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% ID, the identity obtained by nucleotide database search; Species, the species with which sequence similarity was highest; LPZ: higher mRNA expression in the lesion projection zone; R: higher mRNA expression in remote peripheral visual cortex; numbers in parenthesis: number of identical nucleotides over total length of the cDNA sequence

% ID, the identity obtained by nucleotide database search; Species, the species with which sequence similarity was highest; LPZ: higher mRNA expression in the lesion projection zone; R: higher mRNA expression in remote peripheral visual cortex; numbers in parenthesis: number of identical nucleotides over total length of the cDNA sequence

2a. Involvement of Several Transcription Factors in Cortical Plasticity

Various transcription factors have already been implicated in retinotopic map plasticity and brain plasticity in general (Kaczmarek & Nikolajew, 1990; Kaplan et al., 1996, Kaczmarek & Chaudhuri, 1997, Obata et al., 1999, Arckens et al., 2000b, Pinaud, 2004). Members of four distinct transcription factor families have been unambiguously correlated to cortical plasticity. With c-fos belonging to the AP-1 family, zif268 to the zinc finger family, creb to the ATF/CREB family and mef2 to the MADS box family of transcription factors, the extent by which the regulation of gene expression is significantly altered after sensory deprivation as induced by retinal lesions became apparent. Some of these transcription factors involved in map plasticity also participate in other protein expression-dependent forms of brain plasticity like long-term potentiation and long-term memory (Hughes and Dragunow, 1995; Herdegen and Leah, 1998). Next to being markers for cortical neuronal activity, transcription factors emerge as components in a cascade of events that leads to long-term changes in cortical circuitry. Evidently, the complex and overlapping expression patterns of the different transcription factors further suggest that they do not function individually in neurons. Rather they will work in concert with a network of transcription factors to exert specific functions (for review see Hughes and Dragunow, 1995; Herdegen and Leah, 1998).

CREB (Obata et al., 1999) and the MEF2 transcription factors (Leysen et al., 2004) showed augmented expression in the LPZ of primary visual area 17. In contrast, the immediate early genes c-fos and zif268 displayed a clearly decreased expression within the LPZ (Arckens et al., 2000b; Van der Gucht et al., 2003). Without doubt, charting the expression of the specific target genes of each of these transcription factors, the so-called 'late-response genes', will contribute to our understanding of the molecular mechanisms of adult brain plasticity. As an example, several neuron-specific genes, like synapsin 2, neurogranin, and VIP, contain putative MEF2 sites in their upstream regulatory regions and some have indeed been shown to be activated by MEF2, e.g. the NR1 subunit of NMDA type glutamate receptors and the cytoskeleton neurofilament protein (Chin et al., 1994; Sato et al., 1995; Hahm and Eiden, 1998, Krainc et al., 1998; Skerjanc and Wilton, 2000). Recently, Western blotting experiments already provided evidence for plasticity-related changes in NR1 subunit expression in the LPZ of area 17 in adult cat (Van Damme et al., 2002). Obata et al. (1999) also showed a clear effect of retinal lesions on synapsin 1 and 2 immunoreactivity within 3 days. Here zif268 could also come into play since Thiel and colleagues (1994) showed that zif268 too could regulate synapsin 1 gene expression.

Remarkable about the modulation of the transcription factor expression in the LPZ of area 17 of animals with retinal lesions is the sustained deviation of c-fos, zif268 and CREB expression levels and the emergence of changes in the phosphorylation state of CREB as late as two years after trauma (Obata et al., 1999; Arckens et al., 2000b). These long-term effects on the regulation of gene transcription may indicate that the related brain regions remain in demand of dynamic adjustments for a long post-traumatic time, an observation also made for the auditory system upon acoustic trauma (Michler and Illing, 2003).

