This leads to exocytosis which releases the neurotransmitter acetylcholine from the synaptic vesicles into the synaptic cleft. Acetylcholine molecules then diffuse across the synaptic cleft and bind to ACh acetylcholine receptors in the membrane of the postsynaptic neuron. The binding opens ion channels, and the membrane depolarizes.
If this depolarization brings the initial segment of the postsynaptic neuron to threshold, it will result in an action potential. Individual giant interneurons can be identified by their unique morphological characteristics and localization [ 42 , 43 , 49 - 51 ].
Iontophoretic injection of ACh onto the finer branches of giant interneurons resulted in a dose-dependent depolarization of a giant interneuron accompanied by a decrease in membrane resistance [ 43 , 50 , 51 ]. It was interesting to note that the minimal quantity of ACh required to generate a depolarizing response of giant interneuron GI 1 was estimated to 3.
A similar action of ACh through activation of postsynaptic nAChRs could be also identified at the monosynaptic connections between the trochanteral hairplate afferents and motoneuron D s of the metathoracic ganglion [ 57 , 58 ], and in embryonic drosophila neurons in vitro [ 59 ].
In the conventional synapse, ACh released from the presynaptic vesicles, targets the postsynaptic nAChRs and opens ion gates. On the other hand, receptor sites were far from the release site. Terms of presynaptic receptors were defined as receptors at or near the nerve terminal Fig. In several studies, the sodium channel blocker, tetrodotoxin TTX has been used to define presynaptic nAChRs because in the presence of this toxin, action potentials arriving at the presynaptic terminals were eliminated [ 5 , 61 ].
In the chick midbrain, accessory motoneurons extend their axons to the ciliary ganglion where they terminate in large calyces on ciliary cells [ 61 - 63 ]. Bath application of nicotine induced inward currents in the calyces capable of generating action potentials APs.
The limited examples of excitatory or inhibitory ACh transmission through insect presynaptic nAChRs have prompted the hypothesis that the receptors serve other functions. Thus, intracellular microelectrode recording and ionophoretic application of carbamylcholine CCh were used to compare the cholinergic sensitivity of postsynaptic dendrites of giant interneuron 3 GI 3 with that of presynaptic cholinergic axon of the lateral filiform hair sensory neuron LFHSN in the first-instar cockroach Periplaneta americana [ 64 ].
From their studies, Blagburn and Sattelle [ 64 ] suggested that presynaptic nAChRs could be present in the axon membrane. They propose three possible locations of the ACh receptors on LFHSN: 1 postsynaptic to cholinergic input synapses onto the axon, 2 presynaptic to cholinergic output synapses made by the axon and 3 extrasynaptic [ 64 ].
Unfortunately, no clear evidence of insect presynaptic nAChRs has been demonstrated, and it was currently admitted that the postsynaptic neurons possess receptors that have an essentially nicotinic pharmacology mediating fast excitatory postsynaptic potentials EPSPs and that presynaptic receptors have only a muscarinic profile [ 46 , 65 , 66 ].
Evidence involving muscarinic receptors in synaptic function was obtained with the locust, Locusta migratoria , synaptosome preparation. It was found that these muscarinic receptors were similar to the mammalian M 2 subtype receptor [ 67 ].
Electrophysiological studies performed on cockroach cercal nerve-giant fiber preparation have identified two distinct muscarinic receptor subtypes. The first subtype was present on the cercal afferent terminal with a pharmacological profile similar to the vertebrate M2 receptor, and the second muscarinic receptor was found on the membrane of the postsynaptic giant interneurons [ 65 , 68 - 70 ].
It was suggested that the presynaptic muscarinic receptors acted as autoreceptors regulating the release of ACh [ 65 , 66 , 68 ] while postsynaptic muscarinic receptors reduced the giant fiber spike threshold [ 71 ].
Comparable studies have been performed in other insects such as the locust Schistocerca gregaria [ 66 ] and the tobacco hornworm, Manduca sexta [ 46 , 72 ].
