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Activation of brain serotonin metabolism by heat: Role of midbrain raphe neurons
BRAIN RESEARCH
37
A C T I V A T I O N OF BRAIN S E R O T O N I N M E T A B O L I S M BY HEAT: ROLE OF M I D B R A I N R A P H E N E U R O N S
BRIAN L. WEISS* AND GEORGE K. AGHAJANIAN
Department of Psychiatry, Yale University School of Medicine, and Connecticut Mental Health Center, New Haven, Conn. 06519 (U.S.A.) (Accepted August 27th, 1970)
INTRODUCTION
An elevated ambient temperature induces an increase in brain serotonin (5hydroxytryptamine; 5-HT) turnover in the rat9, 2z. This increase in 5-HT turnover can be blocked entirely by parenterally administered D-lysergic acid diethylamide (LSD) 5. It had previously been shown that under basal environmental conditions (i.e., at room temperature) LSD produces an increase in the concentration of brain 5-HT 12 and a concomitant decrease in the principal 5-HT metabolite, 5-hydroxyindoleacetic acid (5-HIAA) 23. These various findings suggest that there is a decrease in the turnover of brain 5-HT after LSD and this has been directly demonstrated by studies employing isotopically labeled tryptophan TM. The mechanism by which LSD produces a decrease in 5-HT turnover is not known. However, it has been found that LSD inhibits the spontaneous firing of 5-HT containing neurons 2. Electrical stimulation of these neurons, most of which are situated in the midbrain raphe nuclei 10, causes an increase in 5-HT turnover, as indicated by the fact that there is a marked elevation in the concentration of 5-HIAA but only a small decrease in 5-HT concentration in both whole brain and forebrainZ, z4. Taken together, these observations suggest the possibility that elevated ambient temperatures could induce an increase in 5-HT turnover via an activation of the firing of 5-HT containing neurons. Furthermore, since LSD depresses the firing of neurons containing 5-HT, this action could account for the drug's ability to prevent the heat-induced increase in 5-HT turnover. In terms of this hypothesis, two different experimental procedures have been carried out. First, to evaluate the importance of the perikarya of 5-HT containing neurons in mediating the heat-induced increase in 5-HIAA, lesions were placed in the midbrain raphe. The raphe lesions were made 3 h prior to a period of incubation at an elevated ambient temperature; upon completion of the incubation period, the forebrain, which is the principal projection area of the midbrain raphe neurons 6, was * Present address: Department of Psychiatry, New York University Medical Center, New York, N.Y. 10016.
Brain Research, 26 (1971) 37-48
38
I3. k. WEISS AND G. K. AGIIAJANIAN
assayed for 5-H|AA and the midbrain was examined histologically to determine the precise location and extent of the lesion. Secondly, since body temperature is increased by incubation 5, we monitored the rate of firing of single units in the raphe nuclei while rats were being heated to see if an increase in body temperature is associated with an altered rate of firing of serotonin-containing neurons. Some animals were given LSD during this procedure. METHODS
Lesion experiments Sprague-Dawley (Charles-River, C.D.) male rats weighing from 200 to 300 g were used. The animals were separated into 3 major groups: (1) non-lesion animals: (2) raphe-lesion animals, in which the lesion destroyed a large portion of the midbrain raphe nuclei; and (3) non-raphe lesion animals, where the lesion spared most or all of the midbrain raphe nuclei. In the non-lesion rats no electrode was placed in the brain. Of these, one group of rats consisted of room temperature control animals that were decapitated in a guillotine device and their brains taken immediately after removal from the animal room. As a further control, some non-lesion rats were anesthetized with chloral hydrate (400 mg/kg) during the incubation procedures since this anesthesia was employed for the single unit recordings (see below). Another group of non-lesion animals received intraperitoneal injections of either LSD, 500 #g/kg as the bitartrate salt, or an equivalent volume of saline free of pyrogen, immediately prior to a 45 min period of incubation at 40°C. Upon completion of the incubation period all rats were decapitated and their brains removed. In the raphe-lesion groups, lesions were made which destroyed large areas of the midbrain raphe nuclei. After chloral hydrate anesthesia, as described above, the animals were placed in a stereotaxic instrument. An insulated 0.25 mm diameter steel electrode with an 0.5 mm exposed pointed tip was then lowered through a burr hole into the midbrain raphe. A 2.5 mA constant anodal current was passed for l0 sec through the electrode at depths from the skull surface of 6.5, 7.5, and 8.5 mm. Animals with sham lesions received no current, the electrode placement being otherwise identical with that in the experimental animals. The electrodes were removed immediately after the making of a lesion or a sham placement. After the 3 h recovery period, the rats (awake and in individual cages) were either placed in an incubator at 40°C for 45 min or allowed to remain at 23° C for 45 min. The rats were then decapitated and their brains removed for histology and for assay of indoles. Midline lesions, 2-3 mm in diameter, destroying the central portions of the areas ventral to the aqueduct (dorsal raphe nucleus) and ventral to the decussation of the superior cerebellar peduncle (median raphe nucleus), encompassing A620-AI60 (after K6nig and KlippeP6), were defined as 'total midbrain raphe lesions'. A group designated 'partial anterior raphe lesion' was composed of rats whose lesions destroyed only the extreme anterior portions (i.e., A620 and anterior) of both nuclei. Brain Research, 26 (1971) 37-48
SEROTONIN METABOLISMAND RAPHE NEURONS
39
The 'non-raphe lesion' groups consisted of those rats whose lesions were primarily outside the midbrain raphe nuclei. The lesions were made as described above, except for the placement of the electrode. A rat was classified in the group of 'pontine lesions' if the lesion was posterior to A160; in some cases such lesions were in the pontine raphe area. A group of 'reticular lesions' was comprised of those rats in which the midline remained intact and the medial edge of the lesion was at least 2 mm from the midline. The final subgroup was the 'median tegmentum' group, in which the lesions were midline but completely anterior to both nuclei, so that neither nucleus was damaged. All the non-raphe lesion rats were incubated for 45 rain at 40 ° C after 3 h had elapsed from the time of lesion placement. Following decapitation and removal of the brains from all rats, the brains were sectioned just anterior to the superior colliculus. The forebrains were frozen immediately and later assayed for 5-H|AA and 5-HT by a fluorimetric method 24 that incorporated modifications of the procedures of Bogdanski et al. 7, Udenfriend et al. ~7,
Fig. 1. Histologicalsection illustrating a typical total midbrain raphe lesion. The dorsal raphe nucleus and the median raphe nucleus have both been destroyed. Cresyl violet stain.
Brain Research, 26 (1971) 37-48
40
I~. L. WEISS A N D G . K. AGI-tAJANIAN
and Wise 28. The midbrains of animals with lesions were fixed in 5 ~,~glutaraldehyde (in 0.15 M sodium phosphate buffer, pH 7.4). Serial frozen sections of midbrain were cut and stained with cresyl violet to identify site of lesion. Fig. 1 illustrates histology of a typical lesion in which there is total destruction of the midbrain raphe nuclei.
