The Chemistry of Perpetual Memory

I. Short-, Long-, and Ultralong-Term Forms of Learning

II. Use of Invertebrate Preparations to Study Simple Forms of Learning

III. Short-Term Facilitation in Aplysia is Mediated by Changes in the Levels of Intracellular Second Messengers

Thus, Reaction Category 1: Altered Levels of Second Messengers

IV. Intermediate-Term Facilitation in Aplysia Involves Altered Gene Expression and Persistent Protein Kinase Activation—A Second Category of Reaction

Thus, We Have Reaction Category 2: Generation of Long Half-Life Molecules

V. Long-Term Synaptic Facilitation in Aplysia Involves Changes in Gene Expression and Resulting Anatomical Changes

VI. Three Attributes of Chemical Reactions Mediating Memory

A. Long-Term Memory in Mammals

B. Long Half-Life Reactions

C. Ultralong-Term Memory: Mnemogenic Chemical Reactions

VII. Summary: A General Chemical Model for Memory

Almost no human has a good intuitive grasp of the ephemeral nature of bio-molecules. Proteins and metabolic intermediates turn over at amazingly fast rates in a mammalian cell, including in a neuron in the CNS. Biochemical bonds are generally quite labile things, and the ongoing breakdown and resynthesis of the constituent molecules of the cells of your body occurs at what is, relatively speaking, breakneck speed. It is difficult to truly grasp this fact in the face of what appears to be such stability and consistency of both our bodies and our minds.

Neuroscientists are not immune to this lack of intuition. The apparent stability of synapses, cells, behavioral patterns, and CNS morphology in our everyday experiments tends to deceive us in our thinking about neuronal function. LTP is long-lasting and stable over the course of a day. Memories are measurably preserved over a significant fraction of an animal's lifetime. This constancy and durability of CNS-based phenomena obscures the underlying rapid turnover of most of the constituent molecules that provide their molecular underpinnings.

FIGURE 1 Hypothetical graph of 32P-PO4 reaching steady state. Steady-state is the point at which the rate of incorporation of the radioactive label equals the rate of breakdown of phosphate bonds in labeled proteins, RNA, or DNA. Isotopic equilibrium is the point at which all phosphate-containing molecules throughout the cell have achieved steady-state labeling. See text for additional discussion.

FIGURE 1 Hypothetical graph of 32P-PO4 reaching steady state. Steady-state is the point at which the rate of incorporation of the radioactive label equals the rate of breakdown of phosphate bonds in labeled proteins, RNA, or DNA. Isotopic equilibrium is the point at which all phosphate-containing molecules throughout the cell have achieved steady-state labeling. See text for additional discussion.

I come from a background in signal transduction, where issues of molecular turnover are dealt with on a more daily basis. I will use one example from my own experiments to illustrate my point of rapid molecular turnover in cells, although the biochemistry and signal transduction literature is full of thousands of similar examples.

Biochemists are fond of using radioactive tracer compounds to track specific molecular events in cells. A typical experimental design is, for example, to introduce 32-P labeled inorganic phosphate (32PO4-) into the culture medium surrounding a neuron maintained in vitro. It is then taken up and incorporated into phosphate-containing molecules in the cell. This radioactive label can then be used to measure the extent of phosphate incorporation into cellular proteins by measuring their level of radioactivity. This is a direct measure of protein phosphorylation by kinases in the cell, or, more accurately stated, it is a direct measure of the steady-state ratio of kinase to phosphatase activity acting on a specific substrate at a specific time point.

The control experiment that one has to do in order to validate this type of approach is to demonstrate that the 32P isotope has reached isotopic equilibrium in the cell. This simply means that the 32P-PO4 must have completely dispersed itself throughout all the relevant pools of phosphate that already existed in the cell, which are, of course, not initially radioactive. One needs to know that a change in radioactive content is truly a reflection of a change in phosphate content in, for example, a substrate protein. One demonstrates this by showing experimentally that the 32P has reached isotopic equilibrium, that is that the labeled compound has come to a random distribution throughout all the nonradioactive phosphate that was previously there.1 Thus, a change in 32P content is truly a reflection of a change in phosphate content in a protein.

There are several ways to demonstrate that the cells under study have reached

1DNA is likely not at equilibrium in these types of experiments.

isotopic equilibrium in the pool of molecules you are investigating. The simplest, if you are interested in protein phos-phorylation, is to show that the total 32PO4 in cellular proteins has reached a plateau level (see Figure 1). This means that the incorporation of label into cellular proteins has achieved a steady-state level—the rate of increase in label in proteins has now been matched by the rate of decrease in label in proteins. The radiolabel has reached equilibrium and is no longer showing either a net increase or a net decrease. One can also specifically measure the 32PO4 content of cellular ATP, ADP, and AMP and4 show that they are at a steady-state level as well. This means that the phosphates in the alpha, beta, and gamma positions of all these adenine nucleotides has undergone turnover at least once and that the net rate of 32P incorporation is matched by the net rate of 32P loss.

