C.Jane Roberts

The pharmacology of Limulus central neurons

1. Intracellular recordings have been made from neurons in the central nervous system of the horse-shoe crab, Limulus polyphemus. Neurons possess resting potentials between -40 and -60 mV, with action potentials ranging from 2-3 mV up to 60 mV in amplitude. Neurons also have excitatory and inhibitory postsynaptic potentials. 2. All the neurons studied are inhibited by GABA and excited by cholinomimetics. The GABA response is chloride mediated and reversibly antagonised by picrotoxinin but not by bicuculline or bicuculline methochloride or methoiodide. The cholinergic response is nicotinic and blocked by pentolinium, hexamethonium, chlorisondamine and dihydro-beta-erythroidine. 3. L-Glutamate can excite some cells, inhibit others and have a biphasic action, inhibition followed by excitation, on other cells. The inhibitory effect is chloride mediated and blocked by picrotoxinin. Ibotenate mimics the action of glutamate both in terms of inhibition and excitation but kainate and quisqualate only mimic the excitatory action of L-glutamate. 4. Dopamine, octopamine, 5-hydroxytryptamine and histamine excite some neurons while inhibiting others or have a biphasic action. Dopamine and octopamine normally have different effects on the same cell, suggesting they act via different receptors. Octopamine shows stereospecificity for the (-) isomer which is more than 100 times more active than the (+) isomer and octopamine is reversibly antagonised by phentolamine and cyproheptadine. 5. Proctolin has an excitatory action on these neurons and this effect is long lasting and can be potentiated by dibutyl cyclic AMP. 6. The pharmacology of Limulus central neurons is compared to the pharmacology of insect and crustacean central neurons. It is concluded that GABA and acetylcholine are central transmitters throughout the arthropods. It is also probable that L-glutamate and octopamine have a physiological role in the arthropod central nervous system. Proctolin appears to modify neuronal and muscle activity in the arthropods and has a modulatory or transmitter function.

The actions of L-glutamate and putative glutamate agonists on the central neurons of Limulus polyphemus

Abstract 1. 1. Intracellular recordings were made from unidentified neurons in the abdominal nerve cord of the horse-shoe crab, Limulus polyphemus. Out of over 100 neurons examined, L -glutamate excited 47.6%, inhibited 20.4% and gave a biphasic response in 15.5%; the remaining 16.5% failed to respond. GABA inhibited all neurons examined. 2. 2. D-glutamate and L - and D -aspartae were all less potent than L -glutamate. Ibotentate was approximately equipotent with L -glutamate on both inhibitory and excitatory responses, but N- methyl- DL -aspartate was inactive. Kainate had no effect on inhibitory glutamate receptors but was about 14 times more potent than L -glutamate on the excitatory receptors. Dihydrokainate, α-ketokainate and allo-α-ketokainate were potent excitants but were devoid of inhibitory effects on glutamate inhibitory receptors. Quisqualate strongly excited cells inhibited by L -glumate, while on cells excited by L -glutamate it was 250 times more potent than glutamate. 3. 3. Three compounds, ibotenate, D -glutamate and cis-1-amino-1,3-dicarboxycyclopentane, produced a biphasic response on neurons which were only excited by L -glutamate. It is proposed that the preferred form for L -glutamate at the inhibitory receptor may be in the extended form, while at the excitatory receptor it may be in a folded form. 4. 4. The glutamate inhibitory response was due to an increase in chloride conductance. This effect had a reversal potential of around −60 mV and was reversibly blocked by picrotoxinin.

Studies on the action of GABA, muscimol and related compounds on Periplaneta and Limulus central neurons

Abstract 1. Intracellular recordings were made from central neurones of Periplaneta americana and Limulus polyphemus. All the neurons tested were found to respond to γ-aminobutyric acid (GABA). 2. A structure-activity study was performed on these GABA receptors using conformationally restricted GABA agonists. Dihydromuscimol was the most potent agonist found, being some 14 times more potent than GABA on both preparations. The introduction of a sulphur group greatly reduced the potency of the agonists, for example, thiomuscimol and piperidine-4-sulphonic acid were far less active than muscimol and isonipecotic acid respectively. 3. It is concluded that the preferred form of GABA for optimal interaction with the receptor is probably similar in both species, that is, in a partially extended conformation possibly in a shape reflected by the dihydromuscimol molecule.

