The major goals of our experiments were to dissect the relationship between the serotoninergic and tachykininergic systems in the ileum and to assess the effect of 5-HT, a common transmitter in ileal myenteric system. We integrated morphological and pharmacological data by performing immunostainings and in vitro pharmacological experiments in rabbit ileal muscle strip model.
A particular aim of our research was to study the tachykininergic system, which is also involved in the organization of gut motility. The main source of tachykinins is the intrinsic enteric neurons that supply the ganglia and the smooth muscle (Shuttleworth and Keef,1995). In mammalian species, tachykinin-immunoreactivity was detected in Dogiel type I neurons with either short filamentous processes or many lamellar dendrites and in Dogiel II cells (Ekblad et al.,1987; Sang and Young,1996,1998; Brookes,2001; Shimizu et al.,2008). Functionally, tachykinins are released from enteric excitatory motorneurons innervating the smooth muscle of gut, intrinsic primary afferent neurons, and last but not least, orally and anally directed interneurons (Ekblad et al.,1987; Brookes et al.,1991,1992,1997; Johnson et al.,1996,1998). Tachykinins utilized as transmitters from enteric neurons target numerous enteric elements through NK1, NK2, or NK3 receptors. NK2 receptors occur primarily on muscle, whereas NK3 receptors are involved in neuroneuronal transmission (Johnson et al.,1996,1998; Portbury et al.,1996a; Jenkinson et al.,1999,2000; Lecci et al.,2002). NK1 receptors have been shown acting on both muscle and enteric neurons. More precisely, functional classes of neurons that expressed NK1 receptors were NOS-IR inhibitory motorneurons, ACh/SP-IR excitatory motorneurons, secretomotor neurons, and ChAT/calbindin-IR sensory neurons (Portbury et al.,1996b; Johnson et al.,1998; Lomax et al.,1998; Lecci et al.,2002; Thornton and Bornstein,2002).
Data that indicate functional relationship between 5-HT and tachykininergic system are rare and to date none has been obtained in rabbit. Work of Bucheit et al. (1985) on guinea pig ileum provided data that SP antagonist reduced the effect of 5-HT; however, they did not specify which tachykinin receptor was involved. Also, Briejer and Schuurkes (1996) found that 5-HT due to 5-HT3 and 5-HT4 receptors had stimulatory effects in the guinea pig colon and this action was mediated through activation of the cholinergic and tachykininergic neurons.
A Possible Enteric Circuit Underlying Serotonin–Tachykinin Interaction in Rabbit Ileum
In our previous article, data had been published about the serotoninergic system in rabbit (Dénes et al.,2003). Briefly, the 5-HT synthesizing enzyme could be detected in the myenteric plexus but before revealing a rich varicose 5-HT-IR fiber meshwork, we had to incubate the ileum with 5-HT. After preincubation with 5-HT, an extensive fiber meshwork was seen in the myenteric plexus forming numerous pericellular baskets as well as running to the muscle layer. No 5-HT-IR neurons were observed in the rabbit myenteric plexus; however, 5-HT stimulated movements of the longitudinal muscle layer by direct and indirect ways in the rabbit ileum. The indirect effect of 5-HT was partially elicited through cholinergic pathway via 5-HT2 receptors, whereas the direct action of 5-HT was mediated by 5-HT4 receptors (Dénes et al.,2003). In this study, a more refined analysis of the effect of 5-HT was performed.
Tachykinins are thought to be major excitatoric transmitters in enteric neurons (Costa et al.,1985; Grider,1989). Therefore, we used NK1, NK2, and NK3 receptor antagonists to analyze contractions evoked by 5-HT and 5-HT2 receptor agonist. Considering that the natural ligand of NK1 receptors is SP whereas NK2 receptors are preferred by NKA (Holzer and Holzer-Petsche,1997), we assume that 5-HT stimulates both SP and NKA release through 5-HT2 receptors. From the consecutive and reversed administration of drugs, it is clear that ACh has a prominent role in the residual contraction after application of NK1 antagonist. At the same time, treatment with ATR could prevent the effect of the 5-HT-evoked tachykinin release. It is reasonable to assume that 5-HT could release SP from interneurons rather than from motor neurons. Our result of with HEX treatments indicates that cholinergic interneurons could be also involved in this circuit.