2b. Altered Neurotransmitter Systems - Changes in the Excitation/inhibition Balance

A variety of studies have suggested that the balance between excitation and inhibition modulates plastic responses in the central nervous system (CNS)

after peripheral or central injuries (Garraghty et al., 1991; Jones, 1993; Feldman et al., 1999; Arckens et al., 1998, 2000a, Myers et al., 2000; Boroojerdi et al., 2001; Tighilet & Lacour, 2001; Tremere et al., 2001; Zepeda et al., 2004). Reductions of cortical inhibition through a decrease in the availability of GABA or an increase in glutamate transmission, or a combinatorial effect, have been suggested as possible mechanisms underlying functional reorganization. Changes at the amino acid level could reflect the activation of adaptive pre-synaptic mechanisms, whereas modification in the receptor subunits could be related to post-synaptic responses that lead to changes in the excitation-inhibition neurotransmission balance. Only few studies correlate time-dependent changes in neurotransmitter systems with functional recovery, although they may reflect biochemical adjustments involved in shaping neuronal receptive fields during functional reorganization.

Following retinal lesions, the release and uptake of inhibitory and excitatory neurotransmitters in cat primary visual cortex are re-regulated in a complex and multifaceted manner. Microdialysis revealed reduced glutamate levels within the LPZ of area 17 (Qu et al., 2003, Massie et al., 2003a,b). This decrease of the excitatory amino acid appeared specific because contents of non-neurotransmitter amino acids, as measured in parallel, did not change in a similar magnitude, suggesting that retinal lesions do not affect all amino acids in the same way, as would be expected if changes in general metabolism were responsible. The reduced neurotransmitter levels were the result of a decreased synthesis/release of the amino acid glutamate in the first weeks post-lesion. Indeed, an effect on neurotransmitter uptake was only observed in animals that survived longer (Massie et al., 2003a), as revealed through the investigation of the expression of glial glutamate transporter molecules, and uptake inhibition experiments in which these transporters were blocked with L-trans-pyrrolidine-3,4-dicarboxylic acid (PDC), a transportable glutamate analogue that produces the most selective and potent inhibition of glutamate uptake (Bridges et al., 1991; Thomsen et al., 1994).

Within the first weeks, altered glutamate receptor subunit expression was also observed in the LPZ. The number of AMPA receptors appeared reduced, since both the GluR1 and GluR2 subunit levels decreased shortly after making the retinal lesions, with a partial restoration of normal expression levels at 30 days post-lesion (Van Damme et al., 2002). Alternatively, the GluR2 down-regulation may be interpreted as an increase in Ca2+-permeable AMPA subunit receptors in the LPZ (Hollmann et al., 1991). Increased Ca2+ influx through GluR2-negative AMPA receptors could then control the phosphorylation of AMPA subunits, thereby triggering long-lasting changes in synaptic efficacy or connectivity (Jonas et al., 1994).

Different observations were made for the NMDA type glutamate receptors in cat area 17 after the induction of retinal lesions. The subunit composition of the NMDA receptors changed drastically with post-lesion survival time. The total number of NMDA receptors looked lower in the LPZ due to a general down-regulation of NR1, the mandatory subunit of the NMDA

receptor. We observed the lowest level for NR1 at 14 days post-lesion, which coincides in time with the presence of a substantial functionally silent LPZ. In contrast, the NR2A subunit expression peaked at 14 days post-lesion in the LPZ, whereas the NR2B subunit did at 30 days corresponding to the phase in the reorganization in which glutamate levels had already substantially recovered (Arckens et al., 2000a; Massie et al., 2003a; Qu et al., 2003) This parallel increase in glutamate probably reflects excitatory adjustments at the pre- and postsynaptic level, pointing to an increased excitability within the LPZ from 2 to 4 weeks post-lesion on. The restoration of approximately normal NR1 levels 30 days post-lesion also correlates well in time with such a progression of the functional reorganization.

Thus, the excitatory neurotransmitter system, including the amino acid glutamate, the glial glutamate transporters and the NMDA and AMPA receptors, participates intimately in the synaptic changes that contribute to functional reorganization after peripheral lesions.

