Periodically Oscillating Neurone Research Articles

Modulation by APGW-Amide, an Achatina Endogenous Inhibitory Tetrapeptide, of Currents Induced by Neuroactive Compounds on Achatina Neurons: Peptides

1. Modulatory effects of APGW-amide (Ala-Pro-Gly-Trp-NH 2), proposed as an inhibitory neurotransmitter of Achatina neurons, perfused at 3×10 −6 M on the currents induced by neuroactive peptides, ejected by brief pressure, were examined by using Achatina giant neuron types, v-RCDN (ventral-right cerebral distinct neuron) and PON (periodically oscillating neuron), under voltage clamp. 2. Outward current ( I out) caused by FMRFamide (Phe-Met-Arg-Phe-NH 2) on v-RCDN, which was probably K + dependent, was inhibited with membrane conductance ( g) increase by APGW-amide. From the dose (pressure duration)-response curves of FMRFamide and a Lineweaver-Burk plot of these data, the inhibition caused by APGW-amide was mainly in an uncompetitive manner. 3. I out caused by APGW-amide on v-RCDN, which was probably K + dependent, was inhibited with g increase by APGW-amide. The inhibition caused by APGW-amide was partly in a competitive manner and partly in a noncompetitive manner. 4. I out caused by [Ser 2]- Mytilus inhibitory peptide, [Ser 2]-MIP (Gly-Ser-Pro-Met-Phe-Val-NH 2) on v-RCDN, which was probably K + dependent, was inhibited with g increase by APGW-amide. Because the modulation of this current was not so marked, a dose-response study of this compound was not carried out. I in induced by oxytocin on PON was not affected by APGW-amide. 5. From the dose-response curves of APGW-amide, perfused consecutively, the inhibitory effects of APGW-amide on the I out caused by APGW-amide were stronger than those on the I out caused by FMRFamide. 6. The inhibition of the APGW-amide–induced I out on v-RCDN by APGW-amide was partly due to the competition in the receptor sites and partly to the g increase. The inhibition by APGW-amide on the I out induced by FMRFamide and [Ser 2]-MIP would be partly due to the g increase. In addition, we consider that APGW-amide affects intracellular signal transduction systems or ionic channels, thus modulating these currents. 7. The currents modulated by APGW-amide were different from those modulated by achatin-I, another Achatina endogenous neuroexcitatory peptide. We consider that the mechanisms underlying the modulatory effects of APGW-amide are different from those of achatin-I.

Synthesis of achatin-I (Gly- d-Phe- l-Ala- l-Asp) analogs having dehydrophenylalanine or aminoisobutyric acid residue at position 2, and their effects on Achatina giant neurons

1. 1. Achatin-I (Gly- d-Phe- l-Ala- l-Asp), a neuroactive tetrapeptide having a d-phenyl-alanine residue, has been proposed to be an excitatory neurotransmitter of Achatina giant neurons. It was revealed that the d-Phe 2 residue is essential for bioactivity of achatin-I, which seems to adopt β-turn conformation. In the present study, in order to investigate the structure-activity relationships of achatin-I and its derivatives, the two highly constrained analogs of achatin-I, [Δ ZPhe 2]achatin-I (Gly-Δ ZPhe- l-Ala- l-Asp) (Δ ZPhe: (Z)-α,β-dehydrophenylalanine) and [Aib 2]achatin-I (Gly-Aib- l-Ala- l-Asp) (Aib: α-aminoisobutyric acid), were synthesized, and their effects on the two identifiable Achatina giant neuron types, PON (periodically oscillating neuron) and v-RCDN (ventral-right cerebral distinct neuron), were examined in comparison with those of achatin-I under voltage clamp. 2. 2. Achatin-I ( n = 6), ejected onto the neurone by brief pneumatic pressure (2 kg/cm 2, 400 ms, 10 −3 M, at 10-min intervals), produced an inward current ( I i ) on PON. The I i value (mean ± SEM) was 0.44±0.03 nA. The interval between the achatin-I ejection and the I i peak was 14.74±3.15 s ( n=6). [Δ ZPhe 2]achatin-I ( n=6) and [Aib 2]achatin-I ( n=6) had no effect on this neuron type. 3. 3. On the other hand, achatin-I ( n=10) and [Δ ZPhe 2]-achatin-I ( n=10), ejected by brief pressure, produced an I in on v-RCDN. The I in values were 0.85±0.07 nA for achatin-I and 0.48±0.05 nA ( p<0.01, compared with that of achatin-I by Student's t-test for paired data) for [Δ ZPhe 2]achatin-I. The intervals between the compound ejection and the I in peak were 5.95±0.33 s for achatin-I and 8.70±0.81 s ( p<0.05, compared with that of achatin-I) for [Δ ZPhe 2]achatin-I. [Aib 2]achatin-I ( n=10) had no effect on this neuron type.

