Biofeedback and Self-Regulation, Vol. 19, No. 2, 1994 ... Rats: A Pilot Study I. Jorge Gallego, 2 Samia Benammou, Jean-Louis Miramand, and Guy Vardon.
Biofeedback and Self-Regulation, Vol. 19, No. 2, 1994
Ventilatory Conditioning by Self-Stimulation in Rats: A Pilot Study I J o r g e G a l l e g o , 2 Samia Benammou, Jean-Louis Miramand, and Guy Vardon
Faculté de Médecine de Paris-Sud Nicole EI-Massioui
Université de Paris-Sud Chantal Pacteau
Université René-Descartes Pierre Perruchet
Universit~ de Bourgogne
This article describes an experimental attempt to condition breathing pattern in rats. In this experiment, a freely moving rat was first rewarded by an electrical stimulation of the medial forebrain bundle whenever inspiratory duration (TI) exceeded 300 ms. A bidirectional control was then used: TIs longer than 400 ms were rewarded, and then TIs shorter than 300 ms were rewarded. The frequency of TIs longer than 300 ms increased when this event was rewarded, further increased when TIs above 400 ms were rewarded, and decreased during reversal conditioning (TI < 300 ms). A t the beginning of the experiment, stimulation caused increased arousal and motor activity, but after prolonged conditioning, the brain stimulation was associated with quiet wakefulness. Although the general procedure appears to be well-suited to the experimental 1We are grateful to Professor Claude Gaultier and to Professor Vincent Bloch for their support, to Pascale Leblanc for her invaluable assistance, and to Gerard Dutrieux and Michel Vigouroux for their technical contribution. This work was supported by the Université de Paris-Sud (Grant AI-9023) and by the Institut National de la Sante et de la Recherche Médicale (CJF 89-09). 2Address all correspondence to Jorge GaUego, Faculté de Médecine de Paris-Sud, Laboratroire de Physiologie, 63 rue Gabriel Péri, 94276 Le Kremlin-Bicêtre, Cedex, France. 171 0363-3586/94/0600-0171507.00/0© 1994 Plenum Publishing Corporation
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study of voluntary breathing, some possible improvements are suggested for further, more extensive investigations. Deseriptor Key Words: conditioned breathing patterns; conditioned breathing and brain stimulation; medial forebrain stimulation and breathing; voluntary breathing and conditioning; operant control of breathing.
Voluntary control of breathing and learned changes in breathing pattern are part of the treatment of many respiratory disorders. In chronic obstructive pulmonary diseases (COPD) and asthma, patients are trained to adopt a slow and deep breathing pattern to increase alveolar ventilation (Donner & Howard, 1992; Singh, Wisniewski, Britton, & Tattersfield, 1990). In mechanically ventilated patients, feedback-assisted control of tidal volume and mean inspiratory flow contributes to reduced weaning time in hard-to-wean patients (Holliday & Hyers, 1990). In addition to its use in obstructive disorders, learning to decrease ventilatory frequency has been shown to improve hyperventilation syndrome and panic attacks, and to reduce psychophysiological arousal (Cappo & Holmes, 1984; Clark, Salkovskis, & Chalkley, 1985; Grossman & Wientjes, 1989). More generaUy, it is often assumed that changing breathing pattern may influence physiological and psychological states (e.g., in yoga), but this assumption has only rarely been addressed in experimental research (Cappo, & Holmes, 1984; McCaul, Solomon, & Holmes, 1984; Gallego & Perruchet, 1993). In chronic disorders, the aim of these therapeutic breathing procedures is to induce permanent changes in breathing pattern. This is based on the implicit assumption that practice may transform initially voluntary breathing pattern into an automatic, spontaneous one, as it may occur in many motor acts. However, the generalization of this general property of motor control to breathing is not supported by experimental evidence. By and large, voluntary breathing is similar to most motor acts in many aspects, including learning (Gallego, Ankaoua, Lethielleux, Chambille, Vardon, & Jacquemin, 1986; Gallego & Perruchet 1988; Gallego & Camus, 1989; Gallego & Perruchet, 1991), and, accordingly, one might expect that learning conditions that yield the automatization of learned motor acts will also permit the automatization of learned breathing patterns. On the other hand, contrary to the learned motor skills that have been investigated in experimental conditions, automatic breathing is controlled by genetically prewired structures: the bulbo-pontine respiratory centers. Because of this peculiarity of breathing, considered as a motor act, changing spontaneous breathing pattern by learning processes, if possible, may be very difficult.
