Hormonal regulation of delta opioid receptor immunoreactivity in interneurons and pyramidal cells in the rat hippocampus - PubMed (original) (raw)

Hormonal regulation of delta opioid receptor immunoreactivity in interneurons and pyramidal cells in the rat hippocampus

Tanya J Williams et al. Neurobiol Learn Mem. 2011 Feb.

Abstract

Clinical and preclinical studies indicate that women and men differ in relapse vulnerability to drug-seeking behavior during abstinence periods. As relapse is frequently triggered by exposure of the recovered addict to objects previously associated with drug use and the formation of these associations requires memory systems engaged by the hippocampal formation (HF), studies exploring ovarian hormone modulation of hippocampal function are warranted. Previous studies revealed that ovarian steroids alter endogenous opioid peptide levels and trafficking of mu opioid receptors in the HF, suggesting cooperative interaction between opioids and estrogens in modulating hippocampal excitability. However, whether ovarian steroids affect the levels or trafficking of delta opioid receptors (DORs) in the HF is unknown. Here, hippocampal sections of adult male and normal cycling female Sprague-Dawley rats were processed for quantitative immunoperoxidase light microscopy and dual label fluorescence or immunoelectron microscopy using antisera directed against the DOR and neuropeptide Y (NPY). Consistent with previous studies in males, DOR-immunoreactivity (-ir) localized to select interneurons and principal cells in the female HF. In comparison to males, females, regardless of estrous cycle phase, show reduced DOR-ir in the granule cell layer of the dentate gyrus and proestrus (high estrogen) females, in particular, display reduced DOR-ir in the CA1 pyramidal cell layer. Ultrastructural analysis of DOR-labeled profiles in CA1 revealed that while females generally show fewer DORs in the distal apical dendrites of pyramidal cells, proestrus females, in particular, exhibit DOR internalization and trafficking towards the soma. Dual label studies revealed that DORs are found in NPY-labeled interneurons in the hilus, CA3, and CA1. While DOR colocalization frequency in NPY-labeled neuron somata was similar between animals in the hilus, proestrus females had fewer NPY-labeled neurons that co-labeled with DOR in stratum oriens of CA1 and CA3 when compared to males. Ultrastructural analysis of NPY-labeled axon terminals within stratum radiatum of CA1 revealed that NPY-labeled axon terminals contain DORs that are frequently found at or near the plasma membrane. As no differences were noted by sex or estrous cycle phase, DOR activation on NPY-labeled axon terminals would inhibit GABA release probability equally in males and females. Taken together, these findings suggest that ovarian steroids can impact hippocampal function through direct effects on DOR levels and trafficking in principal cells and broad indirect effects through reductions in DOR-ir in NPY-labeled interneurons, particularly in CA1.

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Figures

Fig. 1

Light microscopic distribution of delta opioid receptor (DOR) immunoreactivity (-ir) in the rat hippocampal formation. (A) Representative light photomicrograph from a male showing DOR labeling in hilar neurons and diffuse DOR-ir visible over the granule cell layer (GCL). (B) In CA1 of a proestrus female, DOR labeling is found in interneurons (arrows) in stratum oriens (SO) as well as within pyramidal cell somata in the pyramidal cell layer (PCL) and proximal dendrites in stratum radiatum (SR). (C) Quantitative densitometric analysis revealed that females had significantly less DOR-ir in the granule cell layer of the dentate gyrus (DG) than male rats (*p<0.05). (D) When proestrus and diestrus females were grouped together, females showed similar numbers of DOR-labeled hilar neurons in comparison to males (p>0.05). (E) When proestrus and diestrus females were analyzed separately, however, proestrus females displayed a significantly increased number of DOR-labeled hilar neurons in comparison to diestrus females and males (*p<0.05, both comparisons). (F) In the CA1, proestrus females displayed decreased DOR-ir in the pyramidal call layer in comparison to males (*p<0.05). Scale bars = 100 m.

Fig. 2

Immunofluorescent images illustrating co-localization of DOR (A, D, G) with NPY (B, E, H) in the hippocampus. (A-C) Dual-labeled cells in the hilus (Hil) of the dentate gyrus (DG), where A and B are unmerged images of C. (D-F) Dual-labeled cells in stratum oriens (SO) of CA3, where D and E are unmerged images of F. (G-I) Dual-labeled cells in stratum oriens of CA1, where G and H are unmerged images of I. DOR-labeling is red. NPY-labeling is green. Dual-labeled cells appear yellow and are indicated by arrows. PCL = pyramidal cell layer. Scale bars = 100 m.

Fig. 3

DOR-labeling is found in dendrites and axon terminals in stratum radiatum of the CA1. Representative electron micrographs from a male (A) and proestrus female (B) demonstrate DOR-labeling in both cytoplasmic and plasmalemmal sites in spiny dendrites similar in morphology to pyramidal cells using the immunogold-silver technique. DOR silver-intensified-gold (SIG) particles are found along the plasma membrane (blue circles), near the plasma membrane (red arrows), or in the cytoplasm (blue arrows). Representative electron micrographs from a proestrus female (C) and male (D-E) show DOR-labeling along the plasma membrane (blue circles), near the plasma membrane (red arrows), or affiliated with small clear synaptic vesicl es in the cytoplasm (blue arrows) of axon terminals. den = dendrite, ter = DOR-labeled axon terminal, ut = unlabeled axon terminal. Scale bars = 500 nm.

