Distinct limbic dopamine regulation across olfactory-tubercle subregions through integration of in vivo fast-scan cyclic voltammetry and optogenetics

Abstract

The olfactory tubercle (OT), an important component of the ventral striatum and limbic system, is involved in multi-sensory integration of reward-related information in the brain. However, its functional roles are often overshadowed by the neighboring nucleus accumbens. Increasing evidence has highlighted that dense dopamine (DA) innervation of the OT from the ventral tegmental area (VTA) is implicated in encoding reward, natural reinforcers, and motivated behaviors. Recent studies have further suggested that OT subregions may have distinct roles in these processes due to their heterogeneous DA transmission. Currently, very little is known about regulation (release and clearance) of extracellular DA across OT subregions due to its limited anatomical accessibility and proximity to other DA-rich brain regions, making it difficult to isolate VTA-DA signaling in the OT with conventional methods. Herein, we characterized heterogeneous VTA-DA regulation in the medial (m) and lateral (l) OT in "wild-type," urethane-anesthetized rats by integrating in vivo fast-scan cyclic voltammetry with cell-type specific optogenetics to stimulate VTA-DA neurons. Channelrhodopsin-2 was selectively expressed in the VTA-DA neurons of wild-type rats and optical stimulating parameters were optimized to determine VTA-DA transmission across the OT. Our anatomical, neurochemical, and pharmacological results show that VTA-DA regulation in the mOT is less dependent on DA transporters and has greater DA transmission than the lOT. These findings establish the OT as a unique, compartmentalized structure and will aid in future behavioral characterization of the roles of VTA-DA signaling in the OT subregions in reward, drug addiction, and encoding behavioral outputs necessary for survival.

Keywords: dopamine; fast-scan cyclic voltammetry; olfactory tubercle; optogenetics; ventral tegmental area.

Figure 1.

Experimental timeline. Rats underwent ketamine/xylazine anesthesia and stereotaxic surgery was performed to infuse a virus encoding for Channelrhodopsin-2 (ChR2) into the ventral tegmental area (VTA)-dopamine (DA) neurons. 4 weeks after viral infusion surgery, rats were anesthetized with urethane and FSCV coupled with optogenetic stimulation of VTA-DA neurons was performed to study VTA-DA regulation in the olfactory tubercle (OT) subregions. After anesthetized fast-scan cyclic voltammetry (FSCV) experiments, rats were transcardially perfused and immunofluorescence was performed to verify virus expression. mOT: Medial Olfactory Tubercle; lOT: Lateral Olfactory Tubercle; TH: Tyrosine Hydroxylase.

Figure 2

Co-expression of tyrosine hydroxylase (TH) and enhanced yellow fluorescent protein (EYFP) in the ventral tegmental area (VTA) and olfactory tubercle (OT). (A) Low magnification (10x) and (B) high magnification (63x) confocal micrographs of the VTA showing TH (red), Channelrhodposin-2-enhanced yellow fluorescent protein (ChR2-EYFP, green), and TH + ChR2-EYFP (orange) expression. White box in (A) indicates area enlarged in (B). (C) TH and EYFP expression throughout the striatum following viral infusion of TH-ChR2-EYFP into the VTA. (D) High magnification of the OT terminals as indicated by the white box in (C).

Figure 3

Maps of optically evoked ventral tegmental area (VTA)-dopamine (DA) across the striatum. A schematic diagram shows the coronal sections (Paxinos & Watson 2006) (left) and relative DA concentrations (right) in the medial (m, A) and lateral (l, B) olfactory tubercle (OT) subregions as a function of depth of the neurochemical sensor. The relative concentration is the concentration at a particular depth (C_d^x) divided by the maximum response (C_d^max). The approximate path of the neurochemical sensor is indicated by the dotted line. Representative concentration vs time traces as the neurochemical sensor traverses through the brain in the medial (C) and lateral (D) coordinates. Inset above 6 mm trace (D) from the skull to visualize VTA-DA release in the Caudate Putamen (CPu) with a smaller concentration scale. Insets (bottom): Cyclic voltammograms identifying the analyte as catecholamine in origin. Blue line indicates period of optical stimulation (40 Hz, 6 ms, 60 p). CPu: Caudate Putamen; NAcS: Nucleus Accumbens Shell; NAcC: Nucleus Accumbens Core; mOT: Medial Olfactory Tubercle; lOT: Lateral Olfactory Tubercle. “n” indicates the number of rats used: mOT (left) n = 8 rats; lOT (right) n = 5 rats.

Figure 4

Maps of maximal stimulated dopamine (DA) release ([DA]_max) in the medial olfactory tubercle (mOT) and lateral olfactory tubercle (lOT): (A) Schematic diagram from AP −5.2 mm (Paxinos & Watson 2006) showing the ventral tegmental area (VTA) and approximate track of the optical fiber (dotted line) as in passes through the VTA (left). Maximum relative optically stimulated DA release (40 Hz, 60 p, 6 ms) measured in the mOT and lOT as the fiber was lowered in small increments to and through the VTA (right). The relative concentration is the concentration at a particular depth (C_d^x) divided by the maximum response (C_d^max). (B) Representative individual concentration vs time traces in the mOT (left) and lOT (right) measured at the optical fiber depth in the VTA. Blue line indicates period of optical stimulation (40 Hz, 6 ms, 60 p). “n” indicates the number of rats used.

Figure 5

Comparison of the effects of different optical stimulation conditions on dopamine (DA) release. Representative traces showing different effects of (A) pulse width (at 40 Hz, 60 p), (B) pulse number (at 40 Hz, 6 ms), and (C) frequency (at 60 p, 6 ms) in the medial olfactory tubercle (mOT, left) and lateral olfactory tubercle (lOT, middle). Maximum relative optically stimulated DA release as a function of parameter (right). The relative concentration is the concentration at a particular depth (C_d^x) divided by the maximum response (C_d^max).*p < 0.05, ****p < 0.0001. “n” indicates the number of rats used.

Figure 6

Effects of selective dopamine (DA) D2 autoreceptor and transporter (DAT) inhibitors on optically evoked DA release and uptake in the medial olfactory tubercle (mOT) and lateral olfactory tubercle (lOT): (A) Representative concentration vs time traces for optically evoked (40 Hz, 60 p, 6 ms) DA in the mOT (left) and lOT (right) following systemic administration of the D2 antagonist, raclopride (RA, 2 mg/kg IP) and the DAT inhibitor, GBR 12909 (GBR, 15 mg/kg IP). The blue line under the concentration vs time trace indicates the period of optical stimulation (40 Hz, 6 ms, 60 p). (B) Effect of selective DA drugs on average maximum optically evoked DA concentration ([DA]_max, left) and half-decay time (t_1/2, right) in the mOT and lOT. **p < 0.01, ****p < 0.0001.

Figure 7

Effects of combined systemic administration of the dopamine (DA) D2 antagonist, raclopride (RA) and DAT inhibitor, GBR 12909 (GBR) on naturally occurring DA transient release in the medial olfactory tubercle (mOT, top) and lateral olfactory tubercle (lOT, bottom). Color plots show background‐subtracted cyclic voltammograms collected over 30 s before (left) and after (right) RA (2 mg/kg) and GBR (15 mg/kg) administration, respectively. DA concentration changes were seen in the color plot at the potential (E_app) for DA oxidation (~ 0.65V, dotted white line). The time courses of the DA concentration changes at ~0.65 V are shown below each color plot. Cyclic voltammograms are shown as insets recorded at the time indicated by the inverted red triangles.