Evidence for beta1-adrenergic receptor involvement in amygdalar corticotropin-releasing factor gene expression: implications for cocaine withdrawal

Abstract

We previously showed that betaxolol, a selective beta(1)-adrenergic receptor antagonist, administered during early phases of cocaine abstinence, ameliorated withdrawal-induced anxiety and blocked increases in amygdalar beta(1)-adrenergic receptor expression in rats. Here, we report the efficacy of betaxolol in reducing increases in gene expression of amygdalar corticotropin-releasing factor (CRF), a peptide known to be involved in mediating 'anxiety-like' behaviors during initial phases of cocaine abstinence. We also demonstrate attenuation of an amygdalar beta(1)-adrenergic receptor-mediated cell-signaling pathway following this treatment. Male rats were administered betaxolol at 24 and 44 h following chronic cocaine administration. Animals were euthanized at the 48-h time point and the amygdala was microdissected and processed for quantitative reverse transcriptase-polymerase chain reaction and/or western blot analysis. Results showed that betaxolol treatment during early cocaine withdrawal attenuated increases in amygdalar CRF gene expression and cyclic adenosine monophosphate-dependent protein kinase regulatory and catalytic subunit (nuclear fraction) protein expression. Our data also reveal that beta(1)-adrenergic receptors are on amygdalar neurons, which are immunoreactive for CRF. The present findings suggest that the efficacy of betaxolol treatment on cocaine withdrawal-induced anxiety may be related, in part, to its effect on amygdalar beta(1)-adrenergic receptor, modulation of its downstream cell-signaling elements and CRF gene expression.

Figure 1

Western blots demonstrating PKA Regulatory Subunit immunoreactivity in the amygdala of animals from four different drug treatment groups (see Table 1). PKA Regulatory Subunit immunoreactivity in the amygdala of these animals is expressed as a percentage of the control mean when the control equals 100 (±S.E.M.). PKA regulatory subunit expression is elevated in the amygdala following early cocaine withdrawal, however; this effect is reversed following betaxolol treatment during cocaine withdrawal. Statistical significance between groups of animals is indicated on graph; * p<0.05.

Figure 2

Western blots demonstrating PKA Catalytic Subunit and the nuclear localization marker, TATA Binding Protein (BP), expression in cytoplasmic (top left) and nuclear (top right) amygdalar extracts obtained from four different groups of drug treated animals (see Table 1). In both the cytoplasmic and nuclear extracts, PKA Catalytic Subunit immunoreactivity in the amygdala of these animals is expressed as a percentage of the control mean when the control equals 100 (±S.E.M.). PKA catalytic subunit expression was significantly decreased in amygdalar cytoplasmic extracts and concurrently increased significantly in amygdalar nuclear extracts from animals that underwent cocaine withdrawal. Betaxolol administration during early cocaine withdrawal reversed alterations in PKA catalytic subunit expression in amygdalar cytoplasmic and nuclear extracts to control levels. Statistical significance between groups of animals is indicated on graphs; * p<0.05, ** p<0.01. TATA BP immunoreactivity was used as a nuclear loading control. Cytoplasmic extracts lack immunoreactivity for TATA BP, while nuclear extracts demonstrate TATA BP immunoreactivity. β-actin immunoreactivity was evaluated in cytoplasmic amygdalar extracts (top left) as a loading control.

Figure 3

Western blots demonstrating pCREB, CREB, and the nuclear localization marker, TATA BP, immunoreactivity in the amygdala of animals from four different drug treatment groups (see Table 1). pCREB protein densitometry levels were evaluated relative to CREB protein densitometry levels for each animal. pCREB/CREB immunoreactivity in the amygdala of these animals is expressed as a percentage of the control mean when the control equals 100 (±S.E.M.). CREB phosphorylation is increased in amygdalar nuclear extracts following early cocaine withdrawal, however; betaxolol administration during early cocaine withdrawal blocks this increase. Statistical significance between groups of animals is indicated on graph; * p<0.05.

Figure 4

Effect of betaxolol treatment during cocaine withdrawal on CRF mRNA levels in the amygdala. Quantitative RT-PCR data was analyzed by the comparative C_T method using the formula 2^−ΔΔC_T. Fold change differences in CRF mRNA abundance in the three drug treatment groups (CS, SB and CB; see Table 1) are plotted relative to saline control (SS) when the saline control group equals 1. CRF mRNA expression was significantly increased in amygdalar extracts from animals following early cocaine withdrawal (CS vs. SS); however, this increase was attenuated in amygdalar extracts from animals that were treated with betaxolol following cocaine administration (CB vs. CS). Betaxolol treatment alone did not have any significant effect on amygdalar CRF mRNA expression (SB vs. SS). Statistical significance between groups of animals is indicated on graph; * p<0.05.

