Signal Transduction Mechanisms and Early Events

Scientists believe that long-lasting changes in synaptic function are essential for learning and memory in the mammalian brain. A widely studied example of such synaptic plasticity is long-term potentiation (LTP). The remarkable feature of LTP is that a short burst of synaptic activity can trigger persistent enhancement of synaptic transmission lasting for at least several hours, and possibly weeks or longer. There is great interest in understanding the cellular and molecular mechanisms that underlie this form of synaptic plasticity. First found in the hippocampus, this phenomenon is now known to exist in cerebral cortex and other areas of the mammalian central nervous system (CNS). Indeed, damage to the hippocampus can result in certain defects in memory acquisition (see Milner, Squire, and Kandel, 1998).

Most studies on LTP focus on the synapse between Schaffer collaterals and hippocampal CA1 neurons. In this system, a brief burst of afferent stimulation leads to induction of LTP in postsynaptic CA1 cells through a combination of (1) membrane depolarization and (2) activation of glutamate receptors of the NMDA subtype (e.g., Collingridge, Kehl, and McLennan, 1983). Researchers generally agree that the depolarization relieves Mg²⁺ (magnesium ion) block of NMDA receptor channels and allows a Ca²⁺(calcium ion) influx into dendritic spines that somehow triggers LTP (Nicoll, Kauer, and Malenka, 1988).

Back-Propagating Action Potentials

In hippocampal pyramidal neurons an important component of the membrane depolarization that allows opening of NMDA receptors is back-propagating action potentials. While action potentials are of course triggered in the active zone of the cell body, hippocampal pyramidal neurons along with many other types of CNS neurons can actively propagate action potentials into the dendritic regions. These dendritic action potentials are just like action potentials propagated down axons in that they are carried predominantly by voltage-dependent ion channels such as sodium channels. The penetration of the back-propagating action potential into the dendritic region provides a wave of membrane depolarization that allows for the opening of the voltage-dependent NMDA receptor/ion channels. In fact, the timing of the arrival of a dendritic action potential with synaptic glutamate input appears to play an important part in precise, timing-dependent triggering of synaptic plasticity in the hippocampus (Magee and Johnston, 1997). Moreover, modulatory neurotransmitter systems can regulate the likelihood of action potential back-propagation through controlling dendritic potassium channels, allowing for sophisticated information processing through an interplay of action potential propagation, glutamate release, and neuromodulation (Johnston, Hoffman, Colbert, and Magee, 1999).

Importance of a Rise in Postsynaptic [Ca²⁺]_i

NMDA receptor activation leads to a transient [Ca²⁺]_i increase arising in the postsynaptic neuron, an effect that researchers have measured by the use of fluorescent Ca²⁺ indicator dyes in pyramidal cell dendrites within hippocampal slices (Regehr and Tank, 1990). A variety of experiments have demonstrated the importance of the rise in [Ca²⁺]_i for LTP. Ca²⁺ buffers such as EGTA or BAPTA have been introduced with the aim of suppressing the transient Ca²⁺ increase; such maneuvers are effective in preventing the induction of LTP (Lynch et al., 1983). Moreover, a rise in postsynaptic Ca²⁺. independent of glutamate receptors, has been imposed by photoactivation of a caged Ca²⁺ compound, nitr-5; this method for Ca²⁺ delivery causes a sustained synaptic potentiation (Malenka, Kauer, Zucker, and Nicoll, 1988).

Involvement of Protein Kinases

The key question at this point is how a relatively brief rise in [Ca²⁺]_i can lead to a long-lasting enhancement of synaptic function. One popular hypothesis is that Ca²⁺ acts by activating signal transduction pathways sensitive to the elevation of postsynaptic Ca²⁺. The pathways that have been implicated are quite varied and include: the Ca²⁺-phospholipid dependent protein kinase (protein kinase C, PKC), multifunctional Ca/calmodulin-dependent protein kinase (CaMKII), calcium/calmodulin sensitive adenylyl cyclase and the PKA pathway, the ras/ERK MAP kinase pathway, and calcium-responsive nitric oxide (NO) synthase (Adams and Sweatt, 2002; Lisman and Zhabotinsky, 2001; Lu, Kandel, and Hawkins, 1999; Sweatt, 1999; Hrabetova and Sacktor, 1996).

Involvement of Ca²⁺ -dependent protein kinases has been extensively tested and a wide variety of evidence is compatible with the general hypothesis of a necessity for postsynaptic protein kinase activation as being necessary for triggering LTP (see Figure 1). A necessity for PKC and CaMKII has been tested by intracellular injection of peptides that are potent and selective inhibitors of either PKC or CaMKII (Malinow, Schulman, and Tsien, 1989), and by characterization of kinase-deficient mice generated through gene knockout technology (Abielovich et al., 1993; Silva, Stevens, Tonegawa, and Wang, 1992). Pharmacologic inhibitors of MAP kinase activation have also been shown to block LTP induction (Adams and Sweatt, 2002). Involvement of the PKA pathway has been probed using pharmacologic and transgenic animal approaches, as has the involvement of the NO synthase/cGMP pathway (Lu, Kandel, and Hawkins, 1999; and Sweatt, 1999).

The results obtained with inhibition of PKC, CaMKII, PKA, MAP Kinase, and the NO/cGMP pathway can be interpreted in terms of a network of protein kinases, with protein phosphorylation as a link between the rise in [Ca²⁺]_i and the eventual expression of enhanced synaptic function. However, the topology of the network and the nature of the interactions remain undefined. Indeed, there is no evidence to date to exclude the idea that one or more of these enzymes act in a merely permissive way. At one extreme, background activity of a particular kinase might be necessary only prior to induction, to set the stage for some other signaling mechanism triggered by Ca²⁺.

