Eserine Sulfate Research Articles

Cholinesterase Activity in the Head of Wild Poecilia reticulata from Bahia, Brazil: Biochemical Characterization, Effects of Sample Storage and Normal Range of Activity

In this study, the soluble cholinesterases (ChE) from the head of Poecilia reticulata from a wild population of Bahia, Brazil were characterized using different substrates (acetylthiocoline, butyrylthiocoline and proprionylthiocoline) and selective inhibitors (eserine sulphate, iso-OMPA and BW284C51). Possible effects of time (1, 2, 3, and 4 weeks) and different storage temperatures (freezer storage at ca. –20ºC and –50ºC, and liquid nitrogen system at –196ºC) on ChE activity were also investigated, together with the normal range of ChE activity of non-exposed individuals. The results for the enzymatic characterization indicate that the enzyme present in the head of P. reticulata was mainly acetylcholinesterase. The mean and standard deviation of activity found in non-exposed wild males collected in different periods of the year were 149.71 ± 7.72 SD U/mg protein, respectively. ChE activity significantly decreased after the seventh day of sample storage, independently of the temperature.

Characterization and Malathion Degradability of Carboxylesterase in Wheat Kernels

Malathion residue in wheat kernels is enzymatically degraded into malathion monocarboxylic acids during sample preparation for pesticide residue analysis by the Japanese official method. To investigate whether the hydrolyzing enzyme is identical to carboxylesterase (CE), we compared the effects of various inhibitors against CE activity using p-nitrophenyl acetate as substrate with that against malathion-hydrolyzation activity. Although neither CE nor malathion-hydrolyzation activities were affected by EDTA, they were irreversibly suppressed by several serine esterase inhibitors and reversibly inhibited by cholinesterase inhibitor or sulfhydryl compounds. These inhibitions suggested that characteristics of both enzymes in wheat kernels were close to that of cholinesterase, though the enzymes were not able to be exactly classified by a classification method for mammals. When native polyacrylamide gel electrophoresis (PAGE) with esterase-zymography was performed with the addition of eserine sulfate into the sample and deoxycholic acid into the cathode buffer to prevent aggregation of CE isozymes, three clear bands of the isozymes were observed. These isozymes were confirmed by hydrophobic interaction chromatography (HIC). When malathion was reacted with each isozyme partially purified by chromatography techniques and native PAGE, malathion α-monocarboxylic acid as a major metabolite and malathion β-monocarboxylic acid as a minor metabolite were produced. These results suggested that all isozymes in wheat kernels responsible for the degradation of residual malathion in the kernels, though there are differences among malathion degradability by the isozymes.

Cholinesterase activity in Gammarus pulex (Crustacea Amphipoda): Characterization and effects of chlorpyrifos

The aim of this study was to characterize cholinesterase (ChE) activity in Gammarus pulex, an abundant and ecologically relevant species of the European stream environment. Biochemical and pharmacological properties were tested using different substrates (acetylthiocholine iodide, propionylthiocholine iodide and butyrylthiocholine iodide) and selective inhibitors (eserine sulfate, BW284c51 and iso-OMPA). In a second part, the in vitro and in vivo effects of a widely used organophosphorous pesticide, chlorpyrifos, on ChE activity were investigated. The results suggest that G. pulex possess only one ChE which displays the typical properties of an acetylcholinesterase, since: (1) it hydrolyses to the substrate acetylthiocholine at a higher rate than all other tested substrates and (2) it is highly sensitive to eserine sulphate and BW284c51, but not to iso-OMPA. In vitro and in vivo inhibitions were observed for highly different contamination levels, which suggests that bioaccumulation and biotransformation mechanisms are involved. In vivo AChE inhibition was observed at realistic environmental concentrations, with lethal effects appearing at inhibitions higher than 50%. The results of this study show the value of G. pulex as a sentinel organism for environmental assessment.

