A Nigro-Vagal Pathway Controls Gastric Motility and Is Affected in a Rat Model of Parkinsonism

Abstract

Background & aims: In most patients with Parkinson's disease, gastrointestinal (GI) dysfunctions, such as gastroparesis and constipation, are prodromal to the cardinal motor symptoms of the disease. Sporadic Parkinson's disease has been proposed to develop after ingestion of neurotoxicants that affect the brain-gut axis via the vagus nerve, and then travel to higher centers, compromising the substantia nigra pars compacta (SNpc) and, later, the cerebral cortex. We aimed to identify the pathway that connects the brainstem vagal nuclei and the SNpc, and to determine whether this pathway is compromised in a rat model of Parkinsonism.

Methods: To study this neural pathway in rats, we placed tracers in the dorsal vagal complex or SNpc; brainstem and midbrain were examined for tracer distribution and neuronal neurochemical phenotype. Rats were given injections of paraquat once weekly for 3 weeks to induce features of Parkinsonism, or vehicle (control). Gastric tone and motility were recorded after N-methyl-d-aspartate microinjection in the SNpc and/or optogenetic stimulation of nigro-vagal terminals in the dorsal vagal complex.

Results: Stimulation of the SNpc increased gastric tone and motility via activation of dopamine 1 receptors in the dorsal vagal complex. In the paraquat-induced model of Parkinsonism, this nigro-vagal pathway was compromised during the early stages of motor deficit development.

Conclusions: We identified and characterized a nigro-vagal monosynaptic pathway in rats that controls gastric tone and motility. This pathway might be involved in the prodromal gastric dysmotility observed in patients with early-stage Parkinson's disease.

Keywords: Animal Model; Central Nervous System; Neurology; Vagus.

Figure 1. SNpc provides a monosynaptic input to cholinergic and catecholaminergic neurons of the dorsal motor nucleus of the vagus (DMV) and A2 area

A. Representative micrographs showing: a) The location of the retrograde tracer cholera toxin-B (red) microinjection in the DMV, green=ChAT-IR, N=9. b) Low magnification image showing TH-IR (red, digital color) neurons in the SNpc of the animal shown in (a). c) Same panel as in (b) using filters for identification of the retrograde tracer (white, digital color). d) Enlargement of the dotted area in (c) showing TH-IR neurons of the SNpc (red) co-localized with retrograde tracer (white, arrows). B. Representative micrographs showing: a) The location of the anterograde tracer biotinylated-dextran in the SNpc (red, green=TH-IR, N=5). b) low magnification of the DMV of an animal which received microinjection of the anterograde tracer biotinylated-dextran in SNpc, brown=ChAT-IR. c,d,e) enlargement of the dotted areas in (b) showing labeled fibers (black) apposing ChAT-IR positive DMV neurons. f) TH-IR (red) neurons in the DMV. Following microinjection of the anterograde tracer dextran-fluorescein in the SNpc (N=4), labeled fibers (green) can be seen apposing TH-IR DMV neurons of the A2 area. g) Neurolucida® reconstruction of the biotinylated-dextran labeled fibers shown in (b). DMV: dorsal motor nucleus of the vagus; NTS: nucleus tractus solitarius; XII: hypoglossal nucleus; cc: central canal; SNpc: substantia nigra pars compacta.

Figure 2. Chemical stimulation of SNpc increases c-FOS-IR in the DMV

A. Low magnification micrographs showing the location of ChAT- (brown, left panel) and TH-IR (brown, right panel) in the brainstem. B. Representative micrographs of the intermediate DVC showing cFOS-IR in response to SNpc microinjections of saline (left panel, a) or NMDA (right panel, a). Note that following SNpc microinjection of NMDA, but not saline, cFOS-IR (blue) is increased in ChAT-IR neurons in the DMV (panel b, high magnification of the dotted area in the right panel a) but not in the nucleus ambiguus (c). Similarly, NMDA microinjection in the SNpc increases c-FOS in the A2 area (panel e, high magnification of the dotted area in the right panel, d) but not in neurons of the A1/C1 area (panel f). C. Graphic summaries showing the co-localization of cFOS-IR in ChAT-IR neurons of the DMV, hypoglossus nucleus or nucleus ambiguus (n=6 for saline and NMDA; number of neurons/slice that co-localize cFOS-ChAT, left panel), and in TH-IR neurons of the A2 or A1/C1 areas (n=5-6 for saline and NMDA, respectively; right panel). The cFOS-IR increase occurred bilaterally (cFOS-ChAT co-localization in DMV, right: from 1.9±0.3 to 9.6±1.3, left: 2.3±0.2 to 9.3±1.1; cFOS-TH co-localization in A2 area, right: from 2.6±0.4 to 5.2±0.8, left: from 3±0.5 to 5.6±1.2; p<0.05 vs control for all). *p<0.05.

