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Published as doi: 10.1096/fj.07-8260com.
 (The FASEB Journal. 2007;21:4028-4037.)
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Loss of high-affinity nicotinic receptors increases the vulnerability to excitotoxic lesion and decreases the positive effects of an enriched environment

Alessio Zanardi*, Rosaria Ferrari*, Giuseppina Leo*, Uwe Maskos{dagger}, Jean-Pierre Changeux{dagger} and Michele Zoli*,1

* Department of Biomedical Sciences, Section of Physiology, and Interuniversity Center for the Study of Aging, University of Modena and Reggio Emilia, Italy; and

{dagger} CNRS UA 2182–"Récepteurs et Cognition", Institut Pasteur, 28, rue du Dr. Roux, 75724 Paris Cédex 15, France

1Correspondence: Department of Biomedical Sciences, Section of Physiology, University of Modena and Reggio Emilia, via Campi 287, 41100 Modena, Italy. E-mail: mzoli{at}unimo.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pharmacological activation of nicotinic acetylcholine receptors (nAChRs) exerts neuroprotective effects in cultured neurons and the intact animal. Much less is known about a physiological protective role of nAChRs. To understand whether endogenous activation of β2* nAChRs contributes to the maintenance of the functional and morphological integrity of neural tissue, adult β2–/– mice were subjected to in vivo challenges that cause neurodegeneration and cognitive impairment (intrahippocampal injection of the excitotoxin quinolinic acid), or neuroprotection and cognitive potentiation (2-month exposure to an enriched environment). The excitotoxic insult caused an increased deficit in the Morris water maze learning curve and increased loss of hippocampal pyramidal cells in β2–/– mice. Exposure to an enriched environment improved performance in contextual and cued fear conditioning and object recognition tests in β2+/+, whereas the improvement was absent in β2–/– mice. In addition, β2+/+, but not β2–/–, mice exposed to an enriched environment showed a significant hypertrophy of the CA1/3 regions. Thus, lack of β2* nAChRs increased susceptibility to an excitotoxic insult and diminished the positive effects of an enriched environment. These results may be relevant to understanding the pathophysiological consequences of the marked decrease in nAChRs that occurs in neurodegenerative diseases such as Alzheimer’s disease and Parkinson’s disease.—Zanardi, A., Ferrari, R., Leo, G., Maskos, U., Changeux, J.-P., Zoli, M. Loss of high affinity nicotinic receptors increases the vulnerability to excitotoxic lesion and decreases the positive effects of an enriched environment.


Key Words: nicotinic subunit knockout mice • quinolinic acid • Morris water maze • fear conditioning • hippocampus


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PHARMACOLOGICAL ACTIVATION OF nicotinic acetylcholine receptors (nAChRs) by nicotine and nicotinic agonists exerts neuroprotective effects both in cultured neurons and in the intact animal (reviewed in refs. 1 2 3 ). In vitro, nicotine and nicotinic agonists protect against several kinds of toxicity including excitotoxicity (4 , 5) and toxicity induced by amyloid β protein (Aβ, the amyloidogenic fragment of the amyloid precursor protein) (6 , 7) in primary hippocampal or cortical neurons. In vivo, nicotine can prevent cell death and/or functional impairments induced by a number of neurotoxic insults, including excitotoxicity (8) , ischemia (9) , and knife cuts (10) . These effects are blocked by {alpha}4/β2*- and/or {alpha}7*-selective nicotinic antagonists, suggesting that they are mediated through activation of multiple neuronal nAChRs. Nicotine may also protect against functional and histological consequences of an insult in human diseases as suggested by epidemiological studies showing that smoking is associated with lower incidence of Parkinson’s disease (PD) and perhaps Alzheimer’s disease (AD) (11 , 12) and the evidence of decreased brain levels of Aβ in aged subjects (13) and AD patients (14) who smoke.

Much less is known about a possible functional and histological protective role of nAChR activation by endogenous ligands in the brain. A neuroprotective role of endogenously activated nAChRs would be of pathophysiological relevance, since decreases in brain nAChRs have been observed in several neurological diseases, and include a marked and consistent decrease in high-affinity nicotine binding in cortico-hippocampal regions in several forms of dementia (15 , 16) . Accordingly, it has been hypothesized that loss of nAChRs may contribute to the development of cognitive impairments and neurodegeneration seen in dementia (2 , 17 , 18) .

Knockout mice lacking nAChR subunits can be used to identify the effects of chronic loss of specific nAChR subtypes on cognitive performance and neurodegeneration. In this respect, mice lacking the nAChR subunit β2 (β2–/– mice) are proving to be particularly interesting. β2–/– mice lack most high-affinity nicotine binding (i.e., {alpha}-bungarotoxin (Btx)-insensitive nAChRs) but maintain normal levels of Btx-sensitive nAChRs in the brain (19) . In cortico-hippocampal areas, these mice express no high-affinity nicotine binding and normal Btx binding. During senescence, β2–/– mice show cortico-hippocampal degeneration and impairments in emotional and spatial learning and memory (17 , 20) , suggesting that loss of β2* nAChRs removes a level of protection against neurodegenerative processes during senescence.

