Possible presence of TRPVin shorthorn sculpin(Myoxocephalus scorpius)

Optimization of the immunohistochemical protocol

Jakob Lundin

Degree project for Bachelor of Science inBiology

BIO602 Biology: Degree project 15 hec Spring 2015

Department of Biological and Environmental SciencesUniversity of Gothenburg

Examiner: Elisabeth Jönsson BergmanDepartment of Biological and Environmental Sciences

University of Gothenburg

Supervisor: Catharina OlssonDepartment of Biological and Environmental Sciences

University of Gothenburg

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Contents Abstract ............................................................................................................................................ 4 Sammanfattning ............................................................................................................................... 4 Introduction ...................................................................................................................................... 5

The TRP Family ........................................................................................................................... 5 The roles of TRPs......................................................................................................................... 5 TRPs and thermoregulation.......................................................................................................... 5 The TRPV subfamily ................................................................................................................... 6 Sensory neurons - distribution and function ................................................................................ 7 TRPV in non-mammalians ........................................................................................................... 8 Aims of this study ........................................................................................................................ 8

Material and methods ....................................................................................................................... 8 Preparing tissue ............................................................................................................................ 9 Immunohistochemistry ................................................................................................................. 9 Data Analysis ............................................................................................................................. 10

Results ............................................................................................................................................ 11 General staining of the myenteric plexus ................................................................................... 11 Evaluation of the TRPV1 antibody ............................................................................................ 13 Identification of calbindin positive nerve cells .......................................................................... 14

Discussion ...................................................................................................................................... 16 Conclusion.................................................................................................................................. 17

Acknowledgement.......................................................................................................................... 18 References ...................................................................................................................................... 18

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Abstract Transient receptor potential channels (TRPs) together constitute a very diverse protein ion channel family consisting of seven subfamilies. Their function ranges from sensory perception and monitoring organ function to regulating intestinal motility and nutrient absorption. The TRP of focus in this study is TRPV. The TRPV subfamily includes six forms (TRPV1-6) where 1-4 is involved in heat sensation. The distribution and function of TRPV in mammals is increasingly well known. This creates a base of knowledge which makes it possible to speculate regarding its roles and location in other less known animal groups. The distribution of TRPVs in fish is to date still not well understood as studies have only been performed in zebrafish. This study thus aims to evaluate the presence of TRPV in the gastrointestinal tract of shorthorn sculpin (Myoxocephalus scorpius) using immunohistochemistry. The results showed no immunoreactivity of TRPV in any of the regions of the gastrointestinal tract. Attempts at optimizing the method included using various concentrations and different secondary antibodies. It should be noted that the general nerve markers Hu and AcT showed strong immunoreactivity of both nerve cells and fibers in the tissue. The lack of specific labelling could be due to the primary antibodies (targeting TRPV) not binding or being defect, unoptimized methods or simply the absence of TRPV in the gastrointestinal tract of M. scorpius. However, considering the recent findings regarding TRPV in zebrafish and a diversity of other vertebrates, the latter theory seems unlikely. Repeated studies with another set of antibodies as well as a refinement of the immunohistochemical protocol is suggested in order to further determine the possible presence of TRPV in M. Scorpius, and other fish species as well.

Sammanfattning Transient Receptor Potential-kanaler (TRP) utgör en mycket varierad jonkanalfamilj bestående av sju underfamiljer. Deras funktion varierar från sensorisk perception och övervakning av organfunktion till att reglera tarmmotiliteten och näringsupptag. TRP-underfamiljen i fokus i denna studie är TRPV. TRPV-underfamiljen innefattar sex former (TRPV1-6) där 1-4 är involverade i värmeperception. Utbredningen och funktioner av olika TRPV:er i däggdjur blir alltmer beskrivet. Detta skapar en kunskapsbas som gör det möjligt att spekulera om deras roller och plats i andra mindre kända djurgrupper. Fördelningen av TRPV:er i fisk är hittills fortfarande inte helt förstådda eftersom omfattande studier endast har utförts på zebrafisk. Denna studie syftar till att utvärdera förekomsten av TRPV i mag-tarmkanalen hos rötsimpa (Myoxocephalus scorpius) med hjälp av immunhistokemi. Resultaten visade ingen immunoreaktivitet av TRPV i någon av regionerna i mag-tarmkanalen. Försök att optimera metoden innefattade användning av olika koncentrationer av primära antikroppen mot TRPV och olika sekundära antikroppar. Det bör noteras att de allmänna nervmarkörerna Hu och AcT visade stark immunoreaktivitet av både nervceller och fibrer i vävnaden. Bristen på specifik infärgning kan bero på att de primära antikropparna inte binder in, är defekta, användning av icke optimerade metoder eller frånvaro av TRPV i mag-tarmkanalen hos M. scorpius. Med tanke på de senaste resultaten beträffande TRPV i zebrafisk och en mångfald av andra ryggradsdjur, verkar den senare förklaringen osannolik. Upprepade studier med en annan uppsättning av antikroppar samt en förbättring av det immunhistokemiska protokollet föreslås för att ytterligare fastställa eventuell förekomst av TRPV i M. scorpius, och även andra fiskarter.

