Hugh A. Tilson

Polychlorinated biphenyls and the developing nervous system: Cross-species comparisons

Polychlorinated biphenyls are stable, lipophilic industrial compounds that are present in residue levels in human tissue, wildlife, and freshwater sediment. They are toxic, and are known to cross the placenta and intoxicate the fetus. Two large outbreaks of PCB poisoning have occurred in Asia; women pregnant at or after the exposures had children who were developmentally impaired. Laboratory experiments in rhesus monkeys and rodents, designed to assess neural or developmental effects, show altered activity levels, impaired learning, and delayed ontogeny of reflexes. Children exposed transplacentally to levels considered to be background in the U.S. have hypotonia and hyporeflexia at birth, delay in psychomotor development at 6 and 12 months, and poorer visual recognition memory at 7 months. Allowing for differences in testing, effects are roughly similar across species, but current methods used to calculate allowable or reference doses give results up to 4 orders of magnitude apart, with the lowest level based on the neurotoxicology level coming from the human data.

AF64A, a cholinergic neurotoxin, selectively depletes acetylcholine in hippocampus and cortex, and produces long-term passive avoidance and radial-arm maze deficits in the rat

The behavioral and biochemical effects of AF64A, a presynaptic cholinergic neurotoxin, were investigated. Bilateral administration of this compound into the lateral cerebral ventricles produced transient and dose-related effects on sensorimotor function and long-term impairments of cognitive behavior. Male Fischer-F344 rats dosed with either 15 or 30 nmol of AF64A reacted 29-62% faster than CSF-injected controls in a hot-plate test 14 (but not 1, 7, 21 or 28) days following dosing. The group administered 15 nmol of AF64A was also significantly more active (41%) than controls 28 days following dosing. The activity level of this group was comparable to that of controls at other times and hyperactivity was never observed in the 30 nmol group. Retention of a step-through passive avoidance task, assessed 35 days after dosing, was impaired in both the 15 and the 30 nmol groups. Their step-through latencies were significantly shorter than the control latencies, and they exhibited more partial entries during the 24-h retention test. Radial-arm maze performance, measured 60-80 days following treatment, was markedly impaired in the treated groups. Animals treated with AF64A made fewer correct responses in their first 8 choices, required more total selections to complete the task, and had an altered pattern of spatial responding in the maze. The neurochemical changes produced by AF64A, determined 120 days after dosing, were specific to the cholinergic system and consisted of decreases of ACh in both the hippocampus (15 and 30 nmol groups) and the frontal cortex (30 nmol group). The concentrations of catecholamines, indoleamines, their metabolites and choline in various brain regions were not affected by AF64A. Furthermore, histological analysis revealed that the doses of AF64A used in the present study did not damage the hippocampus, the fimbria-fornix, the septum or the caudate nucleus. These data support the contention that cholinergic processes in the hippocampus and/or frontal cortex play an important role in learning and memory processes. Furthermore, based upon the behavioral and biochemical data presented, it is suggested that AF64A could be a useful pharmacological tool for examining the neurobiological substrates of putative cholinergic disorders such as senile dementia of the Alzheimers type.

Increased dopamine release from striata of rats after unilateral nigrostriatal bundle damage

Dopaminergic control of striatal neurons is retained in rats sustaining lesions of the nigrostriatal bundle (NSB) as long as 10% of the projection remains, suggesting that enhanced efficiency of dopamine (DA) transmission may compensate for the denervation of the striatum. To examine this hypothesis we have studied the extracellular concentration of striatal DA using brain dialysis. In control rats, haloperidol (1 mg/kg, i.p.) or depolarization of striatal tissue with 25 mM KCl increased, and gamma-butyrolactone (500 mg/kg, i.p.) decreased DA and homovanillic acid (HVA) levels in striatal dialysates. Three weeks after unilateral injection of 6-hydroxydopamine (6-OHDA) to substantia nigra, DA content in the ipsilateral striatum was decreased by 60-98%. Nevertheless, extracellular DA concentration in the lesioned striata remained unchanged in rats with 60-90% DA depletions. More extensive lesions (96% DA depletion) were accompanied by 60% reduction in DA release. In contrast, extracellular HVA levels in the lesioned striata decreased proportionally to the depletion of tissue DA, indicating decreased inactivation of extracellular DA. We propose that the capacity of the residual DA terminals to maintain normal levels of extracellular DA after 60-90% NSB lesions may serve to compensate for the partial denervation of the striatal tissue. Disruption of striatal DA functions and postsynaptic supersensitivity after more extensive lesions may be associated with the failure of the NSB to fully compensate for loss of DA terminals. In striata contralateral to the 6-OHDA lesions, increased DA release was also observed. In addition, 60-90% ipsilateral DA depletions were accompanied by 32% and 42% increases in DA and HVA content in contralateral tissue, respectively. The possibility of the contralateral sprouting of DA terminals is discussed.

