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Kainic acid microinjected into the cat raphe dorsal nucleus modulates the somatosensory evoked potentials and their cycles of excitability
NeurofcienceVol. 22, No. 1, pp. 83-89, 1987 Printed in Great Britain
~306~522/87$3.00+ 0.00 Pergamon Journals Ltd Q 1987 IBRO
KAINIC ACID MICR~~NJECTED INTO THE CAT RAPHE DORSAL NUCLEUS MODULATES THE SOMATOSENSORY EVOKED POTENTIALS AND THEIR CYCLES OF EXCITABILITY S. MOYANOVA,*~ S. DIMOV* and A. IVANOVA~~ *Institute of Physiology, Bulgarian Academy of Sciences, Sofia; and ?fBrain Research Institute, Bulgarian Academy of Sciences, Sofia, Bulgaria
Ahatraet-Studies were made on the effect of the neuroexcitatory agent kainic acid, microinjected into raphe dorsal nucleus by glass micropipette and an air pressure system in doses ranging from 0.2 to 24.0 nmol (in volumes from 0.05 ~1 to 0.47 yl), on the somato~nso~ evoked potentials and their cycles of recovery (excitability) obtained from cortex (primary somatosensory and parietal associative), thalamus (ventral posterolateral nucleus and centre median nucleus), mesencephalic reticular formation and raphe dorsal nucleus. Kainic acid in doses higher than 3 nmol exerted an activating effect on the evoked potentials and their recovery cycles especially in thalamus and mesencephalic reticular formation. The analysis of these electrophysiological parameters revealed that the non-specific structures were involved to a larger extent in the activating effect of kainic acid than the specific ones. The morphological changes were not severe and were limited to a part of the raphe dorsal nuckus neurons. Our data indicate that kainic acid injected into raphe dorsal nucleus modulates (in direction of facilitation) the somatosensory evoked potentials and their cycles of excitability obtained in some brain structures. The results suggest that this nucleus is involved in the somatosensory information processing in a non-specific manner.
Much is now known about the raphe dorsal nucleus (NRD) but the functional role of this nucleus belonging to the ascending serotonergic (5-HT) system of the brain is unclear as yet. The raphe system has been suggested as participating in a variety of functions such as sleep, locomotor activity, aggression, sexuality, emotional reactivity, learning, memory, and so on. Among the various functions attributed to the raphe dorsal nucleus, its role in the somatosensory information processing has received little attention. It has been shown that NRD receives somatic sensory inputs which inhibit the majority of its neurons.2*33There are also contradictory data that NRD units respond to peripheral somesthetic stimulation with an increase in their discharge rate.30 In only a few studies were attempts made to investigate the effects of lesion and chemical or electrical stimulation of NRD on the sensory evoked potentials.3J7Jg So, the present knowledge about the participation of NRD in the modulation of the sensory transmission
and in the regulation of the brain excitability level is still incomplete and fragmentary. Intracerebral microinjections of the heterocyclic neuroexcitatory and toxic kainic acid (KA) has rapidly acquired a place as tools in neurobiology for anatomical, chemical, and physiological analysis of the central nervous system.13 The purpose of this study was to elucidate what happens with the sensory transmission through some brain structures after KA was microinjected into NRD. For the experiments described here we used the somatosensory evoked potential technique. The effect of KA on the evoked potentials obtained simultaneously in various brain structures and on the excitability level in the same brain regions estimated by the recovery (excitability) cycles technique was investigated.
EXPERIMENTAL PROCEDURES Animals and surgery
Eighteen adult male cats (3.0-3.5 kg) were used in all experiments. The surgery and implantation of the electrodes were made under ether anaesthesia. Following catheterization of the femoral vein and fixation of the animal in a stereotaxic frame, the cats were immobili~ with pavulon (Pancuronium bromide, Organon) and were artificiaily respirated through a tracheal cannula. The scalp was incised and the fascia overlying the cranium was cleared away. Skin margins were repeatedly infiltrated with 0.5% procaine throughout the experiment. The body temperature was
tTo whom correspondence should be addressed at: Institute of Physiology, Bulgarian Academy of Sciences, AC. G. Bonchev Str., bl.23, 1 I13 Sofia, Bulgaria. Abbr~ia#jo~: As Cx, associative cortex; CM, centre median nucleus; EEG, el~t~n~phalogram; EP, evoked potentiai; 5-HT, S-hydroxytryptamine (serotonin); KA, kainic acid; MRF, mesencephalic reticular formation; NRD, raphe dorsal nucleus; SEP, somatosensory evoked potential; Sm Cx, somatosensory cortex; VPL, ventral posterolateral nucleus. 83
S. M~VAN~VA
84
maintained at about 37°C with a heating blanket and the animals were kept in a normocapnic state. Small holes were drilled into the skull above the location of each proposed electrode site. Electrodes
and stimulation procedure
