The effects of anaesthetic agents on synapses of lymnaea stagnalis (L.)

Woodruff, Kim Therese (2004) The effects of anaesthetic agents on synapses of lymnaea stagnalis (L.). Doctoral thesis, University of Central Lancashire.

[thumbnail of Thesis document] PDF (Thesis document) - Submitted Version
Restricted to Repository staff only
Available under License Creative Commons Attribution Non-commercial Share Alike.

6MB

Abstract

In the mammal, anaesthetics are known to act via two distinct mechanisms, either increasing inhibition via GABAA receptors (eg. Na-thiopentone) or decreasing excitation via NIvIDA receptors (eg. ketamine). The aim of this thesis is to investigate
the effects of both increased inhibition and decreased excitation at the synaptic level within an invertebrate model system, something which cannot readily be done in vertebrate systems. This was achieved by carrying out experiments using both the above mentioned anaesthetics on the whole animal, isolated brain and cultured neurons.
In invertebrates it has been shown that GABA and Glutamate can be both excitatory and inhibitory, and injection of GABA into Lymnaea has been shown to result in behavioural changes in feeding, locomotion, escape reactions, male mating and
respiration. Injection of Na-thiopentone into the whole animal was carried out in this investigation, in order to establish the anaesthetic response of the animal model to this barbiturate. The presence of gamma-aminobutyric acid (GABA) receptors has been demonstrated in a respiratory interneuron (RPeD1) both electrophysiologically and via molecular techniques, however inmiunostaining has proved negative in RPeD1 and follower cells VD2/3 (unidirectional excitatory synapse) and VD4 (mutual inhibitory synapse). This suggests that these neurons are not themselves GABAergic, although this investigation shows the responses of these neurons to bath and direct application of GABA.
Na-thiopentone did not reliably anaesthetise Lymnaea upon injection into the sole of the foot, suggesting that Na-thiopentone binds to proteins within the snail, andlor has a low affininty for the GABAA receptor in Lymnaea. Other anaesthetic studies using propofol and ketaniine have also demonstrated a lack of anaesthetic response.
RPeD1 hyperpolarised and became quiescent in response to the application of high concentrations of GABA (10 3-104M), however at lower doses (1O 8-1O 5M), no effect was observed (p<0.05). Under these conditions simultaneous recordings from VD4 showed hyperpolarisation in response to the application of GABA, whereas VD2 and VD3 exhibited excitatory responses. Presynaptic picoinjection of GABA also resulted in hyperpolarisation and quiescence in RJeD1, but the simultaneous response in VD3 was not observed. Postsynaptic application of GABA directly to 'VD2, and VD4 however, resulted in responses similar to those seen in the whole brain. VD2 and 3 also receive input 2, which hyperpolarises RPeD1 and elicits an excitatory EPSP in VD2 and 3 as this is similar to the response observed in this experiment it is possible that the effects of input 2 on RPeD1 and VD2 & 3 are mediated by GABA. As RPeDI does not stain positively for GABA and hyperpolarises in response to the drug, it seems unlikely that the postsynaptie effects are due to presynaptic release of GABA. RPeD1 has been shown to form reciprocal synapses with VD4 both in vivo and in vitro. When perfused with GABA (lmJ'i4), both cells hyperpolarised reversibly. The postsynaptic response could be due to the action of GABA presynaptically inhibiting RPeDI, or directly on postsynaptic GABA receptors. However VD4 forms connections with other cells in the brain such as input 3 which may also have resulted in this inhibitory response. RPeD1 would however have received a simultaneous excitatory input from this interneuron. Attempts were made to establish the nature of the RPeD1JVD4 synapse in these experiments, but no synapses were evident. These experiments therefore confirm the presence of GABA receptors in RPeD1 and suggest theft presence in VD2, and VD4.
This investigation confirms the findings of previous studies, that injection and bath perfusion of barbiturates does not lead to responses in Lymnaea comparable to that of the mammal.
In addition to it's main target site, ketamine (a frequently used intravenous anaesthetic) has also been shown to act at cholinergic receptors. The effects of ketamine on learning and memory and apoptosis in the mammalian CNS are well recognised. Within the Lymnaea CNS, VD4 and LPeD1 form a unidirectional excitatory cholinergic synapse, and this was chosen to investigate the effects of ketamine on excitatory synaptic transmission, short term potentiation and synapse formation in the invertebrate animal model.
Ketamine decreased synaptic transmission between VD4 and LPeD1 in a concentration dependent manner, but did not significantly affect short term synaptic plasticity (pc0.05). While neurite outgrowth remained extensive, actual sprouting was diminished by all doses of ketamine. Cells exhibited extensive veiling, which was not present in control cells. Percentage chemical synapse formation was reduced by all doses of ketamine, and in some cases inappropriate inhibitory chemical synapses were formed.
Furthermore acute, clinically relevant levels of ketamine reduce excitatory cholinergic transmission between VD4 and LPeD1, but short term plasticity is unaffected. Nerve regeneration was seriously compromised, and formation of appropriate chemical
synapses greatly reduced. This data has serious implications for the clinical - use of ketamine, particularly in pregnant women, children or critical care patients where nerve regeneration and synapse formation are of great importance and long term exposure common practice.
In conclusion, this work supports that of other studies which have showed that invertebrates appear to be relatively insensitive to barbiturates, whereas ketamine appears to effect excitation in a manner similar to that in the mammal


Repository Staff Only: item control page