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Pharmacologically induced changes in the latency of digastric reflexes in 1–7 day old rabbits
Camp. Biochem. Physiol. Vol. 89C, No. 2, pp. 383-387, 1988
0306~4492/88 $3.00 + 0.00 0 1988 Pergamon Press plc
Printed in Great Britain
PHARMACOLOGICALLY INDUCED CHANGES IN THE LATENCY OF DIGASTRIC REFLEXES IN l-7 DAY OLD RABBITS A. J. THEXTON*~, J. D. MCGARRICK* and T. W. STONE? *Department of Physiology, United Medical and Dental Schools (Guy’s Campus), London SE1 9RT and iDepartment of Physiology, St George’s Hospital Medical School, London SW17 ORE, UK (Telephone: (01) 407-7600) (Received
10 April
1987)
Abstract-l. The naturally occurring change in latency of the digastric reflex from long (40-70 msec) to short (15-35 msec) in the first week of life was studied in rabbits using drugs known to affect GABAergic and glycinergic transmission. 2. Usually the long or the short latency reflex response appeared alone but after strychnine both appeared together. 3. The long latency responses were favoured by the GABA blockers bicuculline and picrotoxin; periodically rhythmic activity was also elicited. 4. The short latency responses were favoured by the GABA agonists pentobarbitone and diazepam.
INTRODUCl.ION
to such differences, the presence of some switching
The digastric reflex is a component of the classical jaw opening reflex (Sherrington, 1917) and can be elicited by electrical or mechanical stimulation within the area innervated by the second and third divisions of the trigeminal nerve. There is circumstantial evidence that the reflex is involved in producing the rapid wide opening of the jaw that differentiates feeding on solids from feeding on liquids (Thexton, 1984; Thexton and McGarrick, 1984). It has previously been reported that, in the rat and the rabbit, the latency of the digastric reflex is very long at birth and that the latency shortens over the first 7-10 days of life to approximate to the adult level (Thexton and Griffiths, 1979; Thexton and McGarrick, 1984). The significance of the change in latency of this reflex relates to the fact that the oral exploration (and subsequently the ingestion) of solid food does not occur until shortly after the latency has approached adult values. At ages from 2 to about 5 days, rabbits can exhibit a relatively long latency digastric response of 40-50msec following a single electrical stimulus (30% above reflex threshold) or a relatively short latency response of 15-25 msec; individual animals exhibiting the short latency response can be immediately converted into long latency responders by manipulating the experimental conditions, e.g. increasing stimulus strength or increasing rate of stimulation etc. (McGarrick and Thexton, 1983; Thexton and McGarrick, 1984). Rabbits less than 1 day old show only long latency responses and those older than 6 days show only short latency responses, irrespective of stimulus strength. Since the naturally occurring latency differences are almost entirely due to differences in central delay and the experimentally induced changes (in the 2-5 day old group) due solely SAuthor to whom correspondence
should be addressed. 383
mechanism must be considered. The aim of this study was to clarify the nature of the switching mechanism using pharmacological agents. The report by Sharav et al. (1982) that picrotoxin disclosed a long latency digastric response in the adult animal suggested the involvement of GABAergic mechanisms in the control of trigeminal reflexes and the work of Kidokoro et al. (1968) implicated glycinergic mechanisms. Drugs in these classes were, therefore, tested. MATERIALS AND
METHODS
The experiments were carried out on rabbit pups (New Zealand White) aged from 1 hr to 7 days post natal. The animals were anaesthetised with halothane/oxygen and a large craniotomy performed on the right side. Suction decerebration was performed at the precollicular level under direct vision. The scalp flaps were reflected to expose the cartilaginous external auditory canals which were then cut to allow the insertion of ear bars. The animal was mounted in a miniaturised version of a conventional head frame. This held the head (a) by ear bars (b) by a bar pressing on the frontal bones so as to hold the head down to a bar in contact with the palatal mucosa immediately behind the upper incisors. Stimulating electrodes (gold plated fish hooks) were inserted into the upper lip on the left side. In some cases flexible stainless steel electrodes were inserted into the left side of the tongue. The reflex response was recorded using fine bipolar wire electrodes which were inserted into the anterior digastric muscle (occasionally also into the masseter) ipsilateral to the stimulating electrodes. The electrical signs of reflex activity in muscle were recorded using a Medelec fibre optic recorder with AA6 MkII amplifiers; the bandpass was 400 Hz-3.2 kHz and amplification varied from 20 to lOO~V/cm. Forty five animals survived decerebration. Following decerebration and cessation of anaesthesia, at least 1 hr was allowed to elapse before eliciting the digastric reflex using 0.05-0.2 msec electrical stimuli applied to the upper lip at a strength 30% above reflex threshold (threshold = signal > baseline by 5 pV). Prior to the administration
