The third ventricle in the hypothalamus is lined by specialized glia-like cells called tanycytes. They are highly polarized cells, whose projections reach into different nuclei of the mediobasal hypothalamus and median eminence, responsible for energy homeostasis, food intake, and hormone release. Historically, tanycytes have been distinguished into alpha and beta subtypes according to their morphology and location along the ventricle. However, the classification according to localization alone does not provide information about the function of these cells. Therefore, we modified adeno-associated virus (AAV)-based vectors that target all tanycytes in such a way that they would allow the manipulation of tanycytic subpopulations. We used different small promoter fragments driving a Cre-recombinase combined with a green fluorescent marker. These AAVs were injected into the lateral ventricle of Cre-reporter mice by stereotactic surgery. Using immunofluorescence and microscopy, we analyzed the induced expression pattern of 18 different promoter viruses. This approach helped us identify specific promoters for different tanycytic subpopulations.It is already known that tanycytes modulate neuronal functions. However, whether neurons mediate tanycytic function as well is still poorly investigated. We performed retrograde tracing utilizing a Cre-dependent rabies virus to reveal neuron to tanycyte connectivity, and identified neuron-tanycyte contacts in specific nuclei across the whole brain. To prove the functionality of these synaptoid contacts, we performed calcium or cAMP measurements on primary tanycytes and acute slices.Overall, the data allows us to refine the historical classification of tanycytes and unveils the functionality of specific tanycyte subtypes.