Baculovirus Expression of <I>Bm</I>AChE3, a cDNA Encoding an Acetylcholinesterase of <I>Boophilus microplus</I> (Acari: Ixodidae)

The complete cDNA sequence encoding a Boophilus microplus (Canestrini) (Acari: Ixodidae) acetylcholinesterase (AChE3) was expressed in the baculovirus system. The recombinant AChE3 protein (rBmAChE3) was secreted as a soluble form into the cell culture medium and was identified as a functional AChE by substrate specificity and by inhibition with the AChE-specific inhibitors eserine sulfate and BW284c51. Inhibition kinetics of rBmAChE3, in the presence of the organophosphate paraoxon, revealed sensitivity comparable with that of adult, organophosphate-susceptible neural AChE. To our knowledge, this is the first report of the cloning and successful expression of a functional ixodid AChE.

Characterization of Esterases in a Brazilian Population of Zaprionus Indianus (Diptera: Drosophilidae)

The aim of this study was to characterize esterases in Zaprionus indianus, a drosophilid recently introduced into Brazil. A further aim was study the variation of activity of esterases in the presence of inhibitors and their expression according to sex, sexual activity and age of individual flies. Polymorphisms were detected in two esterase loci (Est-2 and Est-3) and monomorphisms in four others (Est-1, Est-4, Est-5 and Est-6). Biochemical tests using alpha- and beta-naphthyl acetate and the inhibitors malathion, eserine sulphate and PMSF allowed us to classify EST-2 and EST-5 as beta-esterases, both carboxyl-esterases, and EST-1, EST-3, EST-4 and EST-6 as alpha-esterases. EST-1 and EST-3 were classified as carboxyl-esterases and EST-4 and EST-6 as cholinesterases. EST-5 activity was more pronounced in males and EST-2 was restricted to them or to recently copulated females. EST-4, rarely detected, was not characterized. Based on their biochemical characteristics possible roles for these enzymes are suggested.

Esterase profile in a pyrethroid-resistant Brazilian strain of the cattle tick Boophilus microplus (Acari, Ixodidae)

The cattle tick Boophilus microplus causes great damage in livestock and is considered one of the most important tropical ectoparasites. The traditional method of control is based on the intensive use of pesticides, however the indiscriminate use of these compounds over the years has led to the selection of resistant ticks. Hydrolases, particularly esterases (EST), have been reported to be associated with acaricide resistance in B. microplus. We compared the esterase profile of susceptible and cypermethrin-resistant strains of adult B. microplus and a pyrethroid susceptible reference strain (the Mozzo strain) using polyacrylamide gel electrophoresis and specific staining. The electrophoretic profiles of protein extracts revealed the presence of four regions with esterase activity in the cypermethrin-resistant strain and three of these regions in the susceptible strains. The bands were numbered EST-1 to EST-4 in sequence (starting from the anode) according to their decrease in negative charge. The EST-1A and EST-1B enzymes were detected only in the resistant strain. The inhibition studies with eserine sulfate, copper sulfate, p- p-chloromercuribenzoate (pCMB), malathion and phenylmethylsulfonyl fluoride (PMSF) indicated that the EST-1A and EST-1B enzymes belong to the acetylcholinesterase class and are probably associated with resistance to acaricides in this Brazilian resistant strain of B. microplus.

Effects of methamidophos on acetylcholinesterase activity, behavior, and feeding rate of the white shrimp ( Litopenaeus vannamei)

The toxicity of methamidophos on the shrimp Litopenaeus vannamei was evaluated using acetylcholinesterase (AChE) activity, behavior, and feeding rate as effect criteria. The biochemical characterization of the soluble cholunesterase (ChE) present in both muscle and eye tissues of L. vannamei was performed in a first phase of the study. In both tissues, almost full inhibition of enzyme activity by eserine sulfate was found, indicating that the measured activity is mainly from ChE and not from other esterases. The highest rate of substrate hydrolysis was found when acetylthiocholine was used as substrate. To evaluate the effects of methamidophos on L. vannamei AChE, behavior, and feeding rate, shrimps were exposed for 24 h to several sublethal concentrations of methamidophos. Significant effects of the pesticide on behavior and AChE were found, with behavior being a more sensitive endpoint than AChE inhibition. Feeding rate was not a sensible endpoint under conditions tested.