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Introduction The TRP Family TRPs (transient receptor potential channels) constitute a large group of ion channels located on the plasma membrane on various tissues found in various organisms. TRP channels are categorized based on amino sequence rather than ligand selection or function simply because of their vast variety and complexity (Moran et al., 2004).They are divided into seven subfamilies that exhibit different functions. These are the TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin),TRPML (mucolipin),TRPA (ankyrin) and TRPN (no mechanoreceptor potential C) families (Pedersen et al., 2005). Most TRP channels are relatively non-selective for cations, however in some cases Ca2+ and Mg2+ ions are seemingly preferred. The structure varies between the families but they all share a defining structure consisting of at least 6 transmembrane domains (Flockerzi and Nilius, 2007) (Fig. 1).

TRP channels were initially discovered in Drosophila (fruitfly) in 1989 and described as involved in visual transduction (Flockerzi and Nilius, 2007). A mutation in the trp gene locus caused a disruption in the lipid signaling system used for visual excitation in the photoreceptor cells (Montell and Rubin, 1989). Immunolocalization later indicated the existence of a TRP protein located on the rhabdomeric membranes of these cells. The behavior of the trp mutant flies suggested that they could not perceive bright light and electroretinograms detected a quick return to the membrane potential base line even when exposed to continuously bright light. Hence the name Transient receptor potential channel (transient=short lived) as the light-induced signals seemed to be short lived (Montell and Rubin, 1989).

The roles of TRPs The TRP channel superfamily includes receptors which, at least in mammals, respond to a great variety of stimuli: mediator molecules, both intra- and extracellular, chemical (e.g. olfaction and taste), temperature, mechanical but also osmotic pressure (Pedersen et al., 2005) (Fig. 1). Coactivators may also be needed, as in the apparent case of TRPV4 (Watanabe et al., 2002). Generally, activation of TRP channels generates a depolarization of the membrane which in turn may give rise to activation of various other voltage-dependent ion channels in the cell membrane (Gees et al., 2010) or simply generate an action potential if located on a nerve fiber. This activation enables an influx of cations, e.g. Ca2+, which changes the intracellular ion balance. Studies show that TRP channels are also present on intracellular organelles where they act as intracellular Ca2+ release channels (Gees et al., 2010).

The biological roles and functions of these channels are also quite diverse. Examples of functions where TRPs are involved include Ca2+ and Mg2+ absorption, endothelial cell permeability, sensory perception, regulation of organ function, smooth muscle proliferation and gender-specific behavior (Flockerzi and Nilius, 2007; Birder et al., 2001). Some may also have roles in regulating cell cycles. However, members of the TRP family are perhaps most known for their involvement in sensory perception, especially temperature and pain, as noxious stimuli detectors in nociceptors (pain sensory neurons) (Patapoutian et al., 2009).

TRPs and thermoregulation The ability to maintain and regulate internal temperature as well as perceive surrounding temperature is crucial for endotherms as well as ectotherms. Endotherms generate and maintain a constant body temperature by the means of metabolic processes while ectotherms rely on external sources of heat, which they use peripheral thermal sensitivity to find (Huey and Kingsolver, 1989). The essential need for such creatures to stay within certain ranges of internal body temperature is to enable and ensure biochemical processes to operate normally and of course to avoid damage caused by noxiously low or high temperatures (often external causes). Activated thermosensors relay signals via afferent sensory nerve fibers and are processed by either the sensory cortex or the preoptic anterior hypothalamus, depending on if the relayed signals originate from peripheral thermosensors (discriminative sensation) or

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central thermosensors (homestatic control), respectively (Romanovsky, 2006). The signals from thermosensors may also trigger reflexes instead of conscious sensation (Purves et al., 2004).

The ability of thermosensitive neurons to perceive temperature is thought to be engaged by activation of certain members of the TRP ion channel family. The TRPV subfamily (TRPV1-4) is the one most involved in temperature sensation, responding to temperatures between below 30 °C up to above 50 °C (Fig. 1) (Ferrandiz-Huertas et al., 2014). Ion channels belonging to the TRPM (TRPM8, TRPM3) and TRPA (TRPA1) families, however, are also responsible for thermosensation, albeit for a range covering colder temperatures, sometimes called ‘cold thermoTRPs’ (Ferrandiz-Huertas et al., 2014) (Fig.1). Individually, these thermoTRP channels respond only to a limited range of temperatures but the partial overlapping of these ranges results in a wide total range (Fig. 1). Thus, they are efficient in detecting temperature fluctuations in both peripheral and central areas of the body.

