Journal of Neuroendocrinology, Vol. I , No. I 0 1989 Oxford University Press, 0953-8194/89 $3.00

The Perfusion of the Endogenous Neuropeptide, Phe-Met- Arg-Phe-NH, (FMRFamide) through the Gill of Aplysia Potentiates the Gill Withdrawal Reflex Evoked by Siphon Stimulation and Prevents its Habituation

Andrew Higgins, David Cawthorpe and Ken Lukowiak Neuroscience Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada.

Keywords: FMRFarnide, Aplysia, habituation, behavioural state, gill withdrawal reflex.

Abstract

Perfusion of Phe-Met-Arg-Phe-NH, (FMRFamide), an endogenous neuropeptide, through the gill of Apfysia significantly potentiates the amplitude of the gill withdrawal reflex evoked by tactile stimulation of the siphon with or without the central nervous system present. The potentiating effect of FMRFamide is reversible with washout. FMRFamide perfusion of the gill also prevented the gill withdrawal reflex from undergoing habituation when the tactile stimulus was presented repeatedly. Rather than habituate, the gill withdrawal reflex increased in amplitude. FMRFamide continued to prevent habituation in the absence of the parietal-visceral ganglion. We hypothesize that FMRFamide plays a role in the mediation of behavioural state primarily by affecting activity in the peripheral nervous system in the gill.

In order to come to an understanding of how the nervous system mediates adaptive behaviour, it is necessary to have a preparation that is amenable to analysis a t the level of single neurons whilst the behavioural change is occurring. One such model system is the siphon, mantle, gill and parietal-visceral ganglion (PVC) preparation of Aplysiu californica. This preparation which was pioneered by the work of Kandel’s group ( I ) and Peretz ( 2 ) has now been extensively used to study the neuronal mechanisms of both associative and non-associative learning (3-5). This preparation has also proved to be eminently suitable to study the role played by neural active peptides in the mediation or modulation of both adaptive behaviours and the phenomenon of hehavioural state (6-10).

The gill withdrawal reflex (GWR) evoked by tactile stimulation of the siphon or gill is mediated by the integrated activity of the central (CNS) and peripheral (PNS) nervous systems ( 1 1-13). It IS important to point out that the G W R is not a ‘reflex act’ but I.; rather a heterogenous collection of a t least four action patterns ( 14). These action patterns grade into each other, so that it may be difficult to categorize a particular movement (action pattern) unambiguously; thus leading earlier researchers to conclude that this was a simple graded reflex (15). However, because of the copious literature concerning this preparation and the use of the word reflex, we still refer to it as the GWR for the sake of clarity. The action patterns evoked by tactile stimulation of either the gill o r siphon continue to occur in the absence of

the PVG (also referred to as the CNS) (11, 14, 16); indicating the PNS in the gill and siphon is competent to mediate the GWR.

The role played by neural active peptides in the mediation and/or modulation of the GWR has primarily been focused on studies in which the peptide has been superfused over only the PVG (the CNS). These studies have demonstrated that the behavioural state of the preparation can be significantly altered by the various peptides. For example, the superfusion of arginine vasotocin (AVT) over the CNS results in behavioural suppression of the GWR and its associated neuronal activity (9) while SCPB superfusion of the PVG results in a facilitation of the GWR, a n increase in the sensory (LE) motor (L,) EPSP and an increase in motor neuron efficacy (8, 17). However, only recently have studies been undertaken to determine the role played by these peptides in the PNS (18, 19) in the mediation of the GWR. However, in those two studies, the CNS had been removed, only the PNS was present. Thus, it seemed to be appropriate to study the effect of perfusion of an endogenous neuropeptide through the gill on the integrated CNS-PNS and determine how the peptide effected both the GWR amplitude and its habituation. In this study we have examined the effect of FMRFamide perfusion on the GWR and its habituation evoked by tactile stimulation of the siphon both with and without the CNS present. We found that FMRFamide facilitated GWR amplitude and prevented habituation of the GWR whether or not the CNS was present.

