Parasitol Res (2006) 99: 231–237 DOI 10.1007/s00436-006-0128-9

ORIGINA L PA PER

George J. Bikopoulos . Tafazzal Hoque . Rodney A. Webb

Infection with the cestode Hymenolepis diminuta induces changes in acetylcholine metabolism and muscarinic receptor mRNA expression in the rat jejunum Received: 22 September 2005 / Accepted: 26 December 2005 / Published online: 16 March 2005 # Springer-Verlag 2005

Abstract Total and neuron-specific uptake of [3H] choline into smooth muscle/myenteric plexus (SM/MP) preparations from the jejunum of rats infected with five Hymenolepis diminuta for 30 days compared to uninfected rats was significantly increased, as was choline acetyltransferase activity and acetylcholine biosynthesis. Although acetylcholinesterase and total cholinesterase activity levels in SM/MP preparations from infected rats were not significantly different from uninfected animals, pseudocholinesterase activity was significantly elevated in infected rats. Infection resulted in a significant elevation in the relative expression of muscarinic 2 (M2) receptor mRNA in jejunum compared to uninfected rats. Conversely, in rats infected with 50 worms for 30 days, the relative expression of muscarinic 1 (M1) receptor mRNA in the jejunum was significantly depressed, while the expression of M2 receptor mRNA was not significantly different from that in five worm infections. The relative expression of muscarinic 3 receptor mRNA was unaffected by infection. The present study shows that infection of rats with low numbers of an enteric cestode leads to a significant modulation of the cholinergic components of the myenteric plexus and M2 receptor mRNA, and that large number of worms result in suppression in the relative expression of M1 receptor mRNA.

Introduction Acetylcholine is a major neurotransmitter in the enteric nervous system. Cholinergic nerves mediate increased gut activity such as contraction and are associated with mucosal ion transport (Ratcliffe et al. 1998). As noted by Oue et al. (2000), despite the discovery of many excitatory and inhibitory neurotransmitters and neuromodulators, acetylG. J. Bikopoulos . T. Hoque . R. A. Webb (*) Department of Biology, York University, Toronto, ON, Canada, M3J 1P3 e-mail: [email protected] Tel.: +1-416-7365396 Fax: +1-416-7365876

choline remains the primary enteric neuronal regulator of gastrointestinal motility and mucosal secretory processes. Infection of the intestinal tract by invasive nematode parasites such as Trichinella spiralis stimulates remodeling of nerves and changes in the levels of neurotransmitters in the enteric nervous system, which ultimately results in effects on neuronal function (McKay and Fairweather 1997). Depolarization of jejunal smooth muscle/myenteric plexus (SM/MP) preparations from rats infected with T. spiralis resulted in less acetylcholine release compared to preparations from uninfected rats (Collins et al. 1989), an effect that persisted for about 40 days postinfection. Furthermore, Davis et al. (1998) demonstrated that acetylcholine production in SM/MP preparations in rats infected with T. spiralis decreased during the course of infection. Concomitant with these changes, acetylcholinesterase (AChE) activity decreased, while choline acetyltransferase (ChAT) activity increased. Similarly, T. spiralis infection was associated with significant suppression of electrical-field-stimulated or KClevoked release of norepinephrine (Swain et al. 1991). These studies collectively illustrate significant changes in neuronal function and neurotransmitter release following infection with nematodes that invade the lamina propria of the intestine. Conversely, infection of rats with Nippostrongylus brasiliensis, a primarily lumenal dwelling nematode, resulted in no significant change in intestinal muscarinic acetylcholine receptor agonist binding properties of smooth muscle cells or receptor density on enterocytes (Fox-Robichaud and Collins 1986), or the expression level of muscarinic (M)3 receptors of mucosal epithelial cells (Russell et al. 2000). In contrast to the many studies on invasive nematode infections, there have been no studies investigating enteric cholinergic functioning following infection with nontissue invasive enteral cestodes such as the rat tapeworm Hymenolepis diminuta. However, infection of rats with H. diminuta induces mastocytosis, hypertrophy of the enteric smooth muscle, alteration of enteric myoelectric activity, and slowed intestinal transit (Dwinell et al. 1994, 1997, 1998), which indicate changes in enteric nerve functioning.

