Oxytocin

Objective: Both oxytocin (OT) and reel in are particularly significant during development and the absence of either may interfere with normal brain development. In addition, reelin is critical to the development of the GABAergic system and GABA modulates the release of OT. Availability of the reelin haploinsufficient (+/-) reeler mouse (HRM) provides a model for examining the role of reelin in the development of the OT system and especially in the expression of the OT receptor (OTR).

Methods: In this study we used immunocytochemistry and in situ hybridization in HRM versus wild-type (+/+) mice (WTM) to quantify OTR abundance in regions of the brain cortex.

Results: Our findings reveal that the oxytocin receptor (OTR), measured either by immunohistochemistry or in situ hybridization, is significantly lower in HRM. Areas showing significant deficits included the piriform cortex, neocortex, retrosplenial cortex and certain regions of the hippocampus.

Conclusion: Both reelin and OT play a role in regulating affect and mood. Down-regulation of reelin has been strongly correlated with schizophrenia and it is proposed that HRM may serve as a model for neural deficits seen in both schizophrenia and autism. We report that HRM show regionally specific reductions in OTRs, especially in cortical areas, which previously have been implicated in social memory and cognitive functions. These findings offer support for the more general hypothesis that down-regulation of reelin, of either genetic or epigenetic origin, through associated reductions in the OTRs, contributes to the deficiencies in social behavior that are characteristic of both schizophrenia and autism. [Neurol Res 2005; 27: 339-345]

Keywords: Autism; oxytocin; oxytocin receptor; reeler mouse; reelin; schizophrenia

INTRODUCTION

Both oxytocin (OT) and reelin have been implicated in autism, schizophrenia and related neuropsychiatrie disorders. Reelin is a glycoprotein (~400 kDa) secreted by inhibitory GABAergic neurons into the extracellular matrix of the neocortex, hippocampus, olfactory bulb as well as other areas of adult rodent and primate brains and is involved in brain lamination1,2. The gene for reelin (ReIn) was discovered because mice lacking this gene show a characteristic gait, including ataxia and tremors3. Recent findings indicate that the heterozygote (+/-) reeler mouse (haploinsufficient for the reeler gene) shares several neurochemical and behavioral abnormalities with schizophrenia and bipolar disorder with mania4,5. The reelin +/- mouse has ~50% down-regulation of reelin and glutamic acid decarboxylase^sub 67^ (GAD^sub 67^) mRNAs (a precursor of GABA), a decrease in reelin expression in neurons, a decrease in the number of reelin and GAD^sub 67^-containing neurons, a decrease in dendritic spine density and a decrease in cortical thickness6. In post-mortem studies of the brains of schizophrenia patients7,8, there is ~50% down-regulation of reelin, GAD^sub 67^ expression and a reduction in dendritic spine density in certain cortical areas9. Behavioral studies have also identified similarities between reelin +/- mice and schizophrenia patients4. It is interesting to note that reelin also has been implicated as a candidate gene for autism10, as genetic studies indicate that chromosome 7q, where the reelin gene is located, is likely to contain an autism susceptibility locus (AUTS^sub 1^)11. As with reelin, GABA also plays a major role in the regulation of neuropeptide hormones, including OT.

OT is a small, 9 amino acid peptide, synthesized primarily in the paraventricular and supraoptic nuclei of the hypothalamus. Classically associated with functions such as birth and lactation, OT also can influence social behavior, the hypothalamus-pituitary-adrenal axis and may have a role in the regulation of neural development12-15.

OT receptors (OTRs) are seven transmembrane receptors of the G protein family, distributed throughout the nervous system, especially in hippocampal and hypothalamic nuclei. OTRs also tend to initially increase in the postnatal period and, at least in rats, may decline in adulthood in areas such as the cortex16,17, possibly supporting the hypothesis that the effects of OT may differ between developing and adult animals.

OT has been implicated in cellular proliferation and stem cells treated with OT proliferate and begin to express OTRs18. Both OT and reelin are particularly significant during development, and the absence of either may interfere with normal brain development. In addition, reelin is critical to the development of the GABAergic system and GABA modulates the release of OT.

Availability of the reelin haploinsufficient (+/-) reeler mouse (HRM) provides a model for examining the role of reelin in the development of the OT system and especially in the expression of the OTR. In rodents, OT expression increases during the immediate postnatal period. Serum levels of OT are low in certain forms of autism19 and there is one report that OT infusion may reduce repetitive symptoms in autism spectrum disorder (ASD)20.

At present, there are no other published studies directly examining possible interactions between OTR and reelin. In this study we used immunocytochemistry and in situ hybridization in HRM versus wild-type (+/ +; WTM) adult mice to quantify OTR abundance in the piriform cortex, the neocortex, hippocampus (archicortex) and the retrospenial cortex (periallocortex).

