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        Functional Imaging of Neurotransmitters in Hymenolepis diminuta Treated with Senna Plant Through Light and Confocal Microscopy
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        Functional Imaging of Neurotransmitters in Hymenolepis diminuta Treated with Senna Plant Through Light and Confocal Microscopy
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Abstract

Previous studies have shown the anthelmintic efficacy of Senna alata, Senna alexandrina and Senna occidentalis on the zoonotic parasite Hymenolepis diminuta through microscopic studies on morphological structure. The present study is based on the light and confocal microscopic studies to understand if Senna extracts affect neurotransmitter activity of the parasites. A standard concentration (40 mg/mL) of the three leaf extracts and one set of 0.005 mg/mL concentration of the reference drug praziquantel were tested against the parasites, keeping another set of parasites in phosphate buffer saline as a control. Histochemical studies were carried out using acetylthiocholine iodide as the substrate and acetylcholinesterase as the marker enzyme for studying the expression of the neurotransmitter of the parasite and the staining intensity was observed under a light microscope. Immunohistochemical studies were carried out using anti serotonin primary antibody and fluorescence tagged secondary antibody and observed using confocal microscopy. Intensity of the stain decreases in treated parasites compared with the control which implies loss of activity of the neurotransmitters. These observations indicated that Senna have a strong anthelmintic effect on the parasite model and thus pose as a potential anthelmintic therapy.

Footnotes

Cite this article: Ukil B, Kundu S and Lyndem LM (2018) Functional Imaging of Neurotransmitters in Hymenolepis diminuta Treated with Senna Plant Through Light and Confocal Microscopy. Microsc Microanal. 24(6), 734–743. doi: 10.1017/S143192761801526X

Introduction

Neurotransmitters of helminthes are among the principal targets of different anthelmintic drugs. Their expression and concentration in the nervous system would contribute in understanding the organization and functioning of the central nervous system (CNS), and is a prerequisite to the design of novel anthelmintic agents through impediment of the worm’s neuromuscular physiology. The determination of choline, particularly acetylcholine (ACh) is of interest for clinical and analytical purposes. ACh, the first identified neurotransmitter, is found in both peripheral nervous systems (PNS) and CNS in mammals, including humans (Liu et al., 2005), while serotonin or 5-hydroxytryptamine (5-HT) acts both as neurotransmitter in CNS and as a hormone (Yadav, 2013). ACh is synthesized in neurons from choline and acetylcoenzyme-A by choline acetyltransferase (ChAT) and serotonin is synthesized from the amino acid l-tryptophan, which is first converted to 5-hydroxytryptophan by the enzyme tryptophan hydroxylase and then to 5-HT by an aromatic l-amino acid decarboxylase. Since tryptophan hydroxylase is predominant in gastrointestinal tract and in brain, these areas are the major site of serotonin production and storage (Feldman, 2004). In helminthes, ACh and 5-HT are localized in many of the organ systems of trematodes and cestodes (Bennett & Bueding, 1971; Lee et al., 1978; Maule et al., 1990; Samii & Webb, 1990). The recorded responses of platyhelminths to the various neurotransmitter agonists and antagonists are usually limited to an analysis of the neurotransmitter’s effect on the basal motor activity of the parasite. Thus, most helminthes contract and relax at a given rate, but when they are incubated in the presence of 5-HT the rate of this contractile activity increases (Chance & Mansour, 1953; Barker et al., 1966; Pax et al., 1984). Larval motility in Mesocestoides corti and Hymenolepis diminuta incubated in serotonin was found to be increased with a 100 nM concentration and above, and excitatory effects were less pronounced below 100 nM, whereas, ACh concentration pronounced stimulation of motility of tetrahydria at 100 µM, but at higher concentration motility becomes transient (Sukhdeo et al., 1984; Hrčkova et al., 2002). Both ACh and 5-HT have excitatory effects at different concentrations and temperatures in different groups of flatworms. The activity of the enzymes (ChAT and acetylcholinesterase (AChE)) involved in the synthesis and degradation of helminth ACh have been detected in Fasciola hepatica (Sukhdeo et al., 1986) and Schistosoma mansoni (Bueding, 1952) where the activity of the esterase is about 200-fold greater than the transferase. Most of the esterases specific for ACh appeared to be associated with the parasite’s CNS and PNS (Bueding et al., 1966; Ramisz, 1967). AChE inhibitors are usually found to block the enzyme activity leading to the accumulation of synaptic ACh and consequently enhanced stimulation of postsynaptic cholinergic receptors in the CNS/PNS. Irreversible inhibitors on the other hand, have toxicological relevance with ACh accumulation in the synapse and loss of neurotransmission (Ĉolović et al., 2013). Cholinergic receptors and receptors for 5-HT have been the targets for nematocidal, trematocidal, and cestocidal drugs (Geary et al., 1992). Drugs like imidazoles, tetrahydropyrimidine derivatives (pyrantel, oxantel, morantel), tribendimidine, and spiroindoles target nicotinic ACh receptors (Robertson et al., 2002; Kopp et al., 2008; Hu et al., 2009; Sattelle, 2009). Amino-acetonitrile derivatives (monepental) target choline receptors in nematodes (Kaminsky et al., 2008; Sager et al., 2009) while imipramine, citalopram, fluoxetine, fluvoxamine, aroxetine, and sertraline target serotonin transporters in S. mansoni (Crisford et al., 2011).