To help understand a possible role of endogenous β2* nAChR activation in maintenance of functional and morphological integrity in neural tissue, we tested adult β2–/– mice using in vivo challenges which cause neurodegeneration and cognitive impairment (excitotoxic insult) or neuroprotection and cognitive potentiation (enriched environment). As an in vivo model of excitotoxic insult, we studied the behavioral and histological effects of an intrahippocampal injection of the excitotoxin quinolinic acid (21) . It has been shown that rodents kept in an enriched environment have improved cognitive performance and increased resistance to neurotoxic insults (22) . We therefore studied whether loss of β2* nAChRs prevents some positive cognitive and structural effects of an enriched environment.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Male and female wild-type (β2+/+) and knockout (β2–/–) mice lacking the β2 subunit of the nAChR were obtained from Charles River laboratories (France). They were bred from animals back-crossed 19 generations onto the C57BL/6J background. Mice were housed in a colony room maintained at 22°C on a 12:12 light:dark cycle with lights on at 7:00 am. Food and water were available at all times. All animal experiments were conducted in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) Laboratory Animals.

In the experiments on quinolinic acid hippocampal lesions, both male and female 10–12 month-old mice were used. They were group housed in cages with a maximum of 5 mice per cage.

In the experiments on enriched environment, only female mice were used since male C57BL/6 mice housed in an enriched environment often develop aggressive behaviors, which can lead to severe injuries or death of some of the mice (22 , 23) .

Enriched environment
Three-week-old female mice were either housed in a standard environment or an enriched environment. Animals exposed to standard environment were housed in pairs in standard cages (30x16x11 cm). Animals exposed to an enriched environment were housed together in a group of 10 in one of two large cages (36x54x19 cm or 45x25x22 cm with a labyrinth) containing an assortment of objects, including climbing ladders, running wheel, balls, plastic and wood objects suspended from the ceiling, paper, cardboard boxes, and nesting material. Toys were changed every 2–3 days, while the bedding was changed every week. The animals were kept in the enriched or standard environment for 2 months before the start of behavioral tests.

Intrahippocampal excitotoxic lesions
The lesion paradigm was adapted from O’Neill et al. (21) . Quinolinic acid was injected bilaterally into the hippocampus using a 1 µl Hamilton syringe (25 g needle) and the following coordinates: AP: –1.8 mm; L: 1.4 mm; D: –1.8 mm. In a set of preliminary experiments we tested the extent of hippocampal lesion induced by several doses of intrahippocampal quinolinic acid injections (from 5 to 30 nmol/250 nl PBS, pH 7.4) in adult C57BL/6 mice. While 5 nmol/injection quinolinic acid induced only minimal lesions in the CA1 field, 15–30 nmol/injection induced greater than 50% loss of pyramidal neurons in the CA1 field at the level of the injection. We chose the 15 nmol/injection dose for the following experiments. These animals were then tested at the Morris water maze test and Y-maze spontaneous alternation test and shown not to have any significant learning deficits (not shown).

Quinolinic acid or vehicle (sham-operation) were bilaterally injected at the coordinates described above. The animals were allowed to recover from surgery for 7 days before starting behavioral testing.

Behavioral tests
Morris water maze
The Morris maze task was modified from Morris (24) (see also ref. 17 ). To assess spatial learning, animals were placed in a circular pool with a diameter of 125 cm filled with milky water at 19°C and allowed to swim for 90 s or until they found the location of a hidden circular platform with 11 cm diameter. Mice were trained with 4 trials per day (starting from a different quadrant for each trial) with an intertrial interval of 15 min for 11 days and escape latency was recorded and averaged across the 4 trials for each day. On the 12th day the platform was removed and the animals were allowed to swim for 60 s. Time spent in each quadrant as well as overall distance traveled by each mouse was measured.

Fear conditioning
Training took place in a conditioning chamber (23x22x24 cm) with gray Plexiglas walls and ceiling. Scrambled shock was delivered by a shock source to a grid floor made of stainless steel bars, 2 mm in diameter, spaced 0.5 cm apart. This chamber was housed in an Igloo ice chest (60x40x33 cm) with a clear Plexiglas window (60x33 cm) cut into the front of the chest. A speaker was mounted on the back wall, through which a computer-generated tone (1000 Hz) was delivered. A fan, mounted on the back wall of the chest, provided ventilation as well as background masking noise. The chamber was cleaned with 70% ethanol before conditioning and the contextual test and again with 70% isopropyl alcohol before the altered context and cued tests.

Fear conditioning procedures were based on published methods (25) . Mice were transferred to the conditioning chamber and, after an initial acclimatization period of 2 min, were presented with 3 pairings of the tone with foot shock (0.5 mA, 2 s). The tone was presented for 30 s, and the shock was administered during the last 2 s of the tone. Pairings were separated by 2 min, and mice were removed from the chamber 30 s after the last shock presentation.

Approximately 24 h after conditioning, mice were tested for contextual conditioning. Mice were placed into the conditioning chamber for 5 min, and freezing behavior was scored. After all mice were scored in the contextual test, subjects were transferred to an altered context and then tested for freezing to the tone. The altered context consisted of a 20 x 20 x 20 cm clear plastic cage that was covered with a filter lid. A novel odor was added to the context by placing a few drops of orange extract (McCormick) mixed with water into a cup that was placed outside the cage. Mice were scored for freezing in the altered context for 3 min (altered context test) and then for 3 min during presentation of the tone (cued conditioning test). The same procedures were repeated 5 and 10 days after conditioning.

