Brain Research, 163 (1979) 223-234 © Elsevier/North-Holland Biomedical Press
223
EFFECTS OF M E D I A N R A P H E N U C L E U S LESIONS ON H I P P O C A M P A L E E G IN T H E F R E E L Y M O V I N G RAT
EIICHI MARU*, LOREY K. TAKAHASHI* * and SHINKURO IWAHARA*** Department of Psychology, Tokyo University of Education, 3-29-1 Otsuka Bunkyoku, Tokyo and the University of Tsukuba, Ibaraki Pref 300-31 (Japan)
(Accepted June 29th, 1978)
SUMMARY The effects of median raphe lesions on the hippocampal EEG were examined in freely moving rats. First, median raphe lesions, including those restricted to the median raphe nucleus, unequivocally produced hippocampal low-frequency theta activity (5.8 Hz, SD = 0.47 Hz) during relaxed immobility which was not observed under normal conditions. This lesion-induced theta activity during immobility continued for at least 20 days, and was markedly suppressed by atropine sulfate (10 mg/kg, i.p.). On the other hand, reticular formation lesions had little effect on either hippocampal EEG patterns during immobility, movement or PS. Second, the mean frequency of theta activity was significantly reduced during movement and PS on the day following the median raphe lesion. These findings suggest a raphe-hippocampal pathway in which the median raphe nucleus plays a major role in hippocampal desynchronization (irregular pattern) by exerting an inhibitory influence on the hippocompal theta generating or facilitating mechanism. Thus the theta activity will be induced by the disinhibition following median raphe lesions.
INTRODUCTION Recent anatomical studies, using autoradiographic, silver staining and horse* To whom reprint requests should be addressed at: Department of Psychology,University of Tsukuba, Ibaraki Pref. 300-31, Japan. ** Japanese National Research Exchange Fellow. Present address: Department of Psychology, University of Hawaii, Honolulu, Hawaii, U.S.A. *** The untimely death of the third author, Dr. Shinkuro Iwahara, occurred while this work was in progress.
224 radish peroxidase techniques, have shown that the hippocampus receives a monosynaptic projection from the median raphe nucleusZ,4,5,14,is. The majority of the ascending projections from the raphe nuclei has been demonstrated by Conrad et al. 4 to run into the medial forebrain bundle and joining the diagonal band of Broca, pass through the septal nuclei or the cingulum bundle to finally reach the subiculum and the hippocampus. Electrophysiological and neurochemical data 1~, moreover, have indicated that electrical stimulation of the raphe nuclei and application of serotonin (5-HT) iontophoretically, exert a strong inhibitory influence upon the firing of hippocampal pyramidal cells. Since the raphe nuclei contain predominant numbers of 5-HT cell bodies, these findings, in addition to others 1°,17, support the view that serotonin might be an inhibitory neurotransmitter for a raphe-hippocampal neural pathway. Furthermore, Lindsley and co-workers 1,3,11,12 have found that high-frequency electrical stimulation of the raphe nucleus (nucleus raphe centralis superior) or the nucleus reticularis pontis caudalis produces a hippocampal desynchronization (lowvoltage fast activity). This desynchronizing system continues rostrally into the lateral hypothalamic area or medial forebrain bundle. Thus, this hippocampal desynchronizing system may correspond to the serotonin ascending pathway 2,4,5,14,18. In contrast, hippocampal synchronization (rhythmic theta activity) was induced by stimulating diffusely distributed regions of the midbrain and pontine tegmentum, including the nucleus reticularis pontis oralis, the nucleus locus coeruleus, the nuclei of giant cells in the pontine tegmental field and the periaqueductal grey substance of the midbrain 12. This synchronizing theta system ascends through the medial hypothalamic area (the dorsal fasciculus of Schutz). Although there is now little information concerning the interaction of the hippocampal synchronizing and desynchronizing systems, it was suggested that the amplitude of hippocampal theta activity can be enhanced by the destruction of the median raphe nucleus, a part of the desynchronizing system (Ueki, Kyushu University, personal communication, 1975). Furthermore, in our previous study tz, it was shown that the hippocampal theta activity, usually not observed during immobility in normal rats 23, appeared continuously during immobility following large brain stem lesions which included the median raphe nucleus. The following experiments were therefore undertaken to determine whether discrete lesions of the median raphe nucleus would induce the hippocampal theta activity during immobility and to examine the effects of the median raphe lesions on the frequency of hippocampal theta activity during voluntary movement and paradoxical sleep (PS). Furthermore, it has been suggested by Vanderwolf and his coworkers 9,24 that there are two types of hippocampal theta activity in rats and rabbits. Atropinesensitive theta activity occurs in rats during freezing and during the immobility that is produced by midbrain tegmentum stimulation or by certain drugs, i.e. ethyl ether, ethyl urethane, and physostigmine 9,2~,z4,26. In contrast, atropine-resistant theta activity occurs in association with 'voluntary movement 'z4,26 or during PS 9,2~,z4,26.