Surface recordings were led either from a screw fixed over the skull-parietal associative cortex (As Cx, gyrus suprasylvius medianus) or from an Ag-AgCI ball electrode placed epidurally and fixed with dental acrylic cement over the primary somatosensory cortex (Sm Cx, gyrus sigmoideus posterior). For deep recordings a block of three bipolar electrodes was placed unilaterally in the mesencephalic reticular formation-MRF (A2.0, L3.5, H - 2.0) ventral posterolateral nucleus-VPL (A9.0, L7.5, H + 1.0) and the centre median nucleus of the thalamus--CM (A7.0, L3.0, H + 2.0) according to the atlas of Snider and Niemer.‘j The raphe dorsal nucleus was approached through the cerebellum with a holder inclined caudally 30” behind the vertical to avoid the bony tentorium. Stereotaxic coordinates for NRD were determined using the atlas of Berman6 as follows: P0.2, LO.0, H - 0.5. The electrodes were concentric and consisted of stainless steel cannulae of 0.8 mm external diameter insulated except the tip for about 0.2 mm, into which an insulated wire was introduced. Two different types of stimulation were used: (i) single stimuli delivered sporadically by hand about one in every 2 s, and (ii) pairs of stimuli with interstimulus intervals of 40, 80, 120, 160, 240, 320 and 400 ms. The forepaw in the region of the radial nerve was electrically stimulated (square pulses of 0.2 ms duration) via bipolar needle electrodes inserted subcutaneously. An intensity I.5 times higher than the lowest stimulus intensity that could evoke responses in the structures studied, and established in every experiment, was maintained throughout the experiment, Injection procedure Kainic acid (Sigma) was microinjected into NRD after removing the recording electrode. The injection was made through a glass micropipette pulled beforehand by means of a vertical puller and introduced stereotaxically by a microdrive at the same point where the recording electrode was placed. Kainic acid was injected by means of an electropneumatic system which applied pressure air pulses to the back of the micropipette. The procedure has been described in detail previously.28 Briefly, KA was dissolved immediately before use in phosphate-buffered saline (PH 7.38) and 5% solution of methyl blue (Edward Gurr, Ltd.) in equal volumes. The dye was added for better visualization and measuring of volumes injected into immersion oil before the insertion of the micropipette into the brain. Varying the parameters of the pressure system (pressure, duration and number of pressure pulses) and the diameter of the micropipette tip, different for each animal, volumes of the KA solution were injected into NRD, from 0.05 to 0.47 nl which contained 0.2-24.0 nmol of KA. The animals were divided into three groups according to the dose of KA injected: group A (0.2-0.6nmol). five animals; group B (I .2-3.0 nmol), five animals; and group C (3. I-24.0 nmol), eight animals. Recording procedure A control registration was made for at least I h to be sure that a stable functional state was present without any signs of pain and discomfort. During this control period the electroencephalogram (EEG) was characterized by appearance of EEG spindles typical of the quiet waking state and/or drowsiness. The EEG and the evoked potentials (EPs) to radial nerve stimulation were amplified with a Reega Duplex EEG machine. The recorded output was fed into a Bell & Howell tape recorder and stored for subsequent analysis on a Nicolet-1024 computer. After the control registration of EEG and EPs to single and paired
et al
stimuli, an injection of KA into NRD was made. Then the macroelectrode was introduced into the place of the micropipette. The EEG and EP recordings-were made for a minimum of 3 h following the KA injection. The EPs were stored on the tape before the KA injection and at 15,60, 120 and 180 min after the injection. Hisrological procedure After termination of the recording procedures, the animals were perfused under deep thiopental anaesthesia, first with saline, then with 10% formaline. The brains were stored in 4% formal solution until sectioned. After fixation, the tissue containing the injection site was embedded in paraffin and was studied by light microscopy. Paraffin sections of 5 nrn thickness were stained by the Nissl method. The position of the deep macroelectrodes marked by small electrolytic lesions made before the perfusion was verified using a freezing microtome. Recordings with incorrect electrode localization were discarded. Data analysis The somatosensory evoked potentials (SEPs) from each brain region were averaged off-line by means of a Nicolet1024 computer and stored on digital magnetic tape. The EPs were analysed by measuring the amplitude and latency of the two main SEP components. The excitability cycles were estimated as a ratio between the amplitude of the averaged SEP to the second (test) stimulus of the pair-R, and the amplitude of the averaged SEP to the first (conditioning) stimulus-R,. The averaged potential to the test stimulus was obtained by means of the computer by subtraction of the averaged SEP to the conditioning stimulus from the averaged SEP to the paired stimuli. This was done for each interstimulus interval. The recovery cycles were calculated for the sum of the two main SEP components. Averaging of the SEP amplitude and latency to single stimulus and of the excitability cycles was made for all animals in each group, for each recording site and at various post-injection times after the KA injection. A paired Student’s f-test was used for statistical analysis.
RESULTS
Controls The intensities responses
in
different.
