# Glutamatergic transmission in hydra: NMDA/D-serine affects the electrical activity of the body and tentacles of Hydra vulgaris (Cnidaria, Hydrozoa).

INTRODUCTION

Glutamate is the primary excitatory neurotransmitter in the vertebrate central nervous system and the nervous systems of many invertebrates. It functions by binding to receptors on nerve and effector cells that gate ions direcT1y, through channel pores in the receptor (ionotropic glutamate receptors, iGluRs; see Dingledine et al., 1999, for a review), or indirecT1y, through a series of intracellular metabolic steps (metabotropic receptors, mGluRs; see Pin and Duvoisin, 1995, for a review). The receptors are characterized pharmacologically and classified according to their selective binding of the various glutamatergic agonists (Watkins and Olverman, 1987).

In mammalian systems, at least three major iGluR subtypes have been identified: [alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainic acid, and N-meth-yl-D-aspartic acid (NMDA); AMPA and kainate receptors are fast ionic channels that gate [Na.sup.+] and [K.sup.+]; the NMDA receptor gates [Na.sup.+], [K.sup.+], and [Ca.sup.2+]. NMDA receptor channels (NMDARs) have been implicated in neuromuscular transmission in crayfish (Feinstein et al, 1998), and in long-term potentiation (LTP), long-term depression (LTD), and learning in vertebrates and in the opisthobranch Aplysia (Collingridge et al., 1983; Dudek and Bear, 1992; Bliss and Collingridge, 1993, for a review; Roberts and Glanzman, 2003).

NMDAR channels are unique in that, under resting conditions, their channel pore is blocked by the divalent magnesium ion. The block is removed only by a membrane depolarization (Nowak et al., 1984; Mayer and Westbrook, 1987). The depolarization is accomplished by glutamate acting on the fast-acting AMPA or kainate receptors that lie close to the NMDA receptors. Kainate and AMPA receptors, which have overlapping, though not identical, gating properties, are sometimes grouped together as AMPA/kainate receptors (see BetT1er and Mulle, 1995, for a review). The NMDA receptor has binding sites for glutamate and glycine, both of which are simultaneously required for the activation of the receptor and for preventing [Ca.sup.2+] -dependent desensitization (Johnson and Ascher, 1987; Kleckner and Dingledine, 1988). In hydra, NMDA receptors are involved in regulating the duration of reduced-glutathione (GSH)-induced mouth opening (Pierobon et al., 2004a) and nematocyst discharge in the tentacles (Kass-Simon and Scappaticci, 2004; Scappaticci and Kass-Simon, 2008).

Hydra has two main effector systems responsible for its behavioral repertoire: the neuro-epitheliomuscular systems, which control mouth opening and closing and regulate the contraction and elongation of hydra's body and tentacles (Passano and McCullough, 1962, 1963, 1964, 1965; Rush-forth and Burke, 1971); and the nematocyst system, which results in the capture and killing of prey (see Kass-Simon and Scappaticci, 2002, for a review). Although a number of other neurotransmitters, including acetylcholine, catecholamines, serotonin, and numerous peptides, have previously been reported to play a role in hydra's neuroeffector systems (see Kass-Simon and Pierobon, 2007, for a review), recent studies have presented extensive evidence for the involvement of the amino acid transmitters glutamate. [gamma]-amino-butyric acid (GABA), and glycine, in the functioning of both the epitheliomuscular and nematocyst effector systems (Bellis et al, 1991; Pierobon et al., 1995, 2001, 2004a, b; Concas et al, 1998; Kass-Simon et al., 2003; Kass-Simon and Scappaticci, 2004; Ruggeri et al., 2004; Scappaticci et al., 2004; Scappaticci and Kass-Simon, 2008).

Three main pacemaker systems control hydra's epitheliomuscular effectors. (1) Contractions of the body column are elicited by the contraction burst (CB) pacemaker system (Passano and McCullough, 1964). Its longitudinally conducted impulses are thought to be generated in a circumferential nerve ring located just below the hypostome (Passano and McCullough, 1963; Kass-Simon, 1972; Kass-Simon and Passano, 1978; Kinnamon and Westfall, 1981; Koizumi et al, 1992), but may also originate elsewhere in the ectoderm of the body column (Kass-Simon, 1970). (2) Elongation of the body column is caused by the rhythmic potential (RP) pacemaker system, which causes a contraction of circumferentially arranged myonemes (Shibley, 1969). These pulses are initiated in the peduncle (Passano and McCullough, 1962, 1965) and are conducted along the endoderm of the body and through-conducted into the tentacles (Kass-Simon and Passano, 1978). (3) The tentacle pulse (TP) system is responsible for the contraction of the tentacles and has been found to excite the CB system (Rushforth and Burke, 1971; Kass-Simon 1972).

In order to continue to delineate the role of NMDA receptors in the effector systems of Hydra vulgaris, we investigated the effects of NMDA, together with D-serine, the specific glycine agonist at the NMDA receptor (Kleck-ner and Dingledine, 1988; Mothet et al., 2000), on the electrical activity of hydra's epitheliomuscular systems. In addition to the classical pacemaker systems, we also monitored the effects of NMDA/D-serine on non-pacemaker electrical activity in the tentacle and the body column. Our results indicate that NMDA/D-serine modifies the activity of both the tentacle and the body--increasing the output of the tentacle pacemaker system and the non-pacemaker pulses in the tentacle, and potentially decreasing the output of the rhythmic potential pacemaker system and the non-pacemaker pulses in the body column.

The study is part of an ongoing collaboration among the laboratories of A. Concas, L. Hufnagel, G. Kass-Simon, and P. Pierobon.

MATERIALS AND METHODS

Animals

Specimens of Hydra vulgaris were asexually cultured in glass baking dishes containing modified bicarbonate versene culture solution (BVC): 1 X [10.sup.-7] mol [l.sup.-1] [NaHCO.sub.3], 1 X [10.sup.-6] mol [l.sup.-1] Ca[Cl.sub.2], and 1 X [10.sup.-8] [mol [l.sup.-1] EDTA, pH 7.0 [+ or -] 0.2 (Loomis and Lenhoff, 1956; Muscatine and Lenhoff, 1965). All animals were kept in an incubator at 18 [+ or -] 1.0 [degrees]C. Hydra cultures were fed freshly hatched Artemia salina nauplii once every 48 h. To prevent possible effects of feeding on contractile behaviors (Passano and McCullough, 1964; Grosvenor et al, 1996), experimental animals were randomly selected from non-budding hydra that were starved for 24 [+ or -] 2 h.

Recording methods

Electrical recordings were made at 22 [+ or -] 2.0 [degrees]C, under red light (low setting on Fiber-Lite 190 lamp, Dolan-Jenner Industries, Boxborough, MA) at an intensity of 102.5 foot-candles (1103.3 lux). Hydras have been reported to be sensitive to all colors of light with the exception of red (Wilson, 1891; Haug, 1933); their pacemaker output in red light is similar to that found in the absence of light (Passano and McCullough, 1962, 1964). The recording techniques were modified from those of Passano and McCullough (1964; e.g., Kass-Simon et al., 2003; Ruggieri et al., 2004). A polyethylene suction electrode was attached to the tip of a tentacle and another to the base of the peduncle (Fig. 1). During recording, animals were observed through a dissecting microscope to correlate various behaviors with their respective pulses. The signals from each electrode were amplified by an AC differential amplifier (Model 3000, A-M Systems, Sequim, WA), digitally converted by a MacLab AID converter (AD Instruments, Colorado Springs, CO), and recorded on a Macintosh G3 computer (Apple Computers) using Chart 3.6.8 software (AD Instruments). Square pulses from a Grass SD9 stimulator (Grass Instruments, West Warwick, RI) were used to mark visually observed tentacle contractions on a separate channel of the computer record. The following parameters were measured for each 5-min period of an experiment: frequency of rhythmic potentials (RP/min), tentacle pulses (TP/min), and contraction bursts (CB/min), and the average number of pulses per contraction burst (P/CB). RPs are defined as small, regularly occurring pulses, associated with the elongation of the body and conducted throughout the body column and tentacles in the endoderm (Passano and McCullough, 1962; Kass-Simon and Passano, 1978). TPs are defined as pulses visually correlated with the contraction of the tentacle and are not always conducted into the body column or into adjacent tentacles (Rushforth and Burke, 1971; Kass-Simon and Passano, 1978; Grosvenor et al., 1996; Kass-Simon et al., 2003; Ruggieri et al., 2004). CBs are defined as several consecutive pulses, usually arising at the hypostome, and are through-conducted into the tentacles from the body; the pulses are associated with the contraction of the body column (Passano and McCullough, 1964; Kass-Simon, 1972, 1973). CBs were considered to consist of at least four through-conducted, contraction-associated pulses occurring within 15 s of each other. In addition to the classical categories of pacemaker pulses, we recorded and tallied other impulses that have not previously been reported, but that have frequenT1y been seen in many other experiments. These impulses are heterogeneous in shape and probably arise either from neurons or myonemes; they are often likely to be a composite of both. We have placed all such uncorrelated pulses into two classes, those recorded at the tentacle and those at the body, and have designated them small, uncorrected tentacle or body pulses (SUTPs or SUBPs) respectively. They are not associated with any visually discernible behavior. Their amplitude and duration vary over a considerable range (SUTPs: 3.2 mV-82.95 mV, 200 ms-2.15 s; SUBPs 2.8 mV-85.05 mV, 250 ms-1.85 s). Pre-locomotor pulses (PLPs), which are pulses that occur before some contraction bursts (Passano and McCullough, 1964), were included in SUTP and SUBP counts if they were not through-conducted; otherwise they were not tallied. The size and duration of PLPs fall within the abovecited parameters for SUTPs and SUBPs, and therefore could not be distinguished from them.

[FIGURE 1 OMITTED]

At the start of a recording session, hydras were placed in a 10-ml petri dish containing 6 ml of BVC, and connected to a suction electrode. All test substances were made up at 10-fold of their final concentration. They were diluted to the final concentration by adding them to the solution in the culture dish (Kass-Simon et al., 2003; Ruggieri et al., 2004). The first 5 min of recording was used as an acclimation period and was not included in the statistical analysis. The treatment protocol began with the addition of 0.6 ml of concanavalin A (Con A), which constitutes the control period (C). Con A (1 x [10.sup.-15] mol [l.sup.-1]) was used to prevent glutamatergic desensitization (Mayer and Vylicky, 1989; Partin et al., 1993; Kass-Simon et al., 2003). In preliminary experiments in which the responses in Con A were compared to those in BVC (n = 10), no statistically significant differences were found in the monitored parameters. Therefore, we used Con A as the control for all our recordings. Recordings lasted for 20-30 min, depending on the number of substances to be tested. Each recording session was divided into 4, 5, or 6 five-minute periods for statistical analysis. The following recording protocol was used for experiments in which NMDA plus D-serine was the only test substance: the first 5-min recording period was a BVC acclimation period, followed by a Con A control period (C), and a first treatment period (T1) containing NMDA and D-serine; this period was continued for an additional 5 min and then considered the second treatment period (T2). For recordings in which AMPA or kainate preceded treatment with NMDA and D-serine, the following protocol was used: a BVC acclimation period, a Con A control period (C), an AMPA or kainate pre-treatment period (pT), an NMDA plus D-serine first treatment period (T1), continued for a second 5-min treatment period (T2). For recordings in which an antagonist preceded NMDA/D-serine treatment, the following protocol was used: a BVC acclimation period, a Con A control period (C), an AMPA or kainate pre-treatment period (pT), a 5-min blocking period (bT) containing d-2-amino-5-phosphonopentanoic acid (D-AP5), followed by two NMDA plus D-serine treatment periods (T1 and T2). D-AP5 is a competitive antagonist of NMDA (Evans et al., 1982).

