Projections of nucleus accumbens adenosine A2A receptor neurons in the mouse brain and their implications in mediating sleep-wake regulation

Abstract

Adenosine A2A receptors (A2ARs) in the nucleus accumbens (Acb) have been demonstrated to play an important role in the arousal effect of adenosine receptor antagonist caffeine, and may be involved in physiological sleep. To better understand the functions of these receptors in sleep, projections of A2AR neurons were mapped utilizing adeno-associated virus (AAV) encoding humanized Renilla green fluorescent protein (hrGFP) as a tracer for long axonal pathways. The Cre-dependent AAV was injected into the core (AcbC) and shell (AcbSh) of the Acb in A2AR-Cre mice. Immunohistochemistry was then used to visualize hrGFP, highlighting the perikarya of the A2AR neurons in the injection sites, and their axons in projection regions. The data revealed that A2AR neurons exhibit medium-sized and either round or elliptic perikarya with their processes within the Acb. Moreover, the projections from the Acb distributed to nuclei in the forebrain, diencephalon, and brainstem. In the forebrain, A2AR neurons from all Acb sub-regions jointly projected to the ventral pallidum, the nucleus of the diagonal band, and the substantia innominata. Heavy projections from the AcbC and the ventral AcbSh, and weaker projections from the medial AcbSh, were observed in the lateral hypothalamus and lateral preoptic area. In the brainstem, the Acb projections were found in the ventral tegmental area, while AcbC and ventral AcbSh also projected to the median raphe nucleus, the dorsal raphe nucleus, and the ventrolateral periaqueductal gray. The results supply a solid base for understanding the roles of the A2AR and A2AR neurons in the Acb, especially in the regulation of sleep.

Keywords: Cre-Lox; adeno-associated virus; green fluorescent protein; mouse; nucleus accumbens; sleep.

FIGURE 1

Schematic diagram showing the AAV-lox-Stop-hrGFP injection sites in the nucleus accumbens. The dotted areas indicate the injection locations in the core (A–E), medial shell (F–H), and ventral shell (I–J). Each dark dot represents one labeled cell body. Abbreviations are as follows: ac, anterior commissural nucleus; AcbC: nucleus accumbens, core; AcbSh, nucleus accumbens, shell; mAcSh, medial AcbSh; vAcSh, ventral AcbSh; LV, lateral ventricle.

FIGURE 2

Images of hrGFP immunoreactivity (hrGFP-IRc) showing examples of the AAV-lox-Stop-hrGFP injection sites. (A,B) hrGFP-IRc in the Acb visualized by fluorescent and ABC techniques with Nissl-counterstaining. hrGFP-IRc staining, confirming the presence of the A_2AR neurons, was deposited around the Acb and covered most of the core area (arrows) in two labeled adjacent sections of this example (M-7). (C,D) hrGFP-IRc staining, indicating A_2AR neurons in the AcbSh, visualized by fluorescence and ABC techniques. Positive staining with hrGFP-IRc occupied the mAcbSh in (C,D) in two labeled adjacent sections of this case (M-11). (E,F) hrGFP-IRc staining, indicating A_2AR neurons in the vAcbSh, visualized by fluorescence and ABC techniques. Positive staining with hrGFP-IRc occupied vAcbSh in (E,F) in two labeled adjacent sections in this case (M-21). (G,H) A brain section from the wild type mouse with fluorescent staining (G) and ABC techniques with Nissl-counterstaining (H) showing no hrGFP-IRc in Acb. Abbreviations are as follows: AC, anterior commissural nucleus; LV, lateral ventricle; AcbC, nucleus accumbens core; AcbSh, nucleus accumbens shell; mAcbSH, medial AcbSh; vAcbSh, ventral AcbSh. Scale bar = 500 μm in (A); 100 μm in (B); 500 μm in (C); 100 μm in (D); 100 μm in (E); 100 μm in (F); 500 μm in (G); 500 μm in (H).

