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J Appl Physiol 94: 812-818, 2003. First published September 27, 2002; doi:10.1152/japplphysiol.00488.2002
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Vol. 94, Issue 2, 812-818, February 2003

HIGHLIGHTED TOPICS
Plasticity in Respiratory Motor Control
Selected Contribution: Classical conditioning of breathing pattern after two acquisition trials in 2-day-old mice

E. Durand1, S. Dauger1,2, G. Vardon3, P. Gressens1, C. Gaultier1,4, S. de Schonen1,5, and J. Gallego1

1 Laboratoire de Neurologie et Physiologie du Développement, INSERM E9935, 2 Service de Pédiatrie-Réanimation, and 4 Service de Physiologie, Hôpital Robert-Debré, 75019 Paris; 3 URAPC, Université de Picardie, 80036 Amiens; and 5 Groupe de Neuropsychologie Cognitive du Développement, LCD, CNRS, Université Paris V, 92100 Boulogne-Billancourt, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The aim of the present study was to test whether breathing pattern conditioning may occur just after birth. We hypothesized that sensory stimuli signaling the resumption of maternal care after separation may trigger an arousal and/or orienting response accompanied with concomitant respiratory changes. We performed a conditioning experiment in 2-day-old mice by using an odor (lemon) as the conditioned stimulus (CS) and maternal care after 1 h without the mother as the unconditioned stimulus (US). Each pup underwent two acquisition trials, in which the CS was presented immediately before (experimental paired group, n = 30) or 30 min before (control unpaired group, n = 30) contact with the mother. Conditioning was tested by using noninvasive whole body plethysmography to measure the respiratory response to the CS for 1 min. We found significantly stronger respiratory responses to the CS in the experimental group than in the control group. In contrast, somatomotor activity did not differ significantly between groups. Our results confirm the sensitivity of breathing to conditioning and indirectly support the hypothesis that learned feedforward processes may complement feedback pathways during postnatal maturation of respiratory control.

learning; control of breathing; development


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CONDITIONED RESPIRATORY RESPONSES are regarded as an adaptive feedforward mechanism of breathing control that anticipates metabolic needs (3, 15). According to this view, such conditioned behaviors are acquired in the early postnatal period (43). Classical conditioning of the breathing pattern by a sound or odor as the conditioned stimulus (CS) and hypercapnia or hypoxia as the unconditioned stimulus (US) has been found effective in adult animals (32-34) and humans (14, 15). The results of these studies suggest that learning processes may influence breathing control (3, 43, 49), in keeping with earlier data on other physiological functions (11).

However, the ontogenesis of learning has been mainly investigated for response systems other than respiration and in species other than mice. Previous studies have achieved conditioning of somatomotor activities, such as suckling (7), in newborns from several species (but not mice), and no information is available on whether breathing is sensitive to conditioning in newborns (30). Because the mouse is the reference mammalian species for genetic studies, there is a need for evaluating the contribution of learning to the development of breathing control in newborn mice.

The aim of the present study was to test whether breathing pattern conditioning may occur just after birth. Our experiment was conducted in 2-day-old mice. We used an odor as the CS because olfaction is mature at birth, whereas auditory and visual systems are immature at this age. Olfactory CS support conditioning in adult rats (35) and in fetal and newborn rats (31, 41, 42). The US was maternal care after a period of separation from the mother. Separation from the mother is a highly aversive condition, because of cooling and absence of licking, stroking, and nutrition. In newborn rats and guinea pigs, maternal separation activates several stress-responsive systems, including the hypothalamic-pituitary-adrenal axis (23). We posited that maternal care after separation may be an effective appetitive US, mainly consisting in immediate warming, maternal contact, and feeding. Specifically, we hypothesized that sensory stimuli signaling the resumption of maternal care after separation may trigger an arousal and/or orienting response accompanied with concomitant respiratory changes (21). Therefore, the conditioning paradigm used here involved an artificial odor CS, signaling a natural US based on maternal care. Conditioning was tested by measuring the respiratory responses to the olfactory CS after two acquisition trials.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Mice

