Robert M. Pitman

Intracellular citrate or externally applied tetraethylammonium ions produce calcium‐dependent action potentials in an insect motoneurone cell body

1. Electrophysiological observations have been made upon the cell body of an identified motoneurone of the cockroach, Periplaneta americana. Normal responses were compared with those observed after intracellular injection of citrate anions or when the preparation was bathed in solutions containing tetraethylammonium ions (TEA+).

Plateau potentials drive axonal impulse bursts in insect motoneurons

Intracellular recordings were made from the soma of the ‘fast’ coxal depressor motoneuron of the hind leg of the cockroach Periplaneta americana under current- and voltage-clamp. In response to depolarizing current injection, this neuron was able to generate plateau potentials that were able to far outlive the duration of the applied depolarization. These events constitute an inherent membrane property of the neuron because they could be evoked in somata that had been surgically isolated from other parts of the neuron (the soma is devoid of synaptic contacts); these experiments also showed that the soma of this neuron can participate in the generation of plateau potentials. The amplitude of these events at the spike-initiating zone was sufficient to evoke action potentials that correlated 1:1 with axonal impulses recorded extracellularly from the axon of the neuron. The ability to generate plateau potentials is associated with a region of negative slope resistance in the current-voltage relation of the neuron. Ion substitution experiments showed that the plateau potentials recorded from this neuron are calcium dependent. Our observations show for the first time that an insect motoneuron can produce plateau potentials. Accordingly, bistable membrane properties may play a role in shaping motor output in insects.

Octopaminergic modulation of synaptic transmission between an identified sensory afferent and flight motoneuron in the locust.

The role of the biogenic amine octopamine in modulating cholinergic synaptic transmission between the locust forewing stretch receptor neuron (fSR) and the first basalar motoneuron (BA1) was investigated. The amines 5‐hydroxytryptamine (5‐HT, serotonin) and dopamine were also studied. Bath application of octopamine, 5‐HT, and dopamine at concentrations of 10‐4 M reversibly decreased the amplitude of monosynaptic excitatory postsynaptic potentials (EPSPs) evoked in BA1 by electrically stimulating the fSR axon. These effects occurred without any detectable change in either input resistance or membrane potential of BA1. The amines also reversibly decreased the amplitude of responses to acetylcholine (ACh) pressure‐applied to the soma of BA1. The muscarinic antagonist scopolamine (10‐6 M) had no significant effect on the octopamine‐induced decrease in ACh responses. These observations suggest that these amines potentially could physiologically depress cholinergic transmission between fSR and BA1, at least in part, by altering nicotinic rather than muscarinic cholinergic receptor function. Although the octopaminergic agonists naphazoline and tolazoline both mimicked the actions of octopamine, the receptor responsible for octopamine‐mediated modulation could not be characterized since amine receptor antagonists tested on the preparation had complex actions. Confocal immunocytochemistry revealed intense octopamine immunoreactivity in the anterior lateral association center, thus confirming the presence of octopamine in neuropil regions containing fSR/BA1 synapses and therefore supporting a role for this amine in the modulation of synaptic transmission between the fSR and BA1. 5‐HT‐immunoreactivity, conversely, was concentrated within the ventral association centers; very little staining was observed in the dorsal neuropil regions in which fSR/BA1 synapses are located. J. Comp. Neurol. 462:55–70, 2003.

Muscarinic receptor activation modulates ligand-gated ion channels in an insect motoneuron via changes in intracellular calcium

An isolated ganglion preparation has been used to make recordings from an identified cockroach neuron in situ. We have shown that the magnitude of conductance increase of ligand-gated ion channels produced by agonists can be modulated by activation of muscarinic ACh receptors (mAChRs); currents through nicotinic ACh receptors (nAChRs) are suppressed, while GABA currents can be either enhanced or suppressed. mAChR activation causes a rise in [Ca2+]1measured using the fluorescent Ca2+ indicator fluo-3. This rise in [Ca2+]1 appears to trigger modulation of nicotinic ACh and GABA currents, since the effects of mAChR activation are mimicked by brief increases in [Ca2+]1 produced by flash photolysis of the Ca2+ chelator nitr-5. These modulatory effects outlast the rise in [Ca2+]j evoked by mAChR activation or photolytic Ca2+ release by 5—10 min. The prolongation by Li+ of the modulatory effects mediated by mAChRs suggests a possible involvement of the phosphoinositol pathway. These results indicate that mAChRs acting via intracellular messengers can exert a powerful modulatory influence upon nACh or GABA gated ion channels of neurons in which these receptors are co-localized.