All substances were added at the start of the respective treatment period. No animal was used more than once. Experiments were performed without washout periods because of the difficulty of maintaining recording conditions during evacuation of the recording dish.

Ligands

The following agonists and antagonists for the NMDA receptor were used: N-methyl-D-aspartate (NMDA), D-2-amino-5-phosphonopentanoic acid (D-AP5), a-amino-3-hy-droxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate, D-serine, and Con A. NMDA, kainate, Con A, and D-serine were purchased from Sigma-Aldrich Corporation (St. Louis, MO). D-AP5 and AMPA were purchased from Tocris Bioscience (Ellisville, MO). The following combinations of ligands (in final concentrations) were used: (1) NMDA + D-serine (both at 1 x [10.sup.-5] mol [l.sup.-1], 1 x [10.sup.-7] mol [l.sup.-1], and 1 x [10.sup.-9] mol [l.sup.-1]); (2) AMPA 1 x [10.sup.-7] mol [l.sup.-1]) + NMDA + D-serine (both at 1 x [10.sup.-5] mol [l.sup.-1], 1 x [10.sup.-7] mol [l.sup.-1], and 1 x [10.sup.-9] mol [l.sup.-1]); (3) kainate 1 x [10.sup.-7] mol [l.sup.-1]) + NMDA + D-serine (both at 1 x [10.sup.-5] mol [l.sup.-1], 1 x [10.sup.-7] mol [l.sup.-1], and 1 x [10.sup.-9] mol [l.sup.-1]); (4) AMPA 1 x [10.sup.-7] mol [l.sup.-1]) + D-AP5 1 x [10.sup.-4] mol [l.sup.-1]) + NMDA + D-serine (both at 1 x [10.sup.-5] mol [l.sup.-1], and 1 x [10.sup.-7] mol [l.sup.-1]); (5) kainate 1 x [10.sup.-7] mol [l.sup.-1]) + D-AP5 1 x [10.sup.-4] mol [l.sup.-1]) + NMDA + D-serine (both at 1 x [10.sup.-5] mol [l.sup.-1]).

Data analysis

At least six animals were used for each ligand series. Each animal was compared against itself because significant, but unaccounted for, differences occur between animals if experiments are conducted for as long as several months (Kass-Simon et al., 2003; Ruggieri et al., 2004). To ensure that no time-dependent changes occurred during the course of an experiment, a series of preliminary recordings, in BVC alone, lasting 30 min, were performed (n = 6). No statistical differences were found among the 5-min periods within an experiment.

Data analysis is modified from that of Kass-Simon et al. (2003) and Ruggieri et al. (2004). A Friedman two-way analysis of variance (FANOVA; Siegel, 1956) and post hoc test, to be used with FANOVAs (Daniel, 1990), was performed. The post hoc test used the following equation:

[absolute value of [R.sub.j] - [R.sub.j']] [greater than or equal to] z [square root of ([bk(k + 1)/6])]

where [R.sub.j] is the jth rank treatment total, [R.sub.j'] is the j'th rank treatment total, z is a value from a standard distribution table, b is the number of animals used, and k is the number of treatments. The corrected alpha ([alpha]) value was solved for by using the following equation: [[alpha].sub.c] = [alpha]/k(k + 1) where [alpha] is the original error level and k is the number of treatments (Daniel, 1990). The first 20 s of each period, in which a test substance was added, were omitted from statistical analysis in order to avoid including behavioral/electrical changes induced by disturbance of the water during substance administration. RPs, TPs, CBs, SUTPs, and SUBPs were converted to pulses per minute for analysis. Since we used a nonparametric statistic based on rank, TP, CB, SUTP, and SUBP values are presented as medians [+ or -] inter-quartile ranges (m.d. [+ or -] i.q.r.) in the text and tables. However, to permit comparisons with our previous studies, we also give the means and standard deviations (m. [+ or -] s.d.) in the text, tables, and histograms. P/CBs are the average number of pulses per contraction burst and are calculated by adding the number of pulses in each CB and dividing the total by the number of CBs in a given period. CBs that started in one period and continued into the next period were counted in the period in which they originated; the pulses in these CBs were not counted and not averaged in with the P/CBs. In some instances, no CBs occurred in a period, and hence the P/CB parameter was omitted from those experiments; this sometimes resulted in the omission of that parameter from an entire series of experiments (Tables 1 and 2). P/CBs are presented as m.d. [+ or -] i.q.r. and means [+ or -] standard errors (m. [+ or -] s.e.). Because RPs were sometimes difficult to discern, they were tallied twice and the average of the two counts was taken as our value. Therefore, RP values are given as m.d. [+ or -] i.q.r. and m. [+ or -] s.e. Differences were considered statistically significant at P [less than or equal to] 0.05, and non-significant with a strong trend, which we termed "potentially significant," at 0.051 [less than or equal to] P [less than or equal to] 0.08.

RESULTS

Effect on tentacle activity

The tentacle pulse (TP) pacemaker system. NMDA and D-serine (1 X [10.sup.-7] mol [l.sup.-1]), in the presence of AMPA (1 X [10.sup.-7] mol [l.sup.-1]), caused a significant increase in the TP rate (pulses/min) during T2 (2.10 [+ or -] 3.95, m.d. [+ or -] i.q.r.; 3.94 [+ or -] 3.90, m. [+ or -] s.d.) relative to the control peirod (0.86 [+ or -] 1.34, m.d. [+ or -] i.q.r.; 1.20 [+ or -] 1.34, m. [+ or -] s.d.) (Table 1 and Fig. 2a); the increase was abolished by 1 x [10.sup.-4] mol [l.sup.-1] D-AP5 (Fig. 2b). In one case, during the blocking experiment containing 1 x [10.sup.-7] mol [l.sup.-1] AMPA, 1 x [10.sup.-4] mol [l.sup.-1] D-AP5, and 1 x [10.sup.-5] mol [l.sup.-1] NMDA/D-serine, there was a significant increase in the TP rate during T2 (2.60 [+ or -] 3.05, m.d. [+ or -] i.q.r; 2.93 [+ or -] s.d.) relative to the rate in AMPA (pT: 0.43 [+ or -] 0.27, m.d. [+ or -] i.q.r.; 0.43 [+ or -] 0.47, m. [+ or -] s.d.) (Table 1). No other treatments, including that of NMDA and D-serine alone, had a significant effect on the TP pacemaker system (Tables 2 and 3).

Small, uncorrelated tentacle pulses (SUTPs). SUTPs, whose amplitudes ranged from 2.8 mV to 85.05 mV, and whose duration ranged between 200 ms and 2.15 s., were tallied (Fig.3). NMDA and D-serine (1 x [10.sup.-5] mol [l.sup.-1]), in the presence of AMPA (1 x [10.sup.-7] [moll.sup.-1]), caused a potentially significant increase in SUTP rates (pulses/min) during T1 (5.79 [+.or.-] 4.83, m.d. [+ or.-] i.q.r.; 8.15 [+ or -] 5.08, m. [+ or -] s.d.; P [less then or equal to] 0.08) and a significant increase in T2 (5.70 [+ or -] 3.20, m.d. [+ or -] i.q.r.; 8.29 [+ or -] 7.13, m. [+ or -] s.d) relative to the control period (3.22 [+ or -] 0.97, m.d. [+ or -] i.q.r.; 3.73 [+ or -] 1.86, m [+ or -] s.d.) (Table 1 and Fig 2c). The increase in frequencies at 1 x [10.sup.-5] mol [l.sup.-1] NMDA/D-serine was counteracted by 1 x [10.sup.-4] mol [l.sup.-1] D-AP5 (Fig. 2d). No other treatments, including that of NMDA and D-serine alone, significanT1y affected SUTPs (Tables 2 and 3).

[FIGURE 3 OMITTED]

Effect on body column

The endodermal rhythmic potential (RP) system. In one series of experiments, kainate (pT), at 1 x [10.sup.-7] mol [l.sup.-1], caused a significant increase in the RP rate (pulses/min) (2.95 [+ or -] 1.05, m.d. [+.or -] i.q.r.; 3.04 [+ or -] 0.41, m [+ or -] s.e.) relative to control (2.04 [+ or -] 1.88 m.d. [+ or -] m.d. [+ or -] i.q.r.; 2.36 [+ or -] 0.46,m. [+ or -] s.e.). NMDA and D-serine, at a concentration of 1 x [10.sup.-5] mol [l.sup.-1], produced a potentially significant reduction in RP rates during T2 (1.80 [+ or -] 1.08, m.d [+ or -] i.q.r.; 1.97 [+ or -] 0.26, m. [+ or -] s.e.; P [less then or equal to] 0.08) relative to pT, containing 1 x [10.sup.-7] mol [l.sup.-1] kainate. T2 was not significanT1y different from control. However, the kainate-induced increase in RP frequency, observed in this set of experiments, was not reproduced in a subsequent set of experiments in which a blocking period (bT), containing 1 x [10.sup.-4] mol [l.sup.-1] D-AP5, was inserted after pT (Table 2 and Fig. 2e and 2f). No other treatment, including that of NMDA and D-serine alone, had an effect of the RP system (Tables 1 and 3).

Small, uncorrelated body pulses (SUBPs). SUBPs, whose amplitudes ranged from 3.2 mV to 82.95 mV, and whose duration ranged from 250 ms to 1.85 s., were counted (Fig. 4), NMDA and D-serine (1 X [10.sup.-7] mol [l.sup.-1]), in the presence of AMPA (1 X [10.sup.-7] mol [l.sup.-1]), caused a potentially significant decrease in SUBP rates (pulses/min) during T2 (2.50 [+ or -] 1.80, m.d. [+ or -] i.q.r.; 2.50 [+ or -] 1.54, m. [+ or -] s.d.; P [less than or equal to] 0.08) relative to the control period (4.08 [+ or -] 2.76, m.d. [+ or -] i.q.r.; 4.78 [+ or -] 3.06, m. [+ or -] s.d.), but had no effect in T1. The potential decrease in T2 was counteracted by 1 X [10.sup.-4] mol [l.sup.-1] D-AP5 (Table 1 and Fig. 2g and 2h). No other treatment, including that of NMDA and D-serine alone, had an effect on SUBPs (Tables 2 and 3).

[FIGURE 4 OMITTED]

The ectodermal contraction burst system. No treatment had a significant effect on the total number of contraction bursts or the average number of pulses per contraction burst (Tables 1, 2, and 3).

DISCUSSION

In this study we have presented evidence that the glutamatergic agonist, N-methyl-D-aspartate (NMDA), and D-serine, together with [alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), increases tentacle activity by causing an increase in the output of the tentacle pulse pacemaker system and the rate of locally recorded, small, uncorrelated pulses in the tentacle (SUTPs). We also give evidence that NMDA/D-serine together, with AMPA or kainate, may affect the electrical activity of the body, causing a potentially significant decrease in the rate of locally recorded, small, uncorrelated body pulses (SUBPs), and a potentially significant decrease in the output of the endodermal rhythmic potential pacemaker system relative to a previous increase caused by kainate.

