Anatomy of hypothalamic and diencephalic nuclei involved in seasonal fertility regulation in ewes

Abstract

In this study, we describe in detail the anatomy of nuclei involved in seasonal fertility regulation (SFR) in ewes. For this purpose, the intergeniculate leaflet of the visual thalamus, the caudal hypothalamic arcuate nucleus, and suprachiasmatic, paraventricular and supraoptic nuclei of the rostral hypothalamus were morphometrically and qualitatively analyzed in Nissl-stained serial sections, in the three anatomical planes. In addition, data were collected on calcium-binding proteins and cell phenotypes after immunostaining alternate serial sections for calretinin, parvalbumin and calbindin. For a complete neuroanatomical study, glial architecture was assessed by immunostaining and analyzing alternate sections for glial fibrillary acidic protein (GFAP) and ionized calcium-binding adapter molecule 1 (IBA1). The results showed a strong microglial and astroglia reaction around the hypothalamic nuclei of interest and around the whole 3^rd ventricle of the ewe brain. Moreover, we correlated cytoarchitectonic coordinates of panoramic serial sections with their macroscopic localization and extension in midline sagittal-sectioned whole brain to provide guidelines for microdissecting nuclei involved in SFR.

Keywords: GFAP; IBA1; Nissl staining; calbindin; calretinin; parvalbumin.

Figure 1

General scheme of the ewe brain in the sagittal plane inspirated in the neuromeric model for brain organization. SFR nuclei rough position are labeled by black dots: (1) IGL - thalamus (2) ARC- posterior blue area (3) SCh and (4) SO nuclei (5) PVN in the anterior red area. AC, anterior commissure; PHy, peduncular hypothalamus; Thy, terminal hypothalamus; POA, preoptic area.

Figure 2

Nissl-stained sagittal serial sections of the ewe diencephalon. (A) Panoramic view of a lateral longitudinal section showing the intergeniculate leaflet (IGL), crossing ventro-dorsally the lateral geniculate nucleus (LGN). Ventrally, the optic tract (OT) in close apposition with the supraoptic nucleus (SO). (B) Higher magnification of IGL; in the bottom, a pseudocolor image allows us to distinguish the meandering trajectory of IGL (dotted line). (C) More medial section showing all nuclei of interest. Red horizontal and vertical lines defined the level of sectioning corresponding to coronal (C 261 …) or horizontal (H 81 …) serial sections shown in Figures 3, 4. (D) Higher magnification showing the arcuate nucleus (ARC) distributed from the (MB) mamillary body to the stalk of the median eminence (MSE). (E) Detail of the rostral area in which the periventricular nucleus (PVN) can be distinguish as a strongly Nissl-stained band of cells close to the 3^rd ventricle. Ventrally suprachiasmatic nucleus (SCh) is observed between the optic tract and the lateral recess. (F) Lateral most panoramic section. (G) Detail showing the infundibulum (Inf). Pi, Pineal gland; VMH, Ventromedial hypothalamus nucleus. (A, C, F) same magnification.

Figure 3

Nissl-stained coronal serial sections. (A) Caudal section showing the dorsal (LGB d) and ventral (LGB v) divisions of the lateral geniculate body separated by the intergeniculate leaflet (IGL). (B, C) Arcuate nucleus (ARC) located I the caudal hypothalamic red area in Figure 1. (D–F) Three representative sections from the rostral hypothalamus (blue area in Figure 1). The labels in the bottom of each image (C 261 to C 441) match the level of sectioning corresponding with vertical red lines of Figure 2. (G–K) Details of Nissl-stained cells from all nuclei of interest. OX, Optic chiasm; MPO, Medial preoptic area; ME, Medial eminence; VMH, ventro-medial nucleus; IIIV, third ventricle- (A–F) same scale bar.

Figure 4

Serial sections in the horizontal plane ordered from ventral (A) to dorsal (C). (A–C) show the localization of arcuate nucleus (ARC) adjacent to the mamillary body (MB), bordering the 3^rd ventricle (III V) delimited by arrows. The SO nucleus is located laterally in the ventral sections. (A, B) and medially in dorsal sections (C, D), as a consequence of its distribution around the optic tract. Note the localization of paraventricular nucleus (PVN) with respect to the 3^rd ventricle in (D). The labels in the bottom of each image (H 81 to H 181) match the level of sectioning corresponding with horizontal red lines of Figure 2. Ic, internal capsule; GP, Globus pallidus; mt, mammillothalamic tract; OT, Optic tract. (A–D) same magnification.

Figure 5

Calcium-binding protein immunoreactivity of the nuclei of interest (SFR). (A–D) Immunostaining of the IGL. Terminals are positive for calbindin and calretinin (E–G) Immunostaining of arcuate nucleus. Cb and Cr-stained neurons are evident. (H–K) Immunostaining of PVN. Large stellate neurons with thick dendrites are Cb positive. (L, M) Immunostaining of SCh. Cr and Cb positive fibers enter the nucleus from the optic chiasm [this photograph is a wider view of (H)]. (N–P) Immunostaining of SO, showing large Cb and Cr neurons oriented in the main axis of the nucleus.

Figure 6

IBA1 immunoreactivity in serial coronal sections. (A, B) caudal ventromedial hypothalamus. Densest microglial reaction is found in the ARC (dotted line) and around the 3^rd ventricle. (C) POA. Details in squares from 1 to 3 (bottom of the figure) show that reactive microglial cells are highly dense and more concentrated around and inside PVN and SO nuclei. Note the intense staining around the 3^rd ventricle in all levels of sectioning. Mean gray level measured inside the squares (MG – mean gray value).

Figure 7

GFAP immunoreactivity in coronal serial sections. (A, B) caudal sections. Squares (1) and (2), tanycytes at the median stalk eminence (MSE). (C) Rostral section. Higher cell density is observed around the 3^rd ventricle in all levels of sectioning, and around and inside PVN and SO nuclei (dotted lines). Squares 3, 4, and 5 are shown at a higher magnification on the right of the figure. (3) Detail of immunoreactivity in PVN. Neurons encircled by astrocytes are shown as white contours. (4) Interstitial astroglia in the lateral preoptic area (LPO). (5) Non-reactive astrocytes far from the 3^rd ventricle.

Figure 8

Anatomical guide for localizing nuclei on the surfaces of a sagittal midline-sectioned brain. Double headed arrows pointed the correspondence between histological sections and the macroscopic view. (A) Projections in a macroscopic view of coordinates measured in a sagittal Nissl section. The green line tangential to the ventral surface of the mammillary body and optic chiasma defines an optimal reference plane for calculating coordinates for precise tissue extraction. The oval line indicates the macroscopic localization of ARC. The rectangle shows the localization of PVN, and the adjacent small oval (red arrow) indicates the localization of the SCh nucleus. The red, dotted rectangle defines the estimated position of IGL within the visual thalamus. (B) Projections of coordinates from horizontal Nissl-stained sections transferred to a macroscopic ventral surface view of the brain. The oval circle indicates the localization and extension of ARC in a ventral view. The oval, dotted line shows the tentative localization and extension of SO and ARC. MB, Mammillary body; III V, third ventricle.