To supplement the data obtained by in vitro pharmacological experiments, immunostainings were also performed. First, we found a massive tachykinin-IR system in the myenteric plexus. We show a strong immunopositive network in the smooth muscle layer, neurons, and pericellular baskets around both unlabelled and tachykinin-immunopositive somata. The finding suggests that tachykinins could participate in motor as well as integrative functions in the rabbit ileum. By mapping SP-IR neurons, we established that the cells were distributed evenly. The proportion of tachykinin-IR neurons in rabbit ileum is surprisingly high compared with rodents. The total neuron density of ileal myenteric plexus in rabbit was established to be ∼2,500 cells/cm2 (Gábriel et al.,1998). We calculated that ∼52% of all myenteric neurons synthesize tachykinins indicating that these peptides must have dominant roles in organizing ileal functions. In guinea pig, the SP-IR neuron population amounted to 23% of all neurons of the ileal myenteric plexus (Costa et al.,1996). In mice, 29% of the total neuron population in the myenteric plexus was found SP-immunopositive (Sang and Young,1996).
Seeking for anatomical evidence for interconnections among serotoninergic, cholinergic, and tachykinin-containing system, double-label studies were performed. In most mammalian species, both SP and NKA appeared to be involved in neuronal transmission in the enteric nervous system and they coexist with ACh in the same excitatory neurons (Grider,1989; Lippi et al.,1998; Maggi,2000). The proportion of coexistence of these markers, however, varies among species. In guinea pig, all SP-IR neurons were also IR for ChAT, whereas 25–45% of all ChAT-IR cells were SP-immunopositive (Steele et al.,1991). In mice, about 50% of ChAT-IR neuron population contained SP, and 80% of SP-IR cells contained also ChAT. Our double-label experiments in rabbit revealed 20% of ChAT-IR cells containing also tachykinins and ∼50% tachykinin-immunopositive cells were IR for ChAT.
We did not find 5-HT-IR pericellular baskets around either ChAT or tachykinin-IR cell bodies. Those few ChAT or tachykinin-IR cells that were apposed by 5-HT-IR boutons were not prominent. Synaptology and thus the function of serotoninergic fibers emerging from serotoninergic descending interneurons has not been clarified yet since the reports that investigated the problem published different results (Erde et al.,1985; Young and Furness,1995; Neal and Bornstein,2007). Erde et al. (1985) reported that mainly type II/AH neurons considered sensory neurons were contacted by serotoninergic terminals. According to Young and Furness (1995), Dogiel type II neurons received a few serotonin inputs and none of them formed synapses, whereas the highest number of serotonin terminals were related to Dogiel type I neurons. A more recent works provided precisely quantified data claiming that anatomical connection between 5-HT-IR varicosities and the longitudinal muscle motor neurons was not significant. In contrast, all excitatory circular muscle motor neurons and some ascending interneurons, which are tachykininergic, were encircled by 5-HT-IR varicosities (Brookes at al.,1997; Neal and Bornstein,2007). However, it is important to note that having close contacts with 5-HT-IR varicosities do not necessarily mean synaptic specializations (Young and Furness,1995). However, this is not a question in case of rabbit considering the small number of cholinergic and tachykinin-containing neurons, which were apposed by 5-HT-IR fibers. Therefore, evidence of morphological basis for 5-HT/tachykinin and 5-HT/ACh interaction could not be found in rabbit ileal myenteric plexus. Clearly, simple light microscopy used in our study is not an efficient tool to reveal synaptic inputs between two structures. This would require ultrastructural analysis. Besides synaptic transmission, intercellular communication can also occur by volume transmission, which makes communication possible between nerve elements lacking synaptic specializations (Zoli et al.,1999; Sykova,2004; Vizi et al.,2004). 5-HT may be released by nonjunctional varicosities and can diffuse to 5-HT receptors of both neurons and smooth muscle cells. 5-HT might also be secreted by endocrine cells and act as a hormone or through activation of local neural pathways. Numerous studies revealed that 5-HT was released in response to pressure, mucosal stimuli from the intestinal mucosa and evoked peristaltic or secretory reflexes (Kirchgessner et al.,1992; Grider et al.,1996; Zhu et al.,2001; Bertrand,2007; Patel et al.,2007).
