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Keywords:

  • astrocytes;
  • cyclic GMP;
  • granule cell neurons;
  • group II metabotropic receptors;
  • metabotropic glutamate receptor type 3;
  • N-acetylaspartylglutamate

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Metabotropic receptors may couple to different G proteins in different cells or perhaps even in different regions of the same cell. To date, direct studies of group II and group III metabotropic glutamate receptors' (mGluRs) relationships to second messenger cascades have reported negative coupling of these receptors to cyclic AMP (cAMP) levels in neurons, astrocytes and transfected cells. In the present study, we found that the peptide neurotransmitter N-acetylaspartylglutamate (NAAG), an mGluR3-selective agonist, decreased sodium nitroprusside (SNP)-stimulated cyclic GMP (cGMP) levels in cerebellar granule cells and cerebellar astrocytes. The mGluR3 and group II agonists FN6 and LY354740 had similar effects on cGMP levels. The mGluR3 and group II antagonists β-NAAG and LY341495 blocked these actions. Treatment with pertussis toxin inhibited the effects of NAAG on SNP-stimulated cGMP levels in rat cerebellar astrocytes but not in cerebellar neurons. These data support the conclusion that mGluR3 is also coupled to cGMP levels and that this mGluR3-induced reduction of cGMP levels is mediated by different G proteins in cerebellar astrocytes and neurons. We previously reported that this receptor is coupled to a cAMP cascade via a pertussis toxin-sensitive G protein in cerebellar neurons, astrocytes and transfected cells. Taken together with the present data, we propose that mGluR3 is coupled to two different G proteins in granule cell neurons. These data greatly expand knowledge of the range of second messenger cascades induced by mGluR3, and have implications for clinical conditions affected by NAAG and other group II mGluR agonists.

Abbreviations used
cAMP

cyclic AMP

cGMP

cyclic GMP

IBMX

isobutylmethylxanthine

mGluR

metabotropic glutamate receptor

NAAG

N-acetylaspartylglutamate

PDE

phosphodiesterase

SNP

sodium nitroprusside

The metabotropic glutamate receptors (mGluRs) are a subgroup of the seven-transmembrane receptor superfamily (Pierce et al. 2002) that has been classified into three groups based on homology, pharmacology and the second messenger cascades that they affect (Conn 2003; Kew and Kemp 2005). Group I receptors are coupled to phospholipase C, whereas group II and III mGluRs are reported to be negatively coupled to cyclic AMP (cAMP) levels. Activation of mGluR3 receptors in striatal neurons (Manzoni et al. 1992), hippocampal neurons (Schoepp et al. 1992), cerebellar granule cell neurons (Wroblewska et al. 1993) and rat cerebellar astrocytes (Wroblewska et al. 1998) is accompanied by decreases in cAMP levels.

Although homologous receptors generally display similar G-protein coupling, there is ample evidence that individual seven-transmembrane receptors may couple to more than one type of G protein (Xiao 2000; Heubach et al. 2004). In 1995, Nakanishi's laboratory speculated that mGluRs might be negatively coupled to cyclic GMP (cGMP) in retinal neurons (Masu et al. 1995). Indeed, in conflicting reports, hyperpolarization of retinal neurons by glutamate and a mGluR6 agonist was alternatively proposed to be induced by both increases and decreases in cGMP levels via a phosphodiesterase (PDE)-mediated process (Nawy and Jahr 1990, 1991; Dixon and Copenhagen 1997). In a later study, however, no evidence was found to support a direct relationship between mGluR6-induced hyperpolarization and PDE- or transducin-mediated events, much less a direct relationship between mGluR6 and cGMP levels (Nawy 1999). In addition, no data have been published on any other neurons, glia or transfected cell lines that would directly support the hypothesis that any mGluRs are coupled to cGMP levels. It is, however, often difficult to detect such a coupling in intact nervous systems because of the diversity of simultaneous neurotransmitter events and the presence of low basal levels of guanylate cyclase and cGMP in some cells. Further confounding the search for a cGMP response coupled to mGluRs are the low levels of guanylate cyclase in many of the cell lines into which the cDNA for these receptors has been transfected.

