kandel, who won the 2000 Nobel Prize for his discoveries in the neurochemistry of learning.
Dr. Kandel began his groundbreaking work by using Pavlov's dog-conditioning techniques to study neural changes in the California sea snail Aplysia as it learned. Aplysia has only 20,000 neurons, and they are the largest in the animal kingdom, visible to the naked eye. This makes the neural network easy to manipulate and identify, and makes Aplysia to neurobiologists what fruit flies are to geneticists and rats to behavioral psychologists.
He began by stimulating Aplysia’s sensory neurons with electrodes, then used the process of elimination, neuron by neuron, to map out the entire neural circuit controlling gill withdrawal - a simple behavior in which Aplysia adapts and learns from its environment.
Just as you would jerk your hand away after touching a hot stove, Aplysia reflexively withdraws its gills in response to aversive (unpleasant) stimulation. In comparison with yours, its brain is primitive, essentially two neural bundles called ganglia; but just as with more complex animals, it can learn. In Aplysia, just as in humans, "practice makes perfect"; repeating a stimulus converts a short-term memory - lasting minutes - into a long-term one, lasting days, weeks or a lifetime.
In Aplysia, the gill withdrawal reflex is controlled by just 24 sensory neurons which enervate (send signals to) six motor neurons. Between them are "middle managers" - interneurons which act as modulators, either excitatory (dialing up the likelihood of firing) or inhibitory (dialling down the likelihood of firing). Stimulating the tail activates these interneurons, which excrete the neurotransmitter serotonin. This triggers an excitatory response, and the motor neurons fire, causing muscular contractions. The end result is that the animal withdraws its gill in response to a shock.
Aplysia's neurons (like yours) are hardwired - physically and functionally fixed by instructions encoded in DNA - so they aren't capable of significant change, such as increasing in number or changing in function or location. However, the connections between them are extremely flexible. Learning changes their signalling efficiency.
Neurons communicate via these connections, across tiny gaps called synapses, which excrete chemical messengers called neurotransmitters.
Lasting memories are preserved by the growth and maintenance of these synapses. In other words, memories are encoded in the connections between neurons.
Dr. Kandel's early experiments involved training Aplysia in a learned fear response called sensitization, in which repeated exposure to an aversive stimulus makes a creature more sensitive to that stimulus. For example, a war veteran sensitized to sounds like gunfire might jump at the slamming of a car door. Similarly, an Aplysia snail which has become sensitized to shocks on its mouth organ (siphon) will also respond to stimuli applied to its tail. This is because, simply stated, "practice makes perfect" - repeated exposure converts short term memory into long-term memory, via physical growth (protein manufacture).
After tracing the specific neural circuits which control gill withdrawal, Dr. Kandel devised a technique for growing cell cultures in a petri dish from larval snails, creating the absolutely simplest learning circuit - just two live neurons, one sensory, one motor.
He and his colleagues could then substitute tail shocks with a squirt of serotonin, and investigate the specific molecular processes which lead to memory formation.
He discovered there are two separate chemical sequences, one for building short-term, and one for building long-term memories. Short-term memory - which lasts for seconds to minutes - comes from increasing a presynaptic neuron's ability to emit neurotransmitters.
This neurotransmitter increase develops from a six-step chemical sequence called a signalling cascade:
When the snail's tail is given a mild shock, the neurotransmitter serotonin is released by interneurons (intermediate neurons which amplify or dampen sensory neuron input to targets such as motor neurons). This neurotransmitter binds to protein receptors embedded in the membrane of a recipient (postsynaptic) neuron.
These serotonin-activated (serotonergic) receptors prompt the conversion of ATP, the cell's natural "fuel" into a special chemical signal, a secondary messenger called cAMP. cAMP is called a secondary messenger because it transfers an external signal from the membrane to molecular machines inside a cell.
In Aplysia, the cAMP signal activates a protein on-off switch called a kinase (PKA). This kinase migrates back to the membrane and causes a shape change (phosphorylation) in special calcium channels, proteins embedded in the membrane which act like selective gateways.
The shape change temporarily opens these channels to calcium ions, which flow into the synaptic terminals of the neuron.