These findings are consistent with earlier studies indicating that glutamate, acting on its NMDA and AMPA or kainate receptors, affects the body and tentacle effector systems of hydra (Kass-Simon et al., 2003). NMDA/D-serine causes a reduction in the duration of reduced-glutathione (GSH)-induced mouth opening in the presence of concanavalin A (Con A) (Pierobon et al., 2004a) and, in the presence of kainate or AMPA, an increase in stenotele nematocyst discharge (Kass-Simon and Scappaticci, 2004; Scappaticci and Kass-Simon, 2008). Biochemical studies have also indicated the presence of a receptor in membrane fractions of hydra that binds to the NMDA channel blocker, MK-801 (Pierobon et al., 2004a), and immunocytochemical studies provided evidence that NMDA receptors exist on the surface of nerve cells, nematocytes, myonemes of epitheliomuscular cells, and interstitial stem cells (Scappaticci et al., 2004). AMPA and kainate receptors (GluR 2, 3 and GluR 5, 6, 7) have also been observed in macerated cell preparations of the hypostome and tentacles (Hufnagel et al., 1997, 1999).

In our earlier electrophysiological studies, glutamate and kainate increased the frequency of tentacle pulses (TPs), rhythmic pulses (RPs), and contraction bursts (CBs), in the absence of Con A, at higher concentrations than were used in the present study. In the earlier study, glutamate, AMPA, and/or kainate, increased RPs, CBs, and pulses per contraction burst (P/CB), in the presence of Con A (Kass-Simon et al., 2003). In this study, also in the presence of Con A, CBs and P/CB were unaffected; probably because of the lower concentrations of AMPA and kainate we used.

In one series of blocking experiments (containing 1 X [10.sup.-7] mol [l.sup.-1] AMPA, 1 X [10.sup.-4] mol [l.sup.-1] D-2-amino-5-phosphonopentanoic acid (D-AP5), and 1 X [10.sup.-5] mol [l.sup.-1] NMDA/ D-serine) the TP rate in the pre-treatment period (pT) with AMPA was unusually low. The low frequency was significanT1y different from the rate in treatment period two (T2) with NMDA/D-serine after the D-AP5 block (bT). However, the rate in T2 was not significanT1y different from the control rate. The difference between pT and T2 appears to be an anomaly, because in all of our other experiments--in the present study and in our previous experiments (Kass-Simon et al., 2003)--AMPA had no effect on TP rates.

RPs were significanT1y increased in one of our experimental series, but the increase was not reproduced in a second series. The reason for this difference is unclear, but could be the result of different recording or treatment conditions. In the series in which kainate (1 X [10.sup.-7] mol[l.sup.-1]) produced a significant increase in RP rate, NMDA/o-serine (1 X [10.sup.-5] mol [l.sup.-1]) caused a potentially significant reduction of the RP frequency, bringing the rate back to control levels. This effect may hint at an indirect inhibition, produced by NMDA/D-serine exciting inhibitory neurons in the body. In this regard, exposure to NMDA/D-serine, in the presence of AMPA, also caused a potentially significant decrease in SUBPs, further suggesting that inhibitory neurons in the body may have been excited.

In the classical extracellular recording studies of Passano and McCullough (1962, 1963, 1964, 1965) and Rushforth and Burke (1971), and in our own earlier studies (e.g., Kass-Simon and Passano, 1978; Kass-Simon et al., 2003; Ruggeri et al., 2004), the repertoire of reported electrical activity was limited to those impulses that were correlated or associated with observable behavioral changes, generally attributed to pacemaker activity, or to feeding behavior (Grosvenor et al., 1996). Uncorrected impulses, though frequenT1y observed in our laboratory, were never tallied. In this study, because it became evident that treatment with NMDA/D-serine affected this uncorrected activity, we counted the pulses. Although the impulses form a heterogeneous group (i.e., large variability in amplitude and duration), they must nonetheless be considered to arise from either nerve or epithelial cells (including epitheliomuscular cells, nematocytes, and stem cells), or both. Since the pulses are affected by our treatment, they would appear to be the result of the activation of glutamatergic NMDA receptors on these cells. Further, the NMDA receptors appear to be differentially distributed in various regions of the hydra, since only tentacle activity and some body column activity (RPs are endodermal) were affected. The precise location of the receptors, their ionic properties, and their pharmacological definition must await intracellular recordings from specific cell types.

Nevertheless, our results indicate that a vertebrate-like NMDA receptor, requiring membrane depolarization by AMPA or kainate receptors, affects the electrical activity both of the tentacles and of the body column in hydra. NMDA and D-serine, in the presence of AMPA/kainate, increased TP and SUTP rates, and also potentially decreased RP and SUBP rates. Together with previous studies, evidence suggests that the glutamatergic NMDA/ glycine-AMPA/kainate system plays a prominent role in controlling the neuroeffector systems of the hypostome, tentacle, and body column of this early-evolved metazoan.

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J. C. KAY AND G. KASS-SIMON *

Department of Biological Sciences, University of Rhode Island, Kingston, Rhode Island 02881

* To whom correspondence should be addressed. E-mail: kass.simon@ uri.edu

Abbreviations: AMPA, [alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; bT, blocking period; BVC, bicarbonate versene culture solution; CB, contraction burst; Con A, concanavalin A; D-AP5, D-2-amino-5-phosphonopentanoic acid; FANOVA, Friedman two-way analysis of variance; GABA, [gamma]-amino-butyric acid; iGluR, ionotropic glutamate receptor; LTD, long-term depression; LTP, long-term potentiation; m. [+ or -] s.d., mean [+ or -] standard deviation; m. [+ or -] s.d. mean [+ or -] standard error; m.d. [+ or -] i.q.r, median [+ or -] inter-quartile range; mGluR, metabotropic receptor; NMDA, N-methyl-D-aspartate; NMDAR, NMDA receptor; P/CB, average number of pulses per contraction burst; PLP, pre-locomotor pulse; pT, pre-treatment period; RP, rhythmic potential; SUBP, small, uncorrelated body pulse; SUTP, small, uncorrelated tentacle pulse; T1, treatment period 1; T2, treatment period 2; TP, tentacle pulse.

Received 5 September 2008; accepted 2 January 2009.

Glutamate is the primary excitatory neurotransmitter in the vertebrate central nervous system and the nervous systems of many invertebrates. It functions by binding to receptors on nerve and effector cells that gate ions direcT1y, through channel pores in the receptor (ionotropic glutamate receptors, iGluRs; see Dingledine et al., 1999, for a review), or indirecT1y, through a series of intracellular metabolic steps (metabotropic receptors, mGluRs; see Pin and Duvoisin, 1995, for a review). The receptors are characterized pharmacologically and classified according to their selective binding of the various glutamatergic agonists (Watkins and Olverman, 1987).

In mammalian systems, at least three major iGluR subtypes have been identified: [alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainic acid, and N-meth-yl-D-aspartic acid (NMDA); AMPA and kainate receptors are fast ionic channels that gate [Na.sup.+] and [K.sup.+]; the NMDA receptor gates [Na.sup.+], [K.sup.+], and [Ca.sup.2+]. NMDA receptor channels (NMDARs) have been implicated in neuromuscular transmission in crayfish (Feinstein et al, 1998), and in long-term potentiation (LTP), long-term depression (LTD), and learning in vertebrates and in the opisthobranch Aplysia (Collingridge et al., 1983; Dudek and Bear, 1992; Bliss and Collingridge, 1993, for a review; Roberts and Glanzman, 2003).

NMDAR channels are unique in that, under resting conditions, their channel pore is blocked by the divalent magnesium ion. The block is removed only by a membrane depolarization (Nowak et al., 1984; Mayer and Westbrook, 1987). The depolarization is accomplished by glutamate acting on the fast-acting AMPA or kainate receptors that lie close to the NMDA receptors. Kainate and AMPA receptors, which have overlapping, though not identical, gating properties, are sometimes grouped together as AMPA/kainate receptors (see BetT1er and Mulle, 1995, for a review). The NMDA receptor has binding sites for glutamate and glycine, both of which are simultaneously required for the activation of the receptor and for preventing [Ca.sup.2+] -dependent desensitization (Johnson and Ascher, 1987; Kleckner and Dingledine, 1988). In hydra, NMDA receptors are involved in regulating the duration of reduced-glutathione (GSH)-induced mouth opening (Pierobon et al., 2004a) and nematocyst discharge in the tentacles (Kass-Simon and Scappaticci, 2004; Scappaticci and Kass-Simon, 2008).

Hydra has two main effector systems responsible for its behavioral repertoire: the neuro-epitheliomuscular systems, which control mouth opening and closing and regulate the contraction and elongation of hydra's body and tentacles (Passano and McCullough, 1962, 1963, 1964, 1965; Rush-forth and Burke, 1971); and the nematocyst system, which results in the capture and killing of prey (see Kass-Simon and Scappaticci, 2002, for a review). Although a number of other neurotransmitters, including acetylcholine, catecholamines, serotonin, and numerous peptides, have previously been reported to play a role in hydra's neuroeffector systems (see Kass-Simon and Pierobon, 2007, for a review), recent studies have presented extensive evidence for the involvement of the amino acid transmitters glutamate. [gamma]-amino-butyric acid (GABA), and glycine, in the functioning of both the epitheliomuscular and nematocyst effector systems (Bellis et al, 1991; Pierobon et al., 1995, 2001, 2004a, b; Concas et al, 1998; Kass-Simon et al., 2003; Kass-Simon and Scappaticci, 2004; Ruggeri et al., 2004; Scappaticci et al., 2004; Scappaticci and Kass-Simon, 2008).

Three main pacemaker systems control hydra's epitheliomuscular effectors. (1) Contractions of the body column are elicited by the contraction burst (CB) pacemaker system (Passano and McCullough, 1964). Its longitudinally conducted impulses are thought to be generated in a circumferential nerve ring located just below the hypostome (Passano and McCullough, 1963; Kass-Simon, 1972; Kass-Simon and Passano, 1978; Kinnamon and Westfall, 1981; Koizumi et al, 1992), but may also originate elsewhere in the ectoderm of the body column (Kass-Simon, 1970). (2) Elongation of the body column is caused by the rhythmic potential (RP) pacemaker system, which causes a contraction of circumferentially arranged myonemes (Shibley, 1969). These pulses are initiated in the peduncle (Passano and McCullough, 1962, 1965) and are conducted along the endoderm of the body and through-conducted into the tentacles (Kass-Simon and Passano, 1978). (3) The tentacle pulse (TP) system is responsible for the contraction of the tentacles and has been found to excite the CB system (Rushforth and Burke, 1971; Kass-Simon 1972).

In order to continue to delineate the role of NMDA receptors in the effector systems of Hydra vulgaris, we investigated the effects of NMDA, together with D-serine, the specific glycine agonist at the NMDA receptor (Kleck-ner and Dingledine, 1988; Mothet et al., 2000), on the electrical activity of hydra's epitheliomuscular systems. In addition to the classical pacemaker systems, we also monitored the effects of NMDA/D-serine on non-pacemaker electrical activity in the tentacle and the body column. Our results indicate that NMDA/D-serine modifies the activity of both the tentacle and the body--increasing the output of the tentacle pacemaker system and the non-pacemaker pulses in the tentacle, and potentially decreasing the output of the rhythmic potential pacemaker system and the non-pacemaker pulses in the body column.

The study is part of an ongoing collaboration among the laboratories of A. Concas, L. Hufnagel, G. Kass-Simon, and P. Pierobon.