N-acetylaspartylglutamate (NAAG) is the third most prevalent transmitter in the mammalian CNS after glutamate and GABA (reviewed in Neale et al. 2000, Neale 2005). NAAG is a selective agonist of mGluR3 (Wroblewska et al. 1997), with substantially lower affinity for mGluR2 (Schaffhauser et al. 1998; Schweitzer et al. 2000). NAAG activation of mGluR3 in cerebellar granule cells (Wroblewska et al. 1993), cerebellar astrocytes (Wroblewska et al. 1998) and transfected cells (Wroblewska et al. 1997; Schweitzer et al. 2000) reduces cAMP levels via a pertussis toxin (PTX)-sensitive G protein.

NAAG and group II mGluRs have been implicated in diverse nervous system processes ranging from presynaptic inhibition of transmitter release (Zhao et al. 2001; Garrido Sanabria et al. 2004), long-term potentiation (Lea et al. 2001) and long-term depression (Huang et al. 1999; Poschel et al. 2005) to therapeutic effects in anoxia-induced excitotoxicity (Lu et al. 2000; Cai et al. 2002), sensory neuropathy (Zhang et al. 2002), schizophrenia (Olszewski et al. 2004), allodynia (Yamamoto et al. 2004) and traumatic brain injury (Zhong et al. 2005). Although it has been assumed that these actions are mediated by a reduction in cAMP levels, the potential interactions between NAAG, mGluR3 and cGMP have not been explored rigorously. In the present study, we report the coupling of mGluR3 activation with reduction in cGMP levels, a finding that militates in favor of a broader restatement of the consequences of activation of group II mGluRs.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Materials

LY354740 was a generous gift from Eli Lilly & Co. (Indianapolis, IN, USA) LY341495 and NAAG were purchased from Tocris (Ellisville, MO, USA). Arabinofuranoside, trypsin, trypsin soybean inhibitor, sodium nitroprusside (SNP), β-NAAG and isobutylmethylxanthine (IBMX) were from Sigma (St Louis, MO, USA). PTX (Bordatella pertussis) was from List Biological Laboratories Inc. (Campbell, CA, USA). 4,4′-Phosphinicobis-(butane-1,3 dicarboxilic acid) (FN6), a potent inhibitor of cloned rat glutamate carboxypeptidase II (EC50 22 nm) that is also a selective mGluR3 agonist (EC50 39 µm) (Nan et al. 2000), was provided by Dr Alan Kozikowski (Department of Medicinal Chemistry and Pharmacognosy, University of Illinois, Chicago, IL, USA).

Rat cerebellar granule cultures

Primary cultures of rat cerebellar granule cells were prepared from the cerebella of 6–8-day-old Sprague–Dawley rat pups (Taconic, Germantown, MD, USA) according to the method described by Gallo et al. (1987). Cerebella were removed, trypsinized (0.175 mg/mL trypsin), and treated with soybean inhibitor (0.312 mg/mL) and DNase (0.0107 mg/mL). Cell suspensions were triturated and sedimented through a solution of bovine serum albumin (40 mg/mL). Cells (1.25 × 106 cells/mL, 0.25 mL/well) were grown on 96-well Nunc culture plates in BME (Basal Medium Eagle) medium (Gibco, Rockville, MD, USA) with 25 mm KCl, 2 mm l-glutamine, 10% heat-inactivated fetal bovine serum (Gibco) and 50 µg/mL gentamicin. These culture dishes were pretreated with poly-l-lysine (Sigma). Arabinofuranoside 10 µm was added after 24–48 h to stop the growth of dividing cells. Cerebellar granule cell cultures were grown at 37°C with 5% CO2 for 9 days.

Astrocyte preparation

The procedure described by Gallo et al. (1987) was used, with the following modifications. Cells from 6–8-day-old rat pups were grown in minimal essential medium (Gibco) with gentamicin (50 µg/mL) and 10% fetal bovine serum (Gibco). Cells were inoculated at a density of 0.6–0.8 × 106 cells/mL (0.25 mL/well) and grown on 96-well Nunc culture dishes. Cells were grown at 37°C with 5% CO2. The medium was changed every 3–5 days.