Cholin- and carboxylesterase activities in developing zebrafish embryos ( Danio rerio) and their potential use for insecticide hazard assessment

Insecticides are a potential hazard for non-target organisms like fish particularly at run off events. The study of effects to embryos of the zebra fish Danio rerio is already an accepted tool in waste water monitoring, but effects of various groups of substances (like most pesticides) to the zebrafish embryo remain to be studied. Enzymes are often taken as biomarkers of exposure and effect. Therefore cholinesterase isozymes and carboxylesterase were examined for their suitability as biomarkers of insecticide exposure. The activities of cholinesterase and of carboxylesterase were monitored in the first 48 h post-fertilization (hpf) of zebrafish development. Significant specific activities in the range of 0.5–25 U could be measured from the sixth somite stage (12 h) up to the Long Pec stage (48 h) for different cholinesterases using acetyl-, acetyl-β-methyl-, butyryl- and propionylthiocholin as substrates. The specific activity of carboxylesterase ranged from 4 to 16 U mg −1 protein in the respective developmental stages. Substrate specificity was analysed using specific inhibitors (eserine sulphate, DPDA, BW284c51). The results showed that the observed cholinesterase activities in the whole embryo may be attributed mainly to acetylcholinesterase with a partial capability to use propionylthiocholine as a second substrate. The potential use of cholin- and carboxylesterase as biomarkers was investigated using the organophosphate paraoxon-methyl. A 40% inhibition of enzyme activities was reached by 0.4 μM paraoxon-methyl indicating the possible use of these enzymes as biomarkers of exposure.

Characterization of the cholinesterases present in head tissues of the estuarine fish Pomatoschistus microps: Application to biomonitoring

In recent years biomarkers have been widely used for the assessment of effects and/or exposure to environmental contaminants. One of the most frequently used biomarkers is the inhibition of cholinesterases (ChE), which is a useful indicator of organophosphate and carbamate exposure and/or effects. Recent studies indicated that more than one ChE may be present in tissues of fish and that different forms may vary in their sensitivity to anticholinesterase agents. Cholinesterase activity of the juvenile of the common goby ( Pomatoschistus microps), a widespread fish in estuaries of the Atlantic coast of northwestern Europe, was characterized using four substrates (acetylthiocholine iodide, acetyl- β-metylthiocholine iodide, propionylthiocholine iodide, and S-butyrylthiocholine iodide) and three ChE inhibitors (eserine sulfate, BW284C51, and iso-OMPA) in different tissues of the fish head. In addition, the range of ChE activity that may be considered as “normal” for non-exposed P. microps was determined. The results suggest the presence of two types of ChE in the whole-head homogenate. The present study underscores the relevance of ChE characterization before its use as a biomarker in biomonitoring studies.

Sea-urchin (Paracentrotus lividus) glutathione S-transferases and cholinesterase activities as biomarkers of environmental contamination

