- Email: [email protected]
Thermosensitivity of dorsal raphe neurons in vitro
Brain Research. 41(/(1987) 189- 194 Elsevier
189
BRE 22239
Thermosensitivity of dorsal raphe neurons in vitro C. Larry Keenan and Nai-Shin Chu Department of Neurology, California College of Medicine, University of California at Irvine, lrvine, CA 92717 (U. S. A. ) (Accepted 27 January 1987) Key words: Brainstem; Dorsal raphe; Thermoregulation
Thermosensitivity of raphe neurons was studied in tissue slices of rat brainstems (400-500 !tm). Measurement of activity of single cells in the dorsal raphe region of the slices revealed that the majority of neurons (89%) were sensitive to changes in temperature. Over the range 34 to 42 °C, 3 classes of thermosensitive cells were found: warm (61%), cold (15%) and biphasic type cells (13%). Many dorsal raphe neurons may be intrinsically temperature sensitive and may serve as extrahypothalamic thermodetector components in the integrative process of central thermoregulation. Identification and characterization of central thermoregulatory neurons are important goals in the study of neural substrates underlying thermostasis, a homeostatic function in mammals vital for their survival. Studies over several decades have implied an hierarchical arrangement of parallel centers capable of integrating heat-loss, heat-production and heatconservation mechanisms during the process of thermostasis 2'3'8Aa'15"24'35. The study of these neuronal populations may reveal the processing mechanisms of central neurons that function as integrators or detectors at the interface between peripheral and central thermal inputs to regulate responses of thermoeffectors. The raphe complex in the brainstem is a major extrahypothalamic site proposed to have thermoregulatory functions lt'12A7'21'34'37. Here we report that dorsal raphe (DR) neurons in an in vitro slice preparation 1°'27 retain thermosensitivity in the absence of ascending or descending inputs. A preliminary report appeared in abstract form earlier 1°. Both warm-responding and cold-responding D R cells exhibited linear or non-linear responses to temperature changes. Thermal coefficients and Ql0'S of the cells studied resemble those reported for D R in vivo and hypothalamic temperature-sensitive cells in vivo and in vit-
ro 5'6'1t'18'21'23. To our knowledge, the D R cells are the only other central extrahypothalamic neurons with thermoregulatory functions investigated to date that retain thermosensitivity in vitro. Our findings that the thermosensitivity of these deafferented cells can be manipulated in vitro provide a unique opportunity to further our understanding of the complex, multiple integrating systems involved in thermoregulation in mammals. Although thermosensitivity in a neuron does not necessarily imply that the cell has a role in thermoregulation 9, on the basis of numerous studies, D R neurons are considered candidates for having a thermoregulatory role (for reviews see refs. 3, 15, 28, 35 and 38). Fluorescence histochemistry and selective brain lesion studies have shown that the majority of serotonergic neurons are localized to the brainstem raphe nuclei 16"2°'25'36'37. Lesions of afferent pathways containing axons from spinal and cutaneous thermoreceptors that are presumed to transmit thermosensory information to hypothalamic structures result in degeneration of neurons in the raphe nuclei and the inability of several species to thermoregulate at low ambient temperature ~6. Injection of serotonin (5HT) causes temperature changes, as do several pharmacological manipulations that either enhance or de-
Correspondence: C.L. Keenan. Present address: Beckman Research Institute of the City of Hope, 1450 E. Duarte Rd., Duarte, CA 91010, U.S.A.
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Fig. 1. A: schematic drawing of experimental recording set-up. A, the microelectrode and micromanipulator assembly used to record the single unit activity. The tissue slices (B) are supported by a nylon netting and submerged in ACSF. The left inset is a drawing of a transversely cut brain slice at the level of the inferior colliculus (IC) approximately 600/~m anterior to the locus coeruleus. Electrode placement into the dorsal raphe (DR) nucleus is easily accomplished by visual guidance using a dissecting microscope. Bar = 2.0 mm. The heating element (C) heats the ACSF (D) in the outer chamber (E). A needle valve (F) controls the flow rate of the ACSF which is saturated with 95% 0 2 - 5 % CO 2 (G). The inset to the right is an example of the extracellular action potential recorded from a warmsensitive DR neuron and the characteristic waveform differences (indicated by the arrows and the duration of the a and b components of the potentials) that accompany changes in firing rate (FR) when temperature is changed. B: polygraph display of FR responses of a warm-sensitive DR neuron to temperature changes. The action potentials were amplified (xl00) with frequency filter cut-offs at 100 Hz and 10 kHz and recorded on magnetic tape while the unit activity was monitored on a storage oscilloscope. Simultaneously the spikes were converted to pulses of constant size and duration by a window discriminator (WPI). The output of the discriminator was counted linearly by a ratemeter and recycled every second. The output of the ratemeter was displayed by a vertical deflection on a polygraph (Beckman Dynograph R612) as shown in the top trace of the example. Measurements and analyses were made during or after the experiments from such polygraph records which displayed the FR (spikes/s) obtained, a slope integration of FR obtained via the output of the window discriminator and resetting integrator (Beckman 9837B) (middle trace), and a calibrated DC voltage output (bottom trace) from the bath thermistor (Yellowspring Telethermometer). For each neuron recorded, temperature was varied at least twice in both the ascending and descending directions over the range of 34 to 42 °C. In the example shown, the discontinuity on the descending portion of the temperature change consists of 6.8 min. Rate of temperature change never exceeded 0.02 °C/s, so that the temperature effects were assumed primarily to reflect static responses of thermodetectors rather than dynamic responses, although the latter cannot be completely ruled out and may contribute to the effects.
