- Email: [email protected]
Influence of rostral and caudal brain stem reticular formation on thalamic neurons
Brain Research Bulletin, Vol. 18, pp. 761-765. 0 Pergamon Journals Ltd., 1987.Printed in the U.S.A.
0361-9230/87$3.00 + .OO
BRIEF COMMUNICATION
Influence of Rostra1 and Caudal Brain Stem Reticular Formation on Thalamic Neurons VELAYUDHAN
MOHAN
KUMAR,’ ABDUL ALEEM, AND BALDEV SINGH
GULSHAN
KUMAR
AHUJA
Department o~~~ysiology and Department of Neurology All India Institute of Medical Sciences, New Delhi-110 029, India Received
29 July 1986
MOHAN KUMAR, V., ABDUL ALEEM, G. K. AHUJA AND B. SINGH. Z~~uence ofrosastruf and caudul brain stem reticularformation on thalumic neurons. BRAIN RES BULL 18(6) 761-765, 1987.~Single neuronal activity was recorded from the diffuse thalamic system. Influence of the rostra1 desynchronizing and caudal synchronizing structures of the brain stem reticular formation on these neurons was studied. Rostra1 stimulation produced an increase and caudal stimulation a decrease in the thalamic unit tiring. A possible mechanism by which the brain stem reticular structures influence the cortical neurons is proposed on the basis of these findings. Brain stem reticular formation
Mi~ine thaiamus
Unit activity
THE thalamus forms an important region of the brain for the modulation of the cortical EEG and consciousness 13, 8, 9, 23, 25, 30, 311. This modulation may be influenced by the subcortical inputs to the thalamus 1311. It has been shown recently that the anterior and the posterior regions of the hypothalamus, which have opposing influence on cortical EEG, have opposite effects on the midline thalamic area (MTh) neurons [ 11. Another important subcortical structure which influences the cortical EEG is the brain stem reticular formation. An EEG desynchronizing system in the rostra1 brain stem reticular formation (RRF) was identified by Moruzzi and Magoun [24]. On the other hand, a portion of caudal brain stem reticular formation (CRF) which could bring about stimulus bound EEG synchronization has also been mapped out [21]. These effects might be mediated, at least partially, through the diffuse thalamic projection system 1241. Direct projection from brain stem reticular formation to the intralaminar and MTh areas have been demonstrated using anatomical and physiological techniques [4, 7, 22,30-321. It has been recently reported that some of the long latency responses induced in the cortical neurons by stimulation of RRF and CRF were modified after lesions in the MTh [28]. This further confirms that some of the cortical
EEG
Electrical stimulation
influences exerted by RRF and CRF are routed through MTh. The RRF and CRF, which have opposing influences on the cortical EEG, produce different effects on cortical neurons [I@. It is possible that these two brain stem StNCtures may have different influences on the neurons of MTh also. In this study, alterations in the MTh units were observed after stimulation of the RRF and CRF at different frequencies. METHOD
Experiments were conducted on cats of both sexes weighing between 2.0 and 3.5 kg. The spinal cord was transected at C, or CB level under ether anaesthesia. The en&We isole’ preparation was artificially ventilated and maintained at normothe~ia (37rt 1°C). The artificial ventilation was so adjusted that it was just sufficient to produce an EEG which showed normal spontaneous periodic fluctuations from synchronization to desynchronization and the pupils which generally remained constricted [34]. Rebreathing of expired air was avoided. It was necessary to remove tracheal secretions and to hyperventilate the animal for a few seconds periodi-
‘Requests for reprints should be addressed to Velayudhan Mohan Kumar, Department of Physiology, All India Institute of Medical Sciences, New Delhi- 110 029, India. Tresent address: Neurophysiology Laboratory, Department of Physiology, J. N. Medical College, Aligarh Muslim University, Aligarh 202001, India.
761
162 tally during the course of the experiment. Blood gas concentrations were not routinely analysed but random blood samplings had shown thai this method of adjustment of ventilation was fairly satisfactory. Stainless steel screw electrodes were fixed on either side of the skull, overlying the middle suprasylvian gyrus of the cortex for recording the EEG. An additional screw electrode was fixed on the bone overlying the frontal sinus. This was used as a ground electrode for recording the EEG and unit activity. Two small holes and a small window were made on the skull for the introduction of the stimulating and the recording electrodes respectively. After completion of the surgery, the wounds and pressure points were well perfused with local anaesthetic bupivacaine hydrochloride, Marcaine (Sarabhai Chemicals Ltd., India) while the ether anaesthesia was discontinued. The local anaesthetic was repeated at 2-4 hr intervals [ 1l]. Bipolar stainless steel concentric stimulating electrodes made from insulated 32 gauge wires, passed through insulated 22 gauge tubes, were used for this study. These electrodes with 0.5 mm uninsulated tip and 1 mm intertip distance had 20-40 Ka interelectrode resistance inside the brain. The RRF stimulating electrode was graduaIly lowered in steps of 0.5 mm to A 2.5, L 4.0, H -2.0 (as per the atlas of Snider and Niemer 1291).The electrode was finally fixed at a point which required the least strength of stimulation (3-6 V, 0.4 msec) for obtaining desynchronization on high frequency (100 Hz) stimulation. These points coincided with the formatio reticularis mesencephali [29] in the central tegmental Geld 121. The CRF stimulating electrode was similarly lowered to P 9.0, L 1.5, H -8.0 and fixed at a place which had the lowest threshold (6-8 V, 0.4 msec) for elicitation of EEG synchronization on low frequency (5-6 Hz) stimulation. These electrode tips were later identified in the nucleus reticularis gigantocellularis [29] within the gigantocellular and magnocellular tegmental fields 121.Steel microelectrodes (5-10 MO resistance), lowered through the skull window with the help of a microdrive (Narishige Scientific Instrument Lab., Japan), were used to pick up single unit activity from the MTh (A 9.5, L 0.5, H +2). The recording electrode tips were later identified in and around the nucleus centralis medialis f29]. The signals picked from these units were fed to a high impedence probe and later amplified and displayed on one of the beams of a dual beam oscilloscope. Cortical EEG was displayed on another beam of the oscilloscope. In some experiments unit activity was passed through a window discriminator. The discriminator output and the EEG were simultaneously recorded in the two channels of a polygraph as described earlier [20]. After recording the activity of a stable unit for 5 to 10 minutes the RRF and the CRF were stimulated, in sequence at low frequency (LF) and high frequency (HF), with the same strength of stimulation (6-8 V, 0.4 msec). The duration of stimulus train was kept approximately at 4 seconds, and a period of at least 5-10 minutes was allowed between the trains of stimulus. The stimulus bound responses were photographed along with pre- and post-stimulator records for analysis. The EEG was visually analysed. Unit activity data for only 4 seconds, before, during and after stimulation were considered for analysis. The records were further divided into bins of 200 msec for statistical analysis. The number of units appearing in each of these bins was counted. The values obtained before stimulations were compared with those obtained during and after stimulation, by using Wilcoxon’s two sample rank test to find out the significant change during and after the stimulation.
