A unique multi-synaptic mechanism involving acetylcholine and GABA regulates dopamine release in the nucleus accumbens through early adolescence in male rats
- Department of Translational Neuroscience, Wake Forest University School of Medicine, United States
- Department of Physiology, University of Kentucky College of Medicine, United States
Figures
Figure 1
Early adolescent male rats have decreased stimulated dopamine release in the nucleus accumbens core.
(A) Ex vivo fast scan cyclic voltammetry was used to compare the maximum concentration of evoked dopamine release in the nucleus accumbens core of adult (>P70) and early adolescent (P28-35) male rats. Representative traces of single-pulse dopamine release in the nucleus accumbens core (NAc core) are shown. (B) Maximum concentration of evoked dopamine release was significantly lower in adolescent than adult rats in the NAc core at single-pulse stimulations and (C) across a range of stimulation parameters that model tonic and phasic firing of dopamine neurons (adults: n=7; adolescents: n=10). (D) Dopamine uptake rates can impact stimulated dopamine release levels (Ferris et al., 2013), so maximal rate of dopamine uptake (Vmax) was examined in the NAc core. However, Vmax did not differ between adult and adolescent rats (adults: n=9; adolescents: n=11). (E) Differences in dopamine release probability can also impact the stimulated concentration of dopamine release (Ferris et al., 2013), so we also used paired-pulse ratios to examine differences in release probability. Paired-pulse ratios did not differ between adult and adolescent rats (adults: n=9; adolescents: n=12). In line graphs, symbols represent means ± SEMs. In bar graphs, bars represent means and symbols represent individual data points. Individual data points (n) indicate the number of rats. *p<0.05.
Figure 2
Nicotinic acetylcholine receptors (nAChRs), and in particular α6-containing nAChRs, differentially regulate dopamine release in the nucleus accumbens core of adolescent male rats.
(A) Acetylcholine, signaling through nAChRs located on dopamine terminals, is an important regulator of local dopamine release (Rice and Cragg, 2004). There are three main types of nAChRs found in the nucleus accumbens core: α7, α6β2-containing, and α4β2-containing. A schematic shows the localization of the sub- types of nAChRs in the nucleus accumbens core and lists the action of drugs bath applied to brain slices to examine nAChR mediation of dopamine release. We used antagonists specific to various sub-types of nAChRs to examine which nAChRs were playing a role in mediating the age-related difference in nAChR modulation of dopamine release. (B) MLA, a selective α7 nAChR antagonist, decreased the maximum concentration of dopamine release, but did not differentially affect the concentration of dopamine release in adult and adolescent rats (adults: n=8; adolescents: n=7). Post-MLA dopamine release is normalized as a percentage of single pulse, pre-drug dopamine release in adults and adolescents, respectively. (C) In contrast, α-Ctx, a selective α6-containing nAChR antagonist, facilitated evoked dopamine release in adolescents, but decreased the concentration of dopamine release in adult rats (adults: n=8; adolescents: n=12). Post-α-Ctx dopamine release is normalized as the percentage of single pulse, pre-drug dopamine release in adults and adolescents, respectively. (D) Further antagonism of non-α6 β2-containing nAChRs, using DHβE, additionally decreased evoked dopamine release, but not in an age-specific manner (adults: n=8; adolescents: n=12). Adolescent + α-Ctx data is repeated from panel C. Data is presented as a percentage of single pulse dopamine release at baseline for each group to assess the combined drug effect. (E) Similarly, analyzing DHβE dopamine release as a percent change from the α-Ctx baseline confirmed no age-related differences. (F) Given the differential effect of antagonizing α6-containing nAChRs on adolescent and adult dopamine release, we next examined whether α-Ctx changed dopamine dynamics in an age-specific manner. Maximal rate of dopamine uptake (Vmax) was not differentially altered by α-Ctx in adult and adolescent rats (adults: n=8; adolescents: n=11). (G) Release probability of dopamine, measured by paired-pulse ratio, was increased by α-Ctx, but not in an age-specific manner (adults: n=8; adolescents: n=12). (H) Representative traces show dopamine release at single pulse and 5 pulse 20 Hz stimulations in adult (black) and adolescent (red, green) rats. Colors and lines correspond to each drug condition. Symbols represent means ± SEMs. Individual data points (n) indicate the number of rats. *p<0.05.
Figure 3
Dopamine release and α6-containing nAChR modulation of dopamine release do not differ between mid-adolescence and adulthood in male rats.
