Presynaptic Mechanisms of Lead Neurotoxicity: Effects on Vesicular Release, Vesicle Clustering and Mitochondria Number

Childhood lead (Pb2+) intoxication is a global public health problem and accounts for 0.6% of the global burden of disease associated with intellectual disabilities. Despite the recognition that childhood Pb2+ intoxication contributes significantly to intellectual disabilities, there is a fundamental lack of knowledge on presynaptic mechanisms by which Pb2+ disrupts synaptic function. In this study, using a well-characterized rodent model of developmental Pb2+ neurotoxicity, we show that Pb2+ exposure markedly inhibits presynaptic vesicular release in hippocampal Schaffer collateral-CA1 synapses in young adult rats. This effect was associated with ultrastructural changes which revealed a reduction in vesicle number in the readily releasable/docked vesicle pool, disperse vesicle clusters in the resting pool, and a reduced number of presynaptic terminals with multiple mitochondria with no change in presynaptic calcium influx. These studies provide fundamental knowledge on mechanisms by which Pb2+ produces profound inhibition of presynaptic vesicular release that contribute to deficits in synaptic plasticity and intellectual development

From the Cover: 7,8-Dihydroxyflavone Rescues Lead-Induced Impairment of Vesicular Release: A Novel Therapeutic Approach for Lead Intoxicated Children

Childhood lead (Pb2+) intoxication is a public health problem of global proportion. Lead exposure during development produces multiple effects on the central nervous system including impaired synapse formation, altered synaptic plasticity, and learning deficits. In primary hippocampal neurons in culture and hippocampal slices, Pb2+ exposure inhibits vesicular release and reduces the number of fast-releasing sites, an effect associated with Pb2+ inhibition of NMDA receptor-mediated trans-synaptic Brain-Derived Neurotrophic Factor (BDNF) signaling. The objective of this study was to determine if activation of TrkB, the cognate receptor for BDNF, would rescue Pb2+-induced impairments of vesicular release. Rats were chronically exposed to Pb2+ prenatally and postnatally until 50 days of age. This chronic Pb2+ exposure paradigm enhanced paired-pulse facilitation of synaptic potentials in Schaffer collateral-CA1 synapses in the hippocampus, a phenomenon indicative of reduced vesicular release probability. Decreased vesicular release probability was confirmed by both mean-variance analysis and direct 2-photon imaging of vesicular release from hippocampal slices of rats exposed to Pb2+in vivo. We also found a Pb2+-induced impairment of calcium influx in Schaffer collateral-CA1 synaptic terminals. Intraperitoneal injections of Pb2+ rats with the TrkB receptor agonist 7,8-dihydroxyflavone (5 mg/kg) for 14-15 days starting at postnatal day 35, reversed all Pb2+-induced impairments of presynaptic transmitter release at Schaffer collateral-CA1 synapses. This study demonstrates for the first time that in vivo pharmacological activation of TrkB receptors by small molecules such as 7,8-dihydroxyflavone can reverse long-term effects of chronic Pb2+ exposure on presynaptic terminals, pointing to TrkB receptor activation as a promising therapeutic intervention in Pb2+-intoxicated children

Enduring changes in reward mechanisms after developmental exposure to cocaine: The role of the D2 receptor

During adolescent brain maturation, there are likely sensitive periods where environmental conditions, including drug exposure, may influence development by modifying neuronal connections. Altering neuronal function may produce different phenotypes than expected under normal conditions that may influence subsequent responding to drugs of abuse after the brain is fully mature. Experiment one investigated the relationship between novelty preference and cocaine place preference in adolescent and adult rats. High responding adolescent rats displaying greater free choice novelty exploration (but not forced novelty locomotion) expressed decreased cocaine place conditioning compared to low responding rats. No relationship was found in adult rats. Experiment two evaluated novelty-induced behaviors in adulthood after adolescent cocaine exposure. Repeated cocaine administration produced greater stress and anxiogenic behavioral responses to novelty in adult rats. Repeated alcohol administration produced less-inhibited novelty-induced behaviors in adulthood. Experiment three and four evaluated the consequence of developmental cocaine exposure on the rewarding efficacy of cocaine in adolescence and adulthood. Additionally, the interaction of D2 receptors and the rewarding efficacy of cocaine were investigated. After developmental cocaine exposure, adolescent and adult rats demonstrate decreased rewarding efficacy to cocaine. Importantly, blockade of the D2 receptor prevents cocaine-induced neurochemical changes, potentially regulating the behavioral and neurochemical alterations that occur after repeated drug use that increases the likelihood of dependence. Together, these data implicate both short and long-term behavioral adaptations that occur after developmental cocaine exposure that may result in a predisposition to develop adulthood drug dependence

