Abstract:
Tetanic stimulation at 50 Hz elicited long-term potentiation (LTP) in the hippocampal CA1 region of 28-day-old control animals. In contrast, no significant potentiation was seen following the same tetanus in animals chronically exposed to 1000 ppm of lead acetate during development. Gender differences were observed in animals exposed to 500 ppm of lead. In females, LTP was significantly attenuated, while in males the LTP was identical to that of controls.
Keywords :
Development; Heavy metal; Learning; Memory; Neurotoxicity; Synaptic plasticity
Long-term potentiation (LTP) in the hippocampus is a form of activity-dependent synaptic plasticity that may be the electrophysiological substrate for learning and memory. Previous studies have reported that both acute and chronic lead exposure impair LTP in the hippocampus in vitro and in vivo.
The developing nervous system is more susceptible to lead toxicity than the mature nervous system, and the lowest blood lead levels at which toxic adverse effects occur are lower for children (10-15 mg/dl) than for adults (40 mg/dl).
Experiment introduction
Twenty one pregnant female Harlan Sprague-Dawley rats were randomly and equally divided into three treatment groups, and exposed to lead acetate at 0, 500 or 1000 ppm in the drinking water for one week prior to mating.
On postnatal day 1, all litters were culled to six pups (three per sex) to insure equal access for each pup to the dam's milk.
On postnatal 21, the pups were weaned and housed by sex and treatment group.
Pups were therefore exposed to 0, 500, or 1000 ppm lead acetate throughout develop ment (in utero, during lactation via the milk, and upon weaning via the drinking water).
On postnatal day 28, a male and a female pup from each litter were anesthetized with urethane (1.2-1.5 g/ kg, or to effect, i.p.). Body temperature was maintained between 36 and 37.58C with a heating pad. Each animal was placed in a stereotaxic frame and two craniotomies were made. A concentric bipolar stimulating electrode was lowered into the CA3 region of the hippocampus (3.0 mm posterior to the bregma, 3.5 mm lateral, and 1.3-1.9 mm ventral to the surface of the cerebrum). A glass recording microelectrode filled with 2 M NaCl (1-3 MV at 12 kHz) was placed in the contralateral CA1 region (3.0 mm posterior to the bregma, 2.0 mm lateral, and 1.0-1.5 mm ventral).
The stimulating electrode was adjusted until a stable and maximal population spike was obtained (increasing stimulation strength beyond this 'maximal' voltage produced no further increases in spike potential amplitude). Single, 100 ms long square wave pulses were then delivered at 1 minintervals during a 5-min baseline recording period.
To elicit LTP, stimulation strength was decreased to one-half the maximal single-shock response in a given animal, and tetanic stimulation was delivered at 50 Hz (one 400 ms train of 20 100 ms square-wave pulses). Stimulation was then returned to baseline (maximal) strength and frequency (1/min), and recordings continued for another 4 h post-tetanus.
For data analysis, the population spike heights of the five responses immediately preceding tetanus were averaged, and all post-tetanic responses were expressed as the percent change relative to this pre-tetanic average. The normalized post-tetanic responses in each animal were averaged over the entire experiment (4 h of continuous data combined), and the group averages were compared using ANOVA (3 groups32 sex). Statistical analysis was based on data obtained from seven male and seven female rats with each pair representing a single litter.
The CA3-evoked CA1 baseline (pre-tetanic) population spike amplitudes varied between 3 and 10 mV at maximal stimulation intensities of 70 to 90 V.
Tetanic stimulation at 50 Hz elicited LTP in the population spike of hippocampal CA1 neurons in all control animals (male and female 0 ppm lead acetate exposure groups). There were no differences in the magnitude or time-course of the increase in population spike amplitude between genders In contrast, no significant potentiation in population spike amplitude was seen following tetanic stimulation at 50 Hz in either male or female rats exposed chronically to 1000 ppm of lead acetate.
Interestingly, there was a gender difference in post-tetanic responses of animals chronically exposed to the lower dose (500 ppm) of lead-acetate. In males, the increase in population spike amplitude following the 50 Hz tetanus was essentially identical to that of controls. On the other hand, in females no significant potentiation was observed, and the post-tetanic responses were indistinguishable from those of animals chronically exposed to 1000 ppm of lead acetate Statistically, the 500 ppm lead acetate female group was significantly different from controls (P>0.003), but not from the 1000 ppm lead acetate group.
