![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
NEUROPHARMACOLOGY
Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Wayne State University, Detroit, Michigan
Received August 12, 2004; accepted November 18, 2004.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
).
The Centers for Disease Control and Prevention currently considers elevated blood lead to be greater or equal to 10 µg/dl. Increasingly, lead is being found to affect brain function at levels below 10 µg/dl (Canfield et al., 2003a,b; Lidsky and Schneider, 2003). Lead exposure during postnatal development has been associated with impairments in cognition, learning, and behavioral function (Needleman, 1993). Behavioral alterations such as reduced attention span, learning deficits, impaired cognition, hyperactivity, impulsiveness, and aggressiveness have been found in children exposed to lead (Needleman, 1993; Wakefield, 2002). Studies on the relationship between lead levels and attention deficit disorder have shown that there is a positive relationship between hair lead levels of children and their attention deficit behaviors in the classroom (Minder et al., 1994; Tuthill, 1996). Canfield et al. (2003b) found that blood lead level was negatively associated with focused attention while performing tasks and suggest that very low-level lead exposure may disrupt the dopamine system function in young children.
In the last several decades, many experimental studies have been performed addressing lead neurotoxicity, yet the exact mechanism for the toxic effects on the nervous system remains unclear. Many of the classical neurotransmitter systems, including dopaminergic, cholinergic, GABAergic, and glutamatergic are affected by lead (e.g., see Shao and Suszkiw, 1991; Cory-Slectha, 1997; Guilarte, 1997; Braga et al., 1999; Lasley and Gilbert, 2002). It is likely that there are numerous targets of lead that are associated with various neurotransmitter mechanisms and that these various mechanisms participate in exposure level-dependent pathological responses once specific thresholds have been reached.
The effect of lead on the dopaminergic system has been reported to occur at clinically relevant blood levels (Kala and Jadhav, 1995a; Cory-Slechta, 1997; Tavakoli-Nezhad et al., 2001; Lewis and Pitts, 2004) in the rat. Many neurochemical studies have reported alterations in DA release, DA turnover, and changes in DA receptor numbers (Nation et al., 1989; Lasley, 1992; Kala and Jadhav, 1995a,b; Pokora et al., 1996; Cory-Slechta, 1997; Zuch et al., 1998). The midbrain DA system is involved in cognition, attention and motor function, and has been shown to be an important participant in many neurological and psychiatric disorders. The midbrain DA system may play a role in the etiology of attention deficit disorder, and as indicated above, lead exposure is a probable risk factor in this disorder (Minder et al., 1994; Tuthill, 1996; Canfield et al., 2003b). It has also been suggested that lead intoxication may be a risk factor in Parkinson's disease (Kuhn et al., 1998), a disease associated with the relatively selective loss of DA-containing neurons in the substantia nigra.
Understanding the impact of lead exposure on the electrophysiological activity of the midbrain DA-containing neurons in the ventral tegmental area (VTA), substantia nigra (SN), and on target neurons in the nucleus accumbens is a necessary and integral step toward understanding the mechanism of the lead-induced neurochemical changes. However, to date, there has been only one report examining the effect of lead on the activity of single midbrain DA neurons by Tavakoli-Nezhad et al. (2001). The present study is an extension of the previous study by Tavakoli-Nezhad et al. (2001) and further examines the effects of postnatal lead exposure on both pre- and postsynaptic components of the midbrain DA system using electrophysiological and immunohistochemical techniques.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Extracellular Electrophysiological Recording and Neuron Identification. The rats were anesthetized with either chloral hydrate (400 mg/kg i.p.) for studies of midbrain DA neurons or urethane (1.25 g/kg i.p.) for studies of nucleus accumbens neurons (under Iontophoresis: Nucleus Accumbens Neurons) and placed in a stereotaxic apparatus. Body temperature was maintained at 37 ± 1°C with a heating pad. Single-unit extracellular recordings were made from spontaneously active DA neurons with single-barrel glass micropipettes (1.5-mm diameter) broken to achieve a tip diameter of approximately 1 to 2 µm (five-barrel pipettes were used for iontophoresis, see below). The pipettes were filled with a 2 M NaCl solution containing 2% pontamine sky blue dye. Electrode impedance typically measured 1.8 to 2.5 megohms at 15 Hz in physiological saline.
Extracellular signals were amplified by a high impedance amplifier (EX1 differential amplifier; Dagan, Minneapolis, MN) with bandpass settings of 300 Hz to 3 kHz. The amplified signal was then sent to an analog oscilloscope, an audiomonitor, a window discriminator (WD-2; Dagan), and via a digital interface (M-100; Modular Instruments, Inc., Southeastern, PA) to a Pentium computer. Two animals were recorded on a given experimental day, one lead- and one sodium-treated.