Blocking effects of promethazine, triprolidine and their analogues on the excitation caused by the peptide, achatin-I

An Achatina endogenous tetrapeptide, achatin-I (Gly- d-Phe-Ala-Asp), applied by brief pressure, produced an inward current ( I in) on an Achatina giant neurone type, PON (periodically oscillating neurone). Promethazine, triprolidine and their analogues tested, applied by perfusion, showed a tendency to inhibit the I in, suggesting that the effective structures vary to a wide extent. With respect to promethazine and its analogues, the presence of 2-bromo, 5-oxo, 3-dimethylsulfamido and 2-methoxy weakened the effects. 10-(2-methylamino-2-methylethyl) instead of 10-(2-dimethylamino-2-methylethyl) of promethazine and the azepine ring instead of phenothiazine ring potentiated the effects. From the dose (pressure duration)-response study of achatin-I, the two promethazine analogues, RP 6497 and RP 6549 (the structures are shown in Fig. 1), inhibited the I in in partly competitive and partly noncompetitive manners. Regarding triprolidine and its analogues, the compounds in Z-configuration seemed to be more effective than those in E-configuration. The presence of 4-methyl in 1-phenyl, and 1-(4-pyridyl) instead of 1-(2-pyridyl) potentiated the effects. 3-Dimethylamino instead of 3-pyrrolidino weakened the effects. The two triprolidine analogues, Trip Der 3 and Trip Der 6 (the structures in Fig. 2), inhibited the I in in an uncompetitive manner.

Multiple intracellular signal transduction pathways mediating inward current produced by the neuropeptide, achatin-I

The effects of intracellular signal transduction system inhibitors on the inward current ( I in) caused by achatin-I (Gly- d-Phe-Ala-Asp), an Achatina endogenous tetrapeptide having a d-phenylalanine residue, applied locally onto the neurone tested, were examined under voltage clamp using two identifiable Achatina giant neurone types, v-RCDN (ventral-right cerebral distinct neurone) and PON (periodically oscillating neurone). H-89 ( N-[2-( p-bromocinnamylamino)-ethyl]-5-isoquinolinesulfonamide) (adenosine-3′,5′-cyclic monophosphate (cyclic AMP)-dependent protein kinase inhibitor) markedly suppressed the achatin-I-induced I in on PON, whereas this drug was ineffective on the I in of v-RCDN. Dose (pressure duration)-response study of achatin-I on PON in a physiological solution and in the presence of H-89, and Lineweaver-Burk plot of these data, indicated that H-89 inhibited the I in in a noncompetitive manner. KT5823 ( N-methyl-(8 R∗,9 S∗,11 S∗)-(−)-9-methoxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1 H, 8 H, 11 H-2,7b,11a-triazadibenzo[a,g]cycloocta[ c,d,e]-trinden-1-one) (guanosine-3′,5′-cyclic monophosphate (cyclic GMP)-dependent protein kinase inhibitor) suppressed the achatin-I-induced I in of v-RCDN in mainly noncompetitive and partly uncompetitive manners, but this drug had no effect on the I in of PON. W-7 ( N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide) (calmodulin inhibitor) suppressed noncompetitively the I in of PON, but this drug had no effect on the I in of v-RCDN. IBMX (3-isobutyl-1-methylxanthine) (cyclic nucleotide phosphodiesterase inhibitor) enhanced the achatin-I-induced I in of v-RCDN, but this drug was ineffective on the I in of PON. However, IBMX might have effects on the achatin-I receptor sites on v-RCDN. These findings suggest multiple intracellular signal transduction pathways mediating the achatin-I-induced I in: the I in of PON is via cyclic AMP-dependent and probably Ca 2+ calmodulin-dependent protein kinases, and that of v-RCDN via cyclic GMP-dependent protein kinase. Other signal transduction system inhibitors including calphostin C (2-[12-[2-(benzyloxy)-propyl]-3,10-dihydro-4,9-dihydroxy-2,6,7,11-tetramethoxy-3,10-dioxo-1-peryleny]-1-methylethyl carbonic acid 4-hydroxyphenyl ester) (protein kinase C inhibitor) did not significantly affect the I in of both v-RCDN and PON.

