Distribution and neurochemical characterization of protein kinase C-theta and -delta in the rodent hypothalamus

Abstract

PKC-theta (PKC-θ), a member of the novel protein kinase C family (nPKC), regulates a wide variety of functions in the periphery. However, its presence and role in the CNS has remained largely unknown. Recently, we demonstrated the presence of PKC-θ in the arcuate hypothalamic nucleus (ARC) and knockdown of PKC-θ from the ARC protected mice from developing diet-induced obesity. Another isoform of the nPKC group, PKC-delta (PKC-δ), is expressed in several non-hypothalamic brain sites including the thalamus and hippocampus. Although PKC-δ has been implicated in regulating hypothalamic glucose homeostasis, its distribution in the hypothalamus has not previously been described. In the current study, we used immunohistochemistry to examine the distribution of PKC-θ and -δ immunoreactivity in rat and mouse hypothalamus. We found PKC-θ immunoreactive neurons in several hypothalamic nuclei including the ARC, lateral hypothalamic area, perifornical area and tuberomammillary nucleus. PKC-δ immunoreactive neurons were found in the paraventricular and supraoptic nuclei. Double-label immunohistochemisty in mice expressing green fluorescent protein either with the long form of leptin receptor (LepR-b) or in orexin (ORX) neurons indicated that PKC-θ is highly colocalized in lateral hypothalamic ORX neurons but not in lateral hypothalamic LepR-b neurons. Double-label immunohistochemistry in oxytocin-enhanced yellow fluorescent protein mice or arginine vasopressin-enhanced green fluorescent protein (AVP-EGFP) transgenic rats revealed a high degree of colocalization of PKC-δ within paraventricular and supraoptic oxytocin neurons but not the vasopressinergic neurons. We conclude that PKC-θ and -δ are expressed in different hypothalamic neuronal populations.

Fig. 1

Generation of the Oxt-Cre mice. (A) Map of mouse oxytocin/vasopressin locus. (B) Transgene with Cre recombinase inserted into Oxt gene and vasopressin ATG codon mutated. Double label IHC for: oxytocin neurons (C) and EYFP (D) showed a high degree of colocalization (E). Colocalized neurons are indicated with white arrows. Scale bar=200 μm for (C–E). IGR, intergenic region.

Fig. 2

Distribution of PKC-θ-IR neurons in rat brain. (A) Thionin-stained section denoting the thalamic nuclei (MHb and PVT) of the brain. (B) Adjacent section of the brain shown in (A), showing PKC-θ-IR neurons in the MHb and PVT. (C) Low-power magnification of a thionin-stained section of hypothalamic nuclei. (D) PKC-θ-IR neurons in the LHA and Pfx. (E) PKC-θ-IR neurons in the DMN. 3v, third ventricle; ARC, arcuate hypothalamic nucleus; D3v, dorsal third ventricle; DMN, dorsomedial hypothalamic nucleus; fx, fornix; LHA, lateral hypothalamic area; MHb, medial habenular nucleus; mt, mammillothalamic tract; Pfx, perifornical area; PVT, paraventricular thalamic nucleus. Scale bars=200 μm for (A); 50 μm for (B, D, E); 100 μm for (C).

Fig. 3

Distribution of PKC-θ-IR neurons in the rat hypothalamic arcuate nucleus (ARC). (A) PKC-θ-IR neurons in the ARC. (B) PKC-θ-IR neurons in the DTM and in ventral aspects of the PMV. 3v, third ventricle; DTM, dorsal tuberomammillary nucleus; fx, fornix; PMV, ventral premammillary nucleus. Scale bars=100 μm for (A, B).

