1 Department of Neurology, University of Leipzig, 04316 Leipzig, Germany
2 Division of Biology, California Institute of Technology, Pasadena, California 91125
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ABSTRACT |
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cholinergic excitotoxicity; mouse
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INTRODUCTION |
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This study analyzes the only known instance of dopaminergic cell death caused by chronic activation of nicotinic receptors (17). The pathology occurs in knockin mice carrying a leucine-to-serine mutation at the 9' position of the pore-lining second transmembrane domain (M2) in the 4-subunit. To retain viability, we work with 1) a strain that displays reduced expression of the hypersensitive allele, because of a deliberate insertion of the neomycin selection cassette in an intron adjoining the mutated exon; and 2) with heterozygotes of this strain. These viable and fertile heterozygous mice are termed "L9'S." L9'S mice do not show a significant reduction of dopaminergic neurons during embryonic development but have a reduction of amphetamine-induced locomotion as they age (17). In addition, L9'S mice display increased anxiety. Following low doses of nicotine, L9'S mice display seizures and Straub tail (6, 17).
Here, we show that adult L9'S mice show a moderate and specific loss of dopaminergic neurons in substantia nigra, whereas dopaminergic neurons in the ventral tegmental area (VTA) and noradrenergic neurons in locus coeruleus show no gross abnormalities. Also, there is a significant reduction of dopaminergic innervation to caudate and putamen but not nucleus accumbens. These morphological changes are paralleled by a significant reduction in amphetamine-induced locomotor activity. We believe that the loss of dopaminergic neurons in substantia nigra is associated with an increased excitation during exposure to agonists, as evidenced by recordings from dopaminergic neurons in acute midbrain slices. Thus, although this 4-hypersensitive genotype is viable and fertile, it displays dopaminergic cell death as a consequence of excessive nicotinic stimulation.
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MATERIALS AND METHODS |
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Drugs.
All drugs were purchased from Sigma-Aldrich (St. Louis, MO) and dissolved as suggested by the manufacturer.
Histology and immunocytochemistry.
Each mouse was fixed by cardiac perfusion (0.1 M PBS and 4% formaldehyde), postfixed for 2 h, and dehydrated in 15% sucrose and 30% sucrose. Ten series of cryosections of 30-µm thickness were prepared in coronal orientation. All stainings were performed free floating. Alternate sections were Nissl stained. Brain sections were qualitatively examined at low magnification (50x and 100x) to assess the gross neuroanatomical structure of L9'S and WT mice. Double staining was performed with antisera for tyrosine hydroxylase (TH) 1:100 (Santa Cruz Biotechnologies, Santa Cruz, CA) and 4-nAChR 1:100 (Santa Cruz Biotechnologies) and fluorescent secondary antibodies (1:500, Alexa 594, Alexa 488; Molecular Probes, Eugene, OR), respectively, using one series of sections for
4 and two series for TH staining (every 10th or every 5th section counted).
For cell counts, sections were stained for indirect immunohistochemistry according to the Vectastain ABC Elite kit protocol (Vector Laboratories, Burlingame, CA). Diaminobenzidine was used as chromogen with nickel enhancement. Images were obtained from a total of eight L9'S and eight WT mice. Cell counts were performed in all midbrain sections containing substantia nigra and VTA with fibers of the third cranial nerve separating the two regions. Cell counts were corrected for cell diameter and slice thickness according to the Abercrombie formula (1).
In addition, nonbiased stereological cell counts were performed using the optical dissector method (34, 35) to assess the number of TH-immunoreactive and Nissl-positive cells in substantia nigra (SN). The system consisted of a Zeiss Axiophot photomicroscope equipped with a Zeiss MSP65 computer-controlled motorized stage (Zeiss) and StereoInvestigator software (MicroBrightField). Five series of 40-µm thick cryosections were cut. Each series contained 10 sections with SN. Staining was performed as outlined above, and sections were embedded in Durcupan as described (23). Final section thickness was 3035 µm. TH cells were counted using a 40 x 40-µm counting frame. A 20-µm dissector was placed leaving a 5-µm top guard zone at counting sites located at 100-µm intervals using a random starting point. For analysis of Nissl-stained sections, a counting frame of 30 x 30 µm and a sampling grid of 150 x 150 µm were used.
For semiquantitative assessment of dopaminergic nerve terminals within subregions of the basal ganglia (caudate-putamen and nucleus accumbens), tyrosine hydroxylase (TH) staining is the most appropriate technique. Free-floating sections containing nucleus accumbens and caudate-putamen were stained using rabbit antisera against rat TH (Pel-Freez, 1:500). Secondary antibody reaction and diaminobenzidine with metal enhancer were performed as described above. We used a Nikon Eclipse inverted microscope outfitted with a Hamamatsu ORCA II digital camera. Intensity of nerve terminal TH staining in nucleus accumbens and striatum was determined by tracing circular areas (400 µm diameter) and measuring light transmission using Metamorph 4.1 software (Universal Imaging). To correct for nonspecific staining, for each section, a circular area in the adjacent cortex was also traced and measured. The final intensity value was obtained as (TH-positive value cortex value)/TH-positive value. TH intensity measurements are performed on both hemispheres for each brain section, and three sections each from nucleus accumbens and striatum are analyzed from each mouse.
