Developmental thyroid hormone insufficiency and brain development: a role for brain-derived neurotrophic factor (BDNF)?

Thyroid hormones (TH) are essential for normal brain development. Even modest degrees of TH disruption experienced in utero can result in neuropsychological deficits in children despite normal thyroid status at birth. Neurotrophins have been implicated in a host of brain cellular functions, and in particular, brain-derived neurotrophic factor (BDNF) has a well documented role in development and function of the nervous system. A number of laboratories have reported the effects of TH administration or severe deprivation on neurotrophin expression in brain. This review provides an overview and update of recent developments in the thyroid field as they relate to the nervous system. Secondly, we describe an animal model of low level TH insufficiency that is more relevant for studying the neurological consequences associated with the modest TH perturbations of subclinical hypothyroidism, or that would be anticipated from exposure to environmental contaminants with a mode-of-action that involves the thyroid. Finally, we review the available in vivo literature on TH-mediated alterations in neurotrophins, particularly BDNF, and discuss their possible contribution to brain impairments associated with TH insufficiency. The observations of altered BDNF protein and gene expression have varied as a function of hypothyroid model, age, and brain region assessed. Only a handful of studies have investigated the relationship of neurotrophins and TH using models of TH deprivation that are not severe, and dose-response information is sparse. Differences in the models used, species, doses, regions assessed, age at assessment, and method employed make it difficult to reach a consensus. Based on the available literature, the case for a direct role for BDNF in thyroid-mediated effects in the brain is not compelling. We conclude that delineation of the potential role of neurotrophins in TH-mediated neuronal development may be more fruitful by examining additional neurotrophins (e.g., nerve growth factor), moderate degrees of TH insufficiency, and younger ages. We further suggest that investigation of BDNF invoked by synaptic activation (i.e., plasticity, enrichment, trauma) may serve to elucidate a role of thyroid hormone in BDNF-regulated synaptic function.

Developmental thyroid hormone insufficiency reduces expression of brain-derived neurotrophic factor (BDNF) in adults but not in neonates.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin critical for many developmental and physiological aspects of CNS function. Severe hypothyroidism in the early neonatal period results in developmental and cognitive impairments and reductions in mRNA and protein expression of BDNF in a number of brain regions. The present study examined the impact of modest levels of developmental thyroid hormone insufficiency on BDNF protein expression in the hippocampus, cortex and cerebellum in the neonatal and adult offspring of rat dams treated throughout pregnancy and lactation. Graded levels of hormone insufficiency were induced by adding propylthiouracil (PTU, 0, 1, 2, 3 and 10 ppm) to the drinking water of pregnant dams from early gestation (gestational day 6) until weaning of the pups. Pups were sacrificed on postnatal days (PN) 14 and 21, and -PN100, and trunk blood collected for thyroid hormone analysis. Hippocampus, cortex, and cerebellum were separated from dissected brains and assessed for BDNF protein. Dose-dependent reductions in serum hormones in dams and pups were produced by PTU. Consistent with previous findings, age and regional differences in BDNF concentrations were observed. However, no differences in BDNF expression were detected in the preweanling animals as a function of PTU exposure; yet dose-dependent alterations emerged in adulthood despite the return of thyroid hormone levels to control values. Males were more affected by PTU than females, BDNF levels in hippocampus and cortex were altered but not those in cerebellum, and biphasic dose-response functions were detected in both sexes. These findings indicate that BDNF may mediate some of the adverse effects accompanying developmental thyroid hormone insufficiency, and reflect the potential for delayed impact of modest reductions in thyroid hormones during critical periods of brain development on a protein important for normal synaptic function.

New and evolving concepts in the neurotoxicology of lead.