Calcium ions function as the chemical switch that triggers increased neurotransmitter release. This increased neurotransmitter release is the physical basis of a short-term memory formation.
To convert this short-term memory into a long-term memory, repeating the stimulation triggers an additional chemical cascade which activates physical growth - protein manufacture.
This anatomical change is the sprouting of new synapses and/or synaptic branches called dendrites. Dendrites are tendril-like appendages which reach out to connect with neighboring neurons.
This anatomical change is called long term potentiation, so-called because, over the long term, the potential for a neuron to fire has been enhanced - the postsynaptic neuron has become more sensitive to stimuli and fires more frequently. This phenomenon involves the long-term modification of the synaptic connection.
In 2010, MIT's Gertler Lab created a spectacular film of this growth process in living, cultured mouse neurons. Here you can see the growth of neurites, neural buds which sprout into dendrites.
In Aplysia, repeating the stimulation (mild shocks) creates higher amounts of serotonin release, triggering a release of higher amounts of the secondary messenger cAMP. This more persistently activates the kinase (PKA) switch. Just as in short-term memory formation, this kinase migrates back to the cell membrane and opens protein channels, and calcium ions rush into the synaptic terminals, triggering neurotransmitter release.
However, the repeated stimulation also results in a second action, that ultimately results in physical growth of the neuron, through protein synthesis.
A subunit of the kinase moves into the neuron's cell nucleus, where it activates a special protein called a transcription factor (CREB protein or cAMP Responsive Element Binding protein). This transcription factor binds to specific DNA sequences (genes), activating them to start manufacturing new synapses, from proteins building blocks like neurexin and neuroligin.
Genes are segments of the DNA molecule arranged in specific sequences to guide the synthesis of messenger RNA (mRNA).
mRNA is a molecule which copies the genetic code from DNA and uses it as a template to guide the manufacture of proteins - the most complex molecules on Earth, which carry out virtually all biological functions, from forming tissues to carrying out chemical processes vital for life.
To accomplish this, mRNA peels off from its parent DNA, then travels outside the cell nucleus to a special region of the cell body - a mazelike series of tubes called the endoplasmic reticulum. Here the mRNA is used by mini protein factories called ribosomes as a blueprint for assembling short building blocks (amino acids) into proteins.
Dr. Robert Singer and colleagues at the Albert Einstein College of Medicine developed a means of filming this neural mRNA synthesis. They attached harmless flourescent chemical tags to mRNA molecules so they could be filmed within live mouse neurons. Here is the world's first film of a memory being formed in real-time: https://www.youtube.com/watch?v=6MCf-6It0Zg
Mammals like mice have evolved a special memory-creating and storing brain structure called a hippocampus, named after the Greek word for seahorse, because of its curly shape.
Dr. Singer's team stimulated neurons in the mouse's hippocampus, where "episodic" and "spatial" memories are formed and stored (episodic memories are the conscious mental record of our life events and the sequences in which they occur, while spatial memories constitute navigational guides through an organism's environments).
The hippocampus acts as a sort of amplifying loop, receiving signals from the cortex - the area where conscious thought and ultimate brain control resides - and sending signals back. As memories are consolidated - stabilized into potentially lifelong memories - they are gradually transferred from the hippocampus to the cortex during sleep.
Dr. Singer's team targeted the mRNA which carries the code for a structral protein called beta-actin, central to long-term memory formation. In mammals, beta-actin proteins strengthen synaptic connections by building and altering dendritic spines.
Within 10 to 15 minutes of stimulation, beta-actin mRNA began to emerge, proving their neural stimulation had triggered transcription of the beta-actin gene. The film shows these fluorescently glowing beta-actin mRNA travelling from neural nuclei to their destinations in the dendrites, where they will be used to synthesize beta-actin protein.
Neural mRNA uses a unique mechanism the Einstein team calls "masking" and "unmasking", which allows beta-actin protein synthesis only when and where it is needed. Because neurons are comparatively long cells, the beta-actin mRNA molecules have to be guided to create beta-actin proteins only in specific regions at the ends of dendrite spines.