~~ -

( orrespondence fo K Lukowiak, Neuroscience Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada

30 FMRFamide prevents habituation in Aplysia

Results

The amplitude of the siphon evoked GWR relative to the amplitude of spontaneous gill movements (SGM’s) are shown in Fig. I . These data are from two preparations. In each case, PVG was intact. Cumulative data showing the absolute amplitude of the siphon evoked GWR with the PVG intact and removed is shown in Fig. 2. These data once again confirm previous findings that the PVG exerts a suppressive influence upon the GWR (11-13, 20).

A

0

SGM GWR

I I

- 2 s

FIG. 1. (A) and (6) Relative amplitude of spontaneous gill movements (SGM) and siphon evoked gill withdrawal reflexes (GWR) from two preparations. Note difference in time base of spontaneous and evoked gill con tractions.

In preparations used in this study with the PVG present the perfusion of FMRFamide through the gill (10 nM) resulted in a significant facilitation of the siphon evoked GWR amplitude. Representative data from one such experiment are shown in Fig. 3, upper trace, while the cumulative data (n = 6) are shown in Fig. 4 A . As is readily apparent, FMRFamide is a potent facilitator of the GWR. The peptide was only perfused through the gill until such time as it began to elicit small spontaneous contractions (Fig. 3), then artificial seawater (ASW) was perfused. The siphon was only stimulated when the gill became quiescent. The effect of FMRFamide was reversible with washout (Fig. 3, upper trace and Fig. 4A). A previous study showed that in the isolated gill (i.e. no CNS) that FMRFamide perfusion facilitated the GWR

[ I ~ S I P H O N E V O K E D GW WITH ABDOMINAL GANGLION I N T A C T S IPHON EVOKED G W WITH ABDOMINAL GANGLION REMOVED

* p < 0 2 5

* In N

I

I

T

FIG. 2. Cumulative data (n = 6 ) showing absolute amplitude of siphon evoked gill withdrawal reflex (GWR) (millimeters, mm) with the parietal- visceral ganglion (PVG) present (open square) and removed (filled square at the top of histogram).

amplitude and prevented habituation of the GWR evoked by tactile stimulation of the gill (19). We therefore tested whether or not FMRFamide perfusion of the gill also potentiated the GWR evoked by siphon stimulation without the CNS present. We found (Fig. 3, lower trace and Fig. 46) that even following removal of the CNS, FMRFamide perfusion of the gill significantly potentiated GWR amplitude. Notice however that the percentage increase in GWR amplitude brought about by FMRFamide was less follow- ing PVG removal. We believe this is due to the fact that the control amplitude of the GWR without the PVG is larger than with the PVG present and thus we have a ceiling effect (Fig. 2). In terms of absolute amplitude of the G W R potentiated with FMRFamide, there is no significant difference with or without the PVG (Fig. 5) . Since perfusion of FMRFamide through the gill potentiated the GWR evoked by siphon stimulation whether the CNS was present or not, we proceeded to determine whether FMRFamide perfusion would also prevent the GWR from habituating. Due to the fact that the perfusion of FMRFamide through the gill elicits contractions of the gill, we could only test the peptide’s effect on habituation after the perfusion of the peptide was terminated and the gill tension returned to baseline. Thus we had to use an interstimulus interval (ISI) of 5 min. We first tested for habituation at this IS1 with ASW perfusion through the gill. As can be seen in the data from two representative preparations, one with the PVG intact (Fig. 6, upper trace) and one following removal of the PVG (Fig. 7, upper trace), repeated stimulation of the siphon resulted in habituation of the GWR. In each experiment following the habituation run, a 3 h rest period was interposed to allow for complete recovery from the effects of habituation (13) before two more control responses were obtained.

C o n t r o l 1

( A S W )

Contro l 2 E x p e r i m e n t a l

( A S W ) ( 1 0 nM F M R F )

W a s h

( A S W )

* IS1 2 0 minutes

! FMRF p e r f u s i o n on J . 5 Q

2 sec I 2 min

I.’IG. 3. FMRFamide perfusion of the gill potentiates the gill withdrawal reflex (GWR) evoked by siphon stimulation both in the presence and absence of the parietal-visceral ganglion (PVG). Control stimuli were delivered to the siphon at an interstimulus interval (ISI) of 20 min. FMRFamide (10 nM) mas perfused through the gill for approximately 3 to 4 min causing phasic contractions, Once washout commenced the siphon was stimulated only after gill rension returned to baseline. Twenty min later, the siphon was again stimulated. Following the last stimulus, the PVG was removed and the preparation I osted 3 h. The experiment was then repeated again in a similar manner. As can be seen FMRFamide reversibly potentiated the amplitude ofthe GWR evoked hy siphon stimulation with and without the PVG present. ASW-(Artificial Seawater).