MATERIALS AND METHODS

Animals

Specimens of Hydra vulgaris were asexually cultured in glass baking dishes containing modified bicarbonate versene culture solution (BVC): 1 X [10.sup.-7] mol [l.sup.-1] [NaHCO.sub.3], 1 X [10.sup.-6] mol [l.sup.-1] Ca[Cl.sub.2], and 1 X [10.sup.-8] [mol [l.sup.-1] EDTA, pH 7.0 [+ or -] 0.2 (Loomis and Lenhoff, 1956; Muscatine and Lenhoff, 1965). All animals were kept in an incubator at 18 [+ or -] 1.0 [degrees]C. Hydra cultures were fed freshly hatched Artemia salina nauplii once every 48 h. To prevent possible effects of feeding on contractile behaviors (Passano and McCullough, 1964; Grosvenor et al, 1996), experimental animals were randomly selected from non-budding hydra that were starved for 24 [+ or -] 2 h.

Recording methods

Electrical recordings were made at 22 [+ or -] 2.0 [degrees]C, under red light (low setting on Fiber-Lite 190 lamp, Dolan-Jenner Industries, Boxborough, MA) at an intensity of 102.5 foot-candles (1103.3 lux). Hydras have been reported to be sensitive to all colors of light with the exception of red (Wilson, 1891; Haug, 1933); their pacemaker output in red light is similar to that found in the absence of light (Passano and McCullough, 1962, 1964). The recording techniques were modified from those of Passano and McCullough (1964; e.g., Kass-Simon et al., 2003; Ruggieri et al., 2004). A polyethylene suction electrode was attached to the tip of a tentacle and another to the base of the peduncle (Fig. 1). During recording, animals were observed through a dissecting microscope to correlate various behaviors with their respective pulses. The signals from each electrode were amplified by an AC differential amplifier (Model 3000, A-M Systems, Sequim, WA), digitally converted by a MacLab AID converter (AD Instruments, Colorado Springs, CO), and recorded on a Macintosh G3 computer (Apple Computers) using Chart 3.6.8 software (AD Instruments). Square pulses from a Grass SD9 stimulator (Grass Instruments, West Warwick, RI) were used to mark visually observed tentacle contractions on a separate channel of the computer record. The following parameters were measured for each 5-min period of an experiment: frequency of rhythmic potentials (RP/min), tentacle pulses (TP/min), and contraction bursts (CB/min), and the average number of pulses per contraction burst (P/CB). RPs are defined as small, regularly occurring pulses, associated with the elongation of the body and conducted throughout the body column and tentacles in the endoderm (Passano and McCullough, 1962; Kass-Simon and Passano, 1978). TPs are defined as pulses visually correlated with the contraction of the tentacle and are not always conducted into the body column or into adjacent tentacles (Rushforth and Burke, 1971; Kass-Simon and Passano, 1978; Grosvenor et al., 1996; Kass-Simon et al., 2003; Ruggieri et al., 2004). CBs are defined as several consecutive pulses, usually arising at the hypostome, and are through-conducted into the tentacles from the body; the pulses are associated with the contraction of the body column (Passano and McCullough, 1964; Kass-Simon, 1972, 1973). CBs were considered to consist of at least four through-conducted, contraction-associated pulses occurring within 15 s of each other. In addition to the classical categories of pacemaker pulses, we recorded and tallied other impulses that have not previously been reported, but that have frequenT1y been seen in many other experiments. These impulses are heterogeneous in shape and probably arise either from neurons or myonemes; they are often likely to be a composite of both. We have placed all such uncorrelated pulses into two classes, those recorded at the tentacle and those at the body, and have designated them small, uncorrected tentacle or body pulses (SUTPs or SUBPs) respectively. They are not associated with any visually discernible behavior. Their amplitude and duration vary over a considerable range (SUTPs: 3.2 mV-82.95 mV, 200 ms-2.15 s; SUBPs 2.8 mV-85.05 mV, 250 ms-1.85 s). Pre-locomotor pulses (PLPs), which are pulses that occur before some contraction bursts (Passano and McCullough, 1964), were included in SUTP and SUBP counts if they were not through-conducted; otherwise they were not tallied. The size and duration of PLPs fall within the abovecited parameters for SUTPs and SUBPs, and therefore could not be distinguished from them.

[FIGURE 1 OMITTED]

At the start of a recording session, hydras were placed in a 10-ml petri dish containing 6 ml of BVC, and connected to a suction electrode. All test substances were made up at 10-fold of their final concentration. They were diluted to the final concentration by adding them to the solution in the culture dish (Kass-Simon et al., 2003; Ruggieri et al., 2004). The first 5 min of recording was used as an acclimation period and was not included in the statistical analysis. The treatment protocol began with the addition of 0.6 ml of concanavalin A (Con A), which constitutes the control period (C). Con A (1 x [10.sup.-15] mol [l.sup.-1]) was used to prevent glutamatergic desensitization (Mayer and Vylicky, 1989; Partin et al., 1993; Kass-Simon et al., 2003). In preliminary experiments in which the responses in Con A were compared to those in BVC (n = 10), no statistically significant differences were found in the monitored parameters. Therefore, we used Con A as the control for all our recordings. Recordings lasted for 20-30 min, depending on the number of substances to be tested. Each recording session was divided into 4, 5, or 6 five-minute periods for statistical analysis. The following recording protocol was used for experiments in which NMDA plus D-serine was the only test substance: the first 5-min recording period was a BVC acclimation period, followed by a Con A control period (C), and a first treatment period (T1) containing NMDA and D-serine; this period was continued for an additional 5 min and then considered the second treatment period (T2). For recordings in which AMPA or kainate preceded treatment with NMDA and D-serine, the following protocol was used: a BVC acclimation period, a Con A control period (C), an AMPA or kainate pre-treatment period (pT), an NMDA plus D-serine first treatment period (T1), continued for a second 5-min treatment period (T2). For recordings in which an antagonist preceded NMDA/D-serine treatment, the following protocol was used: a BVC acclimation period, a Con A control period (C), an AMPA or kainate pre-treatment period (pT), a 5-min blocking period (bT) containing d-2-amino-5-phosphonopentanoic acid (D-AP5), followed by two NMDA plus D-serine treatment periods (T1 and T2). D-AP5 is a competitive antagonist of NMDA (Evans et al., 1982).

All substances were added at the start of the respective treatment period. No animal was used more than once. Experiments were performed without washout periods because of the difficulty of maintaining recording conditions during evacuation of the recording dish.

Ligands

The following agonists and antagonists for the NMDA receptor were used: N-methyl-D-aspartate (NMDA), D-2-amino-5-phosphonopentanoic acid (D-AP5), a-amino-3-hy-droxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate, D-serine, and Con A. NMDA, kainate, Con A, and D-serine were purchased from Sigma-Aldrich Corporation (St. Louis, MO). D-AP5 and AMPA were purchased from Tocris Bioscience (Ellisville, MO). The following combinations of ligands (in final concentrations) were used: (1) NMDA + D-serine (both at 1 x [10.sup.-5] mol [l.sup.-1], 1 x [10.sup.-7] mol [l.sup.-1], and 1 x [10.sup.-9] mol [l.sup.-1]); (2) AMPA 1 x [10.sup.-7] mol [l.sup.-1]) + NMDA + D-serine (both at 1 x [10.sup.-5] mol [l.sup.-1], 1 x [10.sup.-7] mol [l.sup.-1], and 1 x [10.sup.-9] mol [l.sup.-1]); (3) kainate 1 x [10.sup.-7] mol [l.sup.-1]) + NMDA + D-serine (both at 1 x [10.sup.-5] mol [l.sup.-1], 1 x [10.sup.-7] mol [l.sup.-1], and 1 x [10.sup.-9] mol [l.sup.-1]); (4) AMPA 1 x [10.sup.-7] mol [l.sup.-1]) + D-AP5 1 x [10.sup.-4] mol [l.sup.-1]) + NMDA + D-serine (both at 1 x [10.sup.-5] mol [l.sup.-1], and 1 x [10.sup.-7] mol [l.sup.-1]); (5) kainate 1 x [10.sup.-7] mol [l.sup.-1]) + D-AP5 1 x [10.sup.-4] mol [l.sup.-1]) + NMDA + D-serine (both at 1 x [10.sup.-5] mol [l.sup.-1]).

Data analysis

At least six animals were used for each ligand series. Each animal was compared against itself because significant, but unaccounted for, differences occur between animals if experiments are conducted for as long as several months (Kass-Simon et al., 2003; Ruggieri et al., 2004). To ensure that no time-dependent changes occurred during the course of an experiment, a series of preliminary recordings, in BVC alone, lasting 30 min, were performed (n = 6). No statistical differences were found among the 5-min periods within an experiment.

Data analysis is modified from that of Kass-Simon et al. (2003) and Ruggieri et al. (2004). A Friedman two-way analysis of variance (FANOVA; Siegel, 1956) and post hoc test, to be used with FANOVAs (Daniel, 1990), was performed. The post hoc test used the following equation:

[absolute value of [R.sub.j] - [R.sub.j']] [greater than or equal to] z [square root of ([bk(k + 1)/6])]

where [R.sub.j] is the jth rank treatment total, [R.sub.j'] is the j'th rank treatment total, z is a value from a standard distribution table, b is the number of animals used, and k is the number of treatments. The corrected alpha ([alpha]) value was solved for by using the following equation: [[alpha].sub.c] = [alpha]/k(k + 1) where [alpha] is the original error level and k is the number of treatments (Daniel, 1990). The first 20 s of each period, in which a test substance was added, were omitted from statistical analysis in order to avoid including behavioral/electrical changes induced by disturbance of the water during substance administration. RPs, TPs, CBs, SUTPs, and SUBPs were converted to pulses per minute for analysis. Since we used a nonparametric statistic based on rank, TP, CB, SUTP, and SUBP values are presented as medians [+ or -] inter-quartile ranges (m.d. [+ or -] i.q.r.) in the text and tables. However, to permit comparisons with our previous studies, we also give the means and standard deviations (m. [+ or -] s.d.) in the text, tables, and histograms. P/CBs are the average number of pulses per contraction burst and are calculated by adding the number of pulses in each CB and dividing the total by the number of CBs in a given period. CBs that started in one period and continued into the next period were counted in the period in which they originated; the pulses in these CBs were not counted and not averaged in with the P/CBs. In some instances, no CBs occurred in a period, and hence the P/CB parameter was omitted from those experiments; this sometimes resulted in the omission of that parameter from an entire series of experiments (Tables 1 and 2). P/CBs are presented as m.d. [+ or -] i.q.r. and means [+ or -] standard errors (m. [+ or -] s.e.). Because RPs were sometimes difficult to discern, they were tallied twice and the average of the two counts was taken as our value. Therefore, RP values are given as m.d. [+ or -] i.q.r. and m. [+ or -] s.e. Differences were considered statistically significant at P [less than or equal to] 0.05, and non-significant with a strong trend, which we termed "potentially significant," at 0.051 [less than or equal to] P [less than or equal to] 0.08.