Freezing was scored using a time sampling procedure in which every 10 s, a determination was made whether or not a mouse was freezing. Freezing was defined as the absence of all movement except for respiration for a minimum of 1 s (26) . Scoring began ~15 s after the mouse was placed into the chamber. Mice were scored for 30 intervals during the context test, 18 intervals during the altered context test, and 18 intervals during the cued test.

Y-maze
Spontaneous alternation performance was assessed using a symmetrical Y-maze as described in Sarter et al. (27) . The maze was constructed of black plastic. Each arm was 22 cm long x 10 cm high x 7 cm wide, and the three arms were connected through a symmetrical 3-way central corridor. Arms were randomly designated as A, B, or C. Mice were allowed to roam freely through the maze during an 8-min trial, and the series of arm entries was recorded. Alternation was defined as entries into all three arms consecutively (i.e., ABC, ACB, CAB, etc.). The maximum number of alternations was, therefore, the total number of arm entries minus two and the percentage of alternation was calculated as (actual number of alternations/maximum number of alternations) x 100.

Object recognition
Object recognition was assessed as described in Vaucher et al. (28) with slight modifications. Briefly, the animals were tested in a clear plastic cage with sloping side-walls to prevent shadows from overhead illumination (36x22 cm, 1 cm sawdust on floor). Habituation consisted of a 5 min period in the empty cage the day before the test. On the first day of testing, the mice were exposed for 5 min to one pair of objects selected from a set of four objects that differed in shape, surface color, contrast, and texture. The four objects were previously shown to induce approximately equal time of exploration in C57BL/6 mice. Three hours after the initial exposure, mice were re-exposed for 5 min to one of the original objects and a member of the second pair of objects. Twenty-four hours after the initial exposure, mice were allowed to explore the other member of the original sample object pair, and the remaining member of the second pair, for 5 min. In all tests, time spent exploring each object was recorded. A mouse was considered to be engaging in exploratory behavior if the animal touched the object with its forepaw or nose or sniffed at the object within a distance of 1.5 cm. Testing was performed by an experimenter unaware of the treatment group. After each exposure, the objects and the cage were wiped with 70% ethanol to eliminate odor cues.

Histology and image analysis
At the end of behavioral testing, the mice were sacrificed and their brains were processed for histological analysis. Neuronal and glial cells were stained using the Nissl method. Series of 100 µm-spaced frozen coronal sections (14 µm thick) were cut at a cryostat following the planes indicated by Paxinos and Franklin atlas (29) and processed according to established protocols (30) . The extent of neuronal degeneration was evaluated in the pyramidal layer of cornu ammonis (CA) 1 and 3 fields and granule cell layer of the dentate gyrus using an image analyzer [KS300 software, Zeiss-Kontron, München, Germany, (31) ]. Briefly, after acquisition of an image of the hippocampal region, several parameters were measured: the length of the pyramidal or granule cell layer with intact or degenerated neurons and the area of the CA1/3 fields or dentate gyrus. Length of neuronal layers and regional areas were measured by means of the DISTANCE and MEASOBJ functions of the image analyzer, respectively. Morphometric analysis was performed at the level of needle insertion (–1.8 mm from bregma) in lesion experiments and at –2.0 mm from bregma in enriched environment experiments. The analysis was performed in two coronal sections/side/animal.

Statistical analysis
Data are presented as mean ± SEM. Statistical analysis was performed by means of the general linear model (repeated measures or general factorial) analysis of variance (ANOVA, statistical package SPSS vers. 10). When a significant interaction was detected, we also performed a one-way ANOVA. P < 0.05 was considered as a threshold for significant difference.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Loss of β2* nAChRs intensifies behavioral deficits and hippocampal lesions induced by quinolinic acid
To test whether the loss of β2* nAChRs influences the behavioral and morphological outcome of quinolinic acid-induced hippocampal lesions we determined whether a bilateral antero-dorsal hippocampal lesion resulted in differential effects in 10–12-month-old β2+/+ and β2–/– mice. This treatment causes relatively localized hippocampal cell loss and no significant behavioral deficit in the Morris water maze or spontaneous alternation in a Y-maze tests in wild-type mice (see Materials and Methods). Sham-operated or lesioned β2+/+ and β2–/– mice were tested for their ability to find a hidden platform in the Morris water maze for 11 days and were subjected to the transfer test 24 h after the last training session. Analysis of the learning curve (Fig. 1 A) by repeated measures, two-way ANOVA showed a significant Genotype x Lesion interaction as well as a significant main effect of Time (indicating an overall improved performance with time) and Lesion [Time: F(10,24)=32.57, P<0.001; Genotype: F(1,24)=2.65, NS; Lesion: F(1,24)=5.16, P=0.032; GenotypexLesion: F(1,24)=5.31, P=0.030]. In view of the significant interaction, we then performed a repeated measures, one-way ANOVA on the 4 groups, showing that the performance of lesioned β2–/– mice was significantly worse than that of all other groups, that in turn were not significantly different between them [F(3,24)=4.37, P=0.014; posthoc LSD test: β2–/– lesioned vs. β2+/+ sham P=0.011, vs. β2+/+ lesioned P=0.010, vs. β2–/– sham P=0.003, all other comparisons NS]. Therefore, both genotype and lesion seem necessary to worsen the performance in the maze. This interpretation is further supported by the evidence that in preliminary experiments (see Materials and Methods), no significant effect of the lesion was observed in adult C57BL/6 mice. These findings indicate that lack of β2* nAChRs significantly worsens the outcome of intrahippocampal quinolinic acid lesion.