Modulation by 5-hydroxytryptamine of nicotinic acetylcholine responses recorded from an identified cockroach (Periplaneta americana) motoneuron.

Recordings from the soma of the cockroach (Periplaneta americana) fast coxal depressor motoneuron (Df) were made while acetylcholine (ACh) was regularly pressure‐applied locally from a micropipette. The modulatory effects upon these nicotinic ACh responses of bath‐applied 5‐hydroxytryptamine (5‐HT, serotonin), dopamine and octopamine were investigated under either current‐clamp or voltage‐clamp conditions. The biogenic amines reversibly suppressed, but never totally abolished, ACh responses, 5‐HT being the most potent, with a threshold near 10−6 m (EC50 = 5 × 10−5 m). Occlusion experiments indicate that the amines share a common mechanism at the level of either receptors or second messenger pathways. The amines also modulated responses to nicotine or carbachol (each of which resists hydrolysis by acetylcholinesterases), indicating that the amines did not act by accelerating ACh degradation. Pharmacological antagonists were used in an attempt to characterize the receptor responsible for amine‐mediated modulation. Although a number of antagonists mimicked the action of amines rather than producing blockade, the antagonistic actions of LSD and RS23597 pointed strongly to a receptor‐mediated mechanism, but did not allow receptor identification. The magnitude of the modulatory effect of 5‐HT was significantly reduced by intracellular guanosine‐5′‐O‐(2‐thiodiphosphate) (GDP‐β‐S), indicating involvement of a G‐protein. Intracellular injection of the calcium chelator BAPTA did not block the modulatory effect of 5‐HT, showing that the amines do not operate through the calcium‐dependent pathway by which muscarinic receptors act on nicotinic currents. The adenylate cyclase inhibitor dideoxyadenosine (DDA), on the other hand, did attenuate the action of 5‐HT, suggesting involvement of cyclic AMP.

Ionic currents in the soma of an identified cockroach motoneurone recorded under voltage-clamp

1. Membrane currents have been recorded from the soma of a bifunctional basalar/coxal depressor motoneurone in the metathoracic ganglion of the cockroach (Periplaneta americana) using a two-electrode voltage-clamp technique. 2. This motoneurone cell body is normally inexcitable when studied under current-clamp. Appropriate depolarizing command steps evoke rapid transient outward currents and late outward currents. 3. Late outward currents are dominated by a Ca-dependent component that confers an N-shaped I-V relationship on the neurone. 4. The Ca-dependent outward current is suppressed by Cd2+ (1 mM), Mn2+ (5 mM) or verapamil (50 microM). 5. Externally applied tetraethylammonium ions (TEA+) (25 mM) block the Ca-dependent current, but also appear to suppress a component of the late outward current that is independent of Ca2+. 6. Aminopyridines cause only minor suppression of late outward currents, but shift the peak in the N-shaped I-V relationship to more negative potentials. 7. The reversal potential of tail currents recorded following pre-pulses to +50 mV were dependent upon the pre-pulse duration; increasing the duration from 10 to 50 msec caused a +17 mV shift in tail current reversal potential. 8. A five-fold increase in the K+ concentration of the solution bathing the preparation only produced small and inconsistent changes in the reversal potential of tail currents. 9. Five-fold reduction in external Cl- caused no change. 10. The dependence of tail current reversal potential upon pre-pulse duration and the limited effect of alterations in the composition of the bathing solution are discussed in the context of restricted ion movements near the external surface of the cell membrane.

Indirect phosphorylation-dependent modulation of postsynaptic nicotinic acetylcholine responses by 5-hydroxytryptamine.