A. J. THEXTONet al.
384
animals responding in a particular way is given. The failure of a proportion of animals to respond to doses (per kg body wt) of drugs that are known to be adequate in the adult rat may be due to species difference, to the use of young animals or to decerebration; this problem was not pursued. The effect on the digastric reflex (in 4 out of 4 animals) of the glycine antagonist strychnine was to increase the amplitude of the short latency (20msec) response by l&SO% and shorten its latency by 2-4msec. More importantly, a longer latency (45 msec) response appeared (Figs 1A and B). The short (20 msec) latency reflex response was suppressed and a longer (40 msec) latency response appeared when GABA antagonists were administered at near convulsant doses (Figs 1C and D). This occurred in 8 out of 11 animals given picrotoxin and in 6 out of 8 animals given bicuculline. Occasionally the late response induced by picrotoxin was followed by bursts of digastric EMG activity at regular intervals of about 100 msec. These were never seen with strychnine. Pentobarbitone opposed the increase in reflex latency produced by repetition of stimuli at 1 Hz and reversed the picrotoxin/bicuculline shift of reflex latency (Fig. 2); these actions were obtained in 11 out of 16 animals. When naturally occurring long latency reflex responses were present in animals over 1 day old, these also were reversed. Attempts were made to test the effect of pentobarbitone on the long latency response of the newborn (< 12 hr) animal. Very few successful experiments were completed as the combination of decerebration and barbiturate was usually lethal in these very young animals. In the 3 successful experiments showing the usual long latency response at birth, there was no convincing evidence of the presence of a short latency response following the
of pentobarbitone a stronger stimulus (up to 1.7 T) was occasionally used to ensure a pre-existing long latency reflex response (McGarrick and Thexton, 1983). Temperature was maintained at 37°C by immersing the animal to the neck in a thermostatically controlled water bath controlled from a rectal thermistor. The bath also served to support the animal whilst giving no purchase when locomotor activity arose. Drugs were administered as follows: strychnine 5 mg/kg,
s.c.; pentobarbitone 10mg/kg, i.p.; diazepam 1 mg/kg, i.m.; bicuculline 10mg/kg, i.p.; picrotoxin 4 mg/kg, i.p. RESULTS The unanaesthetised decerebrate rabbit pup exhibited periods of vigorous locomotor activity. This made it difficult to maintain long term stability of the animal in the head frame as the skull and the bearing points by which it was held were largely cartilaginous. Many desirable experimental procedures such as continuous monitoring of the afferent volleys were, therefore, excluded. However, because urethane, halothane and particularly pentobarbitone (in anaesthetic doses) were found to suppress the latency switching under investigation, the use of anaesthesia was contraindicated. Furthermore, surgical anaesthesia raised the threshold of the jaw opening reflex and could block it completely, contrary to the findings of Cardot and Laugier (1922) in the adult animal. The use of strong electrical stimuli (i.e. >>1.3 T) to overcome this problem was contraindicated because, as previously reported, such stimuli strongly favoured the appearance of long latency response in isolation (McGarrick and Thexton, 1983). The effects of the pharmacological agents subsequently described in this paper all arose 10-20 min after administration; in each case the proportion of A
I
C
Fig. 1. (A) Control responses with a 22 msec latency (elicited by a 1.3 x threshold stimulus) before administering strychnine. (B) The effect of strychnine. Amplification reduced-see calibration bar. The latency of the reflex was slightly shortened, its amplitude increased and an additional burst appeared with a latency of SOmsec. (C) Control responses elicited by a near threshold stimulus, prior to the administration of bicuculline. A short (20 msec) latency appeared on two occasions. (D) The effect of bicuculline. The short latency, short duration response completely disappeared and a longer (40msec) latency, regularly elicited response took its place. Stimuli applied at 5 set intervals. Seven traces were superimposed in each record. The 0.5 mV calibration bar of A applies to C, D; each ramp of the time trace represents 10 msec.