Effects of l-glutamic acid and its agonists on snail neurones

The sensitivities of 22 giant neurone types of an African giant snail ( Achatina fulica Férussac) to threo-β-hydroxy- l-glutamic acid (threo- l-BHGA), a derivative of l-glutamic acid ( l-Glu), applied by brief pneumatic pressure ejection, were examined under current clamp. The 5 neurone types were depolarized by this compound, whereas 2 were hyperpolarized. The 4 neurone types, PON (periodically oscillating neurone), RAPN (right anterior pallial nerve neurone), d-RPLN (dorsal-right parietal large neurone) and RPeNLN (right pedal nerve large neurone) that are excited by threo- l-BHGA and one type, v-LCDN (ventral-left cerebral distinct neurone), inhibited by this compound, were selected to study their pharmacological features in detail. Effects of the stereoisomers of l-Glu and threo- l-BHGA, and mammalian l-Glu receptor agonists, ejected by brief pressure, on the 5 Achatina neurone types were examined under voltage clamp. d-RPLN produced an inward current (I in) by l-Glu and threo- l-BHGA, whereas this neurone type was insensitive to D-Glu and erythro-L-, threo-D- and erythro-D-BHGA. This was also excited by AMPA, indicating that the pharmacological features of the l-Glu receptors in this neurone type were similar to those of the mammalian ionotropic AMPA type L-Glu receptors. RAPN produced I in by l-Glu and threo- l-BHGA. This neurone type was also excited by quisqualic acid and ibotenic acid, indicating that the features of the l-Glu receptors were similar to those of the mammalian metabotropic l-Glu receptors. PON and RPeNLN produced I in, by l-Glu and threo- l-BHGA. These neurone types were also excited by quisqualic acid, AMPA and ibotenic acid, indicating that their l-Glu receptors seemed to be in the mixed type, of the two types mentioned. On the other hand, v-LCDN produced an outward current (I out) by threo- l- and erythro- l-BHGA, but was insensitive to L-Glu, indicating that the receptors activated by l-BHGA were not l-Glu receptors. This neurone type was also inhibited by quisqualic acid.

Suppressing effects of neuroactive peptides on the inward current caused by achatin-I, an Achatina endogenous peptide

1. 1. Modulatory effects of the four molluscan neuroactive peptides. FMRFamide (Phe-Met-Arg-Phe-NH 2), APGW-amide (Ala-Pro-Gly-Trp-NH 2), oxytocin and [SER 2]- Mytilus inhibitory peptide ([SER 2]-MIP) (Gly-Ser-Pro-Met-Phe-Val-NH 2) were examined on the inward current ( I in) caused by achatin-I (Gly- d-Phe-Ala-Asp), which has been isolated from the Achatina ganglia. 2. 2. Two Achatina giant neurone types, v-RCDN (ventral-right cerebral distinct neurone) and PON (periodically oscillating neurone), were used. Achatin-I was applied locally to the neurone tested by brief pneumatic pressure ejection, and the other molluscan neuroactive peptides were perfused around the ganglia. 3. 3. FMRFamide, perfused at 3 μM, suppressed markedly the I in elicited by the achatin-I of both v-RCDN and PON. APGW-amide at 3μM also suppressed the I in of v-RCDN, but did not affect that of PON. Oxytocin at 1 μM suppressed the I in of PON, but did not affect that of v-RCDN. [Ser 2]-MIP at 3 μM did not affect the I in of v-RCDN. 4. 4. The dose-response curves of FMRFamide, APGW-amide and oxytocin, indicated that their respective suppressive effects on the I in of achatin-I were dose-dependent, and that APGW-amide was slightly more potent than the other peptides. The dose (pressure duration)-response curves of achatin-I (1 kg/cm 2, 10 −3M, 5 min interval), obtained by varying the duration of the achatin-I pressure ejection, were measured in the presence and absence of each of the three peptides. These peptides did not affect the E max of the pressure duration for the achatin-I ejection, but markedly lowered the E max of achatin-I, indicating the effects to be non-competitive. The three peptides may act as neuromodulators for achatin-I effects, likewise achatin-I probably acts as a neuromodulator for these peptides as previously reported.

