Delta 9-tetrahydrocannabinol suppresses vomiting behavior and Fos expression in both acute and delayed phases of cisplatin-induced emesis in the least shrew

Abstract

Cisplatin chemotherapy frequently causes severe vomiting in two temporally separated clusters of bouts dubbed the acute and delayed phases. Cannabinoids can inhibit the acute phase, albeit through a poorly understood mechanism. We examined the substrates of cannabinoid-mediated inhibition of both the emetic phases via immunolabeling for serotonin, Substance P, cannabinoid receptors 1 and 2 (CB(1), CB(2)), and the neuronal activation marker Fos in the least shrew (Cryptotis parva). Shrews were injected with cisplatin (10mg/kg i.p.), and one of vehicle, Delta(9)-THC, or both Delta(9)-THC and the CB(1) receptor antagonist SR141716A (2mg/kg i.p.), and monitored for vomiting. Delta(9)-THC-pretreatment caused concurrent decreases in the number of shrews expressing vomiting and Fos-immunoreactivity (Fos-IR), effects which were blocked by SR141716A-pretreatment. Acute phase vomiting induced Fos-IR in the solitary tract nucleus (NTS), dorsal motor nucleus of the vagus (DMNX), and area postrema (AP), whereas in the delayed phase Fos-IR was not induced in the AP at all, and was induced at lower levels in the other nuclei when compared to the acute phase. CB(1) receptor-IR in the NTS was dense, punctate labeling indicative of presynaptic elements, which surrounded Fos-expressing NTS neurons. CB(2) receptor-IR was not found in neuronal elements, but in vascular-appearing structures. All areas correlated with serotonin- and Substance P-IR. These results support published acute phase data in other species, and are the first describing Fos-IR following delayed phase emesis. The data suggest overlapping but separate mechanisms are invoked for each phase, which are sensitive to antiemetic effects of Delta(9)-THC mediated by CB(1) receptors.

Figure 1

Counts of Fos-immunoreactive Nuclei Following Acute or Delayed Phase Emesis in the Least Shrew. Fos-IR nuclei in the dorsal vagal complex (DVC) were analyzed for each nuclear region in the DVC. Induction of Fos (and vomiting) was through treatment with 10 mg/kg CIS (i.p.). Means and standard errors of Fos-IR nuclei are graphed for each pre-treatment condition, each subnucleus of the DVC, and both phases of CIV. Significantly reduced numbers of Fos-immunopositive nuclei (* p < 0.05) were noted after THC injection when compared to either the vehicle or SR141716A+THC conditions. When regions of the DVC having the same treatment conditions were compared between acute and delayed phases, the AP and NTS had significantly fewer († p < 0.05) Fos+ nuclei in the delayed phase than in the acute phase of emesis when treated with vehicle or SR141716A+THC, but not when treated with THC alone (p > 0.1). Numbers of Fos+ nuclei in the DMNX during the delayed phase were not significantly different from the acute phase (p > 0.12). Abbreviations: AP – area postrema; DMNX – dorsal motor nucleus of the vagus nerve; NTS – nucleus of the solitary tract.

Figure 2

Examples of Fos-IR in the Dorsal Vagal Complex of the Least Shrew Following Cisplatin-induced Emesis. Sagittal sections through the DVC demonstrate different numbers of Fos-IR nuclei depending on the emetic phase or drug treatment. Vehicle-injected controls (panel A) were effectively devoid of Fos-IR. Cisplatin-injected shrews demonstrated a robust increase in Fos-IR nuclei (black ovals) following the acute phase of emesis (panel B), and a less robust but still significant increase in Fos-IR following the delayed phase (panel C). Injection of THC produces near-control levels of Fos-IR nuclei (panel D), an effect reversible by blockade of CB₁ receptors with SR141716A (panel E). When a transmitted light image of Fos-IR is overlaid on a confocal image of CB₁ receptor-IR from the same microscopic field of the NTS of shrews following acute phase emesis (panel F), Fos-IR nuclei (pseudocolored white ovals) can be seen within unlabeled somata enmeshed in the CB₁ receptor-IR terminal-like structures (grey dots). Asterisks mark the somata of unlabeled (Fos-/CB₁-IR negative) neurons, arrowheads mark CB₁ receptor-IR terminals apposed to NTS somata, and arrows identify somata with both Fos-IR nuclei and CB₁ receptor-IR structures apposed to them. Abbreviations: AP – area postrema; DMNX – dorsal motor nucleus of the vagus; NTS – nucleus of the solitary tract. Scale bars = 50 μm for A–E; 20 μm for F.

Figure 3

Confocal Micrographs of Cannabinoid Receptor, 5-HT, and Substance P Immunolabeling in the Dorsal Vagal Complex of the Least Shrew. A) Sagittal section similar to that in figure 2C, stained for CB₁ receptor-IR. CB₁-IR was localized mostly to the NTS. B) Coronal section of the DVC demonstrating CB₂-IR, which localizes to the brain surface and choroid plexus (arrows), and to non-neuronal elements within the brainstem that appear vascular in nature (arrowheads). Elements suggestive of neuronal origin were absent. C) Multiple-labeling of 5-HT (blue), CB₁ receptor (green), and SP (red) in the NTS of the shrew demonstrated a variety of interactions. Asterisks demonstrate unlabeled somata apposed to punctate (terminal-like) immunolabeling for 5-HT, CB₁ receptors, and/or SP. While many terminal-like structures were singly-labeled, some showed colocalization of 5-HT/CB₁ receptor (arrows), SP/CB₁ receptor (arrowheads), or 5-HT/SP (double arrow). A single terminal also demonstrated colocalization of all three antigens (double arrowhead). D) Zero-primary coronal DVC section using the anti-rat secondary antibody and Alexa594-conjugated tyramide amplification. E) Zero-primary coronal DVC section using the anti-rabbit secondary and Alexa488-conjugated tyramide. Abbreviations: 12 – hypoglossal nucleus; 4V – 4^th ventricle; AP – area postrema; ChP – choroid plexus; Cu – cuneate nucleus; mlf –medial longitudinal fasciculus; NTS – nucleus of the solitary tract. Scale bars – A, 50 μm; B, 500 μm; C, 10 μm; D and E, 50 μm.