Nicotine Acts on Cholinergic Signaling Mechanisms to Directly Modulate Choroid Plexus Function

Nicotine activates calcium signaling in choroid plexus epithelial cells in vitro. A, Time course of Ca²⁺-dependent fluorescence signal recorded from responding (red) and nonresponding (black) cells following application of 30 μM nicotine. Traces represent average intensity from nine responsive (red) and 40 nonresponsive (black) primary choroid plexus epithelial cells in vitro. Primary choroid plexus obtained from n = 6 rats (five cell culture plates analyzed, resulting in 49 cells quantified). B, Representative images of a responsive cell (center) and nonresponsive cells (bottom right) captured at the time points 1–4 as indicated in A. Increasing cytosolic free Ca²⁺ is represented by warmer colors (as depicted with the color bar) and increasing height of each pixel. Scale bar = 20 μm. C, Average fluorescence signals measured at the peak of each recording obtained during 11 separated trials; ****p < 0.0001. Data represent mean ± SEM. Central tendency (mean) and variation (SEM) values for each are as follows: nonresponsive, –0.054 ± 0.008 and responsive, 0.556 ± 0.167.

nAChR subunit expression in choroid plexus derived from the lateral, fourth, and d3Vs. Choroid plexus was discretely dissected from the ventricles of the brain. A, The outer layer of cortex and hippocampus were gently separated from the midline to allow for visualization and removal of the d3V choroid plexus with visualization via dissection microscope (left image with white arrow), and following removal, the choroid plexus was clearly visualized as completely separate from brain tissue (right image with black arrow). Similar dissections were conducted with microscopic visualization and verification for the lateral and fourth ventricle locations. B–D, The expression of nAChR subunits was examined in choroid plexus tissue from rats (n = 8–15/group, specific number per group denoted on each bar). B, In the lateral ventricle (LV), mRNA expression was found for the α4 (Chrna4), α5 (Chrna5), β2 (Chrnb2), β3 (Chrnb3), and β4 (Chrnb4) nAChR subunits. Putative nAChR subtype schematics are graphically illustrated (insert). Statistical data are as follows [central tendency (mean) ± variation (SEM), lower 95% confidence interval, upper 95% confidence interval]: α4: 3.35 ± 0.34, 2.61, 4.09; α5: 1.44 ± 0.29, 0.77, 2.10; β2: 2.37 ± 0.28, 1.77, 2.97; β3: 0.32 ± 0.32, 0, 1.03; and β4: 1.50 ± 0.42, 0.56, 2.45. C, In the fourth ventricle (4V), only α4 (Chrna4) and β2 (Chrnb2) mRNAs were detected, and thus, the α4β2 nAChR subtype may be present (schematic insert). Statistical data are (mean ± SEM, lower CI, upper CI): α4: 8.93 ± 1.18, 6.38, 11.47 and β2: 2.77 ± 0.38, 1.93, 3.61. D, In the d3V, subunit expression consisted of α4 (Chrna4), α7 (Chrna7), β2 (Chrnb2), and β3 (Chrnb3), which allows for four different putative nAChR subtypes (schematic insert). Statistical data are (mean ± SEM, lower CI, upper CI): α4: 5.86 ± 0.53, 4.71, 7.01; α7: 2.12 ± 0.67, 0.61, 3.62; β2: 5.16 ± 1.19, 2.53, 7.79; and β: 21.12 ± 8.25, 2.09, 40.14. For each nAChR gene above, expression data were normalized to expression of β-actin as the endogenous control. nd = not detected based on the predetermined RT-qPCR criteria (Ct value > 35). Data represent mean normalized values ± SEM.