Changing latency of digastric reflex
385 AFTER FlJRTtER
WTER CONTRCY_
NEHWTRL
Pl~TOXjN
AFTER NEMBUTAL
Fig. 2. The reversibility of the effects of pentobarbitone (nembutal) and picrotoxin. The control records were obtained at 1 set intervals; this is an experimental manipulation that may be used to change the latency of response. The records run sequentially upwards. With successive repetitions of the stimulus at 1 Hz, the short latency (25 msec) response disappeared and a longer (60 msec) latency response appeared. Following (-t-20 min) the administration of pentobarbitone the same stimuli did not elicit the late response but the early response persisted. In contrast, 2Omin after the administmtion of picrotoxin the early response disappeared and a late response appeared. Whereas dual stimuli were used in the two previous records (at start of oscilloscope sweep), in this case only a single stimulus was used because the animal was in a near convulsive state. However, approximately 20min after the administration of further pentobarbitone, the eariy response reappeared as a massive signal irrespective of whether single (lower portion of record) or dual stimuli were used. The records were obtained at approximately 30 min intervals except for last two “further nembutal” records. administration of pentobarbitone. Diazepam also suppressed the long latency response and favoured the appearance of the short latency reflex in 5 out of
7 animals over 1 day old. Afferent volleys capable of eliciting digastric activity also produced digastric inhibition in the occasional animal showing background digastric EMG activity. The period of inhibition corresponded to the interval between the times at which the short and long latency digastric responses would otherwise have occurred; an indication of this inhibition appears in Fig. 3. When, in addition to the digastric activity, the activity in the masseter muscle was recorded in 5 animals less than 2 days old, an excitatory reflex with a short (15-20 msec) latency was evident (Fig. 3); the response was followed 100msec later by a second burst of masseter activity in all cases, In these cases the digastric activity consisted of a small or nearly absent burst with short latency and a longer latency large burst of activity contending to the interval between the two periods of masseter activity. In 3 animals (exhibiting steady background EMG activity in digastric and/or masseter) the preceding pattern was periodically followed by a series of reciprocal rhythmic bursts of EMG activity in digastric and
masseter continuing up to 4 sets.
at regular 100 msec intervals for
DISCUSSION
The results indicate that both giycinergic and GABAergic inhibitor mechanisms operate on the digastric reflex arc in the first week of life in the rabbit. Under normal circumstances (in animals over 1 day old) there appeared to be a measure of antagonism between the early and the late reflex responses in that only one or the other was usually elicited. This antagonism could be present naturally or the change from short to long latency could be provoked by repetition of 1.3 T stimuli at 1 Hz or be induced by increasing stimulus strength. Although this latter manoeuvre suggested that the long latency response might be preferentially elicited by smaller diameter afferents, the problems arising from possible stimulus spread in these small animals reduce the reliance that can be put on this inte~retation. The antagonism between the short and long latency responses could, however, be abolished by strychnine so that large early and late responses coexisted. The balance of antago~sm could, however, be tipped in favour of the production of early responses by pentobarbitone,
A. J. THEXTON et al.
386 CONTROL
MASSETER DIGASTRIC
NEMBUTflL
MASSETER DICASTRIC
S
Fig. 3. The relationship between reflex masseter and digastric activity in a 1.5 day old animal and the effect of pentobarbitone (nembutal). Single responses are shown in each record. In the control situation a 1.3 T stimulus for the digasttic reflex (large stimulus artefact at ‘S’ followed 25 msec later by a small digastric response.) elicited a large masseter response with a similar latency. The masseter activity recurred after a period of approximately 100 msec and a second burst of digastric activity occurred with a latency of 55-60 msec. Note that this animal showed background activity in the digastric muscle-see pre-stimulus period. Following the administration of pentobarbitone, the reflex activity completely disappeared in the masseter but the short latency reflex in the digastric muscle increased markedly; although the second digastric response persisted the latency increased to about 70msec. Each ramp of the time trace represents 10msec; the height of each ramp corresponds to 0.1 mV.
has been shown to potentiate the effects of GABA on central neurones (Barker and Ransom, 1978) and by diazepam, which has a similar central action (Simmonds, 1980). Conversely the reflex activity could be modified in favour of the late responses by the GABA antagonists bicuculline and picrotoxin. These latter results, therefore, confirm the report of the effect of picrotoxin in the adult animal (Sharav et al., 1982). One possible interpretation of the results of this study (Fig. 4) would be that glycinergic neurons were involved in both tonic inhibition of cells in the digastric reflex pathways and in reciprocal inhibition between the two pathways. The administration of strychnine would consequently cause an enhancement of both the early and the late phases of the digastric EMG response. The inhibitory influence of GABA neurones may then be exercised upon glycinergic neurons acting on the short latency pathway and perhaps also upon the excitatory intemeuron pool involved in the long latency activity. In this
which
Fig. 4. Schematised summary of the pharmacological results in rabbit pups over 1 day old. Afferent volleys (not necessarily identical) ate shown as activating those neurons responsible for the short latency (early) response and for the long latency (late) response. The two groups of neurons (early and late) are shown as exercising inhibitory influences on each other via glycinergic interneurons that may be tonically active. The inhibitory pathway from the “late” to the “early” neurons and/or the interneurons on the long latency pathway are then shown as under GABAergic inhibition.
