Genetic Substrates of Memory: Amygdala
Scientists believe that the amygdala plays a critical role in the ability to learn and remember certain types of information. Converging evidence from studies using humans as well as a variety of laboratory species support the idea that the amygdala, together with a network of closely interconnected brain regions, allows people to store long-term memories for events that are emotionally important and to respond appropriately to stimuli in the environment that signal threat. The ability to store long-term memories of this type is likely due to experience-dependent changes in gene expression within amygdala neurons. Many scientists and clinicians believe that understanding the functions of the amygdala may be the key to the treatment and prevention of human anxiety disorders and other psychiatric problems.
Scientific knowledge about how the amygdala works comes largely from the development and widespread use of Pavlovian fear-conditioning with rodents as a model system throughout the late 1980s and 1990s. In a typical fear-conditioning experiment, a rat is placed in a distinctive observation chamber where a simple signal such as a flashing light or a tone (the conditional stimulus, or CS) is repeatedly activated prior to delivery of a mild electric shock to the animal's feet. The rat will learn that the signal predicts shock and will display species-appropriate fear behaviors when presented with the CS alone after training. The animal will also learn to fear the apparatus, or context, in which the training took place. This relatively simple form of learning is rapidly acquired, easy to observe in the laboratory, retained indefinitely, and completely disrupted in animals with significant damage to the amygdala (Davis, 2000; LeDoux, 2000; Maren, 2001).
Fear conditioning experiments have shown that discrete anatomical regions within the amygdala may play selective roles in the learning process. The lateral nucleus and closely related cell groups within the basolateral region of the amygdala receive direct and indirect synaptic input from sensory systems that detect and process stimuli in the animal's environment. On the other hand, the central nucleus of the amygdala appears to play a key role in the expression of behavioral and physiological reactions to fear-provoking stimuli through its extensive connections with the brain stem. The amygdala is important for learning about new danger signals in the environment as well as for producing the appropriate behavioral reactions to those signals (Helmstetter and Bellgowan, 1994). Changes in gene expression within amygdala neurons may accompany both learning and response performance in fear conditioning.
Learning Can Alter Gene Expression in the Amygdala
A number of studies have been able to show that exposure to the training procedures used in Pavlovian fear conditioning will selectively alter the expression of messenger RNA (mRNA) or protein in the amygdala. Immediate early genes (IEGs) tend to be minimally expressed in quiescent cells but are often rapidly and dramatically activated when cells are stimulated by synaptic inputs. Many IEGs encode transcription factors that ultimately regulate other gene products expressed later after stimulation. In one of the first studies of gene expression during fear conditioning, Serge Campeau and colleagues (1991) showed that the amount of mRNA coding c-fos in the amygdala was elevated by exposing rats either to foot shock itself, or to an environment that had been previously paired with foot shock. Therefore, the time-dependent storage of new memory following the shock experience as well as the neuronal activity in the amygdala provoked by exposure to a fear-producing stimulus may increase production of FOS protein. Subsequent experiments have continued to shed light on this and related phenomena. Several different aspects of the fear conditioning procedure appear to activate c-fos transcription and the level of expression of FOS protein correlates with how well animals learn during fear conditioning (Radulovic, Kammermeir, and Spiess, 1998).
J. B. Rosen and colleagues (1998) reported that while mRNA for c-fos was elevated in the amygdala in animals that learned fear conditioning as well as in control subjects that did not learn, another IEG, "early growth response gene 1" (egr-1), showed a pattern of message expression in the lateral nucleus of the amygdala that was specific to learning. Egr-1 mRNA is selectively expressed in the amygdala between fifteen and thirty minutes after learning and only in the region of the amygdala likely to undergo critical synaptic plasticity during the formation of memory. Importantly, simple retrieval of fear memory and performance of fear responses do not alter egr-1 expression supporting the idea that this IEG, unlike c-fos, may play a selective role in the formation of long-term memory (Mackani and Rosen, 2000).