The TRPV subfamily The TRPs of focus in this project is the TRPV subfamily. Like other TRPs, TRPVs are polymodal, meaning they can be activated by many different stimuli. The TRPV subfamily consist of six protein channels (TRPV1-6) where TRPV5-6 act mainly as Ca2+ channels in the intestine and kidney and are not involved in thermoperception as TRPV1-4 (Flockerzi and Nilius, 2007).

“V” stands for “vanilloid”, a group of carbon-based compounds including capsaicin, the reactive substance in chili peppers (Szallasi and Blumberg, 1999). The reason behind capsaicin being the substance defining the name is due to its ability to activate TRPV1, which was the first TRPV to be identified (Caterina et al., 1997). Initially it was called vanilloid receptor type 1 but subsequent discoveries of its relatives in the TRP family resulted in the change of name to TRPV1 (Clapham et al., 2003).

TRPV1 is, as shown above, also activated by relatively high temperatures (Fig. 1). The burning sensation of capsaicin thus depends on activation of TRPV1 channels on the nociceptors of the sensory nervous system. TRPV1-2 is activated at higher temperatures than TRPV3-4 so it is possible they are more involved in peripheral nociception. Activation of TRPV1 starts at 37 °C and reach full activation at 43 °C, while TRPV2 is activated by temperatures (42 °C to 52 °C) (Ferrandiz-Huertas et al., 2014) (Fig. 1).

The complete distribution is not yet fully known but of the four channels the locations of TRPV4 and TRPV1 are the most well-known. TRPV1 has been identified on the somata and peripheral processes of afferent sensory neurons originating from the trigeminal, nodose and dorsal root ganglia (Holzer, 2004). TRPV1 positive afferent nerve fibers innervate the gut and other organs (Holzer, 2004). It is still not clear

Fig 1. The ranges of temperature which activate TRPV1-4, TRPA1 and TRPM8. TRPV1-4 are activated by a specific and narrow range of temperature and certain chemical compounds. The temperature activating TRPVs ranges from 27°C to 52°C. TRPA1 and TRPM8 are identified as “cold receptors” due to the sensation of cold (should the signal reach the brain) as a result of their lower temperature range of activation and certain chemical stimuli (Figure drawn by author).

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if the expression of TRPV1 in the gut occurs on enteric neurons or only on processes of extrinsic afferent neurons (Buckinx et al., 2013). Other cells types have also been reported to express TRPV1 (although in lower levels): neurons in the brain, epithelial cells of the urinary bladder (not just the innervating neurons) and keratinocytes in the skin (Caterina, 2003; Birder et al., 2001).

TRPV4 expression has been found in vascular endothelial cells of the aorta in mouse (Watanabe et al., 2002). This suggests that this channel may have a role in local vascular responses to changes in body temperature (Benham et al., 2003). Expression of TRPV4 has also been reported in the skin (Lee and Caterina, 2005). Moreover, TRPV4 has been located in the anterior hypothalamus (Benham et al., 2003), an area sensitive to changes in the core temperature and regulates it accordingly.

TRPV 3 is expressed mainly in the CNS and sensory neurons but also in the skin (especially in keratinocytes), while the less known TRPV2 appears on sensory neurons whose locations are not yet fully understood (Benham et al., 2003). Overall, these findings support the idea that TRPVs are active in both core temperature regulations and peripheral sensation.

Sensory neurons - distribution and function As mentioned above, TRPV1 is found to a large extent on peripheral sensory (afferent) neurons. Sensory neurons are neurons which function is to relay and transmit sensory information. Sensory input is essential as a means to adapt and respond to the animal´s surrounding and internal environment. The signals are transmitted either to the central nervous system or participate in local reflexes (Purves et al., 2004). The signal is required to reach the brain for it to be perceived as a conscious sensation while reflexes do not reach it. Sensory stimuli reaching the brain’s sensory cortex thus enable the animal to consciously perceive its environment (Purves et al., 2004).

