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Selective activation of central subtypes of the nicotinic acetylcholine receptor has opposite effects on neonatal excitotoxic brain injuries¹

VINCENT LAUDENBACH^*,2, FADIA MEDJA^*, MICHELE ZOLI^,, FRANCESCO M. ROSSI, PHILIPPE EVRARD^*, JEAN-PIERRE CHANGEUX and PIERRE GRESSENS^*

^* Laboratoire de Neurologie du Développement, INSERM E9935, Hôpital Robert Debré, Paris, France;
Laboratoire Récepteurs et Cognition, URA CNRS 2182, Institut Pasteur, Paris, France; and
Department of Biomedical Sciences, Section of Physiology, University of Modena and Reggio Emilia, Italy

²Correspondence: Department of Anesthesiology, Hôpital Robert Debré, 48, Bd Sérurier, 75019 Paris, France. E-mail: vlaudenb{at}infobiogen.fr

SPECIFIC AIMS

Using a murine model of excitotoxic brain injury, we investigated the neuroprotective properties of nicotine in neonates, separating the respective roles of the two dominant isoforms of the central nicotinic acetylcholine receptor (nAChR), namely, ₄ß₂ (heteropentameric) and ₇ (homopentameric), by pharmacological (i.e., specific agonists or antagonists) and molecular (ß₂^-/- and ₇^-/- ‘knockout’ mice) approaches. In vitro excitotoxic challenge experiments were performed in primary cultures of cortical neurons from wild-type (W_t, ß₂^+/+) and mutant (ß₂^-/-) animals.

PRINCIPAL FINDINGS

1. Nicotine prevented neuronal death induced by intracerebral injection of the glutamatergic agonist ibotenate into the developing cortex
Under general anesthesia, 5-day-old mice (PD5) of two different strains, i.e., Swiss (outbred) and W_t C₅₇Bl₆ (inbred), each received a stereotactic injection into the right frontoparietal neopallium of ibotenate (10 µg/2 µl), a glutamatergic agonist acting mainly via N-methyl-D-aspartate (NMDA) receptors. Animals were treated with intraperitoneal (i.p.) S(-) nicotine (0.625, 1.25, 2.5, 5, or 10 mg/kg/12 hx3 injections), or saline (controls) 24 h before the ibotenate injection. On PD10, they were decapitated and their brains were processed for histopathological examination after cresyl violet staining. In controls, mean size of the ibotenate-induced excitotoxic cortical lesion in Swiss, W_t C₅₇Bl₆ (ß₂^+/+), and W_t C₅₇Bl₆ (₇^+/+) mice was 800 ± 52 µm, 914 ± 53 µm, and 880 ± 80 µm, respectively (mean±SE, P=0.3). Pretreatment with S(-) nicotine resulted in a significant dose-dependent reduction in lesion size (-51.8±5.9% with 1.25 mg/kg/12 h in W_t C₅₇Bl₆ (ß₂^+/+) mice, P <0.01) (Fig. 1 A).

View larger version (33K): [in this window] [in a new window]   Figure 1. Influence of the modulation of putative ß2- and 7-containing nAChRs on the ibotenate-induced cortical lesion. 5-Day-old (PD5) mice received an intracerebral (i.c.) injection of ibotenate (NMDA agonist, 10 µg/2 µl) into the right frontoparietal neopallium. On PD10, brains were harvested, cut into 20 µm coronal sections, and stained with cresyl violet. Bars show the mean length (±SE) of the cortical excitotoxic lesion along the sagittal fronto-occipital axis, used as an index of mean lesion size and quantified by two observers unaware of treatment conditions. Numbers in brackets are the numbers of animals in each experimental group. Statistically significant differences between controls and experimental animals: *P < 0.05, **P < 0.01 by ANOVA with Dunnett’s multiple comparison test. A–D) Involvement of ß2-containing (most likely 4ß2) nAChRs in neonatal nicotinic neuroprotection. A) Before i.c. ibotenate injection, mouse pups (Swiss and WT C57Bl6) were treated during 24 h with the following drugs or drug combinations: i.p. S(-) nicotine (0.625, 1.25, 2.5, 5, or 10 mg/kg/12 h), dihydro-ß-erythroidine (DHßE, 0.2 mg/kg/12 h), mecamylamine (Mec, 1 mg/kg/12 h), or saline. D) Animals lacking 4ß2-containing nAChRs (ß2-/-) received S(-) nicotine, DMXB, MLA, or saline. B, C) Aggravating effect of 7-containing nAChRs on neonatal excitotoxic cortical lesion. B) Before i.c. ibotenate injection, Wt Swiss and C57Bl6 mice were given i.p. injections of DMXB (1 mg/kg/12 h), MLA (5 mg/kg/12 h), or saline. C) Animals lacking 7-containing nAChRs (7-/-) received only i.p. saline and i.c. ibotenate; their cortical lesions were compared with those of their WT (7+/+) siblings subjected to the same treatment.