Blockers for excitatory effects of achatin-I, a tetrapeptide having a d-phenylalanine residue, on a snail neurone

Some histamine H 1 receptor antagonists suppressed the inward current (I in) of an Achatina identifiable neurone type, PON (periodically oscillating neurone), caused by an Achatina endogenous tetrapeptide having a d-phenylalanine residue, achatin-I (Gly- d-Phe-Ala-Asp), under voltage clamp. Achatin-I was applied locally to the neurone by brief pneumatic pressure ejection and antagonists were administered by perfusion. The dose-response curves of the effective histamine H 1 antagonists indicated their potency order to suppress the I in as follows: chlorcyclizine, promethazine, triprolidine and homochlorcyclizine > trimeprazine and clemastine > diphenylpyraline. The potent drugs were mostly piperazine and phenothiazine types. The effects of chlorcyclizine, promethazine and triprolidine on the dose (the duration of the pressure ejection)-response curve of achatin-I indicated that these drugs affected the I in caused by achatin-I in a non-competitive manner. The antagonists for the receptors of the small-molecule neurotransmitters other than histamine H 1, such as histamine H 2, acetylcholine, γ-aminobutyric acid (GABA), l-glutamic acid, dopamine, α- and β-adrenalin and 5-hydroxytryptamine, had no effect on the I in caused by achatin-I.

Modulation of neuropeptide effects by achatin-I, an Achatina endogenous tetrapeptide

Achatin-I (Gly-D-Phe-L-Ala-L-Asp), an endogenous tetrapeptide in the ganglia of Achatina fulica Férussac, at 3 × 10 −6 M suppressed both the inward current (I in) of an Achatina giant neurone, PON (periodically oscillating neurone), caused by locally ejected oxytocin, and the outward current (I out) of v-RCDN (ventral-right cerebral distinct neurone) induced by APGW-amide. Dose (pressure duration)-response studies with oxytocin and APGW-amide showed that their ED 50 values were not affected by achatin-I, whereas the E max values were suppressed. The v-RCDN I out caused by FMRF-amide was enhanced by achatin-I, but the same current induced by [Ser 2] Mytilus inhibitory peptide ([Ser 2]MIP) was not affected. Achatin-I suppressed the I out of TAN (tonically autoactive neurone) caused by acetylcholine, but did not affect the I in of v-RCDN elicited by acetylcholine. The I out caused by γ-aminobutyric acid, threo-β-hydroxy-L-glutamic acid and dopamine was not affected by achatin-I. It is considered that achatin-I acts as a neuromodulator and has both suppressing and enhancing actions on the effects of various neurotransmitters, including peptides, in Achatina giant neurones.

Influence of the drugs for membrane excitability modification on the excitation caused by achatin-I

1. The pneumatic pressure ejection of achatin-I (Gly- d-Phe- l-Ala- l-Asp), an endogenous tetrapeptide having a d-phenylalanine residue, produced an inward current ( I in) in an identifiable giant neuron, PON (periodically oscillating neuron), of an African giant snail, Achatina fulica Férussac. The influence of the drugs for membrane excitability modification, applied by perfusion, on the PON excitation caused by achatin-I was examined under voltage clamp. 2. The four channel blocking drugs, tetrodotoxin (TTX), tetraethylammonium chloride (TEA), verapamil and picrotoxin, at 10, −4 M did not affect significantly the PON excitation caused by the peptide. 3. N-β- phenylpropionyl- L-tyrosine (BPLT), a membrane hyperpolarizant, at 10 −6 M and concanavalin A (Con A), which altered the response to l-glutamate, at 100 μg ml −1 were considered to hardly influence the PON excitation caused by achatin-I.

Effects of cyclic AMP, cyclic GMP and IP 3 intracellularly injected into the identifiable Achatina giant neurones

1. The three second messengers, cyclic AMP, cyclic GMP and IP 3, were injected by pneumatic pressure into the soma of the following six neurone types identified in the suboesophageal ganglia of Achatina fulica Férussac: PON (periodically oscillating neurone), TAN (tonically autoactive neurone), RAPN (right anterior palliai nerve neurone), d-RPLN (dorsal-right parietal large neurone), v-VLN (ventral-visceral large neurone) and LPeNLN (left pedal nerve large neurone). 2. All neurones tested were depolarized by cyclic AMP and cyclic GMP, and hyperpolarized by IP 3. The involvement of these second messengers in the neuronal activity of Achatina neurones seems simple; cyclic AMP and cyclic GMP mediate excitation and IP 3, inhibitions, in comparison to effects of neurotransmitter candidates on these neurones.