The monosynaptic connection between sensory neurons and identified proleg motoneuron of the tobacco hornworm, Manduca sexta presents common characteristics with the cockroach cercal nerve-giant fiber.
In fact, sensory neurons associated with a planta hair send an axon into the ganglion of the same segment where the afferent terminals make synaptic contact with interneurons and motoneurons such as proleg motoneuron called PPR [ 46 , 72 ]. In the vertebrates, the availability of stable host cells expressing nAChR subtypes from humans or rats allowed further examination of nAChR pharmacology [ 73 ]. Thus, there was a strong correlation between native and expressed nAChR subtypes. Consequently, the minimum subunit combinations capable of forming functional receptors on expression systems have constrained views of the subunit composition of native neuronal nAChRs.
These results demonstrated that when experimental verification was possible, insect homomeric nAChRs could be pharmacologically distinct from vertebrate homomeric receptors. The most plausible explanation was given from vertebrate nAChRs obtained in transfected cell lines expressing either the human or rat nAChRs. It was observed that upregulation mechanisms differ from one expression system to another [ 82 - 86 ].
These mechanisms could alter functional expression of insect nAChRs in host cells. A second explanation could be that more complex subunit combinations are necessary because evidence for insect neuronal nAChR comprised of three subunits is accruing [ 87 , 88 ].
In all cases, contrary to vertebrate nAChRs, the pharmacological profile of insect nAChRs associated with subunit composition was difficult to perform through host cells. The initial experiments on native insect neurons were performed on the somata of neurons isolated on dorsal unpaired median DUM neurons of the grasshopper, Schistocerca nitens [ 89 , 90 ].
In line with this study, Lane et al. These first results suggested a difference between nAChRs expressed on both DUM neurons and motoneuron D f and likely that more than one type of nicotinic acetylcholine receptors was expressed in the isolated cells. In non-cholinergic neurons such as dopaminergic, GABAergic and glutamatergic neurons, several vertebrate pre-synaptic nAChRs have been identified with a modulatory role on neurotransmission [ 96 - ].
Moreover, it has been proposed that striatal muscarinic receptors mAChRs such as M2 and M4 receptors, by inhibiting ACh release from cholinergic interneurons, modify nAChR activity controlling DA release from dopaminergic neurons, suggesting that in other regions such as striatum and nucleus accumbens, there is evidence for presynaptic muscarinic receptors [ ]. Despite that vertebrate nAChR subunits have been found on the soma of DA neurons [ ] as well as on GABAergic terminals [ , ], there was no clear evidence of specific nAChR subunit on insect non-cholinergic neurons.
Physiological recordings in drosophila, locust and honeybee indicated that Kenyon cells KCs receive olfactory associative information directly from cholinergic projection neurons PNs located in the antennal lobes ALs and indirectly via GABAergic lateral horn neurons [ - ]. Moreover, drosophila neuronal cultures in which cholinergic and GABAergic synapses are functionally formed revealed that DA suppressed cholinergic synaptic currents [ ].
Putative locations of insect nicotinic receptors. A Olfactory sensory neurons OSN extend dorsally toward the brain and synapse in the glomerulus in the antennal lobe AL.
The cell body of projection neuron PN synapses with OSN in the antennal lobes and extend its axon dorsally to make synapses in the mushroom body calyx Ca. Neonicotinoid insecticides represent a relatively new group of chemicals that includes imidacloprid, thiamethoxam, clothianidin and acetamiprid. They are highly efficient in suppressing the overwhelming majority of crop pests.
It was known that the insecticidal activity of neonicotinoids is due to their agonist action on nicotinic receptors. At synaptic level, nenonicotinoid insecticides probably affect postsynaptic nAChRs [ , ] less than presynaptic receptors. In fact, bath application of clothianidin on giant interneuron synapses induced a strong depolarization which was not blocked by muscarinic antagonists suggesting that its effect occurred on postsynaptic nicotinic receptors [ ].