Figure 1
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  		Figure 1. Morris water maze performance in 10–12 month-old β2+/+ and β2–/– mice (8–9 mice/group) with a bilateral quinolinic acid injection in the dorsal hippocampus. A) Learning curve for training to find the hidden platform in the Morris water maze. The latency to find the platform in the 4 sessions of each day was averaged in order to obtain one value/animal/day. Mean ± SEM values are shown. Statistical analysis of the overall learning curve was performed by means of repeated measures two-way ANOVA, Time: F(10,24) = 32.57, P < 0.001; Genotype: F(1,24) = 2.65, NS; Lesion: F(1,24) = 5.16, P = 0.032; Genotype x Lesion: F(1,24) = 5.31, P = 0.030. To interpret two-way ANOVA results we performed a repeated measures one-way ANOVA: F(3,24) = 4.37, P = 0.014; posthoc LSD test: β2–/– lesioned vs. β2+/+ sham P = 0.011, vs. β2+/+ lesioned P = 0.010, vs. β2–/– sham P = 0.003, all other comparisons NS. * = P < 0.05 vs. all other groups. B) Transfer test performance. Quadrants are designated as follows: Training = the quadrant of the maze that contained the submerged escape platform during training, ADJ-L = the quadrant of the maze to the left of the training quadrant, ADJ-R = the quadrant of the maze to the right of the training quadrant. Opposite = the quadrant of the maze directly opposite to the training quadrant. Data are shown as mean ± SEM. Time spent in the quadrant where the platform was placed during the training phase: two-way ANOVA, Genotype: F(1,24) = 2.25, NS; Lesion: F(1,24) = 5.74, P = 0.025; Genotype x Lesion: F(1,24) = 2.95, P = 0.098. To interpret two-way ANOVA results we performed a one-way ANOVA: F(3,24) = 3.66, P = 0.026; posthoc LSD test: β2+/+ lesioned vs. β2+/+ sham P = 0.009, vs. β2+/+ lesioned P = 0.031, vs. β2–/– sham P = 0.010, all other comparisons NS. Overall distance: no significant effect. * = P < 0.05 vs. all other groups.

On day 12, mice underwent the transfer test. Percent time spent by the mouse in the quadrant where the platform was placed during the training phase is considered as an index of spatial memory. The analysis of behavior in the transfer test was carried out by two-way ANOVA and showed a trend for significant Genotype x Lesion interaction [F(1,24)=2.95, P=0.098] and a significant main effect for Lesion [F(1,24)=5.74, P=0.025] but no main effect for Genotype [F(1,24)=2.25, NS] for the percentage of time spent in the correct quadrant (Fig. 1B ). A one-way ANOVA on the four groups showed that % time in correct quadrant of β2–/– lesioned mice was significantly decreased with respect to all other groups that did not differ between them [F(3,24)=3.66, P=0.026; posthoc LSD test: β2+/+ lesioned vs. β2+/+ sham P=0.009, vs. β2+/+ lesioned P=0.031, vs. β2–/– sham P=0.010, all other comparisons NS]. Therefore, the Lesion main effect at the two-way ANOVA is probably not relevant. This is indeed supported by the fact that no significant effect was observed in a preliminary experiment performed in lesioned adult C57BL/6 mice (see Materials and Methods). No significant main effect or interaction in the overall distance covered by the mice was observed (β2+/+ sham 874±71 cm/min, β2+/+ lesioned 894±81 cm/min, β2–/– sham 896±49 cm/min, β2–/– lesioned 931±53 cm/min, not shown), indicating that the changes observed cannot be attributed to motor impairments.

The mice were then tested in the Y-maze, and no significant effect of genotype or quinolinic acid-lesion was observed (% alternation: β2+/+ sham 58.1±3.4, β2+/+ lesioned 56.3±3.3, β2–/– sham 62.0±3.3, β2–/– lesioned 56.3±4.4, NS).

In parallel with the behavioral deficits in the Morris maze, the absence of β2* nAChRs aggravated hippocampal cell loss induced by quinolinic acid (Fig. 2 ). Quinolinic acid caused loss of neurons and shrinkage of CA1 and CA3 hippocampal fields in both β2+/+ and β2–/– mice; however, the histological outcome of the lesion was significantly exacerbated by the absence of β2* nAChRs. Two-way ANOVA of the length of intact pyramidal layer/section in CA1 + CA3 fields of the hippocampal formation showed a significant Genotype x Lesion interaction, as well as Genotype and Lesion main effects [Genotype: F(1,28)=11.88, P=0.002; Lesion: F(1,28)=710.23, P<0.001; GenotypexLesion: F(1,28)=13.50, P<0.001] (Fig. 2B ). At one-way ANOVA, all groups significantly differed between them except sham-operated β2+/+ and β2–/– mice [F(3,28)=245.21, P<0.001, posthoc LSD test: all comparisons P<0.001 except β2+/+ sham vs. β2–/– sham NS]. Therefore, the Genotype main effect at two-way ANOVA does not seem relevant. Overall, the statistical analysis indicates that a significant loss of pyramidal cells occurs in β2+/+ lesioned mice that is significantly increased by the lack of β2 subunit. Indeed, while fewer than half of β2+/+ mice (7/16 hippocampi) had a lesion of the CA3 field, all β2–/– mice had a lesion in the CA3 field (16/16 hippocampi) ({chi}2 test, P<0.01).