AJ-2615, a long-acting Ca 2+ channel antagonist with a novel structure

AJ-2615, a dihydrodibenzothiepin derivative, at 10 −4 M inhibited the peak I Ca amplitude of an identifiable Achatina neurone, PON (periodically oscillating neurone), by nearly a half of the control response 30 min after the start of perfusion (ED 50: 0.96 × 10 −4 M). The I Ca inhibition was still sustained 30 min after washout, indicating tha AJ-2615 has long-lasting activity. The compound inhibited the I Ca over a wide range of membrane potentials depolarized by the voltage pulse (V d), but did not change the membrane potential required to produce the maximal I Ca ( V d = 0 mV). The additional decrease in I Ca amplitude caused by changing from low-frequency (1/5 min) depolarizing pulses to high-frequency (3 / min) depolarizing pulses in the presence of AJ-2615 at 10 −4 M was 39.2±4.5% (mean + S.E.M.; n = 5), indicating use dependence. The steady state inactivation curves were measured in the presence or absence of AJ-2615 at 10 −4 M with a depolarizing prepulse (V d = varied, duration = 30 s) followed by a depolarizing test pulse (V d = +10 mV, duration 80 ms), with 2 ms intervals ( n = 3): the ratio of AJ-2615 dissociation constant in the resting Ca 2+ channel (K r) and in the inactivated Ca 2+ channel (K i), K r/K i, was calculated to be 9.6:1, indicating voltage dependence.

Slow inward current induced by achatin-I, an endogenous peptide with a D-Phe residue

Following a preliminary report on the isolation of a neuroactive tetrapeptide, achatin-I (Gly-D-Phe-L-Ala-L-Asp) that has a D-phenylalanine residue, from the Achatina fulica ganglia, the pharmacological features of this peptide on Achatina giant neurones were now worked out in detail. Of the eight possible stereoisomers, only achatin-I markedly, and [D-Ala 3]achatin-I slighly, induced a slow inward current (I in) with an increase in membrane conductance (g) of the identifiable neurones, tonically autoactive neurone (TAN), dorsal-right cerebral distinct neurone (d-RCDN) and periodically oscillating neurone (PON) which had been tested previously. Of 23 types of neurones tested, 10 types including the three mentioned were excited by achatin-I wwhereas no neurone was inhibited. The ED 50 of achatin-1 for the neurones tested were 0.2–2.7 × 10 −5 M, and that for PON was the lowest. The Hill coefficients of achatin-1, 0.62–0.80, deviated from 1.0. E max values of achatin-I for producing I in, 4.2–6.3 nA, were significantly greater than those of [D-Ala 3]achatin-I, 1.8–3.4 nA. I in of TAN and d-RCDN induced by achatin-I was blocked in the Na +-free state, but unaffected in the Ca 2+-free (replaced with Co 2+), Cl −-free or K +-enriched (3.0X) state, indicating that the current was produced by the g increase in response to Na +. However, the I in was partially blocked by tetrodotoxin 10 −4 M. We propose that achatin-I is an excitatory neurotransmitter on Achatina neurones.

Isolation of achatin-i, a neuroactive tetrapeptide having a d-phenylalanine residue, from Achatina ganglia, and its effects on Achatina giant neurones

1. We have isolated a neuroexcitatory tetrapeptide having a D-phenylalanine (Gly- D-Phe-L-Ala-L-Asp) from the ganglia of Achatina fulica Férussac. This peptide was termed achatin-I (Kamatani el al., 1989). In the present report, we shall present highlights from the original paper concerning the process of peptide isolation and the examination of its effects. 2. From the ganglia of about 30,000 animals, we obtained 50μg of achatin-I and 17 μg of its stereoisomer consisting of only L-amino acid residues (Gly- L-Phe- L-Ala- L-Asp) which was termed achatin-II. The data of instrumental analyses ( 1H-NMR, SIMS, CD and HPLC) of isolated achatin-I and achatin-II were identical to those of synthetic ones. 3. Achatin-I showed marked excitatory effects on the three Achatina giant neurones, PON (periodically oscillating neurone), TAN (tonically autoactive neurone) and V-RCDN (ventral-right cerebral distinct neurone), whereas achatin-II had no effect. Among their stereoisomers, ( D-Ala 3]-achatin-I (Gly- D-Phe- D-Ala- L-Asp) had slight excitatory effects on the Achatina neurones tested. Amide derivatives of achatin-I and achatin-II were ineffective. 4. Dose-response curves of achatin-I and [ D-Ala 3]-achatin-I for producing the inward current of PON were measured under voltage clamp at a holding membrane voltage ( V h) of −50 mV. The results obtained were: 0 ED 50 = 2.29 × 10 −6 M , its 95% confidence limit = 1.67 − 3.14 × 10 −6 M, and E max ( mean ± SEM) = 5.53 ± 0.28 nA for achatin-I (N = 6); and ED 50 = 5.37 × 10 −5 M , its 95% confidence limit = 3.68 − 7.84 × 10 −5 M, and E max = 0.84 ± 0.19 nA for [ D-Ala 3]- achatin-I (N = 3) . ED 50 and E max of achatin-I were significantly different from those of [ D-Ala 3-achatin-I. The Hill coefficient of achatin-I was calculated as 0.58 (coefficient for linear Hill plot: 0.991), which deviates from 1.0. 5. The current-voltage relations ( I–V curves) of PON membrane were measured by superimposing a depolarizing ramp voltage on V h = −100 mV. The inward current was produced by achatin-I in a wide range of membrane voltage in the physiological state. However, the inward current was hardly produced by the same peptide in the Na +-reduced state, indicating that the effects of achatin-I were due to membrane permeability increase to sodium ions. 6. We propose that this endogeneous peptide is a neurotransmitter of Achatina neurones. We predict that some neuroactive peptides having a D-amino acid residue, like achatin-I, exist and play important functions in the mammalian nervous system.