This synaptic effect could account for neonicotinoid symptoms described in the cockroach [ ]. In fact, Tan et al. The first subgroup includes molecules resulting in strong excitation symptoms with uncoordinated quivering, hyper-excitability and rapid spontaneous movements of cockroaches, while there was no excitation symptoms in the second subgroup [ ].
Although several electrophysiological studies have shown that neonicotinoid insecticides are likely to be low toxic in humans [ - ], several cases of acute poisoning have been associated with the development and the use of these insecticides [ , ]. They share a similar mechanism of toxicity and therefore presenting patients have comparable symptoms such as respiratory, gastrointestinal, cardiovascular and central nervous system effects [ ].
This apparent neonicotinoid toxicity, if associated with direct effect, suggests that the low affinity of these ligands to mammalians nAChRs must be clarified. In fact, we have recently found that thiamethoxam which was a poor agonist of insect nAChR on isolated cell bodies was able to generate a strong depolarization of the 6 th abdominal ganglion personal observation. This effect suggested that there was a distinct effect of thiamethoxam on nAChRs expressed on isolated cell bodies compared to the one expressed at synaptic level.
However, there were two striking differences. The first was that expressed insect neuronal nAChRs of matching subunit composition can differ markedly from the vertebrate nAChRs in showing lower or higher sensitivity. The existence of multiple classes of insect nAChR found on isolated cell bodies does not preclude the existence of these receptors at synaptic level.
This lack of formal studies allows us to consider that only muscarinic receptors were expressed at insect presynaptic site and consequently account for the modulation of ACh release. In fact, works on cockroach and locust have provided evidence for postsynaptic function of nicotinic receptors and a presynaptic function of muscarinic receptors. It was noted that these postsynaptic nAChRs can be modulated by both muscarinic cholinergic and serotoninergic pathways [ , ]. In this case, muscarinic pathway may act as a feedback mechanism to control ACh excitability to prevent excessive repeated depolarization but also regulate the actions of the inhibitory neurotransmitter GABA.
Considering studies from vertebrate nAChRs showing the involvement of these receptors on presynaptic level and that in all the insects species investigated, a much higher density of CNS nicotinic receptors is detected compared to muscarinic receptors; therefore it would be conceivable to suggest that insect nAChRs could account for the same complex processes at presynaptic site. In conclusion, insects provide material suited to developmental and genetic approaches to the study of nicotinic receptors, their involvement in learning and memory processes and the toxic effect of active compounds such as neonicotinoid insecticides.
This will help in providing new approaches to the chemical control of insect pests of agricultural, veterinary and medical importance. The development of novel nicotinic ligands for the treatment of cognitive deficits or as neonicotinoid insecticides depends on determining the differential roles of various nicotinic receptor subtypes. Key questions for future research: 1 Does insect presynaptic cholinergic neurons express nAChRs and if so, which subtypes?
In fact, nAChRs have also been identified on the cell bodies of several insects [ 28 ] such as honeybee KCs and ALs [ , , , ].
Authors would like to thanks Dr. Monique Gauthier for their help during the manuscript process. National Center for Biotechnology Information , U. Journal List Curr Neuropharmacol v. Curr Neuropharmacol. Author information Article notes Copyright and License information Disclaimer. This article has been cited by other articles in PMC. Abstract Acetylcholine ACh is probably the oldest signalling neurotransmitter which appeared in evolution before the nervous system.
Keywords: Nicotinic acetylcholine receptors, pharmacology, insect, mammal, synapse. Open in a separate window. The action of cholinomimetic substances on impulse conduction in the habenulointerpeduncular pathway of the rat in vitro.
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Chicken neuronal acetylcholine receptor alpha 2-subunit gene exhibits neu-ron-specific expression in the brain and spinal cord of transgenic mice. Differential expression of the alpha 9 nicotinic acetylcholine receptor subunit in neonatal and adult cochlear hair cells. Ultrastructural localization of the alpha4-subunit of the neuronal acetylcholine nicotinic receptor in the rat substantia nigra. Alpha-7 nicotinic receptor expression by two distinct cell types in the dorsal raphe nucleus and locus coeruleus of rat.
