Introduction:The structural correlates of nerve cell function are being studied in the early-evolved coelenterate, Hydra. The studies rely on immunocytochemistry focusing on the nerve cells that innervate the battery cell complexes of the hydra tentacles. Currently, the role of a glutamate receptor, N-methyl-D-aspartate (NMDA), as a transmitter in hydra is under study. NMDA is important in development of the nervous system and synapses in mammals (Gosh, 2003.) Previous findings provide immunocytochemical evidence for NMDA Receptor 1 (NMDAR1) presence in hydra macerates (Scappaticci et al, 2004), specifically in the tentacle/ hypostome region. Amyotrophic lateral sclerosis (ALS), often referred to as Lou Gehrig’s disease, is a progressive disease that affects nerve cells in the brain and spinal cord. There is evidence suggesting probable glutamate excitotoxicity in patients with ALS (Shaw et al, 1997). Glutamate is released when nervous tissue is damaged and too much glutamate over-stimulates neurons and can cause them to die. The NMDA receptor complex is a glutamate receptor subtype and its dysfunction is involved in many neurological disorders associated with aging, chronic pain, depression, stroke and Parkinson's disease. With our study, we are coming closer to a full understanding of Hydra’s neural pathways and allowing the possibility that drug discovery experiments can be conducted on Hydra, rather than costly mammalian cells.

In our current studies we have been working to develop methods to extend our previous studies to whole animals. We have also been conducting experiments to confirm previous findings on isolated cells, using a new batch of antibodies. In addition, we are testing the effects of fixatives on patterns of labeling with the NMDAR1 antibodies and have introduced a two step antibody labeling procedure which avoids the use of our previous 3-step Avidin-biotin procedure, which could be a cause of artifacts due to endogenous biotin.

Materials and Methods:Animals: All experiments were carried out on Swiss Hydra vulgaris. Animals were starved for two days prior to experiment

Macerated HydrasHydras were dissociated on agar-coated slides

using 1:1:7 medium (A Scappaticci et al, 2004). Tentacle/hypostome pieces were fixed with: 4% formaldehyde in Phosphate Buffer Saline (PBS pH 7.4), 1% glutaraldehyde 3.75% formaldehyde in .01M S-Collidine Buffer (pH 7.2), or lavdowsky’s fixative. After buffer rinses cells were labeled with primary antibody followed by Alexa 488 tagged secondary antibody. ProLong Gold antifade

reagent (Molecular Probes) was used as mounting medium.

Whole HydrasTwo to three hydras were processed at the same

time in a 9- well depression plate. After relaxed the hydra were briefly fixed in 50:50 dissociation medium/fixative. They were then fixed overnight. They were rinsed with the appropriate buffer to remove the fixative pre-treated with modified PBS containing BSA as a blocking agent. Lastly cells were labeled with primary antibody followed by Alexa 488 secondary antibody. ProLong Gold antifade reagent by Molecular Probes was used as mounting medium.

Antibodies UsedPrimary:

Sigma Anti Tubulin Monoclonal Antibody (MAB) B512

Chemicom Anti NMDA-R1 MAB 363Secondary:

Molecular Probes Anti-mouse IgG Alexa 488

AcknowledgmentsWe thank Jarren Kay and everyone who helped maintain the hydra.

Results:All of the following results describe only that labeling which was above the low-level background labeling viewed in controls without primary antibody:

MaceratesVery strong labeling was observed in the cytoplasm of the desmoneme and stenotele nematocytes. Moderately strong labeling of myonemes and large condensed nucleoli of several different cell types including Epithelial Muscular Cell’s (EMC’s) and in nematoblasts.

Conclusions:

Mapping hydra’s nervous systemL A Hufnagel1, K Soullier2, S Guertin2, A Wilcox2, M Zompa2, W O’Leary1, O Omoniyi2, G Kass-

Simon2

1Dept of Cell and Molecular Biology and 2Department of Biological Sciences, University of Rhode Island, Kingston, RI 02881

Literature cited Gosh A. 2003. NMDA Related to Development of the Nervous System. Science Vol. 295 pages 449-

451 Scappaticci A.A., Jacques R., Carroll J.E., Hufnagel L.A., Kass-Simon G.( 2004) Immunocytochemical

evidence for and NMDAR1 receptor subunit in dissociated cells of Hydra vulgaris. Cell Tissue Research Vol. 316 pages 263-270

Hufnagel, L.A., L. Studer, and G. Kass-Simon, Localization of binding sites for GluR5,6,7 in macerates and tissue pieces of Hydra. Proc. 7th Int. Workshop on Hydroid Development, Tutzing, Germany, Sept, 1997.

Hufnagel, L.A., B. Spencer, C. Straub, M. Geotis, M. Calagni, G. Kass-Simon, and R. Zackroff, Further immunocytochemical evidence for the use of glutamate as neurotransmitter by hydra. Proc. 8 th Int. Wrkshp. On Hydroid Development, Tutzing, Germany, Sept., 1999

Kass-Simon,G., Pannaccione, A. and Pierobon, p. (2003) GABA and glutamate receptors are involved in modulating pacemaker activity in hydra. Comp. Biochem, Physiol. 329-342

Pierobon P, Tino A, Minei R, Marino G. (2004) Different rols of GABA and glycine in the modulation of chemosensory responses in Hydra vulgaris. Hydrobiologia 530/531: 59-66

RuggieriRD, Pierobon P and Kass-Simon g. (2004) Pacemaker activity in Hydra is modulated by glycine receptor ligands. Comp. Biochem. Physiol. Part A 138:193-202.