Midbrain DA neurons were identified by well established electrophysiological criteria (Grace and Bunney, 1980; Bunney et al., 1991). The electrophysiological characteristics used to identify midbrain DA neurons included the following: 1) biphasic positive-negative action potential (Fig. 1A) located in the ventral midbrain approximately 6.5 to 8.5 mm below the brain surface (see other coordinates relative to lambda and sagittal suture below), 2) a very long duration action potential (range: 2.0 to 4.0 ms) with an initial segment-somatodendritic break on the ascending limb (Fig. 1), 3) a discharge pattern consisting of either an irregular single-spike mode or burst mode with each burst containing three to eight spikes of decreasing amplitude and increasing duration, and 4) a discharge rate of approximately 0.5 to 10 spikes per second (mean 
4.0). The midbrain DA neurons can be easily distinguished from neighboring non-DA neurons (e.g., the GABAergic reticulata neurons of the SN). The non-DA neurons of the SN and VTA generally have a much more narrow spike width (<1.7 ms) that lacks an initial segment-somatodendritic break, a higher average discharge rate, and different discharge patterns (Grace and Bunney, 1979).
|
Nucleus accumbens neurons were studied under urethane anesthesia as described because chloral hydrate appears to suppress the activity of spontaneously active neurons more than urethane. Nucleus accumbens neurons were found approximately 7.5 mm below the cortical surface. Stereotaxic coordinates for recording nucleus accumbens neurons were as follows: 10.2 to 10.8 mm anterior relative to lambda and 0.9 to 1.6 mm lateral to midline. Type I neurons were identified by their negative-positive extracellular waveforms, respectively (Shen et al., 1992) (Fig. 1B).
Cells Per Track Assay: Midbrain DA Neurons. To systematically sample midbrain DA neuron activity, stereotaxic coordinates (see below) were used to define a block of tissue (e.g., see Chiodo and Bunney, 1983; White and Wang, 1983; Tavakoli-Nezhad et al., 2001). Each neuron located was monitored for approximately 3 to 5 min to assess discharge rate and discharge pattern. For each of the nuclei (VTA or SN), mean values were calculated for the number of cells per track and for the average firing rate. The average firing rate was calculated as the mean of all VTA and the mean of all SN DA neurons for each rat.
From the age of 22 days old to 9 weeks old, rats were chronically exposed to either lead- or sodium-acetate via drinking water. In addition, a third group was given haloperidol (HAL) injections (0.5 mg/kg/day i.p.) between 8:00 and 10:00 AM each day over the same time period. To test if depolarization inactivation contributed to changes in the spontaneous activity of DA neurons after lead exposure, apomorphine (50 µg/kg i.v.) was administered to sodium-, lead-, and HAL-treated groups as a "hyperpolarizing" challenge. The HAL-treated group was given a morning injection of haloperidol approximately 2 to 3 h before surgical preparation for the cells per track assay. The cells per track assay was performed before and after apomorphine. In this cell per track study, the number of spontaneous active DA neurons was counted in 12 electrode tracks (six in SN and six in VTA) in a given subject before apomorphine challenge. An additional 12 electrode tracks (six in SN and six in VTA) was then recorded after apomorphine challenge.
Two basic sets of region-targeted coordinates were used in the apomorphine challenge study, which corresponded to the two areas of the midbrain studied, the VTA and SN. The specific coordinates used within a given targeted region (VTA or SN) were also systematically varied in relation to the timing of the apomorphine challenge with the specific coordinates used for a midbrain nucleus changing after apomorphine administration. The coordinates used for the VTA were as follows: 2.8 and 3.2 mm anterior to lambda and 0.4, 0.6, and 0.8 mm lateral to sagittal suture before apomorphine, and 3.0 and 3.4 mm anterior to lambda and 0.4, 0.6, and 0.8 mm lateral to sagittal suture after apomorphine. The coordinates used for the SN before apomorphine were 2.8 and 3.2 mm anterior to lambda and 2.0, 2.2, and 2.4 mm lateral to sagittal suture, and after apomorphine, the SN coordinates were 3.0 and 3.4 mm anterior to lambda and 2.0, 2.2, and 2.4 mm lateral to sagittal suture. Note that the anterior coordinates change according to the status of the apomorphine challenge, not the lateral coordinates. Finally, for a given region (VTA or SN), the sequence of the anterior coordinates that related to the apomorphine challenge was reversed for one half of the animals studied. For example, one half of the animals used 3.0 and 3.4 mm anterior to lambda before apomorphine and 2.8 and 3.2 mm anterior to lambda after apomorphine. The number of spontaneously active cells, discharge rate, and percentage of bursting cell were calculated in both regions with the presence and absence of apomorphine being treated as a within-subjects factor (see Data Analysis below for more details).
Discharge Pattern Analysis: Midbrain DA Neurons. DA neurons were classified as bursting or nonbursting in each cell per track experiment using computer software. Briefly, interspike interval (ISI) histograms consisting of 500 consecutive active potentials were collected from DA neurons as previously described (Tavakoli-Nezhad et al., 2001). To distinguish bursting cells from nonbursting cells, the following criteria were used: 1) bursts were defined to begin within any ISI less than 80 ms and to terminate with the first ISI greater than 160 ms, and 2) a minimum of two, three-spike bursts were encountered within the sample of 500 consecutive action potentials (see Zhang et al., 1996). The mean value was calculated for the proportion of bursting cells found in each animal, and this value was used in inferential statistical tests.
Iontophoresis: Nucleus Accumbens Neurons. Extracellular neuronal signals were recorded through the center barrel of five-barrel micropipettes (ASI Instruments, Inc., Warren, MI) that were broken back to a diameter of approximately 4 to 7 µm. The center barrel was filled with 2 M NaCl and 2% pontamine sky blue. The impedance in the center barrel was typically 2 to 4 megohms. One of the side barrels was used for current balance and contained 4 M NaCl. Tip currents were automatically neutralized by this side barrel. The impedance of the balance barrel was typically 10 to 20 megohms in physiological saline. The other side barrels were filled with DA D1 receptor agonist, (±)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride [(±) SKF-38393 HCl] (10 mM, dissolved in 0.9% saline, pH 4.0).