Lead (Pb) is a xenobiotic metal with no known essential function in cellular growth, proliferation, or signaling. Decades of research characterizing the toxicology of Pb have shown it to be a potent neurotoxicant, especially during nervous system development. New concepts in the neurotoxicology of Pb include advances in understanding the mechanisms and cellular specificity of Pb. Experimental studies have shown that stress can significantly alter the effects of Pb, effects that could potentially be mediated through alterations in the interactions of glucocorticoids with the mesocorticolimbic dopamine system of the brain. Elevated stress, with corresponding elevated glucocorticoid levels, has been postulated to contribute to the increased levels of many diseases and dysfunctions in low socioeconomic status populations. Cellular models of learning and memory have been utilized to investigate the potential mechanisms of Pb-induced cognitive deficits. Examination of long-term potentiation in the rodent hippocampus has revealed Pb-induced increases in threshold, decreases in magnitude, and shorter retention times of synaptic plasticity. Structural plasticity in the form of adult neurogenesis in the hippocampus is also impacted by Pb exposure. The action of Pb on glutamate release, NMDA receptor function, or structural plasticity may underlie perturbations in synaptic plasticity and contribute to learning impairments. In addition to providing insight into potential mechanisms of Pb-induced cognitive deficits, cellular models offer an opportunity to investigate direct effects of Pb on isolated biological substrates. A target of interest is the 78-kDa molecular chaperone glucose-regulated protein (GRP78). GRP78 chaperones the secretion of the cytokine interleukin-6 (IL-6) by astrocytes. In vitro evidence shows that Pb strongly binds to GRP78, induces GRP78 aggregation, and blocks IL-6 secretion in astroglial cells. These findings provide evidence for a significant chaperone deficiency in Pb-exposed astrocytes in culture. In the long term, chaperone deficiency could underlie protein conformational diseases such as Alzheimer's Disease (AD). Lead exposure in early life has been implicated in subsequent progression of amyloidogenesis in rodents during old age. This exposure resulted in an increase in proteins associated with AD pathology viz., beta-amyloid precursor protein (beta-APP), and beta-amyloid (Abeta). These four new lines of research comprise compelling evidence that exposures to Pb have adverse effects on the nervous system, that environmental factors increase nervous system susceptibility to Pb, and that exposures in early life may cause neurodegeneration in later life.

Developmental lead (Pb) exposure reduces the ability of the NMDA antagonist MK-801 to suppress long-term potentiation (LTP) in the rat dentate gyrus, in vivo.

Chronic developmental lead (Pb) exposure increases the threshold and enhances decay of long-term potentiation (LTP) in the dentate gyrus of the hippocampal formation. MK-801 and other antagonists of the N-methyl-D-aspartate (NMDA) glutamate receptor subtype impair induction of LTP. In addition, Pb exposure reduces presynaptic glutamate release and is associated with alterations in NMDA receptor expression. This study examined LTP in Pb-exposed animals challenged with a low dose of MK-801 to assess the sensitivity of this receptor to inhibition. Pregnant rats received 0.2% Pb acetate in the drinking water beginning on gestational day 16, and this regimen was continued through lactation. Adult male offspring maintained on this solution from weaning were prepared with indwelling electrodes in the perforant path and dentate gyrus. Several weeks later, input/output (I/O) functions were collected in awake animals before and after saline or MK-801 administration (0.05 mg/kg, s.c.). LTP was induced using suprathreshold train stimuli 60 min post-drug. Post-train I/O functions were reassessed 1 and 24 h after train delivery. Upon full decay of any induced LTP, drug conditions were reversed such that each animal was tested under saline and MK-801. I/O functions measured 1 and 24 h after train induction as well as immediate post-train responses revealed significant LTP of comparable magnitude that was induced in both control and Pb-exposed animals tested under saline conditions. In contrast, MK-801 reduced LTP in control but not in Pb-exposed animals. The broadening of the excitatory postsynaptic potential evident in responses evoked by train stimuli is NMDA-dependent. Pb exposure attenuated the MK-801-induced reduction in area of this NMDA component by approximately 50%. These findings are consistent with other neurochemical and behavioural observations and suggest that up-regulation of postsynaptic NMDA receptors produces subsensitivity to the inhibitory effects of MK-801 on hippocampal LTP following chronic developmental Pb exposure.

Rat hippocampal glutamate and GABA release exhibit biphasic effects as a function of chronic lead exposure level.