Figure 3. Chemical stimulation of SNpc modulates gastric tone and motility via vagal pathways

A. Left panel: representative recording from the anterior gastric corpus showing that microinjection of NMDA in the left SNpc increases tone and motility. Right panels: Graphic summaries showing the dose-dependent increase in corpus (n=5-15 per each dose) and antrum tone (n=5-13 per each dose) or motility index in corpus (n=3-13 per each dose) and antrum (n=5-14 per each dose). B. Left panel: representative recording from the anterior corpus of a rat that received 6-OHDA in the left SNpc. Following NMDA microinjection in the left SNpc, the gastric excitatory effects of NMDA were negligible indicating that they were mediated by catecholaminergic neurons of the SNpc (upper trace). Conversely, microinjection of NMDA in the right SNpc (untreated side) of the same animal increased gastric tone and motility (lower trace). Right panels: graphic summaries showing the decreased response to NMDA microinjection in the left SNpc in corpus and antrum tone and motility in rats pretreated with 6-OHDA or saline in the SNpc. Tone: corpus, n=11; antrum, n=13. Motility: corpus and antrum, n=13. Conversely, microinjection of NMDA in the right SNpc (untreated side) increased corpus and antrum tone and motility. Tone: corpus, n=7; antrum, n=8. Motility: corpus and antrum, n=8. * p<0.05 vs untreated side. C. Left panel: Representative recording from the anterior corpus of a rat that received a posterior gastric branch vagotomy. Microinjection of NMDA in the left SNpc increased tone and motility (upper trace); after the complete vagotomy, the NMDA microinjection was repeated (lower trace); the excitatory effects of NMDA were prevented. Right panels: Graphic summaries showing the decreased response to NMDA microinjection in the left SNpc in corpus (black bars) and antrum (gray bars) tone and motility upon complete vagotomy. Tone: corpus and antrum, n=5. Motility: corpus and antrum, n=5. * p<0.05 vs control.

Figure 4. The NMDA-induced increase in gastric tone and motility is mediated via DA1 receptors

A. Representative recording from the anterior corpus showing that the increase in gastric motility and tone following microinjection of NMDA in the left SNpc, (left trace). Upon recovery, the DA1 antagonist SCH23390 was applied to the floor of the 4^th ventricle and the NMDA microinjection was repeated (right trace). The excitatory effects of NMDA were attenuated significantly indicating that they were mediated by activation of DA1-like receptors. B. Representative confocal micrograph showing that, upon microinjection of dextran-fluorescein in SNpc, a labeled fiber apposes a DMV neuron immunoreactive for DA1 receptors (arrow). C. Graphic summaries showing the decreased response to NMDA microinjection in the left SNpc in corpus and antrum tone and motility upon pretreatment with SCH23390 (gray bars) but not by pretreatment with the DA2 antagonist L741,626 (white bars). SCH23390, Tone: corpus, n=6; antrum, N=5. Motility: corpus: n=5; antrum, n=4 L741,626, Tone: corpus and antrum, n=7. Motility: corpus: n=6; antrum, n=7. In all panels *p<0.05 vs control. D. Graphic summaries showing that pretreatment with the α1 adrenoceptor antagonist, prazosin (white bars), did not affect the increase in gastric tone or motility in response to left SNpc NMDA microinjection. Conversely, pretreatment with the α2 adrenoceptor antagonist yohimbine (gray bars) reduced the increase in corpus or antrum motility in response to left SNpc microinjection. Prazosin, Tone and motility: corpus and antrum, n=5. Yohimbine, Tone and motility: corpus and antrum, n=5. In all panels *p<0.05 vs control. E. Representative traces of gastric tone and motility from the anterior corpus showing that application of the dopamine reuptake inhibitor benztropine on the 4^th ventricle increases gastric tone and motility (upper trace). Following application of the DA1 receptor antagonist SCH23390, the excitatory effects of benztropine were significantly reduced (lower trace). F. Graphic summaries showing the decreased response to benztropine application on the 4^th ventricle in corpus and antrum tone and motility upon pretreatment of DA1 antagonist SCH23390 (gray bar) Tone and motility: corpus and antrum, n=5. In all panels *p<0.05 vs control.

Figure 5. Optogenetic inhibition of the DVC decreases gastric tone and motility, and attenuates the effects of SNpc stimulation

A. Microinjection of rAAV2/hSyn-eNpHR3.0-EYFP (green) in the SNpc does not alter the neurochemical phenotype of TH-IR (red) neurons in the SNpc. B. Representative recording from the anterior corpus showing that optogenetic inhibition of the DVC with green light decreases baseline tone and motility. C. Representative recording from the anterior corpus showing that optogenetic inhibition of the DVC with green light attenuates the increase in tone and motility resulting from NMDA-mediated (arrow) stimulation of the SNpc. D. Graphic summaries showing the decreased baseline response to green light stimulation in the DVC in corpus (black bars) and antrum (white bars) tone and motility. Tone: corpus, n=3,6 in controls (black and white bars), and opsin (green bars) rats respectively; antrum, n=3,8 in controls (black and white bars), and opsin (green bars) rats respectively. Motility: corpus: n=3,6 in controls (black and white bars), and opsin (green bars) rats respectively; antrum, n=3,7 in controls (black and white bars), and opsin (green bars) rats respectively. E. Graphic summaries showing the decreased response to green laser stimulus in the DVC in corpus (black bars) and antrum (white bars) tone and motility following NMDA-mediated stimulation of the SNpc. Tone: corpus, n=3,5 in controls (black and white bars), and opsin (green bars) rats respectively; antrum, n=3,6,5 in controls (black and white bars), and opsin (green bars) or paraquat-treated (blue bars) rats respectively. Motility: corpus: n=3,7 in controls (black and white bars), and opsin (green bars) rats respectively; antrum, n=3,7in controls (black and white bars), and opsin (green bars) rats respectively. *p<0.05 vs control.