Using
of radial the
brain
nerve
stimulation
structures
a relatively
low intensity
evoking
studied
were
stimulus
it
evoked potentials only in VPL and Sm Cx. With higher intensities SEPs were recorded in the other responsive structures, too. The evoked responses in CM, MRF and As Cx had a similar threshold. In cats in which phosphate buffer or buffer with methyl blue was injected into NRD, the SEPs were rested in the limits of the physiological alterations up to 3 h of registration after the injection. Therefore, the results from KA-treated animals were compared to the pre-drug values. was possible
to observe
somatosensory
Effect of kainic acid microinjected
nucleus on the somatosensory
into the raphe dorsal
evoked potentials
The effect of KA on the SEPs was studied at 15, 60, 120 and 180 min after KA. In all animals of groups B and C, the spontaneous EEG activity after KA was characterized by the appearance of different types of EEG pathological patterns.28 The evoked
85
NRD kainic acid injections and brain excitability
CM % 200 15mm 100
% 200 Mmin
Mmin 100
100
0 %
%
% 200
200 VOmin
1ZOmin
TOO
100
100
0
0
0
% 200
%
H
200
200
IeDmin
K
l@JllWT
100
0
100
t k
100
0 ABC
A
6 2-3
1-2
a
C
0 A
0
A
C
0
ABC
C
1-Z
2-3
l-2
2-3
Fig. 2. The same as in Fig. 1 for CM (a) and VPL (b). higher at 60 min and this increase was accompanied
by a decrease in the latency. In VPL (Fig. 2b) and in Sm Cx (Fig. 3a), a considerable facilitation (about 2-fold) only of the second SEP component began at Sm Cx
potentials obtained accidentally during such high voltage single abnormal EEG patterns in some of the
brain structures studied were excluded from the analysis by preliminary expectation of the records. When seizure episodes occurred, especially at higher doses of KA, such animals were rejected from the analysis only at the time perioids when seizure activity appeared. In group A, there were no significant changes in the SEP amplitude and latency in any of the structures investigated. Such was the case at 15 min for all groups of animals. Only in NRD (Fig. la) was there an increase of the amplitude and a decrease of the latency of the two SEP components significant for the second SEP component in group C. At 60 min these changes were not significant, but later they became pronounced again. In MRF (Fig. 1b) after 60 min there was a significant increase in the amplitude of the SEP second component obtained in groups B and C and this amplitude increase was accompanied by a decrease in the latency. The SEP amplitude increase in group C at 180min was 2.4 times the pre-drug value. The amplitude of the two SEP components recorded in CM (Fig. 2a) in groups B and C became
2-3
b
a
b
Fig. 1.Changes in SEPs obtained in NRD (a) and MRF (b) at 15,60, 120 and 180 min after KA was injected into NRD. The amplitude (open bars) and the latency (batched bars) of two components of SEP were measured (l-2 and 2-3, see the SEPs depicted on the top). (AC) Three groups of animals injected with different doses of KA: (A) 0.24.6nmo1, (B) 1.2-3.0nmo1, and (C) 3.1-24.0nmol. Ordinate: changes (%) in the SEP amplitude and latency as compared to the predrug values taken as 100% (dashed horizontal line). lP < 0.05.
ABC
1-2
As
cx
100
100
0
0
%
H
zoo Wmin 100
200 12Omin 100
0
%
l
200 1Mmin 400
A
B q-2
A
C
a
B 2-3
C
A
B
A
C
l-2
B
C
2-3
b
Fig. 3. The same as in Fig. 1 for Sm Cx (a) and As Cx (b).
86
S. MO~AN~VA
VPL
R2/R,% ! xa
et al.
As Cx
R,, % 5 2m
5-n Cx
R,, oh % I
Fig. 4. Effect of KA injected into NRD on the SEP excitability cycles obtained in six brain structures at 15, 60, 120 and 180min after KA injection. (Group C) Before KA. Ordinate: ratio of R, to R, (%), where R, is the total amplitude of the two components of SEP (peak-to-peak) to the first and R, to the second stimulus of the pair. The interstimulus intervals (ms) are depicted on the third axis. The averaged excitability cycles (eight animals injected with KA doses of 3. I-24.0 nmol) before KA and at the corresponding post-injection times were compared. *P -c 0.05.
for groups B and C in VPL and for group C in Sm Cx. SEPs recorded from As Cx (Fig. 3b) changed in the same manner as those in CM and MRF. 60 min, significant
Effect of kainic acid injected into the raphe dorsal nucleus on the somatosensory evoked potential excitability cycles
In Fig. 4 the SEP excitability cycles in the brain structures studied are presented as a function of time after KA injection. The data concern group C because significant modification of the SEP excitability cycles occurred only at KA doses higher than 3 nmol. In NRD there was a facilitation of the recovery of the SEPs, significant for the interstimulus intervals above 160 ms at 15 min after KA. Later this facilitation disappeared and at 180 min the increased recovery of the SEPs occurred again. In MRF there was an increase of the SEP to the second stimulus of the pair (R2) above the SEP to the first stimulus (R, ) for the interstimulus intervals more than 160ms, which was significant after 120 min. The excitability cycles of the SEPs recorded in CM were facilitated even to a larger extent than in MRF. The SEP recovery for the shorter than 120 ms intervals was not changed up to 180 min. In VPL, a significant facilitation of R2 above R, began at 60 min and continued up to 180min. Facilitation of the recovery for the shorter intervals, i.e. a shortening of the recovery was also observed in this structure. In the same direction as in CM and MRF were the changes in the excitability cycles of the SEPs obtained in As Cx. In Sm
Cx, the cycles were facilitated only at 180min after the KA injection. Morphological changes
Light-microscopic examination of the NRD cells revealed histopathological changes only in three of the animals injected with KA doses higher than 3 nmol (Fig. 5). Some neurons showed different stages of chromatolysis, e.g. displacement of the Nissl substance. No clear relationship was found between the KA doses ranging from 3 to 24 nmol and the degree of the histological alterations in the region of NRD. Even the highest doses of KA failed to degenerate the NRD cells. DlSCUSSlON
The present results indicate that KA injected into the raphe dorsal nucleus alters the somatosensory transmission via the mesencephalic reticular formation, thalamus and cortex. Anatomical and biochemical studies have shown that the raphe dorsal nucleus in cats is directly connected with the sensorimotor and associative parietal cortical areas, with the non-specific intralaminar and midline nuclei of thalamus, with striatum and with the mesencephalic reticular formation.5.7,9,3b This study showed that KA injected into NRD changed the somatosensory evoked potentials in the two main central levels of the somatosensory system, i.e. in the relay thalamic nucleus (ventral posterolateral) and in the primary focus of its cortical
NRD kainic acid injections and brain excitability
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Fig. 5. Raphe dorsal nucleus, 4 h after KA (15.5 nmol) injection. No severe destruction of the tissue was Along with well preserved neurons, few nerve cells showed a diminution of basophil granules in their perikaryon. Paraffiin section (5 pm), Cresyl Violet stain. x 252.