Eight pregnant mice were obtained by mating Swiss-IOPS females overnight (IFFA-CREDO, L'Arbresle, France). The mice were housed at 24°C with a 12:12-h light-dark cycle and were given food and water ad libitum. Each litter was culled to 10 pups (5 males and 5 females). Then the pups were extracted from the litter one by one randomly. Within each gender, they were alternately assigned to the experimental (paired) group or the control (unpaired) group. This led to two gender-balanced groups. Each pup in the experimental or the control group was marked by a spot of red or green food dye on the back. The colors were counterbalanced over the litters to avoid differences in maternal attitude caused by the marks. One litter was discarded because of lack of maternal care during acquisition (see Acquisition, below). For 10 pups (5 experimental and 5 control pups), measurement artifacts precluded calculation of at least 10 breaths within a 15-s period (see Data Analysis, below). These 10 pups were from six of the seven remaining litters. This left 60 pups for the study (30 in each group). Experimental protocols met the animal research guidelines established by the Institut National de la Santé et de la Recherche Médicale (French national institute for health and medical research).

Respiratory Measurements

Breathing variables were measured noninvasively by using whole body, barometric plethysmography based on Drorbaugh and Fenn's principle (10, 12). According to this principle, when an animal breathes in a rigid chamber, the pressure in the chamber increases during inspiration because of addition of water vapor to the inspired gas and of warming of the inspired gas from the temperature in the chamber to that in the alveoli. Conversely, pressure decreases during expiration because of condensation of water vapor and cooling of expired gas. Measurement of these pressure variations was used to calculate breath duration (TTOT, ms), tidal volume (VT, µl), and ventilation (VE, calculated as VT/TTOT, and expressed in µl/s). VT and VE were divided by body weight and expressed in microliters per gram and microliters per gram per second, respectively. Hereafter, VT and VE designate these weight-corrected variables.

The plethysmograph consisted of two 30-ml syringes serving as measurement and reference chambers inserted in a 14 × 8 × 8 cm Plexiglas box filled with water from a thermoregulated bath (Polystat Pro, Bioblock Scientific, Illkirch, France) via a pump (Masterflex, Cole-Parmer Instrument, Vernon Hills, IL). This maintained the temperature inside the syringes between 26 and 28°C. The pressure difference between the measurement and the reference chambers (EFFA transducer, Asnières, France; range ± 0.1 mb) was filtered (bandwidth, 0.05-15 Hz at -3 dB), converted to a digital signal (MacAdios 12-bits converter, GW Instruments, Somerville, MA) at a sample rate of 100 Hz, and processed by custom-written software using Superscope II (GW Instruments). To avoid restraining the pups, body temperature was not measured and was assumed to be stable at 33°C. Calibration was done before each session by injecting 2 µl of air into the measurement chamber from a Hamilton syringe. The pressure rise induced by this injection was of similar magnitude to that induced by the VT of a newborn mouse.

Behavioral Scoring

Throughout the test, the experimenter observed the pup in the plethysmograph and recorded visible somatomotor activities for each of 12 consecutive 5-s periods. In each 5-s period, the score was 0 if there was no somatomotor activity and 1 if there was any kind of somatomotor activity (head movement, neck extension, crawling). Thus the mean behavioral score values in each group during a given period could range between 0 and 1 and reflected the proportion of pups showing somatomotor activity.

Conditioned and Unconditioned Stimuli

The CS was a lemon odor [R-(+)-limonen 97%, Aldrich, Steinheim, Germany]. A small glass filled with cotton wool was impregnated with a saturated solution of limonen. The glass was placed in the middle of an airtight box 1 h before the pup was introduced in the box for CS exposure during acquisition. During the test trial, the odor CS was administered into the plethysmograph by use of a 60-ml syringe containing a cotton disk impregnated with 200 µl of limonen.