Table 1 The effect of N-methyl-D-aspartate (NMDA) and [alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) on the electrical activity of hydra Treatment period (b) Electrical C pT activity (a) Treatment: 1 X [10.sup.-5] mol [l.sup.-1] NMDA/D-serine with 1 X [10.sup.-7] mol [l.sup.-1] AMPA TP (n = 9) 1.07 [+ or -] 0.86 0.21 [+ or -] 0.21 (1.17 [+ or -] 1.48) (1.55 [+ or -] 2.85) SUTP (n = 9) 3.22 [+ or -] 0.97 4.08 [+ or -] 2.47 (3.73 [+ or -] 1.86) (5.22 [+ or -] 2.70) RP (n = 9) 2.58 [+ or -] 1.29 2.90 [+ or -] 0,64 (2.90 [+ or -] 0.44) (2.93 [+ or -] 0.25) SUBP (n = 9) 3.43 [+ or -] 3.38 4.84 [+ or -] 4,86 (7.19 [+ or -] 9.19) (4.89 [+ or -] 3.36) CB (n = 9) 0.21 [+ or -] 0.21 0.21 [+ or -] 0.21 (0.26 [+ or -] 0.14) (0.24 [+ or -] 0.17) P/CB (n = 9) 18.00 [+ or -] 4.25 20.00 [+ or -] 5.00 (21.06 [+ or -] 3.34) (18.78 [+ or -] 4.26) Treatment: 1 X [10.sup.-7] mol [l.sup.-1] NMDA/D-serine with 1 X [10 .sup.-7] mol [l.sup.-1] AMPA TP (n = 10) 0.86 [+ or -] 1.34 1.93 [+ or -] 2.20 (1.20 [+ or -] 1.34) (2.32 [+ or -] 1.92) SUTP (n = 9) 2.15 [+ or -] 2.04 3.11 [+ or -] 2.15 (2.80 [+ or -] 1.71) (3.34 [+ or -] 2,45) RP (n = 9) 1.50 [+ or -] 0.64 1.39 [+ or -] 1.50 (1.75 [+ or -] 0.30) (1.51 [+ or -] 0.27) SUBP (n = 9) 4.08 [+ or -] 2.76 4.40 [+ or -] 2.36 (4.78 [+ or -] 3.06) (3.86 [+ or -] 1.66) CB (n = 10) 0.32 [+ or -] 0.21 0.21 [+ or -] 0.21 (0.34 [+ or -] 0.21) (0.30 [+ or -] 0.11) P/CB (n = 10) 13.00 [+ or -] 5.00 15.00 [+ or -] 6.00 (11.80 [+ or -] 3.07) (15.50 [+ or -] 1.51) Treatment: 1 X [10.sup.-9] M NMDA/D-serine with 1 X [10 .sup.-7] mol [1.sup.-7] mol [l.sup.-1] AMPA TP (n = 9) 2.15 [+ or -] 1.72 1.07 [+ or -] 5.58 ( 1.74 [+ or -] 1.43) (4.27 [+ or -] 5.59) SUTP (n = 9) 2.79 [+ or -] 2.68 3.76 [+ or -] 1.72 ( 3.36 [+ or -] 1.58) (3.71 [+ or -] 1.28) RP (n = 9) 1.18 [+ or -] 0.13 1.29 [+ or -] 0.75 (1.19 [+ or -] 0.13) (1.20 [+ or -] 0.15) SUBP (n. = 9) 4.94 [+ or -] 2.04 2.79 [+ or -] 1.50 (4.43 [+ or -] 1.81) (3.45 [+ or -] 2.12) CB (n = 8) 0.54 [+ or -] 0.21 0.43 [+ or -] 0.21 (0.51 [+ or -] 0.16) (0.32 [+ or -] 0.16) P/CB (n = 8) 19.42 [+ or -] 9.42 13.25 [+ or -] 1.63 (21.17 [+ or -] 4.93) (12.25 [+ or -] 2.08) Treatment: 1 X [10.sup.-5] mol [l.sup.-1] NMDA/D-serine with 1 X [10 .sup.-7] mol [l.sup.-1] AMPA + 1 X [10.sup.4] mol [l.sup.-1] D AP5 TP (n = 8) 1.07 [+ or -] 1.13 0.43 [+ or -] 0.27 (1.07 [+ or -] 0.65) (0.43 [+ or -] 0.47) # SUTP (n = 7) 4.29 [+ or -] 3-76 3.54 [+ or -] 2.41 (5.46 [+ or -] 2.81) (3.91 [+ or -] 1.82) RP (n = 7) 1.18 [+ or -] 0.21 1.18 [+ or -] 0.98 (1.20 [+ or -] 0-11) (1.14 [+ or -] 0.25) SUBP (n = 7) 2.47 [+ or -] 2.25 2.68 [+ or -] 1.45 (2.88 [+ or -] 1.31) (2.77 [+ or -] 1.54) CB (n = 8) 0.32 [+ or -] 0.21 0-21 [+ or -] 0.00 (0.32 [+ or -] 0.11) (0.19 [+ or -] 0.08) P/CB (-) -- -- Treatment: 1 X [10.sup.-7] mol [l.sup.-1] NMDA/D-serine with 1 X [10 .sup.-7] mol [l.sup.-1] AMPA + 1 X [10.sup.4] mol [l.sup.-1] D-AP5 TP (n = 8) 0.43 [+ or -] 0.97 0.21 [+ or -] 0.48 (0.91 [+ or -] 1.44) (0.51 [+ or -] 0.64) SUTP (n = 8) 3.65 [+ or -] 2.92 4.18 [+ or -] 5.95 (5.04 [+ or -] 3.09) (5.70 [+ or -] 3.62) RP (n = 8) 1.18 [+ or -] 0.70 1.18 [+ or -] 0.64 (1.31 [+ or -] 0.26) (1.14 [+ or -] 0.23) SUBP (n = 8) 3.90 [+ or -] 3.22 2.31 [+ or -] 1.42 (3.19 [+ or -] 2.05) (2.72 [+ or -] 2.06) CB (n = 8) 0.21 [+ or -] 0.21 0.21 [+ or -] 0.05 (0.13 [+ or -] 0.11) (0.30 [+ or -] 0.16) P/CB (-) -- -- Treatment period (b) Electrical bT T1 activity (a) Treatment: 1 X [10.sup.-5] mol [l.sup.-1] NMDA/D-serine with 1 X [10.sup.-7] mol [l.sup.-1] AMPA TP (n = 9) NA 0.64 [+ or -] 1.07 (1.03 [+ or -] 1.15) SUTP (n = 9) NA 5.79 [+ or -] 4.83 (8.15 [+ or -] 5.08) # RP (n = 9) NA 2.58 [+ or -] 0.75 (3.03 [+ or -] 0.52) SUBP (n = 9) NA 4.08 [+ or -] 3.35 (4.10 [+ or -] 2.26) CB (n = 9) NA 0.21 [+ or -] 0.21 (0.21 [+ or -] 0.21) P/CB (n = 9) NA 22.50 [+ or -] 12.50 (17.06 [+ or -] 4.80)) Treatment: 1 X [10.sup.-7] mol [l.sup.-1] NMDA/D-serine with 1 X [10 .sup.-7] mol [l.sup.-1] AMPA TP (n = 10) NA 2.36 [+ or -] 3.92 (3.03 [+ or -] 2.59) SUTP (n = 9) NA 3.97 [+ or -] 1.61 (4.01 [+ or -] 1.91) RP (n = 9) NA 1.18 [+ or -] 0.64 (1.16 [+ or -] 0.15) SUBP (n = 9) NA 2.15 [+ or -] 3.33 (3.21 [+ or -] 2.31) CB (n = 10) NA 0.21 [+ or -] 0.00 (0.24 [+ or -] 0.19) P/CB (n = 10) NA 14.00 [+ or -] 5.00 (14.23 [+ or -] 3.22) Treatment: 1 X [10.sup.-9] M NMDA/D-serine with 1 X [10 .sup.-7] mol [1.sup.-7] mol [l.sup.-1] AMPA TP (n = 9) NA 1.29 [+ or -] 8.37 (5.67 [+ or -] 7.34) SUTP (n = 9) NA 3.00 [+ or -] 3.43 (4.53 [+ or -] 2.40) RP (n = 9) NA 0.86 [+ or -] 0.75 (1.14 [+ or -] 0.16) SUBP (n. = 9) NA 2.90 [+ or -] 1.72 (2.77 [+ or -] 1.23) CB (n = 8) NA 0.43 [+ or -] 0.05 (0.40 [+ or -] 0.14) P/CB (n = 8) NA 17.75 [+ or -] 7.13 (18.08 [+ or -] 1.94) Treatment: 1 X [10.sup.-5] mol [l.sup.-1] NMDA/D-serine with 1 X [10 .sup.-7] mol [l.sup.-1] AMPA + 1 X [10.sup.4] mol [l.sup.-1] D AP5 TP (n = 8) 0.64 [+ or -] 1.82 1.29 [+ or -] 2.15 (1.21 [+ or -] 1.44) (1.53 [+ or -] 1.35) SUTP (n = 7) 3.86 [+ or -] 1.50 4.61 [+ or -] 3.97 (4.20 [+ or -] 1.21) (5.47 [+ or -] 2.71) RP (n = 7) 0.64 [+ or -] 0.64 1.07 [+ or -] 1.02 (0.71 [+ or -] 0.14) (1.12 [+ or -] 0.23) SUBP (n = 7) 2.15 [+ or -] 1.07 2.04 [+ or -] 2.47 (1.72 [+ or -] 0.70) (2.62 [+ or -] 1.81) CB (n = 8) 0.21 [+ or -] 0.00 0.11 [+ or -] 0.21 (0.21 [+ or -] 0.11) (0.11 [+ or -] 0.11) P/CB (-) -- -- Treatment: 1 X [10.sup.-7] mol [l.sup.-1] NMDA/D-serine with 1 X [10 .sup.-7] mol [l.sup.-1] AMPA + 1 X [10.sup.4] mol [l.sup.-1] D-AP5 TP (n = 8) 0.75 [+ or -] 0.75 0.97 [+ or -] 2.90 (1.77 [+ or -] 3.12) (1.77 [+ or -] 1.97) SUTP (n = 8) 3.43 [+ or -] 2.39 4.61 [+ or -] 2.47 (4.10 [+ or -] 2.03) (5.61 [+ or -] 2.96) RP (n = 8) 1.02 [+ or -] 0.51 1.23 [+ or -] 0.91 (1.22 [+ or -] 0.25) (1.29 [+ or -] 0.24) SUBP (n = 8) 2.95 [+ or -] 3.00 2.52 [+ or -] 1.77 (3.49 [+ or -] 2.62) (2.92 [+ or -] 1.96) CB (n = 8) 0.21 [+ or -] 0.29 0.00 [+ or -] 0.05 (0.24 [+ or -] 0.18 (0.05 [+ or -] 0.10) P/CB (-) -- -- Treatment period (b) Electrical T2 Significant activity (a) differenced Treatment: 1 X [10.sup.-5] mol [l.sup.-1] NMDA/D-serine with 1 X [10.sup.-7] mol [l.sup.-1] AMPA TP (n = 9) 0.60 [+ or -] 3.20 (1.87 [+ or -] 2.39) SUTP (n = 9) 5.70 [+ or -] 3.20 (8.29 [+ or -] 7.13) # C < T1^, T2 *# RP (n = 9) 2.60 [+ or -] 0.90 (2.44 [+ or -] 0.22) SUBP (n = 9) 4.30 [+ or -] 1.98 (4.18 [+ or -] 1.79) CB (n = 9) 0.20 [+ or -] 0.40 (0.22 [+ or -] 0.19) P/CB (n = 9) 19.50 [+ or -] 5.25 (12.94 [+ or -] 3.66 Treatment: 1 X [10.sup.-7] mol [l.sup.-1] NMDA/D-serine with 1 X [10.sup.-7] mol [l.sup.-1] AMPA TP (n = 10) 2.10 [+ or -] 3.95 (3.94 [+ or -] 3.90) # C < T2 *# SUTP (n = 9) 3.80 [+ or -] 1.70 (3.97 [+ or -] 2.13) RP (n = 9) 0.80 [+ or -] 1.20 (1.34 [+ or -] 0.41) SUBP (n = 9) 2.50 [+ or -] 1.80 (2.50 [+ or -] 1.54) # C > T2^ # CB (n = 10) 0.20 [+ or -] 0.20 (0.28 [+ or -] 0.17) P/CB (n = 10) 13.00 [+ or -] 3.50 (15.03 [+ or -] 2.81) Treatment: 1 X [10.sup.-9] M NMDA/D-serine with 1 X [10 .sup.-7] mol [1.sup.-7] mol [l.sup.-1] AMPA TP (n = 9) 1.00 [+ or -] 4.20 (3.22 [+ or -] 4.00) SUTP (n = 9) 7.00 [+ or -] 3.60 (6.73 [+ or -] 4.92) RP (n = 9) 0.80 [+ or -] 0.40 (1.20 [+ or -] 0.31) SUBP (n. = 9) 2.70 [+ or -] 0.10 (2.64 [+ or -] 1.20) CB (n = 8) 0.40 [+ or -] 0.25 (0.45 [+ or -] 0.21) P/CB (n = 8) 14.00 [+ or -] 3.50 (12,58 [+ or -] 2.24) Treatment: 1 X [10.sup.-5] mol [l.sup.-1] NMDA/D-serine with 1 X [10 .sup.-7] mol [l.sup.-1] AMPA + 1 X [10.sup.4] mol [l.sup.-1] D AP5 TP (n = 8) 2.60 [+ or -] 3.05 (2.93 [+ or -] 2.06) pT < T2 *# SUTP (n = 7) 4.60 [+ or -] 2.10 (4.43 [+ or -] 1.83) RP (n = 7) 0.80 [+ or -] 0.20 (1.34 [+ or -] 0.62) SUBP (n = 7) 1.80 [+ or -] 0.60 (2.43 [+ or -] 2.59) CB (n = 8) 0.20 [+ or -] 0.25 (0.23 [+ or -] 0.17) P/CB (-) - Treatment: 1 X [10.sup.-7] mol [l.sup.-1] NMDA/D-serine with 1 X [10 .sup.-7] mol [l.sup.-1] AMPA + 1 X [10.sup.4] mol [l.sup.