Sensory input originates both from specialized sensory organs (eyes, ears, mouth, etc) but also from more or less specialized nerve endings, e.g. in the skin and various internal organs. The cell bodies of somatic and visceral sensory nerve fibers (transferring mechanical stimuli, temperature, pain etc.) innervating the various body parts reside within the dorsal root ganglia, trigeminal ganglia and nodose ganglia. After receiving a signal, they forward it to the central nervous system via neurons along the spinal cord where it will be processed as sensation or reflex (Purves et al., 2004). The extrinsic sensory neurons of the gastrointestinal tract relay reflex signals via spinal primary afferents passing dorsal root ganglia and vagal primary afferents passing the nodose ganglia. There they participate in regulating blood flow and ion transport but also mucosal repair, protection and inflammation and gastrointestinal nociception and motility (Holzer, 2004).

Neurons relay their signals via release of neurotransmitters. Most neurons contain a combination of neurotransmitters that may vary between different classes of neurons and their location in the body (Costa et al., 1996). In addition, nerves contain other chemical substances (receptors, growth factors etc) that may help in identifying different classes. One commonly found transmitter within the sensory and autonomic nervous system ganglia is ATP (Purves et al., 2004). Other common neurotransmitters in sensory neurons are calcitonin gene-related peptide (CGRP) and substance P (SP).

Also calbindin, a calcium-binding protein, has been used as a marker for certain sensory neurons in the gastrointestinal tract (Costa et al., 1996) while acetylated tubulin and human neuronal protein (Hu C/D) may be used as indicators to identify nerves generally.

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TRPV in non-mammalians The sequence of TRP in Drosophila has, on the basis of similarity, provided the possibility to track and identify complimentary DNA (cDNA) of 28 TRP related proteins in various mammalian species (humans, mouse, rat, cattle, etc) (Flockerzi and Nilius, 2007). TRP-related proteins have also been identified in other fly species, worms like C. elegans (Harteneck et al., 2000) and tunicates (sea squirt) (Flockerzi and Nilius, 2007).

Among TRPVs, TRPV1 is the form identified in most species. TRPV1 has been identified in various mammals like humans, mouse and rabbit (Flockerzi and Nilius, 2007) but also in other animal groups like fish (Gau et al. 2013), reptiles like crocodile, lizards (Seebacher and Murray, 2007) and snakes (McLamb, 2014), birds like zebrafinch (Seebacher and Murray, 2007) and amphibians like frogs (Saito et al., 2011). One TRP-related gene has also been found in yeast while no encounters in plants so far (Flockerzi and Nilius, 2007). These studies suggest that the family of TRP ion channel proteins appears to be evolutionarily quite well conserved.

Knowledge of the functions and distributions of TRPV in fish is far less detailed than in mammals and only recent studies on zebrafish have begun to reveal its role(s) in physiological and behavioral processes (Gau et al., 2013). Studies have revealed that only one type of TRPV is present in this species, a variant resembling both of the mammalian TRPV1 and TRPV2 (Gau et al., 2013). However, other TRPs have also been identified in zebrafish: TRPN (Nilius and Voets, 2005) and TRPM (Kastenhuber et al., 2013). The temperature threshold of TRPV1 has been discovered to be lower in non-mammalian species, like zebrafish (Gau et al., 2013). Still, there is reason to believe that TRPV in fish is possibly similar in function as in mammals but, in order to reach any conclusions, studies on their distribution is needed as a first step.

Shorthorn sculpin is a fitting study animal for such initial studies. It has frequently been used in other studies examining various responses to changing temperature, e.g. changes in motor activity (Matishov et al., 2009). How physiological mechanisms, e.g. in the gastrointestinal tract, respond to changes in temperature and motility patterns in gut and the intestine have also been studied (Gräns et al., 2013; Brijs et al., 2014).

Aims of this study The overall aim of this study was to examine the distribution of TRPV in the teleost shorthorn sculpin (Myoxocephalus scorpius). Distribution and abundance of receptors was examined by the method of immunohistochemistry. Immunohistochemistry takes advantage of antibodies designed to recognize specific protein sequences. The antibodies bind to the sequence in the tissue and may then be detected by either fluorescent molecules bound to them or bound enzymes. Recently, an antibody against zebrafish TRPV1/2 was produced (Thermo Fisher Scientific, OST00070G) and more specifically, the initial aim was to evaluate and optimize the use of this commercially available antibody in adult shorthorn sculpin. In the second step, the aim was to examine the distribution of TRPV immunoreactivity in the gastrointestinal tract as well as in the extrinsic innervations of the gastrointestinal tract (e.g. vagus nerve).

Material and methods Tissue was taken from two shorthorn sculpins (Myoxocephalus scorpius). The fish were caught on the Swedish west coast and kept in tanks with circulating sea water (10 °C) with air supply at Zoologen, Department of Biological and Environmental sciences, University of Gothenburg. The killing procedure involved a sharp blow to the forehead with a baton, followed by dissection and preparation of tissue samples. The experimental procedures were approved by the Animal Ethics Committee of Gothenburg (89-2013).