Alterations in gene expression in the amygdala are not restricted to IEGs nor are they solely related to the initial storage of information. Cyclic AMP responsive-element binding protein (CREB) is a transcription factor that plays a key role in memory formation in a number of species (Silva, Kogan, Frankland, and Kida, 1998). In mice exposed to a simple fear-conditioning protocol, levels of phosphorylated CREB (pCREB) in the amygdala were elevated in trained animals relative to controls during the memory consolidation period after conditioning (Stancui, Radulovic, and Speiss, 2001). Increased phosphorylation and thus activity of CREB is also seen in the amygdala when rats retrieve memories learned in fear conditioning that have already been stored (Hall, Thomas, and Everitt, 2001). Retrieval and/or emotional responding also causes cells in the central nucleus to increase production of mRNA for the neuropeptide enkephalin (Petrovich, Scicli, Thompson, and Swanson, 2000).
Manipulating Gene Expression in the Amygdala Can Affect Learning
Demonstrating that cells in the amygdala express a different quantity or a unique pattern of gene products while animals are learning is an important step toward understanding the mechanisms of long-term memory. However, one can see that just because a particular protein or mRNA appears to change when subjects learn, it does not necessarily follow that this gene product is essential for formation of the memory. In order to know if something is required for memory formation, one must be able to manipulate it directly.
D. J. Bailey and colleagues (1999) took a first step in this direction by asking a more general question. They tested whether or not the general ability of cells in the amygdala to synthesize new mRNA is critical for the formation of long-term memory in fear conditioning. Actinomycin-D, a drug that selectivity prevents cells from copying information from DNA in the nucleus onto new mRNA molecules (transcription), was microinjected into the amygdala of rats prior to training with foot shock. Twenty-four hours after training the animals were returned to the laboratory and tested for memory of what was learned the day before. The results of this experiment are summarized in Figure 1. Suppressing transcription of new mRNA in the amygdala greatly disrupted the rats' ability to remember what happened during training. This experiment was one of the first direct sources of evidence that mRNA synthesis, and by implication the synthesis of new protein, is required in the amygdala during the acquisition of fear learning.
Several other studies have supported the idea that the synthesis of new proteins in the amygdala is critical for memory formation. The molecular events occurring during the period directly after training when long-term memories are consolidated are of particular interest. Blocking the translation of mRNA into new protein in the amygdala during this post-training period prevents long-term retention of fear conditioning, as does selective disruption of the activity of the enzyme protein kinase A (PKA) (Schafe et al., 2000). In taste-aversion learning, where animals associate a specific taste with illness, pretraining amygdala injections of antisense oligodeoxynucleotides (ODNs) that selectively prevent the translation of c-fos mRNA into protein prevent the formation of taste-aversion memory (Lamprecht and Dudai, 1996).
Studies in which mRNA transcription or protein translation is globally affected within amygdala neurons are somewhat limited given their lack of specificity. Suppression of specific proteins with antisense ODNs has several advantages but also suffers from important limitations. One of the most exciting recent technical advances in this area involves the use of viral-vector gene-transfer technology to selectively introduce and promote the expression of target gene products within restricted brain regions. For example, S. A. Josselyn and colleagues (2001) were able to cause the selective overexpression of CREB in rat amygdala neurons prior to training in fear conditioning. Rats that had significantly more CREB being expressed actually showed better memory than control animals. This type of evidence, when coupled with the variety of experiments showing that down regulation of CREB impairs memory, makes a very convincing case for the role of this particular transcription factor in memory formation.
Neuroscientists are developing a clear picture of how the amygdala contributes to memory at the circuit and neural systems level. Comparatively little is known about the molecular events taking place within amygdala neurons as an organism learns. Several gene products have been identified that undergo changes in expression that appear to be related to learning. In some cases, direct manipulation of these proteins can have an effect on the formation of memory. As interest in this area increases and the technology available to scientists improves, new genes that are involved in amygdala-dependent memory phenomena will continue to be described.
