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Molecular profiling of CO2/pH-sensitive neurons in the locus coeruleus of bullfrogs reveals overlapping noradrenergic and glutamatergic cell identity.
Amaral-Silva L
,
Santin JM
.
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Locus coeruleus (LC) neurons regulate breathing by sensing CO2/pH. Neurons within the vertebrate LC are the main source of norepinephrine within the brain. However, they also use glutamate and GABA for fast neurotransmission. Although the amphibian LC is recognized as a site involved in central chemoreception for the control of breathing, the neurotransmitter phenotype of these neurons is unknown. To address this question, we combined electrophysiology and single-cell quantitative PCR to detect mRNA transcripts that define norepinephrinergic, glutamatergic, and GABAergic phenotypes in LC neurons activated by hypercapnic acidosis (HA) in American bullfrogs. Most LC neurons activated by HA had overlapping expression of noradrenergic and glutamatergic markers but did not show strong support for GABAergic transmission. Genes that encode the pH-sensitive K+ channel, TASK2, and acid-sensing cation channel, ASIC2, were most abundant, while Kir5.1 was present in 1/3 of LC neurons. The abundance of transcripts related to norepinephrine biosynthesis linearly correlated with those involved in pH sensing. These results suggest that noradrenergic neurons in the amphibian LC also use glutamate as a neurotransmitter and that CO2/pH sensitivity may be linkedto the noradrenergic cell identity.
Fig. 1. Locus coeruleus neurons of adult bullfrogs increased firing frequency in response to hypercapnia (5% CO2). Neurons in the region comprising the locus coeruleus (A) had firing frequency recorded in control conditions (B, C) and after being exposed to 5% CO2 (C, D). Hypercapnia increased firing frequency of all neurons used in this study (E, n = 19). Results were compared using paired t-test.
Fig. 2. mRNA abundance of neurotransmitter markers and candidate pH sensing ion channels. A) Individual neurons harvested after electrophysiological recordings had gene expression analyzed using RT qPCR (n = 19). B) mRNA transcript abundance for markers of noradrenergic (dopamine beta-hydroxylase; DBH), glutamatergic (vesicular glutamate transporter 2; vGluT2), and GABAergic (glutamate decarboxylase 1; GAD1) transmission. C) mRNA transcript abundance for markers of pH sensors, TASK2 (K+ channel), ASIC2 (cation channel), and Kir5.1 (K+ channel). Bars represent means ± SD. Results were compared using one-way ANOVA on rank (Kruskal-Wallis test) followed by Dunn's posthoc tests.
Fig. 3. Correlations between the noradrenergic marker (dopamine beta-hydroxylase; DBH) and pH sensors (n = 19). Expression of DBH was positively correlated to expression of the pH-sensing markers TASK2 (A) and ASIC2 (B) but not to Kir5.1 (C). A positive correlation was also observed between the abundance of TASK2 and ASIC2 (D).
Fig. 4. There was no correlation between the glutamatergic marker (vesicular glutamate transporter 2; VGluT2) and pH sensors (n = 19). The expression of vGluT2 was not related to the expression of the pH-sensing markers TASK2 (A), ASIC2 (B), or Kir5.1 (C). The expression of glutamatergic (VGluT2) and noradrenergic (dopamine beta hydroxylase; DBH) markers were also not related (D).
Figure S1 – Locus coeruleus neurons slightly decreased firing frequency on average when maintained in control conditions. Neurons in the region comprising the locus coeruleus had firing recorded in control conditions for 20 min (A). We observed a slight decrease in firing frequency over time (n=8). Results were compared using paired t-test.
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