The effect of N-arachidonoyldopamine on distributioh of cannabinoides receptors (type 1 and type2) in dissotiated hyppocampal cultures in acute hypoxia in vitro

The aim of the investigation was to study the effect of the endocannabinoid N-arahidonoyldopamin (N-ADA) on distribution of cannabinoid receptors type 1 and 2 in dissociated embryonic hippocampal primary cultures in acute hypoxia in vitro. For the immunostaining principal cytoskeleton proteins of neurons (MAP2) and astrocytes (GFAP), and cannabinoid receptors type 1 and type 2 (CB1 and CB2) were marked in dissociated hippocampal cultures on the 7th day after acute normobaric hypoxia. It has been shown the morphology of cells in dissociated culture significantly changed by acute normobaric hypoxia. A particular interest was to study the changes in the morphology of astrocytes. Thus the partial dendrites reduction and rounding bodies of neurons was observed. After modeling normobaric hypoxia glial cells acquired an elongated shape, while astrocytes were combined into a single entity. In cultures the glial strands were detected and in this structures it was impossible to distinguish individual cells.

Type 1 cannabinoid receptors are localized on the bodies and processes of neurons. To estimate the effects of acute hypoxia on the distribution of CB1 receptors were characterized such parameters as the number of clusters and the average area of CB1 cluster. It was shown that hypoxic injury did not change the number of clusters CB1, but leads to a statistically significant reduction average size of the clusters CB1 (0,72 ± 0,07 mm2 to 0,51 ± 0,07 mkm2). Application of N-ADA 10 mcM during hypoxia and on the first days after hypoxia maintained the normal size of CB1 receptor clusters. Number of CB1 clusters increased significantly (from 18,95 ± 2,16 to 27,35 ± 2,24 cluster/100 mcm length of neuron-s process).

CB2 receptors are localized mainly on glial cells and detected at some neurons. It is shown that the number of clusters on the CB2 receptor significantly reduced in the posthypoxic period (from 4,92 ± 0,55 to 2,65 ± 0,75 clusters on 100 mcm2), and their size significantly reduced too (from 1,02 ± 0,21 to 0,54 ± 0,09 mkm2). Application of N-ADA 10 mcM during hypoxia and on the first days after hypoxia prevented reduction the size and the number of CB2 receptor clusters. That allows us to conclude N-ADA prevents the changes in cannabinoides receptor distribution during acute normobaric hypoxia.