Neurochemical Effects of Cocaine in Adolescence Compared to Adulthood

Adolescence is a time of high risk behavior and increased exploration. This developmental period is marked by a greater probability to initiate drug use and is associated with an increased risk to develop addiction and adulthood dependency. Human adolescents are predisposed toward an increased likelihood of risk taking behaviors [M. Zuckerman, Sensation-seeking and the endogenous deficit theory of drug abuse. NIDA Res Monogr. 74 (1986) 59-70.], including drug use or initiation. In the present study, adolescent and adult animals were first tested on several behavioral measures (novel environment exploratory behavior, novel object preference, novelty-induced impulsivity and novelty-induced exploration) which were used to categorize them as high- (HR) or low-responders (LR). The purpose of the present study was to characterize the neurochemical responsivity of the nucleus accumbens septi (NAcc) in HR and LR adolescent and adult animals in response to a systemic challenge of cocaine. Regardless of age, animals that were more reactive when placed in a novel environment had greater cocaine-induced increases in dopamine (DA). Several important and complex neurochemical differences existed between adolescent and adult animals. Adolescent animals that rapidly approached the novel object (i.e., HR) were the only group to show greater cocaine-induced responsivity. However, adult animals that spent less time near the novel object (i.e., LR) were the only group to have greater cocaine-induced responsivity. Adolescent animals that approached a novel object faster (HR) showed an increased dopaminergic (DAergic) response to an acute cocaine challenge. In contrast, adolescent animals that spent less time with the novel object had an increased cocaine-induced DAergic response compared to HR adults. Adults that approached the object less had a greater cocaine-induced DA response relative to HR adults. Finally, cocaine yielded a greater DA response in adolescent animals that showed a high novelty-induced exploration and impulsivity response, whereas the opposite was true for adults. The differences in response to cocaine between ages and groups are likely due to ontogeneticdifferences in brain regions that are involved in reward and/or stress responsivity

The Potential Role of Dopaminergic Neuromodulation in Adolescence

Adolescence is a period when the brain is undergoing many complex changes that can exert long-term influences on decision making and cognitive processes. It is also a period of experimentation. Adolescents demonstrate a more abrupt progression of illicit drug use and development of substance use disorders than adults, suggesting that this ontogenetic period renders the adolescent more vulnerable to addiction. Development of the central nervous system (CNS) during adolescence may play a key role in the increased likelihood to initiate drug use. Topics discussed in this chapter include theories of addiction (anhedonia hypothesis; abberant learning theory; and incentive-sensitization theory); novelty preference and impulse control; conditioned place preference; mesolimbic dopamine (DA) pathway and reward; cocaine and mesolimbic DA system; mesolimbic DA pathway and behavior during adolescence; and impact of cocaine during adolescence

Chronic Cocaine or Ethanol Exposure During Adolescence Alters Novelty-Related Behaviors in Adulthood

Adolescence is a time of high-risk behavior and increased exploration. This developmental period is marked by a greater probability to initiate drug use and is associated with an increased risk to develop addiction and adulthood dependency and drug use at this time is associated with an increased risk. Human adolescents are predisposed toward an increased likelihood of risk-taking behaviors [Zuckerman M. Sensation seeking and the endogenous deficit theory of drug abuse. NIDA Res Monogr 1986;74:59–70.], including drug use or initiation. In the present study, adolescent animals were exposed to twenty days of either saline (0.9% sodium chloride), cocaine (20 mg/kg) or ethanol (1 g/kg) i.p. followed by a fifteen-day washout period. All animals were tested as adults on several behavioral measures including locomotor activity induced by a novel environment, time spent in the center of an open field, novelty preference and novel object exploration. Animals exposed to cocaine during adolescence and tested as adults exhibited a greater locomotor response in a novel environment, spent less time in the center of the novel open field and spent less time with a novel object, results that are indicative of a stress or anxiogenic response to novelty or a novel situation. Adolescent animals chronically administered ethanol and tested as adults, unlike cocaine-exposed were not different from controls in a novel environment, indicated by locomotor activity or time spent with a novel object. However, ethanol-exposed animals approached the novel object more, suggesting that exposure to ethanol during development may result in less-inhibited behaviors during adulthood. The differences in adult behavioral responses after drug exposure during adolescence are likely due to differences in the mechanisms of action of the drugs and subsequent reward and/or stress responsivity. Future studies are needed to determine the neural substrates of these long lasting drug-induced changes

Enhanced Nitric Oxide Production during Lead (Pb2+) Exposure Recovers Protein Expression but not Presynaptic Localization of Synaptic Proteins in Developing Hippocampal Neurons