According to Dr. Singer, just after beta-actin mRNA forms in the nuclei of hippocampal neurons, it migrates out to the cells' inner gel (the cytoplasm). At this point, the molecules are packed into granules whose genetic code is inaccessible for protein synthesis.
Stimulating the neuron makes the beta-actin granules fall apart, unmasking the mRNA molecules, and making them available for beta-actin protein synthesis. When the stimulation stops, this protein synthesis shuts off: after synthesizing beta-actin proteins for only a few minutes, the mRNA molecules abruptly repack into granules, returning once again to their default inaccessible mode.
In this way, stimulated neurons activate protein synthesis to form memories, then shut it down. Frequent neural stimulation creates frequent, controlled bursts of messenger RNA, resulting in protein synthesis exactly when and where it's needed to strengthen synapses.
Of course, the process involves more than just a single gene - in fact a dizzingly huge number are involved. Learning involves large clusters of genes within huge numbers of cells.
Florida U. Neuroscience Professor Leonid Moroz has tracked specific gene sequence expression in Aplysia neurons, and estimates that any memory formation event will alter the expression of at least 200-400 genes. This is out of over 10,000 which are active every moment of the simple marine snail's life.
Moroz's team zeroed in on genes associated Aplysia's feeding and defensive reflexes, and found over 100 genes similar to those linked to every major human neurological illness, and over 600 similar genes which control development. This shows these genes emerged in a common ancestor and have remained nearly unchanged for over half a billion years of both human and sea slug evolution.
Human brains contain about one hundred billion neurons, each expressing over 18,000 genes, at varying levels of expression (protein synthesis rates), with over 100 trillion connections between them. Aplysia has a comparatively much simpler nervous system, with only 10,000 easily identifiable, large neurons. However, it is still capable of learning, and its neurons communicate using the same general principles as human neurons.
According to Dr. Moroz, if genes use a chemical alphabet, there is a kind of molecular grammar, or set of rules controlling the coordinated activity of gene expression across multiple neural genes. To understand memory or neurological diseases at a cellular level, scientists need to learn these grammatical rules.
Like the Einstein team, Dr. Eric Kandel also went on to study the mouse hippocampus, and found it makes use of a chemical cascade similar to that of Aplysia neurons. But in mouse - and human - hippocampi, the chemical sequence for memory formation is slightly different:
In the hippocampus the changes which lead to memory formation occur in the receiving (postsynaptic) neuron rather than the sending (presynaptic) neuron. This process occurs in two stages:
During Early LTP, the excitatory neurotransmitter glutamate is released. This glutamate acts upon two types of receptors, NMDA (N-methyl-D-aspartate) and AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors. Normally, only the AMPA receptors respond, but repeated stimulation activates the NMDA receptors as well. These open ion channels in the membrane, allowing calcium ions to flow into the neurons, and this activates a (different) second-messenger kinase, CAMK2A (calcium calmodulin kinase). CAMK2A triggers a chemical cascade that results in the growth of additional AMPA receptors.
In Late LTP, repeated stimulation activates a second system, which releases the modulatory neurotransmitter dopamine.
Like serotonin in Aplysia, dopamine acts upon the hippocampus like a sort of volume switch, dialing up neural activity. (Disruptions in the dopamine system are at the heart of disorders like schizophrenia and Parkinson's disease.)
Dopamine binds to its own receptors on the cell membrane, increasing production of cAMP, activating the PKA-CREB sequence which starts protein synthesis for new dendrite/synapse formation.
Mice are also capable of much more sophisticated memory feats than sea slugs, such as memorizing spatial layouts in a manner much like humans.
In his experiments with mice, Dr. Kandel found three principles at work in learning. First, when two or more neurons converge, stimulating a third neuron, it creates a logic circuit known as a “coincidence detector”. This circuit underlies associative learning - we link A with B, cause with effect, touching a hot stove with pain, etc. Variations of this circuit underlie many forms of mental computation.
Secondly, he found that mice hippocampi use place cells, special pyramidal neurons which fire in response to specific locations. These place cells act as mental markers, firing at the sight of certain landmarks, allowing mice to encode internal navigational maps.
