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J Appl Physiol 103: 1479-1487, 2007. First published July 19, 2007; doi:10.1152/japplphysiol.00384.2007
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Classical eyeblink conditioning during acute hypobaric hypoxia is improved in acclimatized mice and involves Fos expression in selected brain areas

J. C. López-Ramos, P. J. Yi,{dagger} L. Eleore, N. Madroñal, A. Rueda, and J. M. Delgado-García

Neuroscience Division, Pablo de Olavide University, Seville, Spain

Submitted 10 April 2007 ; accepted in final form 18 July 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
This work attempts to evaluate the cognitive aspects of the acclimatization ability of mice submitted to simulated altitude. Critical altitudes were detected by evaluating open field activity, combined or not with object recognition tasks, at different acute simulated altitudes. Results showed impaired cognitive abilities at {approx}3,733 m and above. To evaluate acclimatization capabilities, mice submitted to hypobaric hypoxia at {approx}5,000 m for 1 wk were tested for learning and memory performances with classical eyeblink conditioning at the same altitude or at land altitude. Results showed total acclimatization in mice conditioned at {approx}5,000 m but no improved performance in those conditioned at land altitudes compared with controls. Selected brain sites of conditioned animals were analyzed by immunohistochemistry to detect expression of the protein product of the protooncogene c-fos (Fos) in relation to both motor learning processes and hypobaric conditions. In the nucleus of the solitary tract, a higher expression of Fos was found in the acute hypobaric conditioned animals than in control conditioned and nonconditioned animals. Similar patterns between groups were found in the other brain areas, mainly in the piriform cortex and area 1 of the cingulate cortex and in the hippocampus. Differences between hemispheres were detected only in acute hypobaric animals. The present results show that acclimatization to high altitude prevents the impairment of classical eyeblink conditioning evoked by hypobaric hypoxic conditions but does not improve this task when acquired under land conditions, although it could diminish the activation requirements for its performance.

simulated altitude; learning; memory; immunohistochemistry

THE DECREASE OF TISSUE OXYGENATION induced by hypobaric hypoxia alters many physiological processes in an elevation- and duration-dependent fashion (39). For instance, the exposure of an organism to transient hypoxic stress activates respiratory and circulatory systems as well as the adrenal glands and affects both the release and the effects of neurotransmitters in the central nervous system (39). However, these complex physiological adaptive responses cannot prevent early impairments in the cognitive functions of subjects exposed to hypobaric hypoxic conditions. For example, spatial memory impairments have been described in adult rats exposed to different simulated altitudes (40) and in newborn rats exposed to intermittent hypobaric hypoxic episodes (41). However, it has been shown that transient exposure to hypoxia may not only prevent these disorders but actually improve learning capabilities (45). Together, these data suggest that both the level and the duration of the hypobaric exposure are determinant factors that can induce either adaptation or functional deficits.

The aim of this study was to evaluate brain adaptive capabilities to altitude with various behavioral and cognitive tasks under different conditions of simulated altitude in both control and acclimatized Swiss mice exposed to hypobaric hypoxia as an experimental model. Swiss mice were selected because of our previous experience with their behavioral patterns and because their size was compatible with the dimensions of the hypobaric chamber used here. Open field activity and object recognition tasks were carried out to determine critical altitudes, while classical eyeblink conditioning—a powerful experimental model for the study of the neuronal mechanisms involved in the acquisition of new motor abilities (10, 11, 36, 42, 43, 46)—was used to evaluate learning and memory capabilities of control and acclimatized animals submitted to different simulated altitudes. In short, animals were conditioned with a trace paradigm consisting of a 20-ms tone as a conditioned stimulus (CS) followed 250 ms later by an electrical shock presented to the supraorbital nerve as an unconditioned stimulus (US). It is assumed that trace conditioning is hippocampus dependent and requires a conscious knowledge and/or declarative or explicit memory of relevant relationships between CS and US stimuli, while delay conditioning (assumed to be a cerebellum-dependent motor learning task) does not (10, 11, 36, 42, 43, 46). In accordance with this assumption we selected this paradigm to determine high-altitude effects on cognitive processes. Moreover, mice are capable of acquiring classically conditioned eyelid responses with trace paradigms (9). To detect learning evolution across conditioning sessions, the electromyographic (EMG) activity of the orbicularis oculi muscle was recorded and analyzed off-line. We hypothesized that adaptation to hypobaric conditions might improve learning capabilities, as determined by this conditioning test.

