CNS dopamine transmission mediated by noradrenergic innervation

Abstract

The presynaptic source of dopamine in the CA1 field of dorsal hippocampus is uncertain due to an anatomical mismatch between dopaminergic terminals and receptors. We show, in an in vitro slice preparation from C57BL/6 male mice, that a dopamine (DA) D1 receptor (D1R)-mediated enhancement in glutamate synaptic transmission occurs following release of endogenous DA with amphetamine exposure. It is assumed DA is released from terminals innervating from the ventral tegmental area (VTA) even though DA transporter (DAT)-positive fibers are absent in hippocampus, a region with abundant D1Rs. It has been suggested this results from a lack of DAT expression on VTA terminals rather than a lack of these terminals per se. Neither a knockdown of tyrosine hydroxylase (TH) expression in the VTA by THsiRNA, delivered locally, by adeno-associated viral vector, nor localized pharmacological blockade of DAT to prevent amphetamine uptake into DA terminals, has any effect on the D1R synaptic, enhancement response to amphetamine. However, either a decrease in TH expression in the locus ceruleus (LC) or a blockade of the norepinephrine (NE) transporter prevents the DA-mediated response, indicating LC terminals can release both NE and DA. These findings suggest noradrenergic fibers may be the primary source of DA release in hippocampus and corresponding DA-mediated increase in synaptic transmission. Accordingly, these data imply the LC may have a role in DA transmission in the CNS in response to drugs of abuse, and potentially, under physiological conditions.

Figure 1.

Pharmacological and endogenous activation of D1Rs increases glutamate transmission. A, Plot and representative traces show application of SKF increases glutamate transmission in extracellular fEPSP recordings, which is blocked by SCH23390 (SCH) treatment (SKF, 159 ± 2%; n = 10; SKF + SCH, 104 ± 2%; n = 6; p < 0.0001). All voltage traces presented in the right column are 10 sweep averages baseline (red) and post-treatment (black) of fEPSP evoked responses (50 ms scale bar) and their associated downward slopes (20 ms scale bar). B, Plot and representative traces show application of AMPH increases glutamate transmission in extracellular fEPSP recordings which is blocked by SCH treatment (AMPH, 132 ± 4%; n = 8; AMPH + SCH, 110 ± 5%; n = 7; p < 0.003). C, Plot and representative traces show AMPH application increases the total charge transfer in whole-cell voltage-clamp experiments at −70 mV (n = 10). The second excursion is in response to a 5 mV, 20 ms voltage step (to test input impedence). D, Plot and representative traces show prazosin and propanolol (included in all in vitro recordings) prevent a NE-mediated alteration in glutamate transmission, indicating NE does not activate D1Rs (103 ± 6% of baseline; n = 3). Error bars indicate SEM.

Figure 2.

TH-positive staining is observed throughout the CA1 region of hippocampus while DAT-positive staining is sparse. Aa, Ab, Ac, Confocal images acquired using a 20× objective show dense TH fiber staining throughout the CA1 region of hippocampus (SP, stratum pyramidale; SO, stratum oriens; SR, stratum radiatum; SLM, stratum lacunosum moleculare), overlying parietal cortex (PCtx) and alveous (Alv) with DAT staining observed only in PCtx and Alv. Ad, Image shows TH and DAT staining in the PFC region. Bars represent 0.5 mm (20× magnification). B, Left, Plot shows total DAT-positive fiber counts across AP coordinates −1.58 to −2.98 from CA1, CA3, dentate, and subiculum (SUB) with the most dense staining observed in SO region of CA1 (SO, 9 ± 2; SR, 1.7 ± 1.2; SLM, 2.3 ± 1.9; Dentate, 4.7 ± 2.2; CA3, 1.3 ± 0.7; SUB, 3.3 ± 0.9; n = 3 mice). B, Right, Coronal sections from AP −1.58 to −2.98. C, Total TH and DAT-positive fiber counts at each AP coordinate from −1.58 to −2.98 from the SO region of CA1 show TH fibers surpass DAT fiber density by ∼100× (TH, 127 ± 4 fibers; DAT, 1 ± 0.3 fibers; p < 0.0001; n = 3 mice). Error bars indicate SEM. D, Confocal images of CA1 show MAP2 (blue), DAT (green), and dopamine D5 receptors (D5Rs; red) at 20×, 63×, and 63× × 150% (digital) magnification. Bars represent 0.5 mm (20× magnification) and 0.15 mm (63×). Images indicate D5R staining is observed throughout the CA1 region and is localized to sites adjacent to CA1 pyramidal cell dendrites such as postsynaptic spines while DAT staining is isolated to the overlying cortical region.