Octopamine receptors on limulus and periplaneta central neurons

Abstract 1. 1. Intracellular recordings were made from neurons in the mid-dorsal region of the abdominal ganglia of Periplaneta americana and from unidentified neurons in the abdominal ganglia of Limulus polyphemus . (±)-Octopamine hyperpolarized Periplaneta neurons and in some cases this hyperpolarization was followed by depolarization, that is, a biphasic response. (±)-Octopamine hyperpolarized Limulus neurons without an excitatory component. 2. 2. D (—)-Octopamine and L -(+)-octopamine are approximately equipotent on Periplaneta neurons, the (+)-isomer being 2.5 times less potent than the (—)-isomer. While on Limulus neurons, D (—)-octopamine is more than 100 times more potent than L (+)-octopamine. 3. 3. Both phentolamine and cyproheptadine, 2–3 × 10 −6 M, reversibly blocked the hyperpolarization component of the octopamine response on both Periplaneta and Limulus neurons. In the presence of cyproheptadine, the octopamine response of Periplaneta neurons is reversed to one of excitation.

Occurrence of noradrenaline, dopamine and 5-hydroxytryptamine in the nervous system of the horse-shoe crab, Limulus polyphemus

Abstract 1. The levels of dopamine, noradrenaline and 5-hydroxytryptamine in the nervous system of the horse-shoe crab, Limulus polyphemus, were determined fluorimetrically. 2. The amine levels in the brain were 528, 1539 and 1948 ng/g wet wt for noradrenaline, dopamine and 5-HT, respectively. 3. The amine levels in the abdominal ganglia were 1487, 2298 and 9326 ng/g wet wt for noradrenaline, dopamine and 5-HT respectively. 4. The levels for noradrenaline and 5-HT were significantly different in the two regions, while that for dopamine was not. 5. The dopamine and noradrenaline levels in the brain were also significantly different. 6. The present findings are discussed in relation to the occurrence of these amines in the nervous tissue of other anthropods.

The action of seven convulsants as antagonists of the GABA response of Limulus neurons.

Abstract 1. Intracellular recordings were made from neurons in the abdominal ganglia of Limulus polyphemus . GABA was inhibitory on all the neurons tested. 2. Picrotoxinin was approximately equipotent with picrotoxin as a reversible antagonist of the GABA inhibitory response. 3. Picrotin, the inactive component of picrotoxin, reversibly antagonized the GABA inhibitory response but was 19 times less potent than picrotoxinin. 4. Both t -butylbicyclophosphate and tetramethylenedisulphotetramine reversibly antagonized the GABA inhibitory responses, but were 2410 and 2705 times respectively less potent than picrotoxinin. 5. Bicuculline methochloride and p -tolyl silatrane had no blocking action on the GABA inhibitory responses of these neurons.

The action of gamma-aminobutyric acid (GABA) and ethylenediamine (EDA) on Limulus and Helix central neurones and rat cerebellar and sympathetic ganglion neurones

Intracellular recordings were made from central Limulus and Helix neurones and extracellular recordings from rat cerebellar Purkinje cells and sympathetic ganglia. The actions of gamma-aminobutyric acid (GABA) and ethylenediamine (EDA) and related analogues on these preparations were investigated. On Limulus neurones inhibited by GABA, EDA and piperazine were 81 and 186 times respectively less potent than GABA. Both the GABA and EDA events were chloride mediated, having similar reversal potentials and were reversibly antagonised by picrotoxinin. The EDA response persisted in high magnesium Ringer. On Helix neurones inhibited by GABA, EDA was 92 times less potent while on neurones excited by GABA, EDA was 9.25 times less potent. The other analogues tested had little or no GABA-like effect on either preparation. On rat cerebellar Purkinje cells, EDA was equipotent with GABA and both compounds were antagonised by bicuculline. Flurazepam only potentiated the action of EDA on 3 out of 23 cells tested while the GABA response of all 23 cells was potentiated by the benzodiazepine. Diaminopropionic acid was a weak inhibitor of cerebellar Purkinje cell firing but flurazepam potentiated this response in 6 out of 10 cells tested. On rat cervical ganglion neurones, EDA was half the potency of GABA and likewise the other analogues were less potent than GABA as depolarising agents. Incubation with glutamic acid decarboxylase inhibitors had no effect on the EDA response. Cross desensitisation between GABA and EDA was demonstrated using the ganglion preparation.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for γ-aminobutyric acid (GABA) as an inhibitory transmitter in the central nervous system of limulus polyphemus