Single unit experiments Rats used for the single unit recordings were anesthetized with chloral hydrate and mounted in a stereotaxic instrument. A tungsten microelectrode with a tip diameter of approximately 1 # m was then lowered with a hydraulic microdrive through a burr hole into various regions of the midbrain and pons. Electrode signals were passed into a high impedance amplifier and then displayed on an oscilloscope. The rate of firing of cells was followed with a triggered electronic counter whose analog output was plotted on a strip chart potentiometric recorder; the counter automatically reset to zero every 10 sec giving a histogram appearance to the records. Raphe units are characterized by their regular rhythm, slow spontaneous rate of firing (approximately 1 spike/sec), and inhibition by low doses of LSD 2,4, Units not in the raphe region usually fire at a faster rate and/or have an irregular rhythm. In addition, the non-raphe units do not respond to LSD with a cessation of activity. When a raphe or other unit was identified, a baseline of spontaneous activity was obtained over a span of several minutes. Body temperature was recorded by means of a colonic thermistor. The head and tail regions were shielded by an asbestos screen and a 150 W infrared lamp, placed 12 in. from the rat, was used to increase body temperature. In some animals LSD was administered intravenously through the tail vein during or after exposure of the rat to heat. After completion of the recording, rats were perfused with fixative and midbrains sectioned and stained for determination of site of electrode tip as previously described~k RESULTS
Lesion experiments As can be seen from Table I, there was a 37 ~,, increase (as compared to room temperature controls) in the concentration of 5-HIAA in the forebrains of incubated control animals kept at 40°C for 45 min. The finding of an increase in brain 5-HIAA concentration after incubation at an elevated ambient temperature is in accord with previous data 5,2°,2~. Table I shows that the increase in 5-HIAA is entirely blocked by the prior injection of 500 #g/kg of LSD; this result is in agreement with our earlier findings 5. A slight but significant depression of 5-HIAA concentration occurs in the animals pretreated with LSD (Table I). The heat-induced increase in 5-HIAA concentration is completely prevented by a total lesion of the dorsal and median midbrain raphe nuclei (Table I). In contrast, the incubated sham-lesion animals had a increase of over 56 ~ in 5-HIAA concentration. Some increase in 5-HIAA occurred even when sham-lesion animals were not
Brain Research, 26 (1971) 37-48
SEROTONIN METABOLISM AND RAPHE NEURONS
41
TABLE I EFFECT OF PRIOR LESIONS OR
LSD
PRETREATMENT ON C O N C E N T R A T I O N OF FOREBRAIN
5-HIAA
IN RATS
EXPOSED TO AN ELEVATED AMBIENT TEMPERATURE ( 4 0 ° C ) *
Group
Procedure
(n)
Ambient temp. (' C)
5-HIAA % Change P** (ng/g) ( ± S.E.M.)
Non-lesion
Control Control LSD Saline
(8) (14) (5) (7)
23 40 40 40
244 355 215 323
Raphe lesion
Total raphe Total raphe Partial raphe (anterior) Sham Sham
(14) (6)
40 23
253 i 267 ±
(9) (17) (4)
Pontine Tegmental Reticular
(7) (5) (4)
Non-raphe lesion
~ 9 ± 6 ± 7 ± 4
---37 --12 I 32
--: 0.001 -~0.05 < 0.001
6 6
I 4 -- 9
N.S. N.S.
40 40 23
282 :] 9 38l ~-_ 8 260 ± 22
i 16 F56 +48
• 0.01 < 0.001 < 0.001
40 40 40
322 ± 11 316 ± 15 357 ± 20
I 32 ~ 30 -46
0.001 ~ 0.001 -~. 0.001
* In all cases periods of exposure were 45 min. ** P values were determined using Student's t test. TABLE II EFFECT OF PRIOR TOTAL RAPHE LESIONS O N FOREBRAIN
5-HT
C O N C E N T R A T I O N AFTER EXPOSURE TO AN
AMBIENT TEMPERATURE OF 4 0 ° C *
Group
(n)
Ambient temp. ('C)
5-HT (ng/g) ( ± S.E.M.)
°o Change
P**
Control Control Sham Total raphe
(10) (5) (4) (5)
23 40 40 40
338 346 354 395
-÷ 2 i 5 + 17
-N.S. N.S. --~ 0.02
± 10 ~ 7 ± 5 ~ 17
* Period of incubation was 45 min. ** P values were determined using Student's t test. incubated. 5-HIAA
In unincubated
concentrations
animals with total lesions of the midbrain
raphe, the
d i d n o t differ s i g n i f i c a n t l y f r o m t h o s e t o t a l r a p h e
lesion
a n i m a l s k e p t a t 40°C f o r 45 m i n . L e s i o n s m a d e e l s e w h e r e in t h e m i d b r a i n (e.g. r e t i c u l a r f o r m a t i o n ) o r ports, s p a r i n g all o r m o s t o f t h e m i d b r a i n r a p h e nuclei, d i d n o t p r e v e n t t h e h e a t - i n d u c e d i n c r e a s e in 5 - H I A A ; 5 - H I A A v a l u e s i n t h e s e n o n - r a p h e l e s i o n a n i m a l s a r e n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h e i n c u b a t e d c o n t r o l a n i m a l s ( T a b l e l). T w o o f t h e 3 p o n t i n e l e s i o n s w e r e in t h e p o n t ± h e r a p h e a r e a b u t t h e 5 - H I A A levels in t h e s e i n s t a n c e s d i d n o t differ f r o m t h e o t h e r n o n - r a p h e l e s i o n a n i m a l s . Measurement of the concentration of 5-HT in the forebrains of incubated animals with total midbrain raphe lesions demonstrates that 5-HT values are not de-
Brain Research, 26 (1971) 37-48
g
I
b.a O~
.R"
...........
5 MIN
!
T
--36
--37
---38
m39
,--40
o
C
Fig. 2. Response of a raplae unit to an increase in body temperature. This unit has a spontancous rate of about 40 spikc~q-J~in~wio~ ~c, ~i~cappiica~,ion of heat (ON arrow). The transient decline in firing rate immediately following the application of heat has been noted in sexera~ other raphe ttnh recordings. A rise in body temperature is associated with an increase in firing rate; at a body temperature of 39.5' C, the firing rate is about 100 spikes/rain. Intravenous injection of LSD (arrow; dose 10/~g/kg) during the period of peak body temperature resulted in an inhibition of firing, The record consists of consecutive 10-see samples of the analog output of a counter triggered by the unit spikes,
I ¸
B.'L
> 7, >
>
"r
>
>
[]
t'-
4a. i,..a
I iOM N
I
~34
35
36
37
38 o C
39
40
Fig. 3. Record of response of a raphe unit to both rise and fall in body temperature (B.T.). At its maximum, the increase in firing rate is approximately two-fold. A drop in body temperature is associated with a decrease in rate of firing. LSD (arrow; dose 10 t~g/kg), administered intravenously while body temperature was falling, completely inhibited the firing of the unit.
gO
Z
¢"~l.J
B.I".
