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Further analysis of the effects of harmine on the electrical activity of the rabbit brain
,Nuerophormucoloyv Vol 17.pp 295-298 %, PergamonPress Ltd 1978Prmledm GreatBrlta~n
FURTHER ANALYSIS ON THE ELECTRICAL
OF THE EFFECTS OF HARMINE ACTIVITY OF THE RABBIT BRAIN
A. SCOTTI DE CAROLIS, V. FLORIO* and V. G. LONGS Laboratorio di Farmacologia. Istituto Superiore di SanitB, Roma. Italy (Accepted
6 September
1977)
Summary-The effects of harmine on the electrical activity of the red nucleus. nucleus ventralis anterior of the thalamus, head of nucleus caudatus and hypothalamus. have been studied in the rabbit. Harmine induced, at the level of the red nuclei. synchronous waves at 10 c/s. These patterns were not modified by a previous treatment with diazepam (I mg/kg, i.v.). Further studies with diazepam have indicated that the drug induced a modification of the electrical activity of the red nucleus, consisting of a diminution in frequency and an increase in voltage of the waves. The same patterns could be obtained after administration of barbiturates and meprobamate but not with diphenylhydantoin.
Previous work from this laboratory has considered the effects of harmine on the electrical activity of the brain (Fuentes and Eongo, 1971; de Trujillo, Scotti de Carolis and Longo, 1977). It has been demonstrated that harmine induces in the rabbit activation of the cortical EEG, bursts of spikes at 10 c/s in the cerebellar vermis, accompanied by synchronous waves in the spinal (lumbar enlargement) leads. According to Llinas and Volkind (1973) and de Montigny and Lamarre (1973). the 10 c/s spiking originates in the olivary complex; they recorded, by means of microelectrodes, the same activity from the cerebellar cortex and the bulbar reticular nuclei. To date, no analysis of the degree of involvement of other nuclei has been made and no description can be found of the resultant EEG changes. In the present investigation the effects of harmine on the electrical activity of some deep sites of the brain situated rostrally to the cerebellum, namely, the red nucleus, the nucleus ventralis anterior of the thalamus. the head of nucleus caudatus and the hypothalamus (tractus mammillo-thalamicus) were studied in the rabbit. The influence of diazepam on the changes induced by harmine were also examined. Since alterations in the electrical activity recorded from the red nucleus have been described previously for barbiturates and meprobamate (Schimmerl and Stumpf, 1958) the effects of diazepam have been compared with those of phenobarbital, pentobarbital, meprobamate and diphenylhydantoin. METHODS The experiments were carried out in the rabbit; a total of 67 animals were used. Implantation of superficial and concentric deep electrodes was carried out under ether anesthesia according to a technique described elsewhere (Longo, 1962). Histological examinations confirmed the electrode location. Recording
sessions started l-2 hr after discontinuation of ether. In some cases, the animals were immobilized with gallamine and artificially ventilated through a tracheal cannula. The following drugs were used: harmine HCl. diazepam. phenobarbital sodium, pentobarbital sodium, meprobamate. diphenylhydantoin sodium; the drugs were dissolved in water, with the exception of diazepam for which the commercial ampoules (Valium) were employed, and administered by slow intravenous injection. RESULTS Typical control recordings from cortical and subcortical sites are depicted in Figures I and 2. In the sensorimotor cortex leads, EEG states of rest and arousal were easily distinguishable by the presence respectively of spindles and slow waves and by a lowvoltage 2&25 c/s activity. Also the EEG activity recorded from the caudate and from the nuclrus cantralis anterior of the thalamus showed different patterns during rest and during arousal. In the thalamus, the record during rest consisted of an irregular activity made up of large (lo(f200 pV) waves at 3-4 c/s, intermingled with lower voltage (50~1V) spiky waves which appeared concomitantly with the cortical spindles and had the same frequency (8-12 c/s); during arousal; regular sinusoidal waves at 4 c/s appeared. with superimposed a lower voltage (20 LLV) rapid (20-30 c/s) activity. In the caudate, during rest, spindles and slow waves were present. while during arousal a lower voltage (20 pV) rapid activity (2C-30 c/s) appeared. In the hypothalamus waves of 2&30 PV at 20 c/s were present. The electrical activity recorded from red nucleus consisted of low-voltage (I@20 pV) waves, at a frequency of around 35 c/s. Only minor alteration of these patterns were observed during the state of rest and arousal in the hypothalamic and red nucleus leads.