2. Putative ₄ß₂-containing nAChRs mediated nicotine-related neuroprotection in neonates and putative ₇-containing nAChRs mediated detrimental effects
In both Swiss and W_t C₅₇Bl₆ mice, the protective effect of i.p. S(-) nicotine was abolished by coadministration of dihydro-ß-erythroidine (DHßE, a nicotinic antagonist with relative selectivity for ₄ß₂ nAChRs, 0.2 mg/kg/12 hx3 injections) (Fig. 1 A). On the other hand, pretreatment with 3-[2,4-dimethoxybenzylidene] anabaseine (DMXB, an agonist of ₇-containing nAChRs, a.k.a. GTS-21, 1 mg/kg/12 hx3 injections) increased the size of the cortical ibotenate-induced lesion by 37.1 ± 12.2% (P<0.01) and 19.9 ± 5.8% (P<0.05) in Swiss and W_t C₅₇Bl₆ animals, respectively. Conversely, treatment with methyllycaconitine (MLA, a putative antagonist of ₇-containing nAChR, 5 mg/kg/12 hx3 injections) significantly reduced median lesion size (-46.2±7.5% compared with controls in W_t C₅₇Bl₆, P<0.01). Coinjection of DMXB with MLA had no significant effect vs. control animals (Fig. 1 B).

3. Animals lacking ß₂- or ₇-containing nAChRs displayed altered sensitivity to intracerebral ibotenate
Ibotenate was injected on PD5 to W_t (₇^+/+ and ß₂^+/+) and mutant (₇^-/- and ß₂^-/-) C₅₇Bl₆ mice (n=8–21/group; see Principal Finding 1). On PD10, ₇-Null mutant mice had significantly smaller ibotenate-induced cortical lesions than their W_t siblings (620±39.3 µm vs. 880±80 µm, P<0.05) (Fig. 1C ). However, ß₂^-/- animals (which lack ß₂-containing heteropentameric nAChRs and central high-affinity binding sites for nicotine) displayed a lower sensitivity to ibotenate than W_t controls (lesion size, 540±62.4 vs. 914±53 µm, P<0.01) (Fig. 1D ). This unexpected result prompted us to investigate nicotinic neuroprotection in ß₂^-/- animals both in vivo and in vitro.

4. The nAChR ß₂-null mutation resulted in loss of the neuroprotective properties of nicotine
Primary cortical neuron cultures were prepared from E14.5 ß₂^+/+ and ß₂^-/- mouse embryos. Reverse transcriptase PCR on total neuronal RNA was used to assess expression of the various nAChR subunits in cultured neurons. After 10 days in vitro, the neurons were incubated in S(-) nicotine (10 µM), DMXB (0.1, 1, or 10 µM), mecamylamine (a nonspecific neuronal nAChR antagonist, 1 µM), DHßE (100 nM), MLA (100 nM), or control, alone or in combination, for 12 h before an excitotoxic challenge by exposure to NMDA (300 µM, 60 mn). Eight hours later, the neurons were fixed and fluorescent chromatin staining was performed using bis-benzimide. Counts of nuclei showing features of cell death (i.e., pyknosis) were used as an indicator of neuron viability. Preincubating ß₂^+/+ neurons in 10 µM S(-) nicotine reduced the number of pyknotic nuclei after NMDA application by 38.1 ± 2.3% (P<0.01). Coincubation with S(-) nicotine and DHßE or mecamylamine produced levels of neuronal death not significantly different from those induced by NMDA alone. In ß₂^-/- cultures, S (-) nicotine was not protective. In accordance with these results, S(-) nicotine had no significant effect on the mean size of ibotenate-induced excitotoxic cortical lesions in ß₂^-/- mutant animals compared with saline (Fig. 1D ). Finally, DMXB, previously shown to prevent neuronal death in vitro, displayed a similar dose-response relationship in ß₂^+/+ and ß₂^-/- cultured neurons.

5. Autoradiography binding experiments showed similar baseline expression of cortical NMDA and ₇ receptors in nAChR ß₂^+/+ and ß₂^-/- mouse pups
To look for neurochemical correlates of our findings on spontaneous and pharmacologically induced neuroprotection in ß₂^+/+ and ß₂^-/- mice, we used autoradiography to study cortical densities of NMDA and ₇ receptors. We found no significant differences in the levels of labeling for ³H-MK-801 (NMDA ligand) or ¹²⁵I--bungarotoxin (₇ nAChR ligand) between 5-day-old mutant animals and their W_t siblings. In a second set of experiments, we investigated the influence of treatment with DMXB and/or ibotenate on the density of ¹²⁵I--bungarotoxin binding sites in both strains. We found that i.p. DMXB, but not intracerebral ibotenate, increased by 20% the density of putative cortical ₇ nAChR in ß₂^+/+ mice. In contrast, neither treatment altered ¹²⁵I--bungarotoxin binding in ß₂^-/- animals.