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To test the hypothesis that enteral cestode infection of the rat small intestine modulates the cholinergic portion of the enteric nervous system, we compared synthesis, release, and breakdown of acetylcholine from the SM/MP of uninfected rats with rats infected with five H. diminuta and the relative expression of M1, M2, and M3 receptor mRNA in uninfected rats and rats infected with five or 50 worms.

Materials and methods Host animals and parasite infection Male Wistar rats of the same age, weighting approximately 200 g, were infected with five or 50 cysticercoids of the tapeworm H. diminuta as described previously (Webb 1991) and fed ad libitum on Purina laboratory chow with unlimited access to water. To study acetylcholine metabolism, rats infected with five worms for 4 weeks and uninfected age-matched controls were lightly anesthetized and killed by cervical dislocation. The jejunum was rapidly removed and cut into four 8-cm segments. Each segment was passed over a glass rod. A dulled scalpel blade was used to score the jejunum on the serosal surface, and the SM/MP was carefully peeled from the underlying tissue using a cotton swab. Preparations were maintained in Krebs buffer aerated with 95% oxygen and 5% carbon dioxide. To study the expression of muscarinic mRNA expression, rats infected with five or 50 worms for 30 days were killed by cervical dislocation, and tissues from the jejunum were collected, frozen in liquid nitrogen, and stored in −80°C until used. Age-matched uninfected rats were killed, and the tissues were collected and stored as above. Choline uptake Preparations of SM/MP were equilibrated for 15 min in Krebs buffer containing the following: 120.9 mM NaCl, 5.9 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 15.5 mM NaCO3, 1.2 mM NaH2PO4, 11.1 mM glucose, and 0.1 mM eserine. The preparations were then aerated with 95% oxygen and 5% carbon dioxide. [Methyl-3H] Choline, 0.2 μM (80 Ci mmol−1) and [14C] polyethylene glycol (PEG) (0.1 μCi ml−1, 0.4% PEG) were added, and preparations were incubated at 37°C for 1 h. Following incubation, the preparations were rapidly washed three times with choline-free buffer and solubilized with 0.5 ml solvable tissue solubilizer (New England Nuclear). Ten milliliters of ACS (Amersham Corp) was added, and the samples were counted for 3H. The amount of [3H] choline taken up by the tissues was determined using [14C] PEG to estimate choline diffusing into the extracellular spaces, as described by Webb (1991). Highaffinity neuron-specific uptake of [3H] choline was inhibited by preincubation for 30 min with Krebs buffer containing 10 μM hemicholinium-3 followed by incubation at 37°C for 1 h in [methyl-3H] choline, 0.2 μM (80 Ci mmol−1), [14C] PEG (0.1 μCi ml−1, 0.4% PEG) in Krebs buffer containing 10 μM hemicholinium-3.