Previous work has suggested that the lead (Pb) exposure-induced decrease in K(+)-evoked hippocampal glutamate (GLU) release is an important factor in the elevated threshold and diminished magnitude reported for hippocampal long-term potentiation (LTP) in exposed animals. In addition, complex dose-effect relationships between Pb exposure level and LTP have been reported. This investigation was conducted to determine the effects of Pb on hippocampal GLU and GABA release as a function of exposure level. Rats were continuously exposed to 0.1, 0.2, 0.5, or 1.0% Pb in the drinking water beginning at gestational day 15-16. Hippocampal transmitter release was induced in adult males by perfusion of 150 mM K(+) in the presence of Ca(+2) (total release) through a microdialysis probe in one test session, followed by perfusion through a contralateral probe in the absence of Ca(+2) (Ca(+2)-independent release) in the second session. Chronic exposure produced decreases in total K(+)-stimulated hippocampal GLU and GABA release at exposure levels of 0.1-0.5% Pb. Maximal effects were seen in the 0.2% group (blood Pb = 40 microg/100 ml), and changes in total release could be directly traced to alterations in the Ca(+2)-dependent component. However, these effects were less evident in the 0.5% group and were no longer present in the 1.0% Pb group, thus defining U-shaped dose-effect relationships. Moreover, in the absence of Ca(+2) in the dialysis perfusate, K(+)-induced release was elevated in the 2 highest exposure groups, suggesting a Pb(+2)-induced enhancement in evoked release. This pattern of results indicates the presence of 2 actions of Pb on in vivo transmitter release: a more potent suppression of stimulated release seen at lower exposure levels (27-62 microg/100 ml) combined with Ca+2-mimetic actions to independently induce exocytosis that is exhibited at higher exposure levels (> or =62 microg/100 ml). Furthermore, significant similarities in the dose-effect relationships uncovered in measures of evoked GLU release and hippocampal LTP (M. E. Gilbert et al., 1999b, Neurotoxicology 20, 71-82) reinforce the conclusion that exposure-related changes in GLU release play a significant role in the Pb-induced effects seen in this model of synaptic plasticity.

Long-term consequences of developmental exposure to lead or polychlorinated biphenyls: Synaptic transmission and plasticity in the rodent CNS.

Exposure to lead (Pb) or polychlorinated biphenyls (PCBs) during early development has been associated with deficits in cognitive function in children (Pediatrics 87 (1991) 219; N. Engl. J. Med. 335 (1996) 783). These effects persist in the child long after exposure has ceased and body burdens have diminished. Despite intensive research, no consensus on the mechanisms of neurotoxicity of these chemicals has resulted. As the primary neurotoxic action of these agents is to impair cognitive ability, a number of laboratories have examined and reported on the detrimental the effects of Pb or PCBs on hippocampal synaptic transmission and long-term potentiation (LTP) in animals exposed during the perinatal period. Use-dependent synaptic plasticity, of which hippocampal LTP is the primary model system, is a fundamental property of neuronal function. In forebrain structures such as amygdala and hippocampus, LTP and related processes are purported to represent a physiological substrate for memory. During brain ontogeny, this type of plasticity guides the establishment and maintenance of synaptic connections in cortical structures based on sensory input. We postulate that the actions of PCBs and Pb in the developing nervous system perturb activity-dependent plasticity and promote organizational changes in brain. Aberrant connectivity derived from perturbations in activity-dependent plasticity during development may manifest as impaired LTP and cognitive ability in the adult organism.

Rat hippocampal NMDA receptor binding as a function of chronic lead exposure level.

Chronic developmental lead (Pb) exposure is known to impair cognitive ability in children and young animals. These findings have led to research examining exposure effects on long-term potentiation (LTP), a model of synaptic plasticity, and on NMDA receptor function. This study determined the changes occurring in hippocampal 3H-MK-801 binding as a function of exposure level for comparison to changes in LTP previously reported from this laboratory. Dams were exposed to 0.1%, 0.2%, 0.5% and 1.0% Pb in the drinking water beginning at parturition, and male offspring were weaned to the same solutions as their dams and maintained on these regimens until assessment as adults. A crude membrane fraction was prepared from hippocampal tissue, and Scatchard analysis conducted in the presence of saturating concentrations of glutamate and glycine. NMDA receptor density was elevated as a result of Pb exposure with significant increases in the 0.2% (38%) and 0.5% (30%) groups compared to control group values. No changes were observed in the 0.1% and 1.0% animals, thus constituting a biphasic dose-effect relationship. These findings are an approximate reflection of analogous relationships reported for hippocampal LTP and glutamate release, suggesting that the diminished glutamate release is one cause of the receptor up-regulation. However, since increases in receptor number were uncovered, it is unlikely that changes in NMDA receptor density constitute a primary mechanism whereby Pb impairs hippocampal LTP.

Glutamatergic components underlying lead-induced impairments in hippocampal synaptic plasticity.