Locomotion measurements.
Before drug administration, animals were allowed to habituate for 30 min. Single events represent disruption of two distinct light beams 10 cm apart in the cage (San Diego Instruments). Amphetamine (5 mg/kg D-amphetamine sulfate dissolved in 0.9% saline) was injected intraperitoneally in a volume of 100200 µl (100 µl/20 g body wt). Control animals were WT littermates of the heterozygous mice. Data were analyzed within and between subjects by ANOVA using Origin (OriginLab, Northhampton, MA).
Recordings from dopaminergic neurons in midbrain slices.
WT or L9'S mice aged P12P25 were used for electrophysiological experiments. Animals were anesthetized using halothane. The brains were immediately removed, rinsed, and transferred to ice-cold artificial cerebrospinal fluid (ACSF), which had been equilibrated for 1 h. Slices (300-µm thick) were cut on a vibratory microtome (Leica or Dosaka) in coronal orientation. After sectioning, slices were recovered in oxygenated ACSF containing 1 mM kynurenic acid at 32°C for 0.5 h and subsequently for at least 0.5 h in ACSF without kynurenic acid in an oxygenated holding chamber at room temperature and then transferred into a recording chamber. They were completely submerged in continuously flowing ACSF at 35°C, pH 7.4. This solution contains (in mM) 126 NaCl, 2.5 KCl, 1 MgCl2, 1.25 NaH2PO4, 2 CaCl2, 25 glucose, and 26 NaHCO3, gassed with 95% O2 and 5% CO2, pH 7.4 (16).
The recording chamber was mounted on the stage of an upright microscope (Zeiss or Olympus) equipped with infrared video microscopy (Hamamatsu). While individual dopaminergic neurons were visualized and approached, positive pressure was applied to the pipette. Pipettes were made from borosilicate glass (1.5 mm, World Precision Instruments or Science Instruments) and pulled with a horizontal (Sutter Instruments) or vertical puller (Narishige). The resistance was between 4 and 8 M when filled with a standard solution containing (in mM) 144 potassium gluconate, 3 MgCl2, 10 HEPES, and 0.2 EGTA, pH 7.3 (16). Whole cell recordings are performed with an Axon Multipatch or Axopatch 1D amplifier (Axon Instruments).
Dopaminergic neurons were identified by anatomy, morphology, and a hyperpolarization-activated cation current (Ih), which is characteristic for these cells in midbrain slices (33). All cells that displayed Ih currents of at least 200 pA at 130 mV were classified as dopaminergic.
Drug application was performed using a gravity-driven 8-channel perfusion system (BioMedical Instruments) for 5 s. Responses to nicotine and GABA were measured during 5 s starting 10 s before drug application and during 5 s starting 5 s after the onset of application each. In addition, we employed local pressurized drug application using a Picospritzer II (General Valve/Parker) to investigate the effects of local and fast drug application. All drugs were purchased from Sigma-Aldrich.
Data are displayed as means ± SE.
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RESULTS |
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As another control region in the brain stem, we studied the locus coeruleus, which also bears a high density of catecholaminergic (noradrenergic) neurons. The locus coeruleus of L9'S mice displayed no reduction of Nissl-stained (data not shown) or TH-immunoreactive cells (Fig. 2). Thus the loss of dopaminergic neurons in substantia nigra seems to be fairly specific among catecholaminergic neurons in the brain stem. Previous surveys in several other brain areas of L9'S mice also indicated no major changes in neuronal cell numbers as detected by Nissl stain (6).
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Locomotor effects of dopaminergic deficits.
Systemic administration of amphetamine induces release of dopamine. This dopamine release induces a robust increase in locomotor behavior, and quantification of this amphetamine-induced locomotor behavior may represent an indirect measure of the amount of dopamine stored in the vesicles of nigrostriatal dopaminergic nerve terminals. The initial report on the L9'S 4 knockin mice found a nonsignificant reduction of amphetamine-induced locomotion in L9'S mice compared with their WT littermates (17). Here, we present data of a large cohort of L9'S mice and their WT littermate controls showing a significant increase of locomotion during the first 5 min when mice adjusted to the novel environment and a significant reduction of amphetamine-stimulated locomotion 3045 min after injection (Fig. 4).