MATERIALS AND METHODS

Animals

Our institute has established a heterozygous reeler mouse-breeding colony (mice originally obtained from The Jackson Laboratory, B6C3Fe strain). Heterozygous reeler mice (HRM) are haploinsufficient, expressing a normal and a defective reelin allele with a deletion of ~150 kb at the 3' end. Genotyping of the offspring of the HRMs was done by PCR as described previously4,6. WTM and HRM males (n=6 of each, ~3 months old and weighing ~30 g average) were randomly chosen from several contemporaneous litters.

Immunolabeling

The protocol was performed as described previously6, with the following modifications. Cryosections (30 µM thick) were incubated for 30 minutes at room temperature in 3% normal goat serum+ 3% normal rabbit serum (NRS) +1% BSA in PBS, followed by incubation for 18-36 hours at 4°C in OTR antibody (N-19, Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:400 in 1% NRS +1% NRS +1% BSA in PBS (diluent). Then sections were incubated for 1 hour in 1:250 biotinylated rabbit anti-goat IgG (Vector Laboratories, Burlingame, CA, USA), and subsequently with avidin-biotin complexes (ABC Elite, Vector Laboratories, Burlingame, CA, USA) for 1 hour. sections were visualized with 3,3'diaminobenzidine (DAB) (Sigma, St Louis, MO, USA). As a negative control, the primary antibody was omitted.

In situ hybridization (mRNA)

The protocol was performed as described previously21, with the following modifications. All PBS was made in diethyl pyrocarbonate-treated H2O. Hybridization was carried as described by Resold et al.21 with 30 µM thick cryosections. Digoxigenin-labeled oligoprobe (50 pmol/ ml) was used for OTR (2301-2324; 1801-1824; 2071-2094; 24 bases scrambled sequence; Integrated DNA Technologies, Inc., Coralville, IA, USA). After hybridization, the sections were incubated overnight at 4°C in 1:1000 anti-digoxigenin antibody (Roche Diagnostic GmbH, Mannheim, Germany). The sections were then immersed in 1:250 biotinylated rabbit anti-sheep secondary antibody (Vector Laboratories, Inc. Burlingame, CA, USA) for 1 hour at room temperature. Finally, sections were developed in the same DAB solution as described above. As a negative control, the digoxigenin-labeled oligoprobe for OTR was omitted and a scrambled sequence was used.

Areas of the brain examined

Our experiments focused on cortical tissue in the brain areas encompassing Bregma -0.94 through to -1.34 mm (Figs 39 - 42 in ref. 22). Microscopic examination concentrated on qualitative differences in immunostaining and in situ hybridization localization patterns and cell numbers. A quantitative analysis using computer-assisted methods has been conducted to count the cells within a defined 300 × 300 µm area (which is the maximum and reasonable area projected to the screen by video camera that shows the studied regions described below). There were six WTMs and six HRMs. From each animal, four sections (two for immunostaining and two for in situ) for each four regions (motor cortex, hippocampus, piriform cortex and retrosplenial cortex) were studied. Positive OTR immunostaining and OTR in situ-stained cell bodies were counted. Two defined areas were counted in each region of each staining in four regions (Table 1).

RESULTS

Examination by light microscopy of the brain sections from the WTM, revealed OTR mRNA and protein throughout the cortical regions of the brain including the neocortex, allocortex and archicortex. In the HRM brain we observed a down-regulation of OTR mRNA and protein in most of the areas examined when compared with the WTM mouse (Table 1). The number of OTR-positive cells of immunolabeling and in situ hybridization methods in four different brain areas in WTM are very significantly higher than that number in HRM.

In the neocortex of the WTM mouse, OTR immunoreactivity is ubiquitously distributed in cells throughout layers M to IV. However, when compared with the cells in layers IV and V and to cells in other areas of the cortex, the intensity of immunolabeling appears to be increased in the cell bodies of layers II and III of the primary motor cortex (Figure 1A). Similar results were obtained with the localization of OTR mRNA in the WTM by in situ hybridization, which was also suggestive of an increase in OTR mRNA in this area. In all layers of the motor cortex of the WTM mouse, OTR immunoreactivity is associated with the neuronal cell bodies as can be seen in Figure 1A. Examination of the same area (M1, 2) in the HRM mouse, immunostained for OTR, is remarkably different (Figure 76). Layers I through to IV are almost completely lacking OTR immunoreactivity in some areas and greatly decreased in others. Results from OTR in situ hybridization in the HRM (Figure 1D) show that there is a significant overall decrease of OTR mRNA in the motor cortex, when compared with the WTM (Figure 1C). Inset images in (A) and (C) are the higher magnification of the boxed areas in (A) and (C), respectively. OTRs are present in the soma and apical dendrites.

The allocortex, including the piriform cortex, consists of regions that have fewer than six cellular layers and are structurally and functionally associated with the limbic system and/or olfaction. OTRs are present in the soma and apical dendrites in all layers of the piriform cortex, as evidenced by the presence of the OTR immunoreactive reaction product (Figure 2A). A significant change, however, is seen in the HRM (Figure 2B). In layer III of the piriform cortex, immunolabeled cells are very few and scattered. OTR mRNA is intensely expressed in the many cell bodies and layers of the piriform cortex in the WTM, comparable with the distribution of the OTR protein (Figure 2Q. OTR mRNA labeling is greatly reduced as are the number of OTR immunoreactive cells in the HRM (Figure 2D). Inset images in (A) and (C) are the higher magnification of the boxed areas in (A) and (C), respectively. OTRs are present in the soma and apical dendrites.