These experiments focused on determining the sensitivity of D1 receptors to the D1 agonist, SKF-38393. SKF-38393 ejection currents (e.g., 20, 40, 60, and 80 nA) were applied after an initial warm up period, which was established with low currents. A positive current was used for the ejection of SKF-38393. A retaining current of -10 nA was applied to the drug barrels between periods of drug application. The DA agonist was ejected for a period of 70 s at each applied current. The D1 DA selective agonist was used to compare the sensitivity of D1 receptors in lead- and sodium-treated animals. A fixed number of cells (i.e., one to three) was studied per animal, and a total number of about 25 neurons (e.g., 12 animals) was examined in a given experimental group.
Tyrosine Hydroxylase Immunohistochemistry. Since it was possible that some DA neurons recorded in electrophysiological experiments might be undergoing apoptotic or necrotic processes following lead exposure, animals used for immunohistochemical studies were treated for longer periods than that used in electrophysiological studies, 7 to 13 weeks rather than 3 to 6 weeks. The immunohistochemical experiments were designed to test the influence of lead on DA neuron viability.
Animals were given an overdose of chloral hydrate at the end of the exposure period, and just prior to complete cessation of respiration the thoracic cavity was opened surgically. A syringe needle was inserted into the left ventricle of the heart, the right atrium was severed, and the animal was then perfused with ice-cold phosphate-buffered saline until the blood exiting the right atrium ran clear. The perfusate was then changed to a chilled phosphate-buffered paraformaldehyde solution (4% prepared fresh each day). The brains were postfixed in phosphate-buffered saline for an hour and then stored in a 20-ml vial containing 10% sucrose in paraformaldehyde solution overnight at 4°C.
Tissue sections for lead-exposed and control animals were processed at the same time to minimize variation in the staining procedure. Forty-micron sections of midbrain were made on a freezing microtome, and the sections floated in chilled Tris-buffered saline (TBS). All sections were then incubated in TBS with 10% methanol and 3% hydrogen peroxide for 5 min to block endogenous peroxidase. This was followed by three 10-min rinses in TBS. The rinsed sections were incubated overnight in TBS/10% normal goat serum (NGS) to block nonspecific binding at 4°C. The next day the sections were rinsed with TBS and then incubated in a monoclonal primary antibody (1:2000) for tyrosine hydroxylase (TH) overnight at 4°C (in TBS/2% NGS with 0.3% Triton X-100). Following three 10-min rinses in TBS, the sections were incubated in the secondary antibody, goat anti-mouse IgG, for 3 h (in TBS/2% NGS with 0.3% Triton X-100). The incubation with secondary antibody is followed by the standard TBS rinses. The sections were then incubated in mouse peroxidase-antiperoxidase for 1 h (TBS/NGS/Triton X-100) to tag the TH enzyme with peroxidase activity. This was followed by one 10-min rinse in TBS and two 10-min rinses in Tris buffer. The sections were next incubated with a diaminobenzidine solution until the dopamine-containing neurons become visible (approximately 15 min). The reaction was stopped by placing the sections in Tris buffer. Midbrain sections were then mounted on slides.
Light Microscopy and Cell Counts. Sections of the midbrain containing TH-positive [TH (+)] cells in the VTA and SN were examined using a light microscope equipped with an eyepiece grid. Slides were selected by specific anatomical criteria for each animal to represent three standard sections containing the midbrain DA neurons (Shen et al., 1999; Tavakoli-Nezhad et al., 2001), approximately 4.2, 3.6, and 3.0 mm anterior to the interaural line (Paxinos and Watson, 1986). These sections represented the anterior, middle, and posterior parts of the SN and VTA. Due to the distance between the selected sections (i.e., 0.6 mm), the same cells are not represented on these adjacent sections.
Sections were obtained from the brains of sodium- and lead-exposed animals that were age-matched. The anatomical features used to distinguish the SN and VTA in these selected sections included: basal cerebral peduncle, mammillary body, oculomotor nerve and roots, and medial lemniscus (e.g., see Paxinos and Watson, 1986). The division between VTA and SN was considered to be a ventral-dorsal line approximately 1.5, 1.2, and 1.2 mm lateral from midline corresponding to the 4.2, 3.6, and 3.0 mm sections, respectively (described above). The cells were counted at 200x magnification. The total number of SN and VTA TH (+) cells in a given midbrain section were counted in the two areas defined by the ventral-dorsal division described above. For each rat, TH (+) cells in the SN and VTA area of one side of the midbrain was counted in the three sections described above. The TH (+) counts for each nucleus were expressed in results as the average number of cells per section (i.e., average of three sections) corresponding to the SN or VTA. This procedure generated two mean cell counts per animal that corresponded to the SN and VTA regions, respectively.
Determination of Blood Lead Level. The blood lead level was measured by an electrolytic voltametric stripping method using a LeadCare blood lead testing system from ESA, Inc. (Chelmsford, MA). The lower limit of detection for this instrument is 1.4 µg/dl. The group of animals used to determine blood lead levels was not used in electrophysiological experiments. This cohort of animals was treated in a manner identical to that of animals used in electrophysiological studies.
