The influence of binocular deprivation on the developmental gene andprotein expression profiles in the central and peripheral visual fieldrepresentation in the primary visual areas of the cat

Long-term congenital cataract, in contrast to delayed onset cataract, results in a severe impairment in global motion perception if left untreated. In cat binocularly deprived from pattern vision (BD), which is an animal model for human cataract, timing and duration of BD were recently identified as two determinants of the severity of this behavioral outcome. These behavioral impairments occur together with permanent changes inthe number and dendritic tree stratification of Y-type alpha retinal ganglion cells. These observations made me hypothesize that BD early in life might particularly disrupt the normal development of the motion-sensitive primary cortical areas, area 18 and peripheral area 17. Mystudy was based on analyzing the developmental profiles of gene and protein expression in the central and peripheral visual field representation in primary visual areas of the cat. To assess the timing of the maturation of normal gene and protein expression patterns I compared the visual cortex of 1, 2, 4 and 6-month-old (1N, 2N, 4N, 6N) and adult cats reared under normal visual conditions. To scrutinize the outcome of BD, fourdifferent deprivation strategies were compared, including early onset BD from birth and lasting for 2, 4 or 6 months (2BD, 4BD, 6BD), and late onset BD for 2 months upon 2 months of normal vision (2N2BD), as animal models of congenital and delayed onset cataract respectively. On the last day of treatment, all animals were maintained overnight in total darkness followed by 1-hour light stimulation to induce maximal visually-driven immediately early gene expression in the visual cortex.In the first part of my study, in situ hybridization was applied for measuring changes in the visually-driven expression of the activity reporter gene zif268 , to visualize possible differences in activation of the central and peripheral region of primary visual cortices 17 and 18 as a function of age and visual manipulation. Decreasing zif268 mRNA levels between month 2 and 4 characterized the normal maturation of the (supra)granular layers of central and peripheral area 17 and 18. In general, all BD conditions had higher than normal zif268 levels. In area 17, early BD showed a delayed decrease in zif268 signal, beginning later in its peripheral than in its central region. In contrast, the decrease occurred between month 2 and 4 in both central and peripheral area 18. Lack of pattern vision stimulation during the first 4 months of life therefore has a different impact on the development of area 17 and 18. As centro-peripheral differences in zif268 expression were visible only in area 17, I was further interested whether such a difference may be related to other genes characterized by light-induced expression. Therefore, I applied quantitative real-time RT-PCR to measure levels of BDNF mRNA expression.During normal development BDNF mRNA levels did not change remarkably. Higher BDNF mRNA levels as compared to age-matched controls were observed only in central area 17 of 2BD and 6BD animals. BDNF overexpression was shown previously to rescue the visual cortex fromthe maturational delay effect of dark rearing. In central area 17, the high BDNF mRNA levels induced by the 1-hour light stimulationat the end of the BD period may activate a mechanism by which normal development is restored.In the next part of the study I screened for maturation-related proteomic changes in central and peripheral area 17 under normal visual stimulation and binocular pattern deprivation in 2- and 4-month-old normal and BD animals. Intracellular protein expression was judged by means of a functional proteomics approach (Two-Dimensional Difference Gel Electrophoresis, 2-D DIGE and mass spectrometry). Thirty six differentially expressed proteins were identified. Ingenuity pathway analysis associated the BD affected proteins with processes of neurite outgrowth, synaptic transmission, clathrin-mediated endocytosis, and energy and mRNA metabolism. In normal kittens there were fewer proteins withan expression change in central compared to peripheral area 17, whereasin BD animals proteomic changes occurred more frequently in the centralcompared to the peripheral region. Since zif268 indicated a delay in cortical maturation of BD animals I next compared 4BD to younger 2N animals. In central and peripheral area 17 respectively, 69% and 97% of the identified proteins did not show an expression difference between 4BD and 2N kittens. Therefore, early onset BD seems to prevent the necessary developmental changes in protein expression more in the peripheral than in the central visual field representation. The proteomic study showed that CRMP4 was the only member of the CRMP family upregulated under BD. As CRMP4 expression is specific for parvalbumin-positive interneurons in adult cat visual cortex, I assumed that the effectof BD was specifically directed onto the functioning of inhibitory transmission. Thus, I further applied Western blot analysis to reveal possible changes in the expression of proteins involved in GABAergic transmission due to different onset and duration of BD. I chose to focus on GABA synthesizing enzymes and proteins regulating neurotransmission identified in the 2-D DIGE screening. This analysis involved: GAD (Glutamate decarboxylase) isoforms: GAD67 (the enzyme producing the main cellular GABA pool) and GAD65 (the GABA synthesizing enzyme at the synapse, synaptic vesicle-associated isoform); septin 5, which co-localizes with presynaptic GABAergic vesicles and acts as an inhibitor of neurotransmitter release; heat shock cognate 71 kDa protein (Hsc70) involved in clathrin-mediated endocytosis of receptors; and alpha-synuclein, which promotes SNARE-complex assembly during exocytosis in a presynaptic terminal andmay enhance clathrin-mediated endocytosis after neuronal stimulation. GAD65 was downregulated, whereas septin 5 upregulated in BD kittens, suggestive of a negative regulation of inhibitory transmission, which normally should increase with age to open and subsequently close the critical period for ocular dominance plasticity. Expression of alpha-synuclein and Hsc70 was decreased in BD kittens, possibly pointing towards negative regulation of neurotransmission and clathrin-mediated endocytosis of receptors. In conclusion, both the light-induced zif268 activation patterns and the proteomic study reveal that early onset BD delays area 17 maturation and that this effect is stronger for peripheral area 17. The protein expression changes indicate that BD results in a negative regulation of inhibitory transmission during area 17 development and possibly of specific structural remodeling of connections formed by inhibitory interneurons. The observations for BDNF suggest that the mechanism of recovery upon ending the deprivation period may differ between regions of primary area 17.status: publishe