Neonatal exposure to parathion alters lipid metabolism in adulthood: Interactions with dietary fat intake and implications for neurodevelopmental deficits.

Organophosphates are developmental neurotoxicants but recent evidence also points to metabolic dysfunction. We determined whether neonatal parathion exposure in rats has long-term effects on regulation of adipokines and lipid peroxidation. We also assessed the interaction of these effects with increased fat intake. Rats were given parathion on postnatal days 1-4 using doses (0.1 or 0.2mg/kg/day) that straddle the threshold for barely detectable cholinesterase inhibition and the first signs of systemic toxicity. In adulthood, animals were either maintained on standard chow or switched to a high-fat diet for 7 weeks. We assessed serum leptin and adiponectin, tumor necrosis factor-alpha (TNFalpha) in adipose tissues, and thiobarbituric acid reactive species (TBARS) in peripheral tissues and brain regions. Neonatal parathion exposure uncoupled serum leptin levels from their dependence on body weight, suppressed adiponectin and elevated TNFalpha in white adipose tissue. Some of the effects were offset by a high-fat diet. Parathion reduced TBARS in the adipose tissues, skeletal muscle and temporal/occipital cortex but not in heart, liver, kidney or frontal/parietal cortex; it elevated TBARS in the cerebellum; the high-fat diet again reversed many of the effects. Neonatal parathion exposure disrupts the regulation of adipokines that communicate metabolic status between adipose tissues and the brain, while also evoking an inflammatory adipose response. Our results are consistent with impaired fat utilization and prediabetes, as well as exposing a potential relationship between effects on fat metabolism and on synaptic function in the brain.

Neonatal parathion exposure disrupts serotonin and dopamine synaptic function in rat brain regions: modulation by a high-fat diet in adulthood.

The consequences of exposure to developmental neurotoxicants are influenced by environmental factors. In the present study, we examined the role of dietary fat intake. We administered parathion to neonatal rats and then evaluated whether a high-fat diet begun in adulthood could modulate the persistent effects on 5HT and DA systems. Neonatal rats received parathion on postnatal days 1-4 at 0.1 or 0.2 mg/kg/day, straddling the cholinesterase inhibition threshold. In adulthood, half the animals in each exposure group were given a high-fat diet for 8 weeks. We assessed 5HT and DA concentrations and turnover in brain regions containing their respective cell bodies and projections. In addition, we monitored 5HT1A and 5HT2 receptor binding and the concentration of 5HT presynaptic transporters. Neonatal parathion exposure evoked widespread increases in neurotransmitter turnover, indicative of presynaptic hyperactivity, further augmented by 5HT receptor upregulation. In control rats, consumption of a high-fat diet recapitulated many of the changes seen with neonatal parathion exposure; the effects represented convergent mechanisms, since the high-fat diet often obtunded further increases caused by parathion. Neonatal parathion exposure causes lasting hyperactivity of 5HT and DA systems accompanied by 5HT receptor upregulation, consistent with "miswiring" of neuronal projections. A high-fat diet obtunds the effect of parathion, in part by eliciting similar changes itself. Thus, dietary factors may produce similar synaptic changes as do developmental neurotoxicants, potentially contributing to the increasing incidence of neurodevelopmental disorders.

Consumption of a high-fat diet in adulthood ameliorates the effects of neonatal parathion exposure on acetylcholine systems in rat brain regions.

BACKGROUND: Developmental exposure to a wide variety of developmental neurotoxicants, including organophosphate pesticides, evokes late-emerging and persistent abnormalities in acetylcholine (ACh) systems. We are seeking interventions that can ameliorate or reverse the effects later in life. OBJECTIVES: We administered parathion to neonatal rats and then evaluated whether a high-fat diet begun in adulthood could reverse the effects on ACh systems. METHODS: Neonatal rats received parathion on postnatal days 1-4 at 0.1 or 0.2 mg/kg/day, straddling the cholinesterase inhibition threshold. In adulthood, half the animals were switched to a high-fat diet for 8 weeks. We assessed three indices of ACh synaptic function: nicotinic ACh receptor binding, choline acetyltransferase activity, and hemicholinium-3 binding. Determinations were performed in brain regions comprising all the major ACh projections and cell bodies. RESULTS: Neonatal parathion exposure evoked widespread abnormalities in ACh synaptic markers, encompassing effects in brain regions possessing ACh projections and ACh cell bodies. In general, males were affected more than females. Of 17 regional ACh marker abnormalities (10 male, 7 female), 15 were reversed by the high-fat diet. CONCLUSIONS: A high-fat diet reverses neurodevelopmental effects of neonatal parathion exposure on ACh systems. This points to the potential for nonpharmacologic interventions to offset the effects of developmental neurotoxicants. Further, cryptic neurodevelopmental deficits evoked by environmental exposures may thus engender a later preference for a high-fat diet to maintain normal ACh function, ultimately contributing to obesity.