Shaw PJ, Ince PG (1997) Glutamate, excitotoxicity and amyotrophic lateral sclerosis. J Neurol. May;244

For further informationPlease contact Linda Hufnagel Department of Cell and Molecular Biology University of Rhode Island, Kingston, RI 02881 Tel: (401) 874-5914 E-mail: lhufnagel@uri.edu. More information on this and related projects can be obtained at http://www.uri.edu/cels/cmb/people/faculty/lhufnagel/

To view this poster online go to: http://home.comcast.net/~dispach/kristen/Hydra.ppt

Fig. 1 Hydra vulgaris: tentacle/hypostome region (dotted line indicates where we macerated the hydra)

Fig. 4 Moderately labeled nucleolus in an EMC. Orange structures seen the phase contrast image are remnants of the food organism, brine shrimp, and seem to be surrounded by brightly fluorescent, antibody-staining material.

Fig. 5 Nerve cells with very weakly labeled cell bodies and very little label on neurites.

Fig. 3 Labeled myonemes (linear, parallel structures) and cytoplasm at the bases of several nematocytes (brighter areas) in battery cell complexes.

Fig. 15 (a) Phase contrast of control shown in b (b) Fluorescence image, primary antibody was omitted.

Fig. 6 Isolated stenotele nematocytes with labeled cytoplasm.

Fig. 8 Two large I-cells (early neuroblasts?), connected by a thin strand of cytoplasm. Each contains a pair of labeled nucleoli and a labeled region in the cytoplasm.

Fig. 2 (a) Phase contrast and (b) fluorescence images of a stenotele in situ in a battery cell complex.

Whole Animals

Fig. 16 (a) Phase contrast- A group of battery cell complexes, containing myonemes and embedded nematocytes. (b) Fluorescence image of same region primary antibody was omitted. Note absence of label on any structures.

ControlsIn photographs taken under comparable exposure conditions, macerates and whole hydra that were not treated with primary antibody showed no detectable labeling.

Distinctive labeling was seen in the myonemal layer throughout the tentacles and body column. Slightly less labeling was seen in the endodermal myonemal layer. There appeared to be strong labeling in the cytoplasm of some ectodermal stenotele nematocytes, and in the nucleoli of a few cells fortuitously exposed at the edges of tissue tears. No regional

differences were seen in labeling.

Fig. 11 (a) Phase contrast side view of the tentacle, (b) endodermal cells labeled also labeling in the cytoplasm surrounding the stenotele

Macerates

Whole hydra

•Observations on macerates confirmed findings of our previous studies on cell macerates.•Labeling of the myonemal layer and the cytoplasm of stenotele nematocysts was confirmed in whole hydra. Preliminary results suggest there may be localized labeling of tentacle buds in budding hydra. Otherwise, no evidence was found for regional differences in labeling intensity in adult hydra.•Further studies and experiments need to be conducted on whole animals that are budding to follow the complete development of hydra•The new two step labeling procedure proved to be successful for labeling both macerates and whole hydras. This indicates that labeling patterns previously seen were not artifacts due to the presence of endogenous biotin. •The genetic evidence shows that cnidaria have NMDA receptor homologues and that the antibody we are using, to mammalian NMDA R1, recognizes an epitope that is also found in NMDA receptors of cnidarians (Nematostella.).

Goals: •To develop fluorescence-based methods for labeling NMDA receptors in whole Hydra. We are looking for the animals to be fixed in a relaxed state, with good penetration of antibodies, preservation of the antigen, and minimize the background fluorescence.•To establish that antibodies to mammalian NMDA receptors recognize the same receptors in Hydra by determining weather the immunogenic sequence used in making the mammal-specific antibody to NMDAR1 is also represented in the cnidarian genome and exclusively in a homologue to the mammalian NMDAR1 gene•To characterize the regional distribution of NMDA receptors in whole Hydra, relative to the different regions of the animal i.e. tentacles, hypostomal region, midbody, peduncle, basal disk.

Fig. 9 Cluster of battery cell complexes from a hydra tentacle photographed at the focal plane of the myonemes. The myonemes show strong labeling. Very intense labeling is also evident in the basal cytoplasm of various nematocytes present in the complexes.

Fig. 12 (a) Darkfield image, showing the acellular mesoglea between the ectodermal layer and endodermal layer as a dark band. (b) Fluorescence image, revealing a labeled layer that is above the mesoglea and thus in the myonemal layer of the ectoderm

Fig. 13 Brightly fluorescent region is at the base of several nematocytes, which are associated closely with the ectodermal myoneme layer (also fluorescent.)

Fig. 14 Fluorescent labeling of a nucleolus of a cell in the body column, which correlates well with previous finding that nucleoli often label in cells of macerates labeled with the NMDAR1 antibody.

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Fig. 7 Two stenotele nematoblasts with weakly labeled nucleoli.

Fig. 10 (a) Phase contrast of myonemes, (b) labeling in myonemal layer and around stenotele capsules