observed.
projection. In addition, the SEPs changed in the mesencephalic reticular formation, in the non-specific thalamic nucleus (centre median) and in the associative parietal area of the cortex. The results on changes in SEPs recorded in VPL should be interpreted with caution because of the lack of anatomical data about a direct pathway from NRD to VPL.” Stimulation of NRD has been reported to be without effect on VPL neurons.34 However, our results suggest modulation influence of NRD on the processing of the somatosensory information in VPL. The possibility that some other structure or structures dispatch NRD messages to VPL cannot be excluded. The second point to be discussed is the direction of these alterations in the evoked potentials and their recovery cycles. The increase of the SEP amplitude with a concomitant decrease in its latency is an expression of enhanced reactivity of the neuronal elements of the structure the SEPs are registered from. The finding that in the non-specific structures such as MRF, CM and As Cx, the two components of the SEPs were increased, while in the somatosensory specific structures such as VPL and Sm Cx,
the late negative component was involved to a larger extent in this facilitation than the earlier positive component, might be interpreted as a result of a prevailing participation of the non-specific structures as compared to the specific ones in this activating effect of KA microinjected into NRD. The increased excitability level in some of the structures investigated estimated by the excitability cycles of the SEPs after KA was injected into NRD suggests that KA, being a neuroexcitatory agent,14 triggers neuronal circuits of increased excitability, especially those of the thalamus and mesencephalic reticular formation. Kainic acid when injected into various brain structures, particularly those of the limbic system, provokes focal and distal epileptiform discharges which might become independent of the primary focus.4,‘7.37 The mechanism of this spread of the paroxysmal activity is not yet established, although it is considered to be an electrophysiological and neuropathological phenomenon rather than a direct spread of the effect of KA.37 In our experiments, epileptiform spontaneous discharges and even an epileptic state
88
S. MOYANOVA et al.
provoked by intraraphedorsal kainic acid. This is described in detail in our previous paper.” A great deal of research has shown that the sensory responsiveness of the brain is increased during seizures in many convulsive disorders or following various convulsant drugs given intravenously.23 A neuronal response enhancement during seizures has been observed to a larger extent in certain non-primary sensory nuclei and in the brain stem reticular formation as compared to other brain structures such as the corpus geniculatum laterale.‘5.‘6 Our results might be compared to these data although the drugs employed and the route of drug administration are different. We failed to find any paper dealing with electrophysiological effects of KA injected into NRD. Relevant to our findings are the data about L-glutamate injections into NRD, as KA is a rigid structural analogue of this amino acid.24 The administration of L-glutamate in the raphe dorsal nucleus accelerates the firing rate of the 5-HT neurons”* and stimulates the release of [3H]5-HT in the caudate nucleus.20 The increased 5-HT release from the terminals is an expression of the activation of the serotonergic system of NRD and is concomitant with the state of wakefulness when the discharge rate of the putative serotonergic cells of NRD is much higher as compared to that in sleep. ‘“,26,32,3* Arousal or activating effects have been observed following activation of the serotonergic system of NRD by high-frequency electrical stimulation.“~‘8.27 All these data are inconsistent with the hypothesis that serotonin release initiates and/or maintains slow-wave sleep.2’ According to a recently published hypothesis,22 serotonin cannot be considered a hypnogenic neurotransmitter. Rather it might be a transmitter released during wakefulness and probably necessary for the biosynthesis of some other factor responsible for the slow-wave sleep. were
Thus, this new hypothesis22 rejects the serotonergic theory of sleep.*’ In the present study, KA did not show an appreciable neurotoxic effect on the neurons of the raphe dorsal nucleus. Reviewing the enormous literature on the effects of KA injected into various brain structures, a conclusion could be drawn that the sensitivity of the different cell groups or systems to the KA toxicity varies within very large limits. There even exist brain structures which are totally resistant to KA toxic action, such as locus coeruleus, and certain hypothalamic and mesencephalic nuclei.‘2z25 If the NRD neurons are not destroyed by KA 3 h after the injection, they will be expected to be excited by this time. The increase of the amplitude with a concomitant decrease in the latency of the SEPs obtained at 15 min after injection of KA in NRD and later on, up to 180 min, as well as their facilitated cycles of excitability, showed that the neuronal elements of NRD were probably excited by KA. The excitatory effect of KA on neurons in many brain structures is well established.‘4,‘9 Our results on increased excitability level in the mesencephahc reticular formation, thalamus and cortex as a result of the probable excitation of the NRD cells by KA, support the hypothesis of Cespuglio et al.” that the functions of NRD are closer to those of the ascending activating system than to those of the hypnogenic bulbar structures. However, the mechanism of this increased excitability level is still unclear. No matter how our results may be interpreted, two main conclusions emerge: first, the raphe dorsal nucleus is probably involved in the processing of the somatosensory information in a nonspecific manner, and second, KA microinjected into this nucleus increases the excitability level in the mesencephalic reticular formation, thalamus and cortex.
