Inhibition of the hypercapnic ventilatory response by adenosine in the retrotrapezoid nucleus in awake rats

Abstract

The brain regulates breathing in response to changes in tissue CO₂/H⁺ via a process called central chemoreception. Neurons and astrocytes in the retrotrapezoid nucleus (RTN) function as respiratory chemoreceptors. The role of astrocytes in this process appears to involve CO₂/H⁺-dependent release of ATP to enhance activity of chemosensitive RTN neurons. Considering that in most brain regions extracellular ATP is rapidly broken down to adenosine by ectonucleotidase activity and since adenosine is a potent neuromodulator, we wondered whether adenosine signaling contributes to RTN chemoreceptor function. To explore this possibility, we pharmacologically manipulated activity of adenosine receptors in the RTN under control conditions and during inhalation of 7-10% CO₂ (hypercapnia). In urethane-anesthetized or unrestrained conscious rats, bilateral injections of adenosine into the RTN blunted the hypercapnia ventilatory response. The inhibitory effect of adenosine on breathing was blunted by prior RTN injection of a broad spectrum adenosine receptor blocker (8-PT) or a selective A1-receptor blocker (DPCPX). Although RTN injections of 8PT, DPCPX or the ectonucleotidase inhibitor ARL67156 did not affected baseline breathing in either anesthetized or awake rats. We did find that RTN application of DPCPX or ARL67156 potentiated the respiratory frequency response to CO₂, suggesting a portion of ATP released in the RTN during high CO₂/H⁺ is converted to adenosine and serves to limit chemoreceptor function. These results identify adenosine as a novel purinergic regulator of RTN chemoreceptor function during hypercapnia.

Keywords: Adenosine; Breathing; Central chemoreflex; Purinergic signaling; RTN.

Figure 1. Adenosine in the RTN attenuates the hypercapnic ventilatory response in anesthetized vago-denervated rats

Changes in A and B) diaphragm (Dia_EMG) and C and D) abdominal (Abd_EMG) electromyography activities after bilateral injection of vehicle or increasing doses of ADO in the RTN under hypercapnic condition. E) Photomicrograph showing typical sites of bilateral injections in the RTN region. The latex microspheres are located ventral to the caudal portion of the facial nucleus in the RTN region. Computer-generated plot of bilateral injections of vehicle or ADO that were confined to the RTN region (bregma level −11.12 to −11.6 mm according to the Paxinos and Watson (1998). *Different from vehicle (Two-way ANOVA; Newman-Keuls’ test, p < 0.05, N = 5–7/group of rats). Abbreviations: py, pyramidal tract; Sp5, spinal trigeminal tract; VII, facial motor nucleus. Scale bar in E = 1 mm.

Figure 2. Adenosine in the RTN attenuates the hypercapnic ventilatory response in awake rats

A) Photomicrograph of a coronal section showing the site of a bilateral injection in the RTN (arrows) and a computer-generated plot of bilateral injections of vehicle or ADO that were confined to the RTN region. B) Recordings showing the effect of bilateral injection of vehicle or ADO (10 mM - 100 nL) into the RTN region under normocapnia and hypercapnia (7% CO₂). Changes in C) tidal volume (V_T, ml/kg), D) respiratory frequency (f_R, bpm) and E) minute ventilation (V_E, ml/kg/min) elicited by vehicle or ADO bilaterally injected into the RTN region under hypercapnic condition. *different from vehicle (Student t-test, p < 0.05); N = 5/group of rats. Abbreviations: py, pyramidal tract; Sp5, spinal trigeminal tract; VII, facial motor nucleus. Scale in A = 1 mm.

Figure 3. Respiratory effects elicited by blockade of the adenosine receptors in the RTN

A) Representative recording of (Dia_EMG) and abdominal (Abd_EMG) electromyography activities after vehicle, ADO (10 mM), 8-PT (100 mM) and 8-PT+ADO bilaterally injected into the RTN. Changes in B) Dia_EMG frequency, C) Dia_EMG amplitude, D) Abd_EMG frequency and E) Abd_EMG amplitude elicited by bilateral injection of vehicle, ADO, 8-PT or 8-PT+ADO into the RTN under hypercapnia (10% CO₂) challenge. F) Computer-generated plot of bilateral injections that were confined to the RTN region. *Different from vehicle, ⁺Different from ADO (One-Way ANOVA, Newman-Keuls’ test, p < 0.05, N = 6/group of rats). Abbreviations: py, pyramid; Sp5, spinal trigeminal tract; VII, facial motor nucleus. Scale bar in F = 1 mm.

Figure 4. Blockade of the adenosine A1 receptors in RTN blocks the inhibitory effect on the hypercapnic ventilatory response in anesthetized vago-denervated rats

A) Representative recording of (Dia_EMG) and abdominal (Abd_EMG) electromyography activities after vehicle, ADO (10 mM), DPCPX (5 μM) or DPCPX+ADO bilaterally injected into the RTN under hypercapnic condition. Changes in B) Dia_EMG frequency, C) Dia_EMG amplitude, D) Abd_EMG frequency and E) Abd_EMG amplitude elicited by bilateral injection of vehicle, ADO, DPCPX or DPCPX+ADO into the RTN under hypercapnia (10% CO₂) challenge. F) Computer-generated plot of bilateral injections that were confined to the RTN region. *Different from vehicle, ⁺Different from ADO (One-Way ANOVA, Newman-Keuls’ test, p < 0.05, N = 6/group of rats). Abbreviations: py, pyramidal tract; Sp5, spinal trigeminal tract; VII, facial motor nucleus. Scale bar in F = 1 mm.

Figure 5. Blockade of the adenosine A1 receptors in RTN blocks the inhibitory effect on the hypercapnic ventilatory response in awake rats

A) Recordings showing the effect of bilateral injection of vehicle or DPCPX (5 μM - 100 nL) into the RTN region under normocapnia and hypercapnia (7% CO₂). Changes in B) tidal volume (V_T, ml/kg), C) respiratory frequency (f_R, bpm) and D) minute volume (V_E, ml/kg/min) elicited by bilateral injection of vehicle or DPCPX injection in the RTN region under normocapnia and hypercapnia. E) Computer-generated plot of bilateral injections that were confined to the RTN region. *Different from normocapnia, +Different from vehicle + CO₂ (One-way ANOVA, Newman-Keuls’ test, p < 0.05, N = 4/group of rats). Abbreviations: py, pyramidal tract; Sp5, spinal trigeminal tract; VII, facial motor nucleus. Scale bar in E = 1 mm.

Figure 6. Inhibition of the ectonucleotidase in the RTN blocks the inhibitory effect on the hypercapnic ventilatory response in awake rats

A) Recordings showing the effect of bilateral injection of vehicle or ARL67156 (200 μM -100 nL) into the RTN region under normocapnia and hypercapnia (7% CO₂). Changes in B) tidal volume (V_T, ml/kg), C) respiratory frequency (f_R, bpm) and D) minute ventilation (V_E, ml/kg/min) elicited by bilateral injection of vehicle or ARL67156 injection in the RTN region under normocapnia and hypercapnia. E) Computer-generated plot of bilateral injections that were confined to the RTN region. *Different from saline + vehicle, +Different from saline + CO₂ (One-way ANOVA, Newman-Keuls’ test, p < 0.05, N = 6/group of rats). Abbreviations: py, pyramidal tract; Sp5, spinal trigeminal tract; VII, facial motor nucleus. Scale bar in E = 1 mm.