Data Analysis. In the electrophysiological studies, the data from lead-exposed offspring were compared with sodium-exposed controls matched daily for their age and duration of treatment. In the electrophysiological studies of midbrain DA neurons, both anatomical regions (SN and VTA) were examined in each rat exposed to lead- or sodium-acetate. The mean values for number of cells per track, discharge rate, and percentage of cells with burst discharges calculated for each animal were analyzed as dependent variables for treatment effects using a three-way analysis of variance (ANOVA) with repeated measures on two variables, cell type (SN or VTA), and apomorphine challenge (before or after). Subsequent planned comparison of means was conducted using contrast analysis or the Fisher's LSD test. In the iontophoretic study, a difference between the mean basal firing rates of the cells sampled from the two treatment groups was observed. The iontophoresis results were therefore analyzed using repeated measures analysis of covariance (ANCOVA) with ejection current as a repeated measures factor, treatment as a between-subjects factor, discharge rate as the dependent variable, and basal discharge rate as the covariate.
In the immunohistochemical study, two mean values (one for SN and one for VTA) were analyzed for treatment effects using ANCOVA with repeated measures (i.e., cell type). The two factors in this analysis were treatment (sodium/lead) and cell type (SN/VTA). The covariate was duration of sodium/lead exposure. In a similar fashion, a multivariate analysis of covariance was used to simultaneously analyze the two dependent variables body weight and brain weight using duration of exposure as a covariate. Blood lead levels were analyzed using ANCOVA with duration of exposure as a covariate. The use of ANCOVA in the analysis of electrophysiological data has been previously described by Pitts et al. (1990).
Drugs and Chemicals. Apomorphine hydrochloride, haloperidol, (±) SKF-38393 hydrochloride, normal goat serum, and goat anti-mouse IgG were purchased from Sigma-Aldrich (St. Louis, MO). Apomorphine hydrochloride was dissolved in 0.9% saline for i.v. administration. Haloperidol was dissolved in dimethyl sulfoxide (0.5 mg/200 µl) for i.p. administration (Zhang et al., 1996). The monoclonal primary antibody (LNC1) for TH was a gift from Dr. Greg Kapatos, Wayne State University.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
2%). The blood lead level of controls was found to be below detection (<1.4 µg/dl, n = 7) or at a very low level of 2.1 ± 0.4 µg/dl (n = 5) relative to lead-treated animals. ANCOVA indicated that lead treatment significantly (P < 0.001) elevated blood lead levels to a mean value of 30.8 ± 1.2. There was not a significant relationship between the covariate, duration of exposure, and the blood lead level (P > 0.30). A one-way ANOVA examining only the lead-treated group did not reveal any significant main effect of treatment duration (3 to 6 weeks) on blood lead levels (P > 0.20). This analysis of the blood lead levels indicates that blood levels were relatively stable between the 3rd and 6th week of exposure.
|
Electrophysiological Study of Midbrain DA Neurons. Electrophysiological studies were conducted to determine the number of spontaneously active midbrain dopamine DA-containing neurons (Fig. 1A) per electrode track in sodium/lead-treated animals before and after an acute apomorphine (APO) challenge dose (50 µg/kg i.v.). Three different post-weaning treatment groups examined are sodium treated (250 ppm, n = 15), lead treated (250 ppm, n = 15), and haloperidol treated (500 µg/kg i.p., n = 14). Figure 2 illustrates the effect of apomorphine treatment on the number of spontaneously active DA neurons in the SN (panel A) and VTA (panel B), respectively. Three-way ANOVA indicated that there was a significant APO main effect (P < 0.05) and a significant treatment (sodium/lead/HAL) x APO interaction (P < 0.001). Fisher's LSD test indicated that lead treatment, prior to APO challenge, significantly decreased the number of spontaneously active DA neurons per electrode track in both the VTA (P < 0.05) and the SN (P < 0.05). In the sodium-treated animals, the APO challenge significantly reduced the number of spontaneously active DA neurons in both the SN (P < 0.05, Fisher's LSD) and VTA (P < 0.001, Fisher's LSD). APO did not significantly alter the number of spontaneously active VTA DA neurons (P > 0.50, Fisher's LSD) or SN DA neurons (P > 0.40) in the lead-treated animals relative to the within-subject pre-APO control. However, in the HAL-treated animals, APO challenge did result in a significant increase in the number of spontaneously active VTA DA neurons (P < 0.05, Fisher's LSD), but not SN VTA neurons (P > 0.50) relative to within-subject pre-APO control.
|
The cells per track analysis indicates that both lead and HAL treatment significantly decreased the number of spontaneously active VTA DA neurons, but APO only "reversed" the effects on the VTA DA neurons in the HAL group. Although the number of spontaneously active SN DA neurons before APO challenge was significantly decreased by lead, the number of active neurons was not significantly reduced by HAL (P > 0.20 relative to sodium-treated, Fisher's LSD). APO did not significantly affect either lead- or HAL-treated neurons relative to their within-subject pre-APO challenge control in the SN.
The effect of apomorphine challenge on the discharge rate of spontaneously active DA neurons in lead/sodium/HAL-treated animals before and after APO challenge in SN and VTA is shown in Fig. 2, panel C and D, respectively. A three-way analysis of variance indicated that there was a significant treatment main effect (P < 0.05) but no other significant main effects or interactions. When ignoring cell type and APO challenge as factors, contrast analysis indicated that HAL treatment significantly increased discharge rate relative to lead treatment (P < 0.05) but not sodium treatment (P > 0.05). Contrast analysis applied in a similar manner did not detect any significant differences between lead and sodium treatment (P > 0.30). There was a trend for a treatment (sodium/lead/HAL) x APO x cell type interaction (P < 0.08).
