Involvement of the cholinergic mechanism in DDT and dieldrin action in the insect central nervous system

The effects of DDT and dieldrin on cholinergic neurotransmission were studied using the sixth abdominal ganglion of the cockroach. Spontaneous electrical discharges in the ganglion recorded with an extracellular electrode were augmented by 0.1 mM DDT and 1 microM dieldrin. This stimulating action was partly blocked by 0.5 mM d-tubocurarine and 0.1 mM hemicholinium-3 and disappeared in a high Mg2+-low Ca2+ medium. DDT and dieldrin increased both ACh release and ACh content in the ganglion. These results suggested that DDT and dieldrin stimulate both ACh release and synthesis.     

Ethylcholine mustard aziridinium blocks the axoplasmic transport of acetylcholinesterase in cholinergic nerve fibres of the rat

A cholinotoxin, ethylcholine mustard aziridinium ion, (AF64A) specifically and irreversibly blocks the intraaxonal transport of acetylcholinesterase in the rat. Impairment of the transport of this enzyme in the septo-hippocampal cholinergic fibres and in the sciatic nerve has been studied, using different doses of AF64A. It is demonstrated that the effect on the axonal transport is dose-dependent, but is not related to the mode of drug application. AF64A thus may exert its neurotoxic effects on cholinergic neurons at several target sites of action. In addition to the localized presynaptic mechanisms, it may also be compromising cholinergic function by inhibiting axonal transport in vivo.     

Ethylcholine mustard aziridinium blocks the axoplasmic transport of acetylcholinesterase in cholinergic nerve fibres of the rat

Summary A cholinotoxin, ethylcholine mustard aziridinium ion, (AF64A) specifically and ireversibly blocks the intraaxonal transport of acetylcholinesterase in the rat. Impairment of the transport of this enzyme in the septo-hippocampal cholinergic fibres and in the sciatic nerve has been studied, using different doses of AF64A. It is demonstrated that the effect on the axonal transport is dose-dependent, but is not related to the mode of drug application. AF64A thus may exert its neurotoxic effects on cholinergic neurons at several target sites of action. In addition to the localized presynaptic mechanisms, it may also be compromising cholinergic function by inhibiting axonal transport in vivo.     

Selective Presynaptic Cholinergic Neurotoxicity Following Intrahippocampal AF64A Injection in Rats

Compound AF64A, ethylcholine mustard aziridinium ion (0.4-8 nmol) was stereotaxically administered into rat dorsal hippocampus, and neurochemical changes were determined 5 days later. AF64A treatment, over an almost 10-fold dose range, resulted in a significant (up to 70%) decline in choline acetyltransferase activity. In the same tissue samples, Na+-dependent choline transport activity was also lowered, with most decreases ranging between 10 and 50% of controls; however, there was no significant correlation (r = 0.39) between these two parameters. Acetylcholinesterase activity was not affected by AF64A treatment when assayed by either histochemical or enzymatic methods. AF64A reduced acetylcholine levels by 43%, but did not alter norepinephrine content or serotonin uptake. These results demonstrate that AF64A can induce a specific, long-term reduction of cholinergic presynaptic biochemical markers in rat hippocampus. Thus, AF64A can serve as a useful new tool to study the cholinergic system and as an important agent to help develop animal models representing disorders of central cholinergic hypofunction.     

The pharmacology of an insect ganglion: actions of carbamylcholine and acetylcholine.

1. Methods for presenting dose-response data for the ganglionic actions of cholinergic agonists (e.g. carbamylcholine) are compared, using the mannitol-gap technique for electrophysiological recording of synaptic events at the cercal nerve, giant fibre synapse of the sixth abdominal ganglion of the cockroach Periplaneta americana. 2. At concentrations around 10(-5)M, carbamylcholine has no effect on ganglionic polarization but potentiates the monosynaptic EPSP. At 10(-4)M and higher concentrations, ganglionic depolarization is accompanied by a reduction of EPSP. 3. Pretreatment with eserine (10(-6) M) considerably shifts the dose-response curve for acetylcholine so that synaptic transmission is consistently sensitive to 10(-6) M acetylcholine.     