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This difficulty has been raised by physiologists (e.g., Cherniack, Chonan, & Altose, 1988), and by therapists concerned with breathing retraining. A recent report (Donner & Howard, 1992) suggested that practicing breathing retraining in COPD should be stopped until definitive evidence is available on the possibility of inducing permanent changes in breathing pattern. Unfortunately, long-term studies with control groups in patients raised numerous practical difficulties, and long-term changes in breathing pattern cannot be attempted in healthy subjects for ethical reasons. The starting point of our work is that animal studies might offer a unique opportunity to document the body of knowledge on voluntary control and learning of breathing pattern. This report focuses on methodological aspects of a conditioning study, in which one animal was trained to increase the duration of inspiration (TI) during several months. Tl was chosen as a rewarded variable because it is the most active part of breathing, compared with expiration. The animal was rewarded by electrical stimulation of the medial forebrain bundle (GaUistel, Shizgal, & Yeomans, 1981; Olds & Fobes, 1981). The changes in T1 were schedule-dependent, and we interpreted them as the result of a purposeful control of breathing pattern.
METHOD
Animal Preparation The ventilatory measurements and behavioral tests were performed on Sprague-Dawley hooded rats weighing 250 g at the time of the surgery, housed individually and red standard rat chow and water ad libitum. The rats were anesthetized intraperitoneally with Pentobarbital (60 mg/kg) and stereotaxically implanted with a bipolar stimulating electrode made from 0.25-mm nichrome twisted wires, insulated except at the cross-sectional area of the tips. The electrode was aimed at the medial forebrain bundle, using the following coordinates (Paxinos & Watson, 1986): 4.8 mm posterior to bregma, 0.6 mm lateral to the midline, and 7.9 mm ventral from the cortex. The electrode was secured to a socket fixed with two small screws and covered with dental cement. Five days after the implantation, behavioral tests were performed in a self-stimulating box. The rewarding stimuli were 350-ms trains of square pulses of 0.5 ms with a frequency of 100 Hz, delivered by a home-made standard stimulator via a multichannel rotating collector. The intensity of stimulation was determined by training the animals on a bar-press task. Each contact with the bar was followed by a stimulation. The intensity was pro-
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gressively increased from 100 ItA until the rat consistently self-stimulated. The intensity was then decreased to the minimal level at which this conditioned behavior was observed. Each rat was run up to three times during a period of about 30 minutes. Animals that could not be conditioned at levels of stimulation lower than 1000 ItA were discarded. These tests for the rewarding effect of stimulation were short enough to minimize the risk of latent inhibition during ventilatory conditioning. We discarded food and nociceptive reinforcers. The delay and the duration of food reinforcers made it poorly suitable for rapid sequences of operant and nonoperant responses. Nociceptive stimuli deeply alter the animal's state when they are delivered repeatedly. One week later, the rats that had been successfully conditioned to the bar-press task were anesthetized as before. After standard tracheotomy, a flexible Silastic T-tube (2.41 mm OD, 1.57 mm ID, 8 mm long) provided
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with a perpendicular side arm (1.02 mm ID, 2.16 mm OD, 7.5 mm long) was placed within the trachea with the side arm protruding through the anterior tracheal wall between the fourth and the fifth tracheal rings (Figure 1). This side arm was threaded beneath the skin to an opening on the top of the skull, where it was secured to the electrode socket. After about orte week, the rats that did not fully recover from the surgical trauma, or displayed low tolerance to the catheter, or did not display normal motor and ventilatory activity, were discarded. Tolerance to the intratracheal catheter was idiosyncratic. It reached several months, and, more exceptionally, one year.
Ventilatory Measurements The flow transducer was composed of a heating element (500 ~tm diameter) placed between two thermocouples (100 I.tm diameter, Mesurix, France), 1 mm apart, inside a ceramic cylinder (8 mm long, 3 mm OD). The three components were electrically connected to an external signal conditioner. During inspiration, the heating element (80°C) heated the inner thermocouple, whereas during expiration, it heated the outer one. The differential signal between the two thermocouples was amplified by a factor of 10,000, low-pass filtered (10 Hz, -3 dB), and shaped into a square signal synchronous with breathing. The calculations of inspiratory and expiratory times were performed by a Macintosh Ilfx computer equipped with an analog-to-digital converter Mac Adios II.
Protocol Three animals fulfilled the above conditions for undergoing the ventilatory conditioning procedure, but two of them died before completing the protocol (one from obstructive syndrome due to the intratracheal catheter, the other for unknown reasons). During the first phase of the experiment, the animal was trained to increase T1 over 300 ms. The two following phases served as bidirectional control procedure (Furedy, 1987). During Phase II, rewarding stimulations were delivered for TI longer than 400 ms, and during Phase III, rewarding stimulations were delivered for TI shorter than 300 ms. The number of sessions was determined as a function of the rat's state. It increased from two to ten 20-min sessions per day. Between each phase, the animal underwent shaping sessions to adapt to the new operant. The total length of conditioning was about 600 hours over five months. The animal was constantly observed during conditioning sessions.