Comparison with precious evidence
In agreement with previous reports, the data indicated that exposure to environmentally relevant lead concentrations (1000 ppm) significantly modified LTP in both male and female rats exhibiting clinically relevant blood lead levels (.40 mg/dl). The association between lead exposure and LTP impairment in young rats may be, at least in part, a result of the interaction between the heavy metal and protein kinase C (PKC). This enzyme family is involved in numerous cellular processes , and some of its isozymes appear to play an important role in learning and memory because of their critical contribution to LTP . Lead could directly affect PKC function through complex interactions by binding to the molecule at multiple sites . We have recently reported that the normal developmental increase in hippocampal PKC activity was significantly attenuated in animals chronically exposed to 1000 ppm of lead acetate . In addition, developmental lead exposure may adversely affect neuronal differentiation and growth, astrocyte development, cholinergic receptors, and the glutamatergic system in the hippocampus . Consequently, the impairment of normal hippocampal function may be one important outcome of developmental lead neurotoxicity.
Chronic exposure to 500 ppm of lead resulted in genderdependent effects. In males, the magnitude and duration of LTP were identical to those in controls, while in females LTP was significantly attenuated. These results differ from our study in adult rats in two ways . First, previously we had not observed gender differences in the post-tetanic responses of adult animals chronically exposed to 500 ppm of lead acetate. Second, in both male and female adults (unlike in young males) the 500 ppm dose of the heavy metal significantly attenuated the late (maintenance) phase, while having no effect on the early (induction) phase of LTP.
Discussion
It is unclear why young females appear more sensitive to chronic lead exposure at 500 ppm. Blood lead level measurements indicated that females carried similar amounts of lead per volume of blood as males, rendering unlikely the possibility that females carried a heavier body burden because they were smaller. Literature searches have been unsuccessful in documenting significant differences between males and females in terms of absorption, distribution and excretion of lead, as well as any major differences in brain or hippocampal protein content or function. It may be that during maturation hormonal influences alone contributed to the gender-dependent effects. However, this presently remains only a vague speculation.
Similarly, it is unclear why the full expression of LTP in young males was unaffected by lead exposure at 500 ppm while it was impaired in the adult. One possible explanation is that there are developmental factors affecting the presence or function of protein kinases contributing to LTP.
Tetanic stimulation at 50 Hz elicited long-term potentiation (LTP) in the hippocampal CA1 region of 28-day-old control animals. In contrast, no significant potentiation was seen following the same tetanus in animals chronically exposed to 1000 ppm of lead acetate during development. Gender differences were observed in animals exposed to 500 ppm of lead. In females, LTP was significantly attenuated, while in males the LTP was identical to that of controls.
Keywords :
Development; Heavy metal; Learning; Memory; Neurotoxicity; Synaptic plasticity
Long-term potentiation (LTP) in the hippocampus is a form of activity-dependent synaptic plasticity that may be the electrophysiological substrate for learning and memory. Previous studies have reported that both acute and chronic lead exposure impair LTP in the hippocampus in vitro and in vivo.
The developing nervous system is more susceptible to lead toxicity than the mature nervous system, and the lowest blood lead levels at which toxic adverse effects occur are lower for children (10-15 mg/dl) than for adults (40 mg/dl).
Experiment introduction
Twenty one pregnant female Harlan Sprague-Dawley rats were randomly and equally divided into three treatment groups, and exposed to lead acetate at 0, 500 or 1000 ppm in the drinking water for one week prior to mating.
On postnatal day 1, all litters were culled to six pups (three per sex) to insure equal access for each pup to the dam's milk.
On postnatal 21, the pups were weaned and housed by sex and treatment group.
Pups were therefore exposed to 0, 500, or 1000 ppm lead acetate throughout develop ment (in utero, during lactation via the milk, and upon weaning via the drinking water).
On postnatal day 28, a male and a female pup from each litter were anesthetized with urethane (1.2-1.5 g/ kg, or to effect, i.p.). Body temperature was maintained between 36 and 37.58C with a heating pad. Each animal was placed in a stereotaxic frame and two craniotomies were made. A concentric bipolar stimulating electrode was lowered into the CA3 region of the hippocampus (3.0 mm posterior to the bregma, 3.5 mm lateral, and 1.3-1.9 mm ventral to the surface of the cerebrum). A glass recording microelectrode filled with 2 M NaCl (1-3 MV at 12 kHz) was placed in the contralateral CA1 region (3.0 mm posterior to the bregma, 2.0 mm lateral, and 1.0-1.5 mm ventral).
