Heterogeneous extracellular dopamine regulation in the subregions of the olfactory tubercle

Abstract

Recent studies show that dense dopamine (DA) innervation from the ventral tegmental area to the olfactory tubercle (OT) may play an important role in processing multisensory information pertaining to arousal and reward, yet little is known about DA regulation in the OT. This is mainly due to the anatomical limitations of conventional methods of determining DA dynamics in small heterogeneous OT subregions located in the ventral most part of the brain. Additionally, there is increasing awareness that anteromedial and anterolateral subregions of the OT have distinct functional roles in natural and psychostimulant drug reinforcement as well as in regulating other types of behavioral responses, such as aversion. Here, we compared extracellular DA regulation (release and clearance) in three subregions (anteromedial, anterolateral, and posterior) of the OT of urethane-anesthetized rats, using in vivo fast-scan cyclic voltammetry following electrical stimulation of ventral tegmental area dopaminergic cell bodies. The neurochemical, anatomical, and pharmacological evidence confirmed that the major electrically evoked catecholamine in the OT was DA across both its anteroposterior and mediolateral extent. While both D2 autoreceptors and DA transporters play important roles in regulating DA evoked in OT subregions, DA in the anterolateral OT was regulated less by the D2 receptors when compared to other OT subregions. Comparing previous data from other DA rich ventral striatum regions, the slow DA clearance across the OT subregions may lead to a high extracellular DA concentration and contribute towards volume transmission. These differences in DA regulation in the terminals of OT subregions and other limbic structures will help us understand the neural regulatory mechanisms of DA in the OT, which may elucidate its distinct functional contribution in the ventral striatum towards mediating aversion, reward and addiction processes.

Keywords: dopamine; dopamine D2 receptors; dopamine transporters; fast-scan cyclic voltammetry; olfactory tubercle; ventral tegmental area.

Figure 1.

Maps of electrically evoked catecholamine (CA) responses in the dorsal (CPu), ventral striatum (NAc), ventral pallidum (VP), and the amOT (a), alOT (b), and pOT (c) subregions as a function of the depth of the carbon-microelectrode. Panels (a)-(c) show coronal sections schematically (left, from Paxinos and Watson (Paxinos & Watson 2007)). The approximate path of the microelectrode (left, dotted line) was aimed at the different subregions of the OT (black: amOT, red: alOT, blue: pOT)(left). Relative response of catecholamine in the OT subregions are shown on the right. Mean relative response (I^x_d/I^max_d) of catecholamine in response to VTA electrical stimulation (60 Hz, 60 pulses) in the amOT (n=6), alOT (n=5), and pOT (n = 8) subregions at different depths of the microelectrode where I^x_d is the response at a particular depth divided by the maximal response, I^max_d of catecholamine. Panels (d)-(f) show representative CA concentration versus time traces at different depths of the microelectrode for each OT subregion before, during and after the electrical stimulation; insets show the distinct cyclic voltammogram for CA at the peak concentration values. Red bar denotes electrical stimulation of the VTA/SN interval (60 Hz, 60 pulses). Abbreviations used: CPu, caudate-putamen; NAc, nucleus accumbens; OT, olfactory tubercle; VP, ventral pallidum.

Figure 2.

Effect of selective NE and DA autoreceptor and transporter inhibitors on electrically evoked release and uptake in the subregions of the OT. The NE drugs, idazoxan (IDA, 3 mg/kg, dotted blue line), and IDA + desipramine (DMI, 15 mg/kg, solid read line) did not alter release or uptake in the representative examples of evoked release in all of the subregions of the OT (a). In contrast, a D2 receptor antagonist, raclopride (RAC, 2m/kg, dotted blue line) increased both [CA]_max and t_1/2 in the representative examples of evoked release in all of the OT subregions (b). Subsequent administration of a DAT inhibitor, GBR 12909 (GBR, 15 mg/kg, solid red line) potentiated both [CA]_max and t_1/2 in the subregions of the OT. Red bar indicates the duration of the electrical stimulation of the VTA (60 Hz, 60 p). Effect of the these agents on the average maximum evoked catecholamine concentration ([CA]_max) and half-decay time (t_1/2) in the OT subregions; the NE drugs idazoxan (IDA, 3 mg/kg, i.p.) and IDA + desipramine (DMI, 15 mg/kg, i.p.) had no significant effect on either the [CA]_max (c, left) or the t_1/2 (d, left). Raclopride (RAC, 2 mg/kg, i.p.) significantly increased evoked [CA]_max ((c, right), amOT t₄=7.61, alOT t₅=3.16, pOT t₇=8.94, *, all p < 0.0001) and half-decay time ((d, right), amOT t₄=4.54, alOT t₅=2.66, pOT t₆=4.37, all p < 0.05) from pre-drug control levels (dashed line). Subsequent administration of GBR 12909 (GBR, 15 mg/kg, i.p.) after RAC further increased maximum evoked release from baseline levels. Fisher post-hoc tests revealed that the [CA]_max after RAC was greater in the amOT than in the alOT (#, p < 0.05), and that the effect of RAC+GBR on maximum evoked release was greater than RAC alone for all OT subregions (†, p < 0.05). GBR after RAC also increased t_1/2 from baseline levels (two-way repeated measures ANOVA, main effect of drug F_1,15=41.9, p < 0.0001), although this effect was not statistically different across OT subregions.

Figure 3.

Combined inhibition of DA uptake and D2 antagonism induces spontaneous DA transients in all subregions of the OT. Color plot representations of background-subtracted cyclic voltammograms collected over 30s before (left) and after (right) raclopride (RAC, 2 mg/kg, i.p.) and GBR 12909 (GBR, 15 mg/kg, i.p.) administration. DA concentration changes were seen in the color plot at the potential for DA oxidation (~ 0.6 – 0.7 V, dashed white line). The time courses of the DA concentration changes are shown below each color plot. Cyclic voltammograms are shown as insets recorded at the time indicated by the asterisk (*).

Figure 4.

Comparison of electrically evoked DA regulation in subregions of the OT. Representative examples of the change in DA concentration in the OT (amOT (a), alOT (b), pOT (c)) after stimulation (60 Hz, 60 pulses) of the VTA. Upper panels are color plots for the voltammetric data shown for each example, comprised of all background-subtracted cyclic voltammograms recorded for 5 s before and 10 s after stimulation, with current changes encoded in false color. Changes in DA concentration occurred at the potential for its oxidation (∼+0.6 - +0.7 V, dotted line) and reduction (∼ −0.3 - −0.2 V, solid line). Lower plots show changes in DA concentration before, during and after stimulation, shown at the potential where DA is oxidized. Background-subtracted cyclic voltammograms are shown as insets at the maximum of evoked release. The red bars indicate the period of electrical stimulation.

Figure 5.

Comparison of representative DA responses in the amOT, alOT and pOT subregions as a function of frequency (a) and pulse number (b). Representative evoked DA concentration traces at 5 Hz and 10 pulses are shown as insets, in (a) and (b), respectively. Maximal mean DA responses in the subregions of the OT as a function of frequency (c) and pulse number (d) as shown as relative response (monitored DA response (I^x)/maximal response (I^max)). Significantly different response between the alOT and amOT (*), amOT and pOT (†), alOT and pOT (‡) subregions (p < 0.05).