Figure 2
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  		Figure 2. Nissl staining of the dorsal hippocampal formation (coronal level –1.8 mm, ref. 29 ) of 10–12 month-old β2+/+ and β2–/– mice with a bilateral quinolinic acid injection in the dorsal hippocampus. A) Representative sections are shown of β2+/+ or β2–/– sham-operated mice, β2+/+ or β2–/– quinolinic acid lesioned mice. B) Quantification of hippocampal lesion. The length of intact pyramidal layer/side/section of the CA1–3 fields was used to assess the extent of quinolinic acid lesion. Data are shown as mean ± SEM, n = 8 mice/group. Statistical analysis was performed by means of two-way ANOVA, Genotype: F(1,28) = 11.88, P = 0.002; Lesion: F(1,28) = 710.23, P < 0.001; Genotype x Lesion: F(1,28) = 13.50, P < 0.001. To interpret two-way ANOVA results we performed a one-way ANOVA: F(3,28) = 245.21, P < 0.001, posthoc LSD test: all comparisons P < 0.001 except β2+/+ sham vs. β2–/– sham NS. ** = P < 0.01 vs. all other groups, ++ = P < 0.01 vs. sham-operated groups.

These results demonstrate that the absence of β2* nAChRs increases the vulnerability of hippocampal tissue to excitotoxicity both at the behavioral (impairment in the Morris water maze learning curve) and histological (increased size of hippocampal lesion) levels. Indeed, these data suggest that the increased loss of neurons observed in quinolinic acid-lesioned β2–/– mice may contribute to the behavioral impairment observed in the Morris maze.

Loss of β2* nAChRs abolishes part of behavioral improvements and prevents neural tissue hypertrophy induced by an enriched environment
Exposure to an enriched environment has positive effects on performance of several cognitive tasks (22 , 23) and induces hypertrophy of cortico-hippocampal regions (32) . The aim of this set of experiments was to investigate whether β2* nAChRs contribute to the effects of an enriched environment on cognitive abilities and hippocampal remodeling. We tested β2+/+ and β2–/– mice exposed to either a standard environment or an enriched environment for 2 months after weaning, in a battery of cognitive tests, including the Morris water maze (Fig. 3 ), fear conditioning (Fig. 4 ), Y-maze test, and object recognition test (Fig. 5 ).


Figure 3
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  		Figure 3. Morris water maze performance in β2+/+ and β2–/– mice (10 mice/group) exposed to a standard (SE) or an enriched (EE) environment. Learning curve for training to find the hidden platform in the Morris water maze. The latency to find the platform in the 4 sessions of each day was averaged in order to obtain one value/animal/day. Mean ± SEM values are shown. Statistical analysis of the overall learning curve was performed by means of repeated measures two-way ANOVA, Time: F(7,36) = 40.82, P < 0.001; Genotype: F(1,36) = 1.91, NS; Environment: F(1,36) = 24.51, P < 0.001; Genotype x Environment: F(1,36) = 0.01, NS. ** = P < 0.01 enriched environment vs. standard environment.


Figure 4
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  		Figure 4. Conditioned fear in β2+/+ and β2–/– mice (9–10 mice/group) exposed to a standard (SE) or an enriched (EE) environment. A) Contextual test. The three test sessions were carried out 1, 5, and 10 days after conditioning. Data are expressed as mean ± SEM percent freezes. Statistical analysis was performed by means of repeated measures two-way ANOVA. Time: F(2,35) = 6.48, P = 0.003; Genotype: F(1,35) = 11.44, P = 0.002; Environment: F(1,35) = 22.01, P < 0.001; Genotype x Environment: F(1,35) = 4.56, P = 0.040. * = P < 0.05 for Genotype x Environment interaction. To interpret two-way ANOVA results we performed a repeated measures one-way ANOVA: F(3,35) = 12.11, P < 0.001, posthoc LSD test: β2+/+ enriched vs. β2+/+ standard P < 0.001, vs. β2–/– standard P = 0.001, vs. β2–/– enriched P < 0.001, all other comparisons NS. B) Cued test. The three test sessions were carried out 1, 5 and 10 days after conditioning. Data are expressed as mean (±SEM) percent freezes. Statistical analysis was performed by means of repeated measures two-way ANOVA. Time: F(2,35) = 15.41, P < 0.001; Genotype: F(1,35) = 56.29, P < 0.001; Environment: F(1,35) = 14.51, P < 0.001; Genotype x Environment: F(1,35) = 6.35, P = 0.016. * = P < 0.05 for Genotype x Environment interaction. To interpret two-way ANOVA results we performed a repeated measures one-way ANOVA: F(3,35) = 24.75, P < 0.001, posthoc LSD test: β2+/+ enriched vs. all other groups P < 0.001, β2+/+ standard vs. β2–/– standard P = 0.001, vs. β2–/– enriched P = 0.012, other comparisons NS. *, ** = P < 0.05, 0.01 vs. all other groups.