191 c r e a s e b r a i n 5 - H T levels 2°29'3°. Single unit studies in
g u l a t i o n and a direct i n f l u e n c e of t h e r m o s e n s i t i v e
v i v o h a v e s h o w n that e i t h e r p e r i p h e r a l o r local t e m -
D R n e u r o n s on the t h e r m o r e g u l a t o r y cells of t h e hy-
p e r a t u r e c h a n g e s alter firing rates of d o r s a l and o t h e r r a p h e n e u r o n s 1U2'15'17'21. L o c a l t h e r m a l s t i m u l a t i o n
pothalamus.
of dorsal r a p h e cells in v i v o alters firing rates of ther-
t h e r m o s e n s i t i v e , the effect of t e m p e r a t u r e on the fir-
m o s e n s i t i v e units in the h y p o t h a l a m i c r e g i o n s k n o w n
ing rates of 57 D R cells w e r e s t u d i e d e l e c t r o p h y s i o -
to be i n v o l v e d in t h e r m o r e g u l a t i o n while the o p p o -
logically in b r a i n s t e m slices o b t a i n e d f r o m adult rats.
site e x p e r i m e n t has no effect o n r a p h e t h e r m o s e n s i tive n e u r o n s 1t'13'31'32. T h e s e results suggest the par-
T h e slices w e r e p r e p a r e d using p r e v i o u s l y d e s c r i b e d
ticipation of r a p h e s e r o t o n e r g i c n e u r o n s in t h e r m o r e -
uously p e r f u s e d with artificial c e r e b r o s p i n a l
A°
B.
T o i n v e s t i g a t e w h e t h e r D R cells are intrinsically
t e c h n i q u e s 1°'27, and m a i n t a i n e d in a c h a m b e r contin-
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Fig. 2. A: responses of thermosensitive DR neurons to temperature changes. Top: thermosensitivities of two cells with bell-shaped curves. Middle: thermosensitive responses of two DR cells illustrate the increase in firing rate as temperature was increased. Bottom: responses of two neurons which increased firing rates as temperature was lowered. Mean Frs were calculated for each degree Celsius (Tx) over the temperature range from TX- 0.5 °C to Tx + 0.5 °C from polygraph records as in Fig. 1B and the data plotted as a function of temperature. B: FR/temperature functions of warm-sensitive cells (top) and cold-sensitive cells (bottom). Mean FRs as functions of temperature (T i, the initial, lowest temperature used; Tx, any temperature in the arithmetic progression Ti, Ti÷ 1, Ti+2 . . . Ti÷(,_l )) have been plotted as individual points and then fitted by least-squares regression analysis using a microcomputer and a curve-fitting program (solid lines). The majority of warm-sensitive neurons respond to temperature as illustrated by the curves in the top graph obtained from 3 cells. The curves are exponentials of the form FR = Aekrwith A = slope, k = constant, and T = (T x- Ti) x 10-1 (°C). The majority of the cold-sensitive neurons have sigmoid functions as illustrated by 3 cell responses to temperature in the bottom graph. The functions are described by the equation FR = A x (1 + BkT)-1. The value B = expected y-intercept. Other values have the same meanings as in the equation for the warm-responding cells.