Fig. 4

Ovarian hormones influence the distribution of DOR-SIG particles in CA1 stratum radiatum dendrites. (A) Females display fewer DOR-SIG particles per dendrite than males (*p<0.05). (B) Both proestrus and diestrus females show fewer DOR-SIG particles per dendrite than males (*p<0.05, both comparisons). (C) Females have reduced plasma membrane density of DOR-SIG particles in comparison to males (*p<0.05). (D) Proestrus females exhibit reduced plasma membrane density of DOR-SIG particles in comparison to males as well as increased cytoplasmic density of DOR-SIG particles in comparison to diestrus females (*p<0.05, all comparisons). (E) The number of DOR-SIG particles is decreased on the plasma membrane of females in comparison to males (*p<0.05). (F) The number of DOR-SIG particles is increased in the cytoplasm of proestrus females in comparison to males (*p<0.05).

Fig. 5

Ovarian hormones influence DOR-SIG particle distribution in small (average diameter <1.03 m) and large (average diameter 1.03 – 2.86 m) dendrites in SR of CA1. (A) Females display fewer DOR-SIG particles per dendrite than males in small (*p<0.05) but not large dendrites. (B) Both proestrus and diestrus females show fewer DOR-SIG particles per dendrite than males in small dendrites (*p<0.05, both comparisons). Diestrus females show fewer DOR-SIG particles per dendrite than proestrus females and males in large dendrites (*p<0.05, both comparisons). (C) No significant differences were found in plasma membrane density of DOR-SIG particles between groups in either small or large dendrites. (D) Diestrus females exhibit decreased cytoplasmic density of DOR-SIG particles in comparison to proestrus females and males in large (*p<0.05, both comparisons) but not small dendrites. (E) No significant differences were found in the number of DOR-SIG particles on the plasma membrane of either small or large dendrites between groups. (F) The number of DOR-SIG particles is increased in the cytoplasm of small but not large dendrites of proestrus females in comparison to males (*p<0.05).

Fig. 6

DOR-labeling is found in NPY-immunoreactive axon terminals in stratum radiatum of CA1. (A, B) Representative electron micrographs demonstrate NPY-ir in single and dual-labeled axon terminal profiles. In dual-labeled axon terminals, DOR-SIG particles are frequently found along the plasma membrane (blue circles) or near the plasma membrane (red arrows). DOR-labeling is often affiliated with small clear synaptic vesicles in the cytoplasm of axon terminals. Moreover, NPY-labeled axon terminals also found in contact with DOR-labeled dendrites. As aforementioned, DOR-labeling within dendrites is frequently cytoplasmic (blue arrows). den = dendrite, ter = NPY-labeled axon terminal, ut = unlabeled axon terminal. Scale bars = 500 nm.

Fig. 7

Schematic diagram summarizing ovarian hormone influences on DOR levels and distribution in dendrites and axon terminals within stratum radiatum of CA1. (A) In males, DORs are found on (or near) the plasma membrane and in the cytoplasm of small and large dendrites. DORs are also found in the cytoplasm and infrequently on the plasma membrane of axon terminals. NPY-labeled axon terminals sometimes contact DOR-labeled dendrites and occasionally contain DORs that are frequently found at or near the plasma membrane. (B) In comparison to males, diestrus (low estrogen) females have fewer DORs in both small and large dendrites. In addition, the cytoplasmic density of DORs is selectively reduced in large dendrites. No differences from males are observed in DOR-ir within NPY-labeled or unlabeled axon terminals. (C) In comparison to males, proestrus (high estrogen) females display fewer DORs as well as trafficking of DORs away from the plasma membrane and into the cytoplasm of small dendrites. Although proestrus females also have more DOR-labeled axon terminals, no differences from males or diestrus females are observed in DOR-ir within NPY-labeled or unlabeled axon terminals. In comparison with diestrus females, proestrus females exhibit increased DORs in large dendrites. Taken together, these results suggest that (1) females generally have fewer DORs in pyramidal cell dendrites and (2) when estrogen levels are high, DORs are internalized from the plasma membrane of distal dendrites and trafficked towards the pyramidal cell soma.

References

1. Adams MM, Oung T, Morrison JH, Gore AC. Length of postovariectomy interval and age, but not estrogen replacement, regulate N-methyl-D-aspartate receptor mRNA levels in the hippocampus of female rats. Exp.Neurol. 2001;170:345–356. - PubMed
1. Afify EA, Law PY, Riedl M, Elde R, Loh HH. Role of carboxyl terminus of mu-and delta-opioid receptor in agonist-induced down-regulation. Brain Res.Mol.Brain Res. 1998;54:24–34. - PubMed
1. Ali AB, Thomson AM. Facilitating pyramid to horizontal oriens-alveus interneurone inputs: dual intracellular recordings in slices of rat hippocampus. J.Physiol. 1998;507(Pt 1):185–199. - PMC - PubMed
1. Bao G, Kang L, Li H, Li Y, Pu L, Xia P, et al. Morphine and heroin differentially modulate in vivo hippocampal LTP in opiate-dependent rat. Neuropsychopharmacology. 2007;32:1738–1749. - PubMed
1. Bausch SB, Chavkin C. Changes in hippocampal circuitry after pilocarpine-induced seizures as revealed by opioid receptor distribution and activation. J.Neurosci. 1997;17:477–492. - PMC - PubMed

Hormonal regulation of delta opioid receptor immunoreactivity in interneurons and pyramidal cells in the rat hippocampus - PubMed (original) (raw)