M uch of the pharmacological and genetic evidence highlighted thus far, points to an involvement of gene expression in L-LTP. Following extracellular stimulation such as that provided by growth factors or neurotransmitters, a small subset of genes undergo a rapid and transient increase in expression that is protein synthesis independent. These are called im m ediate early genes (lEGs). W hat are the candidate lEG s expressed in response to L-LTP induction, and what role might they play (if any) in
the consolidation of long term m em ory? In 1987, M organ et a l reported that
following the injection of a seizure inducing drug, mRNA levels of the transcription factor c-fos were increased in mouse brain, within 60 minutes, declining to baseline levels over 3 hours. Similarly, a transient increase in c-fos protein immunoreactivity was seen in the dentate gyrus of rats within 30 minutes of an electrically-induced seizure produced by kindling (Dragunow and Robertson, 1987). Following these
studies, a number of mRNAs from genes encoding for the transcription factors zif268,
c-jun, and ju n -B , in addition to c-fos were found to be induced in hippocam pal neurons as well as other brain areas in the rat, in response to drug induced seizure
(Saffen et a l, 1988). The mRNA increases were rapid and transient, occurring within
1 hour and returning to baseline within 2 hours. Furthermore, zif268 (also known as
Krox-24, EGR-1, or NGFl-A) mRNA levels in the dentate gyrus of anaesthetised rats, were found to increase in response to high frequency (but not low frequency)
stim ulation that could induce LTP (Cole et a l , 1989). The increases in zif2 6 8
expression, only occurred in the ipsilateral hippocampus and were blocked by NMDA receptor inhibitors (Cole et a l, 1989). Initially, the levels of zif268 (and to a lesser extent c-jun, ju n -B , jun-D and fo s related mRNAs) induction in the dentate gyrus of awake rats following LTP had been shown to be highly correlated with the duration of
the LTP rather than with the magnitude of the initial LTP. (Richardson et a l, 1992;
Abraham et a l , 1993). However, other studies have shown that the link between LTP
and the induction of these transcription factor lEGs is not quite so straightforward.
W isden et a l (1990), reported that LTP in the dentate gyrus of anaesthetised rats did
not result in the induction of mRNA for c-fos, c-jun, jun-D and a num ber of other lEGs. A lthough zif268, and to a lesser extent, ju n -B induction was seen following NM DA dependent LTP, non-specific induction of all the lEGs examined occurred in
response to forms of stimulation that activated granule cells but did not induce LTP. In addition, no changes in lEG induction were seen in animals trained for spatial
learning in the water-maze (Wisden et aL, 1990). Consistent with these results, c-fos
induction in unanaesthetised rats did not correlate with LTP induction (Dragunow et
a l, 1989).
Despite the ambiguity of the early findings, recent evidence does suggest that zif268
is necessary for both L-LTP and long-term memory (Jones et aL, 2001). In vivo
recordings from awake mutant mice with a targeted disruption of zif268, showed that
although LTP was unaffected for the first hour after induction, it was absent when measured 24 and 48 hours post-tetanus. Furthermore, these mice displayed deficits in long-term but not short-term memory for a number of spatial and non-spatial learning
tasks (Jones et aL, 2001). The finding that both zif268 and c-fos contain cAMP
response elements (CREs) in their promotor sequences (Sassone-Corsi et aL, 1988;
Sakamoto et aL, 1991), suggests a possible role for these transcription factor lEGs in
the CREB mediated regulation of expression of, yet unknown, late effector genes, that
could govern the duration of L-LTP. However, although it appears that zif268 is
necessary for the induction of L-LTP it has still not been determ ined whether this gene is produced constitutively, or is specifically induced during L-LTP induction.
In addition to the transcription factor lEGs, there is also a small set of lEGs that have the potential to directly m odulate changes involved in LTP. One of the best characterised of these lEGs is tissue-plasminogen activator (tPA), an extracellular
serine protease that converts plasminogen into plasmin. In situ hybridisation showed
that tPA is rapidly induced (within 1 hour) in the whole brain in response to injection with seizure inducing drugs, or increased throughout the hippocampus in response to
kindling stim ulation in the perforant path (Qian et aL, 1993). Furtherm ore, tPA
expression was found to rapidly increase (within 1 hour), specifically in the ipsilateral
dentate granule neurons, in response to an LTP inducing tetanus delivered to the
perforant path. The tPA increase was transient (decreasing at 6 hours and returning to
baseline levels at 24 hours) and blocked by the NM DA receptor inhibitor MK801 (Qian et aL, 1993).
