In the photoreceptors of the vertebrate retina, absorption of a quantum of light triggers a number of processes, called phototransduction, starting with activation of the visual pigment molecule, rhodopsin, and ending with changes in the permeability of the plasma membrane and photoreceptor hyperpolarization. In vertebrates, the secondary messenger of phototransduction is cGMP, the level of which in the cytoplasm is relatively high in the dark and decreases during light stimulation. It is known that the operation of the phototransduction cascade is regulated by calcium, but numerous phenomenological data indicate that there may be other regulatory signaling pathways in the phototransduction cascade for which there is no corresponding mechanism in the classical phototransduction scheme.
Several indirect data indicate that cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3), and diacylglycerol (DAG) could also have a regulatory effect on the phototransduction cascade. Obviously, a necessary condition for the presence of such a regulatory effect is a change in the concentration of the signaling molecule during the development of the regulated process. It was shown earlier that the concentration of cAMP and IP3 in photoreceptors can vary in response to changes in light conditions with characteristic times of more than one hour, which points to the involvement of these signaling molecules in the regulation of circadian rhythms and slow light or dark adaptation processes. However, it remains unclear whether cAMP, IP3 and DAG are involved in the regulation of the phototransduction cascade during the development of the photoreceptor response to a short light stimulus or the response to switching on or off a long light stimulus. The characteristic times of these processes are less than a second. Considering that traditional fluorescence methods cannot be applied to the measurement of concentrations of any signal molecules in the retina, we have created a setup allowing to perform cryofixation of retinal samples at the required rate. The complex allows to fix up to 6 samples in one series with a delay of less than 100 ms after light stimulation. The concentration of signal molecules is measured by high-performance liquid chromatography with high-resolution tandem mass spectrometry. Pilot experiments show an increase in the concentration of all three signal molecules studied within 400 ms after the presentation of a saturating light stimulus of 10 ms or 2000 ms. These data suggest the involvement of cAMP, IP3, and DAG in the regulation of the phototransduction cascade at the timescale corresponding to the development of a rapid photoresponse.

Funding
Supported by Russian Scientific Foundation grant RSF #22-25-00656