‘A. SCWTI t>t
296
CAROLIS. V.
FLORIOand V. G. LONGO
2 set Fig. I. ERects of harmine on the EEG of the rabbit. The control tracing was recorded under basic conditions in the curarized rabbit (gallamine 5 mg/kg. iv.). At the arrow, an acoustic stimulus was applied. The harmine record was taken 3 min after the administration of the drug. The cortical spindles and slow waves were substituted by low-voltage rapid activity; synchronous waves at 10 c/s appear in the leads from the red nucleus. Leads: ASM--anterior sensorimotor cortex; PSM-posterior sensorimotor cortex: OPT-ootic cortex: HYP-tractus mamillo thalamicus: LNR---left red nucleus: RNR-right red nucleus.
E&cts
of hartnine
A total of 23 animals was treated with harmine. and on the basis of results obtained in previous experiments a dose of 2 mg/kg was administered. A few minutes after administration. in the leads from the red nucleus, bursts of synchronous waves at 10 c/s appeared. Eventually the bursts evolved into sustained rhythmic activity at the same frequency (Fig. 1). In two cases only, spikes were observed in the thalamic record. No changes were observed in the hypothalamic record. An arousal pattern appeared in the cortical cerebral leads and in the caudate. Return to normal occurred in 20-30 min.
The drug was administered in doses of 1 mg/kg to 14 rabbits. This dose, in previous experiments (de Trujillo et al., 1977) antagonized the tremors induced by harmine and prevented the spinal 10 c/s synchronous waves induced by this drug. A few minutes after injection, synchronization of the electrocorticogram of the caudate and of the thalamic records, consisting of an increase in slow (2-3 c/s) waves and spindles occurred together with a diminution in frequency and an increase in voltage of the waves recorded from the red nucleus (Fig. 2). Return to nor-
mat was observed in 30-30 min. This drug was without effect on the hypothalamic ekctrical activity. In 6 of these animals, 2 mg,kg of harmine was injected 15 min after diazepam. Diazepam pretreatment did not prevent the appearance of the 10 c/s synchronous waves in the red nucleus leads, but blocked the desynchronization which harmine induced at the level of the cortical leads.
l$>cts of harhiturutrs, wzeproharnute and diphmyfhydarltoirl A total of 30 animals was used. Subnarcotic doses of phenobarbital (50 mg/kg) and pentobarbital (10 mg/kg) were administered. The two drugs induced analogous changes in the EEG. consisting of a synchronization of the cortical, caudate, and thaldmic records, together with an increase in voltage and a diminution in frequency of the electrical activity recorded from the red nucleus (Fig. 2). Meprobamate (100 mg/kg) also induced changes in the red nucleus electrical activity similar to that observed after barbiturates. After this drug, there was a prevalence of high-voltage slow (2-3 c/s) waves in the cortical leads. On the other hand, diphenylhydantoin (10. 20 and 30 mg/kg) induced only a synchronization of the cortical, caudate and thalamic EEG. without influencing the record from the red nucleus.
297
DISCUSSION The present
results
demonstrate
that
after
harmine,
in previous experiments (de Trujillo et al., 1977) at the level of cerebellar vermis and spinal cord is also recorded in the red nucleus. It is known from anatomo-physiological research that the red nucleus is part of a rubro-olivo-cerebellorubral loop. From the studies of Papez and Stotler (1940) this loop includes the denate nuclues, the red
the
10 c/s activity
observed
nucleus, the reticular nuclei, the inferior olive and the neocerebellar cortex. This circuit therefore seems distinct from the pathway where the harmine spikes were recorded by Llinas and Volkind (1974) (inferior olive, paleocerebellum and fastigial nucleus). In all probability. these two loops are functionally interconnected. In favour of this hypothesis are the present results, indicating that after harmine the 10 c/s activity is present also in the red nucleus, and the data of Larochelle, Bedard, Poirier and Sourkes, (1971) who demonstrated that, in the monkey, electrolytic
298 lesions
A.