Cochicine-induced granule cell loss in rat hippocampus: Selective behavioral and histological alterations

Bilateral injection of 3.5 micrograms of colchicine into two levels of the dentate gyrus produced a selective loss of dentate granule cells and persistent behavioral effects in male Fischer rats. Histological analysis confirmed that this dosage of colchicine resulted in the selective loss of most granule cells in both superior and inferior blades of the dentate gyrus near the injection sites, while sparing pyramidal cells in CA1, CA2, CA3, and CA4, and GABAergic interneurons throughout the hippocampus. Rats injected with colchicine were significantly more active than cerebrospinal fluid-injected controls 2, 7, 14, 21 and 28 days after treatment. Behavioral reactivity, assessed by the magnitude of the acoustic startle response and the latency to respond in a hot-plate test, was not affected at any of these time points. Retention of a step-through passive avoidance task was impaired in the colchicine group one month after surgery. Their step-through latencies were significantly shorter than control latencies, and they exhibited more partial entries during the retention test. Acquisition and performance in a radial-arm maze, measured up to 3 months after surgery, were also impaired by colchicine. Animals injected with colchicine required more trials to acquire the task and were less accurate in the task even after their performance had stabilized. These data suggest that the hippocampus modulates motor behavior and cognitive function. The results of these experiments also support the use of colchicine as a means of defining the functional and anatomical consequences following selective destruction of the granule cell population of the dentate gyrus.

Influence of nigrostriatal dopaminergic tone on the biosynthesis of dynorphin and enkephalin in rat striatum.

The purpose of this study was to obtain direct evidence that the nigrostriatal dopamine (DA) pathway modulates the metabolism of striatal dynorphin and [Met5]-enkephalin. This was achieved by repeated injections of apomorphine (APO) or D-amphetamine (AMP) in unilateral nigral 6-hydroxydopamine (6-OHDA)-lesioned rats. Three weeks after a 6-OHDA lesion, dynorphin A(1-8)-like immunoreactivity (DN-LI) and the level of mRNA encoding prodynorphin in the striatum on the lesioned side were decreased compared with the contralateral control side. Activation of DA receptors by 7 daily injections of APO (5 mg/kg, Bid, s.c.), however, caused a large increase (3- to 4-fold of saline control) in striatal levels of DN-LI and prodynorphin mRNA on the 6-OHDA lesioned side, which is far greater than the increase on the contralateral side (2-fold of saline control). Presumably, the potentiated effect of APO in 6-OHDA lesioned rats is due to hypersensitivity of DA receptors resulting from DA denervation. Seven daily injections of AMP (5 mg/kg, Bid, s.c.), a DA-releasing agent, increased striatal DN-LI (187% of saline control) on the non-lesioned side, but not on the 6-OHDA-lesioned side. Taken together, the data indicate that the nigrostriatal pathway exerts a tonic excitatory influence over the biosynthesis of dynorphin and that this influence is not maximal since an additional increase in dopaminergic tone further increases the expression of dynorphin. In contrast, [Met5]-enkephalin-like immunoreactivity (ME-LI) in the striatum was increased by a 6-OHDA-lesion (145% of contralateral control), which was blocked by repeated administration of APO but not AMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Interlaboratory comparison of motor activity experiments: implications for neurotoxicological assessments.

Motor activity is an important functional measure used in neurotoxicology. The effects of chemicals on motor activity, however, may depend on variables such as type of measurement apparatus, physical and environmental testing conditions, and many other experimental protocol and organismic variables. Due to the increasing use of motor activity in neurotoxicology, a major question concerns the potential for differences in experimental findings due to variations in sensitivity and reliability between different laboratories and devices used to measure motor activity. This study examined historical data from a number of laboratories that employed different devices and experimental protocols to measure motor activity. Four aspects of the motor activity data were compared: 1) within-laboratory control variability across time; 2) within-laboratory replicability of control data; 3) between-laboratory variability in the effects of chemicals; and 4) between-laboratory comparison of the control rates of habituation. The analyses indicated that there was a relatively restricted range of within-laboratory variability and reliability in control values, and that these ranges were comparable across laboratories. Similar profiles of habituation were also seen across the different laboratories. Moreover, in virtually every case, all laboratories were capable of detecting qualitatively similar changes in motor activity following acute exposure to a variety of chemicals. These data indicate a high degree of comparability in the data generated by the different devices and experimental protocols.