Furthermore, the expression of a marker of cell activation, the protein product of the protooncogene/immediate-early gene c-fos (Fos), was studied to determine which cerebral structures are involved in the acquisition of eyelid conditioned responses (CRs) in control animals and in animals under hypobaric hypoxic conditions. Previous studies have used Fos induction as a tool to determine the circuitry associated with the acquisition of CRs. Indeed, an increase in Fos production has been reported in the motor cortex (19), cerebellum (14), inferior olive (34), and other brain stem structures (7, 17) during different types of motor learning, including the generation of eyelid CRs (14, 17). Similar studies regarding Fos expression in cerebral cortical areas during classical conditioning of eyelid responses have been carried out in rabbits (13, 18). In other respects, it has been described that the expression of c-fos is affected in hypobaric/hypoxic conditions (35, 25, 24), particularly in the nucleus of the solitary tract (4, 5, 12, 15), a neural center involved in ventilatory control. In the present study, Fos expression was quantified in the nucleus of the solitary tract, the piriform cortex, area 1 of the cingulate (Cg1) cortex, and the hippocampus of both control and experimental mice to detect changes in expression evoked by the hypobaric conditions and/or the classical eyeblink conditioning.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Subjects.   Studies were carried out on male 2-mo-old Swiss mice obtained from an official supplier (Animal House of the University of Granada, Granada, Spain). A total of 103 animals were kept under standard conditions of temperature with a 12:12-h light-dark cycle and allowed ad libitum access to commercial mouse chow and water. All experiments were carried out during the light cycle and according to European Union guidelines for the use of animals for biochemical research and chronic behavioral experiments (86/609/EU). Protocols were reviewed and approved by professionals credited by Consejería de Agricultura y Pesca, Junta de Andalucia, Spain.

Open field activity.   To detect activity changes during hypobaric hypoxia, animals (n = 6) were exposed to a progressive decompression from 35 m ({approx}757 mmHg) to 9,674 m ({approx}226 mmHg). This level of hypobaric hypoxia was achieved in 15 min and was followed by an analogous recompression until restoration of initial conditions. During this whole process, the individual motor activity was recorded each minute with the help of an infrared ray actimeter (Cibertec, Madrid, Spain) adapted to the hypobaric chamber. With the aim of determining the "critical altitude" at which the open field activity is decreased, the motor activity of the mice (n = 5) was recorded each minute for 10 min at pressures of 35, 2,650, 3,733, and 5,000 m ({approx}757, 550, 470, and 394 mmHg, respectively). All the decompressions and recompressions were achieved at a rate of 600 m ({approx}40 mmHg)/min. A diagram of the experimental design illustrating the two paradigms used here to evaluate open field activity is shown in Fig. 1, A and B.


Figure 1
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  		Fig. 1. Open field activity during a hypobaric hypoxic episode. A and B: diagrams of the experimental design detailing the 2 paradigms used here to evaluate the open field activity. C: evolution of an open field activity task carried out during 30 min of progressive change in simulated altitude from {approx}1,279 to {approx}9,674 m and back to land level. Data are mean ± SE motor activity in arbitrary units; data shown were collected from 6 control mice. Dotted line represents quadratic polynomial tendency curve best fitted to the data. D: open field activity recorded at {approx}35, 2,650, 3,733, and 5,000 m. Values are mean ± SE arbitrary motor activity units recorded for 10 min (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001 (1-way ANOVA).

 
Object recognition.   For object recognition, mice not submitted previously to simulated altitude (n = 16) were first habituated individually to a round open field (27 x 25 cm) adapted to the hypobaric chamber. Sessions lasted 10 min and were repeated for 3 days. During the training session, two unknown objects were placed into the open field, and the animals were allowed to explore them for 10 min. The time spent exploring each object and the total exploration time were quantified. During retention sessions 60 min after the training session, animals were placed back into the same open field and one of the familiar objects was replaced by a novel object while the other was substituted by an identical object, to avoid odor recognition. Mice were allowed to explore freely for 10 min. The time spent exploring each object and the total exploration time were again quantified. The preference index for the familiar and novel objects (estimated as % of the total exploration) was used to measure recognition memory. Training sessions were carried out at a normal pressure, while half of the mice were submitted to a simulated altitude of 3,733 m during the retention sessions to evaluate the effect of the hypobaric hypoxia on this task. During all sessions, the open field activity of the mice was recorded with the aforementioned system. The recognition session was not carried out at 5,000 m, because at that simulated altitude the lack of attention and activity made it impossible to implement the task (data not shown).