The US consisted in maternal care when the pups were returned to the mother after a 1-h separation. The dams in this situation typically retrieved their pups, groomed them, crouched over them, and exposed their nipples. The pups responded by rooting for the nipples and suckling.

Design

Each pup underwent two acquisition trials followed by one test trial. All trials (including the tests) were performed on the same day between 9 AM and 6 PM, i.e., during the light phase.

Acquisition. First, the 10 pups in each litter (5 in the experimental group and 5 in the control group) were removed from the mother's box and put in Petri dishes, with two or three pups of the same group per dish. The dishes were placed in a neutral environment. The pups usually fell asleep, as assessed by behavioral criteria, after 2-3 min. After 30 min of separation from the mother (minute 30), the control (i.e., the explicitly unpaired CS-US) pups were wakened by gently moving the dish, then placed for 5 min in the airtight box containing the olfactory CS. Then (minute 35), they were returned to the neutral environment for 25 min. At minute 60, they were wakened and displaced to a neutral place without further exposure to the CS and were returned to their mother at minute 65. The experimental (i.e., paired CS-US) pups underwent the same procedure in terms of wakening, displacement, total time without the mother (65 min), and exposure to the CS (5 min). The only difference from the control pups was that exposure to the olfactory CS occurred at minute 60, immediately before contact with the mother, instead of at minute 30. Thus the experimental pups were kept in a neutral environment without their mother for 30 min, wakened, placed elsewhere for 5 min to mimic the control condition, and returned to the former neutral environment at minute 35 for 25 min. At minute 60, they were wakened, placed in the box with the CS for 5 min, then returned to their mother at minute 65.

After the 65-min separation, the pups in both groups were left with their mother for 1 h. During this period, the experimenter recorded maternal care, i.e., retrieval of the pups, grooming, crouching over the litter, and feeding. Then, a second acquisition test was run under the same conditions just after the first test. This was based on preliminary experiments in which one acquisition trial did not yield significant results. If no maternal care was observed within a period that we arbitrarily set at 30 min after either trial, we considered that the pups had not received the US (this occurred with one mother, i.e., 10 pups). In a separate litter independent from the experiment, we found that weight during 1-h separation nonsignificantly decreased from 2.25 ± 0.20 to 2.24 ± 0.20 g, increased to 2.26 ± 0.20 g after 1 h with the mother, and decreased to 2.24 ± 0.21 g after a second 1-h separation.

Test. The test was identical in the experimental and control pups. The pups in the two groups were tested in alternation after the second acquisition trial. Each pup was isolated for 60 min then placed in the plethysmograph. The pup was allowed 1 min to become familiar with this new environment. Then, 40 ml of room air (without the olfactory CS) was injected with a syringe into the plethysmograph for baseline recording of the breathing pattern. The injection took 15 s and precluded recording of the respiratory signal. After the end of the injection, breathing was recorded for 1 min. Forty-five seconds later, a second injection was done with the CS odor (extinction trial), and the breathing pattern was recorded again for 1 min. Then, the pup was removed from the plethysmograph and weighed. In addition, we measured mouth temperature in separate samples of pups exposed to the same treatment as the experimental and control pups. Mouth temperatures just before and after plethysmographic measurements were practically identical in the two groups, from 29.8°C before to 29.2°C after plethysmography.

Data Analysis

Respiratory data free from measurement artifacts were checked visually over the 1-min test duration. Sequences of valid breaths were used after exclusion of apneas, defined as ventilatory pauses longer than twice the duration of the preceding breath (1). Apnea type (central or obstructive) could not be determined. We calculated the number and total duration of apneas after air and CS exposures. We averaged TTOT, VT, and VE over four successive 15-s periods (P1 to P4) to examine the time course of the respiratory response to the 1-min CS exposure in the test trial. Because ~40 breaths usually occur in 15 s, data of pups with fewer than 10 breaths within a 15-s period (a finding that can reflect measurement artifacts) were excluded. Five experimental and five control pups were excluded for this reason. To control for possible confounding effects of the gas injection per se, the TTOT, VT, and VE responses to the CS were expressed as the difference between CS and room air values (designated Delta -variable).