Despite intensive investigation of the inhibitory neurotransmitter system in animals with retinal lesions, the findings for GABA in the LPZ are much less striking as compared to glutamate. GABA immunocytochemistry, GABAA and GABAB receptor autoradiography or analysis of extracellular fluid GABA concentrations could not reveal any substantial GABA-related changes within the LPZ (Rosier et al., 1995; Arckens et al., 2000a, Massie et al., 2003b). Only GAD, the enzyme that synthesizes GABA out of glutamate, displayed a lesion-dependent immunoreactivity pattern. The decrease in detectable GAD-immunoreactive terminals was interpreted as a decreased synaptic inhibition, possibly through a hampered axonal transport of the enzyme, since the number of GAD-immunoreactive cell bodies simultaneously increased. With GAD65 being responsible for the GABA synthesis-on-demand at synaptic sites, such a restrain on the transport of GAD65 could indeed lead to local, subtle changes in pre-synaptic, vesicular GABA supply and thus inhibition (Soghomonian and Martin, 1998). In accordance, Engel and colleagues (2001) have recently shown that GABA metabolism can determine inhibitory synaptic strength. Therefore pre-synaptic GABA content might be a regulated mechanism for synaptic plasticity.

The critical role of the central neuromodulatory systems in maintaining and shaping the neuronal network of the cerebral cortex is not yet clearly understood. The catecholamine and serotonin content of the LPZ in area 17 of animals with retinal lesions has been investigated by reverse phase high performance liquid chromatography and electrochemical detection (Qu et al., 2000). The dopamine metabolites homovallinic acid (HVA) and dihydrox-yphenilacetic acid (DOPAC) appeared reduced 14 days post-trauma, which could point to a change in dopamine metabolism in the LPZ. Levels of serotonin and its metabolite 5-HIAA were also decreased in the LPZ. Lack of knowledge on the temporal aspect of the neuromodulator changes and on how the specific receptors react to retinal lesions makes it difficult to speculate on the specific mechanisms by which dopamine and serotonin might modulate cortical reorganization. Since in normal animals their respective receptors are distributed throughout supra- and infragranular layers in sensory cortices (Dyck and Cynader, 1993; Rakic and Lidow, 1995), serotonin and dopamine might modulate the strength of cortico-cortical projections. Serotonin modulates LTP/LTD-like processes in developing visual cortex (Kojic et al., 2000, Edagawa et al., 2001). Likewise, transient changes in the serotonin levels in the LPZ could thus facilitate LTP/LTD-like adaptations of synaptic contacts upon sensory deprivation. Indeed, in primary auditory cortex dopamine-induced remodelling of the sound-frequency representations has also been described (Stark and Scheich, 1997, Bao et al., 2001).

2c. Growth Factors

Lesion-induced changes in cortical topography are furthermore accompanied by a rapid increase in the expression of neurotrophins, including BDNF and NT-3, and relevant receptors (Obata et al., 1999). Neurotrophins are important regulators of synaptic development and plasticity in both the central and peripheral nervous system. Neurotrophins can modulate synaptic transmission at the pre- and postsynaptic level in a target-specific fashion. In the visual cortex, BDNF can influence GABAergic intracortical inhibitory neurons and serotonergic afferents from the Raphe nucleus (Berardi et al., 2003). The observation that the elevation of the neurotrophin levels is sustained for up to two years after induction of retinal lesions may reflect the fact that even though visually driven activity restores in the cortical scotoma, the level of activity never fully returns to that of the surrounding cortex and some imbalance of activity persists (Das and Gilbert, 1995b).

2d. Synapse Efficacy, Axon Growth, Dendritic Sprouting

The sculpting, maintenance and remodelling of axonal and dendritic arbors happens under influence of an amalgam of intracellular and extracellular factors. Synapsin I has been implicated to play a role in the establishment of synaptic contacts during the development of the mammalian CNS as well as during the process of re-innervation within the adult brain after partial deaf-ferentation (De Camilli et al., 1983, Brock and Callaghan, 1987, Moore and Bernstein, 1989, Melloni et al., 1994). The detection of changes in synapsin I levels has been used as parameter for changes in the number of nerve terminals within a certain brain region. Moreover it has been reported that the appearance of detectable synapsin I levels in developing and sprouting synapses would coincide with the acquisition of function by those synapses. The persistent upregulation of synapsin immunoreactivity in the LPZ thus seems to correlate with ongoing changes in synaptic density and activity (Obata et al., 1999; Walaas et al., 1988, Moore and Bernstein, 1989, Melloni et al., 1994).