Epidemiological investigations have established the relationship between chronic developmental lead (Pb) exposure and cognitive impairments in young children, defining Pb neurotoxicity as a significant pediatric health problem. Exposed animals have proven to be effective models of this condition, exhibiting similar sensitivity to the actions of Pb and replicating abnormal learning behaviors in exposed children. Research has extended these observations in animals to identifying the processes underlying the cognitive dysfunction, utilizing the long-term potentiation (LTP) paradigm as a correlate of learning ability. Results from these studies have been in widespread agreement in reporting impairments in synaptic plasticity. Exposure-related changes consist of increases in LTP induction threshold, decreases in magnitude of potentiation, and shortened LTP duration. Furthermore, while LTP may be more readily affected by Pb during early development, exposure initiated after weaning also potently affects synaptic plasticity. Biphasic dose-effect relationships also appear in which impaired LTP is observed at intermediate exposure levels (27-62 microg/100 ml), but not at higher exposures. Investigation of the synaptic processes underlying LTP has provided additional insight into the bases of the impaired potentiation and diminished cognitive ability. Biochemical and neurophysiological approaches have found stimulated glutamate release to be diminished in hippocampus at blood Pb values where deficits in LTP have been observed. Multiple actions of Pb may be involved at this exposure level since animals exposed postweaning exhibited similar decrements in evoked glutamate release to those exposed continuously from conception, similar to the observations in measures of LTP. A biphasic dose-effect relationship was also found in which stimulated glutamate release in hippocampus was decreased at intermediate exposures, but not at higher levels. A direct inhibitory effect of Pb2+ on NMDA receptor function does not appear to occur at environmentally relevant exposure levels, but both exposure-induced increases and decreases in receptor density have been reported by different workers. Evidence from behavioral and neurophysiological investigations can be explained by increased NMDA receptor density on the bases of increased sensitivity to agonists and decreased sensitivity to antagonists. From this body of findings it is apparent that decreases in stimulated glutamate release are a significant contributing factor to the exposure-related changes seen in LTP. Furthermore, despite general agreement on the actions of Pb on synaptic plasticity, reports of exposure effects on NMDA receptor function have been relatively variable, suggesting either that the nature of the receptor changes are dependent on exposure conditions or that the receptors are secondarily affected by Pb actions produced at signal transduction or cellular loci.

Bridging the gap between in vitro and in vivo models for neurotoxicology.

In vitro systems are widely used for investigation of neurotoxicant-induced perturbations of cellular functions. A variety of systems exist that demonstrate certain similarities to neurotoxicant-induced events in the intact animal are discussed, including single-cell types, systems that consider endpoints relevant in toxicology, and systems that consider heterogeneous cell interactions. Relationships between the in vitro and in vivo systems are examined in which ethanol, lead, polychlorinated biphenyl compounds, and organophosphate insecticides are examples. Situations in which the in vitro systems have been used to advantage are provided, along with cautions associated with their use.

Lead inhibits the rat N-methyl-d-aspartate receptor channel by binding to a site distinct from the zinc allosteric site.

Due to the importance of the NMDA receptor in cognitive function and in models of synaptic plasticity, the effect of Pb(+2) on this receptor has been one focus of attempts to define the bases of Pb-induced cognitive impairments seen in young children. The following study was performed to identify the effects on access to the NMDA receptor channel of acute exposure to free Pb(+2) in vitro. Cerebrocortical membranes were prepared from adult male Sprague-Dawley rats, and binding was measured in 50 mM Tris-acetate with (3)H-MK-801 in the presence of saturating concentrations of glutamate and glycine. The potency of Pb(+2) to inhibit access to the receptor channel (IC(50) = 0.55 microM) was greater than that of Zn(+2) (IC(50) = 1.30 microM). Dissociation of MK-801 from its binding site exhibited two-component kinetics, and both rate constants were significantly slowed in the presence of Pb(+2) or Zn(+2). To directly address the question of whether Pb(+2) inhibited the receptor channel by binding to the Zn(+2) modulatory site, changes in inhibitory potency for the receptor channel were measured when both metals were present. The results demonstrate that multiple levels of Pb(+2) produce a concentration-dependent downward shift of the Zn(+2) inhibition curve, indicating a noncompetitive inhibition of MK-801 binding by Pb(+2) with respect to that of Zn(+2). Moreover, Zn(+2) IC(50) values significantly decreased as a function of increasing Pb(+2) concentrations. Analogous results were obtained when Pb(+2) inhibition curves were determined in the presence of multiple levels of Zn(+2). These findings indicate that the inhibitory properties of free Pb(+2) and Zn(+2) on the NMDA receptor channel are similar in nature but are exerted via independent allosteric binding sites.

