DEMANDA

Although a growing body of data suggests the presence of dendritic mRNA and their local translation, there is at present no evidence that this local protein synthesis is required for hippocampal LTP. However, in the last five years, local protein synthesis has been shown to be involved in a number of different forms of synaptic plasticity. In 1996 Kang and Schuman showed that local protein synthesis was involved in neurotrophin induced synaptic plasticity in the hippocampus. Application of either BDNF or NT-3 produced a slow onset enhancement of the field EPSP slope of C A l pyramidal cells in hippocampal slices, that lasted for at least 3 hours. In addition, this enhancement was immediately and significantly attenuated by the protein synthesis inhibitors anisom ycin and cyclohexim ide, suggesting a local rather than somatic protein synthesis. Using this preparation, the authors were able to examine the locus of neurotrophin induced protein synthesis. They found that when either the

neuropil, the enhancement was unaffected (Kang & Schuman, 1996). These results indicate that the protein synthesis is not somatic and strongly suggest a dendritic origin.

Recently, a form of chemically induced hippocampal long-term depression (LTD) has been shown to be dependent on local protein synthesis. A rapid onset depression of the field EPSP slope in the C A l region of rat hippocam pal slices, occurred in response to application of the selective group 1 mGluR agonist DHPG. This LTD was blocked by the mGluR antagonist LY341495 and was dependent on translation but

not transcription (H uber et al., 2000). To confirm this requirem ent for mRNA

translation, postsynaptic cells were loaded with an mRNA cap analogue (m^GpppG) prior to application of DHPG. This cap analogue competes with endogenous capped RNA for the cap binding protein eIF-4E (a translation initiation factor) thereby in h ib itin g tran slatio n . Follow ing m^GpppG ap p licatio n , D H PG produced a

décrémentai LTD that returned to baseline levels over 1 hour (Huber et ah, 2000).

Finally, DH PG induced depression also occurred in dendrites that had be separated from their cell bodies. Taken together, these results suggest that chemically induced mG luR LTD, is dependent on the local postsynaptic translation of pre-existing,

dendritically localised mRNAs (Huber et al., 2000). Although the data from the two

previous studies is encouraging, sim ilar results showing no requirem ent for nuclear involvem ent, have yet to be shown with electrically induced homosynaptic LTP. Indeed, in the C A l region of hippocampal slices, tetanic stimulation has been shown to fail to induce L-LTP (potentials returned to pre-tetanus levels over 4-5 hours),

when dendrites were separated from their cell bodies (Frey et al., 1989). However, the

results from Frey et al. (1989), although attributed to an involvem ent of somatic

protein synthesis in L-LTP could equally be due to a block in mRNA trafficking to the dendrites.

To date, the m ost com pelling evidence for local protein synthesis in synaptic

p lasticity com es from a non-m am m alian system. In 1997 M artin et al. used a

m odified A p ly s ia , sensory to m otor neuron culture system , in which a single

bifurcated sensory neuron made synaptic contact with two separate motor neurons. Topical application of 5 pulses of serotonin to one of these sensory-m otor synapses

induced a specific long-term synaptic facilitation (LTF) in that synapse, whilst the other remained unaffected. This synapse-specific facilitation (lasting at least 24 hours) was dependent on transcription and on CREB activation and was accompanied by

grow th of new synaptic connections (M artin et al., 1997a). In addition, local

application of the protein synthesis inhibitor emetine to one synapse prior to, and during the application of 5 pulses of serotonin, blocked LTF specifically within that synapse. This branch specific local protein synthesis was shown to have a presynaptic locus and could occur in the neurites of sensory cells that had been separated from their cell bodies, increasing 3 fold in response to application of serotonin (Martin et al., 1997a). M ost importantly, application of one pulse of serotonin to a synapse that would normally produce a short-term facilitation could be converted into a long-term facilitation, if preceded by 5 pulses of serotonin applied to the other synapse on the same cell. Therefore a single serotonin pulse serves to m ark or tag, this synapse enabling the ‘capture’ of products induced by gene expression in response to 5 pulses of serotonin at the other synapse. These ‘synaptic capture’ results are similar to those seen in the synaptic tagging experiments of Frey and Morris (Frey & Morris, 1997). Finally, synapse-specific facilitation lasting 24 hours was found to require protein

synthesis; while synaptic capture lasting 24 hours did not (M artin et al., 1997a).