Activities of glutathione S-transferases (GST) and cholinesterase (ChE) from Paracentrotus lividus were investigated as possible biomarkers of environmental contamination in the coastal zone. In the first phase of the study, the activity of both enzymes was determined in various tissues in order to select the most appropriate ones to be used in the following assays. In the second phase, the ChEs present in ambulacra were characterized using different substrates and selective inhibitors. In the next phase, laboratory bioassays were performed with dilutions of water-accommodated fraction of #4 fuel-oil (WAF) and benzo[a]pyrene (BaP) to determine the response of those enzymes to these pollutants and, finally, the activity of both enzymes was determined during a year in indigenous specimens from six sites on the Northwest coast of Portugal, with different pollution levels, to determine basal values and seasonal variations of ChE and GST activities. Among the several tissues tested, ambulacra and the anterior portion of the intestine were selected for ChE and GST assays, respectively. The determination of ChE in ambulacra tissue may be performed in a non-destructive way. Ambulacra ChE hydrolysed acetylthiocholine preferentially to propionylthiocholine and butyrylthiocholine and, inhibition by excess of substrate was observed. Enzymatic activity was almost fully inhibited by eserine sulfate (>98%) at concentrations equal or higher than 6.25 microM. Sensitivity to both BW284C51 (reaching 98% at 200 microM) and iso-OMPA (73% at 8 mM) was found. In laboratory bioassays, GSTs activity was inhibited by WAF and induced by BaP, whereas ChE activity was not affected by any of these environmental contaminants. Seasonal variations in enzymatic activities were found. For example, in a reference site, ChE values changed from 52.2 +/- 9.3 U mg(-1) protein in autumn to 71.8 +/- 13.3 U mg(-1) protein in spring, while GST activity changed from 129.9 +/- 29.8 U mg(-1) protein in winter to 279.0 +/- 48.0 U mg(-1) protein in autumn. Sea-urchins from reference sites presented significantly higher values of both ChE and GST than animals from contaminated sites in all seasons. In conclusion, the results of this study indicate that (i) ambulacra and the anterior portion of intestine are the most suitable tissues to measure ChE and GST activities, respectively; (ii) only one form of ChE seems to be present in ambulacra, showing properties of both typical acetylcholinesterase (AChE) and pseudocholinesterase (PChE); (iii)P. lividus GST is sensitive to both WAF and BaP even after acute exposures while ChE is not, and (iv) in spite of the significant seasonal variations observed in both enzymes in the field, P. lividus ChE and GST were capable of discriminate sites with different contamination levels and, thus, they are suitable for use as biomarkers in biomonitoring studies in the coastal zone.

Biochemical studies on sheep body louse Bovicola ovis (Schrank) (Phthiraptera: Trichodectidae): comparison of pyrethroid and organophosphate resistant and susceptible strains

Strains of sheep louse Bovicola ovis (Schrank) with various levels of resistance to pyrethroid and one strain with high degree of resistance to organophosphate (OP) insecticides were used to investigate the biochemical mechanisms of insecticide resistance, i.e., enhanced levels of general esterases, specific acetylcholinesterases (AChE), glutathione S-transferase (GST), and mixed function oxidases. Native gel electrophoresis combined with quantitative enzyme assays showed analogous expression profiles of several esterase isozymes in all the strains tested. The determination of the sensitivity of each esterase isozyme to five inhibitors (acetylthiocholine iodide, butyrylthiocholine iodide, paraoxon eserine sulfate, and pCMB) led to the identification of nine esterases in the B. ovis strain. Gel electrophoresis results are supported by enzyme assay studies where, except for the OP resistant strain, no differences in esterase activities were detected in all the pyrethroid resistant and susceptible strains assayed. Statistical analyses demonstrated that some strains have elevated GST activities compared to the susceptible reference strain.

Effect of dichlorvos on cholinesterase activity of the European sea bass ( Dicentrarchus labrax)

In this study, the acute toxicity of the organophosphorous pesticide (OP) dichlorvos and both in vitro and in vivo effects of dichlorvos on cholinesterase (ChE) activity of the European sea bass ( Dicentrarchus labrax) were investigated. The characterisation of ChE and the “normal” range of activity in brain and muscle of non-exposed fish were determined in a first phase of the study. Acetylthiocholine was the substrate preferred of both brain and muscle ChE. Eserine sulphate and BW284C51 significantly inhibited the brain and muscle enzyme activity at low concentrations (μM range). Iso-OMPA had a significant effect in muscle, but not in brain tissue. These results suggest that acetylcholinesterase (AChE) is the predominant ChE form in brain tissue. In contrast, both acetylcholinesterase and butyrylcholinesterase seem to exist in muscle. Using acetylthiocholine as substrate, the “normal” range of fingerling head and muscle ChE were 58.05±2.11 and 118.03±8.67 U/mg protein, respectively. Corresponding values for juveniles were 43.32±4.42 and 19.44±2.44 U/mg protein for brain and muscle, respectively. Dichlorvos significantly inhibited the activity of ChE in the selected tissues, both in vitro and in vivo conditions. Differences in ChE sensitivity were found in relation to the age of the fish and the tissue analysed. The present study also showed that fingerlings of the European sea bass are relatively resistant to in vivo acute (96 h) dichlorvos exposure to concentrations between 0.125 and 1 mg/L, being able to tolerate high percentages of head ChE inhibition (37% and 76%) without lethal effects.