situation, agents such as pentobarbitone and diazepam, which potentiate the action of GABA would, therefore, tend, like strychnine, to suppress some of the glycine mediated inhibitions but would strongly favour the appearance of the shorter latency response. GABA antagonists then would have the contrary effect of enhancing glycinergic inhibition but this would be exerted primarily on the short latency pathway (if the long latency excitatory interneurons were normally subject to GABA inhibition, they would then also be protected from that inhibition). The result would be that the long latency response would appear in isolation. Inability to shorten the long latency response at birth suggests that the circuitry involved in short latency activity only matures after day 1, possibly correlating with myelination of faster conducting axons. It is also tempting to speculate that the naturally occurring shift of digastric reflex latency from long to short in the first few days of life may be related to the appearance and development of GABA containing neurones reflected in the increases in GABA levels found in this period (Vernadakis and Woodbury, 1962). It may, however, be inappropriate to regard the reciprocal relationship between short and long latency responses as reflecting purely a direct reciprocity between simple reflexes. The fact that repetitive discharges sometimes occurred in association with the long latency digastric response could indicate that the response was analogous to that occurring when a central locomotor rhythm generator is excited by flexor reflex afferents (Jankowska et al., 1967; Viala et al., 1974). Suppression of the short latency response by activation of the long latency response/pattern generator would then be entirely consistent with the suggestion (Olsson et al., 1986) that activation of the masticatory pattern generator in the adult rabbit depresses the jaw opening reflex (short latency digastric response). The finding of a short latency excitatory reflex in the masseter rather than the classically described
Changing latency of digastric reflex inhibition (Sherrington, 1917) was not unexpected. Evidence for such an excitatory reflex arc has been reported previously in adult animals and man (Kidokoro et al., 1968; Goldberg, 1971; Achari and Thexton, 1972; Thexton, 1973, 1974). It did, however, appear that, in this study, the response might be part of a burst of rhythmic activity involving reciprocal activation with the digastric muscle (Fig. 3). Concurrent with the masseter activity there was depression of the short latency digastric reflex; the later digastric reflex then had a latency such that the activity was reciprocally related to masseter activity. Such a pattern has been reported for limb muscles following stimulation of flexor reflex afferents after pharmacological activation of rhythm generating networks (Jankowska et al., 1967; Viala et nl., 1974). The effect of pentobarbitone, in abolishing the masseter response and releasing the short latency digastric response, was to produce a pattern that corresponded to the non activated state in the experiments of Jankowska et al. (1967) and Viala et al. (1974). Diazepam similarly suppressed the late digastric response in favour of the early one. If this also reflects suppression of central pattern generator activity then this could be the obverse of the well known oral automatisms that follow benzodiazepine withdrawal. It is, however, uncertain as to whether, in this study, all the long latency responses in the digastric muscle belonged to a single class; one may, therefore, not be justified in assuming that all such responses are reflections of activity in some central pattern generator. Acknowledgements-This study was supported Medical Research Council of Great Britain.
by the
REFERENCES Achari N. K. and Thexton A. J. (1972) Diencephalic influences on the jaw opening reflex in the cat. Archs oral. Biol. 17, 1073-1080.
Barker J. L. and Ransom B. R. (1978) Pentobarbitone pharmacology of mammalian central neurones grown in tissue culture. J. Physiol. 280, 355-372.
387
Cardot H. and Laugier H. (1922) Le reflex linguomaxillaire. Cr hebd. Seunc. Sot. Biol. 86, 529-530. Goldberg L. J. (1971) Masseter muscle excitation induced by stimulation of periodontal and gingival receptors in man. Brain Res. 32, 369-380. - _ Jankowska E.. Jukes M. G. M.. Lund S. and Lundbere A. (1967) The effect of DOPA on the spinal cord. 5. R&procal organisation of pathways transmitting excitatory action to alpha motoneurones of flexors and extensors. Acta physiol. stand. 70, 369-388.
Kidokoro Y., Kubota K., Shuto S. and Sumino R. (1968) Reflex organisation of cat masticatory muscles. J. Neurophysiol. 31, 695-708.
