Differential activation of neuronal cell types in the basolateral amygdala by corticotropin releasing factor

Abstract

Enhanced corticotropin releasing factor (CRF) release in the basolateral amygdala (BLA) is strongly associated with the generation of behavioral stress responses through activation of the CRF-R1 receptor subtype. Stress and anxiety-like behavior are modulated in part by the balance of peptide actions such as excitatory CRF and inhibitory neuropeptide Y (NPY) receptor activation in the BLA. While the actions of CRF are clear, little is known about the cell type influenced by CRF receptor stimulation. These studies were designed to identify the cell types within the BLA activated by intra-BLA administration of CRF using multi-label immunohistochemistry for cFos and markers for pyramidal (CaMKII-immunopositive) and interneuronal [glutamic acid decarboxylase (GAD65)] cell populations. Administration of CRF into the BLA produced a dose-dependent increase in the expression of cFos-ir. Intra-BLA injection of CRF induced significant increases in cFos-ir in the CaMKII-ir population. Although increases in cFos-ir in GAD65-ir cells were observed, this did not reach statistical significance perhaps in part due to the decreased numbers of GAD65-ir cells within the BLA after CRF treatment. These findings demonstrate that CRF, when released into the BLA, activates projection neurons and that the activity of GABAergic interneurons is also altered by CRF treatment. Decreases in the number of GAD65-ir neurons could reflect either increased or decreased activity of these cells and future studies will more directly address these possibilities. The expression of cFos is associated with longer term regulation of gene expression which may be involved in the profound long term effects of neuropeptides, such as CRF, on the activity and plasticity of BLA pyramidal neurons.

Figure 1. Histological verification of injector placement in the BLA

A) Schematic representation of injector placements as determined by histology. Injector tip placements are illustrated as symbols for each treatment; left side of the atlas section represents placements for CRF dose response experiment; right side illustrates placements for the coexpression study (cFos/CaMKII or cFos/GAD65). Illustrations of coronal brain sections are based on the rat brain atlas of Paxinos and Watson (1998). B) Representative section showing proper probe placement (between arrows) within the borders of the BLA. CeA, central amygdalar nucleus. Scale bar=200µm.

Figure 2. Photomicrographs of cFos-ir in the BLA following CRF delivery

cFos-ir is seen in the first column (green), ToPro (nuclear marker) fluorescence is shown in the second column (blue) and merged images are presented in the third column. cFos-ir nuclei following intra-BLA delivery of A) vehicle (aCSF), B) 2 fmol CRF, C) 20 fmol CRF, D) 200 fmol CRF or E) 2000 fmol CRF are indicated with arrowheads. Scale bar= 20µm.

Figure 3. Total number of cFos-ir cells in the BLA following intra-BLA delivery of increasing concentrations of CRF

Intra-BLA delivery of 20, 200 or 2000 fmol of CRF resulted in a significantly higher number of cFos-ir cells in the BLA compared to vehicle (aCSF) treated control. Data are reported as mean + SEM. One-way ANOVA, followed by Student-Newman-Keuls post-hoc test: *p<0.05, **<0.01 compared to vehicle; +p<0.05 compared to 2 fmol CRF, n=4–7/group.

Figure 4. Photomicrograph of cFos-ir in Pyramidal Cells and Interneurons in the BLA following CRF delivery

Compared to vehicle treated controls (A–C and G–I), intra-BLA delivery of 20 fmol CRF resulted in robust cFos-ir in CaMKII-ir cells (D–F) and GAD65-ir cells (J–L) in the BLA (arrowheads), respectively. CRF treatment also resulted in a reduction in the number of GAD65-ir cells. Single-labeled CaMKII-ir pyramidal neurons and GAD-65-ir interneurons are indicated with horizontal arrows and single-labeled cFos-ir cells with vertical arrows. Scale bar=20µm.

Figure 5. Effect of acute CRF treatment (20 fmol) on total number of CaMKII- and GAD65-ir cell numbers in the BLA

Total cell populations for CaMKII-ir and GAD65-ir cells were detemined in the BLA 90 minutes after injection of either vehicle or CRF (20 fmol). *p<0.05 from corresponding vehicle-treated group, Student’s t test.