Evidence for Presynaptic Expression of LTP

The question of how the kinases act leads to consideration of ongoing debate about the nature of the maintained synaptic enhancement in LTP. There is a variety of evidence to support the view that both increased presynaptic transmitter release and enhanced glutamate receptor function postsynaptically are involved. The results indicating enhancement presynaptically support the idea of a retrograde signal that travels from the postsynaptic cell back to the presynaptic terminal. This line of reasoning has led to a search for specific compounds that might act as the retrograde messenger, with arachidonic acid, nitric oxide, and superoxide being the most widely considered possibilities (Sweatt, 1999). The precise mechanisms by which these retrograde signals might affect neurotransmitter release are unclear, although presynaptic activation of PKC and the cGMP-dependent protein kinase are appealing possibilities.

Little is known for certain about possible pre-synaptic mechanisms that might be set in motion by putative retrograde messengers. A widely considered mechanism for synaptic potentiation involves a persistent enhancement of a presynaptic protein kinase, such as PKC (Linden and Routtenberg, 1989). The maintained expression of LTP can be reversibly blocked by bath application of a relatively nonspecific kinase inhibitor, H-7 (Malinow, Madison, and Tsien, 1988), and biochemical measurements show a sustained enhancement of PKC in hippocampal slices (Klann, Chen, and Sweatt, 1993; Hrabetova and Sacktor, 1996). PKC is known to increase the efficiency of excitation-secretion coupling in many systems, including chromaffin cells, motor nerve terminals, and cultured hippocampal neurons. Further exploration of presynaptic mechanisms is needed to determine which steps leading to exocytosis are enhanced in LTP.

Mechanisms of Postsynaptic Enhancement

In terms of mechanisms for enhancing postsynaptic responsiveness, two types of mechanisms are being actively pursued experimentally. One type of process is referred to as activation of "silent synapses" (Poncer and Malinow, 2001; Isaac, Nicoll, and Malenka, 1999). The concept is that some latent synapses have available only NMDA receptors and thus are inactive in terms of normal baseline synaptic transmission. Activation of NMDA receptors by the mechanisms described above leads to calcium influx and calcium-mediated insertion of AMPA receptors locally. Thus previously "silent" synapses become capable of baseline synaptic transmission and the net result is an increased efficacy of transmission.

A second mechanism under investigation is kinase-dependent regulation of AMPA receptors through direct phosphorylation. It is known that phosphorylation of AMPA receptors by PKC and CaMKII leads to increased conductance of ions through the AMPA channel, and evidence is available that phosphorylation at these regulatory sites increases with LTP-inducing stimulation (Lee et al., 2000; Derkach, Barria, and Soderling, 1999; Barria et al., 1997). Thus it is hypothesized that through increased postsynaptic phosphorylation of AMPA receptors synaptic efficacy is increased by enhancement of AMPA receptor function. The "silent synapse" hypothesis and the enhanced AMPA receptor conductance hypothesis are not mutually exclusive; indeed both could be simultaneously occurring at different synapses between the same cells, or at the same synapses at different points in time.

Potential Roles for Protein Phosphatase Regulation

Finally, while increased protein phosphorylation has been hypothesized to play a key role in triggering LTP for quite some time, studies conducted since the turn of the century highlight a similar importance of tightly regulating protein dephosphorylation as one of the mechanisms controlling the induction of long-term synaptic change and lasting memory (Winder and Sweatt, 2001). In one series of studies, Isabel Mansui and her colleagues (Mallaret et al., 2001) engineered a mouse line in which the activity of the calcium-dependent phosphatase calcineurin (protein phosphatase 2B) could be regulated, in a reversible manner, by tetracycline administration to adult animals. In engineering their mouse they capitalized on the known mechanism of regulation of calcineurin; that is, its regulation by an intrinsic autoinhibitory domain. Mansui's group engineered a mouse in which they could regulate the expression of the isolated autoinhibitory domain of the calcineurin A α subunit. In their mice, treatment of animals with tetracycline led to expression of the calcineurin autoinhibitory domain protein in several brain regions, including the hippocampus. They then investigated long-term potentiation in hippocampal slices, specifically in area CA1 where many of the earlier studies implicating kinases had been performed. Calcineurin inhibition enhanced the magnitude and duration of long-term potentiation in this region when LTP was triggered by high-frequency stimulation. These studies and a variety of others (Winder and Sweatt, 2001) suggest the possibility of an important role for calcium-dependent phosphatases in regulating the magnitude of LTP induction in the hippocampus.

Since the early 1990s, much progress has been made in scientific understanding of the mechanisms involved in LTP induction. A role for protein phosphorylation in LTP induction is clear. Dendritic action potentials were inconceivable in the early 1990s but in the twenty-first century are known to be critical components of information processing in neurons, through their contributions to LTP induction. Many questions remain concerning the targets of protein kinases in LTP, the role of morphological changes and local protein synthesis in early stages of LTP, and the seemingly perpetual question of pre-versus postsynaptic loci of LTP expression. Scientists are hopeful that continued research and progress will shed light on these topics.

See also:GLUTAMATE RECEPTORS AND THEIR CHARACTERIZATION; GUIDE TO THE ANATOMY OF THE BRAIN: HIPPOCAMPUS AND PARAHIPPOCAMPAL REGION; LONG-TERM POTENTIATION: OVERVIEW: COOPERATIVITY AND ASSOCIATIVITY; SECOND MESSENGER SYSTEMS

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Richard W.Tsien

Revised byJ. DavidSweatt