The Case For Altered Gene Expression In Lltp

In this section, I will make the case that there is a uniquely definable stage of LTP that we will call L-LTP. I will begin by describing three unique attributes of L-LTP that make it distinct from E-LTP. This will serve to define more precisely the form of synaptic plasticity that is under discussion. After that we can proceed to discuss L-LTP mechanisms per se.

First, L-LTP is defined as that phase of LTP that is dependent upon altered gene expression and protein synthesis for its induction. As we noted in the last chapter, "altered" is the key word here. In the limit, every cellular process depends upon ongoing gene expression and protein synthesis, if only to replenish mRNA and proteins as they turn over. What defines L-LTP is a requirement, for its induction, of changes in gene expression and protein synthesis, different from a pre-existing baseline.

The maintenance and expression mechanisms for L-LTP are still mysterious, and it is not clear if specifically altered gene expression is necessary for L-LTP maintenance. It is possible that the ongoing maintenance of altered levels of expression of specific genes is necessary for L-LTP maintenance, but this is not clear at this point. At a minimum, however, available data can be interpreted to indicate that alterations in gene and protein expression are necessary for the induction of L-LTP. Thus we will use this as the first and defining attribute of L-LTP for the purposes of the following discussion.

How then is L-LTP maintained? The working model of L-LTP maintenance that I will use posits a local, self-reinforcing alteration of protein synthesis and synap-tic structure (broadly defined) as the maintenance mechanism for L-LTP. I will speculate on some specific possibilities for molecular players in this scenario later in this chapter, and we will return to the issue in a more general sense in Chapter 12. The upshot of all this is that we will discuss hypothetical mechanisms involved in L-LTP maintenance that utilize persisting alterations in protein synthesis, but we will not discuss any scenario involving persistent changes in gene expression.

A second attribute of L-LTP is that it generally is induced, selectively, by multiple trains of tetanic stimuli or in some cases by more prolonged theta-type stimulation or stimulation paired with dopamine or other neuromodulators. The fact that L-LTP can be selectively induced with specific protocols suggests that unique molecular events are associated with its induction. Also, the unique L-LTP inducing stimuli serve a practical purpose in that one can design experiments to see what molecular events are uniquely associated with these stimuli.1

A third attribute of L-LTP is, of course, its "lateness." At the risk of sounding Clintonesque, it's hard to define exactly how late late is. Late LTP is generally held to be LTP beginning at about the 90-minutes post-tetanus time point. This is harder to nail down than you might think because there is not typically a precipitous drop-off of LTP at any specific time point even when L-LTP is blocked with protein synthesis inhibitors (see Figure 1). In addition, L-LTP is studied in a wide variety of preparations and at various temperatures, ranging from room temperature in in vitro slices to 37 degrees in intact animal in vivo recording. There's probably about a fivefold difference in the kinetics of most

1I need to note at this point that, regardless of the induction protocol used to generate the data we will be discussing in this chapter, all the data I will present concern NMDA receptor-dependent L-LTP. I will, however, intermingle data from Schaffer-collateral synapses with data from studies of the perforant path inputs to the dentate gyrus. I am taking this license in order to have an adequate body of literature to draw from.

FIGURE 1 Protein synthesis dependence of L-LTP where L-LTP is disrupted in dominant negative CaMKIV transgenic mice. Late-phase LTP was induced by four trains of tetanic stimulation spaced by 5-minute intervals. The left panel illustrates the effects of application of the protein synthesis inhibitor anisomycin (filled triangles) and genetic suppression of CaMKIV (C34, open circles) on late-phase LTP. Note that later-developing stages of LTP are selectively lost. The right panel illustrates in greater detail the same data, focusing on the first 60 minutes of LTP. Filled triangles indicate L-LTP obtained with wild-type slices in the presence of anisomycin (Aniso). Superimposed representative EPSPs shown were recorded 5 minutes before and 3 hours after L-LTP induction. Calibration bars, 1 mV and 20 ms. As has been routinely observed, only modest effects of protein synthesis inhibitors are observed in E-LTP. Figure and legend adapted from Kang et al. (8).

FIGURE 1 Protein synthesis dependence of L-LTP where L-LTP is disrupted in dominant negative CaMKIV transgenic mice. Late-phase LTP was induced by four trains of tetanic stimulation spaced by 5-minute intervals. The left panel illustrates the effects of application of the protein synthesis inhibitor anisomycin (filled triangles) and genetic suppression of CaMKIV (C34, open circles) on late-phase LTP. Note that later-developing stages of LTP are selectively lost. The right panel illustrates in greater detail the same data, focusing on the first 60 minutes of LTP. Filled triangles indicate L-LTP obtained with wild-type slices in the presence of anisomycin (Aniso). Superimposed representative EPSPs shown were recorded 5 minutes before and 3 hours after L-LTP induction. Calibration bars, 1 mV and 20 ms. As has been routinely observed, only modest effects of protein synthesis inhibitors are observed in E-LTP. Figure and legend adapted from Kang et al. (8).