Cell treatments and cGMP determination

To determine changes in cGMP responses, cerebellar granule cells and astrocytes cells were washed twice with Locke's buffer (154 mm NaCl, 5.6 mm KCl, 1.3 mm CaCl2, 3.6 mm NaHCO3, 5.6 mm glucose, 1.0 mm MgCl2, 5 mm HEPES, pH 7.4 at 37°C) and preincubated with PDE inhibitor IBMX (1 mm) and NMDA receptor antagonist MK801 (1 µm) for 10 min followed by incubation with SNP, IBMX, MK801 and appropriate metabotropic agonists and/or antagonists for 6 min. NAAG is a very low potency agonist at some NMDA receptors in cultured spinal cord neurons (Westbrook et al. 1986) but was found to be neither a significant agonist nor antagonist at NMDA receptors in cerebellar granule cells and had no significant effect on NMDA receptor-mediated spontaneous synaptic currents in these cells (Losi et al. 2004). Nonetheless, in order to preclude any interactions with NMDA receptors in the present study, these experiments were carried out in the presence of the NMDA receptor antagonist MK801. Incubations were terminated by rapid aspiration of cell medium and extraction with 0.1 m HCl.

Levels of cGMP were detected in the cell extracts using an acetylated 125I-labelled cGMP kit (Amerlex-M; Amersham, Piscataway, NJ, USA). The remaining cell membranes were dissolved in 0.1 m NaOH and the bicinchoninic protein assay was used to determine protein content. In some experiments, primary cultures of cerebellar granule cells and cerebellar astrocytes were exposed to PTX (1 µg/mL) overnight (up to 18 h) on day 8 in vitro.

Data analysis

The cGMP levels were assayed in duplicate in each culture. The mean of these duplicate assays from each cell culture represents a separate experimental value. Typically, three cultures were assayed per condition in each set of experiments. Each set of experiments was repeated at least three times, with the exception of some treatments of astrocytes with PTX, which were executed twice with n = 6 and yielded statistical significance at p < 0.05 or lower. Values represent the mean ± SEM of these mean values for cGMP levels (as a percentage of the maximal stimulation with SNP) in individual cultures across all experiments. Student's t-test was used to determine the statistical significance between different treatment groups.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

All experiments were conducted in the presence of the PDE inhibitor IBMX (1 mm) and the NMDA receptor antagonist MK801 (1 µm). SNP increases cGMP levels by acting as a donor of nitric oxide (NO) (Lippton et al. 1982) to soluble guanylate cyclase. SNP increased cGMP formation in cultures of cerebellar granule cells and cerebellar astrocytes (Fig. 1). Basal levels of cGMP in these cells were low (4.0 ± 0.7 and 3.6 ± 0.5 pmol/mg protein for cerebellar granule cells and astrocytes respectively) and so it was difficult to detect reductions in levels of this second messenger in unstimulated cells. Stimulation with SNP (100 µm) led to a 45-fold increase over basal levels in granule cells and a 22-fold increase in cerebellar astrocytes.

image

Figure 1. SNP significantly stimulates increases in cGMP formation in rat cerebellar granule cells and rat cerebellar astrocytes. Cells grown on 96-well culture dishes were washed twice and preincubated for 10 min at room temperature (25°C) with Locke's buffer (1 mm IBMX and 1 µm MK801) and incubated without or with SNP (100 µm) for 6 min. Values are mean ± SEM (n = 24 for each bar). ***p < 0.001 versus basal (Student's t-test).

