Cyto- and chemoarchitecture of the hypothalamic paraventricular nucleus in the C57BL/6J male mouse: a study of immunostaining and multiple fluorescent tract tracing

Abstract

The paraventricular nucleus of the hypothalamus (PVH) plays a critical role in the regulation of autonomic, neuroendocrine, and behavioral activities. This understanding has come from extensive characterization of the PVH in rats, and for this mammalian species we now have a robust model of basic PVH neuroanatomy and function. However, in mice, whose use as a model research animal has burgeoned with the increasing sophistication of tools for genetic manipulation, a comparable level of PVH characterization has not been achieved. To address this, we employed a variety of fluorescent tract tracing and immunostaining techniques in several different combinations to determine the neuronal connections and cyto- and chemoarchitecture of the PVH in the commonly used C57BL/6J male mouse. Our findings reveal a distinct organization in the mouse PVH that is substantially different from the PVH of male rats. The differences are particularly evident with respect to the spatial relations of two principal neuroendocrine divisions (magnocellular and parvicellular) and three descending preautonomic populations in the PVH. We discuss these data in relation to what is known about PVH function and provide the work as a resource for further studies of the neuronal architecture and function of the mouse PVH.

Figure 1

Nissl-stained digital images of seven representative levels through the PVH of the C57BL/6J mouse. A represents level 56, B–F levels 60–64, and G level 66 of the Allen Reference Atlas (ARA; Dong, 2007). Scale bars = 200 µm.

Figure 2

Photomicrographs illustrating our general strategy for characterizing the cyto- and chemoarchitecture of the mouse PVH. A shows a representative photomicrograph of the mouse PVH with fluorogold (FG)-labeled hypothalamic neuroendocrine neurons after tail vein injection of the retrograde tracer. These animals also received stereotaxic injections of the retrograde tracers fast blue (FB) and/or Red Retrobeads (RR) into the spinal cord and/or the DMX, respectively to label the descending preautonomic neurons in the PVH (PVHd). B illustrates a representative injection site of FB restricted to the CGS. Brain sections from these animals were used to perform multiple immunostainings with different combinations to reveal distributions of all neuroendocrine hormonal peptides (OXY, VAS, CRH, SS, and TRH) in the PVH. C shows a multifluorescent, confocal photomicrograph of FG-labeled neuroendocrine neurons (green), FB-labeled CGS-projecting neurons (blue), and OXY (red)- and VAS (magenta)-immunopositive neurons. Note how neurons of these two magnocellular neuroendocrine populations are double labeled with FG, whereas singly labeled FG neurons are presumably parvicellular neuroendocrine neurons. Each neuronal phenotype is displayed separately as shown in a–d. Topological spatial distributions of these different neuronal populations were compared with Nissl-stained cytoarchitecture of the PVH in adjacent histological sections illustrated in D, which allows more precise delineation of different parts of the PVH. E shows a representative level of the PVH containing FG-labeled neuroendocrine neurons (green) and RR-labeled DMX-projecting preautonomic neurons (red). F shows a representative confocal photomicrograph, which simultaneously reveals the entire population of hypothalamic neuroendocrine neurons labeled with FG (green), VAS-immunopositive neurons (magenta), descending IML-projecting neurons labeled with FB (blue), and descending DMX-projecting neurons labeled with RR (red). Scale bars = 200 lm.

Figure 3

Confocal photomicrographs show a series of coronal sections (one in four series, 25 lm thickness) across eight levels of the PVH (experimental case T10; noncolchicine treated) in which four neuronal populations are labeled with different fluorescent markers. The parvicellular neuroendocrine neurons are singly labeled with the retrograde marker FG (green). Two magnocellular neuroendocrine populations are double labeled with FG (green) and OXY (red) or VAS (magenta), whereas CGS-projecting descending neurons are labeled with FB (blue; for injection site see Fig. 2B). Scale bar = 200 lm.

Figure 4

Confocal photomicrographs showing OXY-immunoreactive neurons in the PVHam double labeled with FG (A–C). Note that the vicinity of the PVHam contains very few singly labeled FG neurons (e.g. white arrow in A), which are presumably parvicellular neuroendo-crine cells. D is a digital image of a Nissl-stained section from the ARA (level 56; see Fig. 1A) that represents the corresponding cytoarchitectural reference for the PVHam. Scale bar = 200 µm.

Figure 5

Distributions of two magnocellular neuroendocrine populations, OXY (A–C,G–I) and VAS (D–F,J–L), are compared in two different coronal levels of the PVH (from two different brains of noncolchicine-pretreated mice). The whole population of neuroendocrine neurons is labeled with FG via tail vein injection (as shown in A,D,G,J), whereas OXY (B,H) or VAS (E,K) neurons were immunostained. Note that almost all OXY (C,I)- or VAS (F,L)-immnopositive neurons are neuroendocrine neurons as indicated by their colocalization with FG. At the rostral level (A–F), OXY neurons are distributed in both the PVHmm and the PVHpmm, whereas VAS neurons are located primarily in the PVHpmm. At the caudal level, VAS neurons form a densely packed “ball” in the PVHpml (K,L), and OXY neurons are distributed more medially in the PVHpv (H,I). At these two levels, the PVHpmm contains both OXY and VAS neurons (I,L). Scale bars (D,J) = 200 µm.

