En bloc and transversal slice preparations from perinatal rodents are established in vitro models for studying control of breathing by neurons and neighboring glial cells in the lower brainstem. These neural “respiratory networks in the dish” show features complementary with those in recent in vivo models that enable, for example, optogenetic manipulation for studying behavioral changes in awake animals or modulation of these circuits by higher brain regions during sleep. Contrary, the in vitro models allow respiratory network analysis at the (sub)cellular level. This is currently studied using powerful analytical tools such as quantitative pharmacology and patch-clamp recording of biophysical membrane properties of respiratory interneurons and/or motoneurons (plus neighboring glia). Increasingly, these approaches are combined with fluorescence imaging of both activity and morphology of individual cells or respiratory groups. Our recent work indicates that properties of the isolated respiratory networks depend critically on both their physical dimensions and the composition of superfusates used for their study. Based on this, we recommend to use anatomically “calibrated” rhythmic en bloc and slice preparations that we have developed and to study these models in superfusate that mimics in vivo conditions as closely as possible. We show here how these preparations are generated and present examples for pharmacological and “electrophysiological imaging” analyses that revealed novel properties of neuron–glial networks involved in respiratory control.