We performed separate ANOVAs by using the multivariate approach (8) for each Delta -variable (Delta TTOT, Delta VT, and Delta VE; Superanova Software, Abacus Concepts, Berkeley, CA) and activity score (for ANOVA of binary variables, see Ref. 40) with group (experimental or control) as a between-subject factor and time (periods P1, P2, P3, and P4) as a repeated factor. Gender, litter, and weight had no significant effects and, consequently, will not be discussed further. Contrast analyses and Student's t-tests were used for partial comparisons. To take into account the heterogeneous correlations among the repeated measurements, we adjusted the degrees of freedom by using the Greenhouse and Geisser factor (8). All reported P values are based on these adjusted degrees of freedom. The criterion for conditioning was a significant main effect of group or group-by-period interaction on breathing or activity. Data are presented as group means ± SD in the text and as means ± SE in the figure.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Mean weights in the experimental and control groups (n = 30 in each group) were similar (2.02 ± 0.27 and 2.04 ± 0.29 g, respectively). Behavioral scores showed no evidence of significant conditioning (Fig. 1). In contrast, respiratory responses to the CS differed between the experimental and control pups (Fig. 1). This evidence of a conditioning effect was supported by the significant group-by-period interaction for Delta VT (P < 0.015) and for Delta VE (P < 0.021). Partial comparisons showed that Delta VT was significantly larger in experimental than in control pups during P1 and P2 (Delta VT: P1: P < 0.003; P2: P < 0.03) but not P3 or P4. Delta VE was significantly larger in the experimental group than in the control group during P2 (P < 0.01).


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  		Fig. 1.   Conditioned respiratory response to the olfactory conditioned stimulus. Variables are the difference (Delta ) between the respiratory responses to odor and air in experimental (paired) mice, n = 30, and control (unpaired) mice, n = 30, from the same litters at 2 days of postnatal age. Data are presented for 4 consecutive 15-s periods. TTOT, breath duration; VT, tidal volume; VE, ventilation. Somatomotor activity was scored on a binary basis (0 or 1). Values are means ± SE. * Significant differences between experimental and control groups at P < 0.05.

The analyses for TTOT showed that group-by-period interaction for Delta TTOT did not reach significance (P = 0.089), although Delta TTOT was significantly smaller in experimental than in control pups during P2 (P < 0.04). The conditioning effects for breathing frequency (f; = 1/TTOT) were significant, with borderline P values (group-by-period interaction, P < 0.048). Partial comparisons confirmed that Delta f was significantly higher during P2 (P < 0.025). Taken together, these data revealed a small, marginally significant conditioning effect on time variables (TTOT and f) associated with the clear-cut conditioning effect on VT.

In both groups, the pups had more apneas and longer total apnea duration after air than after CS exposure (P < 0.002 for both comparisons), which possibly reflected activation caused by arousal or orienting responses to the CS. Group had no significant effect on apnea number or duration, whether as a main effect or as interaction with other factors. This suggested that the conditioned changes in breathing variables were not related to changes in apnea number or duration.

The analysis of motor activity showed that 60% of controls and 50% of experimental pups displayed at least one episode of motor activity during the first 15-s phase of CS exposure, as a possible consequence of arousal. These proportions fell to 32 and 48%, respectively, during the second phase. Exploratory ANOVAs with the occurrence of motor activity as a new between-subject factor found no significant effect of this factor on conditioning for either respiratory variable.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The aim of the present study was to test whether breathing pattern conditioning may occur just after birth. Our 2-day-old mice developed a conditioned respiratory response to an olfactory CS previously paired with maternal care. This conditioning effect was observed after two acquisition trials and was not associated with significant changes in somatomotor activity.

Design

As a rule, conditioning is not established until it is shown that the response elicited by the CS is a specific consequence of its pairing with the US. A control group in which the presentations of CS and US are not paired is necessary for this purpose (25). Our control procedure was based on the explicitly unpaired CS-US group, in which the CS and the US were delivered alternately (but not randomly). Other control groups, such as CS-only or US-only groups, have been used (25) but did not provide conclusive information and were not used in the present study.