Senna alata Linn., Senna alexandrina Mill., and Senna occidentalis (L.) Link (Leguminosae) are three plant species of Senna that possess multiple medicinal properties including antimicrobial, antidepressant, antianxiety, and antibacterial (Sermakkani & Thangapandian, 2012; Khare et al., 2017; Harsha et al., 2018). Senna leaves and pods contain sennosides A, B, C, D, and G, rhein, kaemferin, and isorhein in free and compound glycoside form (Srivastava et al., 1983). These three species of Senna plant have flavonoids, terpenoids, anthraquinones, and saponins as their essential ingredients that possess antimicrobial and antifungal as well as laxative potencies (Viswanathan and Nallamuthu, 2012; Odeja et al., 2015; Karthika et al., 2016; Khare et al., 2017). Recently, the extract of these three plants have shown significant alterations in the morphology of the tegument of H. diminuta (Kundu et al., 2012, 2015, 2016). In the absence of an organized body cavity and circulating body fluid, flatworms lack the capability of classical endocrine cellular communication and therefore the coordination of movement, behavior, metabolism, and reproduction must be directed by the nervous system (Ribeiro et al., 2005). With neural organization of anterior cephalization and a centralized nervous system, flatworms have more directive and highly developed motor systems than their radially symmetrical ancestors (Day & Maule 1999). Alteration in the tegument leads to hyperpolarization and reduced excitability that could lead to muscle relaxation and flaccid paralysis (Voge & Bueding, 1980; Leitch & Probert, 1984; Martin, 1985). The musculature of helminthes being innervated by nerves makes a neuromuscular system immensely important for motility and attachment of the parasite within the host (Geary et al., 1992). Thus, early paralysis caused by Senna leaf extracts may signify probable effect of the extracts on the neuromuscular coordination of the worm which thus evokes the necessity of exploring the neurotransmitters.

Understanding the physiology of various neurotransmitters through microscopic techniques facilitated the identification of several classical neurotransmitters with explorations of the facets of their innervations into tegument and musculature (Halton, 2004).

Materials and Methods

Preparation of Plant Extracts

Fresh young leaves of S. alata, S. alexandrina, and S. occidentalis were collected from in and around the University campus. The leaves were weighed and thoroughly washed with distilled water and oven-dried at 50°C. About 250 g of the powdered leaves were extracted with 1 L of ethanol (90%) in a Soxhlet apparatus for 7–8 h. The final crude extract (17.25 g) was recovered using a rotary evaporator and stored at 4°C until further use.

Drugs and Chemicals

Chemicals for histochemical studies were obtained from Merck (NJ, USA). Acetylthiocholine iodide (ATCI) and bovine serum albumin (BSA) were obtained from Hi-Media Laboratories Pvt Ltd. (Mumbai, India). Both primary and secondary antibodies for immunohistochemical studies were procured from Sigma-Aldrich (Bengaluru, India). Ethanol was obtained from Bengal Chemicals (Kolkata, India) and the reference drug praziquantel (Pzq) with the trade name Distocide (composed of 600 mg of Pzq) is a product of Chandrabhaghat Pharma Pvt Ltd. (Mumbai, India).