Two-way analysis of variance (ANOVA) was used to detect significant differences between groups in both object recognition and open field activity tasks.

Surgical preparation for classical eyeblink conditioning.   Under deep anesthesia (35 mg/kg ketamine and 2 mg/kg xylazine ip), animals not exposed previously to simulated altitude (n = 81) were implanted with four electrodes in the upper eyelid of the left eye. Electrodes were made of Teflon-insulated, annealed stainless steel wire (50-µm diameter; WA-98324, A-M Systems, Carlsborg, WA). One pair of electrodes was aimed toward the supraorbitary branch of the trigeminal nerve and served for the application of electrical stimuli. The second pair of wire electrodes was implanted in the ipsilateral orbicularis oculi muscle and served for recording its EMG activity. The four electrodes were connected to a four-pin socket (RS-Amidata, Madrid, Spain) that was fixed to the cranial bone with dental cement. After surgery, animals were kept for 5–7 days in independent cages before the beginning of the experiment, with free access to food and water, for a proper recovery. They were also maintained in individual cages for the rest of the experiment.

Classical conditioning procedures.   For EMG recordings, animals were placed individually in a small (5 x 5 x 10 cm) plastic cage, located inside the hypobaric chamber and covered with a ground-connected metallic sheet to eliminate electrical interference. An adaptation was made to the hypobaric chamber to allow the passage of a four-band wire connecting the animal to the recording/stimulating system.

A trace conditioning paradigm was carried out. For this, animals were presented with a tone (2,400 Hz, 95 dB, 20 ms) as a CS, followed 250 ms later by an electrical stimulation (500 µs, 3x threshold) as an US. CS-US presentations were separated at random by 30 ± 5 s. For habituation and extinction sessions, only the CS was presented, also at intervals of 30 ± 5 s. Over 5 days 200 trials, divided into four 50-trial sessions, were presented to each animal. Distribution of habituation, conditioning, and extinction sessions is schematized in Fig. 2.


Figure 2
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  		Fig. 2. Schematic representation of habituation (H), conditioning (C), and extinction (E) sessions carried out during performance of classical conditioning eyeblink tasks. Each of the 5 days spent in the experiment consisted of 200 trials, divided into 4 sessions of 50 trials each. Sessions analyzed to obtain learning curves, shown in Figs. 5 and 6, are indicated by a number.

 
The EMG activity of the orbicularis oculi muscle was recorded with differential amplifiers and a bandwidth of 1–10 kHz (Grass Technologies, West Warwick, RI). Data were stored directly on a computer through an analog-to-digital converter (CED 1401 Plus, Cambridge, UK) at a sampling frequency of 11–22 kHz and an amplitude resolution of 12 bits. Data were analyzed off-line for quantification of CRs with the help of the Signal Average Program (Cambridge Instruments, Cambridge, UK). We considered a response to be conditioned when the rectified EMG activity during the CS-US period presented the following conditions: 1) the EMG activity lasted >10 ms; 2) the EMG was not preceded by any spontaneous activity in the 200 ms preceding CS presentation; 3) the EMG activity was initiated >50 ms after CS onset; and 4) the integrated EMG activity was at least 2.5 times larger than the activity recorded 200 ms before CS presentation (9).

As indicated in Table 1, six experimental groups (n = 8 animals each) were established: 1) control nonconditioned (NC) mice maintained under land conditions, 2) control (C) mice conditioned at 35 m (i.e., Seville city altitude), 3) mice conditioned at 5,000 m (H), 4) mice conditioned at 7,000 m ({approx}298 mmHg; H7,000), 5) mice acclimatized at 5,000 m for 1 wk and conditioned at 5,000 m (AH), and 6) mice acclimatized under the same conditions as group 5 and conditioned at 35 m (AL). For all groups, habituation sessions were carried out at 35 m. A seventh group of eight animals was pseudoconditioned at 35 m to test the reliability of the task. Finally, five additional groups (n = 5 animals each), identical to groups 1, 2, 3, 5, and 6, received only habituation and conditioning sessions and were used for immunohistochemical analysis.


