Verification of the anti-5-HT_2BR antibody.

<p>(<b>A</b>) (<b>a</b>) The 5-HT_2BR belongs to the family of seven-transmembrane-domain receptors that are coupled to hetero-trimeric guanine-nucleotide-binding protein q (G_q). The transmembrane-domains are indicated by red cylinders (I–VII). We produced a novel monospecific polyclonal antibody against the 5-HT_2BR by selecting a specific amino acid sequence (Cys¹⁴⁶ - Gln¹⁶¹; NH₂-<i>CAISLDRYIAIKKPIQ</i>-COOH) of the second intracellular loop of the rat 5-HT_2BR-sequence. The peptide exhibits 100% homology in mouse. Red letters indicate mismatches in the human-sequence (L→V). (<b>b</b>) Immunoblot analysis of mouse (<b>1</b>) or rat (<b>2</b>) brainstem lysate revealed a specific band at about 48 kDa that corresponds with the predicted relative molecular mass of the 5-HT_2BR. (<b>B</b>) 5-HT_2BR expression in non-transfected (<b>a</b>) and transfected N1E-115 cells (<b>b</b>). The anti-5-HT_2BR antibody-dependent staining indicated a strong labeling of N1E-115 cells that had been transiently transfected with the rat 5-HT_2BR (<b>b</b>). Non-transfected cells expressing the mouse 5-HT_2BR showed a weak neuronal immunofluorescent signal that corresponds with a weak PCR signal (amplicon size 1114 bp) for the mouse 5-HT_2BR-mRNA (<i>Htr2b</i>) (<b>c</b>). Samples without reverse transcription (w/o RT) served as negative controls. (<b>C</b>) (<b>a</b>, <b>b</b>) Immunohistochemistry. Both pyramidal neurons of the cortex and motoneurons of the hypoglossal nucleus (XII) revealed a strong 5-HT_2BR immunoreactivity (-IR) (<b>a</b>) that was effectively blocked after pre-incubation of the antibody with a 50-fold molar excess of the peptide <i>CAISLDRYIAIKKPIQ</i> (+ peptide) that was used for immunization (<b>b</b>). Insets in (<b>a</b>) show labeled neurons at a higher magnification. Immunolabeling was performed using the PAP-method with diaminobenzidine as chromogen. (<b>c</b>) RT-PCR analysis of the rat cortex and hypoglossal nucleus. The 5-HT_2BR-specific mRNA (<i>Htr2b</i>) was detectable in neurons within both the rat cortex and the hypoglossal nucleus (XII) (amplicon size 380 bp).</p

Expression patterns of 5-HT_2A and 5-HT_2BRs within the pontine respiratory network.

<p>Both 5-HT_2A and 5-HT_2B receptors are abundantly expressed in neurons of the internal lateral nucleus of the parabrachial complex (<b>B</b>, <b>E</b>). Within the nucleus Kölliker-Fuse the 5-HT_2BR expression is weak compared to 5-HT_2ARs (<b>A</b>, <b>D</b>). Abbreviations: internal lateral nucleus of the PB (il), lateral crescent nucleus of the PB (cr), nucleus Kölliker-Fuse (KF), parabrachial complex (PB), superior cerebellar peduncle (scp).</p

Expression patterns of 5-HT_2A and 5-HT_2BRs within the medullary respiratory network.

<p>(<b>A</b>) 5-HT_2AR expression pattern in the pre-BötC. The plots (<b>B</b>, <b>C)</b> represent 5-HT_2AR immunoreactivity within the pre-BötC (<b>B</b>) and BötC (<b>C</b>). (<b>D</b>–<b>F</b>) shows the corresponding expression pattern for the 5-HT_2BR. The insets show labeled neurons at a higher magnification. Abbreviations: Bötzinger complex (BötC), nucleus ambiguus (NA), pre-Bötzinger complex (pre-BötC), principal nucleus of the inferior olive (IO_Pr), interpolar spinal trigeminal nucleus (Sp5l).</p

Quantification of expression levels and co-expression of 5-HT_2A and 5-HT_2BRs within the pontine respiratory network.

<p>(<b>A</b>) (<b>a</b>) shows a schematic representation of the dissected pre-BötC and its landmarks: pre-Bötzinger complex (pre-BötC), nucleus of the solitary tract (Sol), nucleus ambiguus (NA), hypoglossal nucleus (XII), principal nucleus of the inferior olive (IO_Pr). (<b>b</b>) shows the specific mRNA of both receptors detected in the pre-BötC. (<b>B</b>) Double labeling of 5-HT_2A and 5-HT_2BRs. 5-HT_2A (Cy5, red) and 5-HT_2BRs (Cy2, green) are strongly co-expressed in pre-BötC-neurons. Immunohistochemical analysis does not reveal the ratio of co-expressed proteins. Therefore, we performed quantitative real-time RT-PCR on four selected nuclei of the respiratory network (<b>C</b>). The bar diagram represents results of quantitative real-time RT-PCR analysis of 5-HT₂R genes (<i>Htr2a</i>, <i>Htr2b</i>) of spinal cord (Spc), inferior olive (IO), pre-Bötzinger complex (pre-BötC), and parabrachial complex (PB). At the RNA level 5-HT_2AR is significantly stronger expressed compared to <i>Htr2b</i> in all regions analyzed. Asterisks indicate significance (*** = p<0.001; ANOVA with Bonferroni's post hoc test).</p

Effects of 5-HT_2AR and 5-HT_2BR agonists on Ca²⁺ fluorescence signals.

*<p>comparing results from single transfected cells stimulated with 5-HT_2AR-agonist or with 5-HT_2BR agonist.</p>**<p>comparing results from both single transfected/stimulated cells against double transfected/double stimulated cells.</p>a<p>only when compared to single transfected cells stimulated with 5-HT_2AR.</p

Respiratory network responses to systemic 5-HT_2AR and 5-HT_2BR activation.

<p>Shown are representative traces of integrated phrenic nerve activity (∫PNA) of 3 different experimental conditions. Black traces represent the ∫PNA under control conditions, while red traces indicate application of agonists and blue traces indicate application of antagonists. The bar diagrams on the right give the averages of amplitude and frequency of least 3 independent experiments of each condition. Statistical analysis (paired t-test) is denoted in the panels on the right side. (<b>A</b>–<b>C</b>) Action of 5-HT_2AR and 5-HT_2BR ligands in the perfused brainstem preparation. (<b>A</b>) Application of the specific 5-HT_2AR agonist TCB-2 (red trace) increased phrenic nerve activity (PNA) by increase of the amplitude, and subsequent application of the specific 5-HT_2AR-antagonist Altanserin (blue trace) reduced the amplitude. PNA frequency decreased below control. (<b>B</b>) Application of the 5-HT_2BR agonist BW 723C86 increased PNA frequency. After subsequent administration of the specific 5-HT_2BR-antagonist LY 272015 phrenic nerve activity returned nearly to baseline level. (<b>C</b>) Simultaneous application of the specific agonists TCB-2 and BW 723C86 only increased the frequency.</p