FMRFamide was then perfused through the gill and its perfusion terminated when the FMRFamide induced contractions occurred. As soon as the gill tension returned to baseline with ASW perfusing the gill, the siphon was stimulated. Stimuli were thus delivered at ;in IS1 of 5 min. As can be seen (Figs. 6 and 7, middle traces), 1:MRFamide perfusion prevented habituation of the GWR both with and without the PVG. With repeated tactile stimulation of the siphon, the GWR amplitude increased whereas with ASW per- fusion, the GWR habituated. Following a further 3 h rest, each preparation habituated in much the same manner as on the first control habituation run (Figs. 6 and 7, lower traces). Cumulative data shown in Fig. 8 demonstrates that FMRFamide (10 nM) can prevent habituation of the siphon evoked GWR when the PVG is present or removed.

Discussion

Previously (19) it had been demonstrated that the endogenous gill neuropeptide (21) FMRFamide facilitated the GWR and prevented its habituation when the G W R was evoked by tactile stimulation of the gill in preparations without the CNS present. Here we have demonstrated that FMRFamide perfusion of the gill facilitated the GWR amplitude and prevented its habituation Mhen evoked by tactile stimulation of the siphon in both the Presence and absence of the CNS. Since the data were similar Mith and without the CNS present, it appears that FMRFamide acts either on the PNS neurons or on gill muscle directly. Presently, we have not been able to distinguish between these possibilities. Experiments utilizing isolated gill muscle fibres in tissue culture (see 2 2 ) may allow us to directly determine the site(s) of FMRFamide action, as well as its mechanism. The data presented in this study

further support our earlier hypothesis that FMRFamide may play an important role in the gill in the mediation of adaptive gill behaviours, especially the facilitated behavioural state (7, 19).

This is not the first instance that the perfusion of a putative neurotransmitter or modulator through the gill affected habitu- ation of the GWR. Ruben and Lukowiak (23) showed that dopamine perfusion facilitated and prevented habituation of the GWR. In that study, however, it appeared that dopamine only acted upon L7’s (a central motor neuron) peripheral terminations in the gill. Dopamine did not affect the other central gill motor neurons nor neurons in the PNS. In this important regard, FMRFamide is much different; it appears to act on the PNS. We concluded that FMRFamide acted primarily on the PNS because there was no difference in FMRFamide’s effect on GWR behav- iours whether or not the CNS was present. If FMRFamide were acting on the peripheral termination of the central motor neurons then we should have seen a change either qualitative or quantitative following CNS removal. Whether or not FMRFamide perfusion of the gill will affect the ability of a central gill motor neuron t o elicit a gill withdrawal response when depolarized needs to be determined. It may well be that in addition to affecting the PNS, FMRFamide also affects all the peripheral termination of the CNS motor neurons. Experiments to answer these questions will be initiated shortly.

FMRFamide perfusion of the gill had the opposite effect to that of SCPB perfusion of the gill. SCP, perfusion leads t o suppression of the GWR amplitude (18, 24) but interestingly enough, does not affect the rate of habituation. This may be due to the fact that SCP, does not in any way affect the processes underlying habituation (e.g. low frequency homo-synaptic de- pression) but only affects a tonic inhibitory input to the muscle

32 FMRFamide prevents habituation in Aplysiu

A. P V G intact

I *

1 C 1 C 2 E l W 1

6. P V G removed

O T - ,

3 2 - I 0 ’ *

- T

I c1 c 2 E l W 1

FIG. 4. (A) Group data (n = 6) showing the effect of FMRFamide on the gill withdrawal reflex (GWR) amplitude evoked by tactile stimulation of the siphon when the parietal-visceral ganglion (PVG) was intact and removed. The data are presented as the ‘/o of control amplitude, which was taken as the GWR on the second 20 min control. Stimuli were presented at an interstimulus interval (ISI) of 20 min. As can be seen FMRFamide significantly potentiated (*p < 0.005) GWR amplitude which was reversible with washout. CI and 2-control; El-FMRFamide 10 nM perfusion; Wl-washout with artificial seawater (ASW) perfusion. (B) Group data from same preparations (n = 6) showing the effect of FMRFamide on G W R amplitude evoked by siphon stimulation following removal of the PVG. As when the PVG was present FMRFamide significantly potentiated GWR amplitude (*p < 0.025) which again was reversed with washout.