SCOTTI DE CAROLIS,
to the rubro-olivo-cerebella-rubral loop induce a postural tremor, which is intensified by harmaline. Centres located more rostrally in the cerebrum, such as the nucleus ventralis anterior of the thalamus, the head of the caudate, the hypothalamus or the cerebral cortex do not exhibit the 10 c/s activity. In these regions, an EEG pattern not dissimilar from that recorded during activation is present. Behavioural investigations have indicated that the rostra1 centres, and in particular the cortex and the striatum, play an important role in the tremorogenic effect of harmine. In mice, removal of the cortex or of the striatum inhibits the appearance of tremors (Hara and Kawamori, 1954). Ontogenetic studies in the rat have shown that harmine and harmaline induce tremors only after the 13th day of age, i.e. at a certain stage of cortical maturation (Henderson and Woolley, 1970). If the 10 c/s activity is the neurophysiological counterpart of the tremors, it seems that the pacemaker centres lie caudal to the mesencephaIon, but regions situated rostrally may provide the major associative link between the pacemaker centres and the motor system. In previous results (de Trujillo et a/.. 1977) as in the present results, it was found that diazepam abated the tremors without significantly influencing the 10 c/s activity at cerebellar or rubral level. Diazepam, however. changed the EEG activation present in the regions rostra1 to the mesencephalon into synchronization. In a previous study (de Trujillo et al., 1977) it was suggested that diazepam acted on the relay reticular nuclei, since the drug blocked the spinal but not the cerebellar seizure due to harmine. It is passible, however, that the blocking effect of diazepam might also be due to an effect on more rostra1 centres responsible for the elaboration of the signals. The results obtained with diazepam have also indicated that the drug induces a modification of the electrical activity of the red nucleus; the same pattern can be obtained after administration of barbiturates and meprobamate but not with diphenylhydantoin. It may be concluded therefore that barbiturates, ben-
1. FLORIOand V. G.
LONCKI
zodiazepines and meprobamate, all induce an increase in voltage and a diminution in frequency of the red nucleus electrical activity, which may reflect a common influence on central mechanisms. Since a role in the maintenance of muscle tonus has been attributed to the rubro-olive-cerebella-rubral loop. it can be hypothesized that this effect could be related to the muscle-relaxation induced by these drugs. This is supported by the fact that diphenylhydantoin, which does not induce muscle relaxation, does not influence the red nucleus electrical activity.
Acknowledgrmmr-The skilful technical assistance of Mr E. Deodati and Mr S. Fortuna is gratefully acknowledged. REFERENCES Fuentes, J. A. and Longo. V. G. (1971). An investigation on the central effects of harmine, harmaline and related P-carbolines. Neuropharmacology IO: 15-23. Hara, S. and Kawamori, K. (1954). Pharmacological studies on the function of the extrapyramidal system. Mechanism of the appearance of tremor due to extrapyramidal poisons. Jap. J. Phnrmac. 3: 1499156. Henderson, G. L. and Woolley. D. E. (1970). Ontogenesis of drug-induced tremor in the rat. .I. Pharmac. up. Ther. 175: 113~120. Larochelle, L.. Bedard. P.. Poirier. L. J. and Sourkes, T. L. (1971). Correlative neuroanatomical and neuropharmacological study of tremor and catatonia in the monkey. Nruropharmacology 10: 273-288. Llinas, R. and Volkind, R. A. (1974). The olivo-cerebellar system: functional properties as revealed by harmalineinduced tremor. Expl Brait Res. 18: 69987. Longo, V. G. (1962). EIrcfroer7cephalographic Atlas for Pharmacological Rrsrarch. Elsevier. Amsterdam. Montigny, C. de and Lamarre. Y. (1973). Rhythmic activity induced by harmaline in the olivo-cerebella-bulbar system of the cat. Brain Rrs. 81: 81--95. Papez. J. W. and Stotler, W. A. (1940). Connectron of the red nucleus. Archs Neural. Psychid.. Lot7d. 44: 776-791. Schimmerl. G. and Stumof. Ch. (1958). Die Tatigkeit des Nucleus ruber nach Verabreichung von Barbituraten und Meprobamat. Naun?n-Schrtriudrhrr/ls Arch. rsp. Path.
Pharmak.
235: 3340.
Trujillo, G. C. de. Scotti de Carolis. A. and Longo. V. G. (1977). Influence of diazepam. L-Dopa and dopamine on the cerebellar and spinal electrical patterns induced by harmine in the rabbit. Nwropharmacoloyy 16: 31 36.