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~306~522/87$3.00+ 0.00 Pergamon Journals Ltd Q 1987 IBRO
KAINIC ACID MICR~~NJECTED INTO THE CAT RAPHE DORSAL NUCLEUS MODULATES THE SOMATOSENSORY EVOKED POTENTIALS AND THEIR CYCLES OF EXCITABILITY S. MOYANOVA,*~ S. DIMOV* and A. IVANOVA~~ *Institute of Physiology, Bulgarian Academy of Sciences, Sofia; and ?fBrain Research Institute, Bulgarian Academy of Sciences, Sofia, Bulgaria
Ahatraet-Studies were made on the effect of the neuroexcitatory agent kainic acid, microinjected into raphe dorsal nucleus by glass micropipette and an air pressure system in doses ranging from 0.2 to 24.0 nmol (in volumes from 0.05 ~1 to 0.47 yl), on the somato~nso~ evoked potentials and their cycles of recovery (excitability) obtained from cortex (primary somatosensory and parietal associative), thalamus (ventral posterolateral nucleus and centre median nucleus), mesencephalic reticular formation and raphe dorsal nucleus. Kainic acid in doses higher than 3 nmol exerted an activating effect on the evoked potentials and their recovery cycles especially in thalamus and mesencephalic reticular formation. The analysis of these electrophysiological parameters revealed that the non-specific structures were involved to a larger extent in the activating effect of kainic acid than the specific ones. The morphological changes were not severe and were limited to a part of the raphe dorsal nuckus neurons. Our data indicate that kainic acid injected into raphe dorsal nucleus modulates (in direction of facilitation) the somatosensory evoked potentials and their cycles of excitability obtained in some brain structures. The results suggest that this nucleus is involved in the somatosensory information processing in a non-specific manner.
Much is now known about the raphe dorsal nucleus (NRD) but the functional role of this nucleus belonging to the ascending serotonergic (5-HT) system of the brain is unclear as yet. The raphe system has been suggested as participating in a variety of functions such as sleep, locomotor activity, aggression, sexuality, emotional reactivity, learning, memory, and so on. Among the various functions attributed to the raphe dorsal nucleus, its role in the somatosensory information processing has received little attention. It has been shown that NRD receives somatic sensory inputs which inhibit the majority of its neurons.2*33There are also contradictory data that NRD units respond to peripheral somesthetic stimulation with an increase in their discharge rate.30 In only a few studies were attempts made to investigate the effects of lesion and chemical or electrical stimulation of NRD on the sensory evoked potentials.3J7Jg So, the present knowledge about the participation of NRD in the modulation of the sensory transmission
and in the regulation of the brain excitability level is still incomplete and fragmentary. Intracerebral microinjections of the heterocyclic neuroexcitatory and toxic kainic acid (KA) has rapidly acquired a place as tools in neurobiology for anatomical, chemical, and physiological analysis of the central nervous system.13 The purpose of this study was to elucidate what happens with the sensory transmission through some brain structures after KA was microinjected into NRD. For the experiments described here we used the somatosensory evoked potential technique. The effect of KA on the evoked potentials obtained simultaneously in various brain structures and on the excitability level in the same brain regions estimated by the recovery (excitability) cycles technique was investigated.
EXPERIMENTAL PROCEDURES Animals and surgery
Eighteen adult male cats (3.0-3.5 kg) were used in all experiments. The surgery and implantation of the electrodes were made under ether anaesthesia. Following catheterization of the femoral vein and fixation of the animal in a stereotaxic frame, the cats were immobili~ with pavulon (Pancuronium bromide, Organon) and were artificiaily respirated through a tracheal cannula. The scalp was incised and the fascia overlying the cranium was cleared away. Skin margins were repeatedly infiltrated with 0.5% procaine throughout the experiment. The body temperature was
tTo whom correspondence should be addressed at: Institute of Physiology, Bulgarian Academy of Sciences, AC. G. Bonchev Str., bl.23, 1 I13 Sofia, Bulgaria. Abbr~ia#jo~: As Cx, associative cortex; CM, centre median nucleus; EEG, el~t~n~phalogram; EP, evoked potentiai; 5-HT, S-hydroxytryptamine (serotonin); KA, kainic acid; MRF, mesencephalic reticular formation; NRD, raphe dorsal nucleus; SEP, somatosensory evoked potential; Sm Cx, somatosensory cortex; VPL, ventral posterolateral nucleus. 83
S. M~VAN~VA
84
maintained at about 37°C with a heating blanket and the animals were kept in a normocapnic state. Small holes were drilled into the skull above the location of each proposed electrode site. Electrodes
and stimulation procedure