MK-801 impedes the acquisition of a spatial memory task in rats

Several studies have reported that MK-801 impairs the acquisition of various learning and memory tasks, while others suggest that MK-801 may interfere with performance rather than having a specific effect on memory. To characterize further the effects of MK-801 on learning and memory, MK-801 (0.05 mg/kg, SC) was administered prior to or immediately after learning trials in a trial-independent water maze task. Since MK-801 may affect nonassociative variables that may influence learning and memory, motor activity and general reactivity measures were also determined for 0.0125, 0.025, or 0.05 mg/kg of MK-801 administered SC. Since MK-801 may also be used to treat children with epilepsy, we investigated the possible persistent cognitive effects on neonates. MK-801 (0.02 mg/kg, SC) was administered at postnatal days 9-15 and tested in the same task as above starting at day 36 of age. There were no persistent effects of neonatal treatment. However, in adult rats, MK-801 impaired the acquisition of the water maze task but did not affect performance during a recall task in the same apparatus. At doses affecting learning, there were no effects on motor activity or general reactivity in adult rats. These results are consistent with the conclusion that MK-801 interferes with acquisition of spatial learning in the rat.

Radial-arm maze deficits produced by colchicine administered into the area of the nucleus basalis are ameliorated by cholinergic agents

Rats were given bilateral injections of colchicine into the area of the nucleus basalis. Colchicine produced dose-dependent alterations in the acquisition of a food-reinforced working-memory task. Colchicine-induced deficits in maze performance were attenuated by cholinergic agents, including physostigmine, RS-86 (2-ethyl-8-methyl-2,8-diazospiro-(4,5)-decan-1,3-dione-hydro bromide) and nicotine. Naloxone and vasopressin did not affect radial-arm maze performance of colchicine-treated rats. Subsequent neurochemical analysis showed that colchicine decreased choline acetyltransferase (ChAT) activity and levels of norepinephrine, dopamine, 3,4-dihydroxyphenylacetic acid, serotonin and 5-hydroxyindoleacetic acid in the neocortex. However, ChAT activity and other neurochemical measures were not altered in the hippocampus or corpus striatum. Histological assessment indicated damage limited to the injection in the area of the nucleus basalis and enlarged cerebrolateral ventricles. These data suggest the possible utility of the colchicine model in the study of cognitive deficits associated with neurodegenerative diseases.

Microinjection of dynorphin into the hippocampus impairs spatial learning in rats

The effect of hippocampal dynorphin administration on learning and memory was examined in spatial and nonspatial tasks. Bilateral infusion of dynorphin A(1-8) (DYN; 10 or 20 micrograms in one microliters) into the dorsal hippocampus resulted in a dose-related impairment of spatial working memory in a radial maze win-stay task. Subsequent experiments found that acquisition of a reference memory task in the water maze was impaired by DYN injections (20 micrograms/microliters) in the dorsal hippocampus, but not in the ventral hippocampus, and that this impairment could be blocked by naloxone. In a nonspatial task, posttraining DYN injections in the dorsal hippocampus had no effect on retention of step-through passive avoidance. These results suggest that dynorphin specifically interferes with spatial learning and memory, and that this effect is mediated by opioid receptors in the dorsal hippocampus.

Neurotoxicity of environmental chemicals and their mechanism of action

Despite a ban on their manufacture in 1977, polychlorinated biphenyls (PCBs) are still found in significant quantities in the environment. Developmental exposure to PCBs and related compounds has been reported to be neurotoxic in human and animals. Research in our laboratory has focused on the possible site(s) and mechanism(s) of PCB-induced developmental neurotoxicity. Recent experiments with rats found that developmental exposure to Aroclor-1254 (ARC) affects the acquisition of a lever press response and produces long-term changes in calcium buffering and protein kinase C (PKC) activity in the brain. In vitro studies in our laboratory have found that ARC increases [3H]phorbol ester binding, an indirect measure of PKC translocation, and inhibits calcium buffering in microsomes and mitochondria. Other experiments indicate that PCB congeners with chlorine substitutions at ortho- or low lateral substitutions are active in vitro, while non-ortho-substituted congeners are less active or inactive. Other research suggests that the lack of coplanarity of the PCB molecule is related to in vitro activity of PCB congeners. These studies indicate that in vivo developmental exposure to PCBs alters behavior and second messenger systems during adulthood, while in vitro experiments indicate that nervous system activity is related to ortho-substituted congeners that tend to be non-coplanar in configuration. Our results are consistent with the hypothesis that developmental neurotoxicity of ARC is due, in part, to the presence of ortho-substituted PCB congeners.