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  		Table 1. Groups involved in conditioning task

 
Data presentation and analysis.   Collected data were quantified, with a purpose-designed Excel worksheet, as the percentage of CRs per session, i.e., the proportion of stimulations within a session of 50 presentations that generated an EMG activity satisfying the above-mentioned criteria (9). Statistical differences between groups were compared across conditioning and extinction sessions by two-way repeated-measures ANOVA performed with the SPSS 13.0 for Windows package (SPSS, Chicago, IL).

Immunohistochemistry.   For tissue processing, we followed experimental procedures described elsewhere (13, 38). Thirty minutes after the end of the fourth conditioning session, mice were deeply anesthetized with chloral hydrate (4%) and perfused transcardially with 5–20 ml of phosphate-buffered saline (PBS) to remove blood and then with 10–150 ml of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (PB). Brains were removed and postfixed for 4 h in the same fixative at room temperature. Afterwards, they were cryoprotected by overnight immersion in 30% sucrose in PB at 4°C.

Tissue blocks were frozen, and serial coronal sections (40 µm) were cut with a cryostat (Leica CM1900, Leica Microsystems, Wetzlar, Germany). The left hemisphere of each brain was marked to allow a posterior comparison with the right hemisphere. Free-floating sections were processed by the avidin-biotin peroxidase complex (ABC) procedure (16, 26, 33) to visualize immunoreactive sites. Briefly, sections were incubated for 30 min in PBS containing 3% normal goat serum (ICN Biochemicals, Cleveland, OH) and 0.2% PBS-Triton X-100. Sections were incubated with specific polyclonal antisera against Fos (Santa Cruz Biotechnology, Santa Cruz, CA), 1:1,000 in the above-mentioned solution, overnight at 4°C. The next day, after several washes in PBS, sections were incubated in biotinylated goat anti-rabbit immunoglobulin (Vector Laboratories, Peterborough, UK) followed by peroxidase-linked ABC (Vector Laboratories). Peroxidase activity was demonstrated by the nickel-enhanced diaminobenzidine procedure (13, 38).

Image analysis of immunostained cells.   For the quantification of Fos-immunostained cells, one of four coronal sections of five brains per group was processed, and the selected areas were photographed with a Leica DC 500 digital camera adapted to a Leica DMRE microscope in transmitted light mode, supported with Leica IM 500 software (Leica Microsystems). The Cg1 motor cortex and piriform cortex were photographed with a x10 objective, the hippocampus with a x5 objective, and the tract of the solitary nucleus with a x20 objective. Special care was taken to maintain the intersection between the dorsoventral and rostrocaudal axes at the same point of each captured image, to allow equal analysis. Following the coordinates of Paxinos and Franklin (30), Cg1 motor cortex photomicrographs were taken from bregma 2.34 to bregma –0.22 (482 analyzed slices), piriform cortex pictures were taken from bregma 1.94 to bregma –2.30 (695 slices), and hippocampus areas were selected between bregma –1.7 and bregma –3.64 (246 slices). The nucleus of the solitary tract was analyzed between bregma –6.24 and bregma –8.24 (110 slices) to quantify Fos-positive cell nuclei. In each section, the desired structures were determined following the schemes of Paxinos and Franklin (30), as detailed in Fig. 7.


Figure 3
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  		Fig. 3. Open field activity recorded during an object-recognition task carried out at {approx}35 and 3,733 m of simulated altitude. Top: % of attention to a familiar (Fam) or novel object. Training sessions were carried out at {approx}35 m, while half of the animals received retention sessions at {approx}35 m and the other half at {approx}3,733 m. Bottom: mean ± SE values of motor activity in arbitrary units during sessions at the indicated altitudes (n = 16). *P < 0.05, **P < 0.01, ***P < 0.001 (1-way ANOVA).

 

Figure 4
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  		Fig. 4. Conditioned eyelid responses collected during exposure to {approx}35 m (C), 5,000 m (H), and 7,000 m (H7,000) of simulated altitude. Ha1–Ha3, data from habituation sessions; Co1–Co4, data from conditioning sessions; Ex1–Ex3, data from extinction sessions. Exposure to the selected simulated altitude was carried out during conditioning and extinction sessions. Values are mean ± SE % of conditioned responses (n = 8). Differences between C and H groups: {circ}P < 0.05, {circ}{circ}P < 0.01. Differences between C and H7,000 groups: {triangledown}P< 0.05, {triangledown}{triangledown}P < 0.01, {triangledown}{triangledown}{triangledown}P< 0.001 (2-way ANOVA).