44
B. l.. WEISS AND G. K. AGtlAJANIAN
creased from control levels. It can be seen from Table 11 that the 5-HT concentration actually is somewhat elevated 3 h and 45 rain after a total lesion of the midbrain raphc nuclei. We observed behavioral changes similar to those noted by Kostowski et al. 17 il~ rats with lesions in the midbrain raphe (e.g. increased spontaneous motor activity. with aimless rotatory movements, and marked hypersensitivity to tactile and auditory stimuli). However, the rats with non-raphe lesions also exhibited such behavior. Single unit e x p e r i m e n t s
Eleven raphe units were studied using microelectrode techniques. Figs. 2 and 3 illustrate that the rate of firing of individual raphe neurons is closely correlated with changes in body temperature induced by heating from an infrared source. In all raphe units studied, it was found that as body temperature rose there was an acceleration in firing rate; conversely, a decline in body temperature was accompanied by a decrease in firing rate. The mean increase in the rate of firing for the I1 units was 2.7-fold (range 1.5-5.0) when body temperature rose from an average baseline of 35.3 C to a peak of 39.5 ° C. The latter body temperature is approximately the same as that seen after incubation at 40 ° C (ref. 5). Little or no change in firing rate occurred merely with the application or termination of the infrared radiation until there was a change in body temperature. Nine units located outside the raphe nuclei demonstrated no obvious correlation between firing rate and body temperature. These non-raphe units were located in either the reticular formation (6 units) or pontine area (3 units). Some non-raphe units responded to the application of heat by an increase in firing rate, while others showed a decrease or combination of effects. Thus a non-raphe unit might alternately accelerate and decelerate while body temperature continued to rise. In contrast to the raphe units, brief changes in the firing rate of the non-raphe units sometimes were correlated with the actual turning off of the heat lamp. The reported inhibitory effect of LSD on the activity of raphe units '~,4 was observed both during the rising (Fig. 2) and falling (Fig. 3) phases of the body temperature curve. LSD did not cause a cessation of activity in the non-raphe units. To control for possible biochemical effects of the anesthesia used during the single unit recordings, forebrain 5-HIAA was measured in rats given chloral hydrate (400 mg/kg)just prior to a 1 h period incubation at 40°C. In anesthetized rats maintained at 23 ° C for I h, the concentration of 5-HI AA in the forebrain was 317 ng/g (-I 5, S.E.M. ; n 6). This value represents a significant (P < 0.001) increase over unanesthetized control rats kept at 23 ° C (@ Table I). After 1 h of incubation at 4if' C, anesthetized rats had a further elevation of forebrain 5-HIAA to 401 ~: 8 ug/g (n 8). DISCUSSION Each of the two different experimental approaches employed suggests that the raphe neurons play a role in the increased turnover of 5-HT induced by an elevated ambient temperature. The first set of experiments, in which lesions were made in the Brain Research, 26 (1971) 37-48
SEROTONIN METABOLISM AND RAPHE NEURONS
45
midbrain raphe nuclei, shows that the heat-induced activation of 5-HT metabolism in the forebrain, as reflected by an increased 5-HIAA concentration, is dependent upon the structural integrity of the raphe nuclei. The second procedure (i.e., microelectrode recordings from individual raphe units) demonstrates that as body temperature increases there is an associated rise in the rate of firing of raphe cells. Both the requirement for structural integrity and the association between body temperature and firing rate are consistent with the hypothesis that the increase in 5-HT catabolism under conditions of increased ambient temperature is at least in part mediated by an increase in the firing rate of the 5-HT containing neurons in the midbrain raphe. An alternative explanation for the increased 5-HT turnover during heat exoosure would be that the synthesis and degradation of 5-HT is directly activated by a rise in body temperatures. Thus increased temperature might result in a direct activation of 5-HT metabolism in the 5-HT terminals within the forebrain. However, this possible mechanism would not by itself explain how acute midbrain raphe lesions could prevent such a direct activation in the forebrai_n. The lesion experiments further demonstrate some specificity of the requirement for destruction of midbrain raphe neuronal perikarya in order to prevent the heatinduced increase in 5-HT turnover. Incubated animals with lesions rostral, lateral, or posterior to the midbrain raphe nuclei all have elevated forebrain 5-HIAA concentrations. Midline lesions destroying only the extreme anterior portions of the raphe nuclei prevent some of the heat-induced rise in 5-HIAA. The probable explanation for the partial blocking effect on the increased turnover of some slightly anterior lesions is that, in addition to the destruction of the anterior portions of the raphe nuclei, 5-HT fiber tracts pro.jectir~g to the forebrain would be destroyed by lesions in this area G,la. Lateral and posterior lesions do not interfere with these projections. In the anterior midbrain these axonal projections to the forebrain deviate from the midline and join the medial forebrain bundle14; this divergence of the fibers from the midline explains the high-5-HIAA concentrations noted with very anterior midline lesions, since the projections course laterally to the lesion and the raphe nuclei are spared. Sham lesions not only fail to prevent the heat-induced increase in 5-H~AA concentration, but even significantly elevate 5-HT turnover above incubated control values. In fact, no incubation is necessary to produce an increased forebrain 5-HIAA concentration in the sham-lesion animals. This increase in turnover may be due to an irritative effect on the raphe neurons, but the actual mechanism has not been determined. The sham lesions provide further confirmation for the specific requirement of raphe neuron destruction to block the heat-related increase in 5-HT turnover. It is significant that the concentration of 5-HT in the forebrain is not depressed 3 h alter midbrain raphe lesions. This observation is in accord with the finding of Moore et al. '21 that after medial forebrain bundle lesions 5-HT does not begin to fall until after 48 h post-lesion. Thus it does not appear likely that at 3 h post-lesion the block of the heat-induced rise in forebrain 5-HIAA concentration can be due to a deficiency of 5-HT. Other studies have demonstrated that early signs of degeneration of 5-HT axons and terminals secondary to lesions of the midbrain raphe nuclei are seen by 48 h post-lesion 1, but no data are available for the 3 h post-lesion point. Brain Research, 26 (I 971 ) 37-48