Figure 5
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  		Figure 5. Object recognition test in β2+/+ and β2–/– mice (9–10 mice/group) exposed to a standard (SE) or an enriched (EE) environment. The two test sessions were carried out 3 and 24 h after presentation of the first pair of objects. Data are expressed as mean ± SEM values. Statistical analysis was performed by means of repeated measures two-way ANOVA. Time: F(1,35) = 33.99, P < 0.001; Genotype: F(1,35) = 4.40, P = 0.043; Environment: F(1,35) = 2.68, NS Genotype x Environment: F(1,35) = 4.17, P = 0.049. To interpret two-way ANOVA results we performed a repeated measures one-way ANOVA: F(3,35) = 3.56, P = 0.024, posthoc LSD test: β2+/+ enriched vs. β2+/+ standard P = 0.015, β2–/– standard P = 0.013, β2–/– enriched P = 0.007, all other comparisons NS. * = P < 0.05 vs. all other groups.

Whereas no significant Environment or Genotype main effect or Genotype x Environment interaction were detected in the Y-maze test (% alternation: β2+/+ standard environment 60.0±2.2, β2+/+ enriched environment 56.0±2.2, β2–/– standard environment 56.5±2.5, β2–/– enriched environment 57.4±2.1, NS) and Morris water maze transfer test (% time in platform quadrant: β2+/+ standard environment 34.3±2.8, β2+/+ enriched environment 42.5±2.5, β2–/– standard environment 37.1±3.2, β2–/– enriched environment 38.4±3.1, NS), two-way ANOVA showed significant effects in the Morris water maze learning curve (Fig. 3) , contextual and cued fear conditioning (Fig. 4) , and object recognition test (Fig. 5) .

For the learning curve in the Morris water maze there was no significant Genotype x Environment interaction, but a highly significant effect of Environment and Time [Time: F(7,36)=40.82, P<0.001; Genotype: F(1,36)=1.91, NS; Environment: F(1,36)=24.51, P<0.001; GenotypexEnvironment: F(1,36)=0.01, NS], indicating that there is a beneficial effect of the enriched environment on the performance in this test for both β2+/+ and β2–/– mice (Fig. 3) .

In the fear conditioning paradigm, 1 or more days after conditioning the mice are put again into the conditioning cage for 5 min (contextual test), then, for 3 min, into a different cage (altered context test) and, in this latter cage, exposed for 3 min to the same tone to which they had been exposed during the conditioning procedure (cued test). By measuring the mouse freezing time (caused by the memory of the shock) in the three tests, it is possible to obtain an index of the strength of the association that occurred during the conditioning, and it stability, between the context and the shock (contextual test) or the tone and the shock (cued test), while the altered context test is a control for non-specific reactivity of the animals.

We performed the altered context test on the first day postconditioning and did not find any significant difference in percent freezing between the experimental conditions (% freezing: β2+/+, standard environment 15.5±2.3, enriched environment 14.8±2.1; β2–/–, standard environment 11.7±2.1; enriched environment 12.2±3.0).

The contextual and cued tests were performed 1, 5, and 10 days after conditioning and analyzed by repeated measures, two-way ANOVA. In the contextual test, there was an overall change in percent freezing with time [F(2,35)=6.48, P=0.003]. Significant Genotype x Environment interaction and Genotype and Environment main effects [Genotype: F(1,35)=11.44, P=0.002; Environment: F(1,35)=22.01, P<0.001; GenotypexEnvironment: F(1,35)=4.56, P=0.040] were detected (Fig. 4A ). At repeated measures, one-way ANOVA, performance of β2+/+ mice exposed to enriched environment significantly differed from performance of all other groups, that in turn did not differ between them [F(3,35)=12.11, P<0.001, posthoc LSD test: β2+/+ enriched vs. β2+/+ standard P<0.001, vs. β2–/– standard P = 0.001, vs. β2–/– enriched P<0.001, all other comparisons NS]. Therefore, both Genotype and Environment main effects do not seem relevant. These results indicate that both β2+/+ genotype and enriched environment are necessary to result in a stronger association between the shock and the context.

In the cued test, there was a decrease in percent freezing with time [F(2,35)=15.41, P<0.001], as well as a significant Genotype x Environment interaction and Genotype and Lesion main effects [Genotype: F(1,35)=56.29, P<0.001; Environment: F(1,35)=14.51, P<0.001; GenotypexEnvironment: F(1,35)=6.35, P=0.016] (Fig. 4B ). At repeated measures, one-way ANOVA, β2+/+ mice exposed to standard or enriched environment significantly differed between them and from both β2–/– groups, that in turn did not differ between them [F(3,35)=24.75, P<0.001, posthoc LSD test: β2+/+ enriched vs. all other groups P<0.001, β2+/+ standard vs. β2–/– standard P=0.001, vs. β2–/– enriched P=0.012, other comparisons NS]. These results indicate that lack of β2 subunit gene by itself impairs performance at the cued test in animals exposed to standard environment. Moreover, β2–/– genotype prevents the stronger and more lasting association between the shock and the tone induced by exposure to enriched environment. Previous data on fear conditioning in β2–/– mice did not show any significant difference between mutant and control mice of 2–3 months of age (20) . This discrepancy is possibly due to differences in housing conditions (two mice/cage for standard environment in present experiments).