 

Figure 5
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  		Fig. 5. Conditioned eyelid responses collected during exposure to {approx}35 m [control nonacclimatized (C) and acclimatized to {approx}5,000 m for 1 wk (AL)] and to {approx}5,000 m [nonacclimatized (H) and acclimatized to {approx}5,000 m for 1 wk (AH)]. Ha1–Ha3, data from habituation sessions; Co1–Co4, data from conditioning sessions; Ex1–Ex3, data from extinction sessions. Exposure to the selected simulated altitudes was carried out during conditioning and extinction sessions. Values are mean ± SE % of conditioned responses (n = 8). Differences between H and C groups: {circ}P < 0.05, {circ}{circ}P < 0.01. Differences between H and AH groups: {triangleup}P < 0.05, {triangleup}{triangleup}P< 0.01, {triangledown}{triangledown}{triangledown}P < 0.001. Differences between H and AL groups: {triangledown}P< 0.05, {triangledown}{triangledown}P < 0.01 (1-way ANOVA).

 

Figure 6
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  		Fig. 6. Quantification of Fos-immunoreactive nuclei in area 1 of the cingulate (Cg1) motor cortex (A), piriform cortex (B), and hippocampus (C) of NC, C, H, AL, and AH groups of mice. Right (open bars) and left (filled bars) hemispheres are shown. Horizontal lines above bars represent significant differences between the respective group and the NC group; dashed lines represent differences between hemispheres in the same group. Data are mean ± SE total inmunostained cell nuclei found in each structure of each analyzed section. Statistical differences: *P < 0.05, **P < 0.01, ***P < 0.001 (2-way ANOVA).

 

Figure 7
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  		Fig. 7. Representative images of brain areas from nonacclimatized and conditioned (at {approx}5,000 m, H group) animals in which the Fos-immunoreactive nuclei were quantified. A and D: photomicrographic reconstruction of Cg1 cortex and hippocampus, respectively. B and C: photomicrographs of the piriform cortex. Left (L) and right (R) hemispheres are indicated. Dashed lines enclose the area quantified for each analysis. Immunoreactive differences between left and right hemispheres are evident in cingulate and piriform cortices, while no differences between left and right hippocampus are distinguishable. Pir, piriform cortex; CA1, CA2, and CA3, cornu ammonis 1, 2, and 3, respectively, of the hippocampus; DG, dentate gyrus. Scale bars: 200 µm.

 
A comparison of the immunoreaction against Fos was carried out between the NC group and the conditioned groups: C, acute 5,000 m hypobaric hypoxic (H), acclimatized and conditioned at 35 m (AL), and acclimatized and conditioned at 35 m (AH). In all cases, except for the nucleus of the solitary tract, the left (L) and right (R) hemispheres were analyzed separately.

Fos-immunostained cell nuclei were identified, and their total numbers per structure and section were quantified with the help of the free Image J image analysis software, maintaining the threshold parameters throughout the analysis of each area. Statistical differences between groups and hemispheres were compared by two-way repeated-measures ANOVA, with post hoc Dunnett t-test comparisons, used to compare all groups with the NC groups. The analysis was performed with the SPSS 13.0 for Windows package. To analyze the nucleus of the solitary tract, the post hoc Bonferroni test was used, with the aim of comparing all the groups.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Open field activity.   Figure 1C illustrates the motor activity of control mice (n = 6) recorded across progressive pressure changes carried out over 30 min, with an altitude evolution from 1,279 to 9,674 m and back to land pressure (35 m). In accordance with the collected data, the evolution of motor activity in control mice was opposite to the experimented decompression, with a delay of {approx}5 min.

As illustrated in Fig. 1D, open field recordings made at different pressures showed a diminution of global motor activity with increase in simulated altitude (from {approx}35 m to {approx}5,000 m). Two-way ANOVA demonstrated significant differences between groups [F(3,49) = 15.19; P ≤ 0.05]. The post hoc Bonferroni test found no significant decrease in motor activity until an altitude of 3,733 m was reached. Data collected at 3,733 and 5,000 m showed the greatest significant differences with those collected at 35 m (land level).

