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. 2019 May 31;13:54.
doi: 10.3389/fnana.2019.00054. eCollection 2019.

Distribution of GABAergic Neurons and VGluT1 and VGAT Immunoreactive Boutons in the Ferret ( Mustela putorius) Piriform Cortex and Endopiriform Nucleus. Comparison With Visual Areas 17, 18 and 19

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Free PMC article

Distribution of GABAergic Neurons and VGluT1 and VGAT Immunoreactive Boutons in the Ferret ( Mustela putorius) Piriform Cortex and Endopiriform Nucleus. Comparison With Visual Areas 17, 18 and 19

Daniela Navarro et al. Front Neuroanat. .
Free PMC article

Abstract

We studied the cellular organization of the piriform network [comprising the piriform cortex (PC) and endopiriform nucleus (EP)] of the ferret (Mustela putorius)-a highly excitable region prone to seizures-and, more specifically, the distribution and morphology of different types of gamma-aminobutyric acid (GABA)ergic neurons, and the distribution and ratio of glutamatergic and GABAergic boutons, and we compared our findings to those in primary visual area 17, and secondary areas 18 and 19. We accomplished this by using cytochrome oxidase and immunohistochemistry for mature neuronal nuclei (NeuN), GABAergic neurons [glutamic acid decarboxylase-67 (GAD67), calretinin (CR) and parvalbumin (PV)], and for excitatory (vesicular glutamate transporter 1; VGluT1) and inhibitory (vesicular GABA transporter; VGAT) boutons. In the ferret, the cellular organization of the piriform network is similar to that described in other species such as cats, rats and opossums although some differences also exist. GABAergic immunolabeling showed similarities between cortical layers I-III of the PC and visual areas, such as the relative distribution of GABAergic neurons and the density and area of VGluT1- and VGAT-immunoreactive boutons. However, multiple differences between the piriform network and visual areas (layers I-VI) were found, such as the percentage of GABAergic neurons with respect to the total number of neurons and the ratio of VGluT1- and VGAT-immunoreactive boutons. These findings are relevant to better understand the high excitability of the piriform network.

Keywords: NeuN; calcium-binding proteins; epilepsy; piriform network; visual cortex.