fibres. In addition, the SCPB effects appear to be mediated via an inhibitory cholinergic mechanism in the gill since nicotinic-like blockers (curare and r-bungarotoxin) prevents SCP,’s suppressive effects from being expressed (24). These findings may be contrasted to the effects of both SCP, and FMRFamide in the CNS. As mentioned above, SCP, when superfused over the PVG results in facilitation of the GWR, sensory (LE), motor (L,) EPSP and motor neuron efficacy (8, 17). In contrast to the central effects of SCPB, FMRFamide has been implicated in the role of mediating presynaptic inhibition associated with behavioural inhibition (25). Again, this is another instance where SCPB and FMRFamide produce opposite effects (8, 18, 26).

0 LD

0 In

0 0

- E -0 n

9 CII

0 N

0 .-

0

aSSIPHON EVOKED G W ‘WITH AG I N T A C T (FMRFornxde 10nM) m S I P H O N EVOKED GW WITH AG REMOVED (FMRFomide 10 nM)

FIG. 5 . Cumulative data (n = 6) showing absolute amplitude of siphon evoked gill withdrawal reflex (GWR) following FMRFamide (10 nM) perfusion. Open square at top of histogram-parietal-visceral ganglion (PVG) intact; filled square-PVG removed. Note: there was no significant difference between these GWR amplitudes.

F M R F - a m t d e b l o c k s hab i tua t ion of the siphon e v o k e d GWR

Control 1 ~ A S W ) H 1 H3 H6

- * 7-J-

Control 2 ~ A S W ) H 1 H3 H6 7 -7r v 1 . 5 .

2 s

IS1 - 5 minutes

H - Habituation trial

3 hr. res t between habituation runs

FIG. 6. FMRFamide reversibly prevents habituation of the gill withdrawal reflex (GWR) evoked by siphon stimulation when the parietal-visceral ganglion (PVG) remains intact. Data from a single representative exper- iment showing the effect of FMRFamide on G W R habituation. Upper row: Habituation of the GWR evoked by repeated tactile stimulation (interstimulus interval (ISI) 5 mi,) of the siphon with artificial seawater (ASW) perfusion of the gill; trials I , 3 and 6 are shown. Following trial 6, a 3 h rest was interposed. Middle row: FMRFamide perfusion during the 5 min IS1 as described in Materials and Methods prevents habituation. Trials I , 3 and 6 are shown. Following trial 6, a 3 h rest was again interposed. Bottom row: Habituation occurs as in first control run. Trials I , 3 and 6 are shown.

Control 1 ( A S W )

FMRFamide prevents habituation in Aplysia 33 FMRF-amide blocks habituation of the siphon evoked GWR

H6

? F Control 2 (ASW)

v "3 %-

_I - 5 g IS1 - 5 minutes

H - Habituation trial

3 hr. rest between habituation runs 2 2 min

FIG. 7. FMRFamide reversibly prevents habituation of the gill withdrawal reflex (GWR) evoked by siphon stimulation following removal of the parietal- visceral ganglion (PVG). Data from a single experiment showing the effect of FMRFamide on GWR habituation. Upper row: The GWR evoked by repeated tactile stimulation (interstimulus interval (ISI) 5 min) of the siphon with artificial seawater (ASW) perfusion of the gill; trials 1.3 and 6 are shown. Following trial 6, a 3 h rest was interposed. Middle row: FMRFamide perfusion during the 5 min IS1 as described in Materials and Methods prevents habituation. Trials I , 3 and 6 are shown. Following trial 6, a 3 h rest was again interposed. Bottom row: As in first control run. Trials 1, 3 and 6 are shown.

We still d o not know whether FMRFamide's effp-t on the GWR Limplitude and the prevention of its habituation are mediated via ;I cyclic AMP pathway as are the phasic contractions produced by FMRFamide perfusion (21). Experiments now in progress should help to clarify whether FMRFamide works via this second messenger pathway.