Choline acetyltransferase activity Homogenates of the SM/MP preparations were homogenized in 10 mM EDTA phosphate buffer (pH 7.4) using a Polytron at 4°C. ChAT activity was then assayed by a modification of the method of Fonnum (1974). The homogenates were activated with 0.5% (v/v) Triton X-100 to solubilize the enzyme. Two microliters of homogenate was added to 5 μl of incubation mixture in a 1-ml centrifuge tube. The incubation mixture contained (final concentration) 20 mM [acetyl-1-14C] coenzyme A (125,000 dpm), 300 mM NaCl, 50 mM sodium phosphate buffer (pH 7.4), 8 mM choline bromide, 20 mM EDTA (pH 7.4), and 0.1 mM eserine. The resulting solution was mixed and incubated for 15 min at 37°C in a water bath. The contents of each tube were transferred to a 20-ml vial containing 5 ml of 10 mM sodium phosphate buffer (pH 7.4) and 2 ml acetonitrile containing 10 mg of sodium tetraphenyl boron. The vial was then vortexed for 10 s, and 10 ml of toluene-based scintillation cocktail was added. This allowed for the differential partitioning of [14C] acetyl–CoA and [14C] acetylcholine, with the latter extracted into the toluene (organic) phase and the former remaining in the aqueous phase, which was discarded. The 14C content was determined by liquid scintillation counting. Results were expressed as picomoles of [14C] acetylcholine per minute per centimeter of tissue. Acetylcholine production Smooth muscle/myenteric plexus preparations were incubated at 37°C with [3H] choline as described above. Tissues were washed with choline-free buffer and homogenized in 0.7 ml of 3-heptanone tetraphenyl boron (10 mg ml−1) and 0.35 ml HCl (1 M) containing eserine (0.1 mM) using a handheld glass homogenizer. The samples were centrifuged (9,800×g) for 10 min at 2°C. The aqueous phase was evaporated to dryness in a vacuum centrifuge. The dried extracts were dissolved in 30 μl of water that contained adenosine triphosphate (ATP) (0.8 mM), dithiothreitol (5 mM), MgCl2 (12.5 mM), glycylglycine (25 nM, pH 8.3), and choline kinase (0.005 U) and incubated at 37°C for 15 min to phosphorylate free choline. The labeled acetylcholine was then extracted using 2 ml of 3-heptanone tetraphenyl boron (10 mg ml−1) in 5 ml of 10 mM sodium phosphate buffer (pH 7.4). The organic phase recovered contained [3H] acetylcholine, whereas the aqueous phase retained the [3H] free choline as choline phosphate. Aliquots of the organic and aqueous phases were separately transferred to 20-ml scintillation vials containing 10 ml of ACS (Amersham Corp) so that the tritium content of acetylcholine and free choline in the preparations could be determined. Acetylcholinesterase activity Total cholinesterase and AChE activity was assayed. Smooth muscle/myenteric plexus preparations were homogenized in 3 ml sodium phosphate buffer (pH 7.4) using a handheld glass homogenizer in an ice bath. For determination of total

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cholinesterase activity 0.5 ml of 3 mM 5,5′-dithio-bis (2-nitrobenzoic acid) (dissolved in 50 ml of 80 mM Tris– HCl buffer, pH 7.0) was mixed with 1.78 ml of Tris–HCl buffer, pH 7.0. Acetylcholinesterase activity was measured in the presence of tetraisopropylpyrophosphoramide (ISO-OMPA), an inhibitor of butyrylcholinesterase; 0.5 ml of 3 mM dithiobisnitrobenzoic acid (DTNB) and 30 μl ISO-OMPA (10 μM) were added to 1.75 ml Tris–HCl buffer (pH 7.0) followed by 0.1 ml homogenate and vortexed. After 15 min preincubation the reaction was started by the addition of a 0.75-ml acetylthiocholine iodide (1 mM in 50 ml of Tris–HCl buffer, pH 7.0). The change in absorbance (Δ) (405 nm) was recorded every 10 s for 2 min. Measurements were performed in duplicate. A blank (without enzyme) was also assayed to follow any spontaneous changes in absorbance. Total esterase, AChE, and pseudocholinesterase activity was calculated from linear regression of the data and was calculated as Δ absorbance per minute per centimeter tissue. Determination of muscarinic receptor expression by semiquantitative polymerase chain reaction Total RNA was extracted from jejunal tissue and purified using Trizol reagent (Gibco-BRL) following the manufactures’ protocol. Extracted RNA was treated with DNase (Ambion). Integrity of RNA was assessed by analysis of 28S and 18S ribosomal RNAs after electrophoresis. Purified RNA (5 μg) samples were reverse transcribed with Superscript II reverse transcriptase (RT; Invitrogen). Gene-specific M1, M2, and M3 receptor primers were designed using the sequence deposited in the gene data bank. Negative RT controls were performed each time. The primers used for muscarinic receptor amplification were as follows: Target GenBank gene accession # M1 M2 M3