Selective and irreversible inhibitors of aphid acetylcholinesterases: steps toward human-safe insecticides.

Aphids, among the most destructive insects to world agriculture, are mainly controlled by organophosphate insecticides that disable the catalytic serine residue of acetylcholinesterase (AChE). Because these agents also affect vertebrate AChEs, they are toxic to non-target species including humans and birds. We previously reported that a cysteine residue (Cys), found at the AChE active site in aphids and other insects but not mammals, might serve as a target for insect-selective pesticides. However, aphids have two different AChEs (termed AP and AO), and only AP-AChE carries the unique Cys. The absence of the active-site Cys in AO-AChE might raise concerns about the utility of targeting that residue. Herein we report the development of a methanethiosulfonate-containing small molecule that, at 6.0 microM, irreversibly inhibits 99% of all AChE activity extracted from the greenbug aphid (Schizaphis graminum) without any measurable inhibition of the human AChE. Reactivation studies using beta-mercaptoethanol confirm that the irreversible inhibition resulted from the conjugation of the inhibitor to the unique Cys. These results suggest that AO-AChE does not contribute significantly to the overall AChE activity in aphids, thus offering new insight into the relative functional importance of the two insect AChEs. More importantly, by demonstrating that the Cys-targeting inhibitor can abolish AChE activity in aphids, we can conclude that the unique Cys may be a viable target for species-selective agents to control aphids without causing human toxicity and resistance problems.

Is fipronil safer than chlorpyrifos? Comparative developmental neurotoxicity modeled in PC12 cells.

Fipronil, a GABA(A) receptor antagonist, is replacing many insecticide uses formerly fulfilled by organophosphates like chlorpyrifos. Few studies have addressed the potential for fipronil to produce developmental neurotoxicity. We compared the neurotoxicity of fipronil and chlorpyrifos in undifferentiated and differentiating neuronotypic PC12 cells, evaluating indices of cell replication, cell number, differentiation, and viability for short- and long-term exposures. Fipronil inhibited DNA and protein synthesis in undifferentiated PC12 cells and evoked oxidative stress to a greater extent than did chlorpyrifos, resulting in reduced cell numbers even though cell viability was maintained. In differentiating cells, fipronil displayed an even lower threshold for disruption of development, reducing cell numbers without impairing cell growth, and promoting emergence of neurotransmitter phenotypes; superimposed on this effect, the phenotypic balance was shifted in favor of dopamine as opposed to acetylcholine. Differentiation also enhanced the susceptibility to fipronil-induced oxidative stress, although antioxidant administration failed to provide protection from cell loss. At low concentrations maintained for prolonged periods, fipronil had a biphasic effect on cell numbers, increasing them slightly at low concentrations, implying interference with apoptosis, while nevertheless reducing cell numbers at higher concentrations. Our results suggest that fipronil is inherently a more potent disruptor of neuronal cell development than is chlorpyrifos. The neurodevelopmental effects are not predicated on GABA(A) antagonist properties, since PC12 cells lack the GABA(A) receptor. If fipronil is intended to provide greater safety than chlorpyrifos, then this will have to entail advantages from factors that are yet unexamined: exposure, persistence, pharmacokinetics.

Exposure of neonatal rats to parathion elicits sex-selective reprogramming of metabolism and alters the response to a high-fat diet in adulthood.