46
B. L. WEISS AND G. K. AGHAJANIAN
The microelectrode studies show that an increase in body temperature is associated with an increased rate of firing of individual raphe neurons. LSD causes a cessation of activity of raphe neurons whether during conditions of basal 2 or elevated body temperature. LSD also entirely blocks the increased 5-HT metabolism induced by an elevated ambient temperature 5. It should be noted that LSD does not block the rise in body temperature resulting from exposure of rats to elevated ambient temperatureS. These findings imply that LSD might prevent an increase in 5-HIAA concentration by blocking an activation of firing of the 5-HT-containing neurons. Of course, the inhibitory effect of LSD on 5-HT metabolism could be mediated in other ways, such as through an inhibition of monoamine oxidase, by an increased 'binding' of 5-HT, or by inhibition of 5-HT release at terminals 8. However, there are data which indicate that LSD does not inhibit monoamine oxidase 13 and actually interferes with the in vitro binding of 5-HT 19. The observation that an increase in the body temperature of the rat is accompanied by an increased rate of firing of raphe units does not establish any causal relationship between the 5-HT system and the physiological regulation of body temperature. The possible role of serotonin in temperature regulation has been studied extensively elsewhere (e.g., studies on the effects on body temperature of 5-HT injected into the cerebral ventricles 11, brain 5-HT metabolism during exposure to increased ambient temperatureg, 22, the effects on body temperature of pharmacological manipulation of brain serotonin concentrationS5, 26, the temperature effects of the direct electrical stimulation of serotonin-containing neurons 24, and measures of amine metabolism during fever caused by leukocytic pyrogen15). Such studies have not as yet led to any definitive conclusion regarding the possible role of serotonin in temperature regulation. Our investigation was not directly concerned with temperature regulation, and the physiological significance, if any, of the correlation between body temperature and the firing rate of raphe cells remains to be explored. Furthermore, an activation of 5-HT neurons by elevated temperature may involve changes in other physiological functions (e.g., respiration, heart rate, blood pressure, etc.) which were not measured in this study. In conclusion, we have obtained results consistent with the hypothesis that changes in forebrain 5-HIAA concentration are mediated by impulses arising in the cell bodies of the midbrain raphe neurons. The heat-induced activation of these neurons could result in an increased 5-HT turnover in the forebrain in a fashion analogous to that seen after electrical stimulation of the same neurons ~4. Both LSD, which depresses neuronal firing, and midbrain raphe lesions, which destroy these neurons, prevent the heat-induced increase in 5-HIAA concentration. SUMMARY
Two different experimental procedures have been utilized to study the possible role of midbrain raphe neurons in mediating the increase in brain 5-HT catabolism induced by an elevated ambient temperature. In one experimental series, lesions were placed in the midbrain raphe nuclei to Brain Research, 26 (1971) 37-48
SEROTONIN METABOLISMAND RAPHE NEURONS
47
evaluate the i m p o r t a n c e o f the p e r i k a r y a o f 5-HT containing n e u r o n s in m e d i a t i n g the heat-induced increase in the 5-HT metabolite, 5 - H I A A . The r a p h e lesions were m a d e 3 h p r i o r to a p e r i o d o f i n c u b a t i o n at 40 ° C; u p o n c o m p l e t i o n o f the i n c u b a t i o n period, the forebrain, which is the principal p r o j e c t i o n area o f the m i d b r a i n r a p h e neurons, was assayed for 5 - H I A A . Lesions destroying a large p o r t i o n o f the m i d b r a i n r a p h e nuclei were found to completely prevent the heat-induced increase in brain 5 - H I A A concentration. Lesions m a d e outside the m i d b r a i n raphe nuclei did n o t prevent the rise in 5 - H I A A . In a n o t h e r set o f experiments the rate o f firing o f single units in the raphe nuclei was m o n i t o r e d while rats were exposed to infrared r a d i a t i o n in o r d e r to d e t e r m i n e if a rise in body t e m p e r a t u r e is associated with an altered rate o f firing o f the serotoninc o n t a i n i n g neurons in the midbrain. It was found t h a t as body t e m p e r a t u r e rises there is a c o n c o m i t a n t increase in the rate o f firing o f individual r a p h e neurons. U n d e r these conditions the firing o f r a p h e units can be entirely inhibited by LSD. N e u r o n s located outside the r a p h e nuclei (pons; reticular f o r m a t i o n ) do not show any obvious correlation between rate o f firing and b o d y t e m p e r a t u r e and are not inhibited by LSD. It is hypothesized t h a t changes in forebrain 5 - H I A A c o n c e n t r a t i o n induced by elevated a m b i e n t t e m p e r a t u r e s are m e d i a t e d at least in part by an increased rate o f firing o f the 5-HT containing neurons. ACKNOWLEDGEMENTS
We t h a n k the F D A - P H S P s y c h o t o m i m e t i c Agents A d v i s o r y C o m m i t t e e for supplying the LSD used in this study. This w o r k was s u p p o r t e d in p a r t by Public Health Service Research Scientist D e v e l o p m e n t A w a r d 5 K O L MH14459 (to G . K . A . ) .