Achatin-I, an endogenous neuroexcitatory tetrapeptide from achatina fulica férussac containing A d-amino acid residue

A tetrapeptide named achatin-I was purified from the suboesophageal and cerebral ganglia of the African giant snail Achatina fulica Férussac, and evoked a potent neuroexcitatory effect. The amino acid sequence of achatin-I is Gly-D-Phe-Ala-Asp. Achatin-I induced a voltage-dependent inward current, due to Na+, on the identifiable giant neuron, periodically oscillating neuron (PON), of the same snail. All possible isomers of achatin-I were synthesized using the solid-phase method. The sensitivity of the neuron to achatin-I and its isomers was strictly stereospecific; among the various isomers, only achatin-I showed marked effects (ED50 = 2.29 x 10(-6)M), while Gly-D-Phe-D-Ala-Asp, the synthetic D-Ala-isomer, was less than 10(-3) active.

Further study of effects of synthetic peptides on identifiable giant neurones of an african giant snail ( Achatina fulica Férussac)

1. 1. Effects of the following peptides at 10 −4 M on identifiable giant neurones of Achatina fulica Férussac were examined: physalaemin, eledoisin, bradykinin, neurokinin A, neurokinin B, neuromedin B, gastrin releasing peptide decapeptide (neuromedin C), gastrin releasing peptide (14–27), cholecystokinin tetrapeptide, cholecystokinin octapeptide, thyrotropin releasing hormone, Arg-vasotocin, γ-melanocyte stimulating hormone. 2. 2. The six neurones tested were as follows: PON (periodically oscillating neurone), TAN (tonically autoactive neurone), RAPN (right anterior pallial neurone), d-RPLN (dorsal-right parietal large neurone), VIN (visceral intermittently firing neurone) and d-VLN (dorsal-visceral large neurone). 3. 3. Of the peptides examined, only Arg-vasotocin at 10 −4 M produced the excitatory effects on PON, VIN and d-VLN. Physalaemin showed slight inhibitory effects on TAN; this substance was sometimes almost ineffective on the neurone. 4. 4. The other peptides examined were completely ineffective on all of the neurones tested.

Effects of compounds related to β-hydroxyglutamic acid (BHGA) on identifiable giant neurones of an African giant snail (Achatina fulica Férussac)

The present study aimed to further elucidate the pharmacological features, wiht respect to sensitivity to L-BHGA agonists,of the receptors sensitive to β-hydroxy-L-glutamic acid (L-BHGA) in five Achatina giant neurones: PON (periodically oscillating neurone), d-RPLN (dorsal-right parietal large neurone), VIN (visceral intermittently firing neurone), RAPN (right anteior pallial neurone) and v-RCDN (ventrral-right cerebral distinct neurone). Of these neurones, d-RPLN and RAPN were depolarized by L-BHGA, while PON, VIN and v-RCDN were inhibited. Threo-β-hydroxy-DL-aspartic acid markedly depolarized d-RPLN and RAPN (effective potency quotient (EPQ) in relation to the more effective L-BHGA isomer: 1 for d-RPLN and 0.3 for RAPN). This compound produced only slight inhibitory effects on PON, VIN and v-RCDN with EPQs calculated to be<0.03, <0.03 and 0.03, respectively. On the other hand, erythro-β-hydroxy-DL-aspartic acid at 10 −3 M was almost ineffective, except on v-RCDN where it elicited some slight inhibitory effects (EPQ: 0.01). L-Aspartic and D-aspartic acid at 10 −3 M, also had almost no effect except for slight effects of D-aspartic acid on d-RPLN (EPQ: 0.1). N-Methyl-L- and N-methyl-D-aspartic acid were slightly effective only on v-RCDN (EPQ: <0.021 and 0.01 respectively). The other compounds, including β-hydroxypyrroglutamic acid (cyclic BHGA) and proline derivatives, were almost ineffective at 10 −3 M; very weak effects were occasionally observed on some neurones. These results have a two-fold meaning: the relative sensitivities of these neurones to the erythro- and threo-L-BHGA stereoisomers do not correspond to the sensitivities of the stereoisomers of β-hydroxy-DL-aspartic acid and the zwitterionic character of the α-amino group, the presence of a β-hydroxyl group and a C4 or C5 carboxyl group are necessary to produced the effects on these neurones.