MOHAN KUMAR ET AL.
CRF- LF
FIG. I, The figure shows the effect ofstlmul~ltion of the rostra1 brain stem reticular formation (RRF) at high frequency (HF) and caudal brain stem reticular formation (CRF) at low frequency (LF) on the spontaneous discharge of different units (U) recorded from midline thalamus and cortical EEG (EJ. The unit activity shown in the figure is the output of window discriminator. The period of stimulation is marked with arrows indicating the onset and termination of the stimulation. HF stimulation of RRF generally produced excitation which outlasted the period of stimulation (I and 3). The poststimulatory effect was not observed in some (2). The inhibition produced by CRF-LF stimulation was found during stimulatory and post-stimulatory period in 1. It was observed during the period of stimulation in 3 and post-stimulatory period in 2. The response shown in 1 of RRF-HF and 3 of CRF-LF were obtained from the
In most cases it was not possible to record during the period of HF stimulation because of masking of the responses by stimulus artifacts. At the end of the experiment the animal was anaesthetized with Nembutal (Abbot Laboratory, India). The recording and stimulation sites were then marked by passing anodal direct current of 2 to 3 mA for 15 to 20 set and 8 to 10 set respectively through the electrodes. The brain was then perfused through the carotid artery with 100 to 150 ml of 30% formalin containing 2% potassium ferrocyanide which stained the marked sites blue. The brain was removed and preserved in 10% formalin for the histological identi~~ation of the electrode sites as described elsewhere [ 11,203.
BRAIN STEM INFLUENCE
ON THALAMIC
TABLE
NEURONS
763
TABLE 2
1
EFFECT OF STIMULATION OF THE ROSTRAL BRAIN STEM RETICULAR FORMATION (RRF) AND THE CAUDAL BRAIN STEM RETICULAR FORMATION (CRF) ON THE MIDLINE THALAMIC NEURONS RRF
CRF
LF
COMPARISON OF THE NUMBER OF NEURONS OF THE MIDLINE THALAMUS SHOWING DIFFERENT POST-STIMULATION EFFECTS ON HIGH FREQUENCY (HF) STIMULATION OF THE ROSTRAL BRAIN STEM RETICULAR FORMATION (RRF9 AND LOW FREQUENCY (LF) STIMULATION OF THE CAUDAL BRAIN STEM RETICULAR FORMATION (CRF)
LF
RRF-HF
DS
PS
HF-PS
DS
PS
HPPS
I
8
D N T
2 26 36
8 4 24 36
20 4 12 36
3 10 22 35
5 18 12 3.5
6 11 18 35
LF-low frequency stimulation; HF-high frequency stimulation; D!%-during stimulation; PS-post-stimulation; I-increased discharge; D-decreased discharge; N-no change; T-total.
RESULTS
Stimulation of the RRF at LF did not appreciably alter the ongoing EEG. Desynchronization of the EEG that outlasted the period of stimulus train was observed on HF stimulation of this area. During LF stimulation of the CRF there was stimulus bound cortical EEG synchronization. On the other hand, there was either desynchronization or no change on HF stimulation of the CRF. Effects of stimulation of the RRF at LF and HF were studied on 36 neurons of the MTh (Table 1). Only about a third of the neurons responded to LF stimulation of RRF. This induced an increase in the discharge rate of a majority of the responding neurons. The response produced during the period of stimulation continued during the poststimulatory period also. There were two neurons which did not show any response during the period of stimulation, but showed decreased discharge during the post-stimulatory period. A large number of neurons of the MTh (24 out of 36) responded to the HF stimulation of the RRF. HF stimulation also produced an increased discharge on the majority of the MTh neurons (Fig. 1). Thus the same trend in the direction of change was observed on LF and HF stimulation of the RRF. The number of MTh neurons that responded to LF stimulation of the CRF (26 out of 35) was much higher than those that responded to LF stimulation of the RRF. Most of the responsive neurons showed decreased discharge during LF stimulation of the CRF (Table 1, Fig. 1). A majority of them (9 out of 13) showed the same trend of change during the post-stimulatory period also; the remaining few did not show any change during the post-stimulatory period. There were 14 neurons which showed significant alteration in firing only during the post-stimulatory period (Fig. I). Thus the number of neurons which showed a response during the poststimulatory period remained high. About a half of the total number of neurons studied showed altered firing on HF stimulation of the CRF. Eleven out of the 17 neurons which responded to HF stimulation of the CRF, showed the same trend on LF stimulation. Decreased discharge after HF stimulation was shown by fewer neurons (Table I). There was also an increase in the number of neurons which showed increased discharge after RF stimulation of the CRF.
CRF-LF I
D N T I-increased T-total.
