Fig 2: Multiple label immunofluorescence of mGlu3 labeling in astrocytes in layer V of rat PL mPFC. Tissue was labeled with antibodies recognizing the astrocytic protein GFAP (cyan), mGlu3 (red), the dendritic marker Map2 (green), as well as the nuclear Hoechst stain (blue). (A) Expression of mGlu3 in the neuropil of Layer V is evident. There is GFAP positive astrocytic process that contains mGlu3 labeling. (Ai–iv) Magnified area of the region outlined by the yellow box in (A) showing co-localization of mGlu3 (red) and GFAP (cyan) within a bulbous end of a process, without the presence of Map2 (green). (B) This is an example of a GFAP positive astrocytic process overlying likely a blood vessel. (Bi–iv) Magnified area of the yellow box in (B) showing co-localization of mGlu3 and GFAP in a Map2 negative region. (C) Orthogonal sectioning of this region (corresponding region of interest from (B) outlined by the yellow box). Selected z-stack image shows co-localization of mGlu3 and GFAP (red and cyan) across three different planes for one point, as indicated by the crossed dashed lines. The right-side bar demonstrates labeling in the YZ plane, while the bottom bar represents labeling in the XZ plane. Scale bars: 10 µm (A,B); 5 µm (Ai–iv,Bi–iv); 10 µm (C).

Fig 3: Multiple label immunofluorescence of mGlu3 labeling in astrocytes in layer II/III of rat PL mPFC. Tissue was labeled with antibodies recognizing the astrocytic protein GFAP (cyan), mGlu3 (red), the dendritic marker Map2 (green), as well as the nuclear Hoechst stain (blue). (A) A region of layer II/III that is diffusely labeled for mGlu3. Map2 positive dendrites are positive for mGlu3. The yellow box demarcates a GFAP-positive astrocytic process. (Ai–iv) Magnified area outlined by the yellow box in A shows a thin astrocytic process (traced by the white arrows) that is mGlu3 and GFAP positive and Map2 negative. (B) Expression of mGlu3 (red) is evident in pyramidal-shaped neurons, as seen by co-localization of Map2 and mGlu3, and diffusely across the neuropil. There is a small blood vessel with a GFAP positive astrocytic process overlying the vasculature that contains mGlu3 labeling. (Bi–iv) Magnified area of the region delineated by the yellow box in (B) showing co-localization of mGlu3 (red) and GFAP (cyan) and the absence of Map2 (green) and Hoechst (blue), consistent with an astrocytic process. (C) Orthogonal sectioning of this region (corresponding region of interest from (B) outlined by the yellow box). Selected z-stack image shows co-localization of mGlu3 and GFAP (red and cyan) across three different planes for one point, as indicated by the crossed dashed lines. The right-side bar demonstrates labeling in the YZ plane, while the bottom bar represents labeling in the XZ plane. Scale bars: 10 µm (A,B); 2.5 µm (Ai–iv); 5 µm (Bi–iv); 5 µm (C).

Fig 4: Multiple label immunofluorescence of mGlu3 labeling in microglia in layer II/III of rat PL mPFC. Tissue was labeled with antibodies recognizing the microglial protein Iba1 (cyan), mGlu3 (red), the dendritic marker Map2 (green), as well as the nuclear Hoechst stain (blue). (A) There are diffuse Iba1 positive microglial processes across the neuropil in layer III. (Ai–iv) Magnified area of the yellow box in (A), showing an Iba1 positive microglial process (cyan) co-localized with mGlu3 (red), indicated by white arrows. Scale bars: 10 µm (A), 2.5 µm (Ai–iv).

Fig 5: Multiple label immunofluorescence of mGlu3 labeling in neurons in layer V of rat PL mPFC. Tissue was labeled with antibodies recognizing mGlu3 (red), the dendritic marker Map2 (green), as well as the nuclear Hoechst stain (blue). (A,B) In layer V, mGlu3 expression (red) is seen diffusely across the neuropil, with labeling within the perisomatic regions of putative pyramidal neurons as well as the apical dendrites. Two representative neurons are outlined by the yellow dashed boxes in (A,B). (Ai–iv,Bi–iv) Correspond to the boxed areas in (A,B). In both (Aii,iii,Bii,iii) mGlu3 labeling clearly outlines the apical dendrites, as demarcated by Map2 (green) and surrounds the nucleus (blue). Scale bars: 10 µm.