The effect of apomorphine challenge on DA neuron burst discharges in all three groups (HAL/sodium/lead) in the VTA and SN can be seen in panel E and F, respectively, of Fig. 2. There was a significant treatment effect (P < 0.005) and a treatment x cell type interaction (P < 0.01), but no other significant main effects or interactions. When the apomorphine challenge factor is ignored, contrast analysis indicates that there is a significant difference in the percentage of bursting neurons between lead- and HAL-treated animals in both VTA (P < 0.05) and SN (P < 0.05). There is also a significant difference in the percentage of bursting neurons between sodium- and HAL-treated animals in SN (P < 0.001) but not in the VTA (P > 0.05). No significant difference in percentage of bursting neurons between lead- and sodium-treated groups was found (P > 0.10 in both cases).
TH Immunohistochemistry. Table 2 shows the number of TH (+) cells counted in the SN and VTA from a longer duration 250-ppm exposure (i.e., 7 to 13 weeks) than that used in the electrophysiology studies. A two-way ANCOVA was used to evaluate the number of TH (+) cells using duration of exposure as the covariate (7 to 13 weeks). No significant differences in the number of TH (+) cells were found between the lead- and sodium-treated animals (P > 0.50 for treatment main effect, P > 0.20 for treatment x cell type interaction). There was a significant cell type main effect (P < 0.0001) indicating a higher number of TH (+) cells in the VTA.
|
Even when the duration of exposure for the 250-ppm exposure is increased relative to that used in the electrophysiological studies (increased to 7 to 13 weeks), these immunohistochemical results indicate that there are no significant changes in the number of TH (+) neurons. Although there is a trend for lead to reduce histological cell count, the calculated percent of control values for SN and VTA are high at 94% and 95%, respectively. For the purpose of comparison, the calculated percent of control values for spontaneously active DA neurons following lead treatment in the electrophysiology study were approximately 60% for both the SN and VTA.
Effects on Nucleus Accumbens D1 Receptor Sensitivity. The effect of the DA D1 receptor selective agonist SKF-38393 on the firing of type I nucleus accumbens (Nacb) neurons (Fig. 1B) was investigated in lead- and sodium-treated animals. The SKF-38393 ejection currents exerted a current-dependent inhibitory effect. Figure 3 shows examples of ratemeter histograms of type 1 Nacb neurons from a sodium-(panel A) and a lead-treated (panel B) animal. Note the difference between these neurons from treated animals in basal discharge rate and the difference in the slope of the inhibitory response in relation to iontophoretic drug application.
|
Figure 4A shows the mean responses of type I Nacb neurons to the microiontophoretically applied SKF-38393 in lead- and sodium-treated rats. The basal discharge rate of the nucleus accumbens neurons was found to differ between the sodium- and lead-treated groups, with that of the lead-treated group being significantly lower (P < 0.005, t test). The current-response relationship was analyzed using a two-way ANCOVA with basal discharge rate as the covariate. There was a significant interaction between treatment group and current (P < 0.005). This can be observed as a shallow slope for the lead-treated group and a steep slope for the control group. The difference in slopes can also be seen in the inset of Fig. 4A, which shows the iontophoretic data redrawn as mean change scores. This indicates that the nucleus accumbens neurons from the lead-treated group are less sensitive to iontophoretically applied SKF-38393 than those from the sodium-treated group. The D1 receptors on Nacb neurons from the lead-treated group appear to be "down-regulated". Figure 4B shows a plot of the adjusted least square means derived from ANCOVA. Again, note that the relative difference in the slopes of the two current-response curves remains after adjusting for the covariate basal discharge rate.
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
) showing a significant lead-induced decrease in the number of spontaneously active midbrain DA neurons in the SN and VTA. Based on TH immunopositive (TH+) cell counts, Tavakoli-Nezhad et al. (2001) suggested that lead exposure may have "silenced" the activity of some DA neurons without causing cell death. However, some of the physiologically "silent" neurons might have been compromised by lead exposure and were silent because of ongoing pathological responses to the insult (e.g., necrosis or apoptosis). It remained possible that a significant decline in TH (+) neurons might be detected if a later period were evaluated after the initiation of lead exposure. When the lead exposure was extended in duration in the present study, TH (+) cell counts were not found to be significantly different between sodium- and lead-treated groups. Therefore, it seems most probable that the significant lead-induced decrease in spontaneously active DA neurons observed in the electrophysiological studies was not due to cell death. These findings suggest that the predominant effect of the longer lead exposure regimen is an alteration in the physiological and possibly morphological properties of the midbrain DA neurons without lead-induced cell loss.
At least two different pathophysiological mechanisms could be proposed to explain the silenced DA neurons: 1) excess hyperpolarization of DA neurons or 2) excess depolarization of DA neurons (i.e., depolarization inactivation). Haloperidol, a selective DA D2-like receptor antagonist, has been shown to reduce the number of active DA neurons below control in both the SN and the VTA following a 3-week treatment (Bunney and Grace, 1978; Chiodo and Bunney, 1983; White and Wang, 1983). DA neurons in chronic haloperidol-treated animals have been shown to be in a tonic depolarized state (Bunney and Grace, 1978; Chiodo and Bunney, 1983). Therefore, if lead caused depolarization inactivation, systemic administration of the hyperpolarizing DA D2-like agonist apomorphine should restore DA neuron activity (e.g., see Chiodo and Bunney, 1983; White and Wang, 1983; Grace and Bunney, 1986).