Collection of Parasite Samples

Hymenolepis diminuta was maintained in the laboratory following the method obtained from earlier studies (Kundu et al., 2012, 2015, 2016). In brief, apolysed proglottids collected from rat fecal samples were fed to prestarved flour beetle (Tribolium sp.) for development of larval stage cysticercoids. The latter were inoculated to adult male Sprague Dawley rats weighing 150–200 g. Adult H. diminuta were obtained from the intestine of the rats 21 days postinfection. All experiments with animals were approved by the Institutional Animal Ethics Committee of Visva-Bharati University.

Treatment of Worms

Live worms were treated in four different petridishes containing 40 mg/mL concentration of three plant extracts and 0.005 mg/mL of Pzq prepared in 0.1 M phosphate buffer saline (PBS) with 1% dimethylsulfoxide (DMSO), and kept in the incubator at 37°C until paralyzed. These two concentrations were derived from earlier studies (Kundu et al., 2012, 2015, 2016) as the worms paralyzed almost same time with the drug as depicted in Table 1. One set of worms was kept as control with only PBS and DMSO. After the parasites lost their motility, which was assured when the worms showed no movements even after shaking vigorously or keeping in slightly warmer PBS, they were removed from the treatment media and processed for histochemistry and immunohistochemical studies.

Table 1 Effects of Crude Leaf Extracts of Three Senna Plants and Praziquantel on Hymenolepis diminuta.

Modified table adopted from Kundu et al. (2012, 2015, 2016). Each value presented as mean±SD (n=6), PT=paralysis time (h); TM= time of mortality (h). Control showed survivability upto 69.22±0.03 h.

Skinning and Fixation

The control and paralyzed worms were kept in distilled water for 3–4 h at room temperature for skinning (removal of body surface) following the method of Gustafsson (1991). During this incubation period the parasite’s tegument gets disrupted due to osmotic pressure which allows smooth penetration of primary and secondary antibodies which is otherwise difficult in thick skinned tapeworms. The media became turbid, which confirmed that skinning has completed. After skinning, one set of worms were fixed in 10% neutral buffered formalin overnight at 4°C for histochemical study and another set of worms were fixed in 4% paraformaldehyde and kept overnight at 4°C for immunohistochemical study.

Histochemical Studies

Whole Mount Specimen

The AChE expression was observed using ATCI as the substrate following the staining protocol of Kemmerling et al. (2006) with some modifications. In brief the control and treated parasites were incubated in 20 mL of staining medium (ATCI-2.2 mg/mL added to a solution of 0.1 M acetate buffer at pH-6.3, 0.1 M sodium citrate, 5 mM potassium ferricyanide, and 30 mM copper sulfate) for ~2 h. However, time taken for staining (Hatchett’s brown) varies from scolex to proglottids. The following reaction is according to Karnovsky & Roots (1964).

$${\rm ATCI}\buildrel {{\rm AChE}} \over \longrightarrow \,{\rm Thiocholine{\plus}K}_{{\rm 3}} {\rm Fe}\left( {{\rm CN}} \right)_{{\rm 6}} \to{\rm K}_{{\rm 4}} {\rm Fe}\left( {{\rm CN}} \right)_{{\rm 6}} {\rm {\plus}Cu}^{{{\rm {\plus}{\plus}}}} \to{\rm Cu}_{{\rm 2}} \downarrow\,\,\left( {{\rm Hattchet's}\,\,{\rm Brown}} \right)$$

After staining, the parasites were observed under light microscope.

Parasite Sections

Sections of the fixed paralyzed specimen (in cold formalin overnight at 4°C) were cut at thickness of 10–15 µm. Sections were processed following the method of Gomori (1952), using standard incubation medium containing cupric sulfate (0.3 g), glycine (0.38 g), magnesium chloride (1.0 g), maleic acid (1.75 g), 48% aqueous sodium sulfate, and 4% aqueous NaOH (30 mL) as described by Pearse (1968). The sections were incubated for 30 min at pH 6–6.2 at 37±1°C, then washed several times and freshly mounted in glycerine jelly. The AChE activity was monitored by the presence of blackish brown color.

