Detection of endogenous retinoids in the molluscan CNS and characterization of the trophic and tropic actions of 9-cis retinoic acid on isolated neurons

Abstract

Retinoic acid (RA) is an active metabolite of Vitamin A that plays an important role in the growth and differentiation of many cell types. All-trans RA (atRA) is the retinoic acid isomer that has been most widely studied in the nervous system, and can induce and direct neurite outgrowth from both vertebrate and invertebrate preparations. The presence and role of the 9-cis-RA isomer in the nervous system is far less well defined. Here, we used high-pressure liquid chromatography (HPLC) and mass spectrometry (MS) to show for the first time, the presence of both atRA and 9-cis-RA in the CNS of an invertebrate. We then demonstrated that 9-cis-RA was capable of exerting the same neurotrophic and chemotropic effects on cultured neurons as atRA. In this study, significantly more cells showed neurite outgrowth in 9-cis-RA versus the EtOH vehicle control, and 9-cis-RA significantly increased the number and length of neurites from identified neurons after 4 d in culture. 9-cis-RA also extended the duration of time that cells remained electrically excitable in culture. Furthermore, we showed for the first time in any species, that exogenous application of 9-cis-RA induced positive growth cone turning of cultured neurons. This study provides the first evidence for the presence of both atRA and 9-cis-RA in an invertebrate CNS and also provides the first direct evidence for a potential physiological role for 9-cis-RA in neuronal regeneration and axon pathfinding.

Figure 1.

RALDH activity in brain extracts of Lymnaea stagnalis. A, Sil-15 reporter cells stained for β-gal activity after addition of culture medium. B, A similar field of Sil-15 reporter cells stained for β-gal activity after the addition of 25 μl of RALDH reaction product with Lymnaea CNS extract as the substrate. Note a significant increase in the number of β-gal-positive (blue) cells. C, RA release by Lymnaea CNS explants. Cells stained for β-gal activity as in A and B above. D, Sil-15 reporter cells following the addition of 10⁻⁶ m atRA.

Figure 2.

HPLC separation of 9-cis-RA and atRA standards run as a mixture. 9-cis-RA elutes at 14.6 min and atRA elutes at 15.1 min.

Figure 3.

Mass spectra and electrospray ionization of atRA and 9-cis-RA standards. A, AtRA standard molecular weight is determined to be 299.1 m/z. B, Electrospray ionization of atRA. C, 9-cis-RA standard molecular weight is determined to be 299.1 m/z. D, Electrospray ionization of 9-cis-RA.

Figure 4.

A, B, Analytical HPLC profiles showing near baseline separation of atRA and 9-cis-RA in adult Lymnaea stagnalis CNS (A) and hemolymph (B) extracts. In both the CNS and hemolymph profiles, peak 1 corresponds to 9-cis-RA and peak 2 to atRA, respectively.

Figure 5.

Estimated relative concentrations of 9-cis-RA and atRA in the adult Lymnaea CNS and hemolymph.

Figure 6.

9-cis-RA and atRA significantly increased neurite outgrowth. A, Representative examples of identified VF cells showing neurite outgrowth after 2 d in culture (DM) after treatment with 9-cis-RA (i), atRA (ii), and EtOH (iii) vehicle solution (example of a cell with neurite outgrowth). B, Histogram showing that the percentage of cells producing neurite outgrowth in 9-cis-RA and atRA on day 2 (10⁻⁷ m) was significantly greater than the percentage of cells producing outgrowth in vehicle control (EtOH). There was no significant difference between 9-cis-RA and atRA. (***p < 0.001, **p < 0.01; compared with EtOH controls). C, The total length of primary neurites extended was significantly greater on days 2, 3 and 4 in both 9-cis-RA and atRA compared with EtOH controls. D, Also, the number of primary neurites extended was significantly greater on days 2, 3 and 4 in 9-cis-and atRA compared with controls. **p < 0.01 (statistical comparison shown only for day 4 and compared with EtOH control conditions).

Figure 7.

Both 9-cis-RA and atRA lengthened the duration that cells were electrically excitable in culture. Standard intracellular recordings were made from cells treated with 9-cis-RA, atRA, or vehicle control on day 4 of cell culture. A, The RMPs of cells cultured in 9-cis-RA and atRA were not significantly different from each other, although they were significantly more negative than the RMPs of control cells. B, The histogram shows the percentage of cells that were electrically excitable (spontaneous or induced firing) on day 4 of culture. There was no significant difference in the number of cells that fired action potentials between cells cultured in 9-cis-RA or atRA; however, both were significantly different from vehicle controls. C, Representative intracellular electrophysiological recordings from cells cultured in 9-cis-RA (i), atRA (ii), and vehicle control (iii). Following depolarizing current injection, the cells cultured in 9-cis-RA and atRA fired action potentials on day 4. The cell cultured in vehicle control failed to respond to current injection on day 4. Arrowheads indicate the time of current injections. *p < 0.05, **p < 0.01 (compared with control conditions).

Figure 8.

9-cis-RA induced positive growth cone turning. A, A VF cell was cultured in CM for 2 d to allow for neurite outgrowth and then monitored for 1 h to ensure no spontaneous changes in growth cone direction occurred. i, Control image before the directional, pulsatile application of 10⁻⁵ m 9-cis-RA via a micropipette (t = 0 min). ii, Eighteen minutes after the start of RA application, the growth cone turned toward the source of 9-cis-RA. iii, Fourteen hours after the removal of the local source of 9-cis-RA, the growth cone continued to grow in the new direction. B, The histogram depicts the maximum turning angles of growth cones exposed to 9-cis-RA (10⁻⁵ m). Each bar on the histogram represents one growth cone from one cell.