I
D
N
T
3 13 6 22
1 3 1 5
2 8 7 17
6 24 14 44
discharge; D-decreased
discharge; N-no
charge;
Post-stimulator effects after HF stimulation of the RRF and LF stimulation of the CRF were compared in the case of 44 neurons of the MTh (Table 2). An increased and decreased discharge after the CRF and RRF stimulation respectively were the predominant effects observed in this group also. It was found that the neurons which showed increased discharge on RRF stimulation either exhibited decreased discharge or were unaffected on CRF stimulation. Similarly the neurons which showed decreased discharge with CRF generally exhibited either increased or unaltered firing on RRF stimulation. DISCUSSION
The neurons of the MTh generally showed increased firing on RRF stimulation, and decreased tiring on CRF stimulation. A large number of neurons were influenced during HF stimulation of the RRF, which elicited cortical EEG desynchronization [24]. The LF stimulation of the CRF, which induced EEG synchronization [19], also influenced a large number of them. HF stimulation of the CRF which did not produce EEG synchronization, influenced a fewer number of neurons. In addition, inhibition was observed less often after CRF stimulation at HF. Thus, stimulation which was effective in inducing EEG changes, was also more potent in bringing alteration in MTh neuronal firing. Stimulation of the two brain stem structures induced opposite influences even on the same neurons of the MTh. On the other hand, based on the study of single neuronal activity of the brain stem during paradoxical sleep, Steriade et af. 1321 concluded that CRF cells projecting to the MTh may act synergistically with the mesencephalic reticular formation for thalamocortical activation. The RRF and CRF do not influence all the brain structures in the opposite direction. They influence the preoptic neurons in the same direction [12,13]. On the basis of these findings it was suggested that these brain stem influences on the preoptic neurons may not be related to EEG changes [13]. On the other hand, the two brain stem structures do not affect the cortical neurons in the same direction [ 181. the same trend was seen in the case of the ventromedial hypoth~amic neurons also [ 191. So the influence of the RRF and the CRF on areas like MTh, the ventromedial hypothalamus and the cortex may be related to EEG changes.
MOHAN KUMAR ET AL.
764
A convergence of the brain stem influences at the level of the MTh may be inferred from the fact that large number of neurons (20 out of 44) of this area were influenced by both the RRF and CRF. The MTh also received inputs from the two antagonistic areas of the hypothalamus [l]. The posterior hypothalamus, which has a desynchronizing influence on the cortical EEG [5,8], generally has an excitatory influence on the MTh neurons. Similarly the preopticoanterior hypothalamus, which has a synchronizing influence on EEG [26,33], has a predominantly inhibitory influence on the MTh neurons. Thus the MTh may be one of the sites where the antagonistic inputs from various subcortical structures are integrated for cortical EEG changes. At the same time, one cannot rule out the possible role of EEG changes in bringing about alterations in the MTh neuronal firing, as the thalamus is reciprocally connected with the cortex [30,31]. Apart from the possible integration at the level of the thalamus, there may be some interaction between the RRF and the CRF at the brain stem level itself, as they have local interactions [ 14,171. Some contribution of this reciprocal interaction, to the changes induced at the level of the thalamus cannot be ruled out. Increased discharge of the MTh neurons, induced on stimulation of the RRF in this study, is in line with many of the earlier observations on various thalamic neurons [8, 26, 30, 311. The present findings are in contrast to the intracellular study of Mancia et al. [ 15,161 in which they observed IPSP in MTh neurons on LF and HF stimulation of the RRF. A possible reason for the difference in observations is the difference in the sampling of the neurons by the two techniques employed in the studies. It has been reported in an intracellular study that the LF stimulation of the CRF brings about either EPSP or EPSPIPSP sequence on the MTh neurons [ 161. An inhibitory influence on the MTh neurons after HF stimulation has also been reported by them. Although it is difficult to compare the extracellular and intracellular studies, the findings of Mancia
et crl. [16] are not apparently in agreement with the present findings. In the present study it was noticed that many neurons which were inhibited during the post-stimulatory period were unaffected during the stimulus train. The short lasting EPSP preceding the IPSP, which cannot be observed in extracellular study, might have masked the response during the period of stimulus train. The secondary inhibition of long duration might have produced the poststimulation inhibitory response in these neurons. The intracellular study [ 161 provided important information regarding the post synaptic changes, but an additional extracellular study is essential to complete the information regarding the action of the brain stem structures on the MTh neurons. It was reported that lesioning of MTh produced an alteration in the long latency responses induced in the cortical neurons by stimulation of the brain stem structures [28]. The responses of long duration induced in the MTh neurons might have been responsible for the long latency responses in the cortical neurons. There is a fallacy in considering the post-stimulus effects as the stimulus effects (Table 2). The influence obtained during this period may be of the opposite nature to that induced during the period of stimulus train [ 161. Though we cannot rule out this possibility, the chances for such alterations are not high in this case, as the alterations in discharge rate noticed during and after stimulation were mostly in the same direction. The comparison made in Table 2 could be justified also because a majority of the neurons were influenced during this period.
ACKNOWLEDGEMENTS
This work was supported by the research funds of Indian Council of Medical Research. The authors would like to acknowledge the help rendered by Mr. K. R. Sundaram. Department of Biostatistics for statistical analysis of data and Dr. P. S. Rao, Department of Physiology, for critical reading of the manuscript.
REFERENCES 1. Abdul Aleem, V. Mohan Kumar, G. K. Ahuja and B. Singh. Influence of preoptico-anterior and posterior hypothalamus on midline thalamic neurons. Brain Res Bull 16: 545-548, 1986. 2. Berman, A. L. The Brain Stem of the Cut: A Cytoarchitectonic Atlas with Stereotaxic Coordinates. Madison, WI: The University of Wisconson Press, 1968. 3. Dempsey, E. W. and R. S. Morrison. The production of rhythmically recurrent cortical potentials after localised thalamic stimulation. Am J Physiol 135: 293-300, 1942. 4. Edwards, S. B. and J. S. de Olmos. Autoradiographic studies of the projections of the midbrain reticular formation: ascending projections of nucleus cuneiformis. J Comp Neural 165: 417432, 1976. of electrocortical 5. Feldman, S. M. and H. J. Wailer. Dissociation activation and behavioural arousal. Nature 1%: 1320-1322, 1962. of the 6. Fuller, J. H. Brain stem reticular units: some properties course and origin of the ascending trajectory. Bruin Res 83: 349-367, 1975. E., R. D. Skinner, S. A. Gilmore and R. Owings. 7. Gagcia-Rill, Connections of the mesencephalic locomotor regions (MLR). II. Afferents and efferents. Brain Res Bull 10: 63-71, 1983. 8. Hess, W. R. The diencephalic sleep centre. In: Brain Mechanisms and Consciousness, edited by E. D. Adrian, F. Bremer and H. H. Jasper. Oxford: Blackwell, 1954, pp. 117-136.