FURTHER ANALYSIS ON THE ELECTRICAL
OF THE EFFECTS OF HARMINE ACTIVITY OF THE RABBIT BRAIN
A. SCOTTI DE CAROLIS, V. FLORIO* and V. G. LONGS Laboratorio di Farmacologia. Istituto Superiore di SanitB, Roma. Italy (Accepted
6 September
1977)
Summary-The effects of harmine on the electrical activity of the red nucleus. nucleus ventralis anterior of the thalamus, head of nucleus caudatus and hypothalamus. have been studied in the rabbit. Harmine induced, at the level of the red nuclei. synchronous waves at 10 c/s. These patterns were not modified by a previous treatment with diazepam (I mg/kg, i.v.). Further studies with diazepam have indicated that the drug induced a modification of the electrical activity of the red nucleus, consisting of a diminution in frequency and an increase in voltage of the waves. The same patterns could be obtained after administration of barbiturates and meprobamate but not with diphenylhydantoin.
Previous work from this laboratory has considered the effects of harmine on the electrical activity of the brain (Fuentes and Eongo, 1971; de Trujillo, Scotti de Carolis and Longo, 1977). It has been demonstrated that harmine induces in the rabbit activation of the cortical EEG, bursts of spikes at 10 c/s in the cerebellar vermis, accompanied by synchronous waves in the spinal (lumbar enlargement) leads. According to Llinas and Volkind (1973) and de Montigny and Lamarre (1973). the 10 c/s spiking originates in the olivary complex; they recorded, by means of microelectrodes, the same activity from the cerebellar cortex and the bulbar reticular nuclei. To date, no analysis of the degree of involvement of other nuclei has been made and no description can be found of the resultant EEG changes. In the present investigation the effects of harmine on the electrical activity of some deep sites of the brain situated rostrally to the cerebellum, namely, the red nucleus, the nucleus ventralis anterior of the thalamus. the head of nucleus caudatus and the hypothalamus (tractus mammillo-thalamicus) were studied in the rabbit. The influence of diazepam on the changes induced by harmine were also examined. Since alterations in the electrical activity recorded from the red nucleus have been described previously for barbiturates and meprobamate (Schimmerl and Stumpf, 1958) the effects of diazepam have been compared with those of phenobarbital, pentobarbital, meprobamate and diphenylhydantoin. METHODS The experiments were carried out in the rabbit; a total of 67 animals were used. Implantation of superficial and concentric deep electrodes was carried out under ether anesthesia according to a technique described elsewhere (Longo, 1962). Histological examinations confirmed the electrode location. Recording
sessions started l-2 hr after discontinuation of ether. In some cases, the animals were immobilized with gallamine and artificially ventilated through a tracheal cannula. The following drugs were used: harmine HCl. diazepam. phenobarbital sodium, pentobarbital sodium, meprobamate. diphenylhydantoin sodium; the drugs were dissolved in water, with the exception of diazepam for which the commercial ampoules (Valium) were employed, and administered by slow intravenous injection. RESULTS Typical control recordings from cortical and subcortical sites are depicted in Figures I and 2. In the sensorimotor cortex leads, EEG states of rest and arousal were easily distinguishable by the presence respectively of spindles and slow waves and by a lowvoltage 2&25 c/s activity. Also the EEG activity recorded from the caudate and from the nuclrus cantralis anterior of the thalamus showed different patterns during rest and during arousal. In the thalamus, the record during rest consisted of an irregular activity made up of large (lo(f200 pV) waves at 3-4 c/s, intermingled with lower voltage (50~1V) spiky waves which appeared concomitantly with the cortical spindles and had the same frequency (8-12 c/s); during arousal; regular sinusoidal waves at 4 c/s appeared. with superimposed a lower voltage (20 LLV) rapid (20-30 c/s) activity. In the caudate, during rest, spindles and slow waves were present. while during arousal a lower voltage (20 pV) rapid activity (2C-30 c/s) appeared. In the hypothalamus waves of 2&30 PV at 20 c/s were present. The electrical activity recorded from red nucleus consisted of low-voltage (I@20 pV) waves, at a frequency of around 35 c/s. Only minor alteration of these patterns were observed during the state of rest and arousal in the hypothalamic and red nucleus leads.
