Ultrastructural localization of the serotonin transporter in limbic and motor compartments of the nucleus accumbens - PubMed (original) (raw)

Ultrastructural localization of the serotonin transporter in limbic and motor compartments of the nucleus accumbens

V M Pickel et al. J Neurosci. 1999.

Abstract

Extracellular levels of serotonin [5-hydroxytryptamine (5-HT)] in the nucleus accumbens (NAc) can influence both cognitive and motor functions involving extensive connections with the frontal cortex. The 5-HT levels reflect vesicular release and plasmalemmal reuptake through the serotonin transporter (SERT). We used electron microscopic immunocytochemistry to determine the sites for SERT activation in the limbic shell and motor-associated core of the rat NAc. Of the SERT-immunoreactive profiles in each region, >90% were serotonergic axons and axon terminals; the remainder were nonserotonergic dendrites and glia. Axonal SERT immunogold labeling was seen mainly at nonsynaptic sites on plasma membranes and often near 5-HT-containing large dense core vesicles (DCVs). SERT-labeled axonal profiles were larger and had a higher numerical density in the shell versus the core but showed no regional differences in their content of SERT immunogold particles. In contrast, immunoreactive dendrites had a lower numerical density in the shell than in the core. SERT labeling in dendrites was localized to segments of plasma membrane near synaptic contacts from unlabeled terminals and/or dendritic appositions. Our results suggest that in the NAc (1) reuptake into serotonergic axons is most efficient after exocytotic release from DCVs, and (2) increased 5-HT release without concomitant increase in SERT expression in individual axons may contribute to higher extracellular levels of serotonin in the shell versus the core. These findings also indicate that SERT may play a minor substrate-dependent role in serotonin uptake or channel activity in selective nonserotonergic neurons and glia in the NAc.

PubMed Disclaimer

Figures

Fig. 1.

Bar graphs showing the numerical density of different types of SERT peroxidase-labeled profiles by using C611–630 antiserum in the NAc shell (white bars) and core (black bars). These profiles were determined from analysis of thin sections that were taken from one to three coronal vibratome sections through the NAc from four animals. The labeled small unmyelinated axons (axon), axon terminals (ter), dendrites (den), and dendritic spines (spine) show significant (*) shell and core differences in numerical density via ANOVA; p< 0.01.

Fig. 2.

Electron micrographs showing immunoperoxidase localization of SERT and dual labeling for SERT and 5-HT in small axons and terminals in the NAc from acrolein-fixed rat brain. A, B, In the NAc core and shell, respectively, peroxidase labeling for SERT is seen in small axons (SERT-Ax) and terminals (SERT-Te) by using the C611–630 antiserum at 1:20,000 dilution. The peroxidase labeling is prominent on or near the plasma membranes (arrowhead) and more lightly distributed around membranes of small synaptic vesicles (SSVs) at a distance from the asymmetric synapse (curved arrow) that is formed with an unlabeled dendrite (UD) in A. The intense labeling in small axons in large part obscures their content of organelles. The SERT-labeled axons are apposed mainly by small unlabeled axons (UA) or unlabeled dendrites (UD).Asterisks show unlabeled glial processes. C, D, In the NAc core and shell, respectively, small unmyelinated axons are dually labeled (Du-Ax) for SERT and 5-HT. Diffuse peroxidase labeling for 5-HT is seen within the axon, whereas immunogold SERT labeling (small arrows) is localized along the plasma membrane. In addition, a large, presumably dense core vesicle (DCV) in D also contains peroxidase immunoreactivity. The labeled axons are apposed to unlabeled axons (UA) in C and to unlabeled axons and an unlabeled dendrite (UD) in D. Scale bars, 0.5 μm.

Fig. 3.

Immunogold-silver (arrows) localization of SERT in small axons and axon terminals in the NAc from acrolein-fixed rat brain. A, In the shell SERT labeling is seen in an axon (SERT-Ax), which is in continuity with a SERT-immunoreactive terminal (SERT-Te) in tissue that was processed by using the C611–630 antiserum at a 1:1000 dilution. The labeled profile is apposed to an unlabeled axon (UA) and terminal (UT). B, C, In the core SERT labeling is present in small axons and axon terminals, respectively, in tissue that was processed by using the same antiserum but at a 1:5000 dilution. Gold particles are seen along portions of the plasma membrane in contact with large dense core vesicles (DCVs) that are present in a small axon (B) and an axon terminal (C). Contacts are seen between the labeled axons or terminals and other unlabeled axons (UA), unlabeled dendrites (UD) or unlabeled terminals (UT). Scale bars, 0.5 μm.

Fig. 4.

