Ultrastructural characterization of noradrenergic axons and Beta-adrenergic receptors in the lateral nucleus of the amygdala

Abstract

Norepinephrine (NE) is thought to play a key role in fear and anxiety, but its role in amygdala-dependent Pavlovian fear conditioning, a major model for understanding the neural basis of fear, is poorly understood. The lateral nucleus of the amygdala (LA) is a critical brain region for fear learning and regulating the effects of stress on memory. To understand better the cellular mechanisms of NE and its adrenergic receptors in the LA, we used antibodies directed against dopamine beta-hydroxylase (DβH), the synthetic enzyme for NE, or against two different isoforms of the beta-adrenergic receptors (βARs), one that predominately recognizes neurons (βAR 248) and the other astrocytes (βAR 404), to characterize the microenvironments of DβH and βAR. By electron microscopy, most DβH terminals did not make synapses, but when they did, they formed both asymmetric and symmetric synapses. By light microscopy, βARs were present in both neurons and astrocytes. Confocal microscopy revealed that both excitatory and inhibitory neurons express βAR248. By electron microscopy, βAR 248 was present in neuronal cell bodies, dendritic shafts and spines, and some axon terminals and astrocytes. When in dendrites and spines, βAR 248 was frequently concentrated along plasma membranes and at post-synaptic densities of asymmetric (excitatory) synapses. βAR 404 was expressed predominately in astrocytic cell bodies and processes. These astrocytic processes were frequently interposed between unlabeled terminals or ensheathed asymmetric synapses. Our findings provide a morphological basis for understanding ways in which NE may modulate transmission by acting via synaptic or non-synaptic mechanisms in the LA.

Keywords: DBH; astrocyte; electron microscopy; fear; lateral amygdala; norepinephrine; synapse; volume transmission.

Figure 1

Light micrographs showing dopamine beta-hydroxylase (DβH) and beta-adrenergic receptors (βARs) in the amygdala. (A) Dark-field micrograph shows DβH in the amygdala. Trapezoid corresponds to area examined by electron microscopy. (B) Higher-magnification dark-field micrograph illustrates thin, varicose DβH axons. Arrowheads point to varicosities. (C,D) Low-magnification bright-field micrographs show βAR 248 (C), and βAR 404 (D) are distributed homogenously throughout the amygdala. (E) Higher magnification shows that βAR 248 intensely labels somata and some proximal dendrites (arrowheads). The nuclei of some labeled cells are seen in some cells (arrows) but are difficult to distinguish in others. (F) Higher-power Nomarski optics show labeled astrocytic cell bodies (arrowheads) and their radiating processes (arrows). Also shown is an astrocytic process (asterisks) surrounding a blood vessel (BV). Scale bar = 100 μm in A–D, 50 μm in B, E, and F. BLA, basolateral amygdala; Ce, central amygdala; LA, lateral amygdala.

Figure 2

Confocal micrographs show that βAR 248 is localized to both excitatory and inhibitory LA cells. (A) Cells immunopositive for CAMKII (green) are also immunoreactive for βAR 248 (red). Arrows point to dually labeled cells while the asterisk indicates a cell singly labeled for βAR 248. (B) GABAergic cells (green) also contain βAR 248 (red). Arrows point to cells dually labeled for βAR 248 and GABA; asterisks indicate βAR 248 singly labeled cells.

Figure 3

Electron micrographs show DβH-terminals in LA. (A) A DβH – terminal (DBH) apposes a dendritic spine (sp) and an unlabeled terminal (ut) forming an asymmetric synapse (asterisks) onto a spine (sp). (B) A DβH-terminal forms a symmetric synapse (arrows) with a dendrite (d). (C) A DβH-terminal forms a synapse (arrowheads) onto a dendrite (d) that also receives a synapse (arrows) from an unlabeled terminal (ut). Glial processes (g and asterisk) are also shown. (D) A DBH-terminal forms a symmetric synapse (arrows) onto a dendritic (d) whose spine (sp) receives a synapse (arrowheads) from an unlabeled terminal (ut). Also shown is a glial process (g). (E) A DβH-terminal is apposed to an unlabeled terminal (ut₁) that forms a symmetric synapse (arrows) on a dendrite (d). An unlabeled terminal (ut₂) forms an asymmetric synapse (arrowheads) on the dendrite's spine (sp). Unlabeled glial processes (g and *) are also shown. (F) A DβH-terminal (DBH₁) forms a synapse (arrows) onto a dendritic shaft (d₁), whose spine (sp) receives an asymmetric synapse (arrowheads) from a second DβH-terminal (DBH₂). DβH₂ apposes unlabeled terminals (ut_1–2) forming asymmetric synapses (arrowheads) with a spine (sp) and a dendrite (d₂). Scale bars = 0.500 μm.

Figure 4

DβH-profiles and glial processes immunoreactive for βAR 404 are associated with blood vessels. (A) A DβH-axon (LAx) follows the contours of a blood vessel. The plasma membrane of the DβH-terminal (DβH) shows parallel alignment with the plasma membrane (small arrows) of an unlabeled terminal (ut). The basal lamina (asterisks) and an endothelial cell (End) separate the DβH-terminal from the blood vessel (BV). (B) A βAR 404-labeled glial process (LG) encircles a blood vessel (BV), which is bounded by an endothelial cell (end) and the basal lamina (asterisks). The βAR 404 astrocyte apposes unlabeled terminals (ut), one of which forms an asymmetric synapse (arrowheads) onto a dendritic spine (sp). Scale bars = 0.500 μm.

Figure 5

Graphs show synaptic targets and specializations made by DβH terminals that form synapses. (A) The vast majority of DBH symmetric synapses occurred on dendrites but a small proportion were made onto spines and somata. (B) Most DBH asymmetric synapses occurred on spines though some were formed on dendrites.

Figure 6

Both LA neurons and glia are immunoreactive (ir) for βAR. (A) Unlabeled terminals (ut) form asymmetric synapses onto βAR 248-ir spines (LSp). The spinous portion (LSp) of a dendrite (d) is βAR 248-ir. (B) An unlabeled terminal (ut) synapses (arrowheads) onto a βAR 248-ir spine (LSp). βAR 248-labeled terminals (LT_1–2) synapse (arrowheads) onto unlabeled spines (usp). (C) The βAR 248 reaction product in a dendrite (LD) is concentrated at the synapse (arrowheads) formed by an unlabeled terminal (ut) but also rims the microtubules (mt) and mitochondria (m). Also shown are a βAR 248-ir terminal (LT) and a small labeled dendrite (LD). (D) βAR 404-ir glial processes (LG, asterisks) are interposed between two unlabeled terminals (ut_1–2). Also shown are an unlabeled terminal (ut₃) synapsing (arrowheads) onto a βAR 404 ir spine (LSp) and a βAR 404-ir terminal (LT) synapsing (arrowheads) onto an unlabeled spine (usp). (E) A βAR 404-ir glial process (LG) surrounds a terminal (ut) that synapses (arrowheads) onto a spine (usp). Asterisks denote βAR 404-ir along the plasma membrane. (F) A βAR 404 ir glial process (LG, asterisks) surrounds an unlabeled terminal (ut) that forms an asymmetric synapse (arrowheads) onto a βAR 404-ir spine (LSp). Scale bars = 0.500 μm.