Vagally mediated gastric effects of brain stem α2-adrenoceptor activation in stressed rats

Abstract

Chronic stress exerts vagally dependent effects to disrupt gastric motility; previous studies have shown that, among other nuclei, A2 neurons are involved in mediating these effects. Several studies have also shown robust in vitro and in vivo effects of α2-adrenoceptor agonists on vagal motoneurons. We have demonstrated previously that brainstem vagal neurocircuits undergo remodeling following acute stress; however, the effects following brief periods of chronic stress have not been investigated. Our aim, therefore, was to test the hypothesis that different types of chronic stress influence gastric tone and motility by inducing plasticity in the response of vagal neurocircuits to α₂-adrenoreceptor agonists. In rats that underwent 5 days of either homotypic or heterotypic stress loading, we applied the α₂-adrenoceptor agonist, UK14304, either by in vitro brainstem perfusion to examine its ability to modulate GABAergic synaptic inputs to vagal motoneurons or in vivo brainstem microinjection to observe actions to modulate antral tone and motility. In neurons from naïve rats, GABAergic currents were unresponsive to exogenous application of UK14304. In contrast, GABAergic currents were inhibited by UK14304 in all neurons from homotypic and, in a subpopulation of neurons, heterotypic stressed rats. In control rats, UK14304 microinjection inhibited gastric tone and motility via withdrawal of vagal cholinergic tone; in heterotypic stressed rats, the larger inhibition of antrum tone was due to a concomitant activation of peripheral nonadrenergic, noncholinergic pathways. These data suggest that stress induces plasticity in brainstem vagal neurocircuits, leading to an upregulation of α₂-mediated responses. NEW & NOTEWORTHY Catecholaminergic neurons of the A2 area play a relevant role in stress-related dysfunction of the gastric antrum. Brief periods of chronic stress load induce plastic changes in the actions of adrenoceptors on vagal brainstem neurocircuits.

Keywords: catecholamines; neurogastroenterology; stomach; stress; vagus.

Fig. 1.

Characterization of the fecal pellet output and immunocytochemistry of the A2 area. A: graphical summary illustrating the number of fecal pellets expelled during the periods of stress loading. Note that the number of fecal pellets is decreased over time in both chronic homotypic stress (CHo) (N = 27) and chronic heterotypic stress (CHe) (N = 32) rats; however, at day 5 CHe rats had a significantly higher number of pellets. *P < 0.05 vs. CHo. B: representative micrographs showing dopamine-β-hydroxylase-immunoreactive (DβH-IR) (left, blue) and tyrosine hydroxylase-immunoreactive (TH-IR) (right, brown) neurons in area A2 in control (top), CHo (middle), and CHe (bottom) stress rats. All slices were obtained at similar brainstem levels. C: summary graphic showing the number of DβH-IR and TH-IR neurons in A2 area of control (N = 6 slices/5 rats; open bar), CHo (6 slices/4 rats; red bar), and CHe (6 slices/5 rats; blue bar) rats. Note the increased DβH-IR in rats that underwent CHe, but not CHo, stress. Data were analyzed with 1-way ANOVA followed by Tukey’s test. *P < 0.05 vs. control and CHo.

Fig. 2.

Dorsal motor nucleus of the vagus (DMV) neurons from the chronic heterotypic stress (CHe) group are more excitable. A: representative traces of single action potentials evoked in DMV neurons from control, chronic homotypic stress (CHo), and CHe groups. Neurons were current clamped at −60 mV before injection of a short (16 ms) depolarizing current pulse of intensity sufficient to evoke a single action potential at its offset. Note that the amplitude and duration of the action potential and the amplitude and time constant of decay of the afterhyperpolarization (AHP) were similar among groups. B: representative traces showing the response of DMV neurons from control, CHo, and CHe rats following injection of 30 (left) and 150 pA (right) DC (400 ms long). C: frequency-response curves for DMV neurons from control (N = 24 neurons from 12 rats), CHo (24 neurons from 11 rats), and CHe (26 neurons from 14 rats) stress groups. Note that neurons from the CHe stress group are more excitable and fire more action potentials than neurons from the other groups. Holding potential = −60 mV. Data were analyzed with 1-way ANOVA followed by Tukey’s test. *P < 0.05.

Fig. 3.

Stress uncovers the α₂-adrenoceptor-mediated decrease in miniature inhibitory postsynaptic current (mIPSC) frequency. A: representative traces of mIPSCs recorded in a gastric-projecting dorsal motor nucleus of the vagus (DMV) neuron from a control rat. Perfusion with the α₂-adrenoceptor agonist UK14304 (1 µM) did not change the frequency or amplitude of mIPSCs. Holding potential = −50 mV. B: representative traces of mIPSCs recorded in a gastric-projecting DMV neuron from a chronic homotypic stress (CHo) rat. Perfusion of the slice with UK14304 reduced the frequency of mIPSCs. Holding potential = −50 mV. C: graphic representation of the percentage of neurons in control (solid bar; N = 0/9), CHo stress (red bar; N = 7/7), or chronic heterotypic stress (CHe) stress (blue bar; N = 4/13) in which perfusion with UK14304 decreased mIPSC frequency. D: graphic representation of the UK14304-induced modulation of the mIPSC frequency in DMV neurons from rats in control, CHo, and CHe stress load groups. In the CHo and CHe groups, only responsive neurons are depicted. Data were analyzed with a χ² test. *P < 0.05 vs. baseline.

