Immuno-EM characterization of vasopressin neurons in macaques by use of formaldehyde-fixed tissues stored for several years

Translational research often requires the testing of experimental therapies in primates, but research in non-human primates is now stringently controlled by law around the world. Tissues fixed in formaldehyde without glutaraldehyde have been thought to be inappropriate for use in electron microscopic analysis, particularly those of the brain. Here we report the immunoelectron microscopic characterization of arginine vasopressin (AVP)-producing neurons in macaque hypothalamo-pituitary axis tissues fixed with 4% formaldehyde and stored at –25°C for several years. The size difference of dense-cored vesicles between magnocellular and parvocellular AVP neurons was detectable in their cell bodies and perivascular nerve endings located, respectively, in the posterior pituitary and median eminence. Furthermore, glutamate and the vesicular glutamate transporter 2 were colocalized with AVP in perivascular nerve endings of both the posterior pituitary and the external layer of the median eminence, suggesting that both magnocellular and parvocellular AVP neurons are glutamatergic in primates. Both ultrastructure and immunoreactivity can therefore be sufficiently preserved in macaque brain tissues stored long-term for light microscopy. Taken together, these results suggest that this methodology could be applied to the human post-mortem brain and be very useful in translational research. ### Competing Interest Statement The authors have declared no competing interest. * ANOVA : analysis of variance AVP : arginine vasopressin CRF : corticotrophin-releasing factor dcv : dense-cored neurosecretory vesicle HRP : horseradish peroxidase NP : neurophysin PB : phosphate buffer PBS : phosphate-buffered saline PFA : paraformaldehyde PVDF : polyvinylidene difluoride PVN : paraventricular nucleus SEM : standard error of the mean SON : supraoptic nucleus Tris-HCl : trishydroxymethyl-aminomethane-HCl TBST : Tris-HCl-buffered saline VGLUT2 : vesicular glutamate transporter 2