225 Thus the effect of atropine sulfate on hippocampal theta activity following median raphe lesions will be examined. MATERIALSAND METHODS
Animals Fifty-one male Wistar albino rats, weighing 250--390 g at the start of the experiment, were used. Animals were individually housed, and were given ad libitum access to both food and water.
General procedure Animals were given a 7-day recovery period following surgery and on the 8th day, all animals were tested for hippocampal EEGs. Only those animals which showed clear hippocampal theta activity during movement were used for the experiments. EEG recordings during various behaviors including PS were taken both before and after the brain stem lesion. All rats received either a median raphe or a unilateral midbrain reticular formation lesion. The animals which showed motor disturbances or abnormal postures with continuous muscle twitchings following these brain stem lesions were discarded. Immediately after the lesion, cortical and hippocampal EEGs were recorded during various behaviors except for PS and again after 24 h, EEG recordings during various behaviors including PS were taken in the same manner as in the pre-lesion recording session. In addition, 3 raphe-lesioned animals were observed at 3, 6, 11, and 20 days after the lesion.
Surgery, lesion and histology Animals were anesthetized with sodium pentobarbital (35 mg/kg, i.p.) and placed in a stereotaxic instrument. Bipolar electrodes were implanted in the dorsal hippocampus. A monopolar electrode was placed in either the median raphe nucleus or the reticular formation. Both hippocampal and brain stem electrodes consisted of glass-coated stainless steel wire 150/zm in diameter, exposed 0.5 mm at the tip. Implantation coordinates were followed according to the atlas of Pellegrino and Cushman 15. Coordinates used were: dorsal hippocampus: AP 3.3, ML 2.5, DV +2.0; median raphe nucleus: AP 0.0, ML 0.0, DV 4.5; midbrain reticular formation (MRF): AP 0.0, ML 2.0, DV 4.5 or--3.0. Cortical and reference electrodes consisted of miniature stainless steel screws 1 mm in diameter. The cortical electrode was placed over the sensory motor area. The reference electrode was attached to the nasal bones. Electromyographic (EMG) recordings were taken from stainless steel wires, 150 #m in diameter, chronically implanted in the neck muscles bilaterally. All lead wires were soldered to miniature connector pins and were fixed to the skull with screw anchors and dental cement.
226
MEDIAN RAPHE LESIONS
~
RETICULAR FORMATION LESIONS A0.6
PO.4
P1.2
O:RPH"39, O: RPH-25
~
:
: -"
'
~
O:RPH-75, O: RPH-77
Fig. 1. Coronal sections displaying the minimal (darkened area) and maximal damage (outlined in black) in the median raphe lesion and in the reticular formation lesion group. Four out of 21 median raphe animals had discrete (minimal) lesions similar to the lesion of RPH-39. The other animals had larger lesions which included the median raphe nucleus and surrounding tissue. There were considerable variations in the location and size of the reticular formation lesion. Atlas was taken from Pellegrino and CushmanlL Immediately after the pre-lesion recording session, electrolytic lesions were produced by passing a 1.5-2.0 mA DC current, 7-10 sec, through the brain stem electrode. The anode was inserted into the rectum. At the end of the experiment, all animals were overdosed with sodium pentobarbital and perfused with 10 ~ formalin solution. Their brains were then removed
227
AFTER MEDIAN RAPHE LESIONS
BEFORE MEDIAN RAPHE LESIONS
rat.~.-3a
=~. i-s.
DHPC
(blpJ
EMG I
1
I
1
I~- rearing
>1
DHPC (bip.)