Biochemical Studies

AChE level was assayed following the method of Ellman et al. (1961) and Ott et al. (1975), where thiocholine reacts with dithiobis nitrobenzoic acid (DTNB) which was estimated spectrophotometrically.

A 10% tissue homogenate was prepared in 0.2 M sucrose solution with a Potter-Elvehjem motor-driven glass homogenizer (REMI, Mumbai, India) and a Teflon pestle at 0±2°C, centrifuged at 20,000 g at 4°C for 30 min and the supernatant was used as enzyme source. The assay mixture in a final volume of 3 mL contained 150 µM sodium phosphate buffer, pH 7.4, 10 mM ATCI, 1–5 mM DTNB, 0.3% Triton X-100, and 0.1 mL enzyme extract. The reaction mixture was incubated at 37±1°C for 3–5 min in a UV visible spectrophotometer (Beckman, CA, USA). The rate of enzyme reaction recorded at 412 nm and optical density (OD) was calculated and the specific activity of enzyme expressed in terms of nmoles thiocholine produced/min/mg protein. Thiocholine concentration of the sample was calculated using an extinction coefficient of 13,600/M/cm.

Immunohistochemical Studies

The immnunohistochemical study was concerned with the expression of the neurotransmitter serotonin (5-HT). Anti-5-HT antibody bound to the places of activity of the neurotransmitter. The intensity of fluorescence depicts the abundance of the neurotransmitter in the parasite. All the above incubation processes were carried out at 4°C.

Immunohistochemical study was performed following the standard method of Gustafsson (1991) with minor modifications. The fixed worms were kept for 24 h in 0.1 M PBS with 1% BSA, 0.1% Triton X-100, and 0.02–0.05% of sodium azide. Worms were incubated in primary antibody [rabbit anti 5-HT (1:500)] for 5–7 days. They were removed from the medium and washed in the same washing buffer for 24 h. After washing, the worms were kept in secondary antibody, that is, anti-serotonin [fluorescein isothiocyanate conjugated sheep anti-rabbit IgG (1:50)] for 20 h. Parasites were removed from the secondary antibody and washed for ~3 h in the same washing buffer mentioned and mounted in 50% glycerol and observed under Leica TCS SP8 (Hesse, Germany) confocal laser scanning microscope.

Results

Histochemistry of Parasite

In the control worms, deposition of Hatchett’s brown in scolex region was observed at 30 min post-incubation. Within this period, no deposition of stain was observed in the proglottid regions of the worm. However, deposition of Hatchett’s brown stain was noticed in the immature proglottids (proglottids attached to the scolex and neck region) 1.5 h post-incubation and in the mature proglottids 2 h after incubation. The cerebral ganglia were prominently seen at the base of the rostellum. Two pairs of nerve cords, the lateral nerve cords (LNC), the median nerve cords (MNC) as well as the transverse nerve connectives (TNC) stained dark brown. However, in the gravid region, Hatchett’s brown stain was not noticeable.

Scolex and Neck Region

The Hatchett’s brown stain was distinct in scolex and neck regions of control parasites with more deposition of the stain in the cerebral ganglia and the rostellar region. The suckers as well as the LNC, MNC, and TNC also took the stain color indicating presence of AChE (Fig. 1a). However, the intensity of the stain in treated parasites varied. Accumulation of dark blackish brown stain occurred in the scolex and nerve cords of Pzq and S. occidentalis treated parasites (Figs. 1b, 1e). While parasites treated with S. alexandrina showed a yellowish brown color different from the control and no deposition of stain in MNC (Fig. 1d) and in S. alata, the Hatchett’s brown stain deposited at the scolex, the LNC and the TNC, while no stain was noticeable in the MNC (Fig. 1c).

Figure 1 Histochemical localization of acetylcholinesterase in the scolex and neck region of H. diminuta. a: Control: brown stain in suckers (S), lateral nerve cords (LNC), median nerve cords (MNC), and transverse nerve connectives (TNC). b: Praziquantel: the sucker took blackish brown stain and discontinuity of stain in the LNC and MNC, and TNC. c: S. alata: dark brown stain in suckers but the stain faded in LNC, MNC, and TNC. d: S. alexandrina: yellowish brown stain in suckers and LNC, but very light brown stain in the MNC and TNC. e: S. occodentalis: dark blackish brown stain in the suckers and the nerve cords showed discontinuity in the stain.

