Expression of cannabinoid CB1 receptors by vagal afferent neurons is inhibited by cholecystokinin

Abstract

Both inhibitory (satiety) and stimulatory (orexigenic) factors from the gastrointestinal tract regulate food intake. In the case of the satiety hormone cholecystokinin (CCK), these effects are mediated via vagal afferent neurons. We now report that vagal afferent neurons expressing the CCK-1 receptor also express cannabinoid CB1 receptors. Retrograde tracing established that these neurons project to the stomach and duodenum. The expression of CB1 receptors determined by RT-PCR, immunohistochemistry and in situ hybridization in rat nodose ganglia was increased by withdrawal of food for > or =12 hr. After refeeding of fasted rats there was a rapid loss of CB1 receptor expression identified by immunohistochemistry and in situ hybridization. These effects were blocked by administration of the CCK-1 receptor antagonist lorglumide and mimicked by administration of CCK to fasted rats. Because CCK is a satiety factor that acts via the vagus nerve and CB1 agonists stimulate food intake, the data suggest a new mechanism modulating the effect on food intake of satiety signals from the gastrointestinal tract.

Figure 1.

RT-PCR indicates CB1 expression in rat nodose ganglia. A, RT-PCR of rat gastric corpus (lane 1), nodose ganglion from fed (lane 2) and fasted (lane 3) rats (pool of 10 ganglia in each case), and template-free control (lane 4) using primers yielding a predicted product of 330 bp from rat CB1 (100 bp size ladder; lane 5). Note the presence of strong bands in lanes 1 and 3. B, Similar data for CB2 using primers yielding a predicted product of 481 bp. C, The abundance of CB1 in extracts of whole nodose ganglion from fasted rats (lane 2) is higher than in fed rats (lane 1) (samples pooled from 6 animals in each case), whereas RT-PCR products corresponding to GAPDH are similar. D, When extracts (pooled from 8 rats) were prepared from the mid and caudal regions of the nodose ganglion of fed rats (lane 1), CB1 (but not GAPDH) was undetectable, whereas there was a strong signal in fasted rats (lane 2).

Figure 2.

Localization of CB1 immunoreactivity in rat nodose ganglion. A, In rats fed ad libitum there is a population of CB1 immunoreactive neuronal cell soma in the rostral part of the ganglion (indicated by direction of arrow), but little immunoreactivity in more caudal parts of the ganglion. B, In rats fasted for 48 hr, CB1-positive neurons in the rostral population are again revealed, together with immunoreactive cell soma in the mid and caudal regions of the ganglion. C, CB1-immunoreactive neuronal cell soma in the mid region of the rat nodose ganglion. D, The same section costained for CCK-1 immunoreactivity; E, the overlay of C and D. F, True Blue fluorescence in the same section after administration of the tracer into the gastric corpus. Circled neurons contain True Blue and express both CB1 and CCK-1 receptors. Scale bars, 100μm.

Figure 3.

Nerve trunk ligation reveals that CB1 receptors are transported toward the periphery in rat vagus nerve. Accumulated CB1 immunoreactivity (open arrow) on the rostral side of a crushing ligature (filled arrow). Scale bar, 50 μm.

Figure 4.

Identification of CB1 expression in human vagus. A, RT-PCR of human vagus nerve (lane 1) and gastric corpus (lane 2) showing bands corresponding to the predicted size of the human CB1 product (291 bp) (lane 3, template-free control; lane 4, 100 bp ladder). B, Similar data for CB2; the predicted human product of 459 bp is found in gastric corpus (lane 2) but not vagus (lane 1). C, D, CB1-immunopositive neurons in neuronal cell soma of human nodose ganglion at low and higher power, respectively. Scale bars, 50 μm.

Figure 5.

RT-PCR identification of CB1, CCK-1, and OX-R1 expression in nodose ganglia from fed and fasted rats. A, By RT-PCR, CB1 product is present in higher abundance in extracts of the whole nodose ganglion of fasted rats compared with rats fed ad libitum (for details, see Fig. 1). B–D, In contrast, in the same extracts there is similar abundance of bands corresponding to RT-PCR products of CCK-1 and OX-R1 receptors, and GAPDH.

Figure 6.

In situ hybridization shows increased abundance of CB1 transcripts in nodose ganglia from fasted rats. A, Probes to CB1 show little or no labeling in cells in the mid and caudal regions of the nodose ganglia rats fed ad libitum (dark-field images; rostral direction indicated by arrow). B, An adjacent section showing cells expressing the CCK-1 receptor revealed by in situ hybridization (open arrows). C, Corresponding regions of rat nodose ganglion after fasting for 48 hr revealing neurons expressing CB1 (open arrows); D, an adjacent section showing CCK-1 expression. E, F, Higher-power fields from A and C, respectively. G, Hybridization of nodose ganglion from a fasted rat with CB1 probes and a 100-fold excess of unlabeled probe. H, I, Higher-power fields from B and D, respectively. J, Hybridization of nodose ganglion from a fasted rat with CCK-1 probe and a 100-fold excess of unlabeled probe. Scale bars: A–D, 100 μm; E–J, 50 μm.

Figure 7.

Refeeding and CCK8 downregulate CB1 expression in rat nodose ganglion. A, In situ hybridization of CB1 in rat nodose ganglion after fasting for 48 hr (dark-field images; rostral direction indicated by arrow; see also Fig. 6). B, In a fasted rat, refed for 2.5 hr, there is virtually complete loss of CB1 transcripts revealed by in situ hybridization. C, Similarly, there is loss of the CB1 signal revealed by in situ hybridization in nodose ganglion after 48 hr fasting and administration of CCK8 (10 nmol, i.p., 2.5 hr before killing). D, Administration of CCK is associated with phosphorylation of CREB in neurons that express CB1 (E). F, Overlay of D and E. G, Higher-power magnification (region indicated by box in F) shows that CB1-expressing neurons (green) respond to administration of CCK8 indicated by colocalization (open arrows) of nuclear phospho-CREB (H, red; I, overlay). J, Deconvolution microscopy of a single neuron showing CB1 immunoreactivity in cytosolic vesicles (filled arrows, red); K, nuclear phospho-CREB (open arrows, green); L, overlay. Scale bars: A–F, 100 μm; G–L, 25 μm.

Figure 8.

CCK mediates the effect of food in downregulating vagal CB1 expression. A, Immunocytochemical localization of CB1 in nodose ganglia after refeeding for 2 hr after fasting for 48 hr and administration of vehicle 15 min before refeeding (rostral direction indicated by arrow). B, In comparison, administration of the CCK-1 antagonist lorglumide 15 min before a refeeding period of 2 hr after fasting for 48 hr rat preserves CB1 immunoreactivity. C, D, In situ hybridization (dark-field images) shows absence of CB transcripts in nodose ganglia after vehicle treatment 15 min before a 2 hr refeeding period after 48 hr fast (see also Fig. 7) (C), but administration of the CCK-1 antagonist lorglumide inhibits the loss of CB1 transcripts (D). E, F, Higher-power dark-field images of C and D (box), respectively; G, H, bright-field images of E, F. Scale bars: A–D 100 μm; E–H, 50 μm.