BACKGROUND: Developmental exposures to organophosphate pesticides are virtually ubiquitous. These agents are neurotoxicants, but recent evidence also points to lasting effects on metabolism. OBJECTIVES: We administered parathion to neonatal rats. In adulthood, we assessed the impact on weight gain, food consumption, and glucose and lipid homeostasis, as well as the interaction with the effects of a high-fat diet. METHODS: Neonatal rats were given parathion on postnatal days 1-4 using doses (0.1 or 0.2 mg/kg/day) that straddle the threshold for barely detectable cholinesterase inhibition and the first signs of systemic toxicity. In adulthood, animals were either maintained on standard lab chow or switched to a high-fat diet for 7 weeks. RESULTS: In male rats on a normal diet, the low-dose parathion exposure caused increased weight gain but also evoked signs of a prediabetic state, with elevated fasting serum glucose and impaired fat metabolism. The higher dose of parathion reversed the weight gain and caused further metabolic defects. Females showed greater sensitivity to metabolic disruption, with weight loss at either parathion dose, and greater imbalances in glucose and lipid metabolism. At 0.1 mg/kg/day parathion, females showed enhanced weight gain on the high-fat diet; This effect was reversed in the 0.2-mg/kg/day parathion group, and was accompanied by even greater deficits in glucose and fat metabolism. CONCLUSIONS: Neonatal low-dose parathion exposure disrupts glucose and fat homeostasis in a persistent and sex-selective manner. Early-life toxicant exposure to organophosphates or other environmental chemicals may play a role in the increased incidence of obesity and diabetes.

Rats gain excess weight after developmental exposure to the organophosphorothionate pesticide, chlorpyrifos.

Pesticides that target molecules with critical roles in brain function deserve careful scrutiny for potential developmental neurotoxicity. In this study, time-pregnant rats were dosed daily by gavage with chlorpyrifos (2.5 mg/kg) from gestational day 7 through the end of lactation on postnatal day 21 (PND 21), and offspring were weighed regularly from birth until brain harvest at PND 22 or young adulthood (PND 95-101). The chlorpyrifos exposure caused excess weight gain in males beginning at PND 45 and reaching levels 10.5% above control by PND 72, while volumetric measurements showed that the exposed males were also 12% larger than controls. The body weight response showed an inverted U-shaped relation to chlorpyrifos dose. These data suggest delayed disturbances in body weight and density as previously unsuspected adverse consequences of developmental exposure to an environmental pesticide. Although we do not regard our findings as definitive evidence that chlorpyrifos exposure is a risk factor for obesity, the potential implications nonetheless deserve serious consideration.

A method to determine precise benchmark doses for carbamate anticholinesterases.

In determining benchmark doses for risk assessment and regulation of carbamate anticholinesterase pesticides like formetanate, oxamyl, and methomyl, one needs to quantitate low levels of cholinesterase inhibition. For improved accuracy while using fewer subjects, we developed an assay based on the recognized ability of carbamates to protect cholinesterase from irreversible inactivation. This assay measures enzyme that survives diisopropylfluorophosphate exposure in vitro and then reactivates by decarbamylation after small molecules are removed with size-exclusion centrifugation. The 99% silencing of unprotected cholinesterase yields a low background. Comparisons of recovered activity with initial activity (representing carbamate-free enzyme) use each sample as its own control. As a result, carbamate-protection assays can demonstrate a statistically significant 2-3% inhibition of brain cholinesterase in a single experimental group of modest size. When applied to brain samples from formetanate-treated rats, such an assay predicted a benchmark dose of 0.19 mg/kg for 10% inhibition (BMD10), with a lower 95% confidence limit of 0.15 mg/kg (BMDL10). Protection assays should enable precise determinations of benchmark doses for other carbamates, as well as accurate assessment of in vivo inhibition half-lives under low-dose scenarios.

Automated measurement of acetylcholinesterase activity in rat peripheral tissues.

The accepted mechanism of toxicity of many organophosphorous and carbamate insecticides is inhibition of acetylcholinesterase activity. In mammals, part of the toxicity assessment usually includes monitoring blood and/or brain acetylcholinesterase inhibition. Other tissues, however, contain cholinesterase activity (i.e. acetyl- and butyryl-cholinesterase), and the inhibition of that activity may be informative for a full appraisal of the toxicity profile. The present group of studies first optimized the variables for extraction and solubilization of cholinesterase activity from various rat tissues and then refined an existing automated method, in order to differentially assess acetyl and butyrylcholinesterase activity in those tissues. All these studies were conducted using tissues from untreated, Long-Evans, adult rats. The first studies determined the effect of Triton X-100 or salt (NaCl) on the extraction and solubilization of cholinesterase activity from retina, brain, striated muscle, diaphragm, and heart: phosphate buffer plus detergent (1% Triton X-100) yielded the highest activity for most tissues. For striated muscle, however, slightly more activity was extracted if the phosphate buffer contained both 1% Triton X-100 and 0.5 M NaCl. It was also noted that the degree of homogenization of some tissues (e.g. striated muscle) must be increased for maximal solubilization of all cholinesterase activity. Subsequent studies developed a method for assessing the level of acetylcholinesterase, butyrylcholinesterase and total cholinesterase activity in these tissues using an automated analyzer. In conclusion, automated assay of acetylcholinesterase activity in cholinergically innervated tissues in the rat (other than brain) is achievable and relatively convenient.