Proglottids

In the proglottids segments of control parasites, all the three nerve cords LNC, MNC, and TNC took up the Hatchett’s brown stain and thus the cord structure became prominent (Fig. 2a). Parasites treated with Pzq showed blackish brown color and there was no uniformity of stain in the LNC, MNC as well as TNC (Fig. 2b). In S. alata treated worms, brown stain was present uniformly exposing the structure of the nervous system (Fig. 2c). In case of S. alexandrina, yellowish brown stain was observed and not the Hatchett’s brown (Fig. 2d). S. occidentalis like Pzq showed a blackish brown color which is not the Hatchett’s stain (Fig. 2e).

Figure 2 Histochemical localization of acetylcholinesterase in the proglottids of H. diminuta. a: Control: Hatchett’s brown stain is in the lateral nerve cords (LNC), median nerve cords (MNC), and transverse nerve connectives (TNC). b: Praziquantel: showed blackish brown stain with no uniformity in the LNC. The MNC was faintly stained and TNC were indistinct. c: S. alata: a uniform distribution of Hatchett’s stain in the LNC and MNC but TNC were light stained. d: S. alexandrina: yellowish brown stain in LNC and MNC. e: S. occidentalis: blackish brown stain color in the LNC with a faded stain in the MNC and absolutely indistinct TNC.

Histochemistry of Cryostat Sections

Brown deposits were observed in the tegument, subtegument, somatic musculature, and inner parenchyma of the control worm (Fig. 3a). But with S. alexandrina, faint stain was observed in the tegument, somatic musculature, and in the inner parenchyma (Fig. 3d). The intensity of the stain faded throughout the tegument and inner parenchyma in S. alata and S. occidentalis (Figs. 3c, 3e). Similar observation was seen in Pzq treated parasites (Fig. 3b).

Figure 3 Acetylcholinesterase expression in cryostat sections of H. diminuta. a: Control: brown stain in tegument, subtegument, and muscular layers. b: Praziquantel: intensity of brown color reduced. c: S. alata: reduction in the stain. d: S. alexandrina: the stain faded inconsistently in the tegumental layers. e: S. occidentalis: depletion of stain and inconsistent deposition in the tegument and subtegument.

Biochemical Activity of AChE

The concentration of the neurotransmitter in the control and treated worms is expressed in graphical representation (Fig. 4) and in tabulated form (Table 2). Parasites treated with S. occidentalis showed 78% depletion in the specific activity compared with the control and almost double the depletion compared with Pzq (42%). S. alata showed about 15% reduction from the control whereas parasites treated with S. alexandrina showed increased enzyme concentration.

Figure 4 Graphical representation showing the concentration level of acetylcholinesterase (AChE) in H. diminuta. Paraistes treated with praziquantel, S. alata and S. occidentalis showed low level of AChE from control while the enzyme level increased in S. alexandrina.

Table 2 Effects of Crude Leaf Extracts of Senna Plants on the Specific Activity and Percentage Reduction of Acetylcholinesterase in Hymenolepis diminuta.

Values are expressed as mean±SD, where n=3.

Immunohistochemistry

Scolex and Neck Region

Confocal microscopic studies revealed the presence of serotonin in the nerve cords of the control parasite with uniform fluorescence (Fig. 5a). However, in drug treated parasites, fluorescence was not uniform and clumping occurred (Fig. 5b). In S. alata treated parasite, expression of the neurotransmitter was seen but inconsistent throughout (Fig. 5c). In S. alexandrina treated parasite, no fluorescence was observed in the scolex region (Fig. 5d), while in S. occidentalis, low fluorescence intensity was seen in the scolex (Fig. 5e). Antibodies specific to the neurotransmitter showed fluorescence in the nerve cords signifying the presence of serotonin in the nervous system of the parasite. Fluorescence was observed uniformly and consistently in the suckers, rostellar region, and cerebral ganglion as well as in the LNC, MNC, and TNC (Fig. 6a). No fluorescence was observed in the cerebral ganglion and suckers even at higher magnification for Pzq, but MNC, LNC, and TNC exhibited mild and discontinuous fluorescence (Fig. 6b). In S. alata treated parasite, fluorescence was clumped at the rostellar region, less fluorescence was observed around the sucker but there was no trace of fluorescence in the cerebral ganglion (Fig. 6c). In S. alexandrina, both rostellum and cerebral ganglia showed no fluorescence (Fig. 6d). While in S. occidentalis, serotonin was observed in the cerebral ganglia and all around the suckers (Fig. 6e).

