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

Farb CR, Chang W, Ledoux JE - Front Behav Neurosci (2010)

Bottom Line: The lateral nucleus of the amygdala (LA) is a critical brain region for fear learning and regulating the effects of stress on memory.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.

View Article: PubMed Central - PubMed

Affiliation: Center for Neural Science, New York University New York, NY, USA.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: Tissue sections designated for EM were processed as previously described (Farb and LeDoux, 1997). In brief, tissue sections containing the amygdala were incubated in 1% osmium tetraoxide/PB, dehydrated in a graded series of alcohols, stained en bloc in uranyl acetate, further dehydrated in acetone and subsequently flat-embedded in EMbed. Portions of the tissue containing the amygdala were cut and glued (Super Glue; Rancho Cucamonga, CA, USA) onto Beem capsules and placed at 60°C for 10 min. Photographs of the amygdala were taken and ultrathin sections (85 nm) were cut from the dorsolateral division of the LA (Figure 1A). Ultrathin sections were collected on 8–12 nickel grids and the tissue was examined on a JEOL 1200EX electron microscope. Photographs were taken using a Hammamatsu digital camera (AMT; Danvers, MA, USA). Electron micrographs were collected from the dorsolateral amygdala of four animals with the best morphological preservation. For each brain, ultrathin sections from at least two vibratome sections containing the AL were examined. Labeled terminals were identified by the presence of peroxidase reaction product within processes and were distinguished from preterminal axons by the presence of vesicles. Immunoreactive terminals without distinct membrane boundaries or whose peroxidase reaction product was too dense to distinguish between it and the post-synaptic density were not included in the analysis. Immunoreactive terminals were characterized as either forming or not forming synaptic contacts by the presence of a post-synaptic membrane specialization, intercleft filaments, and widened (10–20 nm) parallel spacing of plasma membranes (Peters et al., 1991). Labeled terminals with thickened post-synaptic densities and widened synaptic clefts were classified as asymmetric while terminals with thin post-synaptic densities and narrower synaptic clefts were identified as symmetric. Appositions were characterized by close membrane associations not separated by astrocytic processes, the lack of conventional synaptic clefts, intercleft material or dense specializations. Dendritic shafts were arbitrarily characterized as large (i.e. proximal) if their diameter was greater than 0.5 μm, or small (i.e., distal) if their diameter was less than 0.5 μm. Dendritic spines were smaller than dendrites and lacked mitochondria.


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

Farb CR, Chang W, Ledoux JE - Front Behav Neurosci (2010)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2967335&req=5

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.
Mentions: Tissue sections designated for EM were processed as previously described (Farb and LeDoux, 1997). In brief, tissue sections containing the amygdala were incubated in 1% osmium tetraoxide/PB, dehydrated in a graded series of alcohols, stained en bloc in uranyl acetate, further dehydrated in acetone and subsequently flat-embedded in EMbed. Portions of the tissue containing the amygdala were cut and glued (Super Glue; Rancho Cucamonga, CA, USA) onto Beem capsules and placed at 60°C for 10 min. Photographs of the amygdala were taken and ultrathin sections (85 nm) were cut from the dorsolateral division of the LA (Figure 1A). Ultrathin sections were collected on 8–12 nickel grids and the tissue was examined on a JEOL 1200EX electron microscope. Photographs were taken using a Hammamatsu digital camera (AMT; Danvers, MA, USA). Electron micrographs were collected from the dorsolateral amygdala of four animals with the best morphological preservation. For each brain, ultrathin sections from at least two vibratome sections containing the AL were examined. Labeled terminals were identified by the presence of peroxidase reaction product within processes and were distinguished from preterminal axons by the presence of vesicles. Immunoreactive terminals without distinct membrane boundaries or whose peroxidase reaction product was too dense to distinguish between it and the post-synaptic density were not included in the analysis. Immunoreactive terminals were characterized as either forming or not forming synaptic contacts by the presence of a post-synaptic membrane specialization, intercleft filaments, and widened (10–20 nm) parallel spacing of plasma membranes (Peters et al., 1991). Labeled terminals with thickened post-synaptic densities and widened synaptic clefts were classified as asymmetric while terminals with thin post-synaptic densities and narrower synaptic clefts were identified as symmetric. Appositions were characterized by close membrane associations not separated by astrocytic processes, the lack of conventional synaptic clefts, intercleft material or dense specializations. Dendritic shafts were arbitrarily characterized as large (i.e. proximal) if their diameter was greater than 0.5 μm, or small (i.e., distal) if their diameter was less than 0.5 μm. Dendritic spines were smaller than dendrites and lacked mitochondria.

Bottom Line: The lateral nucleus of the amygdala (LA) is a critical brain region for fear learning and regulating the effects of stress on memory.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.

View Article: PubMed Central - PubMed

Affiliation: Center for Neural Science, New York University New York, NY, USA.

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.

No MeSH data available.


Related in: MedlinePlus