REFERENCES 1 AGHAJANIAN,G. K., BLOOM, F. E., AND SHEARD, M. H., Electron microscopy of degeneration within the serotonin pathway of rat brain, Brain Research, 13 (1969) 266-273. 2 AGHAJANIAN,G. K., FOOTE, W. E., AND SI-[EARD,M. H., Lysergic acid diethylamide: sensitive neuronal units in the midbrain raphe, Science, 161 (1968) 706-708. 3 AGHAJANIAN,G. K., ROSECRANS,J. A., ANDSHEARD,M. H., Serotonin: release in the forebrain by stimulation of midbrain raphe, Science, 156 (1967) 402-403. 4 AGHAJANIAN, G. K., SHEARD, M. n., AND FOOTE, W. E., Action of psychotogenic drugs on single midbrain raphe neurons, J. Pharmacol. exp. Ther., 171 (1970) 178-187. 5 AGHAJANIAN,G. K., AND WEISS, B. L., Block by LSD of the increase in brain serotonin turnover induced by elevated ambient temperature, Nature (Lond.), 220 (1968) 795 796. 6 AND~N,N., DAHLSTROM,A., FUXE,K., LARSSON,K., OLSON,L., AND UNGERSTEDT, U., Ascending monoamine neurons to the telencephalon and diencephalon, Acta physiol, scand., 67 (1966) 313 326. 7 BOGDANSKI,D. F., PLETSCHER,A., BRODIE,B. B., AND UDENFRIEND,S., Identification and assay of serotonin in brain, J. Pharmacol. exp. Ther., 117 (1956) 82-88. 8 CHASE, T. N., BREESE, G. R., AND KOPIN, 1. J., Serotonin release from brain slices by electrical stimulation: regional differences and effect of LSD, Science, 157 (1967) 1461 1463. 9 CORRODI,H., FUXE, K., AND HOKFEL'r,T., A possible role played by central monoamine neurones in thermo-regulation, Acta physiol, scand., 71 (1967) 224-232. Braht Research, 26 (1971) 37 48
48
B. L. WEISS AND G. K. A(ilIAJANIAN
10 DAHLSTROM, A., AND FUXE, K., Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Actaphysiol. scand., 62, Suppl. 232 (1965) 1 55. l l FELDaERG, W., AND LOTTI, V. J., Temperature responses to monoamines and an inhibitor of MAO injected into the cerebral ventricles of rats, Brit. J. Pharmacol. Chemother., 31 (1967) 152-161. 12 FREEDMAN, D. X., Effects of LSD-25 on brain serotonin, J. Pharmacol. exp. Ther., 134 (1961) 160-166. 13 FREEDMAN, D. X., AND GIARMAN, N. J., LSD-25 and the status and level of brain serotonin, Ann. N. Y. Acad. Sci., 96 (1962) 98-106. 14 FUXE, K., Evidence for the existence of monoamine-containing neurons in the central nervous system. IV. Distribution of monoamine nerve terminals in the central nervous system. Acta physiol, scand., 64, Suppl. 247 (1965) 41-85. 15 GIARMAN, N. J., TANAKA, C., MOONEY, J., AND ATKINS, E., Serotonin, norepinephrine and fever. Adcanc. Pharrnaeol., 6A (1968) 307--317. 16 K6NIG, J. F. R., AND KLIPPEL, R. A., The Rat Brain, Williams and Wilkins, Baltimore. 1963. 17 KOSTOWSKI, W., GIACALONE, E., GARATTINI, S., AND VALZELLI, L., Studies on behavioural and biochemical changes in rats after lesion of midbrain rapbe, Europ. J. Pharmacol., 4 (1968) 371-376. 18 LIN, R. C., NGAI, S. H., AND COSTA, E., Lysergic acid diethylamide: rolein conversion of plasma tryptophan to brain serotonin (5-hydroxytryptamine), Science, 166 (1969) 237-239. 19 MARCHBANKS, R. M., ROSENBLATT, F., AND O'BRIEN, R. D., Serotonin binding of nerve ending particles of the rat brain and its inhibition by lysergic acid diethylamide, Science, 144 (1964) 1135-1137. 20 McDONALD, R. K., Personal communication. 21 MOORE, R. Y., AND HELLER, A., Monoamine levels and neuronal degeneration in rat brain following lateral hypothalamic lesions, J. Pharmacol. exp. Ther., 156 (1967) 12 22. 22 REID, W. D., VOLICER, L., SMOOKLER, H., BEAVEN, M. A., AND BRODIE, B. B., Brain amines and temperature regulation, Pharmacology (Basel), I (1968) 329-344. 23 ROSECRANS,J. A., LOVELL, R. A., AND FREEDMAN, D. X., Effects of lysergic acid diethylamide on the metabolism of brain 5-hydroxytryptamine, Bioehem. PharmacoL, 16 (1967) 2011- 202 I. 24 SHEARD, M. H., Ar~OAGHAJANIAN, G. K., Stimulation of the midbrain raphe: effect on serotonin metabolism, J. Pharmacol. exp. Ther., 163 (1968) 425-430. 25 SHELLENBERGER, M. K., AND ELDER, J. T., Changes in rabbit core temperature accompanying alteration in brainstem monoamine concentrations, J. Pharmacol. exp. Ther., 158 (1967) 219-226. 26 SOMERWLLE, A. R., AND WHITTLE, B. A., The interrelation of hypothermia and depletion of noradrenaline, dopamine, and 5-hydroxytryptamine from brain by reserpine, p-ehlorophenylalanine and alpha-methylmetatyrosine, Brit. J. Pharmacol. Chemother., 31 (1967) 120-131. 27 UDENFRIEND, S., WEISSBACH, H., AND BRODIE, B. B., Assay of serotonin and related metabolites, enzymes, and drugs. In D. GL1CK (Ed.), Methods of Biochemical Analysis, Vol. 6, lnterscience Publ., New York, 1958, pp. 95-130. 28 WISE, C. D., The fluorimetric determination of brain serotonin, Anal. Biochem., 20 (1967) 369-371.
Brain Research, 26 (1971) 37-48
37
A C T I V A T I O N OF BRAIN S E R O T O N I N M E T A B O L I S M BY HEAT: ROLE OF M I D B R A I N R A P H E N E U R O N S
BRIAN L. WEISS* AND GEORGE K. AGHAJANIAN
Department of Psychiatry, Yale University School of Medicine, and Connecticut Mental Health Center, New Haven, Conn. 06519 (U.S.A.) (Accepted August 27th, 1970)
INTRODUCTION
An elevated ambient temperature induces an increase in brain serotonin (5hydroxytryptamine; 5-HT) turnover in the rat9, 2z. This increase in 5-HT turnover can be blocked entirely by parenterally administered D-lysergic acid diethylamide (LSD) 5. It had previously been shown that under basal environmental conditions (i.e., at room temperature) LSD produces an increase in the concentration of brain 5-HT 12 and a concomitant decrease in the principal 5-HT metabolite, 5-hydroxyindoleacetic acid (5-HIAA) 23. These various findings suggest that there is a decrease in the turnover of brain 5-HT after LSD and this has been directly demonstrated by studies employing isotopically labeled tryptophan TM. The mechanism by which LSD produces a decrease in 5-HT turnover is not known. However, it has been found that LSD inhibits the spontaneous firing of 5-HT containing neurons 2. Electrical stimulation of these neurons, most of which are situated in the midbrain raphe nuclei 10, causes an increase in 5-HT turnover, as indicated by the fact that there is a marked elevation in the concentration of 5-HIAA but only a small decrease in 5-HT concentration in both whole brain and forebrainZ, z4. Taken together, these observations suggest the possibility that elevated ambient temperatures could induce an increase in 5-HT turnover via an activation of the firing of 5-HT containing neurons. Furthermore, since LSD depresses the firing of neurons containing 5-HT, this action could account for the drug's ability to prevent the heat-induced increase in 5-HT turnover. In terms of this hypothesis, two different experimental procedures have been carried out. First, to evaluate the importance of the perikarya of 5-HT containing neurons in mediating the heat-induced increase in 5-HIAA, lesions were placed in the midbrain raphe. The raphe lesions were made 3 h prior to a period of incubation at an elevated ambient temperature; upon completion of the incubation period, the forebrain, which is the principal projection area of the midbrain raphe neurons 6, was * Present address: Department of Psychiatry, New York University Medical Center, New York, N.Y. 10016.