Fig. 4

Distribution of PKC-δ-IR neurons in rat brain. (A) Thionin-stained section denoting the thalamic nuclei (VPM and VPL) of the brain. (B) Adjacent section of the brain shown in (A) at higher magnification, showing PKC-δ-IR neurons in the VPM and VPL. (C) PKC-δ-IR neurons in the PVN. (D) PKC-δ-IR neurons in the SON. 3v, third ventricle; fx, fornix; ic, internal capsule; LV, lateral ventricle; PVN, paraventricular hypothalamic nucleus; SON, supraoptic nucleus; VPL, ventral posterolateral thalamic nucleus; VPM, ventral posteromedial thalamic nucleus. Scale bars=200 μm for (A); 200 μm for (B); 50 μm for (C, D).

Fig. 5

Validation of PKC-δ antibody. (A) Low power magnification of PKC-δ fluorescent IHC in the thalamic nuclei (VPM and VPL) of the mouse brain. (B) Higher power view of boxed area in (A). (C) Lack of staining in section from same atlas level as (B) in PKC-δ-KO mouse. ic, internal capsule; Rt, reticular thalamic nucleus; VPL, ventral posterolateral thalamic nucleus; VPM, ventral posteromedial thalamic nucleus. Scale bars=100 μm for (A); (B, C)=50 μm.

Fig. 6

Double-label IHC for PKC-θ-IR neurons (A, denoted by white arrow heads) and LepR-b-EGFP in mouse hypothalamus (B, denoted by white lines) showed no colocalization (C) in the Pfx. fx, fornix; Pfx, perifornical area. Scale bar=50 μm for (A–C).

Fig. 7

Double label IHC for: PKC-θ-IR neurons (A) and ORX-GFP (B) in mouse hypothalamus showed a high degree of colocalization (C). Colocalized neurons are indicated with white arrows. High density of PKC-θ-IR (D) and ORX-GFP-IR fibers (E) in the PVN. Overlapping PKC-θ-IR and ORX-GFP-IR fibers (F, denoted by white arrows). High density of PKC-θ-IR (G) and ORX-GFP-IR fibers (H) in the VTA. No overlapping distribution pattern of PKC-θ-IR (denoted by white arrowheads) and ORX-GFP-IR fibers were observed (I). The fibers lay adjacent to each other and made several appositions. Points where appositions are made have been indicated by *. 3v, third ventricle; IPF, interpeduncular fossa; LHA, lateral hypothalamic area; MM, medial mammillary nucleus; Pfx, perifornical area; PVN, paraventricular hypothalamic nucleus; VTA, ventral tegmental area. Scale bars=50 μm for (A–C); 100 μm for (D–F); 100 μm for (G–I).

Fig. 8

Double label IHC for: PKC-θ-IR (A, denoted by white arrow heads) and histidine decarboxylase-IR (B, denoted by white lines) in mouse hypothalamus showed several appositions (C). Points where appositions are made have been indicated by *. Scale bar=100 μm.

Fig. 9

Colocalization between Oxt-YFP-IR and PKC-δ-IR neurons in the mouse hypothalamus. Lower power magnification of Oxt-YFP-IR neurons in PVN (A) and SON (B). Double-label IHC for PKC-δ-IR (C) and Oxt-YFP-IR (D) in boxed area of (A) showed a high degree of colocalization in the PVN (E). Double-label IHC for PKC-δ-IR (F) and Oxt-YFP-IR (G) showed a moderate degree of colocalization in the SON (H). Examples of colocalized neurons are indicated by white arrows. 3v, third ventricle; PVN, paraventricular hypothalamic nucleus; SON, supraoptic nucleus. Scale bars=100 μm for (A–G).

Fig. 10

Colocalization between PKC-δ-IR and AVP-IR in mouse hypothalamus. Double-label IHC for PKC-δ-IR neurons (A, denoted by white arrow heads) and AVP-EGFP in mouse hypothalamus (B, denoted by white lines) showed no colocalization (C) in the PVN. There was no PKC-δ-IR at this level of the SCh (D), only AVP-EGFP was observed (E) and hence no colocalization was observed (F). 3v, third ventricle; PVN, paraventricular hypothalamic nucleus; SCh, suprachiasmatic nucleus. Scale bars=100 μm for (A–C); 50 μm for (D–F).