Haloperidol was found to significantly decrease the number of spontaneously active VTA DA neurons in the present study. There was also a nonsignificant decrease in the number of spontaneously active SN DA neurons. Based on previous reports, hyperpolarization induced by systemic apomorphine should relieve DA neurons from depolarization inactivation by repolarizing the membrane and thereby increase the number of spontaneous active DA neurons in the haloperidol-treated rats relative to preapomorphine controls (e.g., see Bunney and Grace, 1978; Chiodo and Bunney, 1983; White and Wang, 1983). Following apomorphine challenge, the number of spontaneously active VTA DA neurons found in the haloperidol-treated group significantly increased relative to the preapomorphine control condition in this study. This result indicates that apomorphine was capable of reversing the effects of haloperidol on the VTA DA neurons. The lack of effect of apomorphine on the SN DA neurons of the haloperidol group is most likely due to the smaller nonsignificant reduction in the number of spontaneously active neurons in the preapomorphine challenge condition. Based on the utility of the haloperidol and apomorphine doses used, the best test of the hypothesis that lead exposure caused significant depolarization inactivation of midbrain DA neurons resides in the evaluation of the effects of apomorphine on VTA neurons from lead-treated animals.
The apomorphine challenge in the lead-treated group did not produce any significant effects. Given the expected significant decrease in the cells per track in the sodium-treated group and the significant apomorphine-induced reversal of the effects of haloperidol on the VTA neurons, it can be concluded that this otherwise active dose of apomorphine (50 µg/kg i.v.) did not affect the lead-treated group. The results of the present study indicate that the significant decrease in the number of spontaneously active VTA DA neurons following 250 ppm lead exposure cannot be readily explained by a depolarization inactivation mechanism. It does, however, remain possible that lead treatment interfered with the function of the somatodendritic autoreceptors, which mediate the hyperpolarizing effects of apomorphine. To the best of our knowledge, the effect of lead on somatodendritic autoreceptors has not been evaluated. It is also possible that this normally strongly hyperpolarizing apomorphine dose was not optimal for repolarizing DA neurons in the lead-treated group that may have been inactivated by a depolarizing mechanism.
The capacity of DA neurons to discharge in the burst mode provides a mechanism for phasic increases in DA levels within the synapse, which is spatially restricted to the synapse as a result of extremely efficient reuptake mechanisms that limit overflow into the extrasynaptic space (Floresco et al., 2003). A model has been described (Grace, 1991, 2000; Floresco et al., 2003), where the "background" or "tonic" level of extrasynaptic DA present in the striatum is regulated by the total number of spontaneously active DA neurons present and by modulatory afferent activity at the level of the forebrain DA terminal. If there is no change in midbrain DA neuron discharge rate and pattern and afferent regulation of terminal release in the forebrain remains unchanged, a decrease in the population of spontaneously active spike-generating DA neurons would be expected to result in a reduction in the tonic release of DA and a reduction in extrasynaptic DA at terminal regions (Floresco et al., 2003).
In both the present and the previous study by Tavakoli-Nezhad et al. (2001), a significant lead-induced reduction in the number of spontaneously active DA neurons was detected, but significant alterations in discharge rate or percentage of bursting cells were not detected. These findings suggest that a lead-induced decrease in forebrain DA release would likely be observed after such an exposure. Although forebrain DA release was not examined in the present study, Kala and Jadhav (1995a) have reported a decrease in basal and K+-induced DA release in the nucleus accumbens induced by 50 ppm lead exposure for 90 days. The time course for the alteration in DA release following low-level lead exposure is apparently not known. Long-term alterations in the tonic level of extrasynaptic DA have been postulated to regulate postsynaptic DA receptor sensitivity through up- and down-regulation (Grace, 1991). Since DA release was not measured in the present study, it is not known if the 250 ppm lead exposure altered DA release in the forebrain.
The basal discharge rate of type I nucleus accumbens neurons was found to be significantly lower in the lead-treated (250 ppm) animals relative to controls. The mechanism for this effect is not known but could be mediated by direct effects on the type I nucleus accumbens neurons or indirectly through altered afferent activity. Our results indicate that lead exposure reduces the sensitivity of type I nucleus accumbens neurons to the inhibitory effects of the selective DA D1 receptor agonist SKF-38393 relative to controls and strongly suggests that lead exposure alters the sensitivity of DA D1 receptors in the nucleus accumbens. Therefore, at the 250-ppm exposure level, lead depressed the discharge rate of type I nucleus accumbens neurons and decreased their sensitivity to DA D1 receptor stimulation. Similarly, Lewis and Pitts (2004) have reported that 250 ppm lead exposure results in a greatly attenuated ability of an acute amphetamine challenge to induce cFOS immunoreactivity in the striatum. Since it is known that DA D1 receptor stimulation is a necessary requirement for the amphetamine-induced increase in cFOS immunoreactivity (Robertson et al., 1991), it is possible that the suppression of amphetamine-induced striatal cFOS expression by lead may involve down-regulation of striatal DA D1 receptors.