FMRFamide perfusion of the gill leads to behaviours resembling [hose termed 'facilitated state' behaviours (7). We have previously hypothesized that the release of peptides both centrally and peripherally may play a role in the mediation of behavioural state (10) and the data presented here are consistent with, and lend support to, this hypothesis. Since the CNS and PNS interact and form an integrated system in the mediation of adaptive gill behaviours (12), FMRFamide effects on the PNS result in a f~i l i ta ted- l ike state. Stimuli or environmental situations which result in the release of FMRFamide in the periphery will produce

facilitated GWR which is resistant to habituation. In summary, the Aplysia model system has received much

attention in attempts by investigators to determine the neuronal mechanisms which underlie adaptive behaviours. Most of these attempts have focused on changes in synaptic efficacy which occur centrally. Here we have shown that the perfusion of an endogenous neuropeptide peripherally alters adaptive GWR behaviours. Thus in any analysis of the neuronal mechanisms which underlie

behavioural change consideration must also be given to events taking place in the gill periphery.

Materials and Methods

Aplysia culijornica (1 10 to 260 g) obtained from Sea Life Supply (Sand City, CA, USA) were kept in a 1200 L aerated aquarium containing ASW (Instant Ocean) at 15 to 17°C.

Prior to dissection, animals were anesthetized by the injection of isotonic MgCI, (33% of body mass). The siphon, mantle, gill and PVG were removed from the anesthetized animals and pinned dorsally to a Sylgard base (Dow-Corning; Midland, MI, USA) of a lucite dish. The gametolytic glands were removed and the preparation was maintained in ASW at 15 "C. The PVG waspinned out ontoa Sylgard platform within a chamber in order to isolate the neurons from any contact with the peptide perfusate. The siphon, branchial, and ctenidial nerves which innervate the mantle organs were left intact; all other nerves and connections were cut.

The gill was then cannulated by an insertion of a glass tube into the afferent vein and perfused with ASW from a gravity flow drip reservoir. The entire system was maintained at 15 "C. The effluent perfusate was removed by continuous suction. Through the use of a switch in the perfusion system, continuous perfusion of either ASW or FMRFamide was made possible. The FMRFamide (Bachem, Torrance, CA, USA) was diluted to desired concentrations from aliquots stored at -70 "C. This dilution was made immediately prior to use and no inhibitors of proteolytic activity were used.

Following cannulation, a steady perfusion rate was maintained and a suture thread, connected to a force transducer (Grass FT03C), was tied

34 FMRFamide prevents habituation in Aplysia

F M R F o m i d e e f f e c t s o n s i p h o n e v o k e d GWR

0

T 0 0 d

0 0

- F l 0 L -2

C 0 V

K O 0 N

-control w i t h o u t P V G control w i t h I 9 8 - 0 +FMRF w i t h 1?

+ +FMRF w i t h o u t 11

_ - --i -1 -

0

1 2 3 4 5 6 H n b i t u o t i o n T r i o 1

FIG. 8. Cumulative data (n = 8) showing that FMRFamide prevents habituation of the gill withdrawal reflex (GWR) evoked by siphon stimulation in the presence and absence of the CNS. The initial GWR amplitude on each run was taken as 100%. With artificial seawater (ASW) perfusion of the gill the GWR habituates over the course of 6 trials to less than 30% of its initial amplitude. When FMRFamide was perfused through the gill, habituation did not occur, rather in each preparation, it increased in amplitude over the 6 trials. (p < 0.005 for habituation FMRFamide perfusion with respect to control habituation in each case). PVG-(Parietal-visceral ganglion).

to a single pinnule. The output of the transducer was displayed on a storage oscilloscope (Hitachi V-134) and on a Grass polygraph. The siphon was stimulated with a force equivalent to 1 g over an area of about 1 mm2 by means of a mechanical tapper (20). (For a figure of the experimental preparation, see ref. 18).

The preparations were allowed to rest for at least 45 min before any experiments were performed. Following this rest, the tactile stimulus was applied to the siphon with an IS1 of 20 min. Only if the control G W R amplitudes were within 10% did the experiment proceed. In these experiments, a total of 17 animals were used. Of these, three did not meet the 10% control criterion and were not included in the results presented here. Significance was determined by a student’s r-test where p < 0.05.

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