Abstract 1. Intracellular recordings were made from neurones in the abdominal ganglia of Limulus polyphemus . All the neurones examined were inhibited by GABA. 2. The inhibitory response to GABA was a chloride event with a reversal potential of −63 mV. Stimulated or spontaneous IPSPs could be recorded from many of the neurones. These IPSPs were also dependent on chloride and had reversal potentials of around −65 mV 3. Picrotoxinin reversibly antagonized both GABA and the IPSPs. 4. A Hill plot suggests that two molecules of GABA interact with the receptor. 5. An analysis of the amino acids present in the nervous tissue shows the presence of 1.5 ± 0.12 nmol/mg tissue GABA. 6. A structure-activity study indicates that the structural requirements for potent GABA-like activity on these neurones is very specific. 7. It is concluded that GABA is likely to be an inhibitory transmitter in the central nervous system of L. polyphemus .

Neuropharmacological studies on the receptors mediating responses to carbachol, amino acids and octopamine on Limulus and Hirudo central neurons

Intracellular recordings were made from central neurons of Limulus polyphemus and from Retizu cells of the leech, Hirudo medicinalis. The effects of carbachol, amino acids and octopamine were examined on these neurons. Octopamine was found to have a mainly inhibitory effect on a few Limulus neurons. The effects of octopamine were mimicked by clonidine and napthazoline but not by xylazine. Both compounds were slightly more potent than octopamine. Yohimbine, metoclopramide, chlorpromazine and chlordimeform failed to antagonize this octopamine response. The excitatory effect of carbachol was blocked by alpha-bungarotoxin, 10(-7)M. Neither this concentration nor higher concentrations of alpha-bungarotoxin had any effect on L-glutamate excitation. m-Carboxyphenyl derivatives of alanine and glycine acted differentially on Limulus neurons responding to L-glutamate. m- Carboxyphenylglycine only inhibited neurones which showed a biphasic response to L-glutamate while m- carboxyphenylalanine only excited these neurons. Both compounds excited leech Retzius cells, with m- carboxyphenylalanine being about 20 times more potent than m- carboxyphenylglycine . The actions of alpha- ketokainate and allo-alpha- ketokainate were compared to kainate, dihydrokainate and L-glutamate on leech Retzius cells. The equipotent molar ratios for kainate, dihydrokainate , alpha- ketokainate and allo-alpha- ketokainate were 0.0029 +/- 0.0004, 0.021 +/- 0.047, 0.029 +/- 0.005 and 0.14 +/- 0.0093 respectively with L-glutamate as one. All the analogues were more potent than L-glutamate. Quinolinic acid had no glutamate-like activity on either Limulus or Hirudo neurons. Methyltetrahydrofolate was inactive on Limulus neurons but excited leech Retzius cells, being slightly less potent than L-glutamate. Dibutyl cAMP terminated the excitatory actions of kainate on both Limulus and Hirudo neurons. Anisatin , a putative GABA antagonist, was a potent antagonist of GABA inhibition on Limulus neurons.

The actions of ibotenate, homoibotenate analogues and AMPA on central neurons of Hirudo, Limulus and Helix

Abstract 1. Intracellular recordings were made from central neurons from Hirudo medicinalis, Helix aspersa and Limulus polyphemus. 2. The actions of L -glutamate, ibotenic acid (IBO) and a series of IBO analogues, in which the structure and position of the amino acid side chain have been changed, were examined. 3. The relative potency of each compound was compared to L -glutamate and a series of Equipotent Molar Ratios obtained. 4. α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and 4-bromo-homo-IBO were over 100 times more potent than L -glutamate on leech neurons but were inactive on the Limulus and Helix neurons tested. 5. 4-Methyl-homo-IBO was about 65 times more potent on leech neurons than L -glutamate but inactive on the Limulus and Helix neurons tested. 6. Homo-IBO was 3 times more active than L -glutamate on leech Retzius cells but 10 times less active on Limulus cells excited by L -glutamate. 7. On Limulus and Helix cells inhibited by L -glutamate, this compound was 4.3 and 10 times less potent respectively. 8. On Helix cells where L -glutamate is excitatory, it was more than 50 times less potent than L -glutamate. 9. IBO was approximately equipotent with L -glutamate of Limulus cells excited or inhibited by L -glutamate or Helix cells inhibited by L -glutamate but was around 15 times more potent than L -glutamate on leech neurons and 8 times less potent on Helix neurons excited by L -glutamate. 10. These results demonstrate that using agonists, there are clear differences between the glutamate receptors on Hirudo, Helix and Limulus central neurons.