Influence of exposure period on in vivo hippocampal glutamate and GABA release in rats chronically exposed to lead.

Previous work has demonstrated that continual exposure to 0.2% lead (Pb) beginning at birth diminishes depolarization-induced hippocampal glutamate (GLU) and GABA release in vivo. The present study sought to extend these findings by examining Pb-induced changes as a function of exposure period. Rats were continually exposed to 0.2% Pb in the drinking water beginning at conception (Gestational-Life, GL) or two weeks after weaning (Wean-Life, WL), while exposure in a third group was begun at conception but terminated at weaning (Gestational-Wean, GW). Hippocampal transmitter release was induced in adult animals by perfusion of 150 mM K+ in the presence of Ca+2 (total release) through a microdialysis probe in one test session, followed by perfusion through a contralateral probe in the absence of Ca+2 (Ca+2-independent release) in the second session. Decreases in total GLU and GABA release were observed in the GL and WL groups compared to controls over the first 20-min after initiation of high K+, decrements that could be attributed to exposure-induced reductions in Ca+2-dependent release. The pattern of Pb-induced changes in the GL group is similar to that observed previously in a group continuously exposed from birth, indicating that gestational exposure did not further enhance the impact of Pb beginning at birth when exposure in both groups extends into adulthood. Similar responses were also found in the WL group, indicating that exposure during early development is not a requirement to induce changes in GLU and GABA release. Pb-induced decreases in response were also seen in the GW group: a decrease in Ca+2-dependent GLU release was observed, while decrements in total and Ca+2-dependent GABA release were similar to those in the GL and WL groups. Thus, exposure limited to early development is also sufficient to produce deficits in evoked transmitter release. In addition, the exposure-induced decreases in GLU responses correspond to Pb-induced impairments in long-term potentiation (LTP) observed in similarly exposed groups (Gilbert et al., 1999), providing further evidence that Pb effects on GLU release are a critical factor in the alterations found in LTP.

The influence of developmental period of lead exposure on long-term potentiation in the adult rat dentate gyrus in vivo.

Previous work has demonstrated an increase in the threshold for induction of long-term potentiation (LTP) in the dentate gyrus of animals chronically exposed to lead (Pb) from birth (Gilbert et al., 1996). The present study sought to extend these findings by evaluating the developmental periods critical for Pb-induced impairment of LTP. Rats were exposed to Pb through maternal milk and/or the drinking water over different developmental intervals: 1) beginning just prior to birth and continuing throughout life (PL); 2) beginning just prior to birth and terminating at weaning (PW); or 3) continuously from the early post-weaning period throughout life (WL). Pregnant dams received 0.2% Pb-acetate in the drinking water on gestational day (GD)16, with male offspring switched to the same solution (PL group) or tap water (PW group) at weaning on postnatal day (PND)21. Postweaning exposure began on PND30 and continued throughout life. As adults (PND130-210), field potentials evoked by perforant path stimulation were recorded in the dentate gyrus under urethane anesthesia, and an ascending series of stimulus trains was administered to induce LTP and to determine its threshold. The magnitude of population spike (PS) LTP was reduced relative to controls in animals exposed throughout life (PL) and in animals exposed after weaning (WL). No impairment in PS LTP was evident in animals removed from Pb at weaning and tested as adults (PW). Similarly, thresholds for induction of PS LTP were elevated relative to controls in the PL and WL groups, but were not affected by Pb exposure limited to the lactational period (PW). Reductions in the magnitude of LTP of the EPSP slope were evident in posttrain I/O functions in all Pb-exposed groups, including the PW group. An elevated LTP threshold was evident in the EPSP slope measure in the continuously exposed group (PL) only. Thus Pb exposure restricted to the lactational period appeared less disruptive to adult LTP in the dentate gyrus than continuous exposure beginning around birth or weaning. However, EPSP slope LTP was impaired in animals exposed to Pb for as little as 30 days in the early postnatal period. An attenuated ability to support neuroplastic change in synaptic function may contribute to cognitive deficits associated with Pb-induced toxicity.

Chronic developmental lead exposure and hippocampal long-term potentiation: biphasic dose-response relationship.