Neural Control of Heart Beat in the African Giant Snail, Achatina Fulica Férussac: I. Identification of the Heart Regulatory Neurones

ABSTRACT Seven heart regulatory neurones (PON, TAN, TAN-2, TAN-3, d-RCDN, d-LCDN and VG1) were identified in the central nervous system of the African giant snail, Achatina fulica Férussac. Among these neurones, the periodically oscillating neurone (PON) was the most effective heart excitor, producing heart excitation at rather low firing frequencies. The tonically autoactive neurones (TAN, TAN-2 and TAN-3) were tonically firing neurones and their spontaneous activity was found to produce tonic heart excitation which supplemented the myogenic heart activity. There was some evidence that two cerebral ganglion cells (the dorsal right and left cerebral distinct neurones, d-RCDN and d-LCDN) were also likely to be heart excitors although the direct connection to the heart was somewhat doubtful in some specimens. No direct inhibitory neurone was found, but the high firing frequency of the visceral ganglion neurone (VG1) usually produced heart inhibition.

Effects of catecholamine and monophenolamine agonists on identifiable giant neurones, sensitive to these amines, of an African giant snail ( Achatina fulica Férussac)

The effects of 23 substances proposed as catecholamine (CA) and monophenolamine (MA) agonists were tested on the five CA-sensitive neurones, periodically oscillating neurone (PON), tonically autoactive neurone (TAN), left-visceral multiple spike neurone (l-VMN), dorsal-right pedal autoactive neurone (d-RPeAN) and visceral intermittently firing neurone (VIN), and the three MA-sensitive neurones, frequently autoactive neurone (FAN), dorsal-left pedal large neurone (d-LPeLN) and dorsal-left cerebral distinct neurone (d-LCDN), of an African giant snail ( Achatina fulica Férussac). Of these neurones, PON, VIN and d-LPeLN were excited by the most effective catecholamine or monophenolamine, i.e. dopamine, epinine or DL-octopamine, whereas TAN, l-VMN, d-RPeAN, FAN and d-LCDN were inhibited by the same substances. Of the five CA-sensitive neurones, PON and VIN were markedly excited by eregometrine (effective potency quotients (EPQs) of these substances as compared with the effective potency of the most effective catecholamine or monophenolamine for these neurones: 3.0 for PON, 1.0 for VIN), methylergometrine (EPQs: 3.0 for PON, 0.3 for VIN) and mescaline (EPQs: 0.3 for PON, 1.0 for VIN). These two neurones were excited slightly by DL-metaraminol (EPQs: 0.03 for both neurones) and DL-neosynephrine (EPQs: 0.03 for PON, 0.1 for VIN); VIN was also slightly sensitive to 3-methoxytyramine (EPQ: 0.03). TAN and l-VMN were inhibited by ergometrine (EPQs: 1.0 for TAN, 0.3 for l-VMN) and methylergometrine (EPQs: 0.3 for TAN, 0.1 for l-VMN), whereas d-RPeAN was inhibited slightty only by DL-neosynephrine (EPQ: 0.01). Of the three MA-sensitive neurones, FAN was insensitive to the substances examined; d-LPeLN was excited slightly only by DL-ethylnorepinephrine (EPQ: 0.03); and d-LCDN was inhibited very slightly by DL-ethylnorepinephrine, DL-neosynephrine and tolazoline (EPQ: 0.003). It was noted that ergometrine and methylergometrine affected most CA-sensitive neurones, DL-ethylnorepinephrine affected most MA-sensitive neurones, and most substances examined had very selective effects on only a few of the catecholamine- and monophenolamine-sensitive neurones.

