Neuropeptide Y signaling in the central nucleus of amygdala regulates alcohol-drinking and anxiety-like behaviors of alcohol-preferring rats

Abstract

Background: The neuropeptide Y (NPY) system of the central nucleus of amygdala (CeA) has been shown to be involved in anxiety and alcoholism. In this study, we investigated the molecular mechanisms by which NPY in the CeA regulates anxiety and alcohol drinking behaviors using alcohol-preferring (P) rats as an animal model.

Methods: Alcohol-preferring rats were bilaterally cannulated targeting the CeA and infused with artificial cerebrospinal fluid (aCSF) or NPY. Alcohol drinking and anxiety-like behaviors were assessed by the 2-bottle free-choice paradigm and light/dark box (LDB) exploration test, respectively. The levels of NPY and related signaling proteins were determined by the gold immunolabeling procedure. The mRNA levels of NPY were measured by in situ RT-PCR. Double-immunofluorescence labeling was performed to observe the colocalization of NPY and Ca(2+)/calmodulin-dependent protein kinase IV (CaMK IV).

Results: We found that NPY infusion into the CeA produced anxiolytic effects, as measured by the LDB exploration test, and also decreased alcohol intake in P rats. NPY infusion into the CeA significantly increased levels of CaMK IV and phosphorylated cAMP responsive element-binding (pCREB) protein and increased mRNA and protein levels of NPY, but produced no changes in protein levels of CREB or the catalytic alpha-subunit of protein kinase A (PKA-Calpha) in the CeA. We also observed that alcohol intake produced anxiolytic effects in P rats in the LDB test and also increased NPY expression and protein levels of pCREB and PKA-Calpha without modulating protein levels of CREB or CaMK IV, in both the CeA and medial nucleus of amygdala. In addition, we found that CaMK IV-positive cells were co-localized with NPY in amygdaloid structures of P rats.

Conclusions: These results suggest that NPY infusion may increase the expression of endogenous NPY in the CeA, which is most likely attributable to an increase in CaMK IV-dependent CREB phosphorylation and this molecular mechanism may be involved in regulating anxiety and alcohol drinking behaviors of P rats.

Figure 1

Representative photomicrographs of low-magnification views of Nissl stained brain sections of P rats (with or without alcohol drinking) infused with aCSF or NPY showing the position of cannula tip, length of probe, and central amygdaloid target regions (WW+aCSF, Water/Water + aCSF; WW+NPY, Water/Water + NPY; WE+aCSF, Water/Ethanol + aCSF; WE+NPY, Water/Ethanol + NPY). Scale Bar = 500 μm.

Figure 2

A) Alcohol drinking (g/kg/day) patterns (7% and 9% ethanol) and effect of NPY infusion into the CeA on alcohol intake (last 3 days of 9% ethanol intake) in P rats (lower panel). No significant differences are observed in the total fluid intake (ml/day) of P rats from different groups (upper panel). Values are mean ± S.E.M. of 7 rats in each group. *Significantly different from P rats infused with aCSF (p < 0.001; repeated measures using a two-way ANOVA followed by the Tukey test). B) The effect of NPY infusion (0.5 μl of 100 pM NPY or aCSF once daily for the last 3 days of 9% ethanol voluntary intake) and alcohol consumption on the total ambulations (upper panel) and percent time spent by the P rats in each of the light and dark compartments (lower panel), during the light/dark box exploration test. Values are the mean ± S.E.M. of 7 rats per group (WW+aCSF, Water/Water + aCSF; WW+NPY, Water/Water + NPY; WE+aCSF, Water/Ethanol + aCSF; WE+NPY, Water/Ethanol + NPY). *Significantly different from the P (WW+ aCSF) rats (p < 0.001; one-way ANOVA followed by the Tukey test).

Figure 3

A) Representative photomicrographs of CREB (upper panel) and p-CREB (lower panel) gold-immunolabeling in CeA structures of P rats infused with aCSF or NPY during voluntary alcohol drinking. These photomicrographs show either CREB- or p-CREB-positive nuclei in the CeA of P rats infused with aCSF or NPY. The inset figures show the immuno-gold particles within a single nucleus (CREB, or p-CREB). Scale bar = 40 μm. B) Effect of NPY infusion into the CeA and voluntary alcohol drinking on CREB or p-CREB protein levels in CeA, MeA, and BLA of P rats. Values are mean ± S.E.M. of 7 rats per group (WW+aCSF, Water/Water + aCSF; WW+NPY, Water/Water + NPY; WE+aCSF, Water/Ethanol + aCSF; WE+NPY, Water/Ethanol + NPY). *Significantly different from the P (WW+ aCSF) rats (p < 0.001; one-way ANOVA followed by the Tukey test).