The permanency of spatial memory varies based upon how much attention is applied to navigation, and levels of attention appear to correspond to levels of the neurotransmitter dopamine. In other words, generally speaking, the more attention (and dopamine) that is applied, the more effectively a route will be memorized. Dopamine modulates the effects of learning - acting like a volume knob that controls how much data is converted from short-term into long-term memory.
Thirdly, Dr. Kandel's team studied motor learning, and concentrated their search in the cerebellum (latin for "Little Brain"), known to be the master coordinator of complex movements.
All animals practice complex sets of coordinated movements until they perfect them, from learning to drive a car (for humans) to catching flies with the tongue (for frogs). Their first attempts may be unsuccessful, but with perseverance, eventually there will be mastery. These are procedural memories, a form of motor learning which is central to animal behaviour. And just as with episodic and spatial memory, procedural memory is also dependent upon changes in synaptic strength.
With repeated practice, there will be growth (of new dendrites and synapses) in a "midline" (superior medial) strip atop the brain's outer surface, the motor cortex, which plans and issues commands for voluntary movements - sequences of muscle contractions.
Coordinating the process, the cerebellum sits behind the brainstem at the top of the spine. It acts as a kind of coach, comparing performance - the actual changing positions of limbs, trunk and head in space - with intentions. Through this "feed forward" mechanism, the cerebellum corrects motion, ensuring smooth timing and orchestration of the many muscle contraction signals in complex movements. Learning to orchestrate these movements depends to a large extent upon special Purkinje cells.
Neurons fire when special protein channels embedded in the cell membranes open, allowing electrically-charged ions to flow inward. Among these, the HCN1 ion channel (Hyperpolarization-activated, Cyclic Nucleotide-regulated nonselective cation), is key to motor learning in Purkinje cells of the cerebellum.
To study the role of HCN1 ion channels in motor learning, Dr. Kandel and his team bred mutant mice with neurons that lacked HCN1 channels in different brain regions.
They then ran their mutant mice (along with a control group of normal mice for comparison) through a series of complex motor tests, which included swimming through water mazes and balancing on rods. These tests required complex, repetitive and coordinated motor output. The mice were also conditioned with simpler motor behaviours like eye blinking.
Mice with no HCN1 channels in their Purkinje cells could still perform simple movements like eye blinking, but had extreme difficulty in performing complex behaviours like swimming and balancing. In contrast, mice without HCN1 channels in their forebrain but with normal cerebella had no problem in performing complex behaviours. This shows that HCN1 channels in Purkinje cells are the key to complex motor learning.
When negative currents were applied to Purkinje cells, those lacking HCN1 channels took longer to return to normal firing activity levels than normal Purkinje cells. This means HCN1 channels stablilize Purkinje cells, allowing them to quickly recover from activation and return to normal functioning. In complex, repeated behaviours, Purkinje cells receive repetitive bursts of input, and this ability to recover quickly is vital to influencing motor activity.
In the end, while it is good to understand the principles by which your brain manufactures memory, here is some more practical advice on how to make the most of what you've got:
1. Sleep at least seven to eight hours at a regular time every night
2. Take fish oil supplements
3. Exercise every day
4. Limit or eliminate your intake of alcohol, tobacco, and fatty or sugary foods
5. Pay full attention to what you want to remember
6. Space your learning out; cramming does not help long-term memory retention, especially all-night study sessions.
7. Think of how the information you want to learn relates to you or to things you already know
8. Study a foreign language and a musical instrument
9. Study in the same environment, at the same time every day
10. Keep your study environment free from clutter and distractions
11. Get up and do something different about every 15 minutes before returning to your studies
12. Spend time in mentally stimulating environments where you have new experiences and interact with many different sorts of people from many cultures and backgrounds
13. Reduce your life stresses to a minimum
If you want to read about the Princeton, MIT, Oxford, Harvard, Yale, Tokyo University and other studies upon which these recommendations are based, I invite you to get a copy of my book, The Path Book II. It also explains the different systems of your body, and the science-supported, most effective nutrition, exercise, love, happiness and success advice found in any book to date.