Brain Research, 26 (1971) 37-48
38
I3. k. WEISS AND G. K. AGIIAJANIAN
assayed for 5-H|AA and the midbrain was examined histologically to determine the precise location and extent of the lesion. Secondly, since body temperature is increased by incubation 5, we monitored the rate of firing of single units in the raphe nuclei while rats were being heated to see if an increase in body temperature is associated with an altered rate of firing of serotonin-containing neurons. Some animals were given LSD during this procedure. METHODS
Lesion experiments Sprague-Dawley (Charles-River, C.D.) male rats weighing from 200 to 300 g were used. The animals were separated into 3 major groups: (1) non-lesion animals: (2) raphe-lesion animals, in which the lesion destroyed a large portion of the midbrain raphe nuclei; and (3) non-raphe lesion animals, where the lesion spared most or all of the midbrain raphe nuclei. In the non-lesion rats no electrode was placed in the brain. Of these, one group of rats consisted of room temperature control animals that were decapitated in a guillotine device and their brains taken immediately after removal from the animal room. As a further control, some non-lesion rats were anesthetized with chloral hydrate (400 mg/kg) during the incubation procedures since this anesthesia was employed for the single unit recordings (see below). Another group of non-lesion animals received intraperitoneal injections of either LSD, 500 #g/kg as the bitartrate salt, or an equivalent volume of saline free of pyrogen, immediately prior to a 45 min period of incubation at 40°C. Upon completion of the incubation period all rats were decapitated and their brains removed. In the raphe-lesion groups, lesions were made which destroyed large areas of the midbrain raphe nuclei. After chloral hydrate anesthesia, as described above, the animals were placed in a stereotaxic instrument. An insulated 0.25 mm diameter steel electrode with an 0.5 mm exposed pointed tip was then lowered through a burr hole into the midbrain raphe. A 2.5 mA constant anodal current was passed for l0 sec through the electrode at depths from the skull surface of 6.5, 7.5, and 8.5 mm. Animals with sham lesions received no current, the electrode placement being otherwise identical with that in the experimental animals. The electrodes were removed immediately after the making of a lesion or a sham placement. After the 3 h recovery period, the rats (awake and in individual cages) were either placed in an incubator at 40°C for 45 min or allowed to remain at 23° C for 45 min. The rats were then decapitated and their brains removed for histology and for assay of indoles. Midline lesions, 2-3 mm in diameter, destroying the central portions of the areas ventral to the aqueduct (dorsal raphe nucleus) and ventral to the decussation of the superior cerebellar peduncle (median raphe nucleus), encompassing A620-AI60 (after K6nig and KlippeP6), were defined as 'total midbrain raphe lesions'. A group designated 'partial anterior raphe lesion' was composed of rats whose lesions destroyed only the extreme anterior portions (i.e., A620 and anterior) of both nuclei. Brain Research, 26 (1971) 37-48
SEROTONIN METABOLISMAND RAPHE NEURONS
39
The 'non-raphe lesion' groups consisted of those rats whose lesions were primarily outside the midbrain raphe nuclei. The lesions were made as described above, except for the placement of the electrode. A rat was classified in the group of 'pontine lesions' if the lesion was posterior to A160; in some cases such lesions were in the pontine raphe area. A group of 'reticular lesions' was comprised of those rats in which the midline remained intact and the medial edge of the lesion was at least 2 mm from the midline. The final subgroup was the 'median tegmentum' group, in which the lesions were midline but completely anterior to both nuclei, so that neither nucleus was damaged. All the non-raphe lesion rats were incubated for 45 rain at 40 ° C after 3 h had elapsed from the time of lesion placement. Following decapitation and removal of the brains from all rats, the brains were sectioned just anterior to the superior colliculus. The forebrains were frozen immediately and later assayed for 5-H|AA and 5-HT by a fluorimetric method 24 that incorporated modifications of the procedures of Bogdanski et al. 7, Udenfriend et al. ~7,
Fig. 1. Histologicalsection illustrating a typical total midbrain raphe lesion. The dorsal raphe nucleus and the median raphe nucleus have both been destroyed. Cresyl violet stain.
Brain Research, 26 (1971) 37-48
40
I~. L. WEISS A N D G . K. AGI-tAJANIAN
and Wise 28. The midbrains of animals with lesions were fixed in 5 ~,~glutaraldehyde (in 0.15 M sodium phosphate buffer, pH 7.4). Serial frozen sections of midbrain were cut and stained with cresyl violet to identify site of lesion. Fig. 1 illustrates histology of a typical lesion in which there is total destruction of the midbrain raphe nuclei.
Single unit experiments Rats used for the single unit recordings were anesthetized with chloral hydrate and mounted in a stereotaxic instrument. A tungsten microelectrode with a tip diameter of approximately 1 # m was then lowered with a hydraulic microdrive through a burr hole into various regions of the midbrain and pons. Electrode signals were passed into a high impedance amplifier and then displayed on an oscilloscope. The rate of firing of cells was followed with a triggered electronic counter whose analog output was plotted on a strip chart potentiometric recorder; the counter automatically reset to zero every 10 sec giving a histogram appearance to the records. Raphe units are characterized by their regular rhythm, slow spontaneous rate of firing (approximately 1 spike/sec), and inhibition by low doses of LSD 2,4, Units not in the raphe region usually fire at a faster rate and/or have an irregular rhythm. In addition, the non-raphe units do not respond to LSD with a cessation of activity. When a raphe or other unit was identified, a baseline of spontaneous activity was obtained over a span of several minutes. Body temperature was recorded by means of a colonic thermistor. The head and tail regions were shielded by an asbestos screen and a 150 W infrared lamp, placed 12 in. from the rat, was used to increase body temperature. In some animals LSD was administered intravenously through the tail vein during or after exposure of the rat to heat. After completion of the recording, rats were perfused with fixative and midbrains sectioned and stained for determination of site of electrode tip as previously described~k RESULTS
Lesion experiments As can be seen from Table I, there was a 37 ~,, increase (as compared to room temperature controls) in the concentration of 5-HIAA in the forebrains of incubated control animals kept at 40°C for 45 min. The finding of an increase in brain 5-HIAA concentration after incubation at an elevated ambient temperature is in accord with previous data 5,2°,2~. Table I shows that the increase in 5-HIAA is entirely blocked by the prior injection of 500 #g/kg of LSD; this result is in agreement with our earlier findings 5. A slight but significant depression of 5-HIAA concentration occurs in the animals pretreated with LSD (Table I). The heat-induced increase in 5-HIAA concentration is completely prevented by a total lesion of the dorsal and median midbrain raphe nuclei (Table I). In contrast, the incubated sham-lesion animals had a increase of over 56 ~ in 5-HIAA concentration. Some increase in 5-HIAA occurred even when sham-lesion animals were not
Brain Research, 26 (1971) 37-48
SEROTONIN METABOLISM AND RAPHE NEURONS
41
TABLE I EFFECT OF PRIOR LESIONS OR
LSD
PRETREATMENT ON C O N C E N T R A T I O N OF FOREBRAIN
5-HIAA
IN RATS
EXPOSED TO AN ELEVATED AMBIENT TEMPERATURE ( 4 0 ° C ) *
Group
Procedure
(n)
Ambient temp. (' C)
5-HIAA % Change P** (ng/g) ( ± S.E.M.)
Non-lesion
Control Control LSD Saline
(8) (14) (5) (7)
23 40 40 40
244 355 215 323
Raphe lesion
Total raphe Total raphe Partial raphe (anterior) Sham Sham
(14) (6)
40 23
253 i 267 ±
(9) (17) (4)
Pontine Tegmental Reticular
(7) (5) (4)
Non-raphe lesion
~ 9 ± 6 ± 7 ± 4
---37 --12 I 32
--: 0.001 -~0.05 < 0.001
6 6
I 4 -- 9
N.S. N.S.
40 40 23
282 :] 9 38l ~-_ 8 260 ± 22
i 16 F56 +48
• 0.01 < 0.001 < 0.001
40 40 40
322 ± 11 316 ± 15 357 ± 20
I 32 ~ 30 -46
0.001 ~ 0.001 -~. 0.001
* In all cases periods of exposure were 45 min. ** P values were determined using Student's t test. TABLE II EFFECT OF PRIOR TOTAL RAPHE LESIONS O N FOREBRAIN
5-HT
C O N C E N T R A T I O N AFTER EXPOSURE TO AN
AMBIENT TEMPERATURE OF 4 0 ° C *
Group
(n)
Ambient temp. ('C)
5-HT (ng/g) ( ± S.E.M.)