The stimulating electrode was adjusted until a stable and maximal population spike was obtained (increasing stimulation strength beyond this 'maximal' voltage produced no further increases in spike potential amplitude). Single, 100 ms long square wave pulses were then delivered at 1 minintervals during a 5-min baseline recording period.
To elicit LTP, stimulation strength was decreased to one-half the maximal single-shock response in a given animal, and tetanic stimulation was delivered at 50 Hz (one 400 ms train of 20 100 ms square-wave pulses). Stimulation was then returned to baseline (maximal) strength and frequency (1/min), and recordings continued for another 4 h post-tetanus.
For data analysis, the population spike heights of the five responses immediately preceding tetanus were averaged, and all post-tetanic responses were expressed as the percent change relative to this pre-tetanic average. The normalized post-tetanic responses in each animal were averaged over the entire experiment (4 h of continuous data combined), and the group averages were compared using ANOVA (3 groups32 sex). Statistical analysis was based on data obtained from seven male and seven female rats with each pair representing a single litter.
The CA3-evoked CA1 baseline (pre-tetanic) population spike amplitudes varied between 3 and 10 mV at maximal stimulation intensities of 70 to 90 V.
Tetanic stimulation at 50 Hz elicited LTP in the population spike of hippocampal CA1 neurons in all control animals (male and female 0 ppm lead acetate exposure groups). There were no differences in the magnitude or time-course of the increase in population spike amplitude between genders In contrast, no significant potentiation in population spike amplitude was seen following tetanic stimulation at 50 Hz in either male or female rats exposed chronically to 1000 ppm of lead acetate.
Interestingly, there was a gender difference in post-tetanic responses of animals chronically exposed to the lower dose (500 ppm) of lead-acetate. In males, the increase in population spike amplitude following the 50 Hz tetanus was essentially identical to that of controls. On the other hand, in females no significant potentiation was observed, and the post-tetanic responses were indistinguishable from those of animals chronically exposed to 1000 ppm of lead acetate Statistically, the 500 ppm lead acetate female group was significantly different from controls (P>0.003), but not from the 1000 ppm lead acetate group.
Comparison with precious evidence
In agreement with previous reports, the data indicated that exposure to environmentally relevant lead concentrations (1000 ppm) significantly modified LTP in both male and female rats exhibiting clinically relevant blood lead levels (.40 mg/dl). The association between lead exposure and LTP impairment in young rats may be, at least in part, a result of the interaction between the heavy metal and protein kinase C (PKC). This enzyme family is involved in numerous cellular processes , and some of its isozymes appear to play an important role in learning and memory because of their critical contribution to LTP . Lead could directly affect PKC function through complex interactions by binding to the molecule at multiple sites . We have recently reported that the normal developmental increase in hippocampal PKC activity was significantly attenuated in animals chronically exposed to 1000 ppm of lead acetate . In addition, developmental lead exposure may adversely affect neuronal differentiation and growth, astrocyte development, cholinergic receptors, and the glutamatergic system in the hippocampus . Consequently, the impairment of normal hippocampal function may be one important outcome of developmental lead neurotoxicity.
Chronic exposure to 500 ppm of lead resulted in genderdependent effects. In males, the magnitude and duration of LTP were identical to those in controls, while in females LTP was significantly attenuated. These results differ from our study in adult rats in two ways . First, previously we had not observed gender differences in the post-tetanic responses of adult animals chronically exposed to 500 ppm of lead acetate. Second, in both male and female adults (unlike in young males) the 500 ppm dose of the heavy metal significantly attenuated the late (maintenance) phase, while having no effect on the early (induction) phase of LTP.
Discussion
It is unclear why young females appear more sensitive to chronic lead exposure at 500 ppm. Blood lead level measurements indicated that females carried similar amounts of lead per volume of blood as males, rendering unlikely the possibility that females carried a heavier body burden because they were smaller. Literature searches have been unsuccessful in documenting significant differences between males and females in terms of absorption, distribution and excretion of lead, as well as any major differences in brain or hippocampal protein content or function. It may be that during maturation hormonal influences alone contributed to the gender-dependent effects. However, this presently remains only a vague speculation.
Similarly, it is unclear why the full expression of LTP in young males was unaffected by lead exposure at 500 ppm while it was impaired in the adult. One possible explanation is that there are developmental factors affecting the presence or function of protein kinases contributing to LTP.
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