The object recognition test is based on the fact that mice preferentially explore a novel object when presented with a pair of objects composed on an already explored and a novel object. Therefore, to find increased exploration of a novel object with respect to an old object means that the animal in fact remembers that the old object has been already explored. Wild-type mice explore less an old object with respect to a novel object 3 h after its initial presentation, but show an equal exploration time at 24 h test.

Performance in this test was analyzed by repeated measures, two-way ANOVA. A significant decrease in the exploration of a novel object with time [F(1,35)=33.99, P<0.001] was observed together with a significant Genotype x Environment interaction and Genotype main effect [Genotype: F(1,35)=4.40, P=0.043; Environment: F(1,35)=2.68, NS Genotypex Environment: F(1,35)=4.17, P=0.049] (Fig. 5) . At repeated measures, one-way ANOVA, performance of β2+/+ mice exposed to enriched environment was increased with respect to the performance of all other groups, that did not differ between them [F(3,35)=3.56, P=0.024, posthoc LSD test: β2+/+ enriched vs. β2+/+ standard P=0.015, β2–/– standard P=0.013, β2–/– enriched P=0.007, all other comparisons NS]. This indicates that the Genotype effect at two-way ANOVA does not seem relevant and that both β2+/+ genotype and enriched environment are necessary to observe improved performance in the novel object recognition test.

In summary, we found that the improvement in performance in contextual and cued fear conditioning and the novel object recognition task induced by an enriched environment depended on the expression of β2* nAChRs, whereas the ability of enriched environment to improve performance in the learning portion of the Morris water maze was independent of mouse genotype.

It has been shown (see, e.g., ref. 32 ) that exposure to an enriched environment causes a hypertrophy, or dendritic remodeling, of cortical and hippocampal regions. As an index of hippocampal hypertrophy we measured the area/side/section of the CA and dentate gyrus regions at Bregma level –2.0 mm in Nissl stained sections (Fig. 6 ). Two-way ANOVA showed a significant Genotype x Environment interaction as well as significant Genotype and Environment main effects for the area/side/section of CA region [Genotype: F(1,35)=11.34, P=0.002; Environment: F(1,35)=6.31, P=0.017; GenotypexEnvironment: F(1,35)=5.54, P=0.024] (Fig. 6B ). At one-way ANOVA, the CA size of β2+/+ mice exposed to enriched environment was significantly larger than CA size of all other groups, that did not differ between them [F(3,35)=7.35, P<0.001, posthoc LSD test: β2+/+ enriched vs. β2+/+ standard P<0.001, β2–/– standard P<0.001, β2–/– enriched P=0.002, all other comparisons NS]. Therefore, both Genotype and Environment main effects at two-way ANOVA do not seem relevant. The results indicate that both β2+/+ genotype and enriched environment are necessary to result in hypertophy of CA fields. No significant difference between the groups was detected in dentate gyrus area/side/section (not shown).


Figure 6
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  		Figure 6. Nissl staining of the dorsal hippocampal formation [coronal level –2.0 mm, (29) ] of β2+/+ and β2–/– mice exposed to a standard (SE) or an enriched (EE) environment. A) Representative sections are shown of β2+/+ or β2–/– mice exposed to SE and β2+/+ or β2–/– mice exposed to EE. B) Quantification of hippocampal hypertrophy in β2+/+ and β2–/– mice (9–10 mice/group) exposed to a standard (SE) or an enriched (EE) environment. The area/side/section of the CA1–3 fields was used as an index of hippocampal hypertrophy. Data are shown as mean ± SEM. Statistical analysis was performed by means of two-way ANOVA, Genotype: F(1,35) = 11.34, P = 0.002; Environment: F(1,35) = 6.31, P = 0.017; Genotype x Environment: F(1,35) = 5.54, P = 0.024. To interpret two-way ANOVA results we performed a one-way ANOVA: F(3,35) = 7.35, P < 0.001, posthoc LSD test: β2+/+ enriched vs. β2+/+ standard P < 0.001, β2–/– standard P < 0.001, β2–/– enriched P = 0.002, all other comparisons NS. ** = P < 0.01 vs. all other groups.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In these experiments, we studied adult animals and showed that the absence of β2* nAChRs aggravates behavioral deficits and neuronal loss subsequent to an excitotoxic insult in the hippocampus and blocks some of the cognitive improvements and hippocampal remodeling induced by exposure to an enriched environment.

Excitotoxicity results from a neurotoxic cascade thought to contribute to the pathogenesis of several types of neurodegeneration in (living) animals and humans including stroke, epilepsy, and AD (33 34 35 36 37) . We tested the effects of hippocampal excitotoxicity in adult β2–/– mice using a subthreshold dose of quinolinic acid, which does not cause significant behavioral deficits in the Morris water maze and Y-maze tests in wild-type mice. We showed that quinolinic acid injection in β2–/– mice causes both an impairment in the learning curve in the Morris water maze and a larger hippocampal lesion than quinolinic acid injection or β2–/– genotype alone. Therefore, the absence of β2* nAChRs confers increased behavioral and cellular susceptibility to an excitotoxic lesion of the hippocampus.