Developmental exposure to lead (Pb) has long been associated with reductions in intellectual function in children and behavioral impairments in animal models of learning and memory. We have used long-term potentiation (LTP) in the dentate gyrus of the Pb-exposed rats to determine the potential of a reduced capacity for synaptic plasticity to contribute to Pb-induced cognitive dysfunction. Previous work demonstrated that developmental exposure resulting in moderate blood concentrations of Pb increase the threshold for induction of long-term potentiation (LTP) in the dentate gyrus in vivo. These findings were also suggestive of reductions in LTP magnitude (Gilbert et al., 1996). The present study was designed to further examine the effects of Pb on LTP magnitude and to determine if lower blood Pb levels commonly encountered in children are also effective in impairing synaptic plasticity in this rodent model. Pregnant dams were exposed to control tap water or 0.1, 0.2, 0.5 or 1.0% Pb-acetate in the drinking water beginning just prior to parturition (gestational day 16, GD16). Male offspring were weaned at 21 days of age (PN21) to the same solution given their dams and continued on this regimen until testing. As adults, animals were anesthetized with urethane and stimulating and recording electrodes placed in the perforant path and dentate gyrus, respectively. Post-train I/O functions taken 1 hour after delivery of a series of six high frequency (400Hz) trains revealed a reduced capacity for LTP of the PS amplitude and EPSP slope in Pb-exposed animals in all but the 1.0% group, indicative of a biphasic dose-effect relationship. The 1.0% Pb exposure was clearly less effective than the lower exposure levels in reducing LTP magnitude, and did not differ significantly from control values. The mechanisms underlying the reduced efficacy of higher exposure levels of Pb to impair LTP are not clear. Blood (26-117 microg/dl) and brain (220-1812 ng/g tissue) concentrations of Pb were elevated as a function of increasing exposure (0.1%-1.0%) and cannot readily account for the lack of an effect in the 1.0% group on LTP. We have observed a similar profile in hippocampal glutamate release employing a similar range of exposure levels, i.e., reduction of glutamate release that is absent at higher concentrations of Pb in the drinking water (Lasley et al., 1998). These and previously reported data suggest that the ability of Pb to diminish presynaptic transmitter release contributes to a reduced capacity for LTP at lower exposure levels. The reversal of the effect of Pb on glutamate release that accompanies higher exposure levels may serve to compensate for the mechanism underlying the LTP impairment and form the basis for the biphasic dose-response pattern seen with chronic developmental exposure.

Chronic developmental lead exposure increases the threshold for long-term potentiation in rat dentate gyrus in vivo.

Chronic developmental lead (Pb) exposure has been long associated with cognitive dysfunction in children and animals. In an attempt to more directly relate the behavioral observations of impaired cognitive ability to Pb-induced effects on neuronal activity, we utilized the long-term potentiation (LTP) model of neural plasticity to assess synaptic function. Male rats were chronically exposed to 0.2% Pb(2+)-acetate through the drinking water of the pregnant dam, and directly through their own water supply at weaning. As adults, field potentials evoked by perforant path stimulation were recorded in the dentate gyrus under urethane anesthesia. LTP threshold was determined by applying a series of stimulus trains of increasing intensities. Baseline testing of dentate gyrus field potentials indicated that input/output functions, maximal response amplitudes, and threshold currents required to evoke a population spike (PS) did not differ for control and Pb-exposed animals. Despite similarities in baseline synaptic transmission, Pb-exposed animals required a higher train intensity to evoke LTP than controls. With maximal train stimulation, however, control and Pb animals exhibited comparable levels of potentiation. These findings suggest that the mechanisms of LTP induction are preferentially impaired by Pb exposure. Although baseline synaptic transmission was not altered in Pb-exposed animals, decreases in glutamate release following high K+ perfusion and reductions in paired pulse facilitation have been reported in the intact animal. Pb-induced reductions in calcium influx through voltage-sensitive or N-methyl-D-aspartate (NMDA) receptor-dependent channels may mediate increases in LTP threshold. It is possible that the threshold changes in the induction of LTP reported here contribute to cognitive impairments associated with Pb exposure.

Presynaptic glutamatergic function in dentate gyrus in vivo is diminished by chronic exposure to inorganic lead.