To further refine the control experiment, you can show that if the cell is stimulated with a neurotransmitter, for example, there is no additional increase in overall phosphate content in all the cellular proteins or in cellular ATP. This means there is no hidden pool of phosphate in the cell that is accessed only under the conditions of stimulation.

I did these types of experiments as part of studies I did in Eric Kandel's lab when we were studying substrate protein phos-phorylation in Aplysia sensory neurons, which we maintained in vitro and labeled with 32PO4 (1). How long does it take for 32P-phosphate to reach isotopic equilibrium in an Aplysia sensory neuron? The answer is: less than 24 hours, a number that is typical for neurons in culture, and mammalian cells in general, when maintained at 37°C.

But think about the implications of this number. It means that essentially every phosphate bond at all three positions in the entire cellular ATP pool, and essentially every phosphate moiety in every cellular phosphate-containing protein, has been broken down and resynthesized in a 1-day time period! As a first approximation every day all the phospho-proteins in your brain have had their phosphate removed and replaced. Any researchers who are considering protein phosphorylation as a mechanism that contributes to information storage for any appreciable period of time must remember that there is continual breakdown and resynthesis of the basic molecular structure underlying the memory.

This high rate of turnover is not limited to phosphorylation events. Protein constituents of neurons are broken down and resynthesized at a rapid rate as well. Andrew Varga in my laboratory has been investigating the turnover of the Kv4.2 potassium channel that we discussed in Chapters 5 and 6 and found that its half-life in a cell is about 4 hours. This means that roughly speaking the entire cellular content of this potassium channel is broken down and resynthesized over a 1-day period. Studies of AMPA receptors done in Rick Huganir's lab have shown that the half-life for this protein in neurons is approximately 30 hours (see reference 2 and Figure 2). These investigations specifically measured the GluR1 cell surface pool, along with the total cellular GluR1. The implication of this finding is that neuronal cell surface AMPA receptors are completely broken down and resynthesized from scratch over the course of a week.

A rapid rate of protein turnover is the rule rather than the exception. This is illustrated by the simple experiment shown in Figure 3. In this experiment, guinea pig hippocampal slices were prepared and labeled in vitro with 35S-methionine for just 30 minutes. Thus, any protein that is labeled with 35S was synthesized de novo from precursor amino acids over the course of this 0.5-hour time frame, or even less because the precursor methionine was added to the extracellular medium and had to cross the cell membrane and be incorporated into methionyl-tRNA before it could be incorporated into a cellular protein. After the labeling period, area CA1

FIGURE 2 Half-life of AMPA receptors. Shown here is the half-life of cell-surface GluRl in spinal cord neuronal cultures at day 4 and day 11 in vitro. Cell-surface molecules were selectively labeled by reacting them with biotin. Plates of spinal cord neurons were biotinylated at day 4 and day 11 and recultured for 0-24 hr, at which time cell extracts were harvested, sonicated, and frozen. Subsequently, these samples were thawed and incubated with streptavidin-linked beads, and the streptavidin-precipitated material was loaded onto gels. (Streptavidin selectively binds biotinylated proteins with very high affinity.) (A) A standard curve including serial dilutions of the t = 0 streptavidin-precipitated material was included on each gel for purposes of quantitation. After transfer, gels were probed with a GluRl-rective antibody in order to quantitate the amount of glutamate receptor remaining from the initial labeling with biotin. (B) The natural log of the percent of remaining surface GluRl was plotted against time, and half-lives were calculated from the regression slopes of the resulting lines. (C) Half-life and percent of receptor on surface experiments is summarized. A paired t test demonstrated a significant increase in the half-life of surface GluRl from day 4 to day 11 (p < .05). Data and figure legend adapted from Mammen, Huganir, and O'Brien (2).

was dissected out, and cellular proteins were separated on the basis of charge and molecular weight using two-dimensional gel electrophoresis. As you can see in Figure 3, at least a couple hundred different protein spots were labeled sufficiently to be detectable using autoradiography of this

2-D gel. Thus, hundreds of proteins in hippocampal area CA1 are being synthesized at a sufficiently rapid rate that they show up using this brief period of pulse-labeling. It is reasonable to infer that, because the cell is at steady state, (i.e., the cells are not growing larger), the rate of breakdown of these same proteins is matching their high rate of synthesis. These data are just a specific example from the hippocampus of what is generally known about protein synthesis—protein half-lives in the cell range from about 2 minutes to about 20 hours, and half-lives of proteins typically are in the 2- to 4-hour time range.