-1] D-AP5 TP (n = 8) 0.60 [+ or -] 2.35 (2.78 [+ or -] 4.41) SUTP (n = 8) 3.60 [+ or -] 3.23 (3.94 [+ or -] 1.93) RP (n = 8) 1.02 [+ or -] 0.43 (1.09 [+ or -] 0.14) SUBP (n = 8) 1.80 [+ or -] 2.38 (2.46 [+ or -] 2.12) CB (n = 8) 0.10 [+ or -] 0.20 (0.10 [+ or -] 0.11) P/CB (-) - Note: Values are pulses/min (median [+ or -] inter-quartile range, with mean and standard deviation in parentheses, except RP and P/CB, where values in parentheses are means [+ or -] standard error). Note: Significant (P [less than or equal to] 0.05) or "potentially significant" (P [less thna or equal to] 0.08) values are indicated in bold. # (a) TP = tentacle pulse; SUTP = small, uncorrected tentacle pulse; RP = rhythmic potential; SUBP = small, uncorrected body pulse; CB = contraction burst; P/CB = average number of pulses per contraction burst; n = number of animals tested. (b) C = Con A control period, pT = AMPA pre-treatment period, bT = D-AP5 blocking period, T1 = first treatment period, T2 = second treatment period. (c) Asterisks (*) denote significant differences. Carets (235) denote "potentially significant" differences. Table 2 The effect of NMDA and D-serine with kainate on the electrical activity of hydra Treatment period (b) Electrical C pT activity (a) Treatment: 1 X [10.sup.-5] mol [l.sup.-1] NMDA/D-serine with 1 X [10.sup.-7] mol [l.sup.-1] kainate TP(n = 10) 0.75 [+ or -] 1.23 0.97 [+ or -] 1.66 (0.90 [+ or -] 1.06) (1.37 [+ or -] 1.38) SUTP(n = 10) 4.56 [+ or -] 6.87 3.86 [+ or -] 0.59 (6.12 [+ or -] 3.98) (5.38 [+ or -] 6.17) RP (n = 10) 2.04 [+ or -] 1.88 2.95 [+ or -] 1.05 (2.36 [+ or -] 0.46) (3.04 [+ or -] 0.41) # SUBP (n = 10) 2.15 [+ or -] 1.15 1.39 [+ or -] 1.82 (2.83 [+ or -] 2.32) (1.56 [+ or -] 1.02) CB (n = 10) 0.43 [+ or -] 0.16 0.32 [+ or -] 0.21 (0.36 [+ or -] 0.10) (0.30 [+ or -] 0.15) P/CB (n = 10) 19.00 [+ or -] 6.50 18.50 [+ or -] 6.00 (23.60 [+ or -] 4.16) (25.35 [+ or -] 5.46) Treatment: 1 X [10.sup.-7] mol [l.sup.-1] NMDA/p-serine with 1 X [10.sup.-7] mol [l.sup.-1] kainate TP (n = 9) 3.00 [+ or -] 2.36 1.93 [+ or -] 3.00 (2.84 [+ or -] 2.54) (3.93 [+ or -] 4.52) SUTP (n = 9) 5.04 [+ or -] 4.6.33 5.36 [+ or -] 2.90 (6.63 [+ or -] 4.83) (5.77 [+ or -] 4.50) RP (n = 9) 3.33 [+ or -] 2.04 2.47 [+ or -] 2.04 (2.67 [+ or -] 0.51) (2.72 [+ or -] 0.48) SUBP (n = 9) 5.90 [+ or -] 5.90 4.51 [+ or -] 2.90 (6.41 [+ or -] 3.99) (6.10 [+ or -] 4.39) CB (n = 9) 0.21 [+ or -] 0.21 0.21 [+ or -] 0.21 (0.29 [+ or -] 0.11) (0.29 [+ or -] 0.15) P/CB (n = 9) 25.00 [+ or -] 12.25 31.25 [+ or -] 28.13 (20.17 [+ or -] 5.14) (21.17 [+ or -] 6.19) Treatment: 1 X [10.sup.-9] mol [l.sup.-1] NMDA/D-serine with 1 x [10.sup.-7] mol [l.sup.-1] kainate TP (n = 9) 0.64 [+ or -] 2.36 2.79 [+ or -] 2.58 (2.27 [+ or -] 2.91) (2.77 [+ or -] 1.79) SUTP (n = 8) 4.56 [+ or -] 2.74 6.01 [+ or -] 5.18 (5.31 [+ or -] 4.09) (6.41 [+ or -] 4.13) RP (n = 8) 1.72 [+ or -] 1.53 1.98 [+ or -] 0.86 (1.98 [+ or -] 0.37) (2.07 [+ or -] 0.32) SUBP (n = 8) 3.70 [+ or -] 3.89 4.24 [+ or -] 2.60 (4.75 [+ or -] 2.40) (4.08 [+ or -] 1.70) CB (n = 9) 0.21 [+ or -] 0.21 0.21 [+ or -] 0.21 (0.24 [+ or -] 0.17) (0.31 [+ or -] 0.16) P/CB (n = 9) 15.50 [+ or -] 2.88 14.25 [+ or -] 3.38 (15.28 [+ or -] 3.48) (10.72 [+ or -] 2.99) Treatment: 1 X [10.sup.-9] mol [l.sup.-1] NMDA/D-serine with 1 X [10.sup.-7] mol [l.sup.-1] kainate + 1 X [10.sup.-4] mol [l.sup.-1] D-AP5 TP (n = 8) 1.18 [+ or -] 0.97 0.64 [+ or -] 1.29 (1.42 [+ or -] 1.20) (0.97 [+ or -] 0.85) SUTP (n = 8) 4.61 [+ or -] 3.65 4.29 [+ or -] 2.76 (4.84 [+ or -] 2.52) (4.99 [+ or -] 2.40) RP (n = 8) 1.02 [+ or -] 0.40 0.70 [+ or -] 0.83 (1.17 [+ or -] 0.23) (0.86 [+ or -] 0.22) SUBP (n = 8) 1.98 [+ or -] 0.80 2.52 [+ or -] 1.64 (2.05 [+ or -] 0.95) (2.68 [+ or -] 1.93) CB (n = 8) 0.21 [+ or -] 0.05 0.21 [+ or -] 0.05 (0.27 [+ or -] 0.19) (0.19 [+ or -] 0.14) P/CB (-) - - Treatment period (b) Electrical bT T1 activity (a) Treatment: 1 X [10.sup.-5] mol [l.sup.-1] NMDA/D-serine with 1 X [10.sup.-7] mol [l.sup.-1] kainate TP(n = 10) NA 1.18 [+ or -] 1.77 (1.95 [+ or -] 2.78) SUTP(n = 10) NA 4.94 [+ or -] 3.03 (7.29 [+ or -] 6.81) RP (n = 10) NA 2.36 [+ or -] 0.94 (2.29 [+ or -] 0.25) SUBP (n = 10) NA 1.77 [+ or -] 1.45 (2.16 [+ or -] 0.97) CB (n = 10) NA 0.21 [+ or -] 0.21 (0.28 [+ or -] 0.14) P/CB (n = 10) NA 18.50 [+ or -] 15.00 (22.30 [+ or -] 5.07) Treatment: 1 X [10.sup.-7] mol [l.sup.-1] NMDA/p-serine with 1 X [10.sup.-7] mol [l.sup.-1] kainate TP (n = 9) NA 1.93 [+ or -] 2.36 (1.93 [+ or -] 1.61) SUTP (n = 9) NA 4.51 [+ or -] 4.61 (5.35 [+ or -] 2.94) RP (n = 9) NA 2.90 [+ or -] 2.04 (2.63 [+ or -] 0.51) SUBP (n = 9) NA 4.29 [+ or -] 2.47 (5.57 [+ or -] 2.42) CB (n = 9) NA 0.21 [+ or -] 0.00 (0.21 [+ or -] 0.15) P/CB (n = 9) NA 27.75 [+ or -] 16.25 (19.33 [+ or -] 6.04) Treatment: 1 X [10.sup.-9] mol [l.sup.-1] NMDA/D-serine with 1 x [10.sup.-7] mol [l.sup.-1] kainate TP (n = 9) NA 1.29 [+ or -] 1.07 (1.48 [+ or -] 1.42) SUTP (n = 8) NA 5.53 [+ or -] 5.80 (5.83 [+ or -] 4.35) RP (n = 8) NA 1.77 [+ or -] 0.56 (2.05 [+ or -] 0.30) SUBP (n = 8) NA 4.08 [+ or -] 3.35 (3.61 [+ or -] 2.75) CB (n = 9) NA 0.21 [+ or -] 0.21 (0.24 [+ or -] 0.17) P/CB (n = 9) NA 17.00 [+ or -] 6.38 (12.50 [+ or -] 3.27) Treatment: 1 X [10.sup.-9] mol [l.sup.-1] NMDA/D-serine with 1 X [10.sup.-7] mol [l.sup.-1] kainate + 1 X [10.sup.-4] mol [l.sup.-1] D-AP5 TP (n = 8) 0.86 [+ or -] 2.25 0.75 [+ or -] 1.66 (1.45 [+ or -] 1.67) (1.61 [+ or -] 2.43) SUTP (n = 8) 4.77 [+ or -] 3.89 3.59 [+ or -] 2.76 (4.53 [+ or -] 2.48) (3.73 [+ or -] 2.14) RP (n = 8) 0.86 [+ or -] 0.48 1.07 [+ or -] 1.02 (0.78 [+ or -] 0.14) (1.29 [+ or -] 0.37) SUBP (n = 8) 1.82 [+ or -] 1.56 1.56 [+ or -] 2.23 (2.17 [+ or -] 0.95) (2.04 [+ or -] 1.34) CB (n = 8) 0.11 [+ or -] 0.21 0.00 [+ or -] 0.27 (0.16 [+ or -] 0.22) (0.13 [+ or -] 0.20) P/CB (-) - - Treatment period (b) Electrical T2 Significant activity (a) differences (c) Treatment: 1 X [10.sup.-5] mol [l.sup.-1] NMDA/D-serine with 1 X [10.sup.-7] mol [l.sup.-1] kainate TP(n = 10) 1.60 [+ or -] 1.65 (1.44 [+ or -] 1.24) SUTP(n = 10) 5.90 [+ or -] 3.90 (6.49 [+ or -] 3.67) RP (n = 10) 1.80 [+ or -] 1.08 (1.97 [+ or -] 0.26) pT > C *, T2 ^# SUBP (n = 10) 1.60 [+ or -] 1.23 (1.75 [+ or -] 0.97) CB (n = 10) 0.40 [+ or -] 0.20 (0.36 [+ or -] 0.16) P/CB (n = 10) 20.00 [+ or -] 8.67 (21.37 [+ or -] 2.70) Treatment: 1 X [10.sup.-7] mol [l.sup.-1] NMDA/p-serine with 1 X [10.sup.-7] mol [l.sup.-1] kainate TP (n = 9) 2.00 [+ or -] 1.80 (3.31 [+ or -] 3.83) SUTP (n = 9) 7.50 [+ or -] 4.50 (5.98 [+ or -] 3.09) RP (n = 9) 3.60 [+ or -] 3.00 (2.84 [+ or -] 0.59) SUBP (n = 9) 6.60 [+ or -] 3.30 (7.07 [+ or -] 3.99) CB (n = 9) 0.20 [+ or -] 0.20 (0.20 [+ or -] 0.24) P/CB (n = 9) 22.17 [+ or -] 8.75 (11.70 [+ or -] 4.46) Treatment: 1 X [10.sup.-9] mol [l.sup.-1] NMDA/D-serine with 1 x [10.sup.-7] mol [l.sup.-1] kainate TP (n = 9) 1.20 [+ or -] 1.00 (1.40 [+ or -] 1.24) SUTP (n = 8) 6.50 [+ or -] 3.95 (6.56 [+ or -] 4.42) RP (n = 8) 1.70 [+ or -] 1.05 (2.01 [+ or -] 0.33) SUBP (n = 8) 3.25 [+ or -] 2.40 (3.41 [+ or -] 1.70) CB (n = 9) 0.20 [+ or -] 0.20 (0.27 [+ or -] 0.17) P/CB (n = 9) 18.50 [+ or -] 1.75 (14.00 [+ or -] 3.14) Treatment: 1 X [10.sup.-9] mol [l.sup.-1] NMDA/D-serine with 1 X [10.sup.-7] mol [l.sup.-1] kainate + 1 X [10.sup.-4] mol [l.sup.-1] D-AP5 TP (n = 8) 2.20 [+ or -] 2.65 (2.25 [+ or -] 1.75) SUTP (n = 8) 3.85 [+ or -] 1.20 (3.59 [+ or -] 1.65) RP (n = 8) 0.80 [+ or -] 0.35 (0.76 [+ or -] 0.12) SUBP (n = 8) 1.65 [+ or -] 2.03 (1.91 [+ or -] 1.32) CB (n = 8) 0.20 [+ or -] 0.25 (0.18 [+ or -] 0.17) P/CB (-) - Note: Values are pulses/min (median [+ or -] inter-quartile range, with mean and standard deviation in parentheses, except RP and P/CB, where values in parentheses are means [+ or -] standard error). Note: Significant (P [less than or equal to] 0.05) or "potentially significant" (P [less than or equal to] 0.08) values are indicated in bold # (a) TP = tentacle pulse; SUTP = small, uncorrected tentacle pulse; RP = rhythmic potential; SUBP = small, uncorrected body pulse; CB = contraction burst; P/CB = average number of pulses per contraction burst; n = number of animals tested. (b) C = Con A control period, pT = AMPA pre-treatment period, bT = D-AP5 blocking period, T1 = first treatment period, T2 = second treatment period. (c) Asterisks (*) denote significant differences. Carets (^) denote "potentially significant" differences.