Dendritic and axonal sprouting has also been associated with MAP2 and GAP-43 expression respectively. MAP2 immunoreactivity has been considered a good marker for dendrites based on experimental evidence that correlates dendritic growth with an increase in MAP2 immunoreactivity (Philpot et al., 1997; Sanchez et al., 2000; Bury and Jones, 2002). At every time point studied, the LPZ in area 17 of retinal lesion cats displayed a modified MAP2 immunolabeling (Obata et al., 1999). The MAP2 upregulation therefore suggests dendritic sprouting as a possible mechanism by which cortical reorganization occurs (Zepeda et al., 2004). GAP43 is mainly present in growth cones. Its expression after injury has been correlated with axonal regeneration (Baekelandt et al., 1994; Stroemer et al., 1995). Nevertheless axonal sprouting does not solely depend on GAP43 levels (Szele et al., 1995). The absence of modulation of GAP43 immunoreactivity cannot rule out that axonal sprouting takes place at some point in the recovery process (Baekelandt et al., 1996; Obata et al., 1999). Indeed axonal remodelling of horizontal connections has been shown to occur in the LPZ of area 17 albeit from 8 months post-lesion on (Darian-Smith and Gilbert, 1994).

2e. Systematic Screening for Plasticity-related Molecules

Differential display is an mRNA fingerprinting technique allowing the systematic screening for differences in gene expression between experimental conditions (Liang and Pardee, 1992). The possibility to detect up- and down-regulated genes makes it an attractive screening method. We have used differential display to compare bi-directionally the repertoire of genes expressed in the LPZ and remote, non-deprived visual cortex of adult cats with small homonymous central retinal lesions (Arckens et al., 2003, Leysen et al., 2004). Differential display qualified as the method of choice since no a priori assumptions had to be made as to the possible identity of the genes of interest. The twenty-seven genes isolated (listed in Table 9.2) execute diverse functions in mammalian brain. Whereas some showed a higher expression level in the LPZ of area 17, even more displayed a lower expression in the LPZ compared to remote visual cortex 3 to 14 days post-lesion. It is tempting to categorize these genes as plasticity activator and suppressor genes, like the memory activator and suppressor genes that either support or put inhibitory constraints on the storage of long-term memory (Abel et al., 1998). For 4 out of 27 genes (Table 9.2), the glial glutamate transporter EAAT2, cyclophilin A, MEF2A and Cu/Zn super oxide dismutase (Cu/Zn SOD), we have gathered detailed experimental evidence on the temporal and spatial regulation of the expression of these genes and their protein products in relation to cortical plasticity (Leysen et al., 2000, Massie et al. 2003a, Arckens et al., 2003, Leysen et al. 2004).

The plasticity-related character of the expression of the other 23 genes reported in Table 9.2 should be interpreted with caution, but based on their cellular functions, several of these genes could act in concert with molecules listed in Table 9.1. SCIP (suppressed camp-inducible POU) or Oct-6 is a POU

domain transcription factor with a temporally and spatially regulated expression during CNS development, and appears highly expressed in cortical layer V neurons, that project to subcortical brain regions (Frantz et al., 1994). Retinoic acid receptors contribute to LTP and LTD, at least in hippocampus, implicating that vitamin A and its derivatives, the retinoids, might contribute to synaptic plasticity (Chiang et al., 1998). The fine regulation of retinoid-mediated gene expression is also important for optimal brain functioning and higher cognition (Etchamendy et al., 2003). Thymosin beta-4 is thought to participate in neurite outgrowth by sequestration of G-actin necessary for growth cone extension (Vartiainen et al., 1996; Carpintero et al., 1999). Beta-adaptin plays a role in receptor-mediated endocytosis (Fergusson, 2001). N-ethylmaleimide-sensitive factor (NSF) interacts directly and selectively with the intracellular C-domain of the GluR2 subunit of the AMPA receptor, and this interaction can be enhanced by BDNF. Blocking NSF results in reduced AMPA receptor-mediated synaptic transmission (Song et al., 1998; Narisawa-Saito et al., 2002). Based on these functions, the genes listed in Table 9.2 have potential as regulators of brain plasticity and thus clearly deserve attention in future research.

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