These results suggest that a retrograde signal travels back to the nucleus to initiate gene transcription and may be dependent on protein synthesis, whilst the tag is not.

Synapse-specific LTF has recently been further investigated in the same laboratory. Using the same cell culture preparation, Casidio et al. (1999) investigated the time course and the nature of the tag. The tag set by a single serotonin pulse could persist for between 4 to 5 hours, whilst the nuclear products of gene expression initiated by 5 pulses of serotonin persisted for between 1 and 4 hours. Furthermore, LTF normally produced by synaptic capture, was blocked at 24 hours post-induction, by the PKA inhibitor Rp-cAM PS, suggesting that initially the tag takes the form of a PKA-

m ediated covalent modification (Casadio et al., 1999). Importantly, extending the

length of tim e over w hich long-term facilitation was follow ed, produced an interesting result. LTF occurring from synaptic capture, although previously shown to

be protein synthesis independent for a least 24 hours (Martin et al., 1997a), was seen

a l , 1999). A dditionally, local application of the im m unosuppresant rapam ycin (known to block serotonin-induced translation), blocked LTF produced by synaptic capture at 72 hours, although synaptic grow th over 24 hours was unaffected. Surprisingly, therefore, rapamycin-sensitive local protein synthesis was not required to initiate synaptic growth but was required for the stabilisation and persistence of that growth. Furthermore, rapamycin did not block synapse-specific facilitation over 24 hours (unlike emetine) but did block it over 72 hours. These results suggest that local

synthesis at the synapse may be required for two different m echanism s: 1) a

rapamycin-insensitive, emetine-sensitive generation of a retrograde signal at the site of LTF, that can travel to the nucleus to initiate gene expression 2) a rapamycin sensitive stabilisation of synaptic growth (maybe due to stabilisation of the initial PKA dependent covalent tag) leading to the persistence of LTF for greater then 72 hours.

In addition to ‘capture’ occurring from synapse-specific facilitation, it has also been shown to occur from cell-wide facilitation. Casidio et al. (1999) showed that 5 pulses

of serotonin applied to the cell bodies of Aplysia cultured sensory neurons, produced a

cell-wide long-term facilitation that was dependent on transcription, translation and CREB activation. This facilitation was different from synapse-specific long-term facilitation, in that it showed no short-term facilitation, was not dependent on synaptic

growth and did not exist beyond 48 hours (Casadio et al., 1999). However, coupling

of this cell-wide facilitation, with a single pulse of serotonin at a specific synapse, could convert this synapse into one that showed growth and in which LTF was persistent for at least 72 hours (Casadio et al., 1999). This situation occurred even in cases in which subthreshold stimulation, that produced no detectable facilitation, was

applied to the cell bodies (Casadio et al., 1999). Around the same time, it was shown

in Aplysia, in vivo, that the duration of serotonin application to either the soma or the synapses could be titrated so as to produce a stimulus that was below the threshold for long-term facilitation. However, if synaptic, as well as somatic serotonin stimulation occurred together, within a stringent time window, LTF occurred (Sherff and Carew, 1999). This coincident LTF was dependent on immediate local protein synthesis at the synapses as well as somatic protein synthesis occurring 1-3 hours later (Sherff & Carew, 1999).

Although a role for local protein synthesis in A plysia long-term facilitation is in no doubt, the mechanisms by which these changes are produced remain unclear. Is local synthesis required to generate a retrograde signal that can travel back to the nucleus? Furtherm ore, are the products trafficked back to the synapses, proteins, mRNAs or both?