Mcdarrick J. and Thexton A. (1983) Digastric reflex latency in the ore-weaning rabbit. J. Phvsiol. 340. 28P-29P. Olsson K: A., Sasamoto K. and Lund J. P. (1986) Modulation of transmission in rostra1 trigeminal sensory nuclei during chewing. J. Neurophysiol. 55, 56-75. Sharav Y., Ta1 M. and Devor M. (1982) Effect of peripheral electrical stimulation on the jaw opening reflex. In Anatomical Physiological and Pharmacological Aspects of Trigeminal Pain (Edited by Matthews B-and Hih R. G.),
pp. 247-260. Excerpta Medica, Amsterdam. Sherrington C. S. (1917) Reflexes elicitable in the cat from pinna, vibrissae and jaws. J. Physiol. 51, 404-431. Simmonds M. A. (1980) A site for the potentiation of GABA mediated responses by benzodiazepines. Nafure 284, 558-560.
Thexton A. J. (1973) Oral reflexes elicited by mechanical stimulation of palatal mucosa in the cat. Archs. oral Biol. 18, 971-980. Thexton A. J. (1974) Jaw opening and jaw closing reflexes in the cat. Brain Res. 66, 425433. Thexton A. (1984) Jaw, tongue and hyoid movement-a question of synchrony? J. Roy. Sot. Med. 77, 1010-1019. Thexton A. J. and Griffiths C. (1979) Reflex oral activity in decerebrate rats of different age. Brain Res. 175, l-9. Thexton A. and McGarrick J. (1984) Maturation of brainstem reflex mechanisms in relation to the transition from liquid to solid food ingestion. Bruin behav. Evol. 25, 138-145. Vernadakis A. and Woodburv D. (1962) Electrolyte and amino acid changes in the rat brain during maturation. Am. J. Physiol.
203, 748-752.
Viala D., Valin A. and Buser P. (1974) Relationship between the “late reflex discharge” and locomotor movements in acute spinal cats and rabbits treated with DOPA. Arch. Ital. Biol. 112, 299-306.
0306~4492/88 $3.00 + 0.00 0 1988 Pergamon Press plc
Printed in Great Britain
PHARMACOLOGICALLY INDUCED CHANGES IN THE LATENCY OF DIGASTRIC REFLEXES IN l-7 DAY OLD RABBITS A. J. THEXTON*~, J. D. MCGARRICK* and T. W. STONE? *Department of Physiology, United Medical and Dental Schools (Guy’s Campus), London SE1 9RT and iDepartment of Physiology, St George’s Hospital Medical School, London SW17 ORE, UK (Telephone: (01) 407-7600) (Received
10 April
1987)
Abstract-l. The naturally occurring change in latency of the digastric reflex from long (40-70 msec) to short (15-35 msec) in the first week of life was studied in rabbits using drugs known to affect GABAergic and glycinergic transmission. 2. Usually the long or the short latency reflex response appeared alone but after strychnine both appeared together. 3. The long latency responses were favoured by the GABA blockers bicuculline and picrotoxin; periodically rhythmic activity was also elicited. 4. The short latency responses were favoured by the GABA agonists pentobarbitone and diazepam.
INTRODUCl.ION
to such differences, the presence of some switching
The digastric reflex is a component of the classical jaw opening reflex (Sherrington, 1917) and can be elicited by electrical or mechanical stimulation within the area innervated by the second and third divisions of the trigeminal nerve. There is circumstantial evidence that the reflex is involved in producing the rapid wide opening of the jaw that differentiates feeding on solids from feeding on liquids (Thexton, 1984; Thexton and McGarrick, 1984). It has previously been reported that, in the rat and the rabbit, the latency of the digastric reflex is very long at birth and that the latency shortens over the first 7-10 days of life to approximate to the adult level (Thexton and Griffiths, 1979; Thexton and McGarrick, 1984). The significance of the change in latency of this reflex relates to the fact that the oral exploration (and subsequently the ingestion) of solid food does not occur until shortly after the latency has approached adult values. At ages from 2 to about 5 days, rabbits can exhibit a relatively long latency digastric response of 40-50msec following a single electrical stimulus (30% above reflex threshold) or a relatively short latency response of 15-25 msec; individual animals exhibiting the short latency response can be immediately converted into long latency responders by manipulating the experimental conditions, e.g. increasing stimulus strength or increasing rate of stimulation etc. (McGarrick and Thexton, 1983; Thexton and McGarrick, 1984). Rabbits less than 1 day old show only long latency responses and those older than 6 days show only short latency responses, irrespective of stimulus strength. Since the naturally occurring latency differences are almost entirely due to differences in central delay and the experimentally induced changes (in the 2-5 day old group) due solely SAuthor to whom correspondence
should be addressed. 383
mechanism must be considered. The aim of this study was to clarify the nature of the switching mechanism using pharmacological agents. The report by Sharav et al. (1982) that picrotoxin disclosed a long latency digastric response in the adult animal suggested the involvement of GABAergic mechanisms in the control of trigeminal reflexes and the work of Kidokoro et al. (1968) implicated glycinergic mechanisms. Drugs in these classes were, therefore, tested. MATERIALS AND
METHODS