More than one cellular site could mediate the DA D1 receptor responses to iontophoretically applied SKF-38393 (Nicola et al., 2000). For example, it is possible that lead affected D1 receptors on glutamatergic afferent terminals (Nicola et al., 2000). Although the exact mechanism for the effect of lead on spontaneously active DA neurons has yet to be determined, the observation of decreased DA D1 receptor sensitivity in the nucleus accumbens would be consistent with at least two different scenarios: 1) a lead-induced increase in forebrain DA neurotransmission resulting in DA D1 receptor down-regulation and/or 2) a direct effect on postsynaptic targets of the midbrain DA system that results in an apparent down-regulation of DA D1 receptors.
It is particularly noteworthy that prenatal or perinatal cocaine exposure (Minabe et al., 1992; Wang and Pitts, 1994), prenatal haloperidol exposure (Zhang et al., 1996), and prenatal ethanol exposure (Shen et al., 1999) have also been found to reduce the number of spontaneously active midbrain dopamine neurons in rats. These results and those of the present study suggest that developmental exposure to specific agents known to affect the midbrain dopaminergic system can result in relatively similar changes in the population activity of midbrain DA neurons. As also suggested by Shen et al. (1999), perturbation of DA neurotransmission during critical periods of development by certain agents/mechanisms may have relatively similar cellular outcomes that are potentially permanent in nature.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
ABBREVIATIONS: DA, dopamine; VTA, ventral tegmental area; SN, substantia nigra; HAL, haloperidol; ISI, interspike interval; SKF-38393, 1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol; TBS, Tris-buffered saline; NGS, normal goat serum; kHz, kilohertz; TH, tyrosine hydroxylase; ANOVA, analysis of variance; LSD, least significant difference; ANCOVA, analysis of covariance; APO, apomorphine; Nacb, nucleus accumbens.
Address correspondence to: Dr. David K. Pitts, Dept. Pharmaceutical Sciences, College of Pharmacy and Health Sciences, Wayne State University, 259 Mack Ave., Detroit, MI 48202. E-mail: pitts{at}wayne.edu
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Braga MF, Pereira EF, and Albuquerque EX (1999) Nanomolar concentrations of lead inhibit glutamatergic and GABAergic transmission in hippocampal neurons. Brain Res 826: 22-34.[CrossRef][Medline]
Bunney BS, Chiodo LA, and Grace AA (1991) Midbrain dopamine system electrophysiological functioning: a review and new hypothesis. Synapse 9: 79-94.[CrossRef][Medline]
Bunney BS and Grace AA (1978) Acute and chronic haloperidol treatment: comparison of effects on nigral dopaminergic cell activity. Life Sci 23: 715-728.[CrossRef][Medline]
Canfield RL, Henderson CR Jr, Cory-Slechta DA, Cox C, Jusko TA, and Lanphear BP (2003a) Intellectual impairment in children with blood lead concentrations below 10 microg per deciliter. N Engl J Med 348: 1517-1526.
Canfield RL, Kreher DA, Cornwell C, and Henderson CR Jr (2003b) Low-level lead exposure, executive functioning and learning in early childhood. Neuropsychol Dev Cogn Sect C Child Neuropsychol 9: 35-53.[Medline]
Chiodo LA and Bunney BS (1983) Typical and atypical neuroleptics: differential effects on the activity of A9 and A10 midbrain dopaminergic neurons. J Neurosci 3: 1607-1619.[Abstract]
Cory-Slechta DA (1997) Relationships between lead induced changes in neurotransmitter system function and behavioral toxicity. Neurotoxicology 18: 673-688.[Medline]
Floresco SB, West AR, Ash B, Moore H, and Grace AA (2003) Afferent modulation of dopamine neuron firing differentially regulates tonic and phasic dopamine transmission. Nat Neurosci 6: 968-973.[CrossRef][Medline]
Grace AA (1991) Phasic versus tonic dopamine release and the modulation of dopamine system responsivity: a hypothesis for the etiology of schizophrenia. Neuroscience 41: 1-24.[CrossRef][Medline]
Grace AA (2000) Gating of information flow within the limbic system and the pathophysiology of schizophrenia. Brain Res Brain Res Rev 31: 330-341.[CrossRef][Medline]
Grace AA and Bunney BS (1979) Paradoxical GABA excitation of nigra dopaminergic cells: indirect mediation through reticulata inhibitory neurons. Eur J Pharmacol 59: 211-218.[CrossRef][Medline]
Grace AA and Bunney BS (1980) Nigral dopamine neurons: intracellular recording and identification with L-DOPA injection and histofluorescence. Science (Wash DC) 210: 654-656.
Grace AA and Bunney BS (1986) Induction of depolarization block in midbrain by repeated administration of haloperidol: analysis using in vitro intracellular recording. J Pharmacol Exp Ther 238: 1092-1100.
Guilarte TR (1997) Glutamatergic system and developmental lead toxicity. Neurotoxicology 18: 665-672.[Medline]
Kala SV and Jadhav AL (1995a) Low level lead exposure decreases in vivo release of dopamine in the rat nucleus accumbens: a microdialysis study. J Neurochem 65: 1631-1635.[Medline]
Kala SV and Jadhav AL (1995b) Region specific alteration in dopamine and serotonin metabolism in brains of rats exposed to low level of lead. Neurotoxicology 16: 297-308.[Medline]
Kuhn W, Winkel R, Woitalla D, Meves S, Przuntek H, and Muller T (1998) High prevalence of parkinsonism after occupational exposure to lead-sulfate batteries. Neurology 50: 1885-1886.