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The mGluR3-selective transmitter NAAG (Wroblewska et al. 1997) and mGluR3-selective agonist FN6 (Nan et al. 2000) added to the incubation buffer in the presence of SNP significantly decreased cGMP levels in both cell types (Fig. 2). Incubation of rat cerebellar granule cells (Fig. 2a) with 100 µm NAAG, 100 µm FN6 or 10 µm LY354740 decreased SNP-stimulated cGMP levels to 24%, 33% and 50% of maximal stimulation in the presence of 100 µm SNP. Similar results were obtained with cultured rat cerebellar astrocytes (Fig. 2b) incubated in the presence of 100 µm SNP and mGluR3 agonists NAAG (100 µm), FN6 (100 µm) and LY354740 (10 µm). Each agonist decreased SNP-stimulated cGMP formation by about 40%.

image

Figure 2. mGluR agonists reduce SNP-stimulated cGMP levels in neurons and astrocytes. (a) In rat cerebellar granule cells (CGC) NAAG (100 µm, n = 24), FN6 (100 µm, n = 24) and LY354740 (10 µm, n = 9) significantly inhibited cGMP formation stimulated by SNP (100 µm, n = 12). (b) In rat cerebellar astrocytes NAAG (100 µm, n = 18), FN6 (100 µm, n = 18) and LY354740 (10 µm, n = 12) significantly reduced cGMP formation stimulated by SNP (100 µm, n = 18). Results are expressed as a percentage of maximal stimulation with 100 µm SNP and are mean ± SEM. **p < 0.01, *p < 0.05 versus SNP (Student's t-test).

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Antagonists of group II mGluRs blocked the effects of mGluR3 agonists on SNP-stimulated cGMP formation in neurons and astrocytes (Fig. 3). In rat cerebellar granule cells (Fig. 3a), the group II mGluR antagonist LY341495 (10 µm) and the mGluR3-selective antagonist β-NAAG (100 µm) (Lea et al. 2001) reversed the effects of NAAG. In rat cerebellar astrocytes (Fig. 3b), β-NAAG (100 µm) and LY341495 (10 µm) blocked the effects of both 100 µm NAAG and FN6 on SNP-stimulated (100 µm) cGMP formation.

image

Figure 3. Antagonists of group II metabotropic receptors prevent the effects of NAAG in neurons and astrocytes. (a) In rat cerebellar granule cells (CGC) β-NAAG (100 µm, n = 11) and LY341495 (10 µm, n = 9), significantly blocked the inhibitory effect of NAAG (100 µm, n = 11) on SNP-stimulated cGMP formation (n = 12). p < 0.001, NAAG + SNP versus NAAG; ***p < 0.001 versus SNP + NAAG (Student's t-test). (b) In rat cerebellar astrocytes β-NAAG (100 µm, n = 12) and LY341495 (10 µm, n = 9) significantly antagonized the effects of NAAG (100 µm, n = 9) and FN6 (100 µm, n = 9) on SNP-mediated cGMP formation (n = 13). p < 0.001, SNP + NAAG versus SNP; p < 0.001, SNP + FN6 versus SNP; **p < 0.01 versus SNP + FN6 or SNP + NAAG (Student's t-test). All results are expressed as a percentage of maximal stimulation with SNP (100 µm) and values are mean ± SEM.

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The mGluR3-induced reductions in cAMP levels in cerebellar granule cells and astrocytes are sensitive to PTX, leading to the conclusion that activation of this receptor is mediated by an inhibitory G protein. To characterize the possible role of an inhibitory G protein in the coupling between mGluR3 and reduction in cGMP levels, granule cells and astrocytes were pretreated with PTX (1 µg/mL). After 18 h of incubation with PTX, agonists of metabotropic glutamate receptors were applied together with SNP. In cerebellar astrocytes (Fig. 4) exposed to NAAG (100 μM), FN6 (100 µm) or LY354740 (10 µm) and SNP (100 μM), the decrease in cGMP formation was blocked by pretreatment with PTX. In contrast, PTX had no apparent effect on the agonistic responses to NAAG (100 µm), FN6 (100 µm) and LY354740 (10 µm) in cerebellar granule cells (Fig. 5). As a positive control for this latter experiment, the same PTX treatment of cerebellar granule cells completely and consistently blocked the mGluR3-induced cAMP response (Wroblewska et al. 1993).

image

Figure 4. PTX prevents the inhibitory effect of group II agonists on cGMP formation in cerebellar astrocytes. In control cultures, NAAG (100 µm, n = 12), FN6 (100 µm, n = 6), and LY354740 (10 µm, n = 6) inhibited cGMP formation stimulated by SNP (100 µm), whereas in astrocytes exposed to PTX (1 µg/mL for 18 h) this inhibition was blocked (SNP + NAAG, n = 12; SNP + FN6, n = 6; SNP + LY354740, n = 6). Results are expressed as a percentage of maximal stimulation with SNP in control cells or cells treated with PTX. Values are mean ± SEM. **p < 0.01, *p < 0.05 versus SNP (Student's t-test).