Sequence

NM_080773 5′-acagctggccaagagaaaga-3′ 5′-gagctgggctactggctatg-3′ AB017655 5′-gaacattgtagcccgcaaatc-3′ 5′-tgccaccttcaaaaagactttt-3′ M62826 5′-ctgcccaaagagcataccaaac-3′ 5′-cagtcactcagttgggcagcta-3′

Location 1041–1060 1201–1020 1074–1095 1332–1353 2043–2064 2229–2250

A semiquantitative RT–polymerase chain reaction (PCR) assay was used to determine the relative amount of mRNA of M1, M2, and M3 receptors in duplicates of the total RNA extracted from the jejunum of rats from the different treatment groups. The PCR products were separated on agarose gel and visualized with ethidium bromide under UV light. The base pair sizes of the PCR products were as expected for each muscarinic receptor mRNA subtype (see Fig. 5). DNA fluorescence in ethidium-bromide-stained gels was densitometrically scanned using a video documentation system and processed with the image analysis software Intelligent Quantifier (BioImage Systems Corp., Ann Arbor, MI).

The mRNA expression levels found in each tissue were normalized to relative mRNA expression levels microgram RNA per microgram total RNA extracted per linear millimeter of intestine. Reagents Unless stated otherwise all reagents and drugs were purchased from Sigma Chemical Co. (Toronto, Canada). The reagents were freshly prepared and diluted to achieve the final concentration indicated. Statistical analysis Results for experiments involving acetylcholine metabolism are expressed as mean±SEM (N=7). Sample means from infected and uninfected animals were analyzed using Student’s t tests following a variance ratio test (F test) that showed the variances of the two populations were not significantly different. Statistical significance was achieved where P was ≤0.05. The relative amounts of mRNA expression of the target gene in all treatments (mean±SEM; N=7) were statistically analyzed by one-way analysis of variance (ANOVA), with a level of significance set at P≤0.05. Where a significant difference was found, the group significantly different from the control was determined by Tukey tests.

Results Choline uptake Total choline uptake into infected SM/MP preparations was significantly higher than that in uninfected preparations (see Fig. 1). This value reflected the neuron-specific and the nonneuronal uptake of choline into the tissue. Incubation of uninfected SM/MP preparations with the high-affinity choline uptake inhibitor hemicholinium-3 caused a significant reduction in the amount of choline taken up to 25% of the total uptake. The neuron-specific choline uptake was therefore approximately 75% of the total uptake. Similarly, incubation of SM/MP preparations from rats infected with five worms with hemicholinium-3 resulted in a reduction in the uptake of choline to 27.5% of the total uptake. The levels of the neuron-specific and nonneuronal choline uptake in SM/ MP preparations from infected rats were significantly greater than that from SM/MP preparations from uninfected rats (P<0.05) (Fig. 1). ChAT activity The levels of ChAT activity in SM/MP preparations were determined from jejuna of uninfected rats and rats infected with H. diminuta. The mean ChAT activity from uninfected preparations was 31.38±5.72 (mean±SEM) pmols min−1 cm−1, while that from preparations derived from tapeworm-infected rats was 61.47±9.28 (mean±SEM ) pmols min−1 cm−1 (P<0.05) (Fig. 2). Thus, tapeworm infection significantly elevated ChAT activity levels in SM/MP preparations. Acetylcholine synthesis The conversion of [3H] choline into [3H] acetylcholine in SM/MP preparations was determined. The proportion of [3H] choline converted to [3H] acetylcholine, expressed as a percentage of total [3H] choline in the