Figure 3.

Loss of TH in the LC is sufficient to prevent the D1-mediated increase in glutamate transmission with AMPH stimulation. A, Left, Top, Confocal images from an AAV-THsiRNA and an AAV-SCR-injected mouse acquired at 5× represent mean fluorescence from each group. TH staining is observed in the LC of AAV-SCR-injected mice but is diminished in mice injected with AAV-THsiRNA. Bubble in left panel removed in Photoshop. Left, Middle, GFP staining shows the precise targeting of virus to the LC. Left, Bottom, TH staining is observed in the VTA region from mice injected with either AAV-THsiRNA or AAV-SCR in the LC. Bubble in right panel removed in Photoshop. Scale bar, 0.5 mm. Right, Coronal drawing depicts bilateral location of LC and surgical targeting adapted from Reference Mouse Brain Atlas: Allen Institute for Brain Sciences. B, Plot shows AMPH increases glutamate transmission in slices from AAV-SCR-injected but not AAV-THsiRNA-injected mice (THsiRNA, 100 ± 4%; n = 10; SCR, 120 ± 8%; n = 5; p < 0.02). C, Plot shows SKF treatment increases glutamate transmission in slices from AAV-THsiRNA-injected mice (AAV-THsiRNA SKF, 126 ± 8%; n = 5; AMPH vs SKF, p < 0.02). AMPH experiments are replotted for comparison. Error bars indicate SEM.

Figure 4.

Loss of TH in VTA does not prevent the D1-mediated increase in glutamate transmission with AMPH stimulation. A, Left, Confocal images from an AAV-THsiRNA and an AAV-SCR-injected mouse acquired at 5× represent mean fluorescence from each group. TH staining is observed in the VTA of AAV-SCR-injected mice but is virtually absent in mice injected with AAV-THsiRNA. Scale bar, 0.5 mm. Right, Coronal drawing depicts bilateral location of VTA and surgical targeting adapted from Reference Mouse Brain Atlas: Allen Institute for Brain Sciences. B, Plot shows AMPH increases glutamate transmission in slices from both AAV-THsiRNA and AAV-SCR-injected mice (THsiRNA, 119 ± 4%; n = 4; SCR, 120 ± 8%; n = 6; p > 0.05). C, Plot shows SKF increases glutamate transmission in slices from AAV-THsiRNA-injected mice (AAV-THsiRNA SKF, 135 ± 1%; p > 0.05; n = 4). AMPH is replotted for comparison. Error bars indicate SEM.

Figure 5.

Activation of NET but not DAT is required for the AMPH-induced increase in glutamate transmission. A, Plot shows application of the NET inhibitor nisoxetine prevents increase in glutamate transmission with AMPH (AMPH, 139 ± 6%; n = 6; AMPH + Nisoxetine, 103 ± 5%; n = 8; p < 0.003). B, Plot shows application of SKF increases glutamate transmission in the presence of nisoxetine even though prior application of AMPH failed to increase transmission (AMPH + Nisox, 104 ± 2%; SKF, 122 ± 6%; n = 6; p < 0.04). C, Plot shows application of the DAT inhibitor GBR12935 does not prevent the AMPH-mediated increase in glutamate transmission (GBR12935, 128 ± 6%; n = 6; AMPH vs AMPH + GBR p > 0.05). Error bars indicate SEM.

Figure 6.

Under normal conditions, DA is converted into NE via DβH in vesicles of noradrenergic terminals. Following action potential stimulation substrate is released. DA and NE are transported back into the presynaptic terminal via NET and once intracellular into vesicles by VMAT2. In the presence of AMPH (AMPH is transported intracellularly into LC terminals by NET) VMAT2 function is prevented and MAO is blocked. The former action prevents concentration of DA in vesicles where DβH is located, preventing formation of NE and increasing intracellular concentrations of DA. Inhibition of MAO prevents the breakdown of DA to DOPAC, which further increases the intracellular concentration of DA. The increased cytosolic concentration of DA may result in reverse transport of DA to the extracellular media via NET down its concentration gradient where it interacts with D1Rs.