pmoles By altering the composition of the bathing saline they determined that the ionic basis of the GABA

response was a substantial increase in the membrane's

permeability to chloride ions and a slight increase in it's

permeability to potassium. In 1970, Pitman and Kerkut

estimated, from the extrapolation of the graph of

response amplitude versus membrane potential, that in

normal saline the reversal potentials of the IPSP and of G A B A were very similar: -78.9 ^2.1 m V for the IPSP; and - 75.4 =^0,6 m V for GABA. They found that the two responses

were reversed, both in chloride free saline and when the

cells were impaled w ith microelectrodes containing IM

potas s i u m chloride. Also, both reversal potentials

were displaced towards the neuron's resting potential

in a high p o t a s s i u m saline. They concluded from these

results that the ionic mechanisms of the G A B A response and

the IPSP were similar. In addition, they also reported

that both responses were bl o c k e d by the G A B A antagonist,

picrotoxin (100 p g / m l ) . Takeuchi and Takeuchi (1969) have

pr o vided evidence, however, that picrotoxin does not in fact interact with the GABA receptor, but rather w ith the chloride ion channels, and so nothing can be deduced

about the receptors involved in the responses. Walker,

General Introduction - Amino Acids page 55 preparation, found that the GABA antagonist, bicuculline

(0.68 - 2.7 blocked both the IPSPs and the GABA

response. They also found, however, that it also blocked the excitatory effect of the ACh agonist c a r b a m y l c h o l i n e . And so nothing can again be said about the receptors i n v olved.

More recently, Roberts, Krogsgarard~Larsen and Walker (1981) carried out a structure - activity study on the GABA

receptors of the above preparation. They used

conformât!onally restricted GABA agonists and determined their potency. The found that dihydromuscimol was the most effective agonist and concluded from the shape of this

molecule that GABA interacts with its receptor in a

partially extended form.

Goodman and Spitzer (1979, 1980) have studied the

development of GABA chemosensitivity in the metathoracic DUM neurones of the grasshopper embryo. They found that neurones first become sensitive to GABA, either iontophoretically or bath applied, between day 8 and day 13 of the e m b r y o ’s life,

before in fact the n e u r o n e s ’ own neurotransmitter is

detectable. This response to GABA reversed at -70 mV, was

abolished in chloride free saline, and was blocked by picrotoxin. These results, unlike those obtained with the

embryonic ACh receptors, are very similar to those of adult

cockroach DUM neurones. The reason for this difference is u n k n o w n .

Using the single fibre oil-gap technique and the cockroach t

(1971a, 1971b) found that by stimulating cereal nerve ten they could evoke IPSPs in the giant fibres. Since IPSP amplitude depended on stimulus strength and the delay between the stimulus and the appearance of the IPSP was about twice that of a single chemical synapse, they decided that the IPSPs were caused by the stimulation of a group of inhibitory interneurones that connected the sensory cereal nerves to the giant fibres. GABA, when either iontophoresed

or bath applied, caused membrane hyperpolarization, a

reduction in EPSP amplitude, and a reduction in membrane input resistance of the giant fibres. The similarity of the GABA response and the IPSP, and the fact that they are both blocked by picrotoxin, has again led to the conclusion that the ionic mechanisms underlying both responses are similar. Hue, Pelhate and Chanelet (1979), have obtained evidence that GABA also acts on the presynaptic cereal afferents, causing a depolarization and a decrease in transmission to

the giant fibres. They reported that this effect is

dependent on the chloride and potassium ion concentrations and that it is blocked by picrotoxin. The underlying ionic mechanism is thus thought to be the same as the one for the GABA postsynaptic effect: a large increase in chloride

conductance accompanied by a smaller increase in the

potassium one.