Figures

Figure 1
Figure 1
Neuronal nuclei (NeuN) immunostaining and cytochrome oxidase histochemistry in the piriform network. Photomicrographs of horizontal (A) and coronal (C–E) sections show NeuN immunostaining in the piriform cortex (PC) and endopiriform nucleus (EP). (B) Cartoon shows the coronal anterior (a), coronal posterior (p) and horizontal (h) planes of cutting. Anterior (C,F) and posterior (D,E,G) sections are shown. (A,C,D) Low magnification photomicrographs show a high density of neurons in PC layers II and upper III, and in EP. (E) High magnification photomicrograph shows small densely packed neurons in PC layer II and EP. (F,G) Plots show the distribution of NeuN-ir neurons. (H) Bar chart shows the percentages of NeuN-ir neurons in PC and EP. A higher percentage of NeuN-ir neurons in PC layer II with respect to other PC layers and EP can be seen. The percentage of neurons was higher in posterior PC layer II and lower in layer III than in the anterior counterparts. (I,J) Cytochrome oxidase staining shows a heavy cytochrome oxidase-stained band in layers II and upper III in the PC (arrow in I). Borders between layers (E,F,G) and limits with adjacent areas (arrowheads in A,C,D,I,J) are indicated. RS, Rhinal sulcus; CS, Cruciate sulcus; SS, Sylvian sulcus; PSS, Pre-sylvian sulcus. Same magnification for (A,C,D,F,G,I,J). Vertical lines in bar charts show SD. #,*P < 0.05, **P < 0.001.
Figure 2
Figure 2
Distribution of glutamic acid decarboxylase-67 (GAD67)-ir neurons in the piriform network. Photomicrographs of horizontal (A) and coronal (B–E) sections show GAD67-ir neurons in the PC and endopiriform nucleus (EP). Anterior (B) and posterior (C–E) sections are shown. (D) At higher magnification, many processes and perisomatic boutons in layer II can be observed. (E) Heavily (arrow) and lightly (arrowhead) stained GAD67-ir neurons in EP can be seen. (F,G) Plots show the distribution of GAD67-ir neurons. (H) Bar chart shows that the percentage of GAD67-ir neurons in PC significantly decreased from layer III to layer I. No other significant differences between layer II and EP, and between anterior and posterior sections were found. Borders between layers (D,F,G) and limits between adjacent areas (arrowheads in A–C) are indicated. Same magnification for (B,C) and (F,G). RS, Rhinal sulcus. Vertical lines in bar charts show SD. **P < 0.001.
Figure 3
Figure 3
Distribution of CR-ir neurons in the piriform network. Photomicrographs of horizontal (A) and coronal (B–E) sections show CR-ir neurons in the PC and endopiriform nucleus (EP). Anterior (B) and posterior (C–E) sections are shown. (D,E) At higher magnification, many processes and boutons in layers I and II can be seen. (D) Most CR-ir neurons in layer II resemble bipolar cells, while in layer III, they are small and large multipolar. (E) In EP, CR-ir neurons are multipolar and fusiform bipolar, and in the neuropil, numerous perisomatic boutons can be observed. (F,G) Plots show the distribution of CR-ir neurons. (H) Bar chart shows a high percentage of CR-ir neurons in PC layers II and III with respect to other layers and EP. No significant differences were found between the anterior and posterior sections. Borders between layers (D,F,G) and limits with adjacent areas (arrowheads in A–C) are indicated. RS, Rhinal sulcus. Same magnification for (B,C,F,G). Vertical lines in bar charts show SD. n.s, not significant differences. **P < 0.001.
Figure 4
Figure 4
Distribution of parvalbumin (PV)-ir neurons in the piriform network. Photomicrographs of horizontal (A) and coronal (B–E) sections show CR-ir neurons in the PC and endopiriform nucleus (EP). Anterior (B,F) and posterior (C–E,G) sections are shown. (D,E) At higher magnification, many processes and boutons in PC layers II and III can be seen. (D) Most PV-ir neurons in layer II resemble bipolar with vertically oriented processes, while in layer III they resemble small and large multipolar. In EP, small multipolar PV-ir neurons can be seen. (E) In PC layers II and upper III, and in EP, numerous terminal-like perisomatic boutons can be seen. (F,G) Plots show the distribution of PV-ir neurons. (H) Bar chart shows a higher percentage of PV-ir neurons (anterior and posterior sections averaged) in layers II and III with respect to layer I and EP. The PV-ir neuron percentage was higher in the posterior than in the anterior EP and was similar in layer I and EP. Borders between layers (D,F,G) and limits with adjacent areas (arrowheads in A–C) are indicated. RS, Rhinal sulcus. Same magnification for (A,B,C,F,G). Vertical lines in bar charts show SD. #,*P < 0.05, **P < 0.001.