9. Jasper, H. H. Functional properties of the thalamic reticular system. In: Brain Mechanismsand Consciousness, edited by E. D. Adrian. F. Bremer and H. H. Jasuer. Oxford: Blackwell. 1954, pp. 376401. IO. Machne, M., 1. Calma and H. W. Magoun. Unit activity of central cephalic brain stem in EEG arousal. J Neurophysiol 18: 547-558, 1955. II. Mallick, B. N., G. S. Chhina, K. R. Sundaram, B. Singh and V. Mohan Kumar. Activity of preoptic neurons during synchronization and desynchronization. Exp Neural 81: 586597, 1983. 12. Mallick, B. N., V. Mohan Kumar, G. S. Chhina and B. Singh. Responses of preoptic neurons to stimulation of caudal and rostral brain stem reticular structures. Brain Res Bull 13: 353-356, 1984. 13. Mallick, B. N., V. Mohan Kumar, G. S. Chhina and B. Singh. Comparison of rostro-caudal brain stem influence on preoptic neurons and cortical EEG. Brain Res Bull 16: 121-125, 1986. 14. Mancia, M., A. Cratyn, C. Broggi and M. Margnelli. Synaptic linkage between mesencephalic and bulbopontine reticular structures as revealed by intracellular recording. Bruin Res 33: 491-494, 1971. 15. Mancia, M. and J. Otero-Costas. Nature of the midbrain influences upon thalamic neurons. Brain Res 49: 20&204, 1973. 16. Mancia, M., M. Margnelli, M. Mariotti, R. Spreafico and G. Broggi. Brain stem-thalamus reciprocal influence in the cat. Brain Re.s 69: 297-314, 1974.
BRAIN STEM INFLUENCE
ON THALAMIC
NEURONS
17. Mancia, M., M. Mariotti and D. R. Spreatico. Caudo-rostra1 brain stem reciprocal influences in the cat. Brain Res 80: 41-51, 1974. 18. Mohan Kumar, V., M. Mariotti, M. Schieppati, D. Esposti and M. Mancia. Postsynaptic changes in sensorimotor cortical neurons during brain stem reticular activation. Bruin Res 163: 156160, 1979. 19. Mohan Kumar, V., S. K. Sikdar, G. S. Chhina and B. Singh. Sensitivity of ventromedial hypothalamic units to rostra1 and caudal brain stem reticular inputs. Brain Res 1%: 530-535, 1980. 20. Mohan Kumar, V., B. N. Mallick, G. S. Chhina and B. Singh. Influence of ascending reticular activating system on preoptic neuronal activity. Exp New-01 86: 40-52, 1984. 21. Mohan Kumar, V., G. S. Chhina and B. Singh. Mapping of areas in the caudal brain stem that produce stimulus bound synchronization in the cortical EEG. Exp New-01 89: 295-303,1985. 22. Morgane, P. J. and W. C. Stern. Chemical anatomy of brain circuits in relation to sleep and wakefulness. In: Advances in Sleep Research, Vol 1, edited by E. Weitzman. New York: Spectrum Publications, 1974, pp. l-131. 23. Morison, R. S. and E. W. Dempsey. A study of thalamocortical relation. Am J Physiol 135: 281-292, 1942. 24. Moruzzi, kG. and H. W. Magoun. Brain stem reticular formation and activation of the EEG. Electroencephalogr Clin Neurophysiol 1: 455-473, 1949. 25. Moruzzi, M. The sleep-waking cycle. Ergeb Physiol64: I-167, 1972. 26. Nauta, N. J. H. Hypothalamic regulation of sleep in rats: An experimental study. J Neurophysiol 9: 285-316, 1946.
765
27. Purpura, D. P., T. L. Frigyesi, J. G. McMurtry and T. Scarff. Synaptic mechanisms in thalamic regulation of cerebellocortical projection activity. In: The Thalamus, edited by D. P. Purpura and M. D. Yahr. New York: Columbia University Press, 1966, pp. 153-172. 28. Schieppati, M., M. Mariotti, V. Mohan Kumar and M. Mancia. Mesencephalic and bulbar reticular influence on somatosensory cortical neurons: short and long latency effects. Sleep 6: 186 195, 1983. 29. Snider, R. S. and W. T. Niemer. A Stereotaxic Atlas of the Cat Brain. Chicago: University of Chicago Press, 1961. 30. Steriade, M., N. Ropert, A. Kitsikis and G. Oakson. Ascending activating neuronal networks in midbrain reticular core and related rostra1 systems. In: Reticular Formation Revisited, edited by J. A. Hobson and M. A. B. Brazier. New York: Raven Press, 1980, pp. 125-167. 31. Steriade, M. and M. Deschenes. The thalamus as a neuronal oscillator. Brain Res 320: l-63, 1984. 32. Steriade, M., K. Sakari and M. Jouvet. Bulbo-thalamic neurons related to thalamo-cortical activating process during paradoxical sleep. Exp Brain Res 54: 463-475, 1984. 33. Sterman, M. B. and C. D. Clemente. Forebrain inhibitory mechanism: Sleep patterns induced by basal forebrain stimulation in the behaving cat. Exp Neural 6: 103-117, 1962. 34. Villablanca, J. Specialized lesions: “cerveau isole” and “encephale isole.” In: Methods in Psychobiology, Vol 2, edited by R. D. Myers. New York: Academic Press, 1972, pp. 285-302.