Object recognition.   Object-recognition tasks evidenced impairment in cognitive capabilities with simulated altitude. Two-way ANOVA showed, during the retention sessions, significant preferences for the novel object over the familiar object only in the control group [F(1,15) = 37.41; P < 0.001; Fig. 3, top]. An open field activity study, evaluated in parallel, showed significant differences between the two situations [F(2,31) = 6.74; P < 0.01], while post hoc Bonferroni test detected a diminution in the motor activity of the group that performed the retention sessions at a simulated altitude of 3,733 m (Fig. 3, bottom).

Classical eyeblink conditioning at different simulated altitudes.   As illustrated in Fig. 4, the percentage of eyelid CRs increased steadily in control (C) mice. In accordance with previous descriptions (9), these animals exhibited a normal rate of CRs with the protocol used in the present study. During the fourth conditioning session, the number of CRs reached by animals was 78.4 ± 8.4% (mean ± SE; n = 8). Acute hypobaric hypoxia impaired the acquisition of CRs across conditioning sessions. The percentage of CRs reached only 32.7 ± 12% (mean ± SE; n = 7) during the fourth conditioning session in the group of mice conditioned at 5,000 m (H) and 3.3 ± 2.9% (mean ± SE; n = 8) in the group conditioned at 7,000 m (H7,000). During extinction, the percentages of CRs presented in C animals were significantly higher than in H and H7,000 animals, reaching values in the third session of 45.5 ± 11.3% vs. 20.1.1 ± 12.5% and 2.2 ± 1.4% (means ± SE), respectively (Fig. 4). Two-way ANOVA applied to the conditioning data indicated that there was a significant difference between groups [F(2,21) = 13.93; P < 0.001]. There was a significant effect of session [F(3,63) = 2.87; P < 0.05] and a significant group-by-session interaction [F(3,6) = 3.89; P < 0.05]. The same analysis applied to the extinction data showed significant differences between the groups [F(2,21) = 13.73; P < 0.001], with no significant overall effect of session [F(2,42) = 1.14; P = 0.34] but with significant group-by-session interaction [F(2,4) = 4.31; P < 0.05]. The percentage of CRs of the pseudoconditioned animals did not exhibit differences between habituation and conditioning, or extinction, sessions during any phase of the experiment [F(9,63) = 1.22;P= 0.32; Fig. 4].

Classical eyeblink conditioning of mice acclimatized to simulated altitude.   Acclimatization at 5,000 m for 7 days restored learning and memory capabilities in mice that, after this period, were conditioned in these same acute hypobaric hypoxic conditions (AH; Fig. 5). The percentage of CRs reached 83.8 ± 6.9% (mean ± SE; n = 8) in the fourth conditioning session. The group acclimatized at 5,000 m for 7 days and conditioned at 35 m (AL) did not exhibit a higher percentage of CRs than those of the AH and C groups. The AL animals reached 76.5 ± 10.5% (mean ± SE; n = 8) in the fourth conditioning session. During extinction, both acclimatized groups (AH, AL) showed significantly higher percentages of CRs than that of the nonacclimatized group conditioned at 5,000 m (H). During the last extinction session, the AH group reached CR values of 50.5 ± 8.8% (mean ± SE; n = 8). For the same session, the AL group reached CR values of 31.6 ± 6.6% (mean ± SE; n = 8). Two-way ANOVA applied to the conditioning data indicated that there was a significant difference between groups [F(3,28) = 6.04; P < 0.01]. There was no significant effect of session [F(3,84) = 1.55; P = 0.15] but significant group-by-session interaction [F(3,9) = 19.27; P < 0.001]. The same analysis applied to the extinction data showed significant differences between the groups [F(3,28) = 6.39; P < 0.01], with no significant overall effect of session [F(2,56) = 1.28; P = 0.28] but with significant group-by-session interaction [F(2,6) = 14.49; P < 0.001]. Figure 5 depicts the learning curves of the two acclimatized groups (AH, AL) compared with both the C and the H conditioned groups. In conclusion, these results show that acclimatization to high altitude prevents the impairment of classical eyeblink conditioning generated under these hypobaric hypoxic conditions but does not improve the same task when performed at land level.

Image analysis and quantification of immunostained cells.   Cell nuclei immunostained with the Fos antibody were found in the Cg1 motor cortex of all groups studied (Fig. 6A). Two-way repeated-measures ANOVA applied to the data indicated a significant difference between groups [F(4,481) = 7.88;P< 0.001]. Post hoc Dunnett t-test found significant differences between the NC group and the C, H, and AH groups (Fig. 6A). Differences between hemispheres were found only in the H group [F(1,100) = 9.95; P < 0.01; Figs. 6A and 7A].