Figure 5
Figure 5
Immunostaining of NeuN-ir, GAD67-ir, CR-ir and PV-ir neurons in visual areas 17, 18 and 19. (A) The schematic upper view of the ferret cerebral hemispheres shows the parasagittal plane of cutting. (B,D,F,H) Low-power photomontages show the borders (arrowheads) between visual areas 17, 18 and 19. (C,E,G,I) Photomicrographs show detail of the distribution of NeuN-ir, GAD67-ir, CR-ir and PV-ir neurons in areas 17, 18 and 19. Borders between layers I through VI (vertical lines) are indicated. (C) Note the clear border between layers of visual areas in NeuN immunostained sections. (H,I) Note the increased PV immunostaining in layers I–IV. (J) High magnification (inset of layer V in area 19) shows numerous perisomatic (arrow) and chandelier-like (arrowheads) boutons. The (J) box is also shown in Supplementary Figure S1.
Figure 6
Figure 6
Distribution of NeuN-ir, GAD67-ir, CR-ir and PV-ir neurons in visual areas 17, 18 and 19. (A,D,G,J) Low-power plots show the radial distribution of NeuN-ir, GAD67-ir, CR-ir and PV-ir neurons (dots) in visual areas 17, 18 and 19 (arrowheads point the limit between visual areas). (B,E,H,K) Detailed plots show the radial distribution of labeled neurons. The borders between cortical layers are indicated by horizontal bars. Note the decreased density of NeuN-ir neurons in layer V and the increased density of CR-ir neurons in layer II. (C,F,I,L) Bar charts show the percentages of labeled neurons across cortical layers in each visual area. Comparing areas 18 and 19 with area 17, note the decrease in GAD67-, CR- and PV-ir neuron percentage in layer IV (F,I,L), the increase in GAD67-ir neuron percentage in layers V and VI (F), and the increase in PV-ir neuron percentage in layer VI (L). The NeuN-ir neuron percentage in layer I was significantly lower than in layers II–VI. These percentages are grouped by visual area in Supplementary Figure S2. (M,N,O) Proportion of GAD67-ir, CR-ir and PV-ir cells in each layer with respect to NeuN-ir neurons in visual areas. Note that the distribution of proportions of CR-ir/NeuN-ir neurons was similar across areas (excluding layer I) and that the PV-ir/NeuN-ir proportion in layer IV was higher in area 17 than in areas 18 and 19. Same magnification for (A,D,G,J) and for (B,E,H,K) Vertical lines in bar charts show SD. n.s., not significant differences. *P < 0.05, **P < 0.001.
Figure 7
Figure 7
Comparison of gamma-aminobutyric acid (GABA)cergic neurons between the piriform network and visual areas 17, 18 and 19. (A) GAD67-ir, (B) CR-ir and (C) PV-ir neuron percentages in layers I–III in PC and visual areas 17, 18 and 19. (B) CR-ir neuron percentages in layers I–III were similar in PC and visual areas, although differences can be perceived in the GAD67-ir and PV-ir neuron percentages between PC layer II and visual layer II. (D) GAD67-ir, CR-ir and PV-ir neuron proportions in layers I–III with respect to the total number of NeuN-ir neurons decrease in PC (see arrows indicating the difference) compared to visual areas. (E–G) GAD67-ir, CR-ir and PV-ir neuron proportions with respect to the total number of NeuN-ir neurons in PC, endopiriform nucleus (EP) and piriform network (PC+EP) were lower than in visual areas (see arrows indicating the difference). Some differences between visual areas 17 and 18 can also be observed for GAD67-ir and PV-ir neuron percentages. Vis, visual. Vertical lines in bar charts show SD. n.s., not significant differences. *P < 0.05, **P < 0.001.
Figure 8
Figure 8