FIGURE 3

Images showing hrGFP-IR-positive neurons in the Acb of the adenosine A_2AR Cre mouse. (A,B) The hrGFP-IR neurons in the core visualized by fluorescent and ABC techniques. Most of the perikarya in the core were observed as multipolar (large arrows) and bipolar (arrowhead) neurons. The dendrites extending from the perikarya were thick, and some of them had punctuate spines (small arrows). The axons were thin, straight fibers (opened arrows). (C) hrGFP-IR neurons in the mAcbSh visualized by fluorescent and ABC techniques. The neurons in this region were similar in shape to those in the core. (D) The neurons in the vAcbSh visualized by ABC techniques. AC, anterior commissural nucleus; LV, lateral ventricle; mAcbSh, medial AcbSh; vAcbSh, ventral AcbSh. Scale bar = 20 μm in (A); 50 μm in (B); 10 μm in (C); 50 μm in (D).

FIGURE 4

Schematic drawings showing the distribution profiles of hrGFP-IR fibers and termini from the AcbC (A1–A12; M-7), medial AcbSh (B1–B8; M-11), and ventral AcbSh (C1–C11; M-21) injections. The hrGFP-IR axons and terminals are indicated by red ellipse dots. The projections from the AcbC and ventral AcbSh extend more caudally than those from medial AcbSh. All abbreviations are defined in the abbreviation section.

FIGURE 5

Fluorescent images showing hrGFP-IR fibers and termini from the AcbC (A,B; M-7), medial AcbSh (C,D; M-11), and ventral AcbSh (E,F; M-21) injections. Axons with varicosities invade the nucleus of the vertical limb of the diagonal band (VDB; A,C,E), the nucleus of the horizontal limb of the diagonal band (HDB; A,D), and the substantia innominate (SI; B,D,F). (A,B) hrGFP-IR axons and termini in the VDB, the HDB, and the SI following the injection of AAV-lox-Stop-hrGFP into the AcbC. (C,D) hrGFP-IR axons and termini in the VDB, the HDB, and the SI following the injection of AAV-lox-Stop-hrGFP into the medial AcbSh. (E,F) hrGFP-IR axons and termini in the VDB and the SI following the injection of AAV-lox-Stop-hrGFP into the ventral AcbSh. Arrowheads indicate varicosities. Scale bar = 100 μm.

FIGURE 6

Fluorescent images demonstrating hrGFP-IR axons and termini from the AcbC (A,B,C; M-7), the medial AcbSh (D,E,F; M-11), and the ventral AcbSh (G,H,I; M-21). Axons invade the anterior part of lateral hypothalamus (LHa), the tuberal part of lateral hypothalamus (LHt), and the mammillary part of lateral hypothalamus (LHm) after AcbC, medial AchSh, and ventral AcbSh injection, respectively. Arrowheads indicate varicosities. Scale bar = 100 μm.

FIGURE 7

Fluorescent images showing hrGFP-IR axons and termini from the AcbC (A–E; M-7) and ventral AcbSh (F–I; M-21) injections. (A–E) Axons with varicosities invade the ventral tuberomammillary nucleus [(A), VTM], the ventral tegmental area [(B), VTA], the medial raphe nucleus [(C), MnR], the dorsal raphe nucleus [(D), DR], and the ventrolateral periaqueductal gray [(E), VLPAG] after injection with AAV-lox-hrGFP into the AcbC (M-7). (F–I) Axons with varicosities invade the ventral tegmental area [(F), VTA], the medial raphe nucleus [(G), MnR], the dorsal raphe nucleus [(H), DR], the ventrolateral periaqueductal gray [(H), VLPAG], and the locus coeruleus [(I), LC] following the injection of AAV-lox-hrGFP into the mAcbSh (M-22). Arrowheads indicate varicosities. Abbreviations: 4V, forth ventricle; Aq, aqueduct; f, fornix. Scale bar = 100 μm.