These results broadly agree with, and complement, the numerous lines of evidence showing neuroprotective effects of nicotine (1 2 3) ; however, while previous pharmacological studies demonstrated the neuroprotective efficacy of pharmacological stimulation of nAChRs, the current results demonstrate that absence of the endogenous, physiological stimulation of nAChRs potentiates neurotoxicity. These data, therefore, support the notion that loss of this class of nAChRs may contribute to the pathogenesis of some types of neurodegeneration.

Much evidence shows that enriched environment potentiates cognitive function, as well as anatomy and function of cortico-hippocampal areas in several animal species. Enriched environment improves performance in many learning tasks, increases size and weight of neocortex and hippocampus, increases the size of neuronal cell bodies and the number of synapses and dendrites in these regions in many species (32) . Similarly, education and cognitive training have positive effects on cognition in aged humans (38) . Indeed, a positive correlation between education and decreased risk of AD or PD dementia has been shown in epidemiological studies (39 , 40) . This suggests that an environment which stimulates learning may confer protection toward neurodegeneration. Further, several instances of increased neuroprotection against neural insults have been demonstrated in animals exposed to an enriched environment (22 , 41) . Interestingly, recent findings show a link between enriched environment and AD pathogenesis. Exposure to enriched environment decreased the deposition of Aβ (42) and diminished cognitive deficits (43) in transgenic mice that overexpress mutated forms of human amyloid precursor protein.

Cholinergic transmission is activated with an enriched environment and may contribute to behavioral and anatomical effects of enriched environment. Choline acetyltransferase, the acetylcholine biosynthetic enzyme, is increased in several telencephalic regions in rats raised in an enriched environment (44) . Performance of rats exposed to an enriched environment in a delayed spatial win-shift discrimination task was facilitated by tacrine (a cholinesterase inhibitor and treatment for AD) and attenuated by atropine (a muscarinic antagonist) (45) . Finally, active manipulation of a toy is paralleled by a significant increase in hippocampal acetylcholine efflux (46) .

The data presented here show that β2* nAChRs are crucial for improvement of performance of some cognitive tasks in young adult mice on exposure to an enriched environment. While lack of β2* nAChRs does not alter hippocampal size in mice raised in standard environment, it does prevent hippocampal hypertrophy induced by an enriched environment. Overall, these findings suggest that enriched environment induces activation of cholinergic transmission acting through β2* nAChRs, which is a necessary factor mediating some of the positive effects of an enriched environment on hippocampal neuronal circuits and cognitive performance.

Developmental compensation for the loss of a targeted gene must always be taken into consideration when studying constitutive knockout mice. In several instances, an acute role for high affinity nAChRs has been demonstrated in β2–/– mice. We showed that re-expression of the β2 subunit in the mesolimbic pathway of adult β2–/– mice restored wild-type phenotypes related to this pathway (47) . Moreover, no change in expression (19) or function (48 , 49) of other nAChR subtypes was observed in these mice. In several brain regions (e.g., thalamus, amygdala, ref. 17 and unpublished observations) and transmitter-identified neuronal populations no cell loss or neurochemical alteration was detected in β2–/– mice (17 , 50) . Even regions that show spontaneous structural alterations in aged mice (cortex and hippocampus) do not have detectable changes at the ages we have investigated in the present experiments. Spontaneous deficits in cognitive tests were only observed between 12 (fear conditioning) and 24 (Morris water maze) months of age (17 , 20) . It should be noted, however, that two instances of developmental alterations have been observed in β2–/– mice: alteration of corticothalamic efferents leading to increased performance in the passive avoidance test (51) and absence of retinal waves in the first postnatal days and alteration in segregation of the retino-thalamic pathway (52) .

Together with previous evidence of spontaneous deficits in aged β2–/– mice and the ability of nicotine to be neuroprotective, the current findings support the notion that β2* nAChRs contribute to the maintenance of cognitive function and neuronal survival during senescence or after an excitotoxic insult, and to potentiation of function after exposure to an enriched environment.

These results may have relevance for human neurodegenerative diseases in which β2* nAChRs are lost. Loss of nAChRs occurs in several forms of dementia such as AD, PD, and Lewy body dementia (16 , 53 54 55) . Interestingly, β2–/– mice reproduce a main feature of AD, since in these patients a marked loss of high-affinity nicotine binding (i.e., β2* nAChRs, ref. 19 ) and β2 subunit immunoreactivity occurs in cortico-hippocampal regions (16 , 55 , 56) . Therefore, present and previous results suggest that loss of β2* nAChRs in neurodegenerative diseases such as AD may contribute to the development of cognitive deficits and favor tissue degenerative processes in several ways, including potentiation of spontaneous, age-related degeneration, increased susceptibility to excitotoxic insult, and blockade of the positive effects of a stimulating environment.


   ACKNOWLEDGMENTS
 
We thank Dr. Roberto D’Amico for help with the statistical analysis. The work was supported by grants Italian PRIN 2005054943 (M.Z.), Telethon D.105 (M.Z.), CARIPLO "Farmacogenetica dei recettori colinergici nicotinici: possibili implicazioni nella malattia di Alzheimer" (M.Z.) and EC project QLK6-CT-2000–00318 (J.P.C., M.Z.). The Authors have no financial interest in or financial conflict with the subject matter or materials discussed in the manuscript.

Received for publication April 5, 2007. Accepted for publication May 31, 2007.


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