°o Change
P**
Control Control Sham Total raphe
(10) (5) (4) (5)
23 40 40 40
338 346 354 395
-÷ 2 i 5 + 17
-N.S. N.S. --~ 0.02
± 10 ~ 7 ± 5 ~ 17
* Period of incubation was 45 min. ** P values were determined using Student's t test. incubated. 5-HIAA
In unincubated
concentrations
animals with total lesions of the midbrain
raphe, the
d i d n o t differ s i g n i f i c a n t l y f r o m t h o s e t o t a l r a p h e
lesion
a n i m a l s k e p t a t 40°C f o r 45 m i n . L e s i o n s m a d e e l s e w h e r e in t h e m i d b r a i n (e.g. r e t i c u l a r f o r m a t i o n ) o r ports, s p a r i n g all o r m o s t o f t h e m i d b r a i n r a p h e nuclei, d i d n o t p r e v e n t t h e h e a t - i n d u c e d i n c r e a s e in 5 - H I A A ; 5 - H I A A v a l u e s i n t h e s e n o n - r a p h e l e s i o n a n i m a l s a r e n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h e i n c u b a t e d c o n t r o l a n i m a l s ( T a b l e l). T w o o f t h e 3 p o n t i n e l e s i o n s w e r e in t h e p o n t ± h e r a p h e a r e a b u t t h e 5 - H I A A levels in t h e s e i n s t a n c e s d i d n o t differ f r o m t h e o t h e r n o n - r a p h e l e s i o n a n i m a l s . Measurement of the concentration of 5-HT in the forebrains of incubated animals with total midbrain raphe lesions demonstrates that 5-HT values are not de-
Brain Research, 26 (1971) 37-48
g
I
b.a O~
.R"
...........
5 MIN
!
T
--36
--37
---38
m39
,--40
o
C
Fig. 2. Response of a raplae unit to an increase in body temperature. This unit has a spontancous rate of about 40 spikc~q-J~in~wio~ ~c, ~i~cappiica~,ion of heat (ON arrow). The transient decline in firing rate immediately following the application of heat has been noted in sexera~ other raphe ttnh recordings. A rise in body temperature is associated with an increase in firing rate; at a body temperature of 39.5' C, the firing rate is about 100 spikes/rain. Intravenous injection of LSD (arrow; dose 10/~g/kg) during the period of peak body temperature resulted in an inhibition of firing, The record consists of consecutive 10-see samples of the analog output of a counter triggered by the unit spikes,
I ¸
B.'L
> 7, >
>
"r
>
>
[]
t'-
4a. i,..a
I iOM N
I
~34
35
36
37
38 o C
39
40
Fig. 3. Record of response of a raphe unit to both rise and fall in body temperature (B.T.). At its maximum, the increase in firing rate is approximately two-fold. A drop in body temperature is associated with a decrease in rate of firing. LSD (arrow; dose 10 t~g/kg), administered intravenously while body temperature was falling, completely inhibited the firing of the unit.
gO
Z
¢"~l.J
B.I".
44
B. l.. WEISS AND G. K. AGtlAJANIAN
creased from control levels. It can be seen from Table 11 that the 5-HT concentration actually is somewhat elevated 3 h and 45 rain after a total lesion of the midbrain raphc nuclei. We observed behavioral changes similar to those noted by Kostowski et al. 17 il~ rats with lesions in the midbrain raphe (e.g. increased spontaneous motor activity. with aimless rotatory movements, and marked hypersensitivity to tactile and auditory stimuli). However, the rats with non-raphe lesions also exhibited such behavior. Single unit e x p e r i m e n t s
Eleven raphe units were studied using microelectrode techniques. Figs. 2 and 3 illustrate that the rate of firing of individual raphe neurons is closely correlated with changes in body temperature induced by heating from an infrared source. In all raphe units studied, it was found that as body temperature rose there was an acceleration in firing rate; conversely, a decline in body temperature was accompanied by a decrease in firing rate. The mean increase in the rate of firing for the I1 units was 2.7-fold (range 1.5-5.0) when body temperature rose from an average baseline of 35.3 C to a peak of 39.5 ° C. The latter body temperature is approximately the same as that seen after incubation at 40 ° C (ref. 5). Little or no change in firing rate occurred merely with the application or termination of the infrared radiation until there was a change in body temperature. Nine units located outside the raphe nuclei demonstrated no obvious correlation between firing rate and body temperature. These non-raphe units were located in either the reticular formation (6 units) or pontine area (3 units). Some non-raphe units responded to the application of heat by an increase in firing rate, while others showed a decrease or combination of effects. Thus a non-raphe unit might alternately accelerate and decelerate while body temperature continued to rise. In contrast to the raphe units, brief changes in the firing rate of the non-raphe units sometimes were correlated with the actual turning off of the heat lamp. The reported inhibitory effect of LSD on the activity of raphe units '~,4 was observed both during the rising (Fig. 2) and falling (Fig. 3) phases of the body temperature curve. LSD did not cause a cessation of activity in the non-raphe units. To control for possible biochemical effects of the anesthesia used during the single unit recordings, forebrain 5-HIAA was measured in rats given chloral hydrate (400 mg/kg)just prior to a 1 h period incubation at 40°C. In anesthetized rats maintained at 23 ° C for I h, the concentration of 5-HI AA in the forebrain was 317 ng/g (-I 5, S.E.M. ; n 6). This value represents a significant (P < 0.001) increase over unanesthetized control rats kept at 23 ° C (@ Table I). After 1 h of incubation at 4if' C, anesthetized rats had a further elevation of forebrain 5-HIAA to 401 ~: 8 ug/g (n 8). DISCUSSION Each of the two different experimental approaches employed suggests that the raphe neurons play a role in the increased turnover of 5-HT induced by an elevated ambient temperature. The first set of experiments, in which lesions were made in the Brain Research, 26 (1971) 37-48
SEROTONIN METABOLISM AND RAPHE NEURONS
45
midbrain raphe nuclei, shows that the heat-induced activation of 5-HT metabolism in the forebrain, as reflected by an increased 5-HIAA concentration, is dependent upon the structural integrity of the raphe nuclei. The second procedure (i.e., microelectrode recordings from individual raphe units) demonstrates that as body temperature increases there is an associated rise in the rate of firing of raphe cells. Both the requirement for structural integrity and the association between body temperature and firing rate are consistent with the hypothesis that the increase in 5-HT catabolism under conditions of increased ambient temperature is at least in part mediated by an increase in the firing rate of the 5-HT containing neurons in the midbrain raphe. An alternative explanation for the increased 5-HT turnover during heat exoosure would be that the synthesis and degradation of 5-HT is directly activated by a rise in body temperatures. Thus increased temperature might result in a direct activation of 5-HT metabolism in the 5-HT terminals within the forebrain. However, this possible mechanism would not by itself explain how acute midbrain raphe lesions could prevent such a direct activation in the forebrai_n. The lesion experiments further demonstrate some specificity of the requirement for destruction of midbrain raphe neuronal perikarya in order to prevent the heatinduced increase in 5-HT turnover. Incubated animals with lesions rostral, lateral, or posterior to the midbrain raphe nuclei all have elevated forebrain 5-HIAA concentrations. Midline lesions destroying only the extreme anterior portions of the raphe nuclei prevent some of