Shortterm Facilitation In Aplysia Is Mediated By Changes In The Levels Of Intracellular Second Messengers

What happens when serotonin is applied to sensory neurons? Serotonin binds to receptors in the neuron's cell surface membrane that are coupled to adenylyl cyclase and phospholipase C, which generate cAMP and DAG, respectively. When a sensory neuron sees a single pulse of serotonin, adenylyl cyclase and phos-pholipase C are activated, cAMP and DAG levels increase, and the activities of PKA and PKC are greatly enhanced. As long as serotonin is present, these enzymatic activities remain elevated. However, after serotonin is removed, metabolic enzymes in the sensory neuron return the cell to its resting state. In this case, phosphodi-esterase breaks down cAMP, diacylglycerol lipase breaks down DAG, and protein phosphatases dephosphorylate the protein kinase substrates. Thus, the duration of facilitation in response to a single application of serotonin is determined by the amount of time serotonin is present, the rate of breakdown of the second messengers, and the rate of reversal of the effects of the protein kinases after serotonin is removed. After a single application of serotonin, these effects are rapidly reversed (usually within a few minutes); therefore, a single application of serotonin gives only short-lasting facilitation.

Thus, Reaction Category 1: Altered Levels of Second Messengers

Thus, Aplysia short-term facilitation of neurotransmitter release provides an example of our first category of memory-forming chemical reaction: transient, stimulus-mediated changes. In this case, the duration of the memory is essentially dependent upon continued release of 5HT onto the neuron.

It is an interesting thought experiment to consider the effects in this system if the breakdown enzymes were removed. Over time, second messengers and phospho-rylated proteins would accumulate, eventually driving the system to saturation. Then, whenever a sensory neuron received a serotonin signal, it would be unable to modulate its intracellular milieu appropriately, and no alteration in synaptic efficacy could be achieved. This thought experiment illustrates an important point; the capacity to dynamically regulate the

FIGURE 6 The Schwartz and Kandel model of short-term and long-term regulation of PKA in Aplysia sensory neurons. This is a mechanism for short- and intermediate-term facilitation of neurotransmitter release. See references 8 and 9 and explanation of pathway in text. PKA shown as tetramer of two regulatory (Reg.) and two catalytic (Cat.) subunits. The catalytic site is shown in yellow. PDE = Phosphodiesterase.

FIGURE 6 The Schwartz and Kandel model of short-term and long-term regulation of PKA in Aplysia sensory neurons. This is a mechanism for short- and intermediate-term facilitation of neurotransmitter release. See references 8 and 9 and explanation of pathway in text. PKA shown as tetramer of two regulatory (Reg.) and two catalytic (Cat.) subunits. The catalytic site is shown in yellow. PDE = Phosphodiesterase.

molecular messengers in a system is critical to the synaptic plasticity that underlies learning.

IV. INTERMEDIATE-TERM FACILITATION IN APLYSIA INVOLVES ALTERED GENE EXPRESSION AND PERSISTENT PROTEIN KINASE ACTIVATION—A SECOND CATEGORY OF REACTION

What happens when the sensory neuron sees repeated applications of serotonin, which elicits long-lasting synaptic facilitation? Repeated applications of serotonin lead to sustained elevations of second messengers, and this sustained elevation elicits activation of a unique and elaborate cascade of biochemical events. Although many mechanistic details have not yet been worked out, several key steps in this cascade have been identified. The long-lasting elevation of cAMP leads to PKA activation and subsequent phosphorylation of the transcription factor CREB. Activation of the ERK MAP kinase cascade is also likely involved as a modulator of CREB activation, specifically acting through disinhibition via repression of negative regulators of CREB. Through mechanisms that are still being investigated, CREB activation leads to regulation of protein breakdown. Specifically, the ubiquitin system is recruited to cause the proteolytic degradation of one subunit of PKA, the PKA regulatory subunit.

An understanding of the consequences of the loss of PKA regulatory subunits becomes clear upon review of the normal control of this enzyme. The cAMP-dependent protein kinase is, of course, a tetramer comprising two regulatory and two catalytic subunits (Figure 6). The two identical regulatory subunits each contain one cAMP binding site; when cAMP binds, the regulatory subunits dissociate from the two (identical) catalytic subunits. The free catalytic subunits are then enzymatically competent and able to phosphorylate their downstream effector proteins. Therefore, proteolytic loss of regulatory subunits results in a decrease in the overall ratio of regulatory to catalytic subunits, promoting an excess of free, active catalytic subunits, and an increase in the phosphorylation of PKA substrates.

In this manner, PKA is persistently activated. Even after cAMP returns to its resting level after serotonin is removed, the excess catalytic subunits remain free of regulatory subunits and active in the sensory neuron. By this clever mechanism, a chain of events is set in motion whereby a biochemical effect that outlasts the initial, triggering elevation of the second messenger cAMP is established in the cell. The PKA will remain activated until compensatory resynthesis of new regulatory subunits occurs, or until the catalytic subunit is degraded. Interestingly, although the mechanism has not yet been worked out, recent evidence indicates that the DAG-responsive effector PKC also is persistently activated after serotonin stimulation of sensory neurons.

Persistent kinase activation is one powerful mechanism contributing to long-lasting facilitation of neurotransmitter release in sensory neurons. Available evidence indicates that the persistent activation of PKA underlies an intermediate stage of facilitation, lasting on the order of many hours after the triggering applications of serotonin are finished. Interestingly, pioneering work on this mechanism was performed using sensitization training in animals, emphasizing the strong likelihood of this mechanism contributing to the underlying cellular basis for the change in the animal's behavior in vivo.

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

Aplysia intermediate-term facilitation of neurotransmitter release provides an example of our second category of memory-forming chemical reaction: generation of long half-life signaling molecules. In this case, the duration of the memory is essentially dependent upon the half-life of the free PKA catalytic subunit. Reversal of the persisting event is dependent upon the half-life of the protein or the rate of synthesis of regulatory subunit. Later in the chapter, I will highlight a few corresponding types of molecular memory traces that have been observed in hippocampal synaptic plasticity, which we have already covered in great detail in Chapter 7.

Before proceeding to Reaction Category 3, it is worth noting how the short- and intermediate-term mechanisms manage to achieve the same final common output of increased synaptic strength. The elegant solution to this problem is inherent in the mechanisms themselves. As both short-term mechanisms and longer-term mechanisms result ultimately in activation of the same kinases, PKA and PKC, the final read-out is the same: increased phospho-rylation of PKA and PKC substrates. Only the mechanisms to achieve the kinase activation are distinct and of different durations.

The substrates affected by PKA and PKC are varied (see Box 2), involving proteins controlling both the electrical properties of the sensory neuron cell membrane and the mechanisms involved in the process of neurotransmitter release. The overall result, though, is an orchestrated set of changes leading ultimately to increased neurotrans-mitter release from the sensory neuron.

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