The pharmacological experiments showing indirect effects of 5-HT through 5-HT2 receptors were also confirmed by immunocytochemistry. We could detect numerous neurons as well as a varicose fiber network that expressed 5-HT2A receptors in the myenteric plexus. Our colocalization studies provided evidence that a small population of tachykininergic neurons possesses 5-HT2A receptors that could be either excitatory motorneurons or interneurons. In addition to this population, there were tachykinin-IR neurons lacking 5-HT2A receptors and many 5-HT2A receptor expressing cells, in fact, did not show tachykinin-immunopositivity.
By comparing our observations with results obtained in other species, we can state that the action of 5-HT on ileal motility in rabbit shares both similarities and dissimilarities to other species like guinea pig, rat, and mouse that are close to rabbits in terms of diet and evolution.
Because the first description that 5-HT potently affects intestinal movements (Gaddum,1953), actions of 5-HT have been investigated in a wide range of animals and from different aspects. Most of the data have been provided from guinea pig small intestine. Two different 5-HT receptors can mediate contractile responses named 5-HT3 and 5-HT4 receptors located on neuronal components (Craig and Clarke,1990; Craig et al.,1990; Eglen et al.,1990; Taniyama et al.,1991). 5-HT acting at 5-HT3 receptors could evoke ascending as well as descending excitatory reflexes (Yuan et al.,1994) through activation of cholinergic motor neurons (Fox and Morton,1990; Monro et al.,2002). 5-HT4 receptors can also mediate contractile effect, increase peristaltic reflex sensitivity (Craig and Clarke,1990,1991; Buchheit and Buhl,1991; Kilbinger and Wolf,1992; Kilbinger et al.,1995; Galligan et al.,2003) like 5-HT3 receptors do. Besides the cholinergic system, evidence was reported that tachykininergic neurons also had roles in contractions induced by 5-HT (Buchheit et al.,1985; Ramirez et al.,1994). In mouse ileum, effect of 5-HT was mediated through neuronally located 5-HT3 receptors (Tuladhar et al.,2000; Chetty et al.,2006). Data obtained in rat ileum are rather controversial. Some studies claimed that 5-HT2A and 5-HT1 receptors located on the smooth muscle were involved in 5-HT-induced contractions, and neither TTX nor atropin caused inhibition suggesting a direct effect on rat ileal muscle (Briejer et al.,1997; Yamano et al.,1997). Meanwhile, the role of 5-HT3 receptors acting through neuronal activation in rat ileum was also proved (Chetty et al.,2006). The latter study also suggested that tachykinins were not mediating 5-HT3-induced contractions.
The main conclusions of our study, therefore, were as follows: (i) in rabbit, 5-HT could stimulate contractions on both indirect and direct ways, whereas in guinea pig, rat, and mouse 5-HT has only one way to act, either direct or indirect. (ii) Cholinergic and tachykinin-containing motorneurons and also interneurons are participating in muscle stimulation in rabbit (Fox and Morton,1990; Briejer and Schuurkes,1996). (iii) In rabbit, movements of longitudinal muscle strips could be evoked potentially via two kinds of 5-HT receptor, 5-HT2 and 5-HT4, whereas 5-HT3, 5-HT4 receptors take parts in mediation of 5-HT-evoked contractions in guinea pig, 5-HT2 receptors in rat and 5-HT3 receptors in mouse, respectively.