The experiments were carried out on rabbit pups (New Zealand White) aged from 1 hr to 7 days post natal. The animals were anaesthetised with halothane/oxygen and a large craniotomy performed on the right side. Suction decerebration was performed at the precollicular level under direct vision. The scalp flaps were reflected to expose the cartilaginous external auditory canals which were then cut to allow the insertion of ear bars. The animal was mounted in a miniaturised version of a conventional head frame. This held the head (a) by ear bars (b) by a bar pressing on the frontal bones so as to hold the head down to a bar in contact with the palatal mucosa immediately behind the upper incisors. Stimulating electrodes (gold plated fish hooks) were inserted into the upper lip on the left side. In some cases flexible stainless steel electrodes were inserted into the left side of the tongue. The reflex response was recorded using fine bipolar wire electrodes which were inserted into the anterior digastric muscle (occasionally also into the masseter) ipsilateral to the stimulating electrodes. The electrical signs of reflex activity in muscle were recorded using a Medelec fibre optic recorder with AA6 MkII amplifiers; the bandpass was 400 Hz-3.2 kHz and amplification varied from 20 to lOO~V/cm. Forty five animals survived decerebration. Following decerebration and cessation of anaesthesia, at least 1 hr was allowed to elapse before eliciting the digastric reflex using 0.05-0.2 msec electrical stimuli applied to the upper lip at a strength 30% above reflex threshold (threshold = signal > baseline by 5 pV). Prior to the administration
A. J. THEXTONet al.
384
animals responding in a particular way is given. The failure of a proportion of animals to respond to doses (per kg body wt) of drugs that are known to be adequate in the adult rat may be due to species difference, to the use of young animals or to decerebration; this problem was not pursued. The effect on the digastric reflex (in 4 out of 4 animals) of the glycine antagonist strychnine was to increase the amplitude of the short latency (20msec) response by l&SO% and shorten its latency by 2-4msec. More importantly, a longer latency (45 msec) response appeared (Figs 1A and B). The short (20 msec) latency reflex response was suppressed and a longer (40 msec) latency response appeared when GABA antagonists were administered at near convulsant doses (Figs 1C and D). This occurred in 8 out of 11 animals given picrotoxin and in 6 out of 8 animals given bicuculline. Occasionally the late response induced by picrotoxin was followed by bursts of digastric EMG activity at regular intervals of about 100 msec. These were never seen with strychnine. Pentobarbitone opposed the increase in reflex latency produced by repetition of stimuli at 1 Hz and reversed the picrotoxin/bicuculline shift of reflex latency (Fig. 2); these actions were obtained in 11 out of 16 animals. When naturally occurring long latency reflex responses were present in animals over 1 day old, these also were reversed. Attempts were made to test the effect of pentobarbitone on the long latency response of the newborn (< 12 hr) animal. Very few successful experiments were completed as the combination of decerebration and barbiturate was usually lethal in these very young animals. In the 3 successful experiments showing the usual long latency response at birth, there was no convincing evidence of the presence of a short latency response following the
of pentobarbitone a stronger stimulus (up to 1.7 T) was occasionally used to ensure a pre-existing long latency reflex response (McGarrick and Thexton, 1983). Temperature was maintained at 37°C by immersing the animal to the neck in a thermostatically controlled water bath controlled from a rectal thermistor. The bath also served to support the animal whilst giving no purchase when locomotor activity arose. Drugs were administered as follows: strychnine 5 mg/kg,
s.c.; pentobarbitone 10mg/kg, i.p.; diazepam 1 mg/kg, i.m.; bicuculline 10mg/kg, i.p.; picrotoxin 4 mg/kg, i.p. RESULTS The unanaesthetised decerebrate rabbit pup exhibited periods of vigorous locomotor activity. This made it difficult to maintain long term stability of the animal in the head frame as the skull and the bearing points by which it was held were largely cartilaginous. Many desirable experimental procedures such as continuous monitoring of the afferent volleys were, therefore, excluded. However, because urethane, halothane and particularly pentobarbitone (in anaesthetic doses) were found to suppress the latency switching under investigation, the use of anaesthesia was contraindicated. Furthermore, surgical anaesthesia raised the threshold of the jaw opening reflex and could block it completely, contrary to the findings of Cardot and Laugier (1922) in the adult animal. The use of strong electrical stimuli (i.e. >>1.3 T) to overcome this problem was contraindicated because, as previously reported, such stimuli strongly favoured the appearance of long latency response in isolation (McGarrick and Thexton, 1983). The effects of the pharmacological agents subsequently described in this paper all arose 10-20 min after administration; in each case the proportion of A
I
C
Fig. 1. (A) Control responses with a 22 msec latency (elicited by a 1.3 x threshold stimulus) before administering strychnine. (B) The effect of strychnine. Amplification reduced-see calibration bar. The latency of the reflex was slightly shortened, its amplitude increased and an additional burst appeared with a latency of SOmsec. (C) Control responses elicited by a near threshold stimulus, prior to the administration of bicuculline. A short (20 msec) latency appeared on two occasions. (D) The effect of bicuculline. The short latency, short duration response completely disappeared and a longer (40msec) latency, regularly elicited response took its place. Stimuli applied at 5 set intervals. Seven traces were superimposed in each record. The 0.5 mV calibration bar of A applies to C, D; each ramp of the time trace represents 10 msec.