Our conclusion that pups underwent conditioning rests on a comparison of results obtained by using the same test procedure in paired (experimental) and unpaired (control) mouse pups. Moreover, the pups were born to the same mothers and, during acquisition, had the same number and duration of CS exposures and identical wakening and displacement periods. Therefore, it can be concluded that the differences in respiratory responses to the olfactory CS between the experimental and control groups were specifically related to pairing of the CS and US. The fact that group differences were significant at the onset of the odor stimulus (the first 30 s) and decreased subsequently further confirmed that the differences in respiratory behaviors between the two groups were specifically related to the stimulus itself rather than to nonspecific stimuli associated with the experimental setting.

We achieved conditioning after only two acquisition trials. Our data do not indicate whether a larger number of acquisition trials would induce stronger conditioned responses, in terms of magnitude or resistance to extinction (which was not studied here). This number is much smaller than in previous respiratory conditioning studies in adult animals (reviewed in Ref. 15). For example, the ventilatory correlates of conditioning to shocks in various species were observed after a number of acquisition trials varying from 120 in rats to 30 in cats or 24 in goats (15). More recent studies in adult rats showed that a respiratory conditioned response was obtained, on average, after 20 pairings of hypercapnic and auditory stimuli (32) or after 15 pairings of hypoxic and odor stimuli (33). This suggests that the postnatal period of maturation, which is crucial for maturation of brainstem respiratory networks (37, 38), may also be characterized by great sensitivity of respiratory control to learning processes.

Unconditioned Stimulus

Separation from the mother did not cause weight loss but certainly triggered a strong thermic response. During separation, the pups were placed by groups of two or three, without contact between groups, at ~22°C. This temperature can be regarded as extreme considering that, in newborn rats, thermogenesis from brown adipose tissue is maximized when air temperature is decreased from the thermoneutral range (35-36°C) to 25°C (5). Furthermore, previous studies showed that 15-day-old rats associated odors with thermotactile contact from the mother rather than with nutritive nursing (2). This suggests that rewarming of previously isolated pups was the determinant factor of the compound US (maternal care) used here.

Although maternal care has not been used previously as a US, artificial equivalents of some components of maternal care, such as tactile stimulation or oral injection of milk, have been used successfully in newborn rats (7). Natural as opposed to artificial US are easy to administer, and there is little room for doubt about their reinforcing properties. However, maternal behavior shows random variations in terms of the number and duration of reinforcing events. In the present study, one of the eight mothers failed to exhibit maternal behavior within 30 min of recovering her pups, so that conditioning could not be evaluated in this litter. This behavior was probably not related to prenatal stress, which was avoided inasmuch as possible and may on the contrary enhance nurturing and nesting behaviors (28). A more likely explanation is that the absence of maternal care from one dam reflected an idiosyncratic response to the experimental conditions.

Respiratory Conditioning

Measurement of breathing in newborn mice raises technical problems that have hampered investigations of the early postnatal development of breathing until recently (38). The measurement device used in the present study was noninvasive and allowed the pup to move freely in the chamber. Although somatomotor activity may hinder the measurement of breathing variables and can be eliminated by restraining the animal (39), restraint is a major confounding stressor, at least in adult mice (9). Consequently, we elected not to restrain the animals in our study.