Numerous cell nuclei immunoreactive to Fos were observed in the piriform cortex of all groups (Fig. 6B). Two-way repeated-measures ANOVA applied to the data indicated a significant difference between groups [F(4,694) = 33.91; P < 0.001]. Post hoc Dunnett t-test found significant differences between the NC group and all other groups (C, H, AL, and AH). Differences between hemispheres were found only in the H group [F(1,148) = 18.2;P< 0.001; Figs. 6B and 7, B and C].

All the areas of the hippocampus showed cells immunostained with the Fos antibody (Fig. 6C). The digital analysis included the whole hippocampus. Two-way repeated-measures ANOVA applied to the data indicated a significant difference between groups [F(4,245) = 4.62;P< 0.01]. Post hoc Dunnett t-test found significant differences only between the NC and AH groups. Differences between hemispheres were not found (Figs. 6C and 7D).

The immunoreaction found in the nucleus of the solitary tract evidenced an influence of the hypoxic hypobaric conditions in the expression of Fos, with the highest values in the acute hypobaric hypoxic group (Fig. 8). Statistical analysis applied to the data indicated significant differences between groups [F(4,109) = 7.671;P< 0.001]; post hoc Bonferroni test showed differences between the H group and both NC and C groups.


Figure 8
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  		Fig. 8. Quantification of Fos-immunoreactive cells in the nucleus of the solitary tract of NC, C, H, AL, and AH groups. Data are mean ± SE total inmunostained cell nuclei found in each structure of each analyzed section. Horizontal bars represent significant differences between indicated groups. **P < 0.01, ***P < 0.001 (1-way ANOVA).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Although there are previous reports addressing the effects of hypobaric episodes on cognitive processes, only a few studies have addressed the effect of acclimatization on learning and memory capabilities during the development of a hypobaric exposure (41, 45, 47). In the present study, open field activity, object recognition, and classical eyeblink conditioning tests were used to evaluate the effect of simulated altitude on motor and cognitive capabilities. To achieve these purposes, a prototype hypobaric chamber described previously (23) was constructed and provided with the necessary equipment to allow each type of experiment.

A proper knowledge of the impairment evoked by hypobaric exposure (40) requires its evaluation at different simulated altitudes. The open field activity task showed that such impairment is progressive, reaching significant values from {approx}3,733 m on. The object-recognition task corroborated these findings. Some studies (40) found a decreased reference and working memory performance from 5,950 m on in rats during water maze tasks. Other specific symptoms, moods, and motor performances are significantly degraded in humans after 4.5 h of exposure to 4,700-m hypobaric hypoxia (39), although another study found no significant differences in word fluency, word association, or lateralized lexical decision in humans at 4,500 m of simulated altitude (29). Moreover, it has been reported that the ability to learn new tasks in humans is not impaired by mild hypoxia at altitudes of up to 3,658 m (28). On the basis of these results, the altitudes selected to carry out the classical eyeblink conditioning were 5,000 and 7,000 m. Results obtained here demonstrate for the first time that both simulated altitudes impaired classical eyeblink conditioning in alert, behaving mice.

Classical eyeblink conditioning at different simulated altitudes showed a diminution in the percentage of CRs compared with control animals, dependent on the altitude reached. These results are in agreement with a previous study on spatial memory carried out at similar altitudes (40). Results obtained with classical eyeblink conditioning of acclimatized mice suggested a restoration of cognitive capabilities, in relation to the same task performed at 5,000 m. Evidence of actual acclimatization has already been described in relation to neuroendocrine (27) and ventilatory (44) functions, in parallel with improvement in erythropoiesis and hemoglobin production (48). Moreover, different patterns of acclimatization have been described in humans (3) that can be seen reflected in metabolic changes, such as those found by other authors (2), who describe unexpectedly increased exhalation of nitric oxide (NO) by chronically hypoxic populations of Tibetans living at 4,200 m and of Bolivian Aymara at 3,900 m. Other cognitive aspects of acclimatization to hypoxia/hypobaria relating to the improvement of adaptive processes based on intermittent exposure to severe hypoxia (20) or the assessment of cognitive states in hypoxia based on speech motor control and comprehension of syntax (21) have also been described.