Figure 6

Confocal photomicrographs showing the distribution of CRH neurons (A–D, with nuclear counterstain DAPI) and their topological spatial relation with OXY and VAS neurons (E–H) in four coronal levels across the PVH (one in four series, 25 lm thickness). Adjacent thionin-stained sections are shown for cytoarchitectural reference (I–K). Although no single neuronal phenotype is restricted to a single subdivision, the same types of neurons do tend to cluster together (i.e., CRH neurons are concentrated in the PVHmpd; the PVHpml contains primarily VAS neurons; the PVHpmm contains both OXY and VAS neurons), allowing further delineation of the PVH. Scale bar = 200 µm.

Figure 7

A and B show the distribution of SS-immunoreative neurons in the PVHpv and periventricular nucleus, compared with the distribution of FG-labeled neuroendocrine neurons. C and D show the spatial correlations of SS (yellow) neurons with OXY (blue in C,D) and VAS (magenta in D) in the PVH. Most SS neurons are medial to the OXY neurons in the PVHpv. E–G show the spatial distribution of TRH-immunopositive neurons (green) in the PVH and their spatial correlation with OXY (red) and VAS (blue) in the PVH. Most of the TRH neurons are distributed in the PVHmpd as shown in F,H. In H, nuclei were revealed with DAPI, to facilitate identification of individual TRH neurons (as shown in the boxed area). All brain sections shown were obtained from mice that had received colchicine injections. Scale bars = 200 µm.

Figure 8

Photomicrographs show the distribution of FG-labeled IML-projecting neurons in the PVH and their spatial correlation with OXY and VAS neurons (A,C,E,G; experimental case T11; noncolchicine pretreated). In the corresponding adjacent sections, Golgi-like neuronal morphology of the FG-labeled descending neurons is revealed with an immunohistochemical method (B,D,F,H). D’ is a magnified enlarged image of a FG-labeled descending neuron in D (within the red box), where arrows indicate dendritic spines. Scale bars = 200 µm.

Figure 9

Photomicrographs show the distribution of Red Retrobeads (RR)-labeled DMX-descending projection neurons and their spatial correlation with FG-labeled neuroendocrine neurons (green) and immunostained OXY neurons (magenta) in three coronal levels of the mouse PVH (A–C). The adjacent Nissl-stained sections show their corresponding cytoarchitectural references (D–F). The red arrowheads point to the same blood vessels in each of the corresponding levels. Please note that the most RR-labeled neurons are distributed in the PVHmpv and its adjacent PVHlp. These photomicrographs are from experimental case SCDMIV2, which was not pretreated with colchicine. Scale bar = 100 µm.

Figure 10

Confocal photomicrographs show spatial distributions of two populations of descending preautonomic neurons that project to the IML (green, FG-labeled neurons) and/or to the DMX (megenta, FB-labeled neurons) at six contiguous coronal levels through the PVH (25 µm thickness; A–F). Arrowheads in A–F indicate double-labeled neurons (white) that presumably send collateral projections to both the IML and DMX. C1–C3 are enlarged images of the boxed area in C to show neurons single labeled with FB (C2) or FG (C3) and double labeled with both tracers (C1). The mouse used in this experiment did not receive a cholchicine injection. A–F are at the same magnification. Scale bars = 50 µm in C3; 100 µm in F.

Figure 11

Schematic drawings illustrating the delineations of the PVH in the mouse brain, which are determined based on distributions of eight neuronal phenotypes in two major neuroendocrine divisions (PVHm, including OXY and VAS; and PVHp, including CRH, SS, and TRH) and three descending preautonomic populations (PVHd) that project to the IML (IML-d), to the CGS (CGS-d), and to the DMX (DMX-d). Schematic drawings of the PVH, based on the present data, were modified from eight coronal levels (levels 57, 59, 60–64, and 66; see Fig. 1 for cytoarchitectural reference) of the ARA (Dong, 2007).

Figure 12

Schematic model of the PVH cyto- and chemoarchitecture in the mouse. In general, the anterior (rostral) two-thirds of the PVH (ARA levels 56–62) contains the vast majority of magnocellular neuroendocrine neurons (PVHm) that project to the posterior pituitary (PP) and parvicellular neuroendocrine neurons (PVHp) that project to the median eminence (ME). The posterior (caudal) one-third of the PVH (ARA levels 63–66) contains most of the descending preautonomic neurons. *indicates the anterior PVHpv, along with the posterior PVHpv, also sends projections to the AP. The line was not drawn here to avoid confusion.