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Voltage-clamp analyses showed that hyperpolarization-induced currents (Ih) did not differ between cells of L9'S and WT mice (Fig. 5B). However, there was a small but significantly (P < 0.05, ANOVA) more negative resting membrane potential (baseline potential of spontaneously spiking neurons) in L9'S (73.8 ± 2.1 mV, n = 18) compared with WT Ih-positive cells (67.0 ± 1.3 mV, n = 31). This hyperpolarization of mutant cells was paralleled by a significantly smaller percentage number of dopaminergic neurons (Ih positive) that showed spontaneous action potentials (21% vs. 79%).
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As a positive control, we applied GABA (10 µM), which induced robust outward currents (Fig. 6D), indicating that the failure to generate nicotine-induced currents was not due to technical factors. We also verified that the responses to GABA in dopaminergic cells displayed no differences between the L9'S and WT genotypes (Fig. 7, C and D).
Recordings in midbrain slices: nondopaminergic cells.
Nondopaminergic L9'S neurons within substantia nigra displayed an increase in spiking frequency at 10 µM nicotine (Fig. 7B), whereas WT neurons displayed transient decreases. This pattern resembles that for dopaminergic neurons. At 100 µM nicotine, both L9'S and WT neurons continued to display nicotine-induced increases in firing frequency, and there was no difference between the two genotypes. We chose not to pursue resting membrane potential measurements for nondopaminergic cells, because the high firing rate of such neurons invalidates the definition of resting potential.
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DISCUSSION |
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It is interesting that only dopaminergic neurons of substantia nigra are affected, whereas dopaminergic neurons in VTA seem to remain intact. This study did not investigate the mechanisms accounting for this differential effect. In Parkinson disease as well as in several other animal models of dopaminergic cell death (e.g., MPTP mouse, weaver mouse), cell death is more extensive in substantia nigra than in VTA. Nicotinic AChRs may be among the genes whose differential expression accounts for this differential vulnerability among dopaminergic neurons.
As expected from the widespread distribution of 4-subunits, we detected immunoreactivity for
4-subunits in areas outside midbrain. Previous studies revealed no cell death outside of the midbrain (7, 17). Although only substantia nigra shows appreciable cell death in the L9'S mice, we detected lower
4 immunoreactivity in nearly all individual
4-immunoreactive cells in nearly all regions of L9'S mice. This additional effect is both understood and expected from previous data. The L9'S mice have approximately fourfold less expression than normal of the mutant allele, because an intact neomycin selection cassette (neo) in a nearby intron interferes with RNA processing or protein expression.
The viability of the neo-intact L9'S genotype studied here depends on this diminished expression of the excitotoxic L9'S 4 subunit. More extensive, prenatal dopaminergic cell death occurs in other, neonatal lethal, genotypes of mice containing 9' leucine-to-serine mutations: 1) homozygous mice with the allele described here; and 2) heterozygotes with nearly physiological expression of the mutated allele, due to excision of neo (17). The loss of dopaminergic neurons is the most prominent pathology in embryonic brain tissue of the two nonviable genotypes (17). A hypersensitive
7 nicotinic strain also shows neuronal death and, in homozygotes, neonatal lethality (25). Therefore severe CNS cholinergic excitotoxicity can occur as a consequence of gain of mutations in at least two distinct nAChR subunits (19).
Circuit-based nicotinic neurotoxicity.
Is the concept of increased cholinergic activation of substantia nigra dopaminergic neurons altering their function or viability relevant to the pathophysiology of Parkinson disease? We believe that this is so. Parkinson disease is characterized by the loss of dopaminergic neurons in substantia nigra, resulting in dopamine deficiency within the striatum. Further downstream, striatal dopamine deficiency leads to increased activity of the subthalamic nucleus and the internal segment of the globus pallidus. In rodents, there are both a direct pathway from the subthalamic nucleus to substantia nigra pars compacta (14) and also an indirect excitatory pathway from subthalamic nucleus via the pedunculopontine nucleus to substantia nigra pars compacta (9). The latter employs both glutamate and acetylcholine as neurotransmitters. In states of dopamine deficiency, one expects the increased activity in subthalamic nucleus to produce an increased cholinergic drive of dopaminergic neurons in substantia nigra. Most interestingly, lesioning the pedunculopontine nucleus has substantial protective effects in MPTP-treated parkinsonian monkeys (29). In summary, cholinergic excitotoxicity potentially represents a self-reinforcing mechanism that arises from basal ganglia circuits and that contributes to the detrimental effects of dopamine deficiency and thus to progressive cell death in Parkinson disease.