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RESULTS At the beginning of the conditioning procedure, the ventilatory data were within the range of values found in the literature (Bartlett & Tenney, 1970; Lai, Tsuya, & Hildebrandt, 1978; Leong, Dowd, & Macfarland, 1964; Nattie, 1977). Figure 2 shows the changes in the percent frequency of TIs longer than 300 ms as a function of the reinforcing schedule. This variable markedly increased over the two first conditioning phases, and decreased during reverse conditioning. This clearly shows that this frequency varied consistently with the reinforcing schedule. The rate of reinforcement (number of stimulations per min) corresponding to the four values displayed in Figure 2 were 2, 29, 4, and 33, respectively. Pairwise comparisons show that ventilatory p e r f o r m a n c e corresponding to similar rates of reinforcements (2 stim/min and 4 stim/min, or 29 and 33) were very different. This further confirmed that the observed changes in the frequency of T I > 300 ms were due to the schedule of reinforcement rather than the stimulation per se. This established ventilatory conditioning. However, we do not rule out the possibility that nonspecific factors such as maturation or habituation to the environment or to the stimuli may have also influenced the increase in long T I frequencies.
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The above changes in the frequency of long TIs were part of more general changes in T1 frequency distribution. Figure 3 shows the complete distribution corresponding to each point of Figure 2. Each histogram displays two modal values of variable respective importance. The first mode (about 70-90 ms) corresponds to sniffing, a behavior commonly observed at the beginning of the experiment (first histogram). The second mode corresponds to "normal" breathing. The reinforcement of long TIs was associated with a decrease in sniffing behavior and a shift of the distribution toward large values. Conversely, reversal conditioning was associated with an increase in sniffing in addition to the increase in intermediate TIs in the 100-300 ms range.
DISCUSSION These results in one animal give some support to the idea that the self-stimulation procedure is well-suited to reinforce conditioned changes in breathing pattern. A bidirectional control procedure was used. The frequency of the operant response varied consistently with reinforcement schedule. Similar levels of stimulations resulted in different breathing patterns, according to what inspiratory duration was rewarded. These observations suggest that conditioning of ventilatory changes occurred in this rat. Obviously, this result should not be considered as established until it is replicated. However, besides this preliminary observation, this study highlights several crucial methodological aspects. Firstly, because breathing frequency is related to motor activity, the changes in breathing may be interpreted either as purposeful changes of breathing, or alternatively, as ventilatory consequences of changes in motor activity. Under this latter hypothesis, motor activity would be the primary conditioned response. Whether conditioning induced a genuine ventilatory conditioning or ventilatory changes mediated by conditioned motor activity is a question that can be answered by a careful observation of the rat's behavior. Here, we observed that the spontaneous reaction to stimulation (an increase in arousal and activity) consistently decreased over sessions and nearly disappeared before Phase I ended. The typical pattern of behavior for high levels of conditioning was quiet wakefulness, interrupted by sleep episodes. This pattern of behavior was maintained during Phase II and, more surprisingly, Phase III, during which TIs tended to decrease. For this reason, the mediation of motor activity in ventilatory changes did not account for the whole pattern of data. This lends support to a learned association between breathing pattern and reward, rather than, or in addition to, an association between motor activity and reward. Accordingly, the changes in breathing may be considered as "purposeful." However, this in-
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terpretation is based on qualitative observations of behavior, and for this reason, it should be considered with caution. Quantitative analysis of behavior should be done in further experiments. A further difficulty stems from the intercorrelations between breathing variables (inspiratory and expiratory durations, tidal volume, etc.). R e inforcing a given inspiratory duration also may have led to reinforcement of a given tidal volume (insofar as the two variables are correlated), and it is quite possible that tidal volume b e c a m e the relevant variable for the animal. Volumes were not measured in this experiment, and it is not possible to estimate w h e t h e r times or v o l u m e s were actually conditioned. Breathing pattern should be more completely analyzed in further studies. A final issue is the slowness of conditioning. Probably, the fact that breathing pattern is mainly an automatic process hinders any association between this activity and reinforcing stimuli. A second factor might be that, contrary to usual operants, breathing is a p e r m a n e n t activity. Because of this, any interruption of the reinforcing p r o g r a m may a m o u n t to cause an extinction procedure. In this experiment, the rat was immediately placed in another cage as soon as conditioning session ended, in order to link the possibility of reward to a specific context, and to prevent the extinction of the c o n d i t i o n e d p a t t e r n . A p e r m a n e n t conditioning p r o g r a m was not adopted here because the technique of measuring breathing m a d e p e r m a nent observation necessary. Whole-body plethysmography, a noninvasive technique for measuring ventilation, is possibly better suited for this kind of experiment. Self-stimulation seems to provide a suitable m e t h o d to study voluntary breathing, but learning procedures should be optimized in future experiments.
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