Dual labeling for SERT and 5-HT in axon terminals within the NAc from brain tissue that was fixed by using glutaraldehyde (A, C) or acrolein (B, D, E). Immunogold labeling for SERT (small arrows) is seen along portions of the plasma membrane (1) away from symmetric axodendritic (open arrowhead in A from the shell and in B from the core) and asymmetric axospinous (curved filled arrow in C from the shell) synapses and (2) near appositional contacts with unlabeled dendrites (UD) and unlabeled terminals (UT) in the shell (D, E). The peroxidase labeling appears as a diffuse precipitate surrounding small synaptic vesicles (SSV), as seen in A, or more discretely localized to membranes of large dense core vesicles (DCVs) as seen in B, D, and E. The gold particles are prevalent near DCVs that contact the plasma membrane at appositional contacts with unlabeled dendrites and terminals.E, The apposed unlabeled terminal forms an asymmetric synapse (curved arrow) with an unlabeled spine (US). Tubulovesicular organelles (TV) also are seen near the plasma membrane and contacted by gold particles. Scale bars, 0.5 μm.

Fig. 5.

Synaptic plasmalemmal localization of SERT in dendrites (SERT-D) and spines (SERT-S) in the NAc shell by using antisera against amino acids N1–85(A, B) and C611–630(C). A, B, Immunoperoxidase reaction product (small arrowheads) for SERT is localized within and near the synapse formed by unlabeled terminals (UT). A, The SERT-D also receives a symmetric synapse (open arrow) from another unlabeled terminal and is apposed to a densely labeled profile resembling an axon (SERT-Ax). B, The unlabeled terminal also provides asymmetric synapses (curved arrows) to two other spines that contain little or no immunoreactivity.C, Immunogold-silver labeling (small arrows) in SERT-D is seen within and near the membrane specialization of a symmetric synapse (open arrow) that is formed by an unlabeled terminal (UT). This terminal also forms a similar contact with an unlabeled dendrite (UD) and is apposed to a small SERT-immunolabeled axon (SERT-Ax). Scale bars, 0.5 μm.

Fig. 6.

Plasmalemmal localization of SERT in dendrites (SERT-D) apposing other unlabeled dendrites (UD) in the NAc shell. A, Immunoperoxidase labeling for mouse N1–85 SERT antiserum at 1:100 dilution in glutaraldehyde-fixed tissue shows reaction product (arrowheads) along apposed and nonapposed surfaces of the plasma membrane. The boxed area in A_is shown at a higher magnification in the inset. In the_inset the arrows indicate the region in which the inner plasma membranes appear to fuse in a manner typical of gap junctions. B, Immunogold labeling for goat anti-C611–630 antiserum at 1:1000 dilution in acrolein-fixed tissue shows immunogold particles (arrows) along the membrane at the appositional contact. In A and B the UD has numerous mitochondria (m), whereas the labeled dendrites have only one within the plane of section. Scale bars, 0.5 μm.

Fig. 7.

Immunoperoxidase localization of SERT in glial processes (SERT-G) in NAc. Shown is an electron micrograph from the shell region in tissue that was processed by using goat anti-C611–630 antiserum at 1:1000 (A, C) or mouse N1–85 antiserum at 1:100 dilution (B). A, The diffuse peroxidase reaction is seen in a glial profile that is apposed to a densely SERT-immunoreactive axon (SERT-Ax) and to an unlabeled terminal (UT) that forms an asymmetric synapse (curved arrow) with a dendrite. B, SERT-G shows intense labeling of the plasma membrane at a gap junction (GJ) with an unlabeled astrocytic process (asterisk) and at appositional contacts with unlabeled axons (UA). C, Micrograph from the NAc core in tissue processed by using goat anti-C611–630 serum shows SERT-G apposed to an unlabeled terminal (UT) that forms an asymmetric synapse (curved arrow) with an unlabeled dendritic spine. Within the neuropil, intense labeling is seen in an axon (SERT-Ax) apposing an unlabeled axon (UA) and other neuronal profiles. Scale bars, 05 μm.

References

1. Austin MC, Bradley CC, Mann JJ, Blakely RD. Expression of serotonin transporter messenger RNA in the human brain. J Neurochem. 1994;62:2362–2367. - PubMed
1. Bal N, Figueras G, Vilaro MT, Sunol C, Artigas F. Antidepressant drugs inhibit a glial 5-hydroxytryptamine transporter in rat brain. Eur J Neurosci. 1997;9:1728–1738. - PubMed
1. Beaudet A. High resolution radioautography of central 5-hydroxytryptamine (5-HT) neurons. J Histochem Cytochem. 1982;9:765–768. - PubMed
1. Beckman ML, Quick MW. Neurotransmitter transporters: regulators of function and functional regulation. J Membr Biol. 1998;164:1–10. - PubMed
1. Blakely RD, Ramamoorthy S, Schroeter S, Qian Y, Apparsundaram S, Galli A, DeFelice LJ. Regulated phosphorylation and trafficking of antidepressant-sensitive serotonin transporter proteins. Biol Psychiatry. 1998;44:169–178. - PubMed

Ultrastructural localization of the serotonin transporter in limbic and motor compartments of the nucleus accumbens - PubMed (original) (raw)