EMG I
223
EFFECTS OF M E D I A N R A P H E N U C L E U S LESIONS ON H I P P O C A M P A L E E G IN T H E F R E E L Y M O V I N G RAT
EIICHI MARU*, LOREY K. TAKAHASHI* * and SHINKURO IWAHARA*** Department of Psychology, Tokyo University of Education, 3-29-1 Otsuka Bunkyoku, Tokyo and the University of Tsukuba, Ibaraki Pref 300-31 (Japan)
(Accepted June 29th, 1978)
SUMMARY The effects of median raphe lesions on the hippocampal EEG were examined in freely moving rats. First, median raphe lesions, including those restricted to the median raphe nucleus, unequivocally produced hippocampal low-frequency theta activity (5.8 Hz, SD = 0.47 Hz) during relaxed immobility which was not observed under normal conditions. This lesion-induced theta activity during immobility continued for at least 20 days, and was markedly suppressed by atropine sulfate (10 mg/kg, i.p.). On the other hand, reticular formation lesions had little effect on either hippocampal EEG patterns during immobility, movement or PS. Second, the mean frequency of theta activity was significantly reduced during movement and PS on the day following the median raphe lesion. These findings suggest a raphe-hippocampal pathway in which the median raphe nucleus plays a major role in hippocampal desynchronization (irregular pattern) by exerting an inhibitory influence on the hippocompal theta generating or facilitating mechanism. Thus the theta activity will be induced by the disinhibition following median raphe lesions.
INTRODUCTION Recent anatomical studies, using autoradiographic, silver staining and horse* To whom reprint requests should be addressed at: Department of Psychology,University of Tsukuba, Ibaraki Pref. 300-31, Japan. ** Japanese National Research Exchange Fellow. Present address: Department of Psychology, University of Hawaii, Honolulu, Hawaii, U.S.A. *** The untimely death of the third author, Dr. Shinkuro Iwahara, occurred while this work was in progress.
224 radish peroxidase techniques, have shown that the hippocampus receives a monosynaptic projection from the median raphe nucleusZ,4,5,14,is. The majority of the ascending projections from the raphe nuclei has been demonstrated by Conrad et al. 4 to run into the medial forebrain bundle and joining the diagonal band of Broca, pass through the septal nuclei or the cingulum bundle to finally reach the subiculum and the hippocampus. Electrophysiological and neurochemical data 1~, moreover, have indicated that electrical stimulation of the raphe nuclei and application of serotonin (5-HT) iontophoretically, exert a strong inhibitory influence upon the firing of hippocampal pyramidal cells. Since the raphe nuclei contain predominant numbers of 5-HT cell bodies, these findings, in addition to others 1°,17, support the view that serotonin might be an inhibitory neurotransmitter for a raphe-hippocampal neural pathway. Furthermore, Lindsley and co-workers 1,3,11,12 have found that high-frequency electrical stimulation of the raphe nucleus (nucleus raphe centralis superior) or the nucleus reticularis pontis caudalis produces a hippocampal desynchronization (lowvoltage fast activity). This desynchronizing system continues rostrally into the lateral hypothalamic area or medial forebrain bundle. Thus, this hippocampal desynchronizing system may correspond to the serotonin ascending pathway 2,4,5,14,18. In contrast, hippocampal synchronization (rhythmic theta activity) was induced by stimulating diffusely distributed regions of the midbrain and pontine tegmentum, including the nucleus reticularis pontis oralis, the nucleus locus coeruleus, the nuclei of giant cells in the pontine tegmental field and the periaqueductal grey substance of the midbrain 12. This synchronizing theta system ascends through the medial hypothalamic area (the dorsal fasciculus of Schutz). Although there is now little information concerning the interaction of the hippocampal synchronizing and desynchronizing systems, it was suggested that the amplitude of hippocampal theta activity can be enhanced by the destruction of the median raphe nucleus, a part of the desynchronizing system (Ueki, Kyushu University, personal communication, 1975). Furthermore, in our previous study tz, it was shown that the hippocampal theta activity, usually not observed during immobility in normal rats 23, appeared continuously during immobility following large brain stem lesions which included the median raphe nucleus. The following experiments were therefore undertaken to determine whether discrete lesions of the median raphe nucleus would induce the hippocampal theta activity during immobility and to examine the effects of the median raphe lesions on the frequency of hippocampal theta activity during voluntary movement and paradoxical sleep (PS). Furthermore, it has been suggested by Vanderwolf and his coworkers 9,24 that there are two types of hippocampal theta activity in rats and rabbits. Atropinesensitive theta activity occurs in rats during freezing and during the immobility that is produced by midbrain tegmentum stimulation or by certain drugs, i.e. ethyl ether, ethyl urethane, and physostigmine 9,2~,z4,26. In contrast, atropine-resistant theta activity occurs in association with 'voluntary movement 'z4,26 or during PS 9,2~,z4,26.