Surface recordings were led either from a screw fixed over the skull-parietal associative cortex (As Cx, gyrus suprasylvius medianus) or from an Ag-AgCI ball electrode placed epidurally and fixed with dental acrylic cement over the primary somatosensory cortex (Sm Cx, gyrus sigmoideus posterior). For deep recordings a block of three bipolar electrodes was placed unilaterally in the mesencephalic reticular formation-MRF (A2.0, L3.5, H - 2.0) ventral posterolateral nucleus-VPL (A9.0, L7.5, H + 1.0) and the centre median nucleus of the thalamus--CM (A7.0, L3.0, H + 2.0) according to the atlas of Snider and Niemer.‘j The raphe dorsal nucleus was approached through the cerebellum with a holder inclined caudally 30” behind the vertical to avoid the bony tentorium. Stereotaxic coordinates for NRD were determined using the atlas of Berman6 as follows: P0.2, LO.0, H - 0.5. The electrodes were concentric and consisted of stainless steel cannulae of 0.8 mm external diameter insulated except the tip for about 0.2 mm, into which an insulated wire was introduced. Two different types of stimulation were used: (i) single stimuli delivered sporadically by hand about one in every 2 s, and (ii) pairs of stimuli with interstimulus intervals of 40, 80, 120, 160, 240, 320 and 400 ms. The forepaw in the region of the radial nerve was electrically stimulated (square pulses of 0.2 ms duration) via bipolar needle electrodes inserted subcutaneously. An intensity I.5 times higher than the lowest stimulus intensity that could evoke responses in the structures studied, and established in every experiment, was maintained throughout the experiment, Injection procedure Kainic acid (Sigma) was microinjected into NRD after removing the recording electrode. The injection was made through a glass micropipette pulled beforehand by means of a vertical puller and introduced stereotaxically by a microdrive at the same point where the recording electrode was placed. Kainic acid was injected by means of an electropneumatic system which applied pressure air pulses to the back of the micropipette. The procedure has been described in detail previously.28 Briefly, KA was dissolved immediately before use in phosphate-buffered saline (PH 7.38) and 5% solution of methyl blue (Edward Gurr, Ltd.) in equal volumes. The dye was added for better visualization and measuring of volumes injected into immersion oil before the insertion of the micropipette into the brain. Varying the parameters of the pressure system (pressure, duration and number of pressure pulses) and the diameter of the micropipette tip, different for each animal, volumes of the KA solution were injected into NRD, from 0.05 to 0.47 nl which contained 0.2-24.0 nmol of KA. The animals were divided into three groups according to the dose of KA injected: group A (0.2-0.6nmol). five animals; group B (I .2-3.0 nmol), five animals; and group C (3. I-24.0 nmol), eight animals. Recording procedure A control registration was made for at least I h to be sure that a stable functional state was present without any signs of pain and discomfort. During this control period the electroencephalogram (EEG) was characterized by appearance of EEG spindles typical of the quiet waking state and/or drowsiness. The EEG and the evoked potentials (EPs) to radial nerve stimulation were amplified with a Reega Duplex EEG machine. The recorded output was fed into a Bell & Howell tape recorder and stored for subsequent analysis on a Nicolet-1024 computer. After the control registration of EEG and EPs to single and paired
et al
stimuli, an injection of KA into NRD was made. Then the macroelectrode was introduced into the place of the micropipette. The EEG and EP recordings-were made for a minimum of 3 h following the KA injection. The EPs were stored on the tape before the KA injection and at 15,60, 120 and 180 min after the injection. Hisrological procedure After termination of the recording procedures, the animals were perfused under deep thiopental anaesthesia, first with saline, then with 10% formaline. The brains were stored in 4% formal solution until sectioned. After fixation, the tissue containing the injection site was embedded in paraffin and was studied by light microscopy. Paraffin sections of 5 nrn thickness were stained by the Nissl method. The position of the deep macroelectrodes marked by small electrolytic lesions made before the perfusion was verified using a freezing microtome. Recordings with incorrect electrode localization were discarded. Data analysis The somatosensory evoked potentials (SEPs) from each brain region were averaged off-line by means of a Nicolet1024 computer and stored on digital magnetic tape. The EPs were analysed by measuring the amplitude and latency of the two main SEP components. The excitability cycles were estimated as a ratio between the amplitude of the averaged SEP to the second (test) stimulus of the pair-R, and the amplitude of the averaged SEP to the first (conditioning) stimulus-R,. The averaged potential to the test stimulus was obtained by means of the computer by subtraction of the averaged SEP to the conditioning stimulus from the averaged SEP to the paired stimuli. This was done for each interstimulus interval. The recovery cycles were calculated for the sum of the two main SEP components. Averaging of the SEP amplitude and latency to single stimulus and of the excitability cycles was made for all animals in each group, for each recording site and at various post-injection times after the KA injection. A paired Student’s f-test was used for statistical analysis.
RESULTS
Controls The intensities responses
in
different.
