Stitched images were taken of the thalamic area as depicted (A) using 20X magnification from an unexposed postnatal day (PND) 7 rat pup (B) and an exposed PND7 rat pup (C). (B–E) We depict Hoechst33342 in blue, caspase 3 in green, and NeuN in red. An increase in the number of caspase 3– positive cells in the sevoflurane exposed PND7 rat pup is noted, indicated by the white arrows comparing representative images of an unexposed thalamus (B) with an exposed thalamus (C). A closer view of a region in the thalamus with a cluster of apoptotic cells is shown (D), allowing for better identification of the nuclei through the Hoechst33342 staining. With higher magnification using 63X oil objective (E), we can clearly see the nuclei of caspase 3–positive cells, appearing irregularly shaped, small rounded structures made up of chromatin bodies indicating the fragmenting DNA of dying cells. These are seen next to other normal appearing nuclei that do not stain for caspase 3. Notice also an abnormally shaped nucleus in a cell staining positive for NeuN and negative for caspase 3; this indicates that the caspase 3 stain is specific for apoptotic cells, only during certain phases of apoptosis. Also notice a cell with an abnormal nucleus staining positive for caspase 3 and negative for NeuN, indicating that nonneuronal cells can also undergo apoptosis and stain positive for caspase 3.