Changing latency of digastric reflex
385 AFTER FlJRTtER
WTER CONTRCY_
NEHWTRL
Pl~TOXjN
AFTER NEMBUTAL
Fig. 2. The reversibility of the effects of pentobarbitone (nembutal) and picrotoxin. The control records were obtained at 1 set intervals; this is an experimental manipulation that may be used to change the latency of response. The records run sequentially upwards. With successive repetitions of the stimulus at 1 Hz, the short latency (25 msec) response disappeared and a longer (60 msec) latency response appeared. Following (-t-20 min) the administration of pentobarbitone the same stimuli did not elicit the late response but the early response persisted. In contrast, 2Omin after the administmtion of picrotoxin the early response disappeared and a late response appeared. Whereas dual stimuli were used in the two previous records (at start of oscilloscope sweep), in this case only a single stimulus was used because the animal was in a near convulsive state. However, approximately 20min after the administration of further pentobarbitone, the eariy response reappeared as a massive signal irrespective of whether single (lower portion of record) or dual stimuli were used. The records were obtained at approximately 30 min intervals except for last two “further nembutal” records. administration of pentobarbitone. Diazepam also suppressed the long latency response and favoured the appearance of the short latency reflex in 5 out of
7 animals over 1 day old. Afferent volleys capable of eliciting digastric activity also produced digastric inhibition in the occasional animal showing background digastric EMG activity. The period of inhibition corresponded to the interval between the times at which the short and long latency digastric responses would otherwise have occurred; an indication of this inhibition appears in Fig. 3. When, in addition to the digastric activity, the activity in the masseter muscle was recorded in 5 animals less than 2 days old, an excitatory reflex with a short (15-20 msec) latency was evident (Fig. 3); the response was followed 100msec later by a second burst of masseter activity in all cases, In these cases the digastric activity consisted of a small or nearly absent burst with short latency and a longer latency large burst of activity contending to the interval between the two periods of masseter activity. In 3 animals (exhibiting steady background EMG activity in digastric and/or masseter) the preceding pattern was periodically followed by a series of reciprocal rhythmic bursts of EMG activity in digastric and
masseter continuing up to 4 sets.
at regular 100 msec intervals for
DISCUSSION
The results indicate that both giycinergic and GABAergic inhibitor mechanisms operate on the digastric reflex arc in the first week of life in the rabbit. Under normal circumstances (in animals over 1 day old) there appeared to be a measure of antagonism between the early and the late reflex responses in that only one or the other was usually elicited. This antagonism could be present naturally or the change from short to long latency could be provoked by repetition of 1.3 T stimuli at 1 Hz or be induced by increasing stimulus strength. Although this latter manoeuvre suggested that the long latency response might be preferentially elicited by smaller diameter afferents, the problems arising from possible stimulus spread in these small animals reduce the reliance that can be put on this inte~retation. The antagonism between the short and long latency responses could, however, be abolished by strychnine so that large early and late responses coexisted. The balance of antago~sm could, however, be tipped in favour of the production of early responses by pentobarbitone,
A. J. THEXTON et al.
386 CONTROL
MASSETER DIGASTRIC
NEMBUTflL
MASSETER DICASTRIC
S
Fig. 3. The relationship between reflex masseter and digastric activity in a 1.5 day old animal and the effect of pentobarbitone (nembutal). Single responses are shown in each record. In the control situation a 1.3 T stimulus for the digasttic reflex (large stimulus artefact at ‘S’ followed 25 msec later by a small digastric response.) elicited a large masseter response with a similar latency. The masseter activity recurred after a period of approximately 100 msec and a second burst of digastric activity occurred with a latency of 55-60 msec. Note that this animal showed background activity in the digastric muscle-see pre-stimulus period. Following the administration of pentobarbitone, the reflex activity completely disappeared in the masseter but the short latency reflex in the digastric muscle increased markedly; although the second digastric response persisted the latency increased to about 70msec. Each ramp of the time trace represents 10msec; the height of each ramp corresponds to 0.1 mV.