The effect of 4-aminopyridine of the synaptic activity in the last abdominal ganglion of the cockroach (Periplaneta americana)]

Synaptic action of 4-aminopyridine (4-AP) has been studied in the sixth abdominal ganglion of the cockroach. Our results show that 4-AP has a strong excitatory effect on the ganglion and its seems that it depolarizes as well the pre and postsynaptic membranes. The threshold concentration is about 10(-5) M.     

AF64A: An Active Site Directed Irreversible Inhibitor of Choline Acetyltransferase

Ethylcholine mustard aziridinium ion (AF64A, MEChMAz) has been proposed as a cholinergic neuron-specific neurotoxin. We report that in further studies on its mechanism of action incubation of the cholinergic neuroblastoma X glioma cell line, NG-108-15, with 100 microM AF64A resulted in a rapid decrease in cellular choline acetyltransferase (ChAT) activity which preceded cytotoxicity. Thus, a 60-85% decrease in ChAT activity was measured within 5 h of AF64A exposure, whereas cell lysis (measured as the release of the cytosolic enzyme lactate dehydrogenase into the medium) did not become apparent until 18 h of AF64A exposure. This led us to examine the effects of AF64A on partially purified ChAT. We report a concentration- and time-dependent inhibition of partially purified ChAT by AF64A that could not be reversed by dialysis but could be prevented by coincubation of the enzyme and AF64A with choline but not with acetyl-coenzyme A. We present kinetic evidence that choline and AF64A compete for the same site on the enzyme. In addition, thiosulfate, which inactivates the aziridinium ion, eliminated AF64A's capacity to inhibit the enzyme. AF64A also irreversibly inhibited partially purified choline kinase and acetylcholinesterase but not lactate dehydrogenase, alcohol dehydrogenase, carboxypeptidase A, or chymotrypsinogen, enzymes that do not use choline as a substrate or product. Thus, the data suggest that AF64A acts as an irreversible active site directed inhibitor of ChAT and possibly other enzymes recognizing choline.     

Immunocytochemical localization of nicotinic acetylcholine receptors in the terminal abdominal ganglion of the cockroach (Periplaneta americana)

A polyclonal, monospecific antiserum raised against a nicotinic acetylcholine receptor protein affinity-purified from insect nervous tissue, was employed to demonstrate the localization of antigenic sites in the neuropile of the terminal (sixth) abdominal ganglion of the cockroach Periplaneta americana. In agreement with previously published autoradiographic mapping of specific [125I]alpha-bungarotoxin binding sites, specific areas of the central neuropile of this ganglion were densely stained, but not the cercal afferent axons. No staining was detected corresponding to the dense, peripheral, partly non-specific binding of alpha-bungarotoxin seen in autoradiographs of the same tissue. Certain peripherally located neuronal cell bodies, including the cell body of giant interneuron 2, contained intracellularly located antigenic sites.     

Ionophoretic application of acetylcholine on to the dendrites of an identified giant interneurone (GI 1) in the cockroach Periplaneta americana

Acetylcholine was ionophoretically applied within the neuropile of the sixth abdominal ganglion of the cockroach Periplaneta americana and depolarizing responses were recorded from an identified giant interneurone (GI 1) using the oil-gap, single-fibre technique. Estimated peak concentrations in the range 1.0 × 10^?11M ? 1.0 × 10^?8 M acetylcholine were present at the receptors, when this putative neurotransmitter was ionophoretically-applied, at threshold doses, to regions of the neuropile containing dendritic branches of GI 1. Using a stepping micro-drive unit for positioning the ionophoretic micropipette, it was shown that profiles of acetylcholine sensitivity correlated with the distribution of dendritic branches of GI 1 revealed by cobalt staining of the cell under test. It is concluded that acetylcholine receptors are widely distributed throughout the dendritic branches of GI 1 in the sixth abdominal ganglion. The results demonstrate the possibility of investigating the effects of locally-applied putative transmitter molecules and pharmacological agents on synaptic membranes of an identified neurone in the central nervous system of an insect.     