The retrosplenial area of the cortex (periallocortex) exhibits pronounced OTR immunoreactivity, particularly evident in the thickly packed cells of layer Il and the pyramidal cells of layer III of the retrosplenial granular area (RSG) (Figure 3A). There is a striking difference in the immunocytochemical labeling results in the HRM, which shows a significant loss of OTR protein in the RSG (Figure 3B). In situ hybridization localizes OTR mRNA primarily to those small cells located at the superficial portion of layer II and the pyramidal cells of the deeper layers (Figure 3Q. As would be expected from the immunocytochemical results, there is a great loss of intensity of the mRNA reaction product in the HRM (Figure 3D). Inset images in (A) and (C) are the higher magnification of the boxed areas in (A) and (C), respectively. OTRs are present in the soma and apical dendrites.

The hippocampus, with the dentate gyrus, is part of the archicortex. In the WTM, OTR immunoreactivity is expressed in the three strata of the dentate gyrus, all four fields (CA1, 2, 3 and 4) of pyramidal cells and the dendrites of the stratum radiatum (Figure 4A,B). In the HRM, immunocytochemical results show notable changes in signal intensity in the hippocampus (Figure 4B). OTR mRNA is also found in CA1 to CA4 and the dentate gyrus of the WTM, but is limited to the cell somata (Figure 4C). The result of OTR in situ hybridization imply declining amounts of OTR mRNA in both the hippocampus and dentate gyrus, with the exception of a small part of CA3 and the hilus (CA4) of the dentate gyrus, which contains scattered, reactive, large, ovoid pyramidal cells (Figure 4D). Inset images in (A) and (C) are the higher magnification of the boxed areas in (A) and (C), respectively. OTRs are present in the soma and apical dendrites.

Quantitative analysis results using computer-assisted methods have been summarized in Table 1. The average number of OTR-positive cells labeled by immunocyto-chemistry (immuno) and in situ hybridization (mRNA) methods from WTM versus HRM are significantly different in all four different brain areas in tissues.

DISCUSSION

Both reelin and OT have been implicated in autism and in schizophrenia. HRM have marked deficits in the expression of OTR as measured by either OTR mRNA or immunocytochemistry. Especially dramatic reductions in OTR in the HRM (compared with WTM) were found in the neocortex and retrosplenial area of the cortex. The retrosplenial area may be implicated in both memory and emotion. In addition, reductions in OTR binding or message were apparent in most areas of the hippocampus, including the dentate gyrus. These findings on the distribution of OTR in the cortex and hippocampus of reelin +/- mice correlate very closely with our earlier findings on the down-regulation of reelin in these same brain regions1'2. Quantification for other OTR-containing brain regions is in progress. Preliminary analyses of other areas containing OTRs (data not shown here), including the reticular thalamus and the central and basolateral amygdala do not show striking differences between WTM and HRM.

In general, cortical and/or hippocampal deficits of either reelin or OTRs might be expected to be associated with reductions in memory and learning, especially in the context of the social environment. A variety of studies have implicated OT in social memory23. In mice both maternal experience, associated with increases in endogenous OT, and treatment of virgin mice with intracerebroventricular injection of OT was associated with improved spatial memory, probably as a result of activity in the hippocampus24. Both reelin and OT play a role in regulating affect and mood. Down-regulation of reelin has been strongly correlated with schizophrenia7'25 and it is proposed that HRM may serve as a model for neural deficits seen in both schizophrenia and autism. Plasma OT levels are lower in some autistic children26. As detailed above, deficiencies both in social behavior and in the functions of the OT system also have been implicated in human autism. Mice with a defective OT gene also show reductions in social behavior and social memory15.

CONCLUSIONS

We report that HRM show regionally specific reductions in OTRs, especially in cortical areas such as the hippocampus and piriform cortex, which previously have been implicated in social memory and cognitive functions. These findings offer support for the more general hypothesis that down-regulation of reel in, of either genetic or epigenetic origin, through associated reductions in the OTRs, contributes to the deficiencies in social behavior that are characteristic of both schizophrenia and autism.

ACKNOWLEDGEMENTS

We are grateful for the assistance of Virginia Kriho. Supported by NIH HD 38490 and NAAR.

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Wensheng Liu*, George D. Pappas*,[dagger] and C. Sue Carter*,[double dagger]

* Department of Psychiatry, [dagger] Anatomy and Cell Biology and [double dagger] Brain-Body Center, University of Illinois at Chicago, 7607 W. Taylor Street, Chicago, IL 60612, USA

* Correspondence and reprint requests to: Dr George D. Pappas, Psychiatric Institute, University of Illinois at Chicago, 1601 West Taylor Street, Chicago, IL 60612, USA [gdpappas@uic.edu] Accepted for publication February 2005.

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