Fig. 4.

Stress and corticotropin-releasing factor (CRF) uncover the α₂-adrenoceptor-mediated decrease of evoked inhibitory postsynaptic current (eIPSC) amplitude. A: representative traces of eIPSCs recorded in a dorsal motor nucleus of the vagus (DMV) neuron from control rat following electrical stimulation of nucleus tractus solitarius (NTS). Note that perfusion with UK14304 (1 µM) did not affect the amplitude of eIPSCs. Holding potential = −50 mV. B: representative traces of eIPSCs recorded in a DMV neuron from a control rat. Note that following perfusion with CRF and recovery to baseline amplitude, a second perfusion with UK14304 reduced the eIPSC amplitude in a concentration-dependent manner. Holding potential = −50 mV. C: representative traces of eIPSCs recorded in a DMV neuron from a chronic homotypic stress (CHo) rat. Perfusion of the slice with UK14304 reduced eIPSC amplitude even without pretreatment of the slice with CRF. Holding potential = −50 mV. D: graphic representation of the percentage of neurons in control (solid bar; N = 0/10), CHo stress (red bar; N = 12/17), or chronic heterotypic stress (CHe) stress (blue bar; N = 5/13) in which perfusion with UK14304 decreased eIPSC amplitude. E: graphic representation of the UK14304-induced modulation of the eIPSC amplitude in control, CHo, and CHe stress groups. In the CHo and CHe groups, only responsive neurons are depicted. Dashed line represents the “threshold” change to classify the neurons as responsive. Data were analyzed with 1-way ANOVA followed by Tukey’s test. P < 0.05 vs. baseline.

Fig. 5.

Microinjection of UK14304 in the dorsal vagal complex (DVC) induced a larger decrease in antrum tone of chronic heterotypic stress (CHe) rats. A: representative recording from the antrum of a control rat showing the decrease in tone upon microinjection of UK14304 (450 pmol/60 nl) in the DVC. B: pretreatment with the α₂-adrenoceptor antagonist, yohimbine (500 pmol/2 µl on the floor of the 4th ventricle), prevented the decrease in tone and motility in response to a second microinjection of UK14304. C: representative trace from the antrum of a CHe rat. Note that the UK14304-induced decrease in gastric tone is larger than the decrease obtained in control animals. D: graphic representation showing the positive correlation between the number of dopamine-β-hydroxylase-immunoreactive (DβH-IR) neurons in the brainstem A2 area and the decrease in antrum tone obtained upon microinjection of UK14304 in the DVC (N = 12). E: summary graph representing the effects of UK14304 microinjection in the DVC on antrum tone in control (N = 8), chronic homotypic stress (CHo) (N = 10), and CHe (N = 11) rats. Note the larger inhibition of tone induced by UK14304 in CHe rats. Pretreatment with the α₂-adrenoceptor antagonist, yohimbine, antagonized the UK14304-induced decrease in antrum tone. Data were analyzed with 1-way ANOVA followed by Tukey’s test. *P < 0.05 vs. control. F: summary graph representing the effects of UK14304 microinjection in the DVC on antrum motility in control rats (N = 8), CHo (N = 11), and CHe (N = 10) rats. No significant variations in antrum motility were observed among the groups. Pretreatment with the α₂-adrenoceptor antagonist, yohimbine, antagonized the UK14304-induced decrease in antrum motility (N = 8). Data were analyzed with 1-way ANOVA followed by Tukey’s test. *P < 0.05 vs. control.

Fig. 6.

Following stress load the UK14304 (UK)-induced effects on antrum tone and motility are mediated by withdrawal of cholinergic tone and activation of nonadrenergic, noncholinergic nitric oxide (NANC-NO) pathway. A: representative traces from a control animal showing that microinjection of 450 pmol/60 nl of UK in the dorsal vagal complex (DVC) decreases antrum tone and motility (left). Upon recovery from the UK-induced relaxation, following intravenous administration of nitro-l-arginine methyl ester (l-NAME) (10 mg/kg), a second microinjection of UK induced a similar inhibition as in naïve rats (middle). Upon recovery and following intravenous administration of bethanechol (50 µg/kg), the inhibitory effects of UK microinjection were prevented (right). Arrows indicate UK microinjection. B: data points showing the antrum tone response of individual animals to microinjection of UK alone (left column), in the presence of l-NAME (middle column), and in the presence of bethanechol (Beth) (right column) for control (left, black; N = 5), CHo (middle, red; N = 6), and CHe (right, blue; N = 6). Bars represent the mean response; data were analyzed with 1-way ANOVA followed by Tukey’s test. *P < 0.05 vs. UK alone. C: data points showing the antrum motility response of individual animals to microinjection of UK alone (left column), in the presence of l-NAME (middle column), and in the presence of bethanechol (right column) for control (left, black; N = 5), CHo (middle, red; N = 6), and CHe (right, blue; N = 6). Bars represent the mean response; data were analyzed with 1-way ANOVA followed by Tukey’s test. *P < 0.05 vs. UK alone.