Our results show neither impairment nor improvement of learning and memory capabilities in acclimatized mice conditioned at land pressure. Other studies of acclimatization or preconditioning to hypoxia show contradictory results. Thus enhanced performance in water maze and eight-arm radial maze tasks has been detected in neonatal mice exposed to intermittent hypoxia (47), and enhanced spatial orientation in rats after brief hypobaric hypoxia has also been reported (45). In contrast, other studies conclude that neonatal brain damage induced by hypobaric hypoxia impairs spatial memory in infant as well as adult rats. In the latter, it has been observed that hypobaric hypoxia delays the maturation of neurons and substantially affects macroglia expression in the cortex, including the hippocampus (41). It therefore seems evident that a hypoxic exposure can promote acclimatization or damage depending on its acuity and duration.

The analysis of Fos expression in the nucleus of the solitary tract showed a significant increment of stained cell nuclei in the acute hypobaric group compared with the animals not exposed to any hypobaric situations (NC and C) and a nonsignificant difference compared with the acclimatized mice (AL and AH), probably due to the acclimatization process. Since all of the animals used in this study were subjected to a similar stressful situation, these results cannot be simply related to the experimental stress. These results are in accord with previous studies (4, 5, 12, 15) and can serve as a control of the hypobaric exposure carried out in each case.

Quantification of immunoreactive nuclei in the Cg1 and piriform cortices and hippocampus showed similar patterns of differences between groups. Thus, in all the areas studied, the highest values were found in the conditioned control (C) and hypobaric acclimatized (AH) groups. Surprisingly, acclimatized groups conditioned under land conditions (AL) showed lower values than might be expected, displaying significant differences with the control nonconditioned (NC) group only in the piriform cortex. Although, as mentioned previously, Fos expression is induced both by acclimatization to hypoxia and by learning episodes, it has not been described how their interaction can affect the expression of Fos. In any case, molecular mechanisms involved in both events are closely related. Thus different studies (32, 22) have demonstrated that adaptation to hypoxia implies elevation of cGMP levels, in a process involving an increase in NO production. Moreover, it is well known that increase in cGMP levels improves learning capabilities (6, 8, 37), and that Fos is regulated by cGMP (31). In accordance with this, we can assume that animals acclimatized to hypoxic conditions and conditioned in a land environment could develop compensatory mechanisms that would explain our results.

Another interesting result found in the quantification of immunoreactive Fos expression is the fact that the only group showing differences between hemispheres was the nonacclimatized group conditioned in hypobaric conditions (HL and HR). This is, moreover, the only group that presented impaired performance in the classical eyeblink conditioning task (the brains of the H7,000 group were not processed). Although it has been described that in rabbits expression of Fos in the cortical areas contralateral to the US presentation side is significantly increased after conditioning (13), we have found that in mice, probably because of the animals’ small size, both trigeminal nerves detect the US presented to one nerve, showing similar (bilateral) patterns of R1 and R2 reflex responses (data not shown). These data could explain most of our results, which did not show any difference in the density of immunoreactive nuclei between the two hemispheres in conditioned animals with an optimal performance. In any case, differences found between HL and HR cortical areas remain unexplained, because no evidence of impaired pain sensitivity or synaptic or axonal conductivity in hypobaric conditions has been found.

In conclusion, the present results strongly suggest that acclimatization to high altitude prevents the impairment of classical eyeblink conditioning evoked by hypobaric hypoxic conditions. In contrast, acclimatization to high altitude does not improve classical eyeblink conditioning compared with control groups checked at land conditions. Nevertheless, acclimatization to high altitude might diminish the activation requirements for the proper performance of acquired eyeblink responses. These findings could be relevant with respect to high-altitude training for the acquisition of new motor skills.


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 ABSTRACT
 MATERIALS AND METHODS
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The present study was supported by a grant from the Andalusian Consejería de Comercio, Turismo y Deporte, Spain.


   ACKNOWLEDGMENTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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REFERENCES
 
The authors are grateful to Roger Churchill for help in the editing of this manuscript. P. J. Yi was a visiting professor from Cayetano Heredia University, Lima, Peru.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. C. López-Ramos, División de Neurociencias, Universidad Pablo de Olavide, Ctra. de Utrera, Km. 1, 41013-Sevilla, Spain (e-mail: jclopram{at}upo.es)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

{dagger} Deceased 26 January 2007. Back


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