Differential expression of α4 and β2 nAChR subunits across choroid plexus sites. A, B, Given that α4 and β2 subunits were found in all choroid plexus sites, their relative expression was compared under baseline conditions (n = 12–15/group, specific number per group denoted on each bar). A, For the α4 nAChR subunit, significantly increased expression was found in choroid plexus from the fourth ventricle compared to tissue from the lateral and d3V. Post hoc corrected for multiple comparisons; *p < 0.05, ***p < 0.001. Central tendency (mean) and variation (SEM) values for each are as follows: LV: 3.350 ± 0.34; 4V: 8.925 ± 1.18; and d3V: 5.861 ± 0.53. For each nAChR gene, expression data were normalized to expression of β-actin. B, With regard to the β2 nAChR subunit, significantly increased expression was found in the d3V choroid plexus as compared to the lateral ventricle (LV). Post hoc corrected for multiple comparisons; *p < 0.05. Central tendency (mean) and variation (SEM) values for each are as follows: LV: 2.371 ± 0.27; 4V: 2.773 ± 0.38; and d3V: 5.156 ± 1.19). 4V: fourth ventricle. For each nAChR gene, expression data were normalized to expression of β-actin. Data represent mean normalized values ± SEM. C–H, Expression of α4 and β2 nAChR subunits with nicotine or saline self-administration (n = 8/group as denoted on each bar). Following saline or nicotine self-administration in rats, normalized expression levels of α4 and β2 nAChR subunits were compared. For the α4 nAChR subunit (left panels), similar mRNA expression levels were found with saline and nicotine self-administration in choroid plexus from the (C) LV, (E) 4V, and (G) d3V. For the β2 nAChR subunit (right panels), differences were not found in mRNA expression in the (D) LV or (H) d3V, whereas a significant decrease with nicotine was found in the (F) 4V choroid plexus; *p < 0.05. Central tendency (mean) and variation (SEM) values (C–H) are as follows: α4 LV - Saline: 3.758 ± 0.46; Nicotine: 4.600 ± 0.60; α4 4V - Saline: 10.60 ± 1.60; Nicotine: 11.74 ± 1.79; α4 d3V - Saline: 5.915 ± 0.87; Nicotine: 7.015 ± 1.28; β2 LV - Saline: 2.978 ± 0.19; Nicotine: 3.310 ± 0.41; β2 4V - Saline: 2.953 ± 0.37; Nicotine: 1.762 ± 0.30; β2 d3V - Saline: 5.319 ± 0.66; and Nicotine: 4.732 ± 1.31. For each nAChR gene, expression data were normalized to expression of β-actin. Data represent mean normalized values ± SEM.

Intravenous nicotine self-administration in rats. Rats were trained in the intravenous nicotine self-administration protocol; access to the acquisition dose of 0.03 mg kg⁻¹ per infusion was provided for 7 d, followed by 0.12 mg kg⁻¹ per infusion on the eighth session. A, The number of nicotine infusions across sessions during nicotine self-administration corresponds to tissue analyzed for studies shown in Figures 4, 6, 7 (n = 33). B, On the final session, the subjects shown in Figure 5A self-administered a mean of 1.1 mg/kg nicotine (±0.09 SEM). C, For the data presented in Figure 6C, groups were tested across doses as described above but were administered either vehicle or mecamylamine before the self-administration session on session 8 (n = 5–6/group). D, For session 8, a significant decrease in the amount of nicotine consumed was found in the mecamylamine group compared to the vehicle group; *p < 0.05. The mean nicotine intake (mg/kg) ± SEM for each group was: Vehicle: 1.248 ± 0.16 and Mecamylamine: 0.62 ± 0.122. Specific subject numbers per group are denoted on the bar graphs. Data represent mean values ± SEM.

Transthyretin expression in the choroid plexus with saline or nicotine self-administration. Expression of choroid plexus specific transthyretin mRNA was examined in rats self-administering saline or nicotine (n = 5–12/group, specific number per group denoted on each bar). A, In the lateral ventricle (LV), differences were not found between groups in transthyretin expression. B, In the fourth ventricle (4V), no differences were found in transthyretin expression. C, In the dorsal third ventricle (d3V), significant upregulation of transthyretin was found with nicotine self-administration, an effect that was reversed with pretreatment of mecamylamine (MEC) before nicotine self-administration; ***p < 0.001. The photomicrograph on the right displays the localization of the choroid plexus (CP) in the d3V, which is in proximity to the habenula (Hb). Central tendency (mean) and variation (SEM) values for each are as follows: LV - Saline: 145.9 ± 32.23; Nicotine: 152.9 ± 36.57; 4V - Saline: 372.8 ± 57.27; Nicotine: 357.3 ± 73.58; d3V - Saline: 214.5 ± 25.81; Nicotine: 455.0 ± 61.43; and Mecamylamine/Nicotine: 214.3 ± 29.21. For all of the above, expression data were normalized to expression of β-actin. Data represent mean normalized values ± SEM.

Nicotine mediated changes in expression of mir-204 in the choroid plexus. The expression of mir-204 was examined following saline or nicotine self-administration for each of the choroid plexus sites (n = 5–9/group, specific number per group denoted on each bar). A, Differences in expression of mir-204 were not found in the lateral ventricle choroid plexus between the nicotine and saline self-administration groups. Central tendency (mean) and variation (SEM) values for each are as follows: Saline: 6.43 ± 1.38 and Nicotine: 8.98 ± 4.01. B, In the fourth ventricle choroid plexus, statistically significant group differences were not found. Central tendency and variation: Saline: 14.32 ± 0.87 and Nicotine: 9.46 ± 2.24. C, In the d3V choroid plexus, nicotine self-administration induced a significant increase in the expression of mir-204 as compared to saline control. Central tendency (mean) and variation (SEM) values for each are as follows: Saline: 9.004 ± 1.32 and Nicotine: 14.49 ± 1.36; *p < 0.05. Data represent mean normalized values ± SEM.