234 100000

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Fig. 1 Uptake of [3H] choline in SM/MP preparations from the jejunum of uninfected and tapeworm-infected animals. The highaffinity uptake inhibitor hemicholinium-3 caused a significant reduction in [3H] choline uptake by blocking the neuron-specific component of choline uptake. Values shown are means±SE (N=7) (P≤0.05)

tissues, was not significantly different between infected [14.14±4.32% (mean±coefficient of variation)] and uninfected [13.83±5.52% (mean±coefficient of variation)] animals. However, the amount of [3H] choline taken up by the infected SM/MP preparations was twice that of the uninfected control, and thus, the total amount of [3H] acetylcholine synthesized in SM/MP preparations from infected animals was significantly higher than that from uninfected animals (Fig. 3). Total cholinesterase, AChE, and pseudocholinesterase activity Total cholinesterase, AChE, and pseudocholinesterase activities in SM/MP preparations from uninfected and infected animals are shown in Fig. 4. The data reveal that 80

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Fig. 3 Acetylcholine content of SM/MP preparations from jejunum of uninfected and infected animals. Values shown are means±SE (N=7) (P≤0.05)

AChE and total cholinesterase activity in uninfected animals was not significantly different from that observed in infected animals. However, pseudocholinesterase activity was significantly higher in parasitized rats. Relative expression of muscarinic receptor mRNA The highest level of relative expression of mRNA for the three muscarinic receptors (M1, M2, and M3) examined in uninfected rats was associated with the M1 receptor, which was approximately 60% of the total, with the balance approximately equally distributed in the M2 and M3 receptor mRNA (Fig. 5). Although infection of rats with five worms did not result in a significant elevation in M1 receptor mRNA expression, infection with 50 worms resulted in a significant diminution in relative expression to less than 50% of that observed in 5-worm infected rats and almost 30% less than that observed in uninfected rats (P<0.01). Conversely, while a significant elevation of M2 0.016 Infected

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Delta-Abs/min/cm

pmols C-Ach/min/cm

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Fig. 2 Choline acetyltransferase activity in SM/MP preparations from rat jejunum. Values shown are means±SE (N=7) (P≤0.05)

Total Cholinesterase

Acetylcholinesterase

Pseudocholinesterase

Fig. 4 Acetylcholinesterase activity in the rat jejunum of uninfected and parasitized animals. Data represent means±SE (N=7) (P≤0.05)

235 Fig. 5 The relative expression of muscarinic M1, M2, and M3 receptor mRNA in the jejunum of uninfected rat and rats infect with five or 50 H. diminuta for 30 days (columns left to right). Statistical data are shown as mean±SEM (N=6). *, P≤0.05; **, P≤0.01; ***, P≤0.001. An illustrative electrophoresis gel shows the relative expression of M1, M2, and M3 receptor mRNA transcripts of the appropriate expected size. The transcripts were visualized with ethidium bromide and scanned densitometrically. Un, uninfected; 5/30, five-worm-infected rats at 30 days postinfection; 50/30, 50-worm-infected rats at 30 days postinfection

receptor mRNA was observed in both five- and 50-worminfected rat jejuna compared to uninfected rats (P<0.05 and P<0.001, respectively), there was no significant difference in the relative levels of expression of M2 receptor mRNA between five- and 50-worm-infected rats (P>0.05). Finally, there were no significant changes in the relative expression of M3 receptor mRNA in infected rats compared to uninfected rats (P>0.05).