Figure 6. Paraquat treatment impairs motor activity, reduces the opsin-mediated inhibition of gastric tone and motility, and promotes α-synuclein formation in DMV and SNpc

A. Schematic representation of the paraquat-treatment schedule of administration and experiments. B. Graphic summary showing the decrease in motor performance of rats treated with paraquat. Note that motor activity was already reduced 2 days after the last injection of paraquat, but continued to decline for at least 2 weeks after paraquat injection. n=10,7,3 in control, 2 days after last injection and 2 weeks after last injection, respectively. *p<0.05 vs baseline. C. Left panel: graphic summary showing the decreased baseline response to green light stimulation in the DVC in corpus (striped bars) and antrum (blue bars) tone and motility. Tone: corpus, n=5; antrum, n=5. Motility: corpus: n=4; antrum, n=5 in controls. Right panel: Graphic summary showing the decreased response to green laser stimulus in the DVC in corpus (striped bars) and antrum (blue bars) tone and motility following NMDA-mediated stimulation of the SNpc. Tone: corpus, n=4; antrum, n=5. Motility: corpus: n=4; antrum, n=5 in controls. *p<0.05 vs opsins in naive rats (figure 5). D. Representative micrographs showing the same area of the DMV of a control (upper) and a paraquat-treated (middle) rat; red=ChAT and green=¹²⁹Ser α-synuclein. Lower panels show ChAT-IR (left), ¹²⁹Ser α-synuclein (middle) and merged (right) images of the dotted area in the middle panels. Arrows indicate DMV neurons with ¹²⁹Ser α-synuclein deposits. E. Representative micrographs showing the same area of the SNpc of a control (upper) and a paraquat-treated (middle) rat; red=TH- and green=¹²⁹Ser α-synuclein. Lower panels show TH-IR (left), ¹²⁹Ser α-synuclein- (middle) and merged (right) images of the dotted area in the middle panels. Arrows indicate SNpc neurons with ¹²⁹Ser α-synuclein deposits. Note that in both panels D and E control animals did not show any ¹²⁹Ser α-synuclein-IR neurons.

Figure 7. The nigro-vagal pathway is impaired in paraquat-treated animals

A. Graphic summary showing that in paraquat-treated animals microinjection of NMDA in the left SNpc increased gastric tone and motility significantly less than in controls. Tone: corpus, n=6,7,5 in control (white bars) and paraquat-treated animals 2 days (light blue bars) and 2 weeks (blue bars) after the last injection of paraquat respectively; antrum, N=6,6,5 in control (white bars) and paraquat-treated animals 2 days (light blue bars) and 2 weeks (blue bars) after the last injection of paraquat respectively. Motility: corpus: n=6,7,6 in control (white bars) and paraquat-treated animals 2 days (light blue bars) and 2 weeks (blue bars) after the last injection of paraquat respectively; antrum, n=5,7,5 in control (white bars) and paraquat-treated animals 2 days (light blue bars) and 2 weeks (blue bars) after the last injection of paraquat respectively. In all panels *p<0.05 vs control. B. Graphic summaries showing that stimulation of the gastric smooth muscle via i.v.administration of bethanecol induced similar increase in gastric tone and motility in paraquat-treated rats. Tone: corpus, n=5,7,4 in control (white bars) and paraquat-treated animals 2 days (light blue bars) and 2 weeks (blue bars) after the last injection of paraquat respectively; antrum, n=5,6,5 in control (white bars) and paraquat-treated animals 2 days (light blue bars) and 2 weeks (blue bars) after the last injection of paraquat respectively. Motility: corpus: n=3,6,4 in control (white bars) and paraquat-treated animals 2 days (light blue bars) and 2 weeks (blue bars) after the last injection of paraquat respectively; antrum, n=3,6,4 in control (white bars) and paraquat-treated animals 2 days (light blue bars) and 2 weeks (blue bars) after the last injection of paraquat respectively. C. Representative micrographs of gastric myenteric ganglia in a control (left) and a paraquat-treated (right) rat. Red=PGP9.5-IR and green=ChAT-IR. D. Graphic summary showing that paraquat treatment did not reduce the proportion of cholinergic neurons in gastric myenteric ganglia.