Reductions in membrane Ca2+ channel currents and depolarization-evoked neurotransmitter release have been repeatedly observed as a result of acute exposure to Pb2+. This study was performed to determine whether hippocampal glutamate and GABA release are impaired in intact animals chronically exposed to lead (Pb). As paired-pulse facilitation in the hippocampus is primarily mediated by an enhancement of glutamate release, this neurophysiological measure was also assessed in the dentate gyrus of Pb-exposed animals. Pregnant dams received 0.2% Pb acetate in the drinking water at parturition, and male offspring were weaned to the same solution as that given their dams. Control animals were maintained on distilled water. As adults, animals had intracerebral dialysis probes inserted through guide cannulae implanted 2-4 days previously and the hippocampal CAI-dentate area was perfused with modified Ringer's solution. Transmitter release was induced by perfusion with 150 mM K+ with half the animals in each group tested with Ca2+ present in the perfusate (total release) and the other half with Ca2+ absent (Ca(2+)-independent release). K(+)-stimulated total glutamate release was reduced in Pb-exposed animals relative to controls. No group differences were observed under Ca(2+)-free conditions, indicating that Ca(2+)-dependent glutamate release was decreased in exposed rats. In contrast no group differences in K(+)-stimulated total GABA release were evident, whereas an augmentation in GABA release under Ca(2+)-free conditions was revealed in Pb-exposed animals. The effects of exposure on the Ca(2+)-dependent components of release are consistent with in vitro evidence indicating an inhibitory action of Pb2+ at voltage-sensitive Ca2+ channels. A separate group of animals was prepared under urethane anesthesia with stimulating and recording electrodes placed in the perforant path and dentate gyrus, respectively. Pairs of stimulus pulses were delivered at interpulse intervals (IPI) of 10-250 ms. Pb exposure induced an increase in paired-pulse depression at the 20 ms 1PI and reduced paired-pulse facilitation at the 30 ms IPI. Decreases in paired-pulse facilitation could not be attributed to the reported effects of Pb2+ on N-methyl-D-aspartate (NMDA) receptors as MK-801 (1.0 mg/kg, s.c.) administration produced an opposing pattern of effects on paired-pulse measures. The Pb-induced suppression of paired-pulse facilitation is consistent with exposure-related decreases in total glutamate release. The impact of these effects of Pb exposure on hippocampal glutamatergic transmission may contribute to the reported effects of Pb on other forms of synaptic plasticity including long-term potentiation, a model of learning and memory.

Diminished potassium-stimulated GABA release in vivo in genetically epilepsy-prone rats.

This experiment was conducted to assess the physiological relevance of observed changes in transmitter amino acid content in severe seizure genetically epilepsy-prone rats (GEPR-9s) by use of microdialysis. Adult male GEPR-9s and non-epileptic control rats were implanted with guide cannulae, and 6 mm (loop) dialysis probes were inserted unilaterally into rostral caudate and perfused with artificial cerebrospinal fluid. Each subject was perfused in the awake state with 100 or 150 mM K+ for 80 min in separate counterbalanced sessions, and 20-min fractions collected. High K+ perfusion increased extracellular fluid GABA and glutamate (GLU) in a concentration-dependent manner in both GEPR-9s and non-epileptic control rats. However, in the presence of 150 mM K+ GABA release was decreased in GEPR-9s relative to controls throughout the stimulation interval. In contrast, the increase in extracellular fluid GLU after high K+ was not different in the two groups. These results suggest an important role for mechanisms underlying GABA release in the seizure susceptibility observed in GEPR-9s.

Chronic exposure to environmental levels of lead impairs in vivo induction of long-term potentiation in rat hippocampal dentate.

This study examined the effects of chronic developmental lead (Pb) exposure in rats on hippocampal long-term potentiation (LTP). Male offspring were exposed to 0.2% Pb acetate continuously from birth until testing at 85-105 days. Excitatory postsynaptic potential (EPSP) and population spike amplitudes were measured in the dentate hilar region in response to stimulation applied to the lateral perforant path. LTP was induced in control animals with an average maximal EPSP potentiation of 41%, which was significantly greater than the increase in EPSP amplitudes (2%) in exposed animals after tetanizing stimulation. Current-voltage curves in controls demonstrated significant increases in EPSPs and population spikes after application of pulse trains to induce LTP, while exposed rats exhibited no discernible change in responses. These findings suggest that induction or development of LTP in the dentate hilar region in vivo is impaired by chronic developmental exposure to environmentally relevant levels of Pb.

The paradoxical effect of NMDA receptor stimulation on electrical activity of the sensorimotor cortex in freely behaving rats: analysis by combined EEG-intracerebral microdialysis.