CONCLUSIONS

Nicotine pretreatment in vivo decreased the mean size of the cortical lesion induced by an intracerebral injection of ibotenate in the newborn mouse. On the contrary, an agonist of ₇ nAChRs potentiated the effect of ibotenate. Thus, two nAChR subtypes had opposite effects. Nicotine-related neuroprotection was counteracted by DHßE in W_t mice and absent in ß₂^-/- mice, suggesting mediation by ß₂-containing (possibly ₄ß₂) nAChRs. The neurotoxic action of DMXB and the protective effect of MLA (a putative selective antagonist for ₇-containing nAChRs) in Swiss and C₅₇Bl₆ W_t mice indicate that the ₇ nAChR subtype plays a deleterious role in this model of neonatal brain injury. Accordingly, animals lacking ₇-containing nAChRs were less susceptible to ibotenate than their W_t siblings. Treatment with ₇-containing nAChR agonists has shown neuroprotective effects in adults. The different effects of ₇-containing nAChR activation in newborn and adult mice may be related to the high permeability to Ca²⁺ of this ligand-gated ion channel, which undergoes a transient expression peak in the cortex during the first postnatal week.

In vitro experiments confirmed that nicotine is neuroprotective against the excitotoxicity elicited by NMDA in primary cortical cultures. In contrast to the in vivo model, both main nAChR subtypes seemed neuroprotective. An involvement of ß₂-containing nAChRs is suggested by our findings that neuroprotection was counteracted by DHßE in ß₂^+/+ neurons and was not detected in cultures from ß₂^-/- mice. The efficacy of DMXB argues in favor of involvement of ₇-containing nAChRs. This partial discrepancy vs. in vivo results may be related to several factors, including differences in the level of ₇-containing nAChR expression or absence of glial cells in our primary cultures.

In addition to abolishing the neuroprotective effects of nicotine in vivo and in vitro, absence of the ß₂ subunit changed the basal susceptibility of cortical tissue to the neurotoxic effect of NMDA agonists. This was unexpected because pharmacological activation of ß₂-containing nAChRs was neuroprotective. Compensatory changes in the development of systems influencing the outcome of ibotenate lesions may possibly occur in the cortex of ß₂^-/- mice. Two factors that may influence NMDA excitotoxicity in ß₂^+/+ and ß₂^-/- mice were assessed: the levels of expression of cortical NMDA receptors and ₇ nAChRs. To find possible difference between the two strains regarding any modulation of ₇ nAChRs in response to pharmacological treatments, ¹²⁵I--bungarotoxin binding experiments were performed in baseline conditions at PD5 as well as in PD6 ß₂^+/+ and ß₂^-/- mice previously treated with DMXB and/or ibotenate. Although no differences in ³H-MK-801 or ¹²⁵I--bungarotoxin binding were detected at PD5, we found that treatment with DMXB during 24 h enhanced the expression of ₇ nAChR in ß₂^+/+ but not ß₂^-/- newborn mice. Ibotenate had no significant effect. Thus, the absence of ß₂-containing nAChRs may alter expression and/or response to pharmacological treatments of 7 nAChRs in the neonatal period, possibly contributing to the absence of DMXB-induced toxicity after ibotenate lesion in ß₂^-/- mice.

In conclusion, our findings confirm the neuroprotective properties of S(-)nicotine against excitotoxic injuries and provide the first evidence that these properties are present during the neonatal period. Our results also highlight the need for careful attention to the subtype of nAChR activated by nicotinic agonists during CNS development: treatment with an ₇-homopentamer agonist may be deleterious at this stage. The murine model of excitotoxic brain injury used here displays histopathological similarities with the brains of human newborns subjected to hypoxic-ischemic or inflammatory insults. Such injuries result in severe disabilities for which no effective prevention is available. Our results suggest that subtype-selective nAChR (possibly ₄ß₂ nAChR) agonists may hold promise for preventing permanent neurological impairments related to neonatal insults.

View larger version (36K): [in this window] [in a new window]   Figure 2. Schematic representation of the modulating effects of central subtypes of the nicotinic acetylcholine receptor (nAChR) on neuronal death induced by agonists of excitatory amino acids receptors within the developing mouse brain.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0532fje; to cite this article, use FASEB J. (January 14, 2002) 10.1096/fj.01-0532fje

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