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Preparing tissue After the fish were killed, the entire gastrointestinal tract was removed and divided in the following pieces: cardiac and pyloric stomach, proximal, middle, distal intestine and rectum. Each piece was cut open and the tissue samples were rinsed in phosphate buffered saline (PBS, 0.1 M, 0.9% NaCl, pH 7.2) and pinned, while stretched, on plates of dental wax. The tissue was fixated during 2 hours at 4 °C while submerged in formalin solution (4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2). The fixation was followed by another wash in PBS (10 minutes repeated 3 times). The tissues were stored in PBS while refrigerated until further processing. The tissues were pinned on silicone coated petri dishes and the mucosa, submucosa and most of the circular muscle layer were peeled off to reveal the myenteric plexus. The pyloric stomach area was not included due to it being too thick and thus unfit for this method. The pyloric stomach tissue sample was stored for possible future cryosectioning. In addition, older tissue samples and frozen sections were also used.

Immunohistochemistry Whole mount preparations were incubated overnight, and I some instances during 36 hours, with primary antibodies, see Table 1. Hu C/D and acetylated tubuline was used in order to see the total neuronal population, and was sometimes used in combination with the antibody against TRPV1. The antibody against calbindin was used to recognize a subset of enteric neurons presumed to be sensory (Olsson, 2011). Afterward, the preparations were washed in PBS and incubated with the appropriate secondary antibodies (Table 1) during a 2 hour period. All antibodies were diluted in 0.1 M PBS containing 2% NaCl, 0.1% bovine serum albumin and 0.2% NaN3. The incubations took place in a moist box at room temperature.

The secondary antibodies are species-specific and carry a fluorophore (or are conjugated to biotin which binds to streptavidin conjugated to a fluorophore). Several secondary antibodies may bind to each bound primary antibody. This result in a stronger labelling, thus making it easier to detect the specific antigen. This method is called indirect marking (R&D systems, n.d.).

The tissues were mounted on slides, with the myenteric plexus upward, using carbonate-buffered glycerol (pH 8.0) and analyzed in a Nikon Eclipse E1000 digital fluorescence microscope equipped with Nikon Digital Camera DXM1200 and the Nikon software, ACT1. The fluorophores used, FITC and CY3, fluoresce in different wavelength (green and red, respectively). In most tests secondary antibodies conjugated to FITC were used to detect AcT and Hu. Different secondary antibodies conjugated to different secondary fluorophores (CY3 included) were used to detect TRPV. In order to see FITC fluorescence a combination of three light filters only letting green light through was used. Likewise, a filter combination for red light was used for CY3.

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Data Analysis To quantify the distribution of possible TRPV immunoreactivity, pairs of pictures covering the same area were taken, using the two different filters. The focus was to take a picture of an area showing Hu and AcT immunoreactivity and then subsequently take a picture of the same area while using the other filter in order to show the TRPV immunoreactivity, without changing focus. The pictures were transferred to a computer for further procession in the program ImageJ (NIH, Bethesda, MD, USA) where the scale of the pictures was set, including adding scale bars. The picture montages were constructed in Microsoft Word.

Table 1. Primary and secondary antibodies used in the study. CY3 = indocarbocyanine (red), FITC = fluorescein isothiocyanate (green).

Primary antibodies

Antigen Host Dilution Code Supplier

Acetylated tubulin (AcT) Mouse 1:1000 T-6793 SIGMA, MO, USA

Calbindin D-28 k Rabbit 1:2000 CB38a Swant, Bellinzona, Switzerland

Human neuronal protein C/D (Hu)

Mouse 1:200 A21271 Molecular Probes, Eugene, OR, USA

TRPV1 Rabbit 1:100-1:400 OST00070G Thermo Fisher Scientific, MA, USA

Secondary antibodies

Antigen Host Dilution Conjugate Code Supplier

Mouse IgG Donkey 1:100 FITC 715-095-150 Jackson ImmunoResearch Lab., West Grove, PA, USA

Rabbit IgG Donkey 1:800 CY3 711-165-152 Jackson ImmunoResearch Lab., West Grove, PA, USA

Rabbit IgG Donkey 1:100 FITC 711-095-152 Jackson ImmunoResearch Lab., West Grove, PA, USA

Rabbit IgG Donkey 1:400 Biotin-SP 711-065-152 Jackson ImmunoResearch Lab., West Grove, PA, USA

Streptavidin Dilution Conjugate Code Supplier

SA-488 1:2000 AF-488 (green)

S-11223 Molecular Probes (Alexa), Eugene, OR, USA

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Results The following results are comprised of the data obtained from an initial test of the TRPV antibody, as well as a few other pilot tests.