Dental unit waterlines: review and product evaluation.

Dental practitioners must be knowledgeable regarding microbial contamination and biofilm formation in dental unit waterlines. Education should stress the need for improvement in the quality of water delivered to patients during treatment. Manufacturers must also play an important role by providing training and education regarding the proper use and maintenance of their systems. Dental facilities, both public and private, need reliable methods to prevent the development of biofilms within DUWs. These methods must be economical and require minimal effort to use on the part of the dental staff. In order for the system to work efficiently, the effluent water that is produced must be compatible with dental materials and be potentially free from toxic or carcinogenic materials. There are numerous models of water filtration units and chemical flushes available to the dental practitioner. However, the Food and Drug Administration have not approved all products currently on the market. Our evaluation of Zerosil, a new waterline-cleaning product, indicates that it is very easy to use and is extremely effective in killing the commonly found microorganisms in dental unit waterlines, as well as eliminating existing biofilms. It is also economical and requires minimum staff time to keep the waterlines clean. Following the initial treatments during week one, the water emanating from the DUWs was free from any viable microorganisms. This effect was present the entire three weeks in which the waterlines were treated. The elimination of viable microorganisms continued into a fourth study week, even though no further treatment of the DUWs was performed. Although the manufacturer recommends weekly treatment of DUWs following the initial treatment regimen, this result indicates that the product has a longer lasting effect than previously thought. Finally, the product can be delivered through any of the commercially available reservoir/bottle water delivery systems. From our study, Zerosil appears to meet the demanding requirements of keeping dental unit waterlines clean. Based on the research that has been done thus far, no universal treatment protocol can be recommended. A combination of approaches may offer the best available assurance of high-quality dental treatment water. Independent water reservoir systems, when used with a periodic chemical treatment protocol, have demonstrated safety and efficacy. Until we reach a point when a recommendation based on thorough evaluations can be made, dental offices should follow current ADA, OSAP, and CDC guidelines: flush waterlines for two to three minutes at the beginning of each day and for 20 to 30 seconds between each patient, and anti-retraction valves should be installed to prevent oral fluids from being drawn into dental waterlines. It is expected that in the near future, the dental practitioner will have a choice of proven systems and products to deal with this issue. Until that time, one should carefully evaluate any product or system being considered to prevent the formation of biofilms in DUWs.

Vulnerable processes of nervous system development: a review of markers and methods.

The susceptibility of the developing nervous system to damage following exposure to environmental contaminants has been well recognized. More recently, from a regulatory perspective, an increased emphasis has been placed on the vulnerability of the developing nervous system to damage following pesticide exposure. The publication of the National Academy of Sciences (NAS) report on Pesticides in the Diets of Infants and Children (1995) and the passage of the Food Quality Protection Act (FQPA) and Safe Drinking Water Act (SDWA) amendments have significantly escalated the scientific debate regarding age-related susceptibility. Key concerns raised in the NAS report include the qualitative and quantitative differences that distinguish the developing nervous system from that of the adult. It was suggested that neurotoxicity testing on adult animals alone may not be predictive of these differences in susceptibility. The age-related susceptibility of the nervous system is compounded by the protracted period of time over which this complex organ system develops. This temporal vulnerability spans the embryonic, fetal, infant, and adolescent periods. Normal development of the nervous system requires the concomitant and coordinated ontogeny of proliferation, migration, differentiation, synaptogenesis, gliogenesis, myelination and apoptosis to occur in a temporally- and regionally-dependent manner. Perturbations of these processes during development can result in long-term irreversible consequences that affect the structure and function of the nervous system and could account for qualitative differences in age-related susceptibility of the developing nervous system as compared to the adult nervous system. A discussion of developmental milestones and the relevance of transient effects on developmental endpoints are presented. Transient effects following developmental perturbations can be missed or dismissed depending on the experimental design or screening strategy employed. This subject is discussed in light of scientific uncertainties regarding perturbation-induced compensation in the developing nervous system. Thus, utilization of age-appropriate tests of these developmental processes may improve the detection and reduce uncertainty about the nature of adverse effects following developmental exposure to environmental neurotoxicants.