has been shown to potentiate the effects of GABA on central neurones (Barker and Ransom, 1978) and by diazepam, which has a similar central action (Simmonds, 1980). Conversely the reflex activity could be modified in favour of the late responses by the GABA antagonists bicuculline and picrotoxin. These latter results, therefore, confirm the report of the effect of picrotoxin in the adult animal (Sharav et al., 1982). One possible interpretation of the results of this study (Fig. 4) would be that glycinergic neurons were involved in both tonic inhibition of cells in the digastric reflex pathways and in reciprocal inhibition between the two pathways. The administration of strychnine would consequently cause an enhancement of both the early and the late phases of the digastric EMG response. The inhibitory influence of GABA neurones may then be exercised upon glycinergic neurons acting on the short latency pathway and perhaps also upon the excitatory intemeuron pool involved in the long latency activity. In this
which
Fig. 4. Schematised summary of the pharmacological results in rabbit pups over 1 day old. Afferent volleys (not necessarily identical) ate shown as activating those neurons responsible for the short latency (early) response and for the long latency (late) response. The two groups of neurons (early and late) are shown as exercising inhibitory influences on each other via glycinergic interneurons that may be tonically active. The inhibitory pathway from the “late” to the “early” neurons and/or the interneurons on the long latency pathway are then shown as under GABAergic inhibition.
situation, agents such as pentobarbitone and diazepam, which potentiate the action of GABA would, therefore, tend, like strychnine, to suppress some of the glycine mediated inhibitions but would strongly favour the appearance of the shorter latency response. GABA antagonists then would have the contrary effect of enhancing glycinergic inhibition but this would be exerted primarily on the short latency pathway (if the long latency excitatory interneurons were normally subject to GABA inhibition, they would then also be protected from that inhibition). The result would be that the long latency response would appear in isolation. Inability to shorten the long latency response at birth suggests that the circuitry involved in short latency activity only matures after day 1, possibly correlating with myelination of faster conducting axons. It is also tempting to speculate that the naturally occurring shift of digastric reflex latency from long to short in the first few days of life may be related to the appearance and development of GABA containing neurones reflected in the increases in GABA levels found in this period (Vernadakis and Woodbury, 1962). It may, however, be inappropriate to regard the reciprocal relationship between short and long latency responses as reflecting purely a direct reciprocity between simple reflexes. The fact that repetitive discharges sometimes occurred in association with the long latency digastric response could indicate that the response was analogous to that occurring when a central locomotor rhythm generator is excited by flexor reflex afferents (Jankowska et al., 1967; Viala et al., 1974). Suppression of the short latency response by activation of the long latency response/pattern generator would then be entirely consistent with the suggestion (Olsson et al., 1986) that activation of the masticatory pattern generator in the adult rabbit depresses the jaw opening reflex (short latency digastric response). The finding of a short latency excitatory reflex in the masseter rather than the classically described
Changing latency of digastric reflex inhibition (Sherrington, 1917) was not unexpected. Evidence for such an excitatory reflex arc has been reported previously in adult animals and man (Kidokoro et al., 1968; Goldberg, 1971; Achari and Thexton, 1972; Thexton, 1973, 1974). It did, however, appear that, in this study, the response might be part of a burst of rhythmic activity involving reciprocal activation with the digastric muscle (Fig. 3). Concurrent with the masseter activity there was depression of the short latency digastric reflex; the later digastric reflex then had a latency such that the activity was reciprocally related to masseter activity. Such a pattern has been reported for limb muscles following stimulation of flexor reflex afferents after pharmacological activation of rhythm generating networks (Jankowska et al., 1967; Viala et nl., 1974). The effect of pentobarbitone, in abolishing the masseter response and releasing the short latency digastric response, was to produce a pattern that corresponded to the non activated state in the experiments of Jankowska et al. (1967) and Viala et al. (1974). Diazepam similarly suppressed the late digastric response in favour of the early one. If this also reflects suppression of central pattern generator activity then this could be the obverse of the well known oral automatisms that follow benzodiazepine withdrawal. It is, however, uncertain as to whether, in this study, all the long latency responses in the digastric muscle belonged to a single class; one may, therefore, not be justified in assuming that all such responses are reflections of activity in some central pattern generator. Acknowledgements-This study was supported Medical Research Council of Great Britain.
by the
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