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Distinction between pyrethroid intoxicants

The paired pulse inhibition paradigm was used for its relevance to toxicity in humans; the ultimate goal of this research being to expand knowledge to ensure humans are safe. The model relies on innate charactersitics of the neuronal networks in the hippocampus, by exploiting the fact that dentate gyrus cells respond to stimulation via the perforant path with a transient phase of lowered excitability [Gilbert et al, 1989] . It is possible to probe the functioning of this central brain structure with electrodes to yield quantitative data.

Previous work has shown that pyrethroids extend this period of inhibition, contrary to what one might expect from their depolarising effects on single cells. This inhibitory phase usually lasts around 20-30 msec; Allethrin (a Type I pyrethroid) extended this to 60-100 msec, and deltamethrin (a Type II) extended it to 200-400 msec. It appears that the duration of inhibition is pyrethroid specific, even at massive doses, and so provides a quantitative way to compare between different compounds in vivo [Joy et al, 1990].

Background of the experimental paradigm

Research into the hippocampus and its workings has been active; the consensus is that this structure is crucial for formation of ego-centric memories, that is those which include a personal reference, also termed 'episodic' memories. Paired-pulse inhibition has been used extensively to investigate the cellular connectivity of the hippocampal formation.

Many researchers have concerned their work with glutamate signaling and the consequential plastic changes in synaptic arrangement. Ideas about long-term potentiation (where a synapse is strengthened through use) and long term depression (where a synapse is desensitised by low level noise) have evolved from electrophysiological experiments in the rodent hippocampus.

Aside from it's role in memory, the hippocampus has attracted research from other quarters. Some forms of epilepsy are associated with run-away excitation originating in the hippocampus, which can spread and cause general seizures.

A neuron being directly probed with a microelectrode; such accuracy is difficult to achieve, and so most in vivo recording is by necessity of fields

The experimental procedure

Stimulating a perforant path neuron with a micro-electrode can evoke an action potential, which passes into the granule cell layers of the hippocampus, to be detected by electrodes in the dentate gyrus. Collateral aborisations synapse on inhibitory inter-neurons (stellate or basket cells), which synapse back onto excitatory neurons in their close vicinity. Hence, stimulating the afferent fibre causes a phase of inhibition, which can be observed by stimulating again in close succession. Subsequent stimulations produce smaller readings from recording electrodes in the granule cell layer, characterised by increased potassium permeability [Kehl and McLennan, 1985]. This effect is described as paired-pulse inhibition, and is thought to underlie important memory formation processes.

Measuring the duration and magnitude of the inhibitory response is possible by delivery pairs of pulses and changing the interval between them while observing the differences between the first and second recorded potential. Work has been carried out analysing the effects of pyrethroids on neural function using this paired pulse inhibition model. Contrary to the general pro-excitatory role of pyrethroids, they have been shown to prolong the inhibitory response, as well as depressing the overall excitability of transmission from the perforant path to dentate gyrus (Gilbert et al, 1989).

One undesirable consequence of using paired-pulse inhibition for quantitative assessment of pyrethroid intoxication is that anaesthesia is required. Anaesthetics increase the effective dose of pyrethroid required. It is also possible that the anaesthetic may interact with the experimentally observed systems in a non-linear manner, complicating the picture. This seems to be the inevitable cost of using electrophysiology in vivo. Malfunction of this recurrent inhibitory circuitry has been linked with epileptiform activity, caused by run-away feedback excitation.

Surmountable inhibition causes the inhibitory effect

The underlying mechanism which produces the transient inhibition in granule cells following brief stimulation is, currently, only speculative. What is known is that the inhibition is mostly unaffected by picrotoxin, a GABA-A receptor antagonist [Joy et al, 1990]. Picrotoxin does cause a slight reduction in the first 10-20 msec of deltamethrin induced depression, while the remaining effect is preserved, suggesting that part of the effect is due to GABA-A receptors operating on chlorine channels. Thus it is likely that a degree of tonic inhibitory activity is present.

Large doses of picrotoxin induce seizures and subsequent pulses applied to the perforant path evoke responses of almost equal amplitude, regardless of the interval between them. So the inhibition is surmountable by blocking inherent GABA-A signaling, contributing to membrane depolarisation. However closer analysis of the interactions between a variety of GABA-A agonists, antagonists and deltamethrin revealed that the inhibitory effect was too robust to be purely GABA-A mediated. More likely the effect is produced by other mechanisms, of which GABA-A receptor has a minor or modulatory role [Joy & Albertson, 1991].

Horizontal section through the medial entorhinal area and Ammon's horn immediately ventral to the plane of the dorsal hippocampal commissure in a 15-day old mouse. Golgi method.
A - medial entorhinal area; B - convergence site for ventral fasicles of the perforant path; C - afferent bundles to the pre-subiculum; D - perforant fibres; E - fibres to the dentate gyrus; F - fibres ending in the dentate gyrus.

Inhibitory inter-neurons in the dentate gyrus

It is supposed that inter-neurons mediate the inhibitory effect through recurrent connections to granule cells. This would explain the latency in response between the initial stimulus, and when the inhibitory phase begins.

Further evidence comes from triple pulses applied to the perforant path, with the second two in close succession. This was initially an accidental observation (bless serendipity) - and curiously the third pulse produced a peak of amplitude close to the first. If pyrethroids were acting to depolarise the granule cell, rendering it unexcitable, the triple-pulse effect wouldn't be possible. Instead the inhibition is not due to refraction in the granule cell, but through some surmountable inhibition acting upon it. Thus it seems that the effect is largely due to the induction or extension of an intrinsic inhibitory process, probably mediated by inter-neurons.

Paradoxical action of pyrethroids

One is still left wondering how pyrethroids, which typically make neurons hyperexcitable by increasing sodium influx, could cause a decrease in excitability. It is possible that neurons within the hippocampus express variants of the sodium channel with differential susceptibility to pyrethroid modification. This could explain the slightly paradoxical effect of paired pulse inhibition in light of the hyperpolarising nature of pyrethroids on single cell preparations [Song et al, 1996]. Currently the expression patterns of sodium channels and other pyrethroid targets in central nervous structures are poorly characterised, leaving us to guess at where exactly pyrethroids are acting.

Diagrammatic representation of the regions targeted by electrodes to do paired-pulse inhibition type experiments in the hippocampus. The perforant path fibres pass from enotorhinal cortex into the dentate gyrus where they bifuricate and synapse with granule cells. Pyramidal cells are not involved but are included for completeness. The detail shows how excitatory cells (G) can form recurrent links with inhibitory interneurons (I).