By comparing the sensitivity of the cockroach giant

interneurone synapses to the amino acids taurine and GABA under varying conditions. Hue, Pelhate and Callec (1981) have obtained indirect evidence for the existence of a specific GABA uptake mechanism. At this synapse, taurine

General Introduction - Amino Acids page 57

mimics the action of GABA. The decrease in the EPSPs and the increase in the membrane conductance due to GABA were enhanced by lowering the temperature of the perfusing saline, by using a sodium pump inhibitor (ouabain), and by using sodium-free salines. None of these conditions caused a significant change in taurine sensitivity. These results suggest the presence of an uptake m e c h a n i s m wh i c h contains within it a sodium sensitive component. They also used two blockers of G A B A uptake mechanisms, L-2,4-diaminobutyric

acid (DABA) and /3-aminobutyric acid (BABA). D ABA

specifically blocks neuronal GABA uptake in mammals, whereas B A B A specifically blocks glial uptake of GABA. Only BABA p r o d u c e d any significant increase in GABA sensitivity and so they concluded that the uptake mecha n i s m was present on the glial cells. This is probably one of the reasons for the low potency of b a t h applied as compared with iontophoretically

applied G ABA (10 m M and 10.5 pmoles, respectively).

V ery little electrophysiological evidence exists for the central effects of the other amino acids. Usher w o o d et al

(1980) found that glutamate hyper p o l a r i z e d freshly

dissociated locust thoracic ganglion neurones, whereas

Steiner and Fieri (1969) found that glutamate usually caused excitation of neurones in the ant protocerebrum.

Kerkut et al (1969b) found that glycine h y p e r p olarized DUM neurones of the m e t a thoraic ganglion of adult cockroaches. This was also the response observed by U s h e r w o o d et al

t,

(1980) with freshly dissociated neurones of adult locusts and cultured neurones from nymphal c o c k r o a c h e s .

The only report so far for the central effects of aspartate was that of Usherwood et al (1980), who found that the

dissociated locust neurones could show either a

page 58a

In the last three sections the evidence for the

neurotransmitter role of acetylcholine, the biogenic amines dopamine, noradrenaline, octopamine and serotonin, and the amino acids glutamate and GABA, in the insect nervous system has been reviewed.

There is more evidence to support the neurotransmitter role of acetylcholine in the insect central nervous system, than for any of the other candidates. It is thought to be released from the central projections of many sensory neurones and may also be used by some interneurones.

It is now generally accepted that the amino acids glutamate and GABA are, respectively, the excitatory and inhibitory neurotransmitters involved in neuromuscular transmission. It is also believed that GABA may act as an inhibitory central neurotransmitter. There is little evidence for the central role of glutamate, although information about this may come from studying the central projections of the excitatory m o t o n e u r o n e s .

Dopamine, octopamine, noradrenaline and serotonin have all been detected within the nervous system of several insect species. In the periphery, it has been shown that octopamine is involved in the modulation of neuromuscular transmission and there is also reasonable evidence for the role of dopamine and serotonin in salivary gland activation. Little is known, however, about their physiological roles in the central nervous system.

since biochemical assays have shown dopamine to be present in significant quantities, but the localization of these neurones in the ventral nerve cord and suboesophageal

ganglia is unknown. The ventral nerve cord and

suboesophageal ganglia are less anatomically complex than the cerebral ganglia and may be better regions to focus on

for intracellular recordings and dye injection.

One of the aims of this study, therefore, was to localize dopamine-containing neurones within wholemount ventral nerve cord and suboesophageal ganglia of the cockroach. This was acheived using a fluorescence-based technique which is able to reveal dopamine, noradrenaline and serotonin. The colour of the fluorescence indicated that no serotonin-containing neurones were detected. Dopamine and noradrenaline could not be distinguished, although other evidence suggests that most, if not all, of the neurones are dopaminergic.

Several attemps were made to further characterize a few of the neurones by combining dye infusion or intracellular dye injection with the fluorescence technique. Although some

success was acheived, especially with fluorescence-dye

infusion combination, difficulties were encountered with the identification and filling of the neurones.

Electrophysiological experiments showed that an identified prothoracic neurone was depolarized in response to pressure ejected and bath applied dopamine and noradrenaline. Some

evidence is p r o vided that catecholamine receptors are

present on the neurone but the possibility that dopamine could be acting at octopamine or acetycholine receptors

page 58c cannot be ruled out. Data is also presented which suggests that, in this case, dopamine does not act through the second messenger, cyclic AMP.

Additional experiments to determine the effect of pressure ejected glutamate and GABA on the membrane potential of an identified metathoracic motoneurone were also carried out. The effects of picrotoxin and 4-acetamido-4'-isothiocyano-

stilbene-2,2'-disulphonic acid on the amino acid responses

and the membrane properties of the neurone somata were i n v e s t i g a t e d .

A preliminary account of some of the above work has previosly been published (Fleming and Pitman, 1983).