0361-9230/87$3.00 + .OO
BRIEF COMMUNICATION
Influence of Rostra1 and Caudal Brain Stem Reticular Formation on Thalamic Neurons VELAYUDHAN
MOHAN
KUMAR,’ ABDUL ALEEM, AND BALDEV SINGH
GULSHAN
KUMAR
AHUJA
Department o~~~ysiology and Department of Neurology All India Institute of Medical Sciences, New Delhi-110 029, India Received
29 July 1986
MOHAN KUMAR, V., ABDUL ALEEM, G. K. AHUJA AND B. SINGH. Z~~uence ofrosastruf and caudul brain stem reticularformation on thalumic neurons. BRAIN RES BULL 18(6) 761-765, 1987.~Single neuronal activity was recorded from the diffuse thalamic system. Influence of the rostra1 desynchronizing and caudal synchronizing structures of the brain stem reticular formation on these neurons was studied. Rostra1 stimulation produced an increase and caudal stimulation a decrease in the thalamic unit tiring. A possible mechanism by which the brain stem reticular structures influence the cortical neurons is proposed on the basis of these findings. Brain stem reticular formation
Mi~ine thaiamus
Unit activity
THE thalamus forms an important region of the brain for the modulation of the cortical EEG and consciousness 13, 8, 9, 23, 25, 30, 311. This modulation may be influenced by the subcortical inputs to the thalamus 1311. It has been shown recently that the anterior and the posterior regions of the hypothalamus, which have opposing influence on cortical EEG, have opposite effects on the midline thalamic area (MTh) neurons [ 11. Another important subcortical structure which influences the cortical EEG is the brain stem reticular formation. An EEG desynchronizing system in the rostra1 brain stem reticular formation (RRF) was identified by Moruzzi and Magoun [24]. On the other hand, a portion of caudal brain stem reticular formation (CRF) which could bring about stimulus bound EEG synchronization has also been mapped out [21]. These effects might be mediated, at least partially, through the diffuse thalamic projection system 1241. Direct projection from brain stem reticular formation to the intralaminar and MTh areas have been demonstrated using anatomical and physiological techniques [4, 7, 22,30-321. It has been recently reported that some of the long latency responses induced in the cortical neurons by stimulation of RRF and CRF were modified after lesions in the MTh [28]. This further confirms that some of the cortical
EEG
Electrical stimulation
influences exerted by RRF and CRF are routed through MTh. The RRF and CRF, which have opposing influences on the cortical EEG, produce different effects on cortical neurons [I@. It is possible that these two brain stem StNCtures may have different influences on the neurons of MTh also. In this study, alterations in the MTh units were observed after stimulation of the RRF and CRF at different frequencies. METHOD
Experiments were conducted on cats of both sexes weighing between 2.0 and 3.5 kg. The spinal cord was transected at C, or CB level under ether anaesthesia. The en&We isole’ preparation was artificially ventilated and maintained at normothe~ia (37rt 1°C). The artificial ventilation was so adjusted that it was just sufficient to produce an EEG which showed normal spontaneous periodic fluctuations from synchronization to desynchronization and the pupils which generally remained constricted [34]. Rebreathing of expired air was avoided. It was necessary to remove tracheal secretions and to hyperventilate the animal for a few seconds periodi-
‘Requests for reprints should be addressed to Velayudhan Mohan Kumar, Department of Physiology, All India Institute of Medical Sciences, New Delhi- 110 029, India. Tresent address: Neurophysiology Laboratory, Department of Physiology, J. N. Medical College, Aligarh Muslim University, Aligarh 202001, India.
761
162 tally during the course of the experiment. Blood gas concentrations were not routinely analysed but random blood samplings had shown thai this method of adjustment of ventilation was fairly satisfactory. Stainless steel screw electrodes were fixed on either side of the skull, overlying the middle suprasylvian gyrus of the cortex for recording the EEG. An additional screw electrode was fixed on the bone overlying the frontal sinus. This was used as a ground electrode for recording the EEG and unit activity. Two small holes and a small window were made on the skull for the introduction of the stimulating and the recording electrodes respectively. After completion of the surgery, the wounds and pressure points were well perfused with local anaesthetic bupivacaine hydrochloride, Marcaine (Sarabhai Chemicals Ltd., India) while the ether anaesthesia was discontinued. The local anaesthetic was repeated at 2-4 hr intervals [ 1l]. Bipolar stainless steel concentric stimulating electrodes made from insulated 32 gauge wires, passed through insulated 22 gauge tubes, were used for this study. These electrodes with 0.5 mm uninsulated tip and 1 mm intertip distance had 20-40 Ka interelectrode resistance inside the brain. The RRF stimulating electrode was graduaIly lowered in steps of 0.5 mm to A 2.5, L 4.0, H -2.0 (as per the atlas of Snider and Niemer 1291).The electrode was finally fixed at a point which required the least strength of stimulation (3-6 V, 0.4 msec) for obtaining desynchronization on high frequency (100 Hz) stimulation. These points coincided with the formatio reticularis mesencephali [29] in the central tegmental Geld 121. The CRF stimulating electrode was similarly lowered to P 9.0, L 1.5, H -8.0 and fixed at a place which had the lowest threshold (6-8 V, 0.4 msec) for elicitation of EEG synchronization on low frequency (5-6 Hz) stimulation. These electrode tips were later identified in the nucleus reticularis gigantocellularis [29] within the gigantocellular and magnocellular tegmental fields 121.Steel microelectrodes (5-10 MO resistance), lowered through the skull window with the help of a microdrive (Narishige Scientific Instrument Lab., Japan), were used to pick up single unit activity from the MTh (A 9.5, L 0.5, H +2). The recording electrode tips were later identified in and around the nucleus centralis medialis f29]. The signals picked from these units were fed to a high impedence probe and later amplified and displayed on one of the beams of a dual beam oscilloscope. Cortical EEG was displayed on another beam of the oscilloscope. In some experiments unit activity was passed through a window discriminator. The discriminator output and the EEG were simultaneously recorded in the two channels of a polygraph as described earlier [20]. After recording the activity of a stable unit for 5 to 10 minutes the RRF and the CRF were stimulated, in sequence at low frequency (LF) and high frequency (HF), with the same strength of stimulation (6-8 V, 0.4 msec). The duration of stimulus train was kept approximately at 4 seconds, and a period of at least 5-10 minutes was allowed between the trains of stimulus. The stimulus bound responses were photographed along with pre- and post-stimulator records for analysis. The EEG was visually analysed. Unit activity data for only 4 seconds, before, during and after stimulation were considered for analysis. The records were further divided into bins of 200 msec for statistical analysis. The number of units appearing in each of these bins was counted. The values obtained before stimulations were compared with those obtained during and after stimulation, by using Wilcoxon’s two sample rank test to find out the significant change during and after the stimulation.