Respiratory signals obtained from barometric plethysmography depend on alveolar temperature (TA): the larger the difference between the alveolar and the chamber temperatures, the larger the pressure variations. In theory, a 1°C error in TA would produce an ~6% error in VT (12). In the present study, TA, which was not measured, was assumed to be stable at 33°C during plethysmographic measurements. This raises the question of whether group differences in breathing were caused by differences in TA, i.e., whether we obtained thermoregulatory rather than respiratory conditioning. Previous studies in 6-day-old rats have shown that body temperature can be conditioned by using a contextual CS paired with a shock (19). In the present experiment, it is unlikely that temperature changes accounted for the observed conditioning effects. First, these effects were also observed for f, which, in contrast to VT, is not subjected to temperature-related errors. Furthermore, if the conditioned increase in VT was related to alveolar temperature, this would imply that the experimental mice rapidly decreased their temperature in response to the CS, an effect that, to our knowledge, has never been reported. Finally, we measured body temperature just before and after plethysmographic measurements in a separate sample of pups exposed to the same treatment as the experimental and control pups of the present study. We found that temperatures were practically identical in the two groups. This further supported similarity of body temperatures in the experimental and control pups, thus militating against a role for this factor in the observed breathing pattern differences.

Sniffing is characterized by a fast breathing rate accompanied with movements of the nostrils and vibrissae and with head-orienting movement. The ontogenesis of sniffing in newborn mice has not been previously described. Sniffing was difficult to analyze in the present study because breathing rate during sniffing in mice pups did not reach the high levels commonly observed in adults (8-10 breaths/min). Furthermore, the movements of vibrissae and nostrils were difficult to detect when the pups were in the plethysmograph. Head movements were analyzed as part of the behavioral scores and breathing rate as one of the breathing variables. The fact that the experimental subjects tended to decrease TTOT in response to the odor CS, whereas the control subjects tended to increase TTOT, may reflect a greater tendency of the experimental pups to sniff, possibly as a result of their arousal response to the CS.

The respiratory conditioned response (CR) was characterized by an increase in ventilation in response to the CS in the experimental pups, contrasting with the tendency to decrease ventilation in the control pups. Conceivably, the conditioned increase in ventilation may have enhanced the perception of odors and therefore localization of the mother. Because there were no significant between-group differences in somatomotor activity, it is unlikely that the respiratory CR was caused by higher metabolic demands in the experimental than in the control pups.

During acquisition, the pups of both groups were wakened before being exposed to the CS. Therefore, the association between the odor and maternal care in experimental pups occurred during wakefulness. On the other hand, it is unclear whether the expression of the conditioned respiratory response during the test occurred during wakefulness. The analysis of movements during CS exposure provided an indirect assessment of arousal. Only some of the pups displayed such movements in either group, and this possible arousal effect was not related to conditioning. Therefore, we cannot rule out that, at least in some animals, the respiratory CR was expressed during sleep. Previous experiments in adult rats showed that wakefulness was not required for the expression of a heart rate conditioned response (26). A similar effect may have occurred in the present experiment.

Previous studies showed that perturbations during sleep in 3-day-old rats impaired sleep breathing during adulthood (44, 47, 48). In a first group, the pups were subjected to repetitive hypoxic, tactile, and auditory stimuli while sleeping during the first 4 wk of life. Auditory stimuli, tactile stimuli, and sleep were considered to be CS (47). In a second group, the pups were subjected to tactile and auditory stimuli only. In a third group, the pups were left undisturbed. After 3.5 mo, the rats in the first two groups showed increased irregularities and apneas during sleep. This apneic breathing was abolished by delivering white noise during sleep without causing wakefulness, suggesting that it was determined, in part at least, by cortical influences (48). Whether this apneic breathing reflected conditioning, i.e., the association between the CS and the US, is unclear. None of the groups studied by Thomas and co-workers (47, 48) incorporated unpaired CS and US, leaving open the possibility that abnormal sleep breathing in experimental rats did not result from associative processes.