This study was designed to determine the effects of N-methyl-D-aspartate (NMDA) receptor stimulation on the electrical activity of neocortex in freely behaving rats. Electroencephalogram (EEG) recording and intracerebral microdialysis were conducted simultaneously in the same site of the sensorimotor cortex, where the basal extracellular concentrations of aspartate and glutamate were 2.1 +/- 0.7 microM and 11.5 +/- 2.4 microM, respectively. Microdialysis with NMDA solutions (ranging from 10.0 microM to 10.0 mM) reduced the amplitude of the EEG activity and decreased the power of all frequency bands, with a virtual elimination of the high frequency waves, in a dose-dependent manner. These EEG changes were reversed after washing out the drug from the microdialysis fluid, and could be effectively antagonized with the competitive NMDA receptor antagonist DL-2-amino-5-phosphonovalerate. Remarkably, the NMDA actions were not associated with epileptiform behavioral or electrographic events. Control studies demonstrated that in the same experimental conditions, cholinergic receptor agonist carbachol caused seizures, and microdialysis with NMDA in the hippocampus readily induced epileptiform spikes. Our study shows that NMDA receptor stimulation in the rat sensorimotor cortex, although excitatory at synaptic level, can depress the local EEG activity. This may indicate that the NMDA receptor-mediated signals are processed by the neocortical network in a different way than by many other brain circuitries including hippocampus.

The combined EEG-intracerebral microdialysis technique: a new tool for neuropharmacological studies on freely behaving animals.

In this study we combined EEG and intracerebral microdialysis techniques in freely behaving rats. Various drugs were delivered into the hippocampus and cerebral cortex by means of microdialysis and, simultaneously, the EEG activity of the dialyzed area was monitored. The microdialysis procedure itself, when artificial cerebrospinal fluid was perfused, did not change the normal hippocampal or cortical EEG pattern. Drug inclusions into the microdialysis fluid, however, caused marked changes in the electrical activity of the dialyzed sites. In this report we present the following examples: (1) the dose-dependent spike-provoking effect of NMDA in hippocampus, (2) the potentiation of this NMDA effect in hippocampus by dibutyryl cyclic AMP, and (3) the EEG depressant effect of high concentration of K+ in the cerebral cortex. The artificial cerebrospinal fluid and drug solutions were alternated in the microdialysis system with a 2-way valve placed outside the test chamber. As a consequence, the drugs were delivered into the brain without interrupting the ongoing behavior, including sleep, of the examined animals. This study shows that the combined EEG-intracerebral microdialysis technique is a useful tool, with many unique advantages, for in vivo neuropharmacological studies.

Regulation of dopaminergic activity, but not tyrosine hydroxylase, is diminished after chronic inorganic lead exposure.

Previous work has indicated that the neurotoxic action of environmentally relevant levels of lead (Pb) on dopaminergic neurons is primarily presynaptic in nature and related to impaired regulation of dopamine (DA) synthesis and decreased DA release. This study was conducted to assess the functional integrity of the regulation of DA synthesis in caudate-putamen (C-P) and nucleus accumbens (NAc) of chronically Pb-exposed rats by measuring tyrosine hydroxylase (TH) activity. A pharmacological paradigm was employed that isolated autoreceptor-mediated regulation of the enzyme. At parturition dams received 0.2% Pb acetate (1090 ppm) in the drinking water while control dams received distilled water. Offspring were weaned to and maintained on the same solution given their dams until termination at 60 or 120 days. Rats were given saline or one of three DA agonists (EMD 23448, CGS 15855A, TL-99) 45 or 60 min before termination followed 15 min later by 750 mg/kg i.p. of gamma-butyrolactone (GBL) or saline. The ability of a DA agonist to prevent the GBL-induced increase in DA content was significantly altered in C-P of exposed rats compared to controls. No effect of Pb on DA content was observed in NAc. Furthermore, no differences in the ability of DA agonists to inhibit GBL-induced activation of TH in Pb-exposed compared to control animals were apparent in either brain region at either age by use of the tritium release method or the accumulation of L-DOPA. On the other hand, concentrations of DA metabolites in exposed rats given GBL and EMD 23448 were significantly lower than those in controls in both C-P and NAc. These findings support previous work suggesting that chronic Pb has multiple actions on CNS dopaminergic neurons consisting of impaired regulation of DA content and decreased DA release. However, these effects cannot be attributed to alterations in autoreceptor-mediated regulation of TH activity.