Discussion The present results clearly show that infection with a low number of noninvasive enteric tapeworms has a marked effect on precursor uptake, acetylcholine synthesis, pseudocholinesterase activity, and the relative expression of muscarinic receptor mRNA in the wall of the host intestine. These statistically significant changes in neurotransmitter synthesis and metabolism on infection of the intestine with a noninvasive cestode are similar to previous work with intestinal dwelling nematodes such as N. brasiliensis, which as adults may penetrate the mucosa, and the more invasive T. spiralis, where infection in rats with these organisms leads to significant changes in the functioning of the enteric cholinergic component (Collins et al. 1989; Davis et al. 1998; Masson et al. 1996). These changes in function include the regulation of intestinal motility and secretory responses (Collins 1996; McKay and Fairweather 1997), and may be reflected through remodeling of enteric nerves (Masson et al. 1996) and/or changes in the levels of neurotransmitters (Davis et al. 1998). The changes in

cholinergic activity in the present study may also reflect the marked changes in host enteric nerve activity and intestinal motility patterns seen during infection with H.diminuta (Dwinell et al. 1994, 1995, 1997, 1998) compared to uninfected rats, which are distinct from the highly propulsive motility associated with invasive nematode parasites (Palmer and Castro 1986). [3H] Choline taken up into myenteric neurons by the high-affinity transporter was significantly greater per unit length of SM/MP preparation from infected rats than those from uninfected rats. Although smooth muscle hypertrophy occurs following infection with H. diminuta (Dwinell et al. 1998), resulting in an increase in the biomass of SM/ MP preparations from infected rats, there is no significant change in intestine length. The increased choline uptake, ChAT activity, and an increase in the relative expression of M2 receptor mRNA per unit length of intestine may therefore reflect either greater cholinergic activity in neurons or cholinergic neuron remodeling. Nerve remodeling following helminth infection has been observed. For example, Stead et al. (1991) used antibodies to neuronspecific enolase (as a marker for all neurons) and to B-50 (as a specific marker for regenerating neurons) and showed that N. brasiliensis infection leads to a significant increase in the number of jejunal neurons. Similarly, nonspecific choline uptake into other cellular components was also significantly greater in SM/MP preparations from H. diminuta-infected rats, which in turn may reflect the increased muscle biomass seen in the infected intestine. Furthermore, the level of ChAT activity in SM/MP preparations was markedly increased during infection,

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which may reflect an increased abundance of ChAT or the appearance of more active isoforms (Cervini et al. 1994), which in turn might translate into increased intracellular synthesis of acetylcholine. This appeared to be the case where, although the rate of choline conversion to acetylcholine was of comparable magnitude between infected and uninfected animals, choline uptake was greater in infected SM/MP preparations, and more acetylcholine was synthesized in infected than in uninfected preparations. Alternatively, this could reflect changes in the turnover of newly synthesized acetylcholine. Unlike the elevation in ChAT activity, AChE and total cholinesterase activity in parasitized animals demonstrated no significant change compared to uninfected animals. In contrast, Davis et al. (1998) demonstrated decreased AChE activity in SM/MP preparations in rats infected with T. spiralis. This may be a reflection of the nematode being highly invasive and stimulating a high level of inflammation, whereas H. diminuta is noninvasive and does not stimulate high levels of inflammation (unpublished observations). However, pseudocholinesterase activity was elevated in SM/MP preparations from infected rats. Although increased levels of this enzyme were observed in rats infected with N. brasiliensis, the increased activity was associated with enterocytes (Russell et al. 2000). The distribution of muscarinic receptors and their respective mRNAs has been investigated in the gastrointestinal tract of mammals. Muscarinic 1 receptor mRNA is found in enteric nerves, with the receptors distributed predominantly if not exclusively on the enteric neurons (North and Surprenant 1985). In contrast, M2 receptors are associated with intestinal smooth muscle and enteric neurons (Takeuchi et al. 2005), while the M3 receptors are associated with intestinal smooth muscle and jejunal epithelium (Przyborski and Levin 1993). Although activation of prejunctional M1 receptors may enhance acetylcholine release (see review by Uchiyama and Chess-Williams 2004), activation of prejunctional M2 receptors leads to autoinhibition of acetylcholine release (Takeuchi et al. 2005). Binding studies have indicated that the uninfected rat intestinal M1 receptor comprises about 65% of the total, with an equal balance of M2 and M3 receptors (see Stadelmann et al. 1998). This compares with the present study, where the relative proportions of the M1 receptor mRNA in uninfected rats are approximately 60%, with the balance found equally in the M2 and the M3 receptor subtype mRNA. There have been relatively few studies on the affect of infection or pathological conditions on the relative abundance of either muscarinic receptors or their mRNAs. For example, viral infection of the lungs decreases prejunctional neuronal M2 receptors, leading to increased sensitivity of airway afferents (Jacoby 2004). In transplantation of segments of the small intestine in rats, M1 receptors decreased from approximately 70% to approximately 45%, while the M3 receptors increased from approximately 15% to about 42% (Stadelmann et al. 1998). However, there appears to be only one study on the effect of helminth infections on intestinal muscarinic receptors where infection