Somatomotor Activity

Respiratory conditioning was not associated with significant behavioral changes, suggesting that breathing may be a more sensitive index of associative learning than somatomotor activity. This may have important practical implications for the postnatal assessment of learning abilities in newborn animals. We cannot exclude the possibility that the absence of somatomotor activity conditioning may reflect inadequate sensitivity of our scoring method for detecting such conditioning. However, an alternative explanation is that the somatomotor and respiratory CRs are mediated by different neuronal pathways that undergo different maturation processes. This view is supported by several previous studies. First, experiments in which rats were exposed to a shock paired with a tone showed that the behavioral CR (freezing) and the autonomic CR (arterial blood pressure change) were mediated by different midbrain pathways. In particular, the lateral hypothalamus was involved in the autonomic but not the behavioral response, whereas the opposite was observed for the periaqueductal gray matter (24). Second, autonomic and behavioral CRs may emerge at different ages. For example, among CR to a tone previously paired with an aversive stimulus, freezing occurs at a younger age than heart rate alterations, which occur at a younger age than startle response potentiation (22).

Neonatal Conditioning in Mice

To our knowledge, this is the first study showing that classical conditioning can be achieved in mouse pups as young as 2 days of age. Respiratory conditioning in newborn mice is of particular interest because this species is the most developed mammalian model for genetic studies. The long-held hypothesis that learning contributes to the development of breathing control has a correlate, which is that transgenic mice with learning impairments may also show abnormal development of respiratory control. Until now, this possibility has not been specifically examined. However, it has been reported that the adult waggler mice (spontaneous mutant mice with a selective deficit in cerebellar granule cell brain-derived-neurotrophic-factor expression without apparent abnormality in cerebellum architecture) show severe impairment in eye-blink conditioning (50), whereas brain-derived-neurotrophic-factor mutant mice show abnormal development of respiratory control in the early postnatal period (4).

Potential Neural Substrates

Our study was not designed to identify neuronal networks underlying respiratory conditioning. Most of the current knowledge on the neural basis of conditioned emotional responses (reviewed recently in Ref. 6) comes from studies in adult animals, especially rats. It is unclear whether these results can be extended to developing mice. As a rule, the cerebellum and the medial prefrontal cortex are closely involved in classical conditioning of motor (18) and cardiac responses (16, 17, 27, 29). Both structures may contribute to the conditioning process examined here. Furthermore, previous lesion studies in 5- or 6-day-old rats exposed to an odor as the CS and to stroking as the US identified the olfactory bulb and amygdala as possible critical components of early associative learning (45). In addition to having a well-established role in fear conditioning (13), the amygdala is involved in appetitive classical conditioning (36). Several arguments support the hypothesis that the amygdala may mediate the respiratory CR. First, previous studies have shown that the amygdala is connected to brainstem respiratory networks (20, 46). Second, in adult cats, the decrease in TTOT elicited by a tone previously paired with shocks was reversibly attenuated by cooling the central nucleus of the amygdala (51). However, because these experiments did not involve control procedures, it is unclear whether the TTOT decrease was a CR or a nonspecific sensitization response caused by repeated exposure to shocks.

In conclusion, in the present study, we used an artificial odor CS and a natural US consisting in maternal care to show that breathing may support conditioning in newborn mice. This had not been previously established in newborn mammals. The present results show that respiratory conditioning can occur soon after birth and, therefore, that the central neuronal networks underlying conditioned respiratory responses are functional in mice soon after birth. However, further experiments are necessary to show that learned feedforward processes actually complement feedback pathways in subordinating respiratory adaptation to metabolic needs.


    ACKNOWLEDGEMENTS

This study was supported by the Institut National de la Santé et de la Recherche Médicale (grant awarded to S. Dauger) and by the Université Paris VII (Legs Poix).


    FOOTNOTES

Address for reprint requests and other correspondence: J. Gallego, Laboratoire de Neurologie et Physiologie du Développement, INSERM-E9935, Hôpital Robert-Debré, 48 Bd Sérurier, 75019 Paris, France (E-mail: gallego{at}idf.inserm.fr).

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.

First published September 27, 2002;10.1152/japplphysiol.00488.2002

Received 3 June 2002; accepted in final form 16 September 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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B. Matrot, E. Durand, S. Dauger, G. Vardon, C. Gaultier, and J. Gallego
Automatic classification of activity and apneas using whole body plethysmography in newborn mice
J Appl Physiol, January 1, 2005; 98(1): 365 - 370.
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