Biochemical detection of esterases in the adult female integument of organophosphate-resistant Boophilus microplus (Acari: Ixodidae).

Esterase activity was present in the integument of adult female Boophilus microplus (Canestrini) ticks that are resistant to organophosphates (OP). Three esterases were purified from adult integument, which hydrolyze the substrates p-nitrophenylacetate and beta-naphthyl acetate after comparison of OP-resistant strain and an OP-susceptible strains. The esterases purified by ion-exchange chromatography were characterized using different esterase inhibitors; eserine sulfate, diethyl p-nitrophenyl phosphate (paraoxon), para-hydroxyl-mercuribenzoate (pHMB), and diisopropylphosphofluoridate (DFP). All of the esterases had a molecular mass of 64 Kd (PAGE), but were characterized based on the esterase inhibitor effects as a B-esterase with beta-naphthyl acetate affinity, a carboxylesterase with beta-naphthyl acetate and p-nitrophenyl acetate affinity, and one A-Esterase (nonspecific esterase) with p-nitrophenyl acetate affinity. The described esterases are an important detoxification mechanism in B. microplus ticks at the integument. We describe also a microplate biochemical assay for the detection of esterase activity in the tick integument, potentially a useful tool to detect esterase-mediated OP resistance in B. microplus ticks.

Characterization of cholinesterase activity in tissues of the grass shrimp ( Palaemonetes pugio)

Inhibition of cholinesterases in estuarine organisms is a useful indicator of organophosphorus insecticide exposure. Recent research has indicated that more than one cholinesterase may be present in tissues of estuarine organisms and that these cholinesterases vary in their sensitivity to organophosphorus insecticides. Baseline cholinesterase activity for adult grass shrimp ( Palaemonetes pugio) was determined with four substrates (acetylthiocholine iodide, acetyl-β-methylthiocholine iodide, propionylthiocholine iodide, S-butyrylthiocholine iodide) and three cholinesterase inhibitors (eserine sulfate, tetraisopropyl pyrophosphoramide, 1,5-bis(4-allyldimethyl-ammoniumphenyl) pentan-3-one dibromide). Cholinesterase activity was the highest with acetylthiocholine iodide at 57.42 nmol/mg P/min and the lowest with acetyl-β-methylthiocholine iodide at 6.17 nmol/mg P/min. The four substrates tested were not inhibited by tetraisopropyl pyrophosphoramide. Eserine sulfate significantly inhibited cholinesterase activity against all substrates except S-butyrylthiocholine iodide. The results of substrate specificity and cholinesterase inhibition in grass shrimp indicate that acetylthiocholine iodide is the most appropriate substrate for assessing cholinesterase inhibition in grass shrimp.

The Scopolamine-Induced Impairment of Spatial Cognition Parallels the Acetylcholine Release in the Ventral Hippocampus in Rats

We investigated the relationship between the induction of spatial cognition impairment in the 8-arm radial maze task and regional changes (ventral hippocampus (VH), dorsal hippocampus, frontal cortex, and basolateral amygdala nucleus) in brain acetylcholine (ACh) release using microdialysis in rats treated with muscarinic (M) receptor antagonists. In a behavioral study, two M1 antagonists, scopolamine (0.5 mg/kg, i.p. and 20 microg, i.c.v.) and pirenzepine (80 microg, i.c.v.), but not an M2 antagonist, AF-DX116 (40-80 microg, i.c.v.), disrupted spatial cognition in the 8-arm radial maze task. In brain microdialysis with Ringer's solution containing 0.1 mM eserine sulfate, scopolamine and AF-DX116, but not pirenzepine, increased ACh release in the VH. Moreover, in the bilateral injection of scopolamine (2 microg/side), the VH and dorsomedial thalamus nucleus were important regions for scopolamine-induced impairment of spatial cognition. A simultaneous determination of the behavioral changes revealed that scopolamine (0.5 mg/kg, i.p.) markedly decreased the ACh contents and also increased the ACh release in all regions tested. Especially, the changes in the ACh release of the VH closely paralleled the induction of the scopolamine-induced impairment of spatial cognition. These results suggest that the blocking balance between M1 and M2 muscarinic receptor in the VH therefore plays a major role in the spatial cognition impairment induced by scopolamine in the 8-arm radial maze task.