‘A. SCWTI t>t
296
CAROLIS. V.
FLORIOand V. G. LONGO
2 set Fig. I. ERects of harmine on the EEG of the rabbit. The control tracing was recorded under basic conditions in the curarized rabbit (gallamine 5 mg/kg. iv.). At the arrow, an acoustic stimulus was applied. The harmine record was taken 3 min after the administration of the drug. The cortical spindles and slow waves were substituted by low-voltage rapid activity; synchronous waves at 10 c/s appear in the leads from the red nucleus. Leads: ASM--anterior sensorimotor cortex; PSM-posterior sensorimotor cortex: OPT-ootic cortex: HYP-tractus mamillo thalamicus: LNR---left red nucleus: RNR-right red nucleus.
E&cts
of hartnine
A total of 23 animals was treated with harmine. and on the basis of results obtained in previous experiments a dose of 2 mg/kg was administered. A few minutes after administration. in the leads from the red nucleus, bursts of synchronous waves at 10 c/s appeared. Eventually the bursts evolved into sustained rhythmic activity at the same frequency (Fig. 1). In two cases only, spikes were observed in the thalamic record. No changes were observed in the hypothalamic record. An arousal pattern appeared in the cortical cerebral leads and in the caudate. Return to normal occurred in 20-30 min.
The drug was administered in doses of 1 mg/kg to 14 rabbits. This dose, in previous experiments (de Trujillo et al., 1977) antagonized the tremors induced by harmine and prevented the spinal 10 c/s synchronous waves induced by this drug. A few minutes after injection, synchronization of the electrocorticogram of the caudate and of the thalamic records, consisting of an increase in slow (2-3 c/s) waves and spindles occurred together with a diminution in frequency and an increase in voltage of the waves recorded from the red nucleus (Fig. 2). Return to nor-
mat was observed in 30-30 min. This drug was without effect on the hypothalamic ekctrical activity. In 6 of these animals, 2 mg,kg of harmine was injected 15 min after diazepam. Diazepam pretreatment did not prevent the appearance of the 10 c/s synchronous waves in the red nucleus leads, but blocked the desynchronization which harmine induced at the level of the cortical leads.
l$>cts of harhiturutrs, wzeproharnute and diphmyfhydarltoirl A total of 30 animals was used. Subnarcotic doses of phenobarbital (50 mg/kg) and pentobarbital (10 mg/kg) were administered. The two drugs induced analogous changes in the EEG. consisting of a synchronization of the cortical, caudate, and thaldmic records, together with an increase in voltage and a diminution in frequency of the electrical activity recorded from the red nucleus (Fig. 2). Meprobamate (100 mg/kg) also induced changes in the red nucleus electrical activity similar to that observed after barbiturates. After this drug, there was a prevalence of high-voltage slow (2-3 c/s) waves in the cortical leads. On the other hand, diphenylhydantoin (10. 20 and 30 mg/kg) induced only a synchronization of the cortical, caudate and thalamic EEG. without influencing the record from the red nucleus.
297
DISCUSSION The present
results
demonstrate
that
after
harmine,
in previous experiments (de Trujillo et al., 1977) at the level of cerebellar vermis and spinal cord is also recorded in the red nucleus. It is known from anatomo-physiological research that the red nucleus is part of a rubro-olivo-cerebellorubral loop. From the studies of Papez and Stotler (1940) this loop includes the denate nuclues, the red
the
10 c/s activity
observed
nucleus, the reticular nuclei, the inferior olive and the neocerebellar cortex. This circuit therefore seems distinct from the pathway where the harmine spikes were recorded by Llinas and Volkind (1974) (inferior olive, paleocerebellum and fastigial nucleus). In all probability. these two loops are functionally interconnected. In favour of this hypothesis are the present results, indicating that after harmine the 10 c/s activity is present also in the red nucleus, and the data of Larochelle, Bedard, Poirier and Sourkes, (1971) who demonstrated that, in the monkey, electrolytic
298 lesions
A.