See also:EMOTION, MOOD, AND MEMORY; GENETIC SUBSTRATES OF MEMORY: CEREBELLUM; GENETIC SUBSTRATES OF MEMORY: HIPPOCAMPUS; GUIDE TO THE ANATOMY OF THE BRAIN: AMYGDALA; HORMONES AND MEMORY; LONG-TERM POTENTIATION: AMYGDALA; NEURAL SUBSTRATES OF CLASSICAL CONDITIONING: FEAR CONDITIONING, FREEZING; NEURAL SUBSTRATES OF CLASSICAL CONDITIONING: FEAR-POTENTIATED STARTLE; NEURAL SUBSTRATES OF EMOTIONAL MEMORY; PROTEIN SYNTHESIS IN LONG-TERM MEMORY IN VERTEBRATES; TASTE AVERSION AND PREFERENCE LEARNING IN ANIMALS
Bailey, D. J., Kim, J. J., Sun, W., Thompson, R. F., and Helmstetter, F. J. (1999). Acquisition of fear conditioning in rats requires the synthesis of mRNA in the amygdala. Behavioral Neuroscience 113, 276-282.
Campeau, S., Hayward, D., Hope, B. T., Rosen, J. B., Nestler, E. J., and Davis, M. (1991). Induction of the c-fos proto-oncogene in rat amygdala during unconditioned and conditioned fear. Brain Research 565, 349-352.
Hall, J., Thomas, K. L., and Everitt, B. J. (2001). Fear memory retrieval induces CREB phosphorylation and Fos expression within the amygdala. European Journal of Neuroscience 13 (7), 1,453-1,458.
Helmstetter, F. J., and Bellgowan, P. S. (1994). Effects of muscimol applied to the basolateral amygdala on acquisition and expression of contextual fear conditioning in rats. Behavioral Neuroscience 108, 1,005-1,009.
Josselyn, S. A., Shi, C., Carlezon, W. A., Neve, R. L., Nestler, E. J., and Davis, M. (2001). Long-term memory is facilitated by cAMP response element-binding protein overexpression in the amygdala. Journal of Neuroscience 21, 2,404-2,412.
Lamprecht, R., and Dudai, Y. (1996). Transient expression of c-fos in rat amygdala during training is required for encoding conditioned taste aversion memory. Learning and Memory 3, 31-41.
LeDoux, J. E. (2000). Emotion circuits in the brain. Annual Review of Neuroscience 23, 155-184.
Malkani, S., and Rosen, J. B. (2000). Specific induction of early growth response gene 1 in the lateral nucleus of the amygdala following contextual fear conditioning in rats. Neuroscience 97, 693-702.
Maren, S. (2001). Neurobiology of Pavlovian fear conditioning. Annual Review of Neuroscience 24, 897-931.
Petrovich, G. D, Scicli, A. P., Thompson, R. F., and Swanson, L. W.(2000). Associative fear conditioning of enkephalin mRNA levels in central amygdalar neurons. Behavioral Neuroscience 114, 681-686.
Radulovic, J., Kammermeier, J., and Spiess, J. (1998). Relationship between fos production and classical fear conditioning: Effects of novelty, latent inhibition, and unconditioned stimulus preexposure. Journal of Neuroscience 18, 7,452-7,461.
Rosen, J. B., Fanselow, M. S., Young, S. L., Sitcoske, M., and Maren, S. (1998). Immediate-early gene expression in the amygdala following footshock stress and contextual fear conditioning. Brain Research 796, 132-142.
Schafe, G. E., Atkins, C. M., Swank, M. W., Bauer, E. P., Sweatt, J. D., and LeDoux, J. E. (2000). Activation of ERK/MAP kinase in the amygdala is required for memory consolidation of Pavlovian fear conditioning. Journal of Neuroscience 20 (21), 8,177-8,187.
Silva, A. J., Kogan, J. H., Frankland, P. W., and Kida, S. (1998). CREB and memory. Annual Review of Neuroscience 21,127-148.
Stanciu, M., Radulovic, J., and Spiess, J. (2001). Phosphorylated cAMP response element binding protein in the mouse brain after fear conditioning: relationship to Fos production. Molecular Brain Research 94, 15-24.