LTM is distinguished from other forms of memory by its sensitivity to protein and mRNA

synthesis inhibitors, demonstrating new protein synthesis and altered gene activity to be essential for its
formation. This is because the range of synaptic and neuronal plasticity required for LTM are facilitated
physically modifications of both the synaptic and membrane complement of proteins 1.

This was demonstrated in Lymnaea, where the expression of new genes from the RPeD1 soma is
required for LTM formation. Sangha at al. have shown that injection of the transcription blocker
actinomycin D after an hour of cooling disrupted the reconsolidation of LTM 2 and of the translation
blocker Anisomycin 2.5 hours before training prevented the establishment of both ITM and LTM 3.
Ultimately, the ablation of the RPeD1 soma before conditioning prevented LTM formation, but not its
retrieval if removed after consolidation4.

More detailed studies have identified the specific genes that are active or up-regulated during
LTM formation and found that they all play a part in facilitating more permanent forms of neural
plasticity, such as LTP, that would perhaps serve as the engram. These genes are involved in intracellular
kinase networks, synaptic function, transcriptional activation and repression, membrane receptors, etc.

In the invertebrate models of aversive operant conditioning, the pathway for inducing memory
formation is postulated as follows: Ca2+ influx through NMDA receptors triggers a signalling cascade that
leads to the generation of cAMP and activation of protein kinase A (PKA), which phosphorylates
mitogen-activated protein kinase pathway (MAKP) and cAMP-response element binding protein (CREB)
to activate the transcription of plasticity-associated genes 5. This pathway appears to be highly conserved
across species in the formation of associative memory across aversive operant conditioning models.
Inhibition of protein kinase C (PKC), MAKP and NMDA receptors have been shown to block LTM and in
some cases ITM formation in Lymnaea6. These results were replicated in a negatively reinforced operant
learning paradigm in Aplysia, where in vivo injections of PKC or PKA inhibitors7and MAPK inhibitors8
were also able to abolish LTM formation. LTM formation after aversive conditioning of Lymnaea aerial
respiratory behaviour caused a significant increase in the expression of mitogen-activated protein kinase
kinase 1 (MEKK1), a signalling molecules in the MAPK pathway, and the novel expression of the epsilon
isoform of PKC9. Aversive operant conditioned Lymnaea exhibited an increase in CREB1 expression
during consolidation of learning into LTM. The subsequent CREB-dependent up-regulation in the
synthesis of presynaptic release mechanisms syntaxin-1 and dynamin-1 most likely facilitate the
increased probability of presynaptic vesicle release and induction of synaptic plasticity 10. CREB1 may also
mediate the cAMP-dependent regulation of a family of cAMP-regulated transcription factors known as
1
Igaz,
2
Sangha, Scheibenstock, Lukowiak, 2003
3
Sangha S, Scheibenstock A, McComb C, Lukowiak K., 2003
4
Scheibenstock, 2002)
5
Robert Waltereit and Michael Weller Molecular Neurobiology Volume 27, Number 1, 99-106, DOI:
10.1385/MN:27:1:99
6
Rosenegger, Lukowiak, 2010
7
Michel, Green, Lyons, Learn. Mem. 2011. 18: 19-23
8
Michel, Green, Eskin, Lyons, Learn. Mem. 2011. 18: 108-117
9
Rosenegger D, Wright C, Lukowiak K: Mol Brain 2010, 3(9):1-17.
10
Guo CH, Senzel A, Li K, Feng ZP.
CCAAT/enhancer-binding protein (C/EBP) , as a CRE site is found upstream of the C/EBP gene 11. LTM
formation in Aplysia was correlated with increased C/EBP expression specifically localized to the nervous
centres controlling feeding 12. A homolog of C/EBP, which is known to activate transcription of
downstream genes that give rise to the growth of new synaptic connections, has also been identified in
Lymnaea13. These cross-species similarities suggest that these processes are fundamental to memory
formation.