General staining of the myenteric plexus The first test aimed to stain the myenteric plexus to get an overall picture of the structure in different regions of the gastrointestinal tract. The tissue was incubated with a mix of antibodies against acetylated tubulin (AcT) and human neuronal C/D (Hu), both produced in mice and recognized by the same secondary antibody. This labels both nerve fibers and nerve cell bodies. The immunoreactivity showed a certain difference in the structure of the fiber network between the three areas. The cardiac stomach exhibits a robust network of thick fiber bundles and nerve cell bodies connected to several nerve fibers however the dendrites are still visible (Fig. 2 A-B). The mid intestine exhibits a more dense weave-like network with less thick bundles and nerve cell bodies not covered in fibers with dendrites clearly visible (Fig. 2 C-D). The myenteric plexus in the rectum is comprised of more linear, less dense thick fibers that run parallel, with less smaller fibers connecting them (Fig. 2 E-F). Uncovered nerve cell bodies with dendrites are visible here as well. The amount of nerve cell bodies differed between the regions, with the mid intestine being the most densely populated while in the rectum and cardiac stomach they were more rarely encountered. All regions also showed nerve fibers located along the longitudinal and remaining circular muscle fibers. The immunoreactivity of these regions seems to be consistent with the results of other studies (Olsson, 2011).

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Fig. 2 Whole-mount preparations of the myenteric plexus of the gastrointestinal tract of M. Scorpius showing Hu and AcT immunoreactivity. Cardiac stomach (A,B), mid intestine (C,D) as well as rectum (E,F) showed strong immunoreactivity regarding both nerve cell bodies (Hu) (arrows) and nerve fibers (AcT). CS contains thicker fiber bundles while MI exhibits a finer network. R express more lone fiber bundles with fewer interconnecting fibers. Nerve cell bodies appear to be at least bipolar and are visible in all three areas. Scale bars = 100 μm

CS

A B

D

F

R

E

MI

C

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Evaluation of the TRPV1 antibody The initial testing of the TRPV1 antibody aimed at determining the optimal dilution, incubation time and use of secondary antibodies. Three dilutions of the primary antibody were used, 1:100, 1:200 and 1:400 during an incubation period of 36 hours. There was no specific immunoreactivity showing nerve fibers or nerve cell bodies, however there are certain differences between the different dilutions regarding the amount of non-specific background fluorescence (Fig. 3). The highest concentration expressed the highest amount of non-specific fluorescence while the higher dilutions showed lower levels of this fluorescence. No major difference between the gastrointestinal regions.

The first test used CY3 as the sole secondary antibody for all three dilutions. The second test compared three different secondary antibodies conjugated to biotin (streptavidin conjugated to fluorophores were later added), FITC or CY3. Incubation duration was 24 hours. Different secondary antibodies may have various affinity to the primary antibody. No specific staining with any of the secondary antibodies was visible in the proximal intestine (Fig. 4) or any other region of the gastrointestinal tract. The amount of non-specific background staining was relatively high (Fig. 4). This seems to be especially prominent with secondary antibodies conjugated to CY3. Autofluorescent blood vessels and blood cells are visible in all filters. Incubation period was 24 hours.

Fig. 3 Whole-mount preparations of the myenteric plexus of the gastrointestinal tract of M. Scorpius showing no TRPV immunoreactivity. The regions showed are the mid intestine (MI) and rectum (R). The use of three different dilutions of the TRPV1 antibody showed no immunoreactivity of either nerve fibers or nerve cell bodies. There is a difference concerning the amount of non-specific background staining between the different dilutions, where the highest dilution expressed the strongest background fluorescence. The intensity of this staining declined with increasing rate of dilution. No specific immunoreactivity could be found in any of the regions. Scale bars = 100 μm

MI

CY3 1:100

R

1:100

1:200

1:200

1:400

1:400

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Identification of calbindin positive nerve cells Double staining of antibodies targeting calbindin (Cb) and nerve cell bodies (Hu) showed the proportion of calbindin positive nerve cells in relation to all nerve cells in the myenteric plexus (Fig. 5). The used secondary antibodies were conjugated to FITC and CY3. This part of the study aimed to compare possible TRPV immunoreactivity with the calbindin positive subpopulation of myenteric nerve cells, suggested to be sensory (Olsson, 2011). Calbindin immunoreactivity was seen in all areas of the gastrointestinal tract except the cardiac stomach (Fig. 5). The nerve cells expressing calbindin varied in size and had clearly visible dendrites as well as axons. The dendrites and axons were not visible using Hu immunoreactivity. The ratio of calbindin positive nerve cells in relation to the total nerve cell population varies between the regions as well as their density. The ratio of calbindin positive nerve cells in relation to Hu positive nerve cells varied between 42 percent (rectum) to 64 percent (proximal intestine) (Fig. 5). The proximal intestine (PI) also expressed the highest density of total nerve cell population while the distal intestine (DI) and rectum (R) expressed the lowest, based on cell count (Fig. 5). The cardiac stomach expressed no calbindin positive nerve cells. These results are yielded from a single tissue sample from a single fish (N=1) with the aim to briefly evaluate to what extent they corresponded to earlier studies (Olsson, 2011).