Using
of radial the
brain
nerve
stimulation
structures
a relatively
low intensity
evoking
studied
were
stimulus
it
evoked potentials only in VPL and Sm Cx. With higher intensities SEPs were recorded in the other responsive structures, too. The evoked responses in CM, MRF and As Cx had a similar threshold. In cats in which phosphate buffer or buffer with methyl blue was injected into NRD, the SEPs were rested in the limits of the physiological alterations up to 3 h of registration after the injection. Therefore, the results from KA-treated animals were compared to the pre-drug values. was possible
to observe
somatosensory
Effect of kainic acid microinjected
nucleus on the somatosensory
into the raphe dorsal
evoked potentials
The effect of KA on the SEPs was studied at 15, 60, 120 and 180 min after KA. In all animals of groups B and C, the spontaneous EEG activity after KA was characterized by the appearance of different types of EEG pathological patterns.28 The evoked
85
NRD kainic acid injections and brain excitability
CM % 200 15mm 100
% 200 Mmin
Mmin 100
100
0 %
%
% 200
200 VOmin
1ZOmin
TOO
100
100
0
0
0
% 200
%
H
200
200
IeDmin
K
l@JllWT
100
0
100
t k
100
0 ABC
A
6 2-3
1-2
a
C
0 A
0
A
C
0
ABC
C
1-Z
2-3
l-2
2-3
Fig. 2. The same as in Fig. 1 for CM (a) and VPL (b). higher at 60 min and this increase was accompanied
by a decrease in the latency. In VPL (Fig. 2b) and in Sm Cx (Fig. 3a), a considerable facilitation (about 2-fold) only of the second SEP component began at Sm Cx
potentials obtained accidentally during such high voltage single abnormal EEG patterns in some of the
brain structures studied were excluded from the analysis by preliminary expectation of the records. When seizure episodes occurred, especially at higher doses of KA, such animals were rejected from the analysis only at the time perioids when seizure activity appeared. In group A, there were no significant changes in the SEP amplitude and latency in any of the structures investigated. Such was the case at 15 min for all groups of animals. Only in NRD (Fig. la) was there an increase of the amplitude and a decrease of the latency of the two SEP components significant for the second SEP component in group C. At 60 min these changes were not significant, but later they became pronounced again. In MRF (Fig. 1b) after 60 min there was a significant increase in the amplitude of the SEP second component obtained in groups B and C and this amplitude increase was accompanied by a decrease in the latency. The SEP amplitude increase in group C at 180min was 2.4 times the pre-drug value. The amplitude of the two SEP components recorded in CM (Fig. 2a) in groups B and C became
2-3
b
a
b
Fig. 1.Changes in SEPs obtained in NRD (a) and MRF (b) at 15,60, 120 and 180 min after KA was injected into NRD. The amplitude (open bars) and the latency (batched bars) of two components of SEP were measured (l-2 and 2-3, see the SEPs depicted on the top). (AC) Three groups of animals injected with different doses of KA: (A) 0.24.6nmo1, (B) 1.2-3.0nmo1, and (C) 3.1-24.0nmol. Ordinate: changes (%) in the SEP amplitude and latency as compared to the predrug values taken as 100% (dashed horizontal line). lP < 0.05.
ABC
1-2
As
cx
100
100
0
0
%
H
zoo Wmin 100
200 12Omin 100
0
%
l
200 1Mmin 400
A
B q-2
A
C
a
B 2-3
C
A
B
A
C
l-2
B
C
2-3
b
Fig. 3. The same as in Fig. 1 for Sm Cx (a) and As Cx (b).
86
S. MO~AN~VA
VPL
R2/R,% ! xa
et al.
As Cx
R,, % 5 2m
5-n Cx
R,, oh % I
Fig. 4. Effect of KA injected into NRD on the SEP excitability cycles obtained in six brain structures at 15, 60, 120 and 180min after KA injection. (Group C) Before KA. Ordinate: ratio of R, to R, (%), where R, is the total amplitude of the two components of SEP (peak-to-peak) to the first and R, to the second stimulus of the pair. The interstimulus intervals (ms) are depicted on the third axis. The averaged excitability cycles (eight animals injected with KA doses of 3. I-24.0 nmol) before KA and at the corresponding post-injection times were compared. *P -c 0.05.
for groups B and C in VPL and for group C in Sm Cx. SEPs recorded from As Cx (Fig. 3b) changed in the same manner as those in CM and MRF. 60 min, significant
Effect of kainic acid injected into the raphe dorsal nucleus on the somatosensory evoked potential excitability cycles
In Fig. 4 the SEP excitability cycles in the brain structures studied are presented as a function of time after KA injection. The data concern group C because significant modification of the SEP excitability cycles occurred only at KA doses higher than 3 nmol. In NRD there was a facilitation of the recovery of the SEPs, significant for the interstimulus intervals above 160 ms at 15 min after KA. Later this facilitation disappeared and at 180 min the increased recovery of the SEPs occurred again. In MRF there was an increase of the SEP to the second stimulus of the pair (R2) above the SEP to the first stimulus (R, ) for the interstimulus intervals more than 160ms, which was significant after 120 min. The excitability cycles of the SEPs recorded in CM were facilitated even to a larger extent than in MRF. The SEP recovery for the shorter than 120 ms intervals was not changed up to 180 min. In VPL, a significant facilitation of R2 above R, began at 60 min and continued up to 180min. Facilitation of the recovery for the shorter intervals, i.e. a shortening of the recovery was also observed in this structure. In the same direction as in CM and MRF were the changes in the excitability cycles of the SEPs obtained in As Cx. In Sm
Cx, the cycles were facilitated only at 180min after the KA injection. Morphological changes
Light-microscopic examination of the NRD cells revealed histopathological changes only in three of the animals injected with KA doses higher than 3 nmol (Fig. 5). Some neurons showed different stages of chromatolysis, e.g. displacement of the Nissl substance. No clear relationship was found between the KA doses ranging from 3 to 24 nmol and the degree of the histological alterations in the region of NRD. Even the highest doses of KA failed to degenerate the NRD cells. DlSCUSSlON
The present results indicate that KA injected into the raphe dorsal nucleus alters the somatosensory transmission via the mesencephalic reticular formation, thalamus and cortex. Anatomical and biochemical studies have shown that the raphe dorsal nucleus in cats is directly connected with the sensorimotor and associative parietal cortical areas, with the non-specific intralaminar and midline nuclei of thalamus, with striatum and with the mesencephalic reticular formation.5.7,9,3b This study showed that KA injected into NRD changed the somatosensory evoked potentials in the two main central levels of the somatosensory system, i.e. in the relay thalamic nucleus (ventral posterolateral) and in the primary focus of its cortical
NRD kainic acid injections and brain excitability
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Fig. 5. Raphe dorsal nucleus, 4 h after KA (15.5 nmol) injection. No severe destruction of the tissue was Along with well preserved neurons, few nerve cells showed a diminution of basophil granules in their perikaryon. Paraffiin section (5 pm), Cresyl Violet stain. x 252.