SCOTTI DE CAROLIS,
to the rubro-olivo-cerebella-rubral loop induce a postural tremor, which is intensified by harmaline. Centres located more rostrally in the cerebrum, such as the nucleus ventralis anterior of the thalamus, the head of the caudate, the hypothalamus or the cerebral cortex do not exhibit the 10 c/s activity. In these regions, an EEG pattern not dissimilar from that recorded during activation is present. Behavioural investigations have indicated that the rostra1 centres, and in particular the cortex and the striatum, play an important role in the tremorogenic effect of harmine. In mice, removal of the cortex or of the striatum inhibits the appearance of tremors (Hara and Kawamori, 1954). Ontogenetic studies in the rat have shown that harmine and harmaline induce tremors only after the 13th day of age, i.e. at a certain stage of cortical maturation (Henderson and Woolley, 1970). If the 10 c/s activity is the neurophysiological counterpart of the tremors, it seems that the pacemaker centres lie caudal to the mesencephaIon, but regions situated rostrally may provide the major associative link between the pacemaker centres and the motor system. In previous results (de Trujillo et a/.. 1977) as in the present results, it was found that diazepam abated the tremors without significantly influencing the 10 c/s activity at cerebellar or rubral level. Diazepam, however. changed the EEG activation present in the regions rostra1 to the mesencephalon into synchronization. In a previous study (de Trujillo et al., 1977) it was suggested that diazepam acted on the relay reticular nuclei, since the drug blocked the spinal but not the cerebellar seizure due to harmine. It is passible, however, that the blocking effect of diazepam might also be due to an effect on more rostra1 centres responsible for the elaboration of the signals. The results obtained with diazepam have also indicated that the drug induces a modification of the electrical activity of the red nucleus; the same pattern can be obtained after administration of barbiturates and meprobamate but not with diphenylhydantoin. It may be concluded therefore that barbiturates, ben-
1. FLORIOand V. G.
LONCKI
zodiazepines and meprobamate, all induce an increase in voltage and a diminution in frequency of the red nucleus electrical activity, which may reflect a common influence on central mechanisms. Since a role in the maintenance of muscle tonus has been attributed to the rubro-olive-cerebella-rubral loop. it can be hypothesized that this effect could be related to the muscle-relaxation induced by these drugs. This is supported by the fact that diphenylhydantoin, which does not induce muscle relaxation, does not influence the red nucleus electrical activity.
Acknowledgrmmr-The skilful technical assistance of Mr E. Deodati and Mr S. Fortuna is gratefully acknowledged. REFERENCES Fuentes, J. A. and Longo. V. G. (1971). An investigation on the central effects of harmine, harmaline and related P-carbolines. Neuropharmacology IO: 15-23. Hara, S. and Kawamori, K. (1954). Pharmacological studies on the function of the extrapyramidal system. Mechanism of the appearance of tremor due to extrapyramidal poisons. Jap. J. Phnrmac. 3: 1499156. Henderson, G. L. and Woolley. D. E. (1970). Ontogenesis of drug-induced tremor in the rat. .I. Pharmac. up. Ther. 175: 113~120. Larochelle, L.. Bedard. P.. Poirier. L. J. and Sourkes, T. L. (1971). Correlative neuroanatomical and neuropharmacological study of tremor and catatonia in the monkey. Nruropharmacology 10: 273-288. Llinas, R. and Volkind, R. A. (1974). The olivo-cerebellar system: functional properties as revealed by harmalineinduced tremor. Expl Brait Res. 18: 69987. Longo, V. G. (1962). EIrcfroer7cephalographic Atlas for Pharmacological Rrsrarch. Elsevier. Amsterdam. Montigny, C. de and Lamarre. Y. (1973). Rhythmic activity induced by harmaline in the olivo-cerebella-bulbar system of the cat. Brain Rrs. 81: 81--95. Papez. J. W. and Stotler, W. A. (1940). Connectron of the red nucleus. Archs Neural. Psychid.. Lot7d. 44: 776-791. Schimmerl. G. and Stumof. Ch. (1958). Die Tatigkeit des Nucleus ruber nach Verabreichung von Barbituraten und Meprobamat. Naun?n-Schrtriudrhrr/ls Arch. rsp. Path.
Pharmak.
235: 3340.
Trujillo, G. C. de. Scotti de Carolis. A. and Longo. V. G. (1977). Influence of diazepam. L-Dopa and dopamine on the cerebellar and spinal electrical patterns induced by harmine in the rabbit. Nwropharmacoloyy 16: 31 36.