biochemical reactions over that temperature range, so Late LTP may start at 3 hours at room temperature in vitro but at 36 minutes in an intact hippocampus in vivo. Of course, some in vivo experiments go for days to weeks as well, and what we monolithically call Late LTP is likely multiple processes. For these various reasons, I greatly prefer to define L-LTP mechanistically in terms of altered gene and protein expression, versus temporally in terms of any particular time point. Nevertheless, we are required to have in hand a reasonable functional definition of L-LTP for experimental purposes (how else to know if it is blocked?), and a working definition of L-LTP is synaptic potentiation in the 90-minutes to 4-hour time range.

With these attributes in mind, we can proceed to consider the case for altered gene expression in L-LTP. Per usual, we will consider this in the context of the block, measure, and mimic criteria (see Table 1). Thus, the hypothesis of a role for altered gene expression in L-LTP predicts that blocking changes in protein and RNA synthesis should block L-LTP induction, that changes in gene and protein expression should occur with L-LTP-inducing stimulation, and that artificially increasing the synthesis of the appropriate genes should lead to an enhancement of synaptic transmission.2

Prediction One: blocking changes in protein and RNA synthesis should block L-LTP induction. In addition to the variety of experiments described in the last chapter demonstrating that protein synthesis inhibitors can block L-LTP (see Figure 1 for a recent example), it also is known that inhibition of RNA synthesis blocks L-LTP. Thus, actinomycin D application blocks

2An additional line of evidence discussed in the literature is based on deduction, and I will only mention it briefly here. L-LTP can last a really long time in vivo, and this implies altered gene expression at some level. This is because the lifetime of L-LTP in vivo encompasses many protein and mRNA half-lives, so the signals mediating that change surely at some level must impinge upon a genomic read-out. This is a compelling but not iron-clad argument because, as mentioned previously, the maintenance mechanism could be restricted to changes in protein synthesis, assuming an adequate baseline level of genomic read-out of the requisite mRNAs. We will return to this topic in the last chapter of the book.

TABLE 1 The Case for Gene Expression in L-LTP Experiment Type Finding References

Block Block of L-LTP with protein synthesis inhibitors (76)

Block of L-LTP with RNA synthesis inhibitors (1, 2, 77)

Loss of L-LTP with CREB knockouts (3-5)

Block of L-LTP with Arc antisense (13)

Loss of L-LTP with CaMKIV knockout (7, 8, 78)

Loss of L-LTP with zif268 knockout (6)

Measure Increased zif268/krox24 mRNA (40)

Increased krox20 (44)

Increased expression of fos, jun IEG mRNAs (40, 42, 43)

Increased CREB phosphorylation (24, 34, 35)

Increased CRE read-out (12, 79)

Increased elk-1 phosphorylation (35)

Increased Arc/Arg3.1 mRNA expression (13, 54, 59)

Increased AMPA receptor protein (51)

Increased BDNF message (46, 80)

Increased tissue plasminogen activator message (47)

Increased C/EBP beta (in long-term memory) (45)

Increased HOMER (11, 53, 72)

Increased MAP kinase phosphatase-1 (35, 50)

Increased SSAT message (49)

Increased MAP2 message (81) Mimic Constitutively active CREB augments L-LTP induction (14)

L-LTP induction (1, 2), although there are some differences in the particulars of the effect in the published reports.3 Similarly, knocking out or inhibiting the transcription factor cAMP-Responsive Element Binding Protein (CREB) and homologous family members can block L-LTP induction, although again there is some disagreement in the reported effects (3-5). (Interpretation of the CREB knockout results is complicated because of compensatory mechanisms that occur with CREB knockouts, and

3Please keep in mind that although I am glibly stating that various processes are necessary for the induction of L-LTP, the same caveats that we discussed in Chapter 5 concerning designing experiments to distinguish between induction, maintenance, and expression of LTP also apply here. This is particularly relevant in the case of knockout mice and irreversible inhibitors like actinomycin D.

there is an apparent specificity of the effects on L-LTP that are dependent on the L-LTP induction paradigm used.) Finally, knocking out the transcriptional regulator zif268, whose message is increased with L-LTP-inducing stimulation, leads to a loss of L-LTP (6). Overall these results indicate that dynamic regulation of gene expression and RNA synthesis is necessary to elicit L-LTP.

There also are more inferential findings that are consistent with a necessity for tran-scriptional regulation for L-LTP induction. Knockout of CaMKIV, a calmodulin-dependent kinase that regulates the activity of the CREB/CREB Binding Protein complex, also leads to a loss of L-LTP (see references 7 and 8 and Figure 1). Given the prominent role of ERK in regulating gene

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