Reversible and Irreversible Inhibition of High-Affinity Choline Transport Caused by Ethylcholine Aziridinium Ion

The effect of ethylcholine aziridinium ion (AF64A) on choline transport in hippocampal, striatal, and cerebrocortical synaptosomes was studied. Synaptosomes prepared from these three brain regions were equally sensitive to AF64A. Low concentrations of AF64A produced a reversible inhibition (IC50 values = 1.35-2.25 microM), whereas higher concentrations produced an irreversible inhibition (IC50 values = 25-30 microM), which started as competitive. The irreversible component of the inhibition was independent of extracellular Na+ concentration, a finding suggesting that the choline transporter is alkylated at its outward position. The kinetics of the inhibition were rapid and similar in the three brain regions examined. The high-affinity choline transport was more sensitive to the toxin than the low-affinity choline transport. Based on these results, we propose a kinetic model that explains the reversible and the irreversible inhibitions induced by AF64A. The possible relationships between the concentrations that in vitro produce reversible and irreversible inhibition and those that in vivo produce selective and nonselective cholinergic hypofunction are discussed.     

A histochemical and electrophysiological study of the action of diazoxon on cholinesterase activity and nerve conduction in ganglia of the cockroach Periplaneta americana L

When sixth abdominal or metathoracic ganglia of the cockroach Periplaneta americana L. were irrigated continuously with diazoxon (O, O-diethyl O-(2-isopropyl-6-methyl-4-pyrimidinyl) phosphate) solution in situ, the log. of the time required to block conduction in certain nerve pathways in the ganglia was directly proportional to the log. of the concentration of diazoxon applied. Inhibition of cholinesterase began peripherally before function was affected, and had begun to affect the neuropile by the time that conduction was first blocked. Longer exposure to diazoxon disrupted nerve function even more, especially in the sixth abdominal ganglion, and inhibited more cholinesterase, but much longer exposure was needed to inhibit nearly all the cholinesterase. Irrigation with saline, begun when block first occurred, failed to restore completely either nerve function or cholinesterase activity. The cholinesterase activity of ganglia from cockroaches treated topically with an LD90 of diazoxon and examined at intervals after treatment decreased steadily to a level similar to that of ganglia treated directly with diazoxon until conduction was just blocked, but rarely became less, even in moribund insects. Nerve function in metathoracic ganglia became badly affected and remained so in all cockroaches that failed to recover, but sixth abdominal ganglia, though usually badly affected for a time, always recovered normal function, even in prostrate cockroaches. The condition of a poisoned insect, therefore, corresponded much more closely to the functional condition of the metathoracic ganglion than to that of the sixth abdominal ganglion. Applying the insecticide close to a ganglion advanced the time of onset of symptoms but affected the final outcome very little. It was calculated that the highest concentration of diazoxon in the haemo-lymph in contact with the nervous systems of cockroaches treated topically with LDgo's of diazoxon was about 10^-5M.     

Serial synapses in Aplysia

Serial synapses occur between small profiles in the neuropil of Aplysia abdominal ganglion. Material was fixed in phosphate buffered OsO₄, embedded in epon, and sections were stained with uranyl acetate and lead citrate. A class of synapses had the following characteristics: (1) synaptic vesicles clustered against the presynaptic membrane, (2) a widened extracellular space of about 20 nm containing electron-dense material, (3) straightening of the pre- and postsynaptic membranes, and (4) no postsynaptic membrane specialization. Some density between the presynaptic membrane and the adjacent synaptic vesicles was occasionally observed. Synapses occurred between small profiles in the neuropil (typical profile diameters were 1–3 m?m). In this sample of approximately 100 synapses, four serial synapses were identified. The serial synaptic profiles were all small. In addition to the finding of serial synapses, 40% of the postsynaptic profiles contained vesicles similar to the synaptic vesicles seen in presynaptic profile. Serial synapses may be the anatomical substrate of presynaptic inhibition and facilitation and of dishabituation.     