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image

Figure 5. PTX does not prevent the inhibitory effect of NAAG, FN6 and LY34740 on SNP-mediated cGMP formation in cerebellar granule cells. NAAG (100 µm, n = 9), FN6 (100 µm, n = 10) and LY354740 (10 µm, n = 9) significantly inhibited cGMP formation stimulated by 100 µm SNP (n = 18) in control granule cells. In cells pretreated for 18 h with PTX (1 µg/mL) all three agonists (NAAG 100 µm, n = 9; FN6 100 µm, n = 14, LY354740 10 µmn = 9) caused significant inhibition of SNP-stimulated cGMP formation (n = 12). Results are expressed as a percentage of maximal stimulation with SNP in control cells or cells treated with PTX and are mean ± SEM. **p < 0.01, *p < 0.05 versus SNP (Student's t-test).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Cerebellar granule cells and cerebellar astrocytes express mGluR3 receptors that are negatively coupled to cAMP via a PTX-sensitive G protein (Wroblewska et al. 1993, 1998), as are cell lines stably transfected with mGluR3 cDNA (Tanabe et al. 1992; Wroblewska et al. 1997). Group II mGluR receptors also are negatively coupled to cAMP levels in striatal neurons (Manzoni et al. 1992) and hippocampal neurons (Schoepp et al. 1992). The data presented here provide the first data directly demonstrating that activation by the mGluR2-selective peptide transmitter NAAG and other group II mGluR agonists reduces SNP-stimulated cGMP levels in neurons and astrocytes; the cGMP coupling in granule cell neurons is mediated by a different G protein from that in astrocytes, the latter being PTX sensitive and the former not sensitive; and mGluR3 is coupled to two different G proteins in cerebellar granule cells, as distinguished by their PTX sensitivity.

There is substantial precedent (Albert and Robillard 2002) for the coupling of individual neurotransmitter receptors to both Gi and Go/s and to different Gi proteins. For example, the β1 but not β2 adrenergic receptor is coupled to both Gi and Gs, whereas the cannabinoid-CB1-i3/IP receptor is coupled to Gi1 and Gi2 and the adenosine-A1/GIRK receptor is coupled to Gi1,2,3 and Go1. As we have not examine this process in single astrocytes or cerebellar granule cells, and these cells may not be completely homogeneous in their qualities, it is not possible to predict from the present data whether mGluR3 is coupled to different cascades in individual cells.

Several results support the conclusion that the cGMP effect is mediated by mGluR3 rather than mGluR2 in cerebellar granule cells and cerebellar astrocytes. In primary cell culture, both cell types express high levels of mGluR3 mRNA and much lower levels of mGluR2 mRNA (Santi et al. 1994; Wroblewska et al. 1998). NAAG is at least 15-fold more potent in activation of mGluR3 than mGluR2 (Cartmell et al. 1997; Wroblewska et al. 1997; Schweitzer et al. 2000). FN6 also is highly selective for mGluR3, with very little effect on mGluR2 even at 1 mm (Nan et al. 2000). Finally, the effect of NAAG was blocked by β-NAAG, a highly selective mGluR3 antagonist (Lea et al. 2001). The agonist LY354740 and antagonist and LY341495 were used at concentrations well above their reported EC50 and IC50 values. Outside of group II mGluRs, the only receptor potentially affected by this dose of LY354740 is mGluR6 and this receptor is not expressed at detectable levels in the cerebellum. In addition, the other agonists used, FN6 and NAAG, do not activate mGluR6. Although the antagonist LY341495 was used at a concentration that would fully block mGluRs 2, 3, 7 and 8, it was tested in these cell cultures against NAAG and FN6, which are selective mGluR3 agonists. Preliminary data in mGluR3-transfected C6 glioma cells also fully support the conclusion that this receptor couples negatively to a PTX-sensitive cGMP cascade in these cell lines (B. Wroblewska, unpublished data).