observed.
projection. In addition, the SEPs changed in the mesencephalic reticular formation, in the non-specific thalamic nucleus (centre median) and in the associative parietal area of the cortex. The results on changes in SEPs recorded in VPL should be interpreted with caution because of the lack of anatomical data about a direct pathway from NRD to VPL.” Stimulation of NRD has been reported to be without effect on VPL neurons.34 However, our results suggest modulation influence of NRD on the processing of the somatosensory information in VPL. The possibility that some other structure or structures dispatch NRD messages to VPL cannot be excluded. The second point to be discussed is the direction of these alterations in the evoked potentials and their recovery cycles. The increase of the SEP amplitude with a concomitant decrease in its latency is an expression of enhanced reactivity of the neuronal elements of the structure the SEPs are registered from. The finding that in the non-specific structures such as MRF, CM and As Cx, the two components of the SEPs were increased, while in the somatosensory specific structures such as VPL and Sm Cx,
the late negative component was involved to a larger extent in this facilitation than the earlier positive component, might be interpreted as a result of a prevailing participation of the non-specific structures as compared to the specific ones in this activating effect of KA microinjected into NRD. The increased excitability level in some of the structures investigated estimated by the excitability cycles of the SEPs after KA was injected into NRD suggests that KA, being a neuroexcitatory agent,14 triggers neuronal circuits of increased excitability, especially those of the thalamus and mesencephalic reticular formation. Kainic acid when injected into various brain structures, particularly those of the limbic system, provokes focal and distal epileptiform discharges which might become independent of the primary focus.4,‘7.37 The mechanism of this spread of the paroxysmal activity is not yet established, although it is considered to be an electrophysiological and neuropathological phenomenon rather than a direct spread of the effect of KA.37 In our experiments, epileptiform spontaneous discharges and even an epileptic state
88
S. MOYANOVA et al.
provoked by intraraphedorsal kainic acid. This is described in detail in our previous paper.” A great deal of research has shown that the sensory responsiveness of the brain is increased during seizures in many convulsive disorders or following various convulsant drugs given intravenously.23 A neuronal response enhancement during seizures has been observed to a larger extent in certain non-primary sensory nuclei and in the brain stem reticular formation as compared to other brain structures such as the corpus geniculatum laterale.‘5.‘6 Our results might be compared to these data although the drugs employed and the route of drug administration are different. We failed to find any paper dealing with electrophysiological effects of KA injected into NRD. Relevant to our findings are the data about L-glutamate injections into NRD, as KA is a rigid structural analogue of this amino acid.24 The administration of L-glutamate in the raphe dorsal nucleus accelerates the firing rate of the 5-HT neurons”* and stimulates the release of [3H]5-HT in the caudate nucleus.20 The increased 5-HT release from the terminals is an expression of the activation of the serotonergic system of NRD and is concomitant with the state of wakefulness when the discharge rate of the putative serotonergic cells of NRD is much higher as compared to that in sleep. ‘“,26,32,3* Arousal or activating effects have been observed following activation of the serotonergic system of NRD by high-frequency electrical stimulation.“~‘8.27 All these data are inconsistent with the hypothesis that serotonin release initiates and/or maintains slow-wave sleep.2’ According to a recently published hypothesis,22 serotonin cannot be considered a hypnogenic neurotransmitter. Rather it might be a transmitter released during wakefulness and probably necessary for the biosynthesis of some other factor responsible for the slow-wave sleep. were
Thus, this new hypothesis22 rejects the serotonergic theory of sleep.*’ In the present study, KA did not show an appreciable neurotoxic effect on the neurons of the raphe dorsal nucleus. Reviewing the enormous literature on the effects of KA injected into various brain structures, a conclusion could be drawn that the sensitivity of the different cell groups or systems to the KA toxicity varies within very large limits. There even exist brain structures which are totally resistant to KA toxic action, such as locus coeruleus, and certain hypothalamic and mesencephalic nuclei.‘2z25 If the NRD neurons are not destroyed by KA 3 h after the injection, they will be expected to be excited by this time. The increase of the amplitude with a concomitant decrease in the latency of the SEPs obtained at 15 min after injection of KA in NRD and later on, up to 180 min, as well as their facilitated cycles of excitability, showed that the neuronal elements of NRD were probably excited by KA. The excitatory effect of KA on neurons in many brain structures is well established.‘4,‘9 Our results on increased excitability level in the mesencephahc reticular formation, thalamus and cortex as a result of the probable excitation of the NRD cells by KA, support the hypothesis of Cespuglio et al.” that the functions of NRD are closer to those of the ascending activating system than to those of the hypnogenic bulbar structures. However, the mechanism of this increased excitability level is still unclear. No matter how our results may be interpreted, two main conclusions emerge: first, the raphe dorsal nucleus is probably involved in the processing of the somatosensory information in a nonspecific manner, and second, KA microinjected into this nucleus increases the excitability level in the mesencephalic reticular formation, thalamus and cortex.
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