Ionotropic nicotinic acetylcholine (ACh) receptors have been shown to be modulated by protein kinase‐mediated phosphorylation in vitro. Here we demonstrate that 5‐hydroxytryptamine (5‐HT) can downregulate postsynaptic nicotinic ACh responses, elicited in an identified arthropod motoneuron in situ, by a mechanism dependent on protein kinase activity. Serotonergic modulation can be mimicked by perfusion with membrane‐permeable analogues of either adenine (cAMP) or guanine (cGMP) cyclic nucleotides, and is prolonged in the presence of phosphodiesterase inhibitors. Furthermore, suppression of the ACh response by 5‐HT is blocked by specific competitive inhibitors of protein kinase A and G, as well as the broad specificity protein kinase inhibitor staurosporine. The protein phosphatase inhibitor cantharidin similarly blocks recovery of the ACh response from suppression mediated by 5‐HT. Thus, it appears that the nicotinic ACh response is modulated by a cAMP‐mediated phosphorylation‐dependent intracellular signalling pathway that is distinct from the direct block of mammalian nicotinic ACh receptors by 5‐HT previously reported in vitro.

Cyclic-AMP regulation of calcium-dependent K channels in an insect central neurone

In the cockroach fast coxal depressor motoneurone, either the muscarinic agonist McN-A-343 or dibutyryl cAMP (Db-cAMP) induced a reduction in voltage-dependent outward current. The response to McN is due to suppression of a calcium-dependent potassium current (IK,Ca) produced secondarily to a reduction in voltage-dependent calcium current (ICa). The response to Db-cAMP was investigated in order to establish whether cAMP might mediate the response to McN. ICa was suppressed by 3-isobutyl-1-methylxanthine (IBMX) but not by Db-cAMP. The effects of IBMX were therefore unlikely to be the result of phosphodiesterase inhibition. Since caffeine also suppressed ICa, the observed effect of IBMX is probably due to release of Ca2+ from intracellular stores. IK,Ca, evoked by injection of Ca2+, was reduced by Db-cAMP or forskolin but not by McN. These results indicate that the electrical response to McN in this neurone is not mediated by changes in cAMP.

Localization of biogenic amine-containing neurones in the ventral nerve cord of the cockroach (Periplaneta americana)

Abstract 1. A modification of the glyoxylic acid histochemical technique has been used to localize biogenic amines in whole-mount preparations of the cockroach ( Periplaneta americana ) ventral nerve cord. 2. Blue-green fluorescent neurones are found in characteristic locations within the suboesphageal, thoracic and abdominal ganglia. 3. The neuropiles of all ventral ganglia contain a dense network of varicose processes. 4. Fluorescent axons run in the interganglionic connectives; the only axons visible in peripheral nerves are a pair in each of the salivary duct nerves and one single faintly fluorescent fibre in each of the cereal nerves. 5. Orange-yellow fluorescence characteristic of indolealkylamines was absent froin thoracic and abdominal ganglia and only seen inconsistently in the suboesophageal ganglion, where it was weak and extremely labile. 6. Fluorescent somata and intraganglionic processes were absent after animals had been pretreated with reserpine. 7. Prior injection with the monoamine oxidase inhibitor nialamide and the DOPAmine precursor l -DOPA produced a modest enhancement in intensity of intraneuronal fluorescence.

Postembryonic development of rectifying electrical synapses in crayfish : physiology

SummaryIn a previous paper we showed that the ultrastructure of the giant fibre to motor giant synapse of crayfish changes in the first few weeks after hatching from having predominantly the appearance of a chemical synapse to having the appearance of an electrical synapse. This is parallelled by a behavioural change from non-giant fibre-mediated to giant fibre-mediated tailflips. In this paper we describe the physiology of the giant fibre to motor giant synapse over this period. We find the following: (1) The giant fibre to motor giant synapse usually transmits spikes 1∶1 from the day of hatching. (2) The synapse operates by electrical transmission from the day of hatching, when no connexons are apparent at the ultrastructural level. (3) The synapse has no detectable chemical component, even at an age when the predominant type of junctional apposition has the ultrastructural appearance of a chemical synapse. (4) Inhibitory chemical synapses occur onto the motor giant at the day of hatching, and these show similar physiological characteristics to those which occur onto the motor giant in adults. (5) In some preparations, the giant fibre to motor giant electrical synapse shows rectification similar to that in the adult, but in most cases both depolarizing and hyperpolarizing current injected into the medial giant spreads to the motor giant. (6) Current spread from the medial giant to the motor giant is increased by hyperpolarizing the motor giant neuron, even when medial giant to motor giant transmission is apparently non-rectifying. (7) Both the giant fibre and the motor giant have resting potentials of about −90 mV. There is no standing difference in resting potential as there is in the adult. This may explain the apparent lack of medial giant to motor giant rectification observed in most preparations.