Cyclic GMP levels are regulated directly by the activity of guanylate cyclase and cGMP PDE and indirectly by calcium–calmodulin activation of NO synthase. NAAG activation of mGluRs reduces voltage-dependent calcium currents (Bischofberger and Schild 1996) and it is thus possible that under some conditions this causes a reduction in NO levels and reduction in activation of guanylate cyclase. In contrast, however, in cerebellar granule cells and cerebellar astrocytes cGMP levels induced by direct stimulation of soluble guanylate cyclase by NO donors, including SNP, were shown decrease in response to increases in intracellular calcium (Baltrons et al. 1997).

In the present study, the reduction in cGMP levels occurred in the presence of 1 mm IBMX, a broad-spectrum PDE inhibitor. This condition would appear to rule out the possibility that the mGluR3-induced reduction in cGMP levels was mediated by activation of cGMP PDEs and militates in favor of Gi-mediated inhibition of guanylate cyclase. However, the PDE(9) is a cGMP-specific PDE that is IBMX insensitive and expressed in brain (van Staveren et al. 2002). Thus, it remains possible that mGluR3 is coupled to a downstream activator of PDE(9) or a PDE(9)-like enzyme that lowers cGMP levels in the presence of IBMX. The Gi coupling to guanylate cyclase in astrocytes is consistent with the conclusion that mGluR3 activation reduces cAMP levels via inhibition of adenylate cyclase (Wroblewska et al. 1993, 1997, 1998).

A difficulty with assessing the efficacy of PTX in blocking the actions of putative Gi coupling in a cell system is the second-order effects of the toxin itself. Different cell types respond differently to PTX treatment owing to variations in the other cell processes that are regulated by PTX-sensitive Gis. In the present study, for example, PTX treatment stimulated basal levels of cGMP in astrocytes but did not affect the levels obtained after stimulation with 100 µm SNP alone. In contrast, PTX treatment had no effect on basal cGMP levels in cerebellar granule cells, but in some experiments inhibited SNP-stimulated levels by about 50% in the absence of mGluR3 agonist. To allow for this effect, the PTX treatment data were expressed as a percentage of the maximal stimulation by 100 µm SNP in the presence of PTX.

These data support the hypothesis that activation of mGluR3 and perhaps other group II and group III mGluRs functions to regulate both cAMP and cGMP levels in neurons and astrocytes. Significantly, the regulation of cGMP levels differs in these two cell types. Understanding the breadth of this coupling gains importance from the role of group II mGluRs in promoting neuronal survival under excitotoxic conditions (Nicoletti et al. 1996; Flor et al. 2002). Agonists of group II metabotropic receptors are neuroprotective in cell culture (Bruno et al. 1998), in a rat model of Huntington's disease (Orlando et al. 1997) and in ischemia/hypoxia (Opitz and Reymann 1993; Harada et al. 2000; Lu et al. 2000). With respect to the role of cGMP, it has been shown that induction of NO by SNP in hippocampal neurons induces programmed cell death, whereas activation of mGluRs significantly limited this process. Inhibition of guanylate cyclase is neuroprotective in cerebellar granule cells in culture (Montoliu et al. 1999). In addition, inhibition of NAAG peptidase activity increases synaptic levels of the peptide and has therapeutic potential via activation of group II mGluRs in animal models of clinical conditions (reviewed in Neale et al. 2005). The discovery that mGluR3 is negatively coupled to cGMP levels greatly extends the range of cellular cascades that may be considered in the future analyses of the mechanisms through which activation of this receptor induces these therapeutic effects.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The authors thank Eli Lilly & Co. for providing LY354740, Alan Kozikowski for providing FN6 and Jarda Wroblewski for review of the manuscript. This research was supported by National Institutes of Health grant NS38080 to JHN.

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  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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