Fig. 4 Whole-mount preparations of the myenteric plexus of the gastrointestinal tract of M. Scorpius showing no TRPV immunoreactivity. The region showed is the proximal intestine (PI). The use of the three different secondary antibodies conjugated to FITC (A), SA-488, (B) and Cy3 (C) proved futile regarding attempting to distinguish presence of TRPV. Strong background autofluoresence and possible non-specific binding is visible. No difference in immunoreactivity could be found in any of the other regions. Scale bars = 100 μm

CY3 C

PI

FITC A SA-488 B

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E2

A2

Fig. 5 Whole-mount preparations of the myenteric plexus of the gastrointestinal tract of M. scorpius showing calbindin (Cb) and Hu immunoreactivity. Cardiac stomach (A1,A2) showed no presence of calbindin, but the proximal intestine (B1,B2), mid intestine (C1,C2), distal intestine (D1,D2) and rectum (E1,E2) showed strong immunoreactivity regarding both nerve cell bodies (Hu) and calbindin positive neurons (Cb). Scale bars = 100 μm

B1 B2

PI

C2 C1

MI

D1 D2

DI

E1

R

E2

A2 Hu Cb A1

CS

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Discussion The initial aim of this study was to examine and evaluate the possible presence of TRPV in the gastrointestinal tract of shorthorn sculpin, and use it as a possible marker for sensory neurons, as well as an initial optimization of the immunohistochemistry protocol for the new TRPV1 antibody. However, the results yielded were influenced by technical disturbances (late arrival of the TRPV antibodies) resulting in limited time regarding carrying out experiments. Hence, this study only contains the initial tests of evaluating the TRPV antibody.

Three main suggestions can be proposed as to explain the absence of immunoreactivity of TRPV: The antibody might not be suitable for use in shorthorn sculpin, the method needs to be further optimized or adjusted in some way in order to obtain an immunoreactive response, or TRPV is simply not present in the gastrointestinal tract.

Considering these suggestions in terms of likelihood the latter may be regarded as the least possible explanation. Studies on zebrafish TRPV has, as mentioned, found a type of TRPV that is similar to the mammalian equivalents of both TRPV1 and TRPV2, with approximately 50 % similarity to rat (Gau et al., 2013). This and several other studies have identified the presence of TRPV in other species from various animal groups (Flockerzi and Nilius, 2007; Seebacher and Murray, 2007; McLamb, 2014; Saito et al., 2011; Ohkita et al. 2012). As mentioned in the introduction, TRPV1 in mammals has been identified in various afferent sensory neurons innervating various organs but also in epithelial cells (the bladder) and in keratinocytes. In frogs, TRPV1 has been identified in dorsal root ganglion neurons and oocytes (Saito et al., 2011; Ohkita et al., 2012). It has also been identified on sperms of a freshwater carp fish (Majhi et al., 2013). In crocodiles, TRPV1 is expressed on liver, muscle and heart tissue (Seebacher and Murray, 2007) and it is also expressed in thermosensory nerve terminals in pit organs of boid and crotaline snakes (McLamb, 2014).

This provides reasons to suspect some form of TRPV should be present in shorthorn sculpin, although not necessarily in the gastrointestinal tract as it has not yet been identified in that area in other fish species, e.g. zebrafish (Gau et al. 2013).

It is perhaps too early to comment on the usefulness of this specific TRPV antibody in studies concerning shorthorn sculpin given the limitations of this study. In order to reach such a conclusion further studies are needed. One suggestion would be to perform western blot tests to determine the specificity of the antibody to sculpin TRPV. Gel electrophoresis would show whether the antibody binds to proteins or peptides of the correct size, different sizes (non-specific binding) or not at all. In time it could also prove useful to compare genetic sequence homologies between sculpin, zebrafish and other species to see if there are TRPV homologs between them. Some studies have already begun comparing TRPV1/2 sequence similarities between certain fish species (Majhi et al., 2013).

