Dopamine Development in the Mouse Orbital Prefrontal Cortex Is Protracted and Sensitive to Amphetamine in Adolescence

Abstract

The prefrontal cortex (PFC) is divided into subregions, including the medial and orbital prefrontal cortices. Dopamine connectivity in the medial PFC (mPFC) continues to be established throughout adolescence as the result of the continuous growth of axons that innervated the nucleus accumbens (NAcc) prior to adolescence. During this period, dopamine axons remain vulnerable to environmental influences, such as drugs used recreationally by humans. The developmental trajectory of the orbital prefrontal dopamine innervation remains almost completely unstudied. Nonetheless, the orbital PFC (oPFC) is critical for some of the most complex functions of the PFC and is disrupted by drugs of abuse, both in adolescent humans and rodents. Here, we use quantitative neuroanatomy, axon-initiated viral-vector recombination, and pharmacology in mice to determine the spatiotemporal development of the dopamine innervation to the oPFC and its vulnerability to amphetamine in adolescence. We find that dopamine innervation to the oPFC also continues to increase during adolescence and that this increase is due to the growth of new dopamine axons to this region. Furthermore, amphetamine in adolescence dramatically reduces the number of presynaptic sites on oPFC dopamine axons. In contrast, dopamine innervation to the piriform cortex is not protracted across adolescence and is not impacted by amphetamine exposure during adolescence, indicating that dopamine development during adolescence is a uniquely prefrontal phenomenon. This renders these fibers, and the PFC in general, particularly vulnerable to environmental risk factors during adolescence, such as recreational drug use.

Keywords: drug use; guidance cue; orbitofrontal cortex; piriform cortex.

Figure 1.

Dopamine varicosity density in the oPFC is protracted across adolescence. A, Timeline of experimental procedures; n = 4 per group. B, A micrograph of a coronal section through the frontal cortex of an adult mouse at low magnification (4×) showing the contour of the oPFC. Scale bar = 500 μm. C, A micrograph of a coronal section of the oPFC of an adult mouse at high magnification (60×) showing TH-immunopositive varicosities. Scale bar = 10 μm. D, The voPFC, loPFC, vaiPFC, and daiPFC respectively, are highlighted in increasingly pale shades of purple. Line drawings were derived from Paxinos and Franklin (2013). E, Stereological quantification of dopamine varicosity density reveals that there are more dopamine varicosities in the adult oPFC than the adolescent. Bars represent mean ± standard error (Extended Data >Fig. 1-1).

Figure 2.

Axons continue to grow to the oPFC during adolescence. A, The dual-viral injection method used to label NAcc-projecting ventral tegmental area neurons with eYFP. B, Timeline of experimental procedures: mice were injected at the start of adolescence (PND22 ± 1) and six weeks later, at which point adolescent mice have reached adulthood, eYFP-expressing dopamine axons in the oPFC were quantified; n = 5. C–E, Micrographs of a coronal section of the oPFC of an adult mouse at high magnification (60×) show (C) TH-immunopositive varicosities, (D) eYFP-expressing varicosities, and (E) an overlay highlighting co-labeled varicosities. Scale bar = 10 μm. F, The voPFC, loPFC, vaiPFC, and daiPFC, respectively, are highlighted in increasingly pale shades of purple. Line drawings were derived from Paxinos and Franklin (2013). G, Stereological quantification of dopamine varicosity density reveals eYFP-expressing dopamine varicosities are present in the oPFC in adult mice that received dual-viral injections in early adolescence (Extended Data Fig. 2-1). H, To ensure the eYFP-expressing dopamine neurons in the oPFC were the result of axon growth and not collaterals, we injected viruses into early adult mice and quantified eYFP-expressing dopamine axons six weeks later; n = 3. I, eYFP-expressing dopamine varicosities are almost entirely absent from the orbital prefrontal cortices of mice that were injected during adulthood (Extended Data Fig. 2-1). G & I, Bars represent mean ± standard error.

Figure 3.

Amphetamine in adolescence alters dopamine connectivity in adulthood. A, Timeline of experimental procedures; n = 4 per group. B, The loPFC, vaiPFC, and daiPFC, respectively, are highlighted in increasingly pale shades of red. Line drawings were derived from Paxinos and Franklin (2013). C, Stereological quantification of dopamine varicosity density reveals that adults exposed to amphetamine during adolescence have about a 40% reduction in dopamine varicosity density in the oPFC compared to saline-treated controls. Bars represent mean ± standard error (Extended Data Fig. 3-1).

Figure 4.

Dopamine connectivity in the piriform cortex is not protracted nor influenced by amphetamine in adolescence. A, The piriform cortex was outlined based on Paxinos and Franklin (2013) and is highlighted in green. B, C, Stereological quantification of dopamine varicosity density reveals that (B) early adolescent mice have a significantly higher density of dopamine varicosities in the piriform cortex compared to adults (Extended Data Fig. 4-1) and (C) that amphetamine exposure during adolescence does not alter the density of dopamine varicosities in the piriform cortex. Bars represent mean ± standard error (Extended Data Fig. 4-2).