Gestational exposure to chlorpyrifos: dose response profiles for cholinesterase and carboxylesterase activity.

This study investigates the in vivo dose response profiles of the target enzyme cholinesterase (ChE) and the detoxifying enzymes carboxylesterase (CaE) in the fetal and maternal compartments of pregnant rats dosed with chlorpyrifos [(O,O'-diethyl O-3,5,6-trichloro-2-pyridyl) phosphorothionate], a commonly used organophosphorus insecticide. Pregnant rats were dosed daily (po) with chlorpyrifos in corn oil (0, 3, 5, 7, or 10 mg/kg) on gestational days (GD) 14-18. Animals were sacrificed 5 h after the last chlorpyrifos dose (time of maximum brain cholinesterase inhibition) for analysis of ChE and CaE activity in maternal blood, liver, brain, placenta, and fetal liver and brain. The in vitro sensitivity (i.e., IC50, 30 min, 26 degrees C) of CaE also was determined by assaying the activity remaining after incubation with a range of chlorpyrifos-oxon concentrations. In vivo exposure to 10 mg/kg chlorpyrifos from GD14-18 caused overt maternal toxicity, with dose-related decreases in ChE activity more notable in maternal brain than fetal brain. Dose-related effects were also seen with chlorpyrifos-induced inhibition of fetal liver ChE and maternal brain CaE activities. Gestational exposure caused no inhibition of placental ChE or CaE, fetal brain CaE, or maternal blood CaE. ChE activities in the maternal blood and liver, as well as fetal and maternal liver CaE, however, were maximally inhibited by even the lowest dosage of chlorpyrifos. The in vitro sensitivity profiles of CaE to chlorpyrifos-oxon inhibition were valuable in predicting and verifying the in vivo CaE response profiles. Both the in vivo and in vitro findings indicated that fetal liver CaE inhibition was an extremely sensitive indicator of fetal chlorpyrifos exposure.

Gestational exposure to chlorpyrifos: comparative distribution of trichloropyridinol in the fetus and dam.

Chlorpyrifos (O,O'-diethyl O-[3,5,6-trichloro-2-pyridyl] phosphorothionate) is a commonly used anticholinesterase insecticide, and therefore the potential for human exposure is high. The present time course and dose response studies were conducted to delineate the toxicokinetics of chlorpyrifos and its metabolites in the pregnant rat and fetus. Time-pregnant, Long-Evans rats were treated orally with chlorpyrifos during late gestation (Gestational Days 14-18). Following euthanasia the level of chlorpyrifos and its metabolites, chlorpyrifos-oxon and 3,5,6-trichloro-2-pyridinol (TCP), were measured in both fetal and maternal brain and liver (limits of quantitation: 59.2, 28.8, and 14.0 ng/g tissue, respectively). In addition, cholinesterase inhibition was also measured in the same tissues for comparison. TCP was the only component detected. The highest level of TCP and the lowest level of cholinesterase activity showed the same time of peak effect: 5 h after the last dose. The concentration of TCP in the maternal liver was approximately fivefold higher than the TCP concentration in fetal liver, but, paradoxically, the concentration of TCP in the fetal brain was two- to fourfold higher than the TCP concentration in the maternal brain. The half-life of the TCP was identical in all tissues examined (12-15 h). These toxicokinetic results suggest that the fetal nervous system may be exposed to a higher concentration of chlorpyrifos than the maternal nervous system when the dam is orally exposed to chlorpyrifos during late gestation.

Biochemical measurement of cholinesterase activity.

The cholinesterases (acetylcholinesterase and butyrylcholinesterase)In this chapter, cholinesterase will be used to refer to both enzymes together (i.e., total cholinesterase), whereas acetylcholinesterase or butyrylcholinesterase will be used when referring to the specific esterase.

Functional knee brace effects during walking in patients with anterior cruciate ligament reconstruction.