Figure 5 Immunohistochemical localization of 5-hydroxytryptamine in the scolex region of H. diminuta. a: Control: intense fluorescence in the suckers (S) and lateral nerve cords (LNC). b: Praziquantel: fainted fluorescence seen only in the suckers. c: S. alata: fluorescence was observed in suckers and LNC. d: S. alexandrina: negligible fluorescence in the suckers and light fluorescence in the LNC. e: S. occidentalis: scattered fluorescence was observed only in suckers.

Figure 6 A magnified view of the scolex and the neck region of H. diminuta for immunohistochmistry. a: Control: rostellum (R), suckers (S), cerebral ganglia (CG), median nerve cords (MNC), lateral nerve cords (LNC), and transverse nerve connectives (TNC) were brightly fluorescent. b: Praziquantel: no stain in rostellum but negligible fluorescent in CG, MNC, and TNC. c: S. alata: rostellum showed fluorescence but no exhibition in the CG, intensity of fluorescence faded in MNC and TNC. d: S. alexandrina: faint fluorescence seen in rostellum and no fluorescence found in CG but light fluorescence seen in MNC, LNC, and TNC. e: S. occidentalis: rostellum and CG showed fluorescence but the MNC, LNC, and TNC faintly stained.

Proglottids

A continuous and bright fluorescence throughout the LNC of the mature proglottids was observed in control parasites (Fig. 7a). While in Pzq and all plant treated parasites, no fluorescence in the mature proglottids, rather a complete dull green fluorescence indicating low serotonin (Figs. 7b–7e). At higher magnification LNC and TNC were prominently distinguished (Fig. 8a). No fluorescence was observed in the different nerve cords of Pzq, S. alata and S. alexandrina treated parasites (Figs. 8b–8d). However, in S. occidentalis, faint and discontinuous expression of serotonin was seen in LNC and TNC (Fig. 8e).

Figure 7 Immunohistochemical localization of 5-hydroxytryptamine in the proglottids of H. diminuta. a: Control: showed uniform dotted fluorescent lines throughout the, lateral nerve cords (LNC) showing expressions of the neurotransmitter. b: Praziquantel (Pzq): no fluorescence was observed throughout the proglottids. c: S. alata, (d) S. aexandrina, and (e) S. occidentalis: showed similar observations as Pzq.

Figure 8 A magnified portion of the proglottids of H. diminuta. a: Control: exhibition of accurate fluorescence in the lateral nerve cords (LNC), median nerve cords (MNC) and transverse nerve connectives (TNC), (b) Praziquantel, (c) S. alata, and (d) S. alexandrina: no fluorescence throughout the proglottid. (e) S. occidentalis: very faint dots of flourescence in the LNC and scattered dots of fluorescence observed in TNC.

In the gravid proglottids of the control parasites, serotonin expression was seen in the cirrus sac along the lateral margin of the proglottid (Fig. 9a). However, no trace of the neurotransmitter was observed in all the treated parasites (Figs. 9b–9e).

Figure 9 Expression of fluorescence in the gravid proglottids of H. diminuta. a: Control: fluorescence in the cirrus sac (C) with no visibility of the nerve cords. b: Praziquantel, (c) S. alata, (d) S. aexandrina, and (e) S. occidentalis: exhibited no fluorescence in the cirrus sac.

Discussion

Confocal microscopy serves as a high resolution optical microscope with an infinite depth of field as described by Hovis & Heuer (2010). It allows perfect imaging with nondestructive segments within whole mount specimens (Johnston et al., 1990). The immunohistochemical observations showed the specific activity of 5-HT as antibodies is more specific than any other staining media. In the present study, histochemical localization of AChE could be visualized and discerned. The whole mounted parasites showed presence of neurotransmitters in a nerve net position. Similar architecture of the cestode parasite H. diminuta was observed by Wilson & Schiller (1969) through light microscopic studies using the enzyme AChE as a marker.