RESULTS

Effect on tentacle activity

The tentacle pulse (TP) pacemaker system. NMDA and D-serine (1 X [10.sup.-7] mol [l.sup.-1]), in the presence of AMPA (1 X [10.sup.-7] mol [l.sup.-1]), caused a significant increase in the TP rate (pulses/min) during T2 (2.10 [+ or -] 3.95, m.d. [+ or -] i.q.r.; 3.94 [+ or -] 3.90, m. [+ or -] s.d.) relative to the control peirod (0.86 [+ or -] 1.34, m.d. [+ or -] i.q.r.; 1.20 [+ or -] 1.34, m. [+ or -] s.d.) (Table 1 and Fig. 2a); the increase was abolished by 1 x [10.sup.-4] mol [l.sup.-1] D-AP5 (Fig. 2b). In one case, during the blocking experiment containing 1 x [10.sup.-7] mol [l.sup.-1] AMPA, 1 x [10.sup.-4] mol [l.sup.-1] D-AP5, and 1 x [10.sup.-5] mol [l.sup.-1] NMDA/D-serine, there was a significant increase in the TP rate during T2 (2.60 [+ or -] 3.05, m.d. [+ or -] i.q.r; 2.93 [+ or -] s.d.) relative to the rate in AMPA (pT: 0.43 [+ or -] 0.27, m.d. [+ or -] i.q.r.; 0.43 [+ or -] 0.47, m. [+ or -] s.d.) (Table 1). No other treatments, including that of NMDA and D-serine alone, had a significant effect on the TP pacemaker system (Tables 2 and 3).

Table 3 The effect of NMDA and D-serine without AMP A or kainate on the electrical activity of hydra Treatment period (b) Electrical C T1 activity (a) 1 X [10.sup.-5] mol [l.sup.-1] NMDA/D-serine TP (n = 7) 2.36 [+ or -] 3.00 0.21 [+ or -] 3.86 (1.47 [+ or -] 2.27) (1.13 [+ or -] 2.06) SUTP (n = 6) 1.18 [+ or -] 1.72 2.15 [+ or -] 1.45 (1.45 [+ or -] 1.29) (2.75 [+ or -] 1.60) RP (n = 6) 2.36 [+ or -] 0.16 2.47 [+ or -] 0.38 (2.16 [+ or -] 0.44) (1.80 [+ or -] 0.26) SUSP (n = 6) 3.65 [+ or -] 4.25 1.61 [+ or -] 2.31 (2.38 [+ or -] 2.39) (1.97 [+ or -] 1.59) CB (n = 7) 0.21 [+ or -] 0.52 0.21 [+ or -] 0.21 (1.43 [+ or -] 0.53) (0.86 [+ or -] 1.07) P/CB (n = 7) 16.25 [+ or -] 17.63 17.00 [+ or -] 10.67 (19.50 [+ or -] 2.99) (11.76 [+ or -] 5.49) 1 X [10.sup.-7] mol [l.sup.-1] NMDA/D-serine TP (n = 10) 0.32 [+ or -] 2.04 1.39 [+ or -] 1.82 (0.75 [+ or -] 1.03) (1.89 [+ or -] 2.18) SUTP (n = 8) 3.97 [+ or -] 4.99 5.63 [+ or -] 4.32 (4.20 [+ or -] 2.55) (5.97 [+ or -] 5.48) RP (n = 8) 1.61 [+ or -] 0.37 1.93 [+ or -] 0.83 (1.73 [+ or -] 0.15) (1.81 [+ or -] 0.21) SUBP (n = 8) 2.20 [+ or -] 3.06 5.53 [+ or -] 3.81 (4.25 [+ or -] 5.40) (4.87 [+ or -] 2.99) CB (n = 10) 0.32 [+ or -] 0.21 0.21 [+ or -] 0.16 (0.36 [+ or -] 0.18) (0.17 [+ or -] 0.14) P/CB (n = 10) 16.50 [+ or -] 4.50 16.00 [+ or -] 4.25 (15.30 [+ or -] 2.09) (10.10 [+ or -] 3.02) 1 X [10.sup.-9] mol [l.sup.-1] NMDA/D-serine TP (a = 9) 0.21 [+ or -] 2.15 0.86 [+ or -] 0.86 (1.14 [+ or -] 1.67) (1.07 [+ or -] 1.32) SUTP (n = 8) 3.43 [+ or -] 1.34 4.56 [+ or -] 1.77 (3.66 [+ or -] 1.26) (5.28 [+ or -] 1.94) RP (n = 8) 1.66 [+ or -] 1.34 1.66 [+ or -] 0.86 (2.49 [+ or -] 0.47) (2.86 [+ or -] 0.55) SUBP (n = 8) 2.84 [+ or -] 1.13 2.25 [+ or -] 1.80 (2.86 [+ or -] 1.15) (2.84 [+ or -] 2.18) CB (n = 9) 0.43 [+ or -] 0.21 0.21 [+ or -] 0.00 (0.29 [+ or -] 0.19) (0.24 [+ or -] 0.13) P/CB (n = 9) 26.00 [+ or -] 11.25 29.25 [+ or -] 8.38 (17.00 [+ or -] 4.47) (20.50 [+ or -] 4.34) Treatment period (b) Electrical activity (a) T2 1 X [10.sup.-5] mol [l.sup.-1] NMDA/D-serine TP (n = 7) 0.00 [+ or -] 3.20 (0.91 [+ or -] 2.33) SUTP (n = 6) 2.45 [+ or -] 1.40 (2.83 [+ or -] 1.87) RP (n = 6) 2.05 [+ or -] 1.08 (2.09 [+ or -] 0.31) SUSP (n = 6) 1.85 [+ or -] 2.05 (2.37 [+ or -] 2.37) CB (n = 7) 0.20 [+ or -] 0.30 (1.14 [+ or -] 0.90) P/CB (n = 7) 19.75 [+ or -] 3.63 (15.64 [+ or -] 4.69) 1 X [10.sup.-7] mol [l.sup.-1] NMDA/D-serine TP (n = 10) 1.80 [+ or -] 1.65 (2.58 [+ or -] 3.12) SUTP (n = 8) 4.20 [+ or -] 5.03 (5.59 [+ or -] 4.24) RP (n = 8) 1.20 [+ or -] 0.83 (1.29 [+ or -] 0.22) SUBP (n = 8) 4.30 [+ or -] 4.15 (4.04 [+ or -] 2.69) CB (n = 10) 0.20 [+ or -] 0.35 (0.24 [+ or -] 0.21) P/CB (n = 10) 19.50 [+ or -] 4.00 (13.62 [+ or -] 3.21) 1 X [10.sup.-9] mol [l.sup.-1] NMDA/D-serine TP (a = 9) 0.60 [+ or -] 2.60 (1.40 [+ or -] 1.89) SUTP (n = 8) 7.05 [+ or -] 6.05 (6.86 [+ or -] 4.32) RP (n = 8) 2.00 [+ or -] 1.35 (2.57 [+ or -] 0.52) SUBP (n = 8) 2.00 [+ or -] 1.33 (1.94 [+ or -] 0.89) CB (n = 9) 0.20 [+ or -] 0.00 (0.20 [+ or -] 0.14) P/CB (n = 9) 22.50 [+ or -] 2.00 (15.72 [+ or -] 3.36) Note: Values are pulses/min (median [+ or -] inter-quartile range, with mean and standard deviation in parentheses, except RP and P/CB, where values in parentheses are mean [+ or -] standard error). (a) TP = tentacle pulse; SUTP = small, uncorrelated tentacle pulse; RP = rhythmic potential; SUBP = small, uncorrelated body pulse; CB = contraction burst; P/CB = average number of pulses per contraction burst; n = number of animals tested. (b) C = Con A control period, T1 = first treatment period, T2 = second treatment period.