We have previously reported that lead (Pb(2+)) exposure results in both presynaptic and postsynaptic changes in developing neurons as a result of inhibition of the N-methyl-D-aspartate receptor (NMDAR). NMDAR inhibition by Pb(2+) during synaptogenesis disrupts downstream trans-synaptic signaling of brain-derived neurotrophic factor (BDNF) and exogenous addition of BDNF can recover the effects of Pb(2+) on both presynaptic protein expression and presynaptic vesicular release. NMDAR activity can modulate other trans-synaptic signaling pathways, such as nitric oxide (NO) signaling. Thus, it is possible that other trans-synaptic pathways in addition to BDNF signaling may be disrupted by Pb(2+) exposure. The current study investigated whether exogenous addition of NO could recover the presynaptic vesicular proteins lost as a result of Pb(2+) exposure during synaptogenesis, namely Synaptophysin (Syn) and Synaptobrevin (Syb). We observed that exogenous addition of NO during Pb(2+) exposure results in complete recovery of whole-cell Syn levels and partial recovery of Syn and Syb synaptic targeting in Pb(2+)-exposed neurons

An Animal Model of Sensation Seeking: The Adolescent Rat

Previous research has established a strong relationship between a rodent\u27s preference for novelty and sensitivity to psychomotor stimulants. Rats with greater sensitivity to the motoric effects of amphetamine exhibit higher preferences for novelty. Additionally, animals with high novelty preference scores are more easily drug conditioned and are more sensitive to, and can more accurately discriminate, amphetamine doses. Novelty preference in animals has been compared to sensation seeking in humans and is strongly correlated with drug use and addiction vulnerability. Thus, the present studies employed a playground maze procedure to measure changes in novelty preference across age following either four or eight habituation trials using eight distinct objects. Early‐adult (postnatal day [PND] 59) animals did not exhibit a significant preference for a novel object regardless of total number of habituation trials. Early‐adolescent animals (PND 34) exhibited a preference for the novel object in fewer than four habituation trials, but exhibited no preference with increased habituation trials. These results are counterintuitive and may demonstrate an overgeneralization of the habituation trials specific to adolescent animals. Given that adolescence is a period of heightened exploration, one would expect adolescent animals to demonstrate an enhanced preference for novel stimuli using this paradigm. However, it is possible that the complexity of the task, as presented, reveals differences in the establishment and behavioral manifestation of associations during adolescence. To address this issue, a separate novelty paradigm was implemented using an open‐field habituation procedure followed by the introduction of a single novel object during the testing period. This revised design provides the foundation needed to better assess novelty‐induced locomotor activity and novelty preference in adolescent rats

Chronic early life lead (Pb2+) exposure alters presynaptic vesicle pools in hippocampal synapses

BACKGROUND: Lead (Pb(2+)) exposure has been shown to impair presynaptic neurotransmitter release in both in vivo and in vitro model systems. The mechanism by which Pb(2+) impairs neurotransmitter release has not been fully elucidated. In previous work, we have shown that Pb(2+) exposure inhibits vesicular release and reduces the number of fast-releasing sites in cultured hippocampal neurons. We have also shown that Pb(2+) exposure inhibits vesicular release and alters the distribution of presynaptic vesicles in Shaffer Collateral – CA1 synapses of rodents chronically exposed to Pb(2+) during development. METHODS: In the present study, we used transmission electron microscopy to examine presynaptic vesicle pools in Mossy Fiber-CA3 synapses and in Perforant Path-Dentate Gyrus synapses of rats to determine if in vivo Pb(2+) exposure altered presynaptic vesicle distribution in these hippocampal regions. Data were analyzed using T-test for each experimental endpoint. RESULTS: We found that Pb(2+) exposure significantly reduced the number of vesicles in the readily releasable pool and recycling pool in Mossy Fiber-CA3 terminals. In both Mossy Fiber-CA3 terminals and in Perforant Path-Dentate Gyrus terminals, Pb(2+) exposure significantly increased vesicle nearest neighbor distance in all vesicular pools (Rapidly Releasable, Recycling and Resting). We also found a reduction in the size of the postsynaptic densities of CA3 dendrites in the Pb(2+) exposed group. CONCLUSIONS: In our previous work, we have demonstrated that Pb(2+) exposure impairs vesicular release in Shaffer Collateral - CA1 terminals of the hippocampus and that the number of docked vesicles in the presynaptic active zone was reduced. Our current data shows that Pb(2+) exposure reduces the number of vesicles that are in proximity to release sites in Mossy Fiber- CA3 terminals. Furthermore, Pb(2+) exposure causes presynaptic vesicles to be further from one another, in both Mossy Fiber- CA3 terminals and in Perforant Pathway – Dentate Gyrus terminals, which may interfere with vesicle movement and release. Our findings provide a novel in vivo mechanism by which Pb(2+) exposure impairs vesicle dynamics and release in the hippocampus