Immunocytochemical staining of central neurones in Periplaneta americana using monoclonal antibodies to choline acetyltransferase

Immunocytochemical mapping of cholinergic neurones in the CNS of the cockroach Periplaneta americana has been attempted using monoclonal antibodies to choline acetyltransferase (ChAT, acetyl-CoA: choline O-acetyltransferase, EC 2.3.1.6). Monoclonal antibodies 11 255 and 1E6 raised against rat brain ChAT and 1C8 raised against Drosophila melanogaster ChAT were ineffective in staining Periplaneta neurones. However, the cytoplasm of certain neuronal cell bodies was stained by monoclonal antibody 4D7 prepared against rat ChAT. Staining of cell bodies by 4D7 was enhanced following in vivo pre-treatment with colchicine. The staining of specific neurones by monoclonal antibody 4D7 indicates that these cockroach cells are rich in a protein with antigenic determinants resembling those of vertebrate ChAT. For some unidentified neurones, 4D7 staining is associated with the presence of acetylcholinesterase indicating that this monoclonal antibody offers a probe for mapping cholinergic neurones in the CNS of Periplaneta americana. The fast coxal depressor motoneurone (D(f)) was not stained by monoclonal antibody 4D7. Some neuronal processes in the sixth abdominal ganglion, and sensory cell bodies in the cerci were lightly stained by monoclonal antibody 4D7 following pre-injection of animals for 36 hr with colchicine.     

Neurotoxic and haemolytic activity of a protein isolated from Bacillus thuringiensis var. israelensis crystals

Abstract Two proteins (protein A and B) were isolated from Bacillus thuringiensis var. israelensis crystals using ion-exchange chromatography. Protein A was found to have haemolytic and neurotoxic activities. Inhibition of nerve conduction in the sixth abdominal ganglion of the American cockroach was observed following application of protein A. Protein A was found to be haemolytic to human and rabbit erythrocytes. Protein B did not exhibit any of these activities.     

Effect of Nerve Growth Factor in Ethylcholine Mustard Aziridinium (AF64A)-Treated Rats: Sensitization of Cholinergic Enzyme Activity in the Septohippocampal Pathway

Abstract: In this study, we examined the effects of nerve growth factor (NGF) administration on cholinergic enzyme activity in both normal and ethylcholine mustard aziridinium (AF64A)-treated rats. Choline acetyltransferase (ChAT) and acetylcholinesterase activity were measured in the hippocampus and septum of rats chronically administered NGF (0.36–2.85 µg/day) into the lateral ventricle for 14 days. In both normal and AF64A-treated rats, NGF increased cholinergic enzyme activity in a dose-dependent manner. Furthermore, although NGF increased ChAT activity in normal rats by 147%, it had a greater effect in AF64A-treated rats, increasing ChAT activity as much as 273%. NGF increased acetylcholinesterase activity in normal rats by only 125% but produced a 221% increase in this activity in AF64A-treated rats. These data indicate that AF64A produces an increased sensitivity to NGF in cholinergic neurons.     

Threonine6-bradykinin in the venom of the wasp Colpa interrupta (F.) presynaptically blocks nicotinic synaptic transmission in the insect CNS

1. The venom of the solitary scoliid wasp Colpa interrupta (F.) shows a kinin-activity, when tested on a cascade of mammalian smooth muscle preparations, and, in addition, a contraction of the rat colon. 2. The venom also irreversibly blocks the nicotinic synaptic transmission from the cercal nerve to a giant interneuron in the sixth abdominal ganglion of the cockroach, Periplaneta americana. 3. The same activities have been found within one HPLC fraction. 4. However, rechromatography of this fraction resulted in four subfractions being active on smooth muscles. 5. One fraction caused contraction of the colon, three other fractions contained kinin-activity. 6. Only the most active kinin fraction blocked synaptic transmission in the insect CNS. 7. This fraction contained threonine-bradykinin. 8. Synthetic Thr-bradykinin causes irreversible presynaptic activation-induced block of transmission in the insect CNS.     