225 Thus the effect of atropine sulfate on hippocampal theta activity following median raphe lesions will be examined. MATERIALSAND METHODS
Animals Fifty-one male Wistar albino rats, weighing 250--390 g at the start of the experiment, were used. Animals were individually housed, and were given ad libitum access to both food and water.
General procedure Animals were given a 7-day recovery period following surgery and on the 8th day, all animals were tested for hippocampal EEGs. Only those animals which showed clear hippocampal theta activity during movement were used for the experiments. EEG recordings during various behaviors including PS were taken both before and after the brain stem lesion. All rats received either a median raphe or a unilateral midbrain reticular formation lesion. The animals which showed motor disturbances or abnormal postures with continuous muscle twitchings following these brain stem lesions were discarded. Immediately after the lesion, cortical and hippocampal EEGs were recorded during various behaviors except for PS and again after 24 h, EEG recordings during various behaviors including PS were taken in the same manner as in the pre-lesion recording session. In addition, 3 raphe-lesioned animals were observed at 3, 6, 11, and 20 days after the lesion.
Surgery, lesion and histology Animals were anesthetized with sodium pentobarbital (35 mg/kg, i.p.) and placed in a stereotaxic instrument. Bipolar electrodes were implanted in the dorsal hippocampus. A monopolar electrode was placed in either the median raphe nucleus or the reticular formation. Both hippocampal and brain stem electrodes consisted of glass-coated stainless steel wire 150/zm in diameter, exposed 0.5 mm at the tip. Implantation coordinates were followed according to the atlas of Pellegrino and Cushman 15. Coordinates used were: dorsal hippocampus: AP 3.3, ML 2.5, DV +2.0; median raphe nucleus: AP 0.0, ML 0.0, DV 4.5; midbrain reticular formation (MRF): AP 0.0, ML 2.0, DV 4.5 or--3.0. Cortical and reference electrodes consisted of miniature stainless steel screws 1 mm in diameter. The cortical electrode was placed over the sensory motor area. The reference electrode was attached to the nasal bones. Electromyographic (EMG) recordings were taken from stainless steel wires, 150 #m in diameter, chronically implanted in the neck muscles bilaterally. All lead wires were soldered to miniature connector pins and were fixed to the skull with screw anchors and dental cement.
226
MEDIAN RAPHE LESIONS
~
RETICULAR FORMATION LESIONS A0.6
PO.4
P1.2
O:RPH"39, O: RPH-25
~
:
: -"
'
~
O:RPH-75, O: RPH-77
Fig. 1. Coronal sections displaying the minimal (darkened area) and maximal damage (outlined in black) in the median raphe lesion and in the reticular formation lesion group. Four out of 21 median raphe animals had discrete (minimal) lesions similar to the lesion of RPH-39. The other animals had larger lesions which included the median raphe nucleus and surrounding tissue. There were considerable variations in the location and size of the reticular formation lesion. Atlas was taken from Pellegrino and CushmanlL Immediately after the pre-lesion recording session, electrolytic lesions were produced by passing a 1.5-2.0 mA DC current, 7-10 sec, through the brain stem electrode. The anode was inserted into the rectum. At the end of the experiment, all animals were overdosed with sodium pentobarbital and perfused with 10 ~ formalin solution. Their brains were then removed
227
AFTER MEDIAN RAPHE LESIONS
BEFORE MEDIAN RAPHE LESIONS
rat.~.-3a
=~. i-s.
DHPC
(blpJ
EMG I
1
I
1
I~- rearing
>1
DHPC (bip.)
EMG I