MOHAN KUMAR ET AL.
CRF- LF
FIG. I, The figure shows the effect ofstlmul~ltion of the rostra1 brain stem reticular formation (RRF) at high frequency (HF) and caudal brain stem reticular formation (CRF) at low frequency (LF) on the spontaneous discharge of different units (U) recorded from midline thalamus and cortical EEG (EJ. The unit activity shown in the figure is the output of window discriminator. The period of stimulation is marked with arrows indicating the onset and termination of the stimulation. HF stimulation of RRF generally produced excitation which outlasted the period of stimulation (I and 3). The poststimulatory effect was not observed in some (2). The inhibition produced by CRF-LF stimulation was found during stimulatory and post-stimulatory period in 1. It was observed during the period of stimulation in 3 and post-stimulatory period in 2. The response shown in 1 of RRF-HF and 3 of CRF-LF were obtained from the
In most cases it was not possible to record during the period of HF stimulation because of masking of the responses by stimulus artifacts. At the end of the experiment the animal was anaesthetized with Nembutal (Abbot Laboratory, India). The recording and stimulation sites were then marked by passing anodal direct current of 2 to 3 mA for 15 to 20 set and 8 to 10 set respectively through the electrodes. The brain was then perfused through the carotid artery with 100 to 150 ml of 30% formalin containing 2% potassium ferrocyanide which stained the marked sites blue. The brain was removed and preserved in 10% formalin for the histological identi~~ation of the electrode sites as described elsewhere [ 11,203.
BRAIN STEM INFLUENCE
ON THALAMIC
TABLE
NEURONS
763
TABLE 2
1
EFFECT OF STIMULATION OF THE ROSTRAL BRAIN STEM RETICULAR FORMATION (RRF) AND THE CAUDAL BRAIN STEM RETICULAR FORMATION (CRF) ON THE MIDLINE THALAMIC NEURONS RRF
CRF
LF
COMPARISON OF THE NUMBER OF NEURONS OF THE MIDLINE THALAMUS SHOWING DIFFERENT POST-STIMULATION EFFECTS ON HIGH FREQUENCY (HF) STIMULATION OF THE ROSTRAL BRAIN STEM RETICULAR FORMATION (RRF9 AND LOW FREQUENCY (LF) STIMULATION OF THE CAUDAL BRAIN STEM RETICULAR FORMATION (CRF)
LF
RRF-HF
DS
PS
HF-PS
DS
PS
HPPS
I
8
D N T
2 26 36
8 4 24 36
20 4 12 36
3 10 22 35
5 18 12 3.5
6 11 18 35
LF-low frequency stimulation; HF-high frequency stimulation; D!%-during stimulation; PS-post-stimulation; I-increased discharge; D-decreased discharge; N-no change; T-total.
RESULTS
Stimulation of the RRF at LF did not appreciably alter the ongoing EEG. Desynchronization of the EEG that outlasted the period of stimulus train was observed on HF stimulation of this area. During LF stimulation of the CRF there was stimulus bound cortical EEG synchronization. On the other hand, there was either desynchronization or no change on HF stimulation of the CRF. Effects of stimulation of the RRF at LF and HF were studied on 36 neurons of the MTh (Table 1). Only about a third of the neurons responded to LF stimulation of RRF. This induced an increase in the discharge rate of a majority of the responding neurons. The response produced during the period of stimulation continued during the poststimulatory period also. There were two neurons which did not show any response during the period of stimulation, but showed decreased discharge during the post-stimulatory period. A large number of neurons of the MTh (24 out of 36) responded to the HF stimulation of the RRF. HF stimulation also produced an increased discharge on the majority of the MTh neurons (Fig. 1). Thus the same trend in the direction of change was observed on LF and HF stimulation of the RRF. The number of MTh neurons that responded to LF stimulation of the CRF (26 out of 35) was much higher than those that responded to LF stimulation of the RRF. Most of the responsive neurons showed decreased discharge during LF stimulation of the CRF (Table 1, Fig. 1). A majority of them (9 out of 13) showed the same trend of change during the post-stimulatory period also; the remaining few did not show any change during the post-stimulatory period. There were 14 neurons which showed significant alteration in firing only during the post-stimulatory period (Fig. I). Thus the number of neurons which showed a response during the poststimulatory period remained high. About a half of the total number of neurons studied showed altered firing on HF stimulation of the CRF. Eleven out of the 17 neurons which responded to HF stimulation of the CRF, showed the same trend on LF stimulation. Decreased discharge after HF stimulation was shown by fewer neurons (Table I). There was also an increase in the number of neurons which showed increased discharge after RF stimulation of the CRF.
CRF-LF I
D N T I-increased T-total.