of rats with N. brasiliensis resulted in no significant difference between levels of expression of M3 receptor mRNA in epithelial cells from infected and uninfected rats (Russell et al. 2000). However, N. brasiliensis infection is known to induce a long-term mast cell hyperplasia which is associated with a structural plasticity of the intestinal wall, mucosal nerve remodeling, and increased excitability of enteric neurons (see Gay et al. 2001). Infection of rats with H. diminuta leads to disruption in the pattern of jejunal migrating myoelectric complexes (MMCs) (Dwinell et al. 1994). Given that acetylcholine remains the primary enteric neuronal regulator of gastrointestinal motility and mucosal secretory processes, it is possible that changes in acetylcholine function mediated by muscarinic receptors may contribute to the disruption in the MMC and the observed changes in intestinal motility. Although changes in receptor functional activity may not be reflected in changes in mRNA levels (see Fayon et al. 2005), changes in the relative expression of mRNA of muscarinic receptors can reflect changes in the expression of physiologically active muscarinic receptors (see, for example, Hao et al. 2005). Therefore, the changes observed in the relative expression of mRNA levels in the present study may reflect changes in muscarinic receptor functioning. The changes in the relative expression of M1 receptor mRNA suggest that infection leads to significant downregulation of expression of messenger or greater turnover of messenger in high-level infections compared to lowlevel infections, which may lead to diminution of M1 receptors in enteric neurons. Given that the initiation of MMC in dogs is under the inhibitory influence of M1 receptors (Fargeas et al. 1987), one may hypothesize that the MMC disruption seen in rats infected with H. diminuta may result from changes in M1 receptors. In contrast, M2 receptors on smooth muscle indirectly mediate an increase in contraction via inhibition of production of cyclic adenosine monophosphate (cAMP), thereby inhibiting adrenergic-stimulated relaxation (see Uchiyama and Chess-Williams 2004). The significant increase in M2 receptor mRNA may indicate changes in M2 receptor functioning activity. However, there were no significant changes in the relative expression of M3 receptor mRNA that leads to production of M3 receptors on smooth muscles where ligand interaction results in direct contraction. Clearly, although the present study shows significant changes in the relative expression of muscarinic receptor mRNA levels, a fuller understanding of these changes and the changes observed in neuron-specific uptake of choline and pseudoacetylcholinesterase requires a parallel examination of the receptor responsiveness of preparations of the SM/MP to receptor-specific agonists and antagonists of acetylcholine. In conclusion, this study is the first to show that low-level infections with the noninvasive enteric cestode H. diminuta lead to changes in enteric cholinergic activity that likely reflect neural functioning. The physiological mechanisms mediating these changes are not yet known, and the ramifications of these alterations are only beginning to be unraveled.

237 Acknowledgements This work was supported in part by a Natural Science and Engineering Research Council of Canada grant (number A6508) to R.A. Webb. The experimental protocols used in this study were approved by the York University Animal Care Committee in accordance with National Animal Care requirements.

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