Characterization of cholinesterase from guppy (Poecilia reticulata) muscle and its in vitro inhibition by environmental contaminants

With a view to using the cholinesterase (ChE) activity from guppy (Poecilia reticulata) muscle as a biomarker, the objectives of this work were: (i) to characterize the soluble cholinesterases present in muscle homogenate using different substrates and specific inhibitors, (ii) to determine the normal range of activity in non-exposed individuals and (iii) to investigate the in vitro effects of two common environmental contaminants, copper sulphate and dodecylbenzene sulphonic acid sodium salt (DBS) on ChE activity. The rate of substrate hydrolysis of P. reticulata ChE decreased in the order acetylthiocholine, propionylthiocholine and butyrylthiocholine. Inhibition by excess of substrate was observed at concentrations higher than 1.28 mM. Furthermore, eserine sulphate and 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one (BW284C51) significantly inhibited the enzyme activity at low concentrations (mM range) and N,N'-diisopropylphosphorodiamic acid (iso-OMPA) had no significant effect up to 8 mM. These findings suggest that the enzyme measured in this study is acetylcholinesterase. The activity determined in non-exposed fish was 145.1 ± 44.7 SD U mg−1 protein. The common environmental contaminants copper and DBS significantly inhibited P. reticulata ChE at concentrations that can be ecologically relevant.

Polyacrylamide Gel Electrophoretic Assessments of Esterases in Cotton Aphid (Homoptera: Aphididae) Resistance to Dichlorvos

The role of esterases in dichlorvos resistance of the cotton aphid, Aphis gossypii Glover was studied by vertical slab polyacrylamide gel electrophoresis by using a susceptible (E-S) and dichlorvos-selected resistant (E-D-R) strain. Results suggested that both resistant and susceptible strains possessed bands E-2, E-11, and E-12, with the latter 2 bands of the resistant strain comigrating slightly faster compared with the susceptible strain. On addition, the resistant strain had additional bands (i.e., E-1, and E-3 to E-10), which probably reflected from changes in conformation. Although bands of both strains exhibited equal affinity (with the shorter naphthyl acetates and indoxyl acetate), none had affinity with naphthyl laurate and myristate. The most elevated in the resistant strain were E-7, E-9, and E-10, which were classified as carboxylesterases. Bands E-11 and E-12 in both strains were classified as eserine sulfate sensitive carboxylesterases. Excessive production of comigrating bands of carboxylesterases in both quantitative and qualitative forms, coupled with modification of these enzymes, may account for dichlorvos resistance in this aphid.

A cluster of esterase genes on chromosome 3R of Drosophila melanogaster includes homologues of esterase genes conferring insecticide resistance in Lucilia cuprina.