The purpose of this study was to compare lower extremity joint kinematics and kinetics during walking with and without a functional knee brace in patients with recent anterior cruciate ligament reconstructions. Seven volunteers walked at 1.26 m/s with and without one of two functional knee braces 3 weeks after surgery. Eleven uninjured subjects were also tested as a control group. Video and ground-reaction data were collected and combined with inverse dynamics to estimate the joint positions, moments, and powers during the stance phase. Patients with ligament reconstructions were more erect with the brace, using 19% less knee flexion compared with walking without the brace. Areas under the internal extensor moment curve (angular impulse) and power curve (work) at the hip increased 40% and 44%, respectively, while walking with the brace. Extensor angular impulse decreased 41% at the knee while using the brace, and plantar flexor angular impulse and work increased 21% and 30%, respectively, at the ankle. While walking with the brace, the patients still had different kinematics, moments, and powers than the control subjects. The reduced extensor moment at the knee in the braced condition indicated that the load on the recently reconstructed ligament was reduced and that the brace protected the ligament during the stance phase of walking. We concluded that functional knee braces may be one means of developing neuromuscular adaptations during gait after anterior cruciate ligament reconstruction surgery.

Gestational exposure to chlorpyrifos: apparent protection of the fetus?

Previous studies have shown that, in general, young, postnatal animals are more sensitive than adults to the toxic effects of anticholinesterase (antiChE) pesticides. Paradoxically, often fetal brain cholinesterase (ChE) is less inhibited than maternal brain after gestational exposure to an antiChE, presumably due to placental and fetal detoxification of the antiChE. The present investigation was designed to study selected toxicokinetic and toxicodynamic factors surrounding the toxicity of chlorpyrifos (CPF; [O,O'-diethyl O-3,5,6-trichloro-2-pyridyl] phosphorothionate) in pregnant rats dosed repeatedly or singly during late gestation. Dams were dosed daily (po) with CPF in corn oil (0 or 7 mg/kg) on gestational days (GD) 14 to 18. Animals were euthanized at 2 to 120 h after the last dose and tissues were collected for enzyme analysis. Using this dosing regimen, we found that (1) the time of maximal ChE inhibition was the same (i.e., 5-10 h after dosing) for both maternal and fetal brain, (2) the degree of fetal brain ChE inhibition was 4.7 times less than maternal brain inhibition, and (3) the detoxification potential (i.e., carboxylesterase and chlorpyrifos-oxonase) of the fetal tissues was very low compared to the maternal tissues. A separate group of experiments showed that if pregnant dams received only one oral dose of 7 or 10 mg/kg CPF on GD18, the degree of ChE inhibition in the fetal brain was comparable to the maternal brain ChE inhibition. Taking into consideration the net increase (more than fourfold) in fetal brain ChE activity from GD14 to 18 in control animals, and the fact that maternal brain ChE was inhibited more than fetal brain ChE only in a repeated-dosing regimen, we conclude that the fetus is not genuinely protected from the toxic effects of a given dose of CPF. We propose that fetal brain ChE is simply able to recover more fully between each dose as compared to maternal brain ChE, giving the illusion that the fetal compartment is less affected than the maternal compartment.

Ontogenetic differences in the regional and cellular acetylcholinesterase and butyrylcholinesterase activity in the rat brain.

Considering the novel functions for both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in the developing nervous system (reviewed in Layer and Willbold, Prog. Histochem. Cytochem., 1995) a quantitative survey of the spatiotemporal developmental profiles of both AChE and BuChE activity in the neonatal rat brain would be extremely useful. To that end, we collected six brain regions at seven developmental time points, (postnatal day 1, 4, 7, 12, 17, 21, adult; n > or = 3) and measured AChE and BuChE activity using both biochemical and histological methods. These results indicated that the developmental pattern of AChE and BuChE activity varied with respect to brain region and age: (1) the ontogeny of either AChE or BuChE specific activity in one region was not necessarily indicative of the developmental pattern of the same cholinesterase in other regions; (2) the AChE developmental profile in a given region did not necessarily predict the BuChE developmental pattern for that same region. The data were also analyzed from a different perspective, i.e., the ratio of BuChE-AChE activity, in order to determine if BuChE activity preceded AChE activity during development as has been proposed for the chick nervous system (Layer, Proc. Natl. Acad. Sci. USA, 1983). Our analysis showed that, in general, the BuChE-AChE ratio decreased as the region matured, data which parallel the pattern of development of these esterases in the chick nervous system.