(A) Maximum concentration of stimulated dopamine release in the nucleus accumbens core did not differ between adult (>P70) and mid- adolescent (P39-42) male rats. (B) α-Ctx, a selective α6-containing nAChR antagonist, decreased the evoked concentration of dopamine release to a similar degree in adult and mid-adolescent rats (adults: n=7; mid- adolescents: n=4 for A and B). Data are shown as a percentage of pre-α-Ctx, single pulse dopamine release in adults and adolescents, respectively. (C) Representative traces show dopamine release at single pulse and 5 pulse 20 Hz stimulations in adult (black) and mid-adolescent (gray) rats with colors and lines corresponding to each drug condition. Symbols represent means ± SEMs. Individual data points (n) indicate the number of rats.
Figure 4
Differential modulation of dopamine release by α6-containing nAChRs in adolescent male rats is mediated through GABA receptor signaling.
(A) A schematic shows localization of nAChRs and GABA receptors in the NAc core. (B) Bath application of bicuculline (BIC; a selective GABAA receptor antagonist) and subsequent application of CGP-52432 (CGP; a selective GABAB receptor antagonist) had no effect on stimulated dopamine release (adults: n=6–8; adolescents: n=5–7). Data are shown as a percentage of pre-drug dopamine release to within each stimulation type (i.e. 20 Hz BIC relative to 20 Hz pre-BIC baseline). (C) Similarly, analyzing BIC and CGP-52432 application to the pre-drug condition confirmed no effect on stimulated dopamine release (adults: n=6–8; adolescents: n=5–7) (D) Prior application of BIC and GCP combined completely blocks the faciliatory effect of α-Ctx on evoked dopamine release in adolescent rats. Effects of each drug on dopamine release are analyzed as a percentage of the pre-drug baseline from their respective stimulation type (e.g. 20 Hz α-Ctx is relative to 20 Hz pre-α-Ctx baseline). Antagonism of GABA receptors did not impact the effect of α-Ctx on dopamine release in adult rats (adults: n=6–8; adolescents: n=5–12). (E) Prior application of BIC and GCP following BIC application is not significantly different than the application of both drugs normalized to the pre-drug condition in 4D, indicating both drugs are responsible for blocking α-Ctx effects. (F) Prior application of GABA did not impact the effects of α-Ctx in adults, but blocked the α-Ctx-induced increase in adolescent dopamine release (adults: n=6–8; adolescents: n=6–12). Adolescent + α-Ctx data as a percent baseline is repeated from panel D. (G) Isolating the effects of GABA on slice prior to α-Ctx application revealed a significant decrease in adolescent dopamine release and an increase in adult dopamine release (adults: 6; adolescents: 5). (H) To further support these findings, we then evaluated the effect of infusing α-Ctx into the NAc on GABA neurotransmission using in vivo microdialysis. Adolescents have higher GABA tone as compared to adults (adults: n=8; adolescents: n=8) as indicated by Age X Drug Interaction and post-hoc multiple comparisons. *p<0.05; Sidak posthoc multiple comparisons test. In line graphs, symbols represent means ± SEMs. In bar graphs, bars represent means and symbols represent individual data points. Individual data points (n) indicate the number of rats. *p<0.05, ****p<0.0001.
Figure 5
Differential effect of α6-containing nAChRs on GABA and dopamine release in the striatum of adult versus adolescent male rats.
A schematic shows the relationship between GABA interneurons, cholinergic interneurons (CINs), and dopamine (DA) varicosities in adult (left column) and adolescent (right column) rats before (top row) and after (bottom row) application of α-Ctx. The green circles represents the primary mechanism of dopamine modulation for each panel. The present study demonstrated that adolescents have higher GABA tone than adults (top right vs left panels) suggesting that dopamine release is reduced in adolescent rats via GABA-mediated inhibition of DA varicosities. Applying α-Ctx on slice (bottom panels) blocks α6-containing nAChRs directly on DA varicosities in adult rats (bottom left, green circles), thus preventing direct facilitation by acetylcholine from CINs. In adolescents (bottom right), the blockade of α6-containing nAChRs reduces GABA tone (green circles) and subsequently disinhibits DA varicosities, which masks the direct effect of acetylcholine on DA varicosities observed in adults. This masked mechanism is revealed in adolescent rats when the influence of α6-mediated decreases in GABA is removed via (1) blocking GABA receptors or (2) rescuing GABA levels with exogenously applied GABA.
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A unique multi-synaptic mechanism involving acetylcholine and GABA regulates dopamine release in the nucleus accumbens through early adolescence in male rats
eLife 13:e62999.
https://doi.org/10.7554/eLife.62999