Confocal microscopic studies on the general body plan, musculature, organs, neuroanatomy, and reproductive system of H. diminuta in the cyst as well as the adult stage were carried out by Rozario & Newmark (2015), using various staining techniques and revealed a series of rings in the rostellar region as was reported by Wilson & Schiller (1969). This was not visible through the synapsin staining technique that mostly reveals frame by frame view of innervations in the adult scolex. The present study concerns with understanding the functional status of the neurotransmitters in the plant treated parasites. Histochemical staining showed the presence of AChE in the rostellum, rostellar rings, and the suckers. In confocal microscopic studies, the cerebral ganglia and the rostellum were markedly distinct, which revealed the nerve composition in the scolex region. The staining intensity was lost in both drug and plant treated parasites signifying depletion of AChE which was also observed biochemically. Specific cholinesterase (ChE) is closely associated with the CNS and PNS of the cestodes (Maule et al., 2002). Depletion of ChE due to exposure to synthetic and natural anthelmintics has been reported by many workers (Tin et al., 1994; Pal & Tandon, 1998). AChE activity was blocked in nematode parasite Trichuris muris and Ancylostoma caninum treated with organophosphate complexes (Sundaraneedi et al., 2018). Thus, anthelmintics including organophosphates and Pzq may have neuromuscular mode of actions (Cox, 1994) by interfering with transmission at nerve–nerve synapse or at neuromuscular junction by inhibiting the enzyme AChE. The paralysis that occurred in the treated parasite may possibly be due to depolarization of neuromuscular blockage and sustained muscle contraction as observed by Coles et al. (1975) and Aubry et al. (1980). As AChE has an inhibitory effect on motility of H. diminuta, as recorded by Sukhdeo et al. (1984) and Thompson et al. (1986), hence, the depletion and absence of stains on the parasite treated with Senna extracts thus describes depletion of neurotransmitters, leading to loss of grip on the site of attachment and hence a vermifugal action may ensue.

Stain as well as fluorescence became scattered in treated parasites. The confocal microscopic study showed either low or absence of serotonin in all the treated parasites throughout the scolex and proglottid regions. This depletion of serotonin may lead to abnormality in neurotransmission which can further alter the motility of the parasites. Such type of abnormal behaviour was also reported in rats treated with serotonin inhibitors (Molina et al., 1987).

Though all the three plants showed low or absence of both AChE and serotonin in the parasite, in some parts of the parasite stain uptake varies. Difference in staining observation could be due to variation in the phytochemical composition of the different plant species.

Conclusion

The study here revealed the advantage of microscopic techniques in identifying the functional status of AChE and 5-HT in the nervous system of the parasite. Due to this advantage, our study on the nervous system of H. diminuta did not remain just on histochemical studies but was also supported by the immunohistochemical observation of neurotransmitters of the parasite using confocal microscopic technique. Such techniques facilitate the identification of the presence of the neurotransmitter serotonin or 5-HT uniformly throughout the whole parasite of H. diminuta from the scolex to the gravid proglottids revealing the physiological function of the neuromuscular system, but after plant treatment, 5-HT was partly seen in the scolex and totally lost in the proglottids, indicating a loss of expression of 5-HT and suggesting the prerequisite site of anthelmintic agents. The same was seen for AChE in the light microscopic study, thus forming an interest for clinical and analytical purpose. Both confocal and light microscopic studies strongly suggest the antagonistic effects of three plants on the two neurotransmitters which are the basal motor activity of the parasite. From the biochemical study, the concentration of AChE was altered in treated plants from the control, which can only add to the suggestion that neurotransmitters are disrupted against treatment with Senna plants. Further, the effects of treatment on the parasite by the three plants varies, in which, S. occidentalis showed the maximum effect followed by S. alata and S. alexandrina strongly supported that each plant contain different phytochemical compounds with different concentrations. Reduction or depletion of the two transmitters AChE and 5-HT on the treated parasites indicates the inhibitiory action of the three species of Senna plants on the neurotransmitters of the parasite and thus showed their anthelmintic potency on the neuromuscular activity, which is a major aspect that allows the survivability of the parasite in the host especially their motility and attachment within the host intestine.

Acknowledgment

The authors acknowledged the University Grants Commission, New Delhi, India for providing financial support to the first author (grant no. F.No. 25-1/2014-15 (BSR)/5-132/2007/(BSR)).

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