Small, uncorrelated tentacle pulses (SUTPs). SUTPs, whose amplitudes ranged from 2.8 mV to 85.05 mV, and whose duration ranged between 200 ms and 2.15 s., were tallied (Fig.3). NMDA and D-serine (1 x [10.sup.-5] mol [l.sup.-1]), in the presence of AMPA (1 x [10.sup.-7] [moll.sup.-1]), caused a potentially significant increase in SUTP rates (pulses/min) during T1 (5.79 [+.or.-] 4.83, m.d. [+ or.-] i.q.r.; 8.15 [+ or -] 5.08, m. [+ or -] s.d.; P [less then or equal to] 0.08) and a significant increase in T2 (5.70 [+ or -] 3.20, m.d. [+ or -] i.q.r.; 8.29 [+ or -] 7.13, m. [+ or -] s.d) relative to the control period (3.22 [+ or -] 0.97, m.d. [+ or -] i.q.r.; 3.73 [+ or -] 1.86, m [+ or -] s.d.) (Table 1 and Fig 2c). The increase in frequencies at 1 x [10.sup.-5] mol [l.sup.-1] NMDA/D-serine was counteracted by 1 x [10.sup.-4] mol [l.sup.-1] D-AP5 (Fig. 2d). No other treatments, including that of NMDA and D-serine alone, significanT1y affected SUTPs (Tables 2 and 3).

[FIGURE 3 OMITTED]

Effect on body column

The endodermal rhythmic potential (RP) system. In one series of experiments, kainate (pT), at 1 x [10.sup.-7] mol [l.sup.-1], caused a significant increase in the RP rate (pulses/min) (2.95 [+ or -] 1.05, m.d. [+.or -] i.q.r.; 3.04 [+ or -] 0.41, m [+ or -] s.e.) relative to control (2.04 [+ or -] 1.88 m.d. [+ or -] m.d. [+ or -] i.q.r.; 2.36 [+ or -] 0.46,m. [+ or -] s.e.). NMDA and D-serine, at a concentration of 1 x [10.sup.-5] mol [l.sup.-1], produced a potentially significant reduction in RP rates during T2 (1.80 [+ or -] 1.08, m.d [+ or -] i.q.r.; 1.97 [+ or -] 0.26, m. [+ or -] s.e.; P [less then or equal to] 0.08) relative to pT, containing 1 x [10.sup.-7] mol [l.sup.-1] kainate. T2 was not significanT1y different from control. However, the kainate-induced increase in RP frequency, observed in this set of experiments, was not reproduced in a subsequent set of experiments in which a blocking period (bT), containing 1 x [10.sup.-4] mol [l.sup.-1] D-AP5, was inserted after pT (Table 2 and Fig. 2e and 2f). No other treatment, including that of NMDA and D-serine alone, had an effect of the RP system (Tables 1 and 3).

Small, uncorrelated body pulses (SUBPs). SUBPs, whose amplitudes ranged from 3.2 mV to 82.95 mV, and whose duration ranged from 250 ms to 1.85 s., were counted (Fig. 4), NMDA and D-serine (1 X [10.sup.-7] mol [l.sup.-1]), in the presence of AMPA (1 X [10.sup.-7] mol [l.sup.-1]), caused a potentially significant decrease in SUBP rates (pulses/min) during T2 (2.50 [+ or -] 1.80, m.d. [+ or -] i.q.r.; 2.50 [+ or -] 1.54, m. [+ or -] s.d.; P [less than or equal to] 0.08) relative to the control period (4.08 [+ or -] 2.76, m.d. [+ or -] i.q.r.; 4.78 [+ or -] 3.06, m. [+ or -] s.d.), but had no effect in T1. The potential decrease in T2 was counteracted by 1 X [10.sup.-4] mol [l.sup.-1] D-AP5 (Table 1 and Fig. 2g and 2h). No other treatment, including that of NMDA and D-serine alone, had an effect on SUBPs (Tables 2 and 3).

[FIGURE 4 OMITTED]

The ectodermal contraction burst system. No treatment had a significant effect on the total number of contraction bursts or the average number of pulses per contraction burst (Tables 1, 2, and 3).

DISCUSSION

In this study we have presented evidence that the glutamatergic agonist, N-methyl-D-aspartate (NMDA), and D-serine, together with [alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), increases tentacle activity by causing an increase in the output of the tentacle pulse pacemaker system and the rate of locally recorded, small, uncorrelated pulses in the tentacle (SUTPs). We also give evidence that NMDA/D-serine together, with AMPA or kainate, may affect the electrical activity of the body, causing a potentially significant decrease in the rate of locally recorded, small, uncorrelated body pulses (SUBPs), and a potentially significant decrease in the output of the endodermal rhythmic potential pacemaker system relative to a previous increase caused by kainate.

These findings are consistent with earlier studies indicating that glutamate, acting on its NMDA and AMPA or kainate receptors, affects the body and tentacle effector systems of hydra (Kass-Simon et al., 2003). NMDA/D-serine causes a reduction in the duration of reduced-glutathione (GSH)-induced mouth opening in the presence of concanavalin A (Con A) (Pierobon et al., 2004a) and, in the presence of kainate or AMPA, an increase in stenotele nematocyst discharge (Kass-Simon and Scappaticci, 2004; Scappaticci and Kass-Simon, 2008). Biochemical studies have also indicated the presence of a receptor in membrane fractions of hydra that binds to the NMDA channel blocker, MK-801 (Pierobon et al., 2004a), and immunocytochemical studies provided evidence that NMDA receptors exist on the surface of nerve cells, nematocytes, myonemes of epitheliomuscular cells, and interstitial stem cells (Scappaticci et al., 2004). AMPA and kainate receptors (GluR 2, 3 and GluR 5, 6, 7) have also been observed in macerated cell preparations of the hypostome and tentacles (Hufnagel et al., 1997, 1999).

In our earlier electrophysiological studies, glutamate and kainate increased the frequency of tentacle pulses (TPs), rhythmic pulses (RPs), and contraction bursts (CBs), in the absence of Con A, at higher concentrations than were used in the present study. In the earlier study, glutamate, AMPA, and/or kainate, increased RPs, CBs, and pulses per contraction burst (P/CB), in the presence of Con A (Kass-Simon et al., 2003). In this study, also in the presence of Con A, CBs and P/CB were unaffected; probably because of the lower concentrations of AMPA and kainate we used.

In one series of blocking experiments (containing 1 X [10.sup.-7] mol [l.sup.-1] AMPA, 1 X [10.sup.-4] mol [l.sup.-1] D-2-amino-5-phosphonopentanoic acid (D-AP5), and 1 X [10.sup.-5] mol [l.sup.-1] NMDA/ D-serine) the TP rate in the pre-treatment period (pT) with AMPA was unusually low. The low frequency was significanT1y different from the rate in treatment period two (T2) with NMDA/D-serine after the D-AP5 block (bT). However, the rate in T2 was not significanT1y different from the control rate. The difference between pT and T2 appears to be an anomaly, because in all of our other experiments--in the present study and in our previous experiments (Kass-Simon et al., 2003)--AMPA had no effect on TP rates.

RPs were significanT1y increased in one of our experimental series, but the increase was not reproduced in a second series. The reason for this difference is unclear, but could be the result of different recording or treatment conditions. In the series in which kainate (1 X [10.sup.-7] mol[l.sup.-1]) produced a significant increase in RP rate, NMDA/o-serine (1 X [10.sup.-5] mol [l.sup.-1]) caused a potentially significant reduction of the RP frequency, bringing the rate back to control levels. This effect may hint at an indirect inhibition, produced by NMDA/D-serine exciting inhibitory neurons in the body. In this regard, exposure to NMDA/D-serine, in the presence of AMPA, also caused a potentially significant decrease in SUBPs, further suggesting that inhibitory neurons in the body may have been excited.

In the classical extracellular recording studies of Passano and McCullough (1962, 1963, 1964, 1965) and Rushforth and Burke (1971), and in our own earlier studies (e.g., Kass-Simon and Passano, 1978; Kass-Simon et al., 2003; Ruggeri et al., 2004), the repertoire of reported electrical activity was limited to those impulses that were correlated or associated with observable behavioral changes, generally attributed to pacemaker activity, or to feeding behavior (Grosvenor et al., 1996). Uncorrected impulses, though frequenT1y observed in our laboratory, were never tallied. In this study, because it became evident that treatment with NMDA/D-serine affected this uncorrected activity, we counted the pulses. Although the impulses form a heterogeneous group (i.e., large variability in amplitude and duration), they must nonetheless be considered to arise from either nerve or epithelial cells (including epitheliomuscular cells, nematocytes, and stem cells), or both. Since the pulses are affected by our treatment, they would appear to be the result of the activation of glutamatergic NMDA receptors on these cells. Further, the NMDA receptors appear to be differentially distributed in various regions of the hydra, since only tentacle activity and some body column activity (RPs are endodermal) were affected. The precise location of the receptors, their ionic properties, and their pharmacological definition must await intracellular recordings from specific cell types.

Nevertheless, our results indicate that a vertebrate-like NMDA receptor, requiring membrane depolarization by AMPA or kainate receptors, affects the electrical activity both of the tentacles and of the body column in hydra. NMDA and D-serine, in the presence of AMPA/kainate, increased TP and SUTP rates, and also potentially decreased RP and SUBP rates. Together with previous studies, evidence suggests that the glutamatergic NMDA/ glycine-AMPA/kainate system plays a prominent role in controlling the neuroeffector systems of the hypostome, tentacle, and body column of this early-evolved metazoan.

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J. C. KAY AND G. KASS-SIMON *

Department of Biological Sciences, University of Rhode Island, Kingston, Rhode Island 02881

* To whom correspondence should be addressed. E-mail: kass.simon@ uri.edu

Abbreviations: AMPA, [alpha]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; bT, blocking period; BVC, bicarbonate versene culture solution; CB, contraction burst; Con A, concanavalin A; D-AP5, D-2-amino-5-phosphonopentanoic acid; FANOVA, Friedman two-way analysis of variance; GABA, [gamma]-amino-butyric acid; iGluR, ionotropic glutamate receptor; LTD, long-term depression; LTP, long-term potentiation; m. [+ or -] s.d., mean [+ or -] standard deviation; m. [+ or -] s.d. mean [+ or -] standard error; m.d. [+ or -] i.q.r, median [+ or -] inter-quartile range; mGluR, metabotropic receptor; NMDA, N-methyl-D-aspartate; NMDAR, NMDA receptor; P/CB, average number of pulses per contraction burst; PLP, pre-locomotor pulse; pT, pre-treatment period; RP, rhythmic potential; SUBP, small, uncorrelated body pulse; SUTP, small, uncorrelated tentacle pulse; T1, treatment period 1; T2, treatment period 2; TP, tentacle pulse.

Received 5 September 2008; accepted 2 January 2009.

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Author: | Kay, J.C.; Kass-simon, G. |
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Publication: | The Biological Bulletin |

Article Type: | Report |

Date: | Apr 1, 2009 |

Words: | 10635 |

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