Vasoactive intestinal peptide (VIP)-like immunoreactivity in the human sympathetic ganglia

Vasoactive intestinal peptide immunoreactive (VIP-IR) nerve fibres and terminals, neurons and small granule containing cells were observed in human lumbal sympathetic ganglia. Electron-microscopically VIP-IR was localized in the large dense-cored vesicles in nerve terminals and on the membranes of the Golgi complexes in the neurons. A small population of principal ganglion cells was surrounded by VIP-IR nerve terminals. Most of these neurons contained acetylcholinesterase (AChE) enzyme but were not tyrosine hydroxylase-immunoreactive (TH-IR). All VIP-IR ganglion cells and most of the nerve fibres contained AChE but not TH-IR. It appears that in human sympathetic ganglia VIP is localized in the cholinergic neurons and nerve fibres and that the VIP-IR nerve terminals innervate mainly the cholinergic subpopulation of the sympathetic neurons.     

Effects of hypoxia on resting potential and transmitter release at cercal afferent,giant interneurone synapses in the cockroach,Periplaneta americana L.

• 1.1. Studies of experimentally induced hypoxia were carried out on synapses in the isolated sixth abdominal (A6) ganglion of the cockroach using electrophysiological methods.
• 2.2. During a break of saline superfusion, oxygen tension (PO₂) decreased and depolarization of presynaptic and postsynaptic membranes was observed.
• 3.3. During hypoxia EPSP amplitude initially increased, but decreased after a few minutes.
• 4.4. Changes in the EPSP and membranes potential resulting from hypoxia were reversed when the saline superfusion was restarted.
• 5.5. Changes in the amplitude of both the EPSP and the depolarization induced by microiontophoretic injections of ACh indicated that ACh release initially increased during hypoxia and decreased during recovery from oxygen deprivation.

     

Maintenance of GABA receptor function of small-diameter cockroach neurons by adenine nucleotides

Small diameter (<20 microm) neurons from the sixth abdominal ganglion of the American cockroach, Periplaneta americana, were enzymatically isolated and responses to exogenously applied gamma-aminobutyric acid (GABA) were recorded using the whole-cell patch clamp technique. With a minimal intracellular medium, responses to repeated applications of GABA decreased to zero within a few minutes. The rate of rundown of GABA responses was decreased by the intracellular inclusion of the phosphatase inhibitors microcystin and okadaic acid, suggesting that phosphorylation is necessary for the maintenance of cockroach GABA receptor function. ATP (5 mM) prevented GABA response rundown. ADP (5 mM) also slowed GABA response rundown, but responses stabilized at a level about half that seen with ATP. In the presence of protein kinase A inhibitory peptide (PKI), ATP was only as efficacious as ADP in slowing rundown. PKI had no effect on the ability of ADP to slow rundown, suggesting that the beta-phosphate of ADP is not involved in PKA-dependent phosphorylation of the GABA receptor. These results suggest that in cockroach neurons, GABA receptor function is maintained intracellularly by adenine nucleotides, not only by phosphorylation, but also possibly by an interaction with a nucleotide recognition site unrelated to PKA-dependent phosphorylation.     

Effects of the venom of Philanthus triangulum F. (Hym. sphecidae) and β- and δ-philanthotoxin on axonal excitability and synaptic transmission in the cockroach CNS

This paper provides answers to the questions which of the toxins present in the venom of the wasp Philanthus triangulum may be responsible for the previously reported blockage of transmission through the sixth abdominal ganglion of the cockroach, and whether this may occur by block of synaptic transmission or by affecting axonal exitability. In current clamp experiments the crude venom induces a slight depolarization of the membrane of the giant axon from the sixth abdominal ganglion of the cockroach and a small and irreversible decrease in the amplitude of the action potential. These marginal effects are not seen with relatively high concentrations of the philanthotoxins β-PTX and δ-PTX. It appears that neither the crude venom nor the toxins significantly affect the excitability of the cockroach giant axon. At a concentration of 20 μg ml^?1 δ-PTX causes a slowly reversible block of synaptic transmission from the cercal nerve XI to a giant interneuron without any change in resting membrane potential, whereas β-PTX is inactive. Iontophoretically evoked acetylcholine potentials of the giant neuron are more sensitive to δ-PTX than excitatory postsynaptic potentials. This suggests that the toxin acts on the postsynaptic membrane.