Lasley SM (1992) Regulation of dopaminergic activity, but not tyrosine hydroxylase, is diminished after chronic inorganic lead exposure. Neurotoxicology 13: 625-635.[Medline]
Lasley SM and Gilbert ME (2002) Rat hippocampal glutamate and GABA release exhibit biphasic effects as a function of chronic lead exposure level. Toxicol Sci 66: 139-147.
Lewis MW and Pitts DK (2004) Inorganic lead exposure in the rat activates striatal cFOS expression at lower blood levels and inhibits amphetamine-induced cFOS expression at higher blood levels. J Pharmacol Exp Ther 310: 815-820.
Lidsky TI and Schneider JS (2003) Lead neurotoxicity in children: basic mechanisms and clinical correlates. Brain 126: 5-19.
Minabe Y, Ashby CR Jr, Heyser C, Spear LP, and Wang RY (1992) The effects of prenatal cocaine exposure on spontaneously active midbrain dopamine neurons in adult male offspring: an electrophysiological study. Brain Res 586: 152-156.[CrossRef][Medline]
Minder B, Das-Smaal EA, Brand EF, and Orlebeke JF (1994) Exposure to lead and specific attentional problems in schoolchildren. J Learn Disabil 27: 393-399.
Nation JR, Frye GD, Van Stultz J, and Bratton GR (1989) Effects of combined lead and cadmium exposure: changes in schedule-controlled responding and in dopamine, serotonin and their metabolites. Behav Neurosci 103: 1108-1114.[CrossRef][Medline]
Needleman HL (1993) The current status of low-level lead toxicity. Neurotoxicology 14: 161-166.[Medline]
Nicola SM, Surmerier J, and Malenka RC (2000) Dopaminergic modulation of neuronal excitability in the striatum and nucleus accumben. Annu Rev Neurosci 23: 185-214.[CrossRef][Medline]
Paxinos G and Watson C (1986) The Rat Brain in Stereotaxic Coordinates, 2nd ed, Academic Press, Inc., New York.
Pitts DK, Kelland MD, Shen R, Freeman AS, and Chiodo LA (1990) Statistical analysis of dose-response curves in extracellular electrophysiological studies of single neurons. Synapse 5: 281-293.[CrossRef][Medline]
Pokora MJ, Richfield EK, and Cory-Slechta DA (1996) Preferential vulnerability of nucleus accumbens dopamine binding sites to low-level lead exposure: time course of effects and interactions with chronic dopamine agonist treatment. J Neurochem 67: 1540-1550.[Medline]
Robertson HA, Paul ML, Moratalla R, and Graybiel AM (1991) Expression of the immediate early gene c-fos in basal ganglia: induction by dopaminergic drugs. Can J Neurol Sci 18: 380-383.[Medline]
Shao Z and Suszkiw JB (1991) Ca2(+)-surrogate action of Pb2+ on acetylcholine release from rat brain synaptosomes. J Neurochem 56: 568-574.[CrossRef][Medline]
Shen R-Y, Asdourian D, and Chido LA (1992) Microiontophoretic studies of the effects of D1 and D2 receptor agonists on type I caudate nucleus neurons: lack of synergistic interaction. Synapse 11: 319-329.[CrossRef][Medline]
Shen R-Y, Hannigan JH, and Kapatos G (1999) Prenatal ethanol reduces the activity of adult midbrain dopamine neurons. Alcoholism Clin Exp Res 23: 1801-1807.[CrossRef][Medline]
Shotyk W, Weiss D, Appleby PG, Cheburkin AK, Gloor RFM, Kramers JD, Reese S, and Van Der Knaap WO (1998) History of atmospheric lead deposition since 12,370 14C yr BP from a peat bog, Jura Mountains, Switzerland. Science (Wash DC) 281: 1635-1640.
Tavakoli-Nezhad M, Barron AJ, and Pitts DK (2001) Postnatal inorganic lead exposure decreases the number of spontaneously active midbrain dopamine neurons in the rat. Neurotoxicology 22: 259-269.[CrossRef][Medline]
Tuthill RW (1996) Hair lead levels related to children's classroom attention-deficit behavior. Arch Environ Health 51: 214-220.[Medline]
Wakefield J (2002) The lead effect? Environ Health Perspect 110: A574-A580.[Medline]
Wang L and Pitts DK (1994) Perinatal cocaine exposure decreases the number of spontaneously active midbrain dopamine neurons in neonatal rats. Synapse 17: 275-277.[CrossRef][Medline]
White FJ and Wang RY (1983) Differential effects of classical and atypical antipsychotic drugs on A9 and A10 dopamine neurons in the rat. Science (Wash DC) 221: 1054-1057.
Zhang J, Wang L, and Pitts DK (1996) Prenatal haloperidol reduces the number of active midbrain dopamine neurons in rat offspring. Neurotoxicol Teratol 18: 49-57.[CrossRef][Medline]
Zuch CL, O'Mara DJ, and Cory-Slechta DA (1998) Low-level lead exposure selectively enhances dopamine overflow in nucleus accumbens: an in vivo electrochemistry time course study. Toxicol Appl Pharmacol 150: 174-185.[CrossRef][Medline]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