The cAMP system plays an especially critical role in Drosophila memory formation, as several
mutations that lead to abnormal cAMP levels impair learning in an operant conditioning task. A new
paradigm of operant conditioning in Drosophila14, in which the flies learn to remain in a particular
portion of an enclosed chamber in order to avoid an increase in temperature if venturing into the
"punished area", has been able to identify several genes involved in memory formation. Mutants of
dunce, which has lesions in the gene that codes for a cAMP-specific phosphodiesterase 15, and rutabaga,
which also has a lesion in the adenylyl cyclase system that makes it less active and decrease the levels of
cAMP16, were able to avoid the heated side of the chamber during training, but showed no avoidance in
the memory test17. In a paradigm where heat was used to condition Drosophila to move a platform, the
amnesiac mutants, who also have a reduced level of adenylyl cyclase activity, showed no memory after
training18. Of particular interest is the ignorant gene coding for the p90 ribosomal S6 kinase (RSK), an
enzyme that is part of a family of serine-threonine kinases that in mammals have been implicated in the
MAPK signalling cascade controlling synaptic plasticity and memory formation 19. Mutant flies with
spatial deletions of the gene are shown to be deficient in the heat box task 20.

Hippocampal gene expression analysis in multi- and single-trial learning tasks in vertebrate
models have identified similar up-regulations in many genes involved in synaptic transmission, cell
signalling and cell-cell interactions after learning. After a single trial contextual fear training, microarray
analysis of hippocampal RNA from trained rats showed increased expression of 14 genes involved in
synaptic transmission and signalling, including CaMKIIa and ERK2 (which are known to be involved in
memory formation and consolidation) and synatxin-1 (which has already been identified in Lymnaea
study to be up-regulated in a CREB-dependent manner during LTM consolidation) 21. Other forms of

11
The cognitive neurosciences By Michael S. Gazzaniga
12
- Levitan D, Lyons LC, Perelman A, Green CL, Motro B, Eskin A, Susswein AJ (2008)
13
Hatakeyama, 2004; Kandel, 2001
14
Wustmann G, Rein K, Wolf R, Heisenberg M:, 1996 1. Journal of comparative physiology A Sensory neural and behavioral physiology (1996)Volume:
179, Issue: 3, Pages: 429-436
15
Byers 1981
16
Livingstone, 1982
17
G Wustmann, K Rein, R Wolf, M Heisenberg
18
Mariath, Volume 31, Issue 10, 1985, Pages 779-787
19
Putz, Bertolucci, Raabe, Zars, Heisenberg
20
- Charactertization of memories and ignorant (S6KII) mutants in operant conditioning in the heat box
(http://www.opus-bayern.de/uni-wuerzburg/volltexte/2003/419/pdf/Thesisgesamt.pdf)
21
Igaz, Bekinschetein, Vianna, Izquierdo, Medina, 2004
avoidance learning tasks such as Morris water maze and stone T-maze have induced similar effects in a
large number of genes, including NMDA receptors, synaptotagmins, C/EBPb transcription factor 22,23.

Knockout of several above mentioned genes in mice have produced marked deficiency in the
acquisition and consolidation of LTM after operant learning tasks. Knockout of the obligatory NR subunit
NR1 in CA1 pyramidal cells in young adult mice lead to impairments in spatial learning acquisitioning
Morris water maze24 and a variety of other classical conditioning trials. Activity-induced CREB
phosphorylation and c-Fos expression were significantly reduced in transgenic mice lacking Ca 2+
stimulated CaMKIV activity in the forebrain, correlated with impairments in the consolidation-retention
phase but not the acquisition phase in LTM and induction of hippocampal L-LTP 25. Finally, transgenic
mice with inhibited forebrain ERK activation exhibited selective LTM impairment in contextual fear
conditioning and protein synthesis-dependent portion of hippocampal L-LTP 26. The ERK pathway is
known to be involved in the phosphorylation of several key transcription factors, now evidence suggests
that it might govern memory consolidation through synaptic changes via its regulation of protein
synthesis in neurons.

22
Cavallaro, 2002
23
Hall et al, 2000
24
McHugh, 1996
25
Tonegawa, 2003
26
Tonegawa, 2003