Confocal immunolabeling of vesicular glutamate transporter 1 (VGluT1)-ir and vesicular GABA transporter (VGAT)-ir boutons in the anterior piriform network. (A–P) Confocal photomicrographs, taken perpendicular to the pial surface, show VGluT1-ir (green) and VGAT-ir (red) boutons in the PC and endopiriform nucleus (EP). (A,E,I,M) Raw confocal images (VGluT1-ir+VGAT-ir). (B,F,J,N) Deconvoluted merged (Dec VGluT1-ir+VGAT-ir). (C,G,K,O) Deconvoluted VGluT1-ir (Dec VGluT1-ir). (D,H,L,P) Deconvoluted VGAT-ir (Dec VGAT-ir). Perisomatic (arrows) and chandelier-like (arrowhead) VGAT-ir boutons are indicated. Same scale for all images.
Figure 9
Figure 9
Confocal immunolabeling of VGluT1-ir and VGAT-ir boutons in the posterior piriform network. Same legend as in Figure 8.
Figure 10
Figure 10
VGluT1-ir and VGAT-ir bouton distribution in the piriform network. (A,B) Bar charts show a higher VGluT1-ir bouton density in PC layer I and in endopiriform nucleus (EP) than in PC layers II and III. No differences were found in VGAT-ir bouton density between PC and EP. (C,D) The percentages of both VGluT1-ir and VGAT-ir boutons were significantly higher in PC layers I and III. (E,F) VGluT1-ir bouton area was lower than VGAT-ir bouton area in both PC and EP. (E) VGluT1-ir bouton area in EP was lower than in PC layers I–III, while (F) VGAT-ir bouton area decreased in PC layer I and EP. (G) VGluT1-ir/VGAT-ir bouton ratio shows 2.8–3.4 times more VGluT1-ir (excitatory) than VGAT-ir (inhibitory) boutons. No differences were found between anterior and posterior regions. #P < 0.07, @P < 0.06, *P < 0.05, **P < 0.001.
Figure 11
Figure 11
VGluT1-ir and VGAT-ir boutons in primary visual area 17. (A,E,I,M,Q,U) Confocal high-power photomicrographs, taken perpendicular to the pial surface, show VGluT1-ir (green) and VGAT-ir (red) boutons for each cortical layer (left). (B,F,J,N,R,V) Deconvoluted merged images (Dec VGluT1-ir/VGAT-ir). (C,G,K,O,S,W) Deconvoluted images show VGluT1-ir bouton profiles (Dec VGluT1-ir). (D,H,L,P,T,X) Deconvoluted images show VGAT-ir bouton profiles (Dec VGAT-ir). Perisomatic (arrows) and chandelier-like (arrowhead) VGAT-ir boutons are indicated. Same scale for all images.
Figure 12
Figure 12
VGluT1-ir and VGAT-ir boutons in secondary visual area 18. Same legend as in Figure 11.
Figure 13
Figure 13
VGluT1-ir and VGAT-ir boutons in secondary visual area 19. Same legend as in Figure 11.
Figure 14
Figure 14
VGluT1-ir and VGAT-ir bouton distribution in visual areas 17, 18 and 19. (A) Bar histograms show higher VGluT1-ir bouton density in layer I of area 19. (B) No differences were found in VGAT-ir bouton density between or within visual areas. (C) VGluT1-ir bouton percentage per layer was higher in layer I of area 19, in layer IV of area 17, and in layer V of areas 17 and 18. (D) VGAT-ir bouton percentage was higher in layer IV of area 17 and lower in layer V of area 19. (E,F) VGluT1-ir bouton area was smaller than the VGAT-ir bouton area. (G) VGluT1-ir/VGAT-ir bouton ratio in the visual areas showed on average 2.6 ± 0.4 times more excitatory than inhibitory boutons. No differences were found between layers. Vertical lines in bar histograms show SD. *P < 0.05, **P < 0.001.
Figure 15
Figure 15
VGluT1-ir and VGAT-ir bouton distribution between the piriform network and visual areas 17, 18 and 19. (A,B) Bar charts show that both VGluT1-ir and VGAT-ir bouton densities were slightly lower in layers I–III of the piriform cortex (PC) than of visual areas, and that both VGluT1-ir and VGAT-ir bouton percentage was lowest (P < 0.001) in PC layer II (C,D) while in visual areas, both were lower (P < 0.001) in layers I and II (C,D). No differences were found in VGluT1-ir bouton areas (E) although it was lower than the corresponding VGAT-ir bouton area (compare E with F). VGAT-ir bouton area in PC layers II–III was lower than in visual areas (F; P < 0.05). No further significant differences were found. (G) VGluT1-ir/VGAT-ir bouton ratio was higher in PC than in visual layers I–III. (H) VGluT1-ir/VGAT-ir bouton ratio in PC, endopiriform nucleus (EP) and piriform network (PC+EP) was also higher than in layers I–VI of visual areas. n.s., not significant differences, *P < 0.05, **P < 0.001.

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