Immunohistochemistry as a method is commonly practiced and other studies report additional measures to use in cases where optimization is needed (Gillett, 2006; Shi et al., 2001). In this study optimization attempts included use of three different dilutions of the antibody as well as three different secondary antibodies, however the results suggest further optimization is needed. In some instances, a pretreatment or anti-gen retrieval step is necessary in order to undue folding or other deformations of the antigen (target protein) that occurred during fixation (Gillett, 2006). Because the antigen has been “hidden” from the antibody, heat or enzymes are used to remodel the antigen’s protein structure into being susceptible to the antibody again and thus enable visualization of it (Gillett, 2006). Heat-mediated antigen retrieval may be performed using a microwave oven. Enzymes like proteases and trypsin are also commonly used to reveal masked antigenic binding sites. No antigen retrieval was performed in this study but it might be useful to include in future studies.

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Other aspects of the method worth consider revising is the duration of the fixation and the type of fixation. Fixation, often with a formaldehyde based solution, is an important step in the procedures practiced when processing tissue samples for examination and preservation and a necessary treatment when performing immunohistochemistry. The treatment helps preserving the cellular structure in the tissue, keeping it from degrading as well as loosening up the tissue to allow the antibody to penetrate and reach into the cells properly and find the target antigens (Thavarajah et al., 2012; Berod et al., 1981).

Many immunohistochemical studies have chosen 4 hours duration for the fixation consisting of 4% PFA (Paraformaldehyde) (C. Olsson, personal communication, June 10, 2015). However, this study only used 2 hours of fixation due to the tissue also being used in other studies were a 2 hour fixation was necessary. Other studies instead utilize a prolonged fixation period of up to 24h (e.g. Olsson, 2011) using another kind of fixative, e.g. Zamboni’s fixative (15% saturated picric acid, 2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.2) in a 4oC condition.

Another factor most likely in need of optimization is the dilution of the TRPV antibody. Generally, the first step of optimization is to determine the optimal dilution. In this study the dilutions 1:100, 1:200 and 1:400 were tested. The stock solution contained 1 μg/μl which means the concentrations in the tests were 0.01, 0.005 and 0.0025 μg/μl respectively. This was at the lower end of the, by the manufacturer, recommended dilution of the TRPV antibody which was 0.01-0.05 μg/µl (Thermo Fisher Scientific, USA). The staining with the highest concentration of TRPV used (1:100) resulted in high non-specific background fluorescence (Fig. 3) and it seemed the antibody had bound to smooth muscle cells. No specifically stained nerve fibers were seen which indicates that the concentration is not the largest concern.

The tests of this study mostly consisted of indirect marking with fluorescent molecules, called fluorophores, conjugated to the secondary antibody (R&D systems, n.d.). The study used three different secondary antibodies against rabbit, conjugated to different fluorophores (FITC and Cy3) or to biotin for further staining by use of streptavidin conjugated to the green fluorophore AlexaFluor 488. No differences in staining were recorded. The same secondary antibody conjugated to Cy3 was used when staining calbindin, showing that this antibody is functional. Secondary antibodies against mouse, conjugated to FITC were used when staining HuC/D and AcT.

However, there are other more sophisticated methods to further improve sensitivity. More sensitive fluorophores, enzyme-reaction complex instead of fluorophores or peroxidase-labeled polymers, which can be used to detect antibody binding and thus increase visualization in tissue containing low levels of the antigen can be used (Gillett, 2006). The latter is based on a sensitive two-step staining using HRP (horseradish peroxidase) labeled polymers conjugated to secondary antibodies. The labeled polymer is not bound to biotin or avidin which reduces the risk of unspecific staining of endogenous avidin-biotin activity which results in a reduction in background fluorescence (Dako, n.d.). These systems are unfortunately expensive so there may be reason to continue optimization of the use of secondary and tertiary indirect staining.

The different duration of incubation during the test of different dilutions of the primary antibody and the test of three different secondary antibodies suggested that 36 hours do not increase staining in comparison to standard 24 hours incubation.

Conclusion Even though the study failed to identify the presence of TRPV immunoreactive nerves in the gastrointestinal tract of the shorthorn sculpin, it provides a base to continue the optimization of the protocol for TRPV immunohiostochemistry. If and when the antibody has proven to work in shorthorn sculpin, it will be very useful to describe the distribution of TRPV positive neurons in relation to the whole myenteric plexus and the possible colocalization with calbindin in order to further characterize these nerve cells. These findings will constitute a first step in providing data and knowledge for future

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studies to ultimately produce reference material with which to use when identifying sensory neurons and hopefully TRPV in the gastrointestinal tract of shorthorn sculpin.

Acknowledgement The author wish to thank Catharina Olsson, professor at the institution for Biological and Environmental Sciences at the University of Gothenburg, for being my supervisor and mentor as well as providing equipment during this study.

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