Predominant NR1-positive puncta with limited PSD95 colocalization and close GluA2-EAAT2 apposition in the...

The preBötzinger complex (preBötC) is a medullary network that generates the inspiratory phase of respiratory rhythm in mammals and depends critically on glutamatergic transmission. To characterize the anatomical organization of excitatory signaling elements within this network, we analyzed the distribution, morphology, and colocalization of the AMPA receptor subunit GluA2 and the NMDA receptor subunit NR1, together with the postsynaptic scaffold protein PSD95. We also used immunofluorescence and confocal microscopy to examine the spatial relationship between GluA2-positive puncta and the astrocytic glutamate transporter EAAT2 in the preBötC of adult male rats. NR1-positive puncta were more abundant and densely distributed than GluA2-positive puncta, whereas GluA2-NR1 colocalized puncta constituted only a small fraction of either receptor population. NR1-positive puncta also showed limited colocalization with PSD95-positive puncta, indicating that a substantial fraction of NR1 immunoreactivity is not associated with PSD95-defined postsynaptic domains under the present imaging conditions. In contrast, GluA2-positive puncta displayed a non-random spatial proximity with EAAT2 astrocytic profiles and were frequently located within submicron distances. Together, these findings reveal a spatially differentiated organization of glutamatergic elements in the preBötC, characterized by abundant non-PSD95-associated NR1-positive puncta and close apposition between GluA2-positive puncta and EAAT2-immunoreactive astrocytic profiles. This organization provides an anatomical framework for future studies testing how glutamate receptor localization and glutamate clearance mechanisms contribute to excitability and respiratory rhythm generation and modulation.