We identify an esterase isozyme in Drosophila melanogaster, EST 23, which shares biochemical, physiological, and genetic properties with esterase E3, which is involved in resistance to organophosphate insecticides in Lucilia cuprina. Like E3, the D. melanogaster EST 23 is a membrane-bound alpha-esterase which migrates slowly toward the anode at pH 6.8. Both enzymes have similar preferences for substrates with shorter acid side chain lengths. Furthermore, on the basis of their high sensitivity to inhibition by paraoxon and their insensitivity to inhibition by eserine sulfate, both enzymes were classified as subclass I carboxylesterases. The activity of each enzyme peaks early in development and, again, in the adult stage. Both enzymes are found in the male reproductive system and larval and adult digestive tissues, the latter being consistent with a role for these enzymes in organophosphate resistance. Fine structure deficiency mapping localized Est 23 to cytological region 84D3 to E1-2 on the right arm of chromosome 3. Moreover, we show that the genes encoding three other esterase phenotypes also map to the same region; these phenotypes involve allozymic differences in EST 9 (formerly EST C), ali-esterase activity, defined by the hydrolysis of methyl butyrate, and malathion carboxylesterase activity, defined by hydrolysis of the organophosphate malathion. This cluster corresponds closely to that encompassing E3 and malathion carboxylesterase on chromosome 4 in L. cuprina, the homologue of chromosome 3R in D. melanogaster.

Acetylcholinesterase activity is associated with efferent endings in the sensory epithelia of the utricle and semicircular canals of the rainbow trout inner ear

The mechanosensory hair cells of the utricle and semicircular canals of the trout inner ear are morphologically similar to type II hair cells of the avian and mammalian vestibular end organs. These cells are innervated by two types of nerve terminals. The nonvesiculated terminals are considered to be afferent, and the vesiculated endings are presumed to be efferent. The presumptive efferent endings contain numerous clear, round vesicles and a few that are dense-cored. Histocytochemical, electron microscopic analysis has localized acetylcholinesterase activity to plasma membranes of vesiculated, presumptive efferent nerve terminals in sensory epithelia of the utricle and semicircular canals. No reaction product was observed at the receptoneural synapse or found in nonvesiculated, presumptive afferent endings. Control specimens incubated in the presence of eserine sulfate, an inhibitor of acetylcholinesterase, were devoid of reaction product. These results support the tenet that vesiculated nerve endings in the sensory epithelia of the utricle and semicircular canals of the trout are cholinergic.

Noradrenergic and cholinergic modulations of corticocerebellar activity modify the gain of vestibulospinal reflexes.

In addition to mossy fibers and climbing fibers, the cerebellar cortex receives noradrenergic and cholinergic afferents. Since the Purkinje (P) cells of the cerebellar vermis (culmen) respond to roll tilt of the animal with a discharge pattern that is out of phase with respect to that of the related lateral vestibular neurons, thus exerting a facilitatory influence on the gain of the vestibulospinal (VS) reflex, we tested the effects of local microinjection into the anterior vermis of noradrenergic and cholinergic agents on these reflexes. In decerebrate cats, unilateral microinjection in the paramedial zone B of the culmen of 0.25 microliters of small doses of alpha 1-, alpha 2-, and beta-noradrenergic agonists (i.e., metoxamine, clonidine, and isoproterenol, respectively) increased the response gain (in impulses/second per deg) of the EMG response of the ipsilateral and to some extent also of the contralateral triceps brachii to animal tilt (at 0.15 Hz, +/- 10 degrees). On the other hand local injection of the corresponding antagonists (i.e., prazosin, yohimbine, and propranolol) either decreased the gain of the ipsilateral triceps brachii to labyrinth stimulation or else prevented the occurrence of the effects induced by the corresponding agonists. An increase in gain of the VS reflexes was also elicited in other experiments by unilateral microinjection either of the nonselective cholinergic agonist carbachol or of the anticholinesterase eserine sulfate. Thus, the effects could be produced by increasing the naturally present amount of acetylcholine. Further experiments indicated that a bilateral increase in the response gain of the triceps brachii to labyrinth stimulation occurred after microinjection of a selective muscarinic (bethanechol) or nicotinic agonist (nicotine), while just the opposite result was obtained after microinjection of the corresponding muscarinic (scopolamine) and nicotinic (hexamethonium, D-tubocurarine) blockers. The effects of the noradrenergic and cholinergic agonists, which persisted for about two hours after the injection, were site specific and dose dependent. It appears, therefore, that the noradrenergic and cholinergic afferents to the cerebellar vermis intervene in the gain regulation of the VS reflexes, possibly by increasing the amplitude of modulation of the P cells to labyrinth stimulation.