Comparative distribution of nicotinic receptor subtypes during development, adulthood and aging: an autoradiographic study in the rat brain. Brain and muscle nicotinic acetylcholine receptors : a gene family. ACh binds to the two a subunits.
The bottom half shows the molecular structure of each a subunit of the nicotinic receptor based on cDNA derived amino acid sequence.
A funnel-shaped internal ion channel is surrounded by the five subunits. Muscarinic receptors, classified as G protein coupled receptors GPCR , are located at parasympathetic autonomically innervated visceral organs, on the sweat glands and piloerector muscles and both post-synaptically and pre-synaptically in the CNS see Table I. The muscarinic receptor is composed of a single polypeptide. Because each of these regions of the protein is markedly hydrophobic, they span the cell membrane seven times as depicted in Figure The fifth internal loop and the carboxyl-terminal tail of the polypeptide receptor are believed to be the site of the interaction of the muscarinic receptor with G proteins see right.
The site of agonist binding is a circular pocket formed by the upper portions of the seven membrane-spanning regions. ACh has excitatory actions at the neuromuscular junction, at autonomic ganglion, at certain glandular tissues and in the CNS. It has inhibitory actions at certain smooth muscles and at cardiac muscle.
The biochemical responses to stimulation of muscarinic receptor involve the receptor occupancy causing an altered conformation of an associated GTP-binding protein G protein. In response to the altered conformation of the muscarinic receptor, the a subunit of the G protein releases bound guanosine diphosphate GDP and simultaneously binds guanosine triphosphate GTP. This hydrolysis terminates the action of the G protein.
The rate of hydrolysis of the GTP thus dictates the length of time the G protein remains activated. Inhibition of Adenylate Cyclase: The muscarinic receptor, through interaction with an inhibitory GTP-binding protein, acts to inhibit adenylyl cyclase.
Reduced cAMP production leads to reduced activation of cAMP-dependent protein kinase , reduced heart rate, and contraction strength. As shown in Figure The DAG activates protein kinase C not shown. Cellular responses are influenced by PKC's phosphorylation of target proteins. This conductance increase increases the resting membrane potential in myocardial and other cell membranes leading to inhibition. ACh binds only briefly to the pre- or postsynaptic receptors.
Following dissociation from the receptor, the ACh is rapidly hydrolyzed by the enzyme acetylcholinesterase AChE as shown in Figure This enzyme has a very high catalysis rate, one of the highest known in biology. AChE is synthesized in the neuronal cell body and distributed throughout the neuron by axoplasmic transport. AChE exists as alternatively spliced isoforms that vary in their subunit composition.
The variation at the NMJ is a heteromeric protein composed of four subunits coupled to a collagen tail that anchors the multi-subunit enzyme to the cell membrane of the postsynaptic cell Figure This four-subunit form is held together by sulfhydryl bonds and the tail anchors the enzyme in the extracellular matrix at the NMJ. Other isoforms are homomeric and freely soluble in the cytoplasm of the presynaptic cell.
In addition, other cholinesterases exist throughout the body, which are also able to metabolize acetylcholine. These are termed pseudocholinesterases. Drugs that inhibit ACh breakdown are effective in altering cholinergic neurotransmission. In fact, the irreversible inhibition of AChE by isopropylfluoroesters are so toxic that they can be incompatible with life—inhibiting the muscles for respiration.
This inhibition is produced because ACh molecules accumulate in the synaptic space, keep the receptors occupied, and cause paralysis. Two notable examples are insecticides and the gases used in biological warfare. The mechanism of action of these irreversible inhibitors of AChE is that they carbamylate the AChE, rendering it inactive.
The carbamylation inactivates both the acetyl and choline binding domains. A recently developed antidote to these inhibitors cleaves the nerve gas so that it will dissociate from the AChE. In contrast to the irreversible inhibitors, the reversible AChE inhibitors are effective in transiently increasing the ACh level and are effective in diseases and conditions where an increased ACh level is desired.
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