Inhibitors o f tPA have been shown to block L-LTP induced by either tetanic
stimulation or forskolin application in both the C A l and CA3 regions (Baranes et aL,
1998). Furtherm ore, application of tPA paired with a stimulus that would normally
induce E-LTP can produce an L-LTP in both these regions (Baranes et aL, 1998). A
possible m echanism involving the production of L-LTP by tPA m ediated synaptic
growth is provided by Baranes et aL (1998). The authors showed that cultured dentate
granule cells secrete tPA and undergo axonal elongation and formation of presynaptic varicosities in response to forskolin application. These changes were blocked by inhibitors o f tPA and could be induced by the application of tPA alone. Furthermore, m utant m ice lacking the tPA gene are im paired in their ability to exhibit L-LTP
(H uang et aL, 1996; Calabresi et aL, 2000), and show reduced potentiation in
response to application of cAMP analogs and D1/D5 agonists (Huang et aL, 1996).
However, not all studies agree. Frey et aL (1996c) found that L-LTP in tPA mutant
mice was unaffected, although L-LTP in these mice, was blocked by inhibition of G A BA ergic transm ission. In addition, conflicting results seen from behavioural studies, have m ade it difficu lt to determ ine w hether tPA is involved in
hippocampal-dependent learning (Huang et aL, 1996; Calabresi et aL, 2000).
A nother lE G that may have a role in the consolidation of L-LTP is the activity
regulated cytoskeletal-associated protein. A rc (also termed A rg 3.1). A rc mRNAs are
enriched in brain and hippocampus after drug or electrically-induced seizures. In addition, LTP-inducing, high-frequency stimulation, applied to the perforant path
resulted in a robust induction o f A r c in the granule cells o f the ipsilateral
hippocampus (Link et aL, 1995; Lyford et aL, 1995). Interestingly, both Arc mRNA
and protein are localised in the soma as well as in the dendrites o f the granule cells (Link et aL, 1995; Lyford et aL, 1995; W allace et aL, 1998). It is thought that Arc may play a role in the activity-dependent response of synapses by modifying the function of existing proteins. Indeed A rc mRNA and protein have been shown to selectively localise to regions of recent synaptic activity in granule cell dendrites
(Stew ard et aL, 1998). M ore recently, using in situ hybridisation and confocal
m icroscopy. A rc mRNA has been visualised in the nucleus within minutes of the exposure of a rat to a specific environment. Furthermore, within 30 minutes this Arc
nuclear to cytoplasmic A rc mRNA could be used to infer the recent environmental
exposure of the animals (Guzowski et a l, 1999). These results provide evidence that
Arc may be induced in response to behaviour in the intact animal. Finally, inhibition
of A r c p ro te in e x p re ss io n , v ia h ip p o c a m p a l in fu s io n o f a n tise n se oligodeoxynucleotides has been shown to block L-LTP and long-term but not
short-term spatial memory in rats (Guzowski et a l, 2000).
In addition to A rc and tPA a number of other lEGs seem to be correlated to LTP induction. The lEG H omer is induced in the dentate gyrus of freely moving rats in response to LTP inducing stimulation. H om er protein appears to target excitatory synapses and dendritic spines. Homer contains a PDZ-like binding domain, and can
bind to ty p el and type 5 metabotropic glutamate receptors (mGluRs) (Brakeman et
aL, 1997). Expression of the lEG form o f H o m e r can m odulate m GluR-induced
intracellular Ca^^ release (decreasing amplitude and increasing latency), suggesting a
mechanism by which it could modulate synaptic function during L-LTP (Tu et al.,
1998). R ecently, a serine/threonine kinase, P im -1 (provirus integration site for
Moloney murine leukem ia virus), has been shown to be consistently, and strongly induced in dentate granule cells in response to LTP inducing high frequency
stimulation, showing both nuclear and dendritic localisation (Konietzko et aL, 1999).
M utant mice deficient for Pim-1 show no L-LTP in the C A l region, whilst E-LTP is
relatively unaffected (Konietzko et aL, 1999). A num ber of other lEG s exist that
encode proteins with the potential to directly m odify synaptic plasticity, such as
BA D 2 (a M APK phosphatase)(Qian et aL, 1994), C ox-2 (an inducible form of
cyclooxygenase), Rheb (a Ras homologue), and N arp (a calcium -dependent lectin),
however less evidence for their involvement in LTP exists (if any), and these are discussed elsew here (for a recent review see L anahan and W orley, 1998). In summary, the evidence for the involvement of a small number of lEGs in L-LTP and long-term memory is promising. However, it remains to be seen whether or not the specific induction of these genes during potentiation is necessary for the maintenance of L-LTP.