I
D
N
T
3 13 6 22
1 3 1 5
2 8 7 17
6 24 14 44
discharge; D-decreased
discharge; N-no
charge;
Post-stimulator effects after HF stimulation of the RRF and LF stimulation of the CRF were compared in the case of 44 neurons of the MTh (Table 2). An increased and decreased discharge after the CRF and RRF stimulation respectively were the predominant effects observed in this group also. It was found that the neurons which showed increased discharge on RRF stimulation either exhibited decreased discharge or were unaffected on CRF stimulation. Similarly the neurons which showed decreased discharge with CRF generally exhibited either increased or unaltered firing on RRF stimulation. DISCUSSION
The neurons of the MTh generally showed increased firing on RRF stimulation, and decreased tiring on CRF stimulation. A large number of neurons were influenced during HF stimulation of the RRF, which elicited cortical EEG desynchronization [24]. The LF stimulation of the CRF, which induced EEG synchronization [19], also influenced a large number of them. HF stimulation of the CRF which did not produce EEG synchronization, influenced a fewer number of neurons. In addition, inhibition was observed less often after CRF stimulation at HF. Thus, stimulation which was effective in inducing EEG changes, was also more potent in bringing alteration in MTh neuronal firing. Stimulation of the two brain stem structures induced opposite influences even on the same neurons of the MTh. On the other hand, based on the study of single neuronal activity of the brain stem during paradoxical sleep, Steriade et af. 1321 concluded that CRF cells projecting to the MTh may act synergistically with the mesencephalic reticular formation for thalamocortical activation. The RRF and CRF do not influence all the brain structures in the opposite direction. They influence the preoptic neurons in the same direction [12,13]. On the basis of these findings it was suggested that these brain stem influences on the preoptic neurons may not be related to EEG changes [13]. On the other hand, the two brain stem structures do not affect the cortical neurons in the same direction [ 181. the same trend was seen in the case of the ventromedial hypoth~amic neurons also [ 191. So the influence of the RRF and the CRF on areas like MTh, the ventromedial hypothalamus and the cortex may be related to EEG changes.
MOHAN KUMAR ET AL.
764
A convergence of the brain stem influences at the level of the MTh may be inferred from the fact that large number of neurons (20 out of 44) of this area were influenced by both the RRF and CRF. The MTh also received inputs from the two antagonistic areas of the hypothalamus [l]. The posterior hypothalamus, which has a desynchronizing influence on the cortical EEG [5,8], generally has an excitatory influence on the MTh neurons. Similarly the preopticoanterior hypothalamus, which has a synchronizing influence on EEG [26,33], has a predominantly inhibitory influence on the MTh neurons. Thus the MTh may be one of the sites where the antagonistic inputs from various subcortical structures are integrated for cortical EEG changes. At the same time, one cannot rule out the possible role of EEG changes in bringing about alterations in the MTh neuronal firing, as the thalamus is reciprocally connected with the cortex [30,31]. Apart from the possible integration at the level of the thalamus, there may be some interaction between the RRF and the CRF at the brain stem level itself, as they have local interactions [ 14,171. Some contribution of this reciprocal interaction, to the changes induced at the level of the thalamus cannot be ruled out. Increased discharge of the MTh neurons, induced on stimulation of the RRF in this study, is in line with many of the earlier observations on various thalamic neurons [8, 26, 30, 311. The present findings are in contrast to the intracellular study of Mancia et al. [ 15,161 in which they observed IPSP in MTh neurons on LF and HF stimulation of the RRF. A possible reason for the difference in observations is the difference in the sampling of the neurons by the two techniques employed in the studies. It has been reported in an intracellular study that the LF stimulation of the CRF brings about either EPSP or EPSPIPSP sequence on the MTh neurons [ 161. An inhibitory influence on the MTh neurons after HF stimulation has also been reported by them. Although it is difficult to compare the extracellular and intracellular studies, the findings of Mancia
et crl. [16] are not apparently in agreement with the present findings. In the present study it was noticed that many neurons which were inhibited during the post-stimulatory period were unaffected during the stimulus train. The short lasting EPSP preceding the IPSP, which cannot be observed in extracellular study, might have masked the response during the period of stimulus train. The secondary inhibition of long duration might have produced the poststimulation inhibitory response in these neurons. The intracellular study [ 161 provided important information regarding the post synaptic changes, but an additional extracellular study is essential to complete the information regarding the action of the brain stem structures on the MTh neurons. It was reported that lesioning of MTh produced an alteration in the long latency responses induced in the cortical neurons by stimulation of the brain stem structures [28]. The responses of long duration induced in the MTh neurons might have been responsible for the long latency responses in the cortical neurons. There is a fallacy in considering the post-stimulus effects as the stimulus effects (Table 2). The influence obtained during this period may be of the opposite nature to that induced during the period of stimulus train [ 161. Though we cannot rule out this possibility, the chances for such alterations are not high in this case, as the alterations in discharge rate noticed during and after stimulation were mostly in the same direction. The comparison made in Table 2 could be justified also because a majority of the neurons were influenced during this period.
ACKNOWLEDGEMENTS
This work was supported by the research funds of Indian Council of Medical Research. The authors would like to acknowledge the help rendered by Mr. K. R. Sundaram. Department of Biostatistics for statistical analysis of data and Dr. P. S. Rao, Department of Physiology, for critical reading of the manuscript.
REFERENCES 1. Abdul Aleem, V. Mohan Kumar, G. K. Ahuja and B. Singh. Influence of preoptico-anterior and posterior hypothalamus on midline thalamic neurons. Brain Res Bull 16: 545-548, 1986. 2. Berman, A. L. The Brain Stem of the Cut: A Cytoarchitectonic Atlas with Stereotaxic Coordinates. Madison, WI: The University of Wisconson Press, 1968. 3. Dempsey, E. W. and R. S. Morrison. The production of rhythmically recurrent cortical potentials after localised thalamic stimulation. Am J Physiol 135: 293-300, 1942. 4. Edwards, S. B. and J. S. de Olmos. Autoradiographic studies of the projections of the midbrain reticular formation: ascending projections of nucleus cuneiformis. J Comp Neural 165: 417432, 1976. of electrocortical 5. Feldman, S. M. and H. J. Wailer. Dissociation activation and behavioural arousal. Nature 1%: 1320-1322, 1962. of the 6. Fuller, J. H. Brain stem reticular units: some properties course and origin of the ascending trajectory. Bruin Res 83: 349-367, 1975. E., R. D. Skinner, S. A. Gilmore and R. Owings. 7. Gagcia-Rill, Connections of the mesencephalic locomotor regions (MLR). II. Afferents and efferents. Brain Res Bull 10: 63-71, 1983. 8. Hess, W. R. The diencephalic sleep centre. In: Brain Mechanisms and Consciousness, edited by E. D. Adrian, F. Bremer and H. H. Jasper. Oxford: Blackwell, 1954, pp. 117-136.