Axonal pathways of giant neurons identified in the right parietal and visceral ganglia in the suboesophageal ganglia of an African giant snail (Achatina fulica Férussac)

1.1. The axonal pathways of thirteen giant neurons identified in the right parietal and the visceral ganglia, found in the suboesophageal ganglia of an African giant snail (Achatina fulica Férussac), were investigated by intracellular injections of Lucifer Yellow, with regard to their axonal projections into the following six peripheral nerves: lap n (left anterior palliai nerve), lpp n (left posterior palliai nerve), int n (intestinal nerve), anal n (anal nerve), rpp n (right posterior palliai nerve) and rap n (right anterior palliai nerve).2.2. These projections were confirmed by the recording of the axonal responses from the nerves.3.3. On the dorsal surface of the right parietal ganglion, the following four giant neurons were identified: PON (periodically oscillating neuron), TAN (tonically autoactive neuron), RAPN (right anterior palliai neuron), and d-RPLN (dorsal-right parietal large neuron).4.4. The PON axonal pathways projected into int n; those of TAN into all of the nerves examined; those of RAPN into lap n, lpp n, int n, anal n and rap n.; and those of d-RPLN into pd nn (pedal nerves) through the pedal ganglia, lpp n, anal n, rap n and sometimes lap n.5.5. On the dorsal surface of the visceral ganglion, the following four giant neurons were also identified: VIN (visceral intermittently firing neuron), FAN (frequently autoactive neuron), INN (intestinal nerve neuron) and d-VLN (dorsal-visceral large neuron).6.6. The VIN axonal pathways, which had no branch into the six nerves examined, went to both the right and the left pedal ganglia, sending a branch into the cerebro-pleural connective; those of FAN projected into lap n, anal n and rap n, and sometimes into lpp n and rpp n; those of INN into int n; and those of d-VLN into pd nn, lap n, lpp n, anal n and rap n.7.7. On the ventral surface of the right parietal ganglion, v-RPLN (ventral-right parietal large neuron) was identified. The axonal pathways went to pd nn, lap n, lpp n, anal n and rap n.8.8. On the ventral surface of the visceral ganglion, the four giant neurons, v-VNAN (ventral-visceral noisy autoactive neuron), v-VLN (ventral-visceral large neuron), r-VMN (right-visceral multiple spike neuron) and 1-VMN (left-visceral multiple spike neuron) were identified.9.9. The axonal pathway of v-VNAN projected into rpp n and rap n; those of v-VLN into pd nn, lap n, anal n, rap n and sometimes to lpp n; those of r-VMN into int n and rpp n; and those of 1-VMN also into int n and rpp n.10.10. The present morphologial investigations of the giant neurons confirmed well the identifications of the neurons previously studied. The axon of the neurons examined here, except for VIN, projected into some of the peripheral nerves, while the VIN axon extended into the cerebro-pleural connective.11.11. The five neurons, PON, TAN, v-VNAN, r-VMN and 1-VMN, formed fine axonal arborizations terminating at the neuropile, while the arborizations of the other neurons were not clearly observed.12.12. Although the anatomical structures of the portion examined of the suboesophageal ganglia are asymmetrical, three pairs of symmetrically-situated neurons, d-RPLN and d-VLN, v-RPLN and v-VLN, and r-VMN and 1-VMN, were found, indicating the existence of symmetrical components in the ganglia.

Effects of synthetic peptides on giant neurones identified in the ganglia of an African giant snail ( Achatina fulica Ferussac)

1. 1. The effects of 13 synthetic biologically active peptides were investigated on the five identifiable giant neurones of an African giant snail (Achatina fulica Férussac), PON (periodically oscillating neurone), TAN (tonically autoactive neurone), d-RPLN (dorsal-right parietal large neurone), VIN (visceral intermittently firing neurone) and d-LCDN (dorsal-left cerebral distinct neurone). 2. 2. The peptides examined were: (1) peptides proposed as neurotransmitters in mammals: metenkephalin, substance P, neurotensin, LH-RH, [Gln 8]-LH-RH, oxytocin and Arg-vasopressin; (2) peptides proposed as neurotransmitters in invertebrates: proctolin and FMRF-amide; and (3) natural venom peptides: bombesin, ranatensin C, vespakinin X and vespakinin M. 3. 3. Of the peptides examined, oxytocin markedly excited PON [minimum effective concentration (MEC): 3 × 10 −7 M], d-RPLN (MEC: 10 −5 M) and VIN (MEC: 10 −6 M). On the other hand, Arg-vasopressin was only slightly excitatory at the concentration of the screening tests (10 −4M) on PON, and was ineffective on d-RPLN and VIN. 4. 4. FMRF-amide markedly inhibited TAN (MEC: 10 −6 M) and d-LCDN (MEC: 3 × 10 −5 M). 5. 5. The other peptides examined were found to be ineffective on the five neurones tested.