Distribution of secretagogin-containing neurons in the basal forebrain of mice, with special reference to the cholinergic corticopetal system

Abstract

Cholinergic and GABAergic corticopetal neurons in the basal forebrain play important roles in cortical activation, sensory processing, and attention. Cholinergic neurons are intermingled with peptidergic, and various calcium binding protein-containing cells, however, the functional role of these neurons is not well understood. In this study we examined the expression pattern of secretagogin (Scgn), a newly described calcium-binding protein, in neurons of the basal forebrain. We also assessed some of the corticopetal projections of Scgn neurons and their co-localization with choline acetyltransferase (ChAT), neuropeptide-Y, and other calcium-binding proteins (i.e., calbindin, calretinin, and parvalbumin). Scgn is expressed in cell bodies of the medial and lateral septum, vertical and horizontal diagonal band nuclei, and of the extension of the amygdala but it is almost absent in the ventral pallidum. Scgn is co-localized with ChAT in neurons of the bed nucleus of the stria terminalis, extension of the amygdala, and interstitial nucleus of the posterior limb of the anterior commissure. Scgn was co-localized with calretinin in the accumbens nucleus, medial division of the bed nucleus of stria terminalis, the extension of the amygdala, and interstitial nucleus of the posterior limb of the anterior commissure. We have not found co-expression of Scgn with parvalbumin, calbindin, or neuropeptide-Y. Retrograde tracing studies using Fluoro Gold in combination with Scgn-specific immunohistochemistry revealed that Scgn neurons situated in the nucleus of the horizontal limb of the diagonal band project to retrosplenial and cingulate cortical areas.

Fig 1

Secretagogin containing neurons in the cholinergic space at various levels of the brain. A) Scgn neurons were found in the lateral part of the MS but not in the central part. They were also found in the AcbSh, AcbC, and in the LS. Neither cell or neuropil positivity was not found in the VP. B) Scgn is strongly expressed in the STM, STL and HDB. C) Scgn is expressed in the STLP and the STLV, as well as in the IPAC. No Scgn-containing profiles can be found in the LPO. D) Large Scgn-containing neurons are found in the EA and in the StHy. E/F) There was no labeling in the LDB but the EA showed strong Scgn expression at every level. Scale bar represents 250 μm in the main figures and 100 μm in the insets.

Fig 2

Co-localization of Scgn with ChAT, Pv, and Cb in neurons of the basal forebrain. A1–A4: Scgn/ChAT: B1–B4: Cr; C1–C4 Cb; D1–D4 Pv; E1–E4 NPY-GFP. Horizontal rows: A1–E1: nucleus accumbens; A2–E2: STMP; A3–E3: extension of the amygdala; A4–E4; IPAC. We found strong co-localizations with ChAT positive neurons in the EA (A–C) as well as in the IPAC regions. In contrast, Scgn seems to be present in completely non-overlapping populations with other CBPs, such as Pv (D–F) and Cb (G–I). Arrows point to cells that contain both Scgn and the relevant marker. Co-localization of the red Scgn with the other green fluorochrome resulted in yellow color in the merged photos. Scale bar represents 100μm.

Fig 3

A–H Distribution of FG- and Scgn-containing neurons in the cholinergic space. The inset demonstrates the FG injection site in the retrosplenial cortex. FG-containing, retrogradely labeled neurons were found in the MS, HDB, and EA areas in the basal forebrain (labeled with black dots), as well as in the IPAC and various thalamic nuclei at the levels examined (Bregma: +0.8−1. 2mm). Scgn-containing neurons are overlapping with the FG-labeled neurons in the HDB and EA (labeled with red dots). I–J. Photographs presented in rows I and J were taken from the area represented by a black rectangle in panels D and E. A small but consistent ratio of Scgn-containing neurons (8.0%) accommodated by the HDB is proved to be projection neurons, sending their axons to the retrosplenial cortex. Double-labeled (Scgn⁺/FG⁺) neurons tend to be localized at the dorsal part of the HDB (marked by white arrows). These neurons are very likely to be cholinergic as well, based on their size and location. Scale bar represents 100 μm.

Fig 4

Average perimeter of Scgn-containing neurons in the shell of the nucleus accumbens (AcbSh; n=79), in the dorsal (STLD; n=91), ventral (STLV;n=60) part of the bed nucleus of stria terminalis, the extension of the amygdala (EA; n=98), and in the horizontal limb of the diagonal band (HDB; n=55) compared to the average cell-size of the cholinergic neurons in the basal forebrain (ChAT; n=123). One-way analysis of variance: p<0.0001; Tukey’s Multiple Comparison Test: p<0.001. The table presents the levels of significance when the average perimeters of the Scgn-containing neurons in the areas of the accumbens (Ac), laterodorsal bed nucleus of stria terminalis (STLD), lateroventral bed nucleus of stria terminalis (STLV), the extension of the amygdala (EA), and the nucleus of the horizontal diagonal band (HDB) were compared with the perimeters of cholinergic neurons of the basal forebrain.