the heat-induced rise in 5-HIAA. The probable explanation for the partial blocking effect on the increased turnover of some slightly anterior lesions is that, in addition to the destruction of the anterior portions of the raphe nuclei, 5-HT fiber tracts pro.jectir~g to the forebrain would be destroyed by lesions in this area G,la. Lateral and posterior lesions do not interfere with these projections. In the anterior midbrain these axonal projections to the forebrain deviate from the midline and join the medial forebrain bundle14; this divergence of the fibers from the midline explains the high-5-HIAA concentrations noted with very anterior midline lesions, since the projections course laterally to the lesion and the raphe nuclei are spared. Sham lesions not only fail to prevent the heat-induced increase in 5-H~AA concentration, but even significantly elevate 5-HT turnover above incubated control values. In fact, no incubation is necessary to produce an increased forebrain 5-HIAA concentration in the sham-lesion animals. This increase in turnover may be due to an irritative effect on the raphe neurons, but the actual mechanism has not been determined. The sham lesions provide further confirmation for the specific requirement of raphe neuron destruction to block the heat-related increase in 5-HT turnover. It is significant that the concentration of 5-HT in the forebrain is not depressed 3 h alter midbrain raphe lesions. This observation is in accord with the finding of Moore et al. '21 that after medial forebrain bundle lesions 5-HT does not begin to fall until after 48 h post-lesion. Thus it does not appear likely that at 3 h post-lesion the block of the heat-induced rise in forebrain 5-HIAA concentration can be due to a deficiency of 5-HT. Other studies have demonstrated that early signs of degeneration of 5-HT axons and terminals secondary to lesions of the midbrain raphe nuclei are seen by 48 h post-lesion 1, but no data are available for the 3 h post-lesion point. Brain Research, 26 (I 971 ) 37-48
46
B. L. WEISS AND G. K. AGHAJANIAN
The microelectrode studies show that an increase in body temperature is associated with an increased rate of firing of individual raphe neurons. LSD causes a cessation of activity of raphe neurons whether during conditions of basal 2 or elevated body temperature. LSD also entirely blocks the increased 5-HT metabolism induced by an elevated ambient temperature 5. It should be noted that LSD does not block the rise in body temperature resulting from exposure of rats to elevated ambient temperatureS. These findings imply that LSD might prevent an increase in 5-HIAA concentration by blocking an activation of firing of the 5-HT-containing neurons. Of course, the inhibitory effect of LSD on 5-HT metabolism could be mediated in other ways, such as through an inhibition of monoamine oxidase, by an increased 'binding' of 5-HT, or by inhibition of 5-HT release at terminals 8. However, there are data which indicate that LSD does not inhibit monoamine oxidase 13 and actually interferes with the in vitro binding of 5-HT 19. The observation that an increase in the body temperature of the rat is accompanied by an increased rate of firing of raphe units does not establish any causal relationship between the 5-HT system and the physiological regulation of body temperature. The possible role of serotonin in temperature regulation has been studied extensively elsewhere (e.g., studies on the effects on body temperature of 5-HT injected into the cerebral ventricles 11, brain 5-HT metabolism during exposure to increased ambient temperatureg, 22, the effects on body temperature of pharmacological manipulation of brain serotonin concentrationS5, 26, the temperature effects of the direct electrical stimulation of serotonin-containing neurons 24, and measures of amine metabolism during fever caused by leukocytic pyrogen15). Such studies have not as yet led to any definitive conclusion regarding the possible role of serotonin in temperature regulation. Our investigation was not directly concerned with temperature regulation, and the physiological significance, if any, of the correlation between body temperature and the firing rate of raphe cells remains to be explored. Furthermore, an activation of 5-HT neurons by elevated temperature may involve changes in other physiological functions (e.g., respiration, heart rate, blood pressure, etc.) which were not measured in this study. In conclusion, we have obtained results consistent with the hypothesis that changes in forebrain 5-HIAA concentration are mediated by impulses arising in the cell bodies of the midbrain raphe neurons. The heat-induced activation of these neurons could result in an increased 5-HT turnover in the forebrain in a fashion analogous to that seen after electrical stimulation of the same neurons ~4. Both LSD, which depresses neuronal firing, and midbrain raphe lesions, which destroy these neurons, prevent the heat-induced increase in 5-HIAA concentration. SUMMARY
Two different experimental procedures have been utilized to study the possible role of midbrain raphe neurons in mediating the increase in brain 5-HT catabolism induced by an elevated ambient temperature. In one experimental series, lesions were placed in the midbrain raphe nuclei to Brain Research, 26 (1971) 37-48
SEROTONIN METABOLISMAND RAPHE NEURONS
47
evaluate the i m p o r t a n c e o f the p e r i k a r y a o f 5-HT containing n e u r o n s in m e d i a t i n g the heat-induced increase in the 5-HT metabolite, 5 - H I A A . The r a p h e lesions were m a d e 3 h p r i o r to a p e r i o d o f i n c u b a t i o n at 40 ° C; u p o n c o m p l e t i o n o f the i n c u b a t i o n period, the forebrain, which is the principal p r o j e c t i o n area o f the m i d b r a i n r a p h e neurons, was assayed for 5 - H I A A . Lesions destroying a large p o r t i o n o f the m i d b r a i n r a p h e nuclei were found to completely prevent the heat-induced increase in brain 5 - H I A A concentration. Lesions m a d e outside the m i d b r a i n raphe nuclei did n o t prevent the rise in 5 - H I A A . In a n o t h e r set o f experiments the rate o f firing o f single units in the raphe nuclei was m o n i t o r e d while rats were exposed to infrared r a d i a t i o n in o r d e r to d e t e r m i n e if a rise in body t e m p e r a t u r e is associated with an altered rate o f firing o f the serotoninc o n t a i n i n g neurons in the midbrain. It was found t h a t as body t e m p e r a t u r e rises there is a c o n c o m i t a n t increase in the rate o f firing o f individual r a p h e neurons. U n d e r these conditions the firing o f r a p h e units can be entirely inhibited by LSD. N e u r o n s located outside the r a p h e nuclei (pons; reticular f o r m a t i o n ) do not show any obvious correlation between rate o f firing and b o d y t e m p e r a t u r e and are not inhibited by LSD. It is hypothesized t h a t changes in forebrain 5 - H I A A c o n c e n t r a t i o n induced by elevated a m b i e n t t e m p e r a t u r e s are m e d i a t e d at least in part by an increased rate o f firing o f the 5-HT containing neurons. ACKNOWLEDGEMENTS
We t h a n k the F D A - P H S P s y c h o t o m i m e t i c Agents A d v i s o r y C o m m i t t e e for supplying the LSD used in this study. This w o r k was s u p p o r t e d in p a r t by Public Health Service Research Scientist D e v e l o p m e n t A w a r d 5 K O L MH14459 (to G . K . A . ) .
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