Abstract
Similar to neurons in the peripheral nervous system, immature CNS-derived RGCs become dependent on target-derived neurotrophic support as their axons reach termination sites in the brain. To study the factors that influence this developmental transition we took advantage of the fact that rat RGCs are born, and target innervation occurs, over a protracted period of time. Early-born RGCs have axons in the SC by birth (P0), whereas axons from late-born RGCs do not innervate the SC until P4-P5. Birth dating RGCs using EdU allowed us to identify RGCs (1) with axons still growing toward targets, (2) transitioning to target dependence, and (3) entirely dependent on target-derived support. Using laser-capture microdissection we isolated ∼34,000 EdU+ RGCs and analyzed transcript expression by custom qPCR array. Statistical analyses revealed a difference in gene expression profiles in actively growing RGCs compared with target-dependent RGCs, as well as in transitional versus target-dependent RGCs. Prior to innervation RGCs expressed high levels of BDNF and CNTFR α but lower levels of neurexin 1 mRNA. Analysis also revealed greater expression of transcripts for signaling molecules such as MAPK, Akt, CREB, and STAT. In a supporting in vitro study, purified birth-dated P1 RGCs were cultured for 24-48 h with or without BDNF; lack of BDNF resulted in significant loss of early-born but not late-born RGCs. In summary, we identified several important changes in RGC signaling that may form the basis for the switch from target independence to dependence.
Highlights
Programmed cell death (PCD) occurs throughout the developing nervous system and is a crucial step in the maturation of neural circuitry
BDNF transcript levels exhibited by far the greatest reduction between growing and targetdependent RGCs, consistent with PCR data from the second laser-capture microdissection (LCM) study
Single-cell laser microdissection is a powerful tool for assessing gene expression in individual cells in situ
Summary
Programmed cell death (PCD) occurs throughout the developing nervous system and is a crucial step in the maturation of neural circuitry. A key element in regulating the onset and distribution of PCD is the availability of neurotrophic factor support In both the CNS and PNS, removal or addition of neurotrophic factors, respectively, increases or decreases the amount of PCD (PNS: Levi-Montalcini and Angeletti, 1968; Thoenen and Barde, 1980; Hamburger and Yip, 1984; Oppenheim 1991; CNS: Cui and Harvey, 1995; Cohen-Cory et al, 1996; Ma et al, 1998; Spalding et al, 1998, 2005). In PNS neurons the transition to target dependency is delayed by the same amount of time in vitro as it would be in vivo (Davies et al, 1987; Davies, 1989; Vogel and Davies, 1991), suggesting it is an intrinsic property of those neurons
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