9. Jasper, H. H. Functional properties of the thalamic reticular system. In: Brain Mechanismsand Consciousness, edited by E. D. Adrian. F. Bremer and H. H. Jasuer. Oxford: Blackwell. 1954, pp. 376401. IO. Machne, M., 1. Calma and H. W. Magoun. Unit activity of central cephalic brain stem in EEG arousal. J Neurophysiol 18: 547-558, 1955. II. Mallick, B. N., G. S. Chhina, K. R. Sundaram, B. Singh and V. Mohan Kumar. Activity of preoptic neurons during synchronization and desynchronization. Exp Neural 81: 586597, 1983. 12. Mallick, B. N., V. Mohan Kumar, G. S. Chhina and B. Singh. Responses of preoptic neurons to stimulation of caudal and rostral brain stem reticular structures. Brain Res Bull 13: 353-356, 1984. 13. Mallick, B. N., V. Mohan Kumar, G. S. Chhina and B. Singh. Comparison of rostro-caudal brain stem influence on preoptic neurons and cortical EEG. Brain Res Bull 16: 121-125, 1986. 14. Mancia, M., A. Cratyn, C. Broggi and M. Margnelli. Synaptic linkage between mesencephalic and bulbopontine reticular structures as revealed by intracellular recording. Bruin Res 33: 491-494, 1971. 15. Mancia, M. and J. Otero-Costas. Nature of the midbrain influences upon thalamic neurons. Brain Res 49: 20&204, 1973. 16. Mancia, M., M. Margnelli, M. Mariotti, R. Spreafico and G. Broggi. Brain stem-thalamus reciprocal influence in the cat. Brain Re.s 69: 297-314, 1974.
BRAIN STEM INFLUENCE
ON THALAMIC
NEURONS
17. Mancia, M., M. Mariotti and D. R. Spreatico. Caudo-rostra1 brain stem reciprocal influences in the cat. Brain Res 80: 41-51, 1974. 18. Mohan Kumar, V., M. Mariotti, M. Schieppati, D. Esposti and M. Mancia. Postsynaptic changes in sensorimotor cortical neurons during brain stem reticular activation. Bruin Res 163: 156160, 1979. 19. Mohan Kumar, V., S. K. Sikdar, G. S. Chhina and B. Singh. Sensitivity of ventromedial hypothalamic units to rostra1 and caudal brain stem reticular inputs. Brain Res 1%: 530-535, 1980. 20. Mohan Kumar, V., B. N. Mallick, G. S. Chhina and B. Singh. Influence of ascending reticular activating system on preoptic neuronal activity. Exp New-01 86: 40-52, 1984. 21. Mohan Kumar, V., G. S. Chhina and B. Singh. Mapping of areas in the caudal brain stem that produce stimulus bound synchronization in the cortical EEG. Exp New-01 89: 295-303,1985. 22. Morgane, P. J. and W. C. Stern. Chemical anatomy of brain circuits in relation to sleep and wakefulness. In: Advances in Sleep Research, Vol 1, edited by E. Weitzman. New York: Spectrum Publications, 1974, pp. l-131. 23. Morison, R. S. and E. W. Dempsey. A study of thalamocortical relation. Am J Physiol 135: 281-292, 1942. 24. Moruzzi, kG. and H. W. Magoun. Brain stem reticular formation and activation of the EEG. Electroencephalogr Clin Neurophysiol 1: 455-473, 1949. 25. Moruzzi, M. The sleep-waking cycle. Ergeb Physiol64: I-167, 1972. 26. Nauta, N. J. H. Hypothalamic regulation of sleep in rats: An experimental study. J Neurophysiol 9: 285-316, 1946.
765
27. Purpura, D. P., T. L. Frigyesi, J. G. McMurtry and T. Scarff. Synaptic mechanisms in thalamic regulation of cerebellocortical projection activity. In: The Thalamus, edited by D. P. Purpura and M. D. Yahr. New York: Columbia University Press, 1966, pp. 153-172. 28. Schieppati, M., M. Mariotti, V. Mohan Kumar and M. Mancia. Mesencephalic and bulbar reticular influence on somatosensory cortical neurons: short and long latency effects. Sleep 6: 186 195, 1983. 29. Snider, R. S. and W. T. Niemer. A Stereotaxic Atlas of the Cat Brain. Chicago: University of Chicago Press, 1961. 30. Steriade, M., N. Ropert, A. Kitsikis and G. Oakson. Ascending activating neuronal networks in midbrain reticular core and related rostra1 systems. In: Reticular Formation Revisited, edited by J. A. Hobson and M. A. B. Brazier. New York: Raven Press, 1980, pp. 125-167. 31. Steriade, M. and M. Deschenes. The thalamus as a neuronal oscillator. Brain Res 320: l-63, 1984. 32. Steriade, M., K. Sakari and M. Jouvet. Bulbo-thalamic neurons related to thalamo-cortical activating process during paradoxical sleep. Exp Brain Res 54: 463-475, 1984. 33. Sterman, M. B. and C. D. Clemente. Forebrain inhibitory mechanism: Sleep patterns induced by basal forebrain stimulation in the behaving cat. Exp Neural 6: 103-117, 1962. 34. Villablanca, J. Specialized lesions: “cerveau isole” and “encephale isole.” In: Methods in Psychobiology, Vol 2, edited by R. D. Myers. New York: Academic Press, 1972, pp. 285-302.