Figure 3

A) Representative photomicrographs of CREB (upper panel) and p-CREB (lower panel) gold-immunolabeling in CeA structures of P rats infused with aCSF or NPY during voluntary alcohol drinking. These photomicrographs show either CREB- or p-CREB-positive nuclei in the CeA of P rats infused with aCSF or NPY. The inset figures show the immuno-gold particles within a single nucleus (CREB, or p-CREB). Scale bar = 40 μm. B) Effect of NPY infusion into the CeA and voluntary alcohol drinking on CREB or p-CREB protein levels in CeA, MeA, and BLA of P rats. Values are mean ± S.E.M. of 7 rats per group (WW+aCSF, Water/Water + aCSF; WW+NPY, Water/Water + NPY; WE+aCSF, Water/Ethanol + aCSF; WE+NPY, Water/Ethanol + NPY). *Significantly different from the P (WW+ aCSF) rats (p < 0.001; one-way ANOVA followed by the Tukey test).

Figure 4

A) Representative photomicrographs of CaMK IV (upper panel) or PKA-Cα (lower panel) gold-immunolabeling in CeA structures of P rats infused with aCSF or NPY during voluntary alcohol drinking. The representative photomicrographs show CaMK IV or PKA-Cα-positive cells in the CeA of P rats infused with aCSF or NPY. The inset figures show the immuno-gold particles within a single nucleus for CaMK IV and within a cell body for PKA-Cα. Scale bar = 40 μm. B) Effect of NPY infusion into the CeA and voluntary alcohol drinking on CaMK IV and PKA-Cα protein levels in CeA, MeA, and BLA of P rats. Values are the mean ± S.E.M. of 5-7 rats per group (WW+aCSF, Water/Water + aCSF; WW+NPY, Water/Water + NPY; WE+aCSF, Water/Ethanol + aCSF; WE+NPY, Water/Ethanol + NPY). *Significantly different from the P (WW+ aCSF) rats (p < 0.001; one-way ANOVA followed by the Tukey test).

Figure 4

A) Representative photomicrographs of CaMK IV (upper panel) or PKA-Cα (lower panel) gold-immunolabeling in CeA structures of P rats infused with aCSF or NPY during voluntary alcohol drinking. The representative photomicrographs show CaMK IV or PKA-Cα-positive cells in the CeA of P rats infused with aCSF or NPY. The inset figures show the immuno-gold particles within a single nucleus for CaMK IV and within a cell body for PKA-Cα. Scale bar = 40 μm. B) Effect of NPY infusion into the CeA and voluntary alcohol drinking on CaMK IV and PKA-Cα protein levels in CeA, MeA, and BLA of P rats. Values are the mean ± S.E.M. of 5-7 rats per group (WW+aCSF, Water/Water + aCSF; WW+NPY, Water/Water + NPY; WE+aCSF, Water/Ethanol + aCSF; WE+NPY, Water/Ethanol + NPY). *Significantly different from the P (WW+ aCSF) rats (p < 0.001; one-way ANOVA followed by the Tukey test).

Figure 5

A) Representative photomicrographs of NPY gold-immunolabeling (upper panel) and NPY mRNA expression (in situ RT-PCR; lower panel) in the CeA of P rats with aCSF or NPY infusion during alcohol drinking. Inset figure for NPY protein shows gold particles in a cell body at high magnification (100x). Scale bar = 40 μm. B) Effect of alcohol consumption with or without central amygdaloid NPY infusion on mRNA and protein levels of NPY in the CeA, MeA and BLA structures of rats. Values are the mean ± S.E.M. derived from 5 P rats per group (WW+aCSF, Water/Water + aCSF; WW+NPY, Water/Water + NPY; WE+aCSF, Water/Ethanol + aCSF; WE+NPY, Water/Ethanol + NPY). *Significantly different from P (WW+ aCSF) rats (p < 0.01-0.001; one-way ANOVA followed by the Tukey test).

Figure 5

A) Representative photomicrographs of NPY gold-immunolabeling (upper panel) and NPY mRNA expression (in situ RT-PCR; lower panel) in the CeA of P rats with aCSF or NPY infusion during alcohol drinking. Inset figure for NPY protein shows gold particles in a cell body at high magnification (100x). Scale bar = 40 μm. B) Effect of alcohol consumption with or without central amygdaloid NPY infusion on mRNA and protein levels of NPY in the CeA, MeA and BLA structures of rats. Values are the mean ± S.E.M. derived from 5 P rats per group (WW+aCSF, Water/Water + aCSF; WW+NPY, Water/Water + NPY; WE+aCSF, Water/Ethanol + aCSF; WE+NPY, Water/Ethanol + NPY). *Significantly different from P (WW+ aCSF) rats (p < 0.01-0.001; one-way ANOVA followed by the Tukey test).

Figure 6

Representative photomicrographs showing the immunofluorescence staining for NPY (green) and CaMK IV (red) in the cells of CeA, MeA and BLA. The colocalization of the NPY and CaMK IV in cells is indicated by the yellow color development (merge). These photomicrographs show that most of the NPY-positive cells co-express CaMK IV (arrows) with a few exceptions (arrowheads). Scale bar = 20 μm.