Okay, you say, that's fine for proteins, but what about "stable" things like the plasma membrane and the cytoskeleton? Neuronal membrane phospholipids turn over with half-lives in the minutes-to-hours range as well (3, 4). The vast majority of actin microfilaments in dendritic spines of hippocampal pyramidal neurons turn over with astonishing rapidity—the average turnover time for an actin microfilament in a dendritic spine is 44 seconds (see reference 5 and Figure 4).

The bottom line of all this is that if you are thinking of a single phosphorylation event or the synthesis of a new protein or the insertion of a membrane receptor or ion channel or even the formation of a new synapse as being capable of storing memory for any appreciable period of time, you must readjust your thinking. As a first approximation, the entirety of the functional components of your whole CNS have been broken down and resynthesized over a 2-month time span. This should scare you. Your apparent stability as an individual is a perceptual illusion.

These considerations apply equally well to anatomical structures. Direct measurements of fractional breakdown rates of skeletal muscle protein indicate that your muscle mass is broken down and resyn-thesized at about 3-4%/day (6). That's equivalent to a complete turnover of what you think of as your "body" in about a month. Development puts everything in

Low V (approx 5 kDa)

FIGURE 3 Rapid rate of protein turnover. In this experiment (Sweatt and Kandel, unpublished), two-dimensional gel analysis of 35S-methionine-labeled proteins from area CA1 of guinea pig hippocampus reveals rapid and extensive labeling of proteins over a very short time period. This implies a fairly rapid breakdown and resynthesis of the labeled proteins. See text for additional explanation of the experiment.

0 50 100 150

FIGURE 4 Rapid basal actin turnover in dendritic spines. The turnover of actin in dendritic spines from neurons grown for 14-16 days in vitro (DIV) was indistinguishable from those grown for 22-24 DIV. Under both conditions, actin microfilaments undergo essentially complete breakdown and re-formation about every 2 minutes. Actin turnover was assessed using fluorescent actin and monitoring recovery from photobleaching. Adapted from Star, Kwiatkowski, and Murthy (5).

its right place, but maintaining that anatomical structure is an active process, and the component molecules are turning over with surprising rapidity.

In pursuing these thoughts, we are bumping up against one of the philosophical discussions concerning the scientific approach that has arisen repeatedly throughout human history. Is the human brain really capable of understanding itself? The fact of the complete turnover of cellular signaling constituents on the time frame we are talking about flies completely in the face of our perception. The facts are at odds with the apparent stability that we perceive in ourselves and others. Our memories last. Our behavior is consistent. Our facial features stay the same. However, our intuition based on our day-to-day perceptions is directly at odds with the available experimental data.

The memory biologist must overcome this cognitive dissonance and come to grips with the rapid turnover of individual molecular components in the nervous system, to be able to begin to understand memory storage in earnest. In this chapter, we will think about memory processes from this perspective. We will think about them as chemical reactions that subserve persisting changes of varying durations. We will develop a generalized chemical categorization of the types of chemical reactions that underlie memory storage. I will describe three types of memory-storing reactions: short-term reactions mediated by transient changes in second messenger levels, long-term reactions mediated by species with long half-lives, and ultralong-term or mnemogenic reactions that can store memory indefinitely, even in the face of ongoing turnover of the molecules involved. Using this framework, I will give some specific examples of the various types of chemical reactions that may and must underlie memory storage in biological systems.

In this chapter, I will use examples from both invertebrate and mammalian learning systems, picking and choosing with relish those examples that I think best illustrate the principles involved. I should note before setting out that some parts of this chapter are adapted from Roberson and Sweatt (7).

In the first part of this chapter, I also will go outside the hippocampus, and even outside the mammalian CNS, and choose several examples from the Aplysia model system. Introducing a whole new model system in the last chapter of a book may seem odd. However, in many ways, the details of the specific molecular mechanisms underlying short, intermediate, and long-term memory are better understood in this system than in any mammalian system. This is particularly true as relates to the mechanisms for transitioning from one memory phase to the next while preserving the same cellular read-out. Thus, because we are trying to talk about specific chemical reactions involved in memory in this chapter, more details of the specific molecules involved is quite helpful.

Also, Aplysia has a long, storied, Nobel Committee-approved status in the memory field. In a sense, no book on memory mechanisms would be complete without some, at least passing, reference to studies using this preparation. In the next few sections, I will give a brief introduction to the Aplysia model to set the framework for the more detailed chemical description that will follow (see references 8-11 for reviews). I also will later in the chapter draw additional parallels to hippocampal molecular information storage processes where appropriate.

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