Figure 5

Proposed cell signaling pathway of noradrenergic influence on CRF transcription. Norepinephrine (NE) in the extracellular space binds to a G-protein coupled adrenergic receptor spanning the neuronal cell membrane (1). This ligand-receptor complex then activates the G protein, causing the alpha subunit to dissociate from the beta and gamma subunits (2). The activated alpha subunit of the G protein functions to activate the plasma membrane bound enzyme, adenylyl cyclase (3). Adenylyl cyclase synthesizes cyclic adenosine monophosphate (cAMP) from adenosine triphosphate (ATP) (4). cAMP binds to the regulatory subunits of cycle-AMP-dependent protein kinase (PKA) (5). This induces a conformational change causing the regulatory subunits to release the catalytic subunits, thereby activating the kinase activity of the catalytic subunits (6). Once the catalytic subunits are freed and active, they translocate into the nucleus of the cell to phosphorylate cAMP response element binding protein (CREB) (7). The regulatory subunits remain in the cytoplasm. Phosphorylated CREB (pCREB) then recruits a transcriptional co-activator called CREB-binding protein (CBP) (8). CBP recognizes the regulatory region of the target gene called the cAMP response element (CRE) and stimulates gene transcription (9). The culmination of this proposed cell signaling pathway is the transcription of CRF (10).

Figure 6

β₁-adrenergic receptors are located on CRF-containing neurons of the amygdala. A and D: High magnification confocal photomicrographs of two separate coronal sections through the CNA labeled for CRF using rhodamine isothiocyanate-conjugated secondary antibody; thin arrows point to CRF-immunoreactive cells, while thick arrows indicate CRF-immunoreactive perikarya containing β₁-adrenergic receptors (see panels C and F). B and E: The same coronal sections as shown in figures A and D, respectively, labeled for β₁-adrenergic receptors (β1-AR) using fluorescein isothiocyanate-conjugated secondary antibody; thick arrows indicate CRF-immunoreactive perikarya containing β₁-adrenergic receptors (β1-AR) (see panels C and F). C and F: Merged images of panels A and B and D and E, respectively. Thick arrows show immunoreactivity for CRF and β₁-adrenergic receptors, while thin arrows indicate single-labeling for CRF only.

Figure 7

Ultrastructural evidence for co-localization of β₁-adrenergic receptors and CRF in the amygdala. A: Electron photomicrograph showing a dendrite containing immunogold-silver labeling (arrowheads) for β₁-adrenergic receptor (β1-AR). A dendrite containing peroxidase labeling (arrows) for CRF (CRF-d) can also be identified in the neuropil. Scale bar: 0.50 μm. B–C: Electron photomicrographs showing CRF (CRF-d; immunoperoxidase) and β₁-adrenergic receptors (β1-AR; immunogold) immunoreactivities co-localized in dendrites of the CNA. Arrowheads indicate immunogold particles for β₁-adrenergic receptors (β1-AR) and peroxidase labeling for CRF (CRF-d; arrows) is identified as electron-dense reaction product. Dendrites of co-localized CRF and β₁-adrenergic receptors form asymmetric (curved arrows) and symmetric (double arrows) synapses with axon terminals. Scale bars: 0.50 μm. Abbreviations: m, mitochondria; ut, unlabeled terminal.

Figure 8

Distribution of β₁-adrenergic receptors in CRF-immunoreactive dendritic processes in the amygdala. A: β₁-adrenergic receptors (β1-AR cyt) are shown located within the cytoplasmic compartment of a CRF-immunoreactive dendrite (CRF-d). B: β₁-adrenergic receptors (β1-AR pm) are shown distributed along the plasma membrane of a CRF-immunoreactive dendrite (CRF-d). C: Some β₁-adrenergic receptors are located within the cytoplasmic compartment, while some are located along the plasma membrane (β1-AR cyt + pm) of a CRF-immunoreactive dendrite (CRF-d). Arrowheads indicate immunogold labeling for β₁-adrenergic receptors within the cytoplasmic compartment, while arrows point to β₁-adrenergic receptors along the plasma membrane. Scale bars: 0.50 μm. Abbreviations: ud, unlabeled dendrite; m, mitochondria; ut, unlabeled terminal.