Administration of nicotine induced prolonged excitation of dopaminergic neurons in substantia nigra of L9'S but not WT mice. Furthermore, there were no morphological changes in an appropriate control region, i.e., locus coeruleus. We suggest that this selective vulnerability of substantia nigra neurons in L9'S mice arises from functional changes due to the hypersensitive nicotinic receptors on the dopaminergic neurons, from possible sustained receptor activation of the hypersensitive receptors by choline in the cerebrospinal fluid (17), as well as the self-reinforcing properties of basal ganglia circuits.
Dopaminergic vs. nondopaminergic neurons.
Our electrophysiological experiments provide evidence that dopaminergic neurons of L9'S mice have an excitatory response to nicotine that is qualitatively different from that of WT neurons. It is possible that under physiological conditions nicotinic agonists mainly act on dopaminergic neurons via stimulation of presynaptic receptors located on GABAergic and glutamatergic nerve terminals projecting into substantia nigra. In fact, the increase of inhibitory postsynaptic currents (IPSCs) and excitatory postsynaptic currents (EPSCs) measured in midbrain dopaminergic neurons represent important effects of administration of nicotine in acute brain slices (21). However, in states of increased cholinergic stimulation, postsynaptic receptors on dopaminergic neurons could be activated and exert excitatory effects on dopaminergic neurons in the midbrain.
Comparison with previous electrophysiological data.
We were initially surprised to find no reproducible excitatory nicotine responses in WT dopaminergic neurons as reported previously (16, 26), although we have experimented with various drug application systems including fast application systems. Instead, in our hands, WT dopaminergic neurons showed a hyperpolarization and reduction in spiking frequency during application of moderate doses of nicotine (10 µM). However, it is not impossible that responses reported in the previous studies represent indirect effects on dopaminergic neurons, since the latency between fast local drug application and change in current was unusually long (16, 26). We have preliminary evidence that nicotinic 4-mediated responses can occur within
100 ms after fast application of nicotine in a new strain of mutant mice (data not shown). However, we are aware that the electrophysiological data presented here do not prove that excitatory effects in L9'S animals are caused by nicotinic receptors located on dopaminergic neurons themselves. This question must be addressed in future studies.
Contrast with the neuroprotective effects of nicotine.
Our findings present a contrast with the potential neuroprotective properties of nicotine. An inverse dose-dependent relation between smoking and Parkinson disease has been established in epidemiological studies (11, 13, 18). The strongest epidemiological evidence supporting this potential neuroprotective effect comes from a study on 71 monozygotic and 90 dizygotic twins showing a significant difference in smoking between affected and unaffected twins (31).
In animal studies, nicotine showed dopamine-releasing effects after acute administration, inducing amelioration of locomotor deficits (4). On the other hand, in rats with a partial hemitransection of nigrostriatal dopaminergic fibers or mice treated with MPTP, continuous chronic treatment with nicotine reduced firing of dopaminergic neurons (12), diminished the loss of TH-immunoreactive neurons in substantia nigra, reduced striatal dopamine content, or ameliorated effects on spontaneous locomotion (10, 15). Thus it is possible that acute administration of nicotine may stimulate dopamine turnover, whereas chronic nicotine application could slow metabolism of dopaminergic neurons, therefore exerting a potential protective effect. How could nicotine protect against neuronal loss in some animal models of Parkinson disease, and what produces the inverse relationship between smoking and Parkinson disease in humans? Exposure to nicotine first induces activation, then desensitization, of nAChRs in native and heterologous expression systems. Chronic treatment with nicotine results in an increased level of binding to receptors with the high nicotine affinity characteristic of the desensitized state, and these receptors appear to be 4ß2 (3, 5, 28).
It is often asked whether the apparent protective effects of nicotine arise directly from the increased activation and metabolic challenge or from subsequent prolonged desensitization, which would lead to a net reduction of cholinergic activation of dopaminergic neurons expressing the desensitized receptors. It is generally found that nAChRs with Leu9'Thr or Leu9Ser mutations desensitize less, for a given level of activation, than do WT receptors (Revah; Labarca), although the point has not been studied systematically for the present 4L9'S receptors. In the present knockin mice with hypersensitive
4-receptors, increased nicotinic activation causes excitotoxic death of dopaminergic neurons rather than protecting them, providing some evidence for the view that desensitization accounts for the protective effects of smoking.
Pathophysiological implications.
Although mutations in nAChR genes have not been identified in Parkinson disease, the L9'S mice may provide an appropriate model for the concept that increased cholinergic drive of dopaminergic neurons in substantia nigra is a consequence, via circuit properties, of dopamine deficiency. Thus these mice may serve to test potentially protective drugs that are designed either 1) to block 4-containing nAChRs on dopaminergic neurons or 2) to exert protective effects at downstream mechanisms.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
Address for reprint requests and other correspondence: J. Schwarz, Liebigstr. 22a, 04103 Leipzig, Germany (E-mail: johannes{at}caltech.edu).
10.1152/physiolgenomics.00012.2004.
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REFERENCES |
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