192 (ACSF) (Fig. 1). Conventional electrophysiological methods were used to record single unit activity (details are given in Fig. 1B). Extracellular action potentials from the neurons were recorded a minimal 10 min at 37 °C to assure stability of each cell and to obtain a baseline firing rate (FR). The majority (74.3%) of DR cells studied in vitro had spontaneous FRs of <6 spikes/s at 37 °C. This compares favorably with discharge rates recorded in vivo and in vitro IA°'26'27. Three types of temperature-sensitive cells were found in the DR (Fig. 2A). Biphasic responses in firing rates were observed in 13% of the cells (Fig. 2A, top). These cells had temperature-response curves that were bell-shaped. In 61% of the D R cells studied, FRs increased as temperature was raised from 34 to 42 °C (Fig. 2A, middle). Increasing discharges were observed in 15% of the cells in response to temperature decreases over the same range (Fig. 2A, bottom). Both warm- and cold-sensitive cells exhibited temperature-dependent changes in action potential waveform. An example of changes recorded from a warm-sensitive cell is shown in the right inset of Fig. 1A. Only 11% of the D R cells studied were considered temperature insensitive because they did not respond to temperature changes or responded with Q10's ~< 2. The low rate of firing in vivo and in vitro was the principle reason for using the al0 effect as the criterion for determining thermosensitivity of a cell. It is for this reason that we report a higher percentage of temperature-sensitive DR neurons (89%) than that reported for preoptic/anterior hypothalamic cells (40-50%) 4'5"32. Thermal coefficients and Ql0's were obtained over the temperature ranges to which the cells were most thermosensitive. Thermal coefficients ranged from 0.1 to 1.0 impulses/s-°C for warm-sensitive cells, and from -0.1 t o - 0 . 8 impulses/s.°C for cold-sensitive cells. Ql0'S ranged from 2 to >100 for warm-respond-
ing cells and 0.006 to 0.5 for cold-responding cells when extrapolated from the thermosensitive ranges of the cells (see Table I for summary). Warm-responding neurons were generally most responsive at temperatures >39 °C and cold-responding cells were most sensitive to temperatures <37 °C. Analysis of the FR functions of the thermosensitive cells using a microprocessor and a curve-fitting program (Jawston, 1982, unpublished) revealed that the majority of the warm-sensitive neurons (28/35) had temperature-dependent FRs best fit by single exponentials of the form FR = Ae kT (Fig. 2B, top). Temperature-dependent FRs of the majority of coldsensitive cells (5/9) had functional relationships best described by sigmoid curves of the form FR = A x (1 + Bkr) -1 (Fig. 2B, bottom). Two cold-sensitive cells had discharge functions that 'decayed exponentially as temperature was increased, while several warmand cold-sensitive cells had linear discharge functions (correlation coefficient > 0.95). The significance of the different discharge functions of the warm- and cold-sensitive cells is not clear. Input-output curves of first and higher order sensory cells have been used in the past as criteria by which to judge whether the cells were primary (involved in the transduction of stimulus intensity) or secondary t6. Although there are many examples of neuronal responses coding stimulus intensity as some power function, there are also sufficient examples of stimulus intensity-frequency functions of sensory cells that are linear, logarithmic, or some higher order polynomial depending on the range of the stimulus or the responsiveness and type of the sensory receptors involved. At present one must conclude that there are no general input-output functions applying to all sensory cells 7. Nevertheless, the similarities between the temperature-dependent discharge functions of DR cells and thermosensitive neurons in the anterior
TABLE I Classification of DR neurons according to FR responses to temperature increases Cell type
Response to T (34-42 °C)
No. of cells
%
Qlo (range)
Thermal coef. (To) (range)
Warm Cold Biphasic Insensitive
Increased FR Decreased FR Maxima/minima FR near 38 °C Small to no change in rate
35 9 7 6
61 15 13 I1
5- 180 0.006-0.5 <2
0.1-1.0 (-0.1)-(-0.8) -0.01 < Tc < (0.1)
193 h y p o t h a l a m u s , in vivo and in vitro, and cutaneous t h e r m o r e c e p t o r s are striking 43H6"23. O u r present results suggest that the D R thermosensitive n e u r o n s are intrinsically capable of t h e r m o d e t e c t i o n . W e cannot, however, rule out the possibility that some of the D R cells are driven by interneurons which are true t h e r m o d e t e c t o r s . F u r t h e r studies in which synaptic activity is blocked have to be c o m p l e t e d to assess this possibility. Thermosensitivity of w a r m - r e s p o n d i n g p r e o p t i c / a n t e r i o r h y p o t h a l a m i c cells in tissue slices is u n c h a n g e d when synaptic mechanisms are b l o c k e d by lowering Ca 2÷ concentration and raising Mg 2+ concentration of the external m e d i u m . Kelso and Boulant 22, and H o r i et al. t9 interpret this as evidence that those cells are p r o b a b l y t h e r m o d e t e c t o r s , and thus are intrinsically thermosensitive, even though an equally compelling a r g u m e n t might be that changes induced by Ca 2+ m a n i p u l a t i o n s simply reflect Ca 2+ involvement in transducer mechanisms t6. Similar experiments may permit further insight into
1 Aghajanian, G.K. and Weiss, B.L., Block by LSD of the increase in brain serotonin turnover induced by elevated ambient temperature, Nature (London), 220 (1968) 795-796. 2 Bligh, J., Neuronal models of mammalian temperature regulation. In J. Bligh and R.E. Moore (Eds.), Essays on Temperature Regulation, Elsevier, New York, 1972, pp. IXXIII. 3 Bligh, J., The control neurology of mammalian thermoregulation, Neuroscience, 4 (1979) 1213-1236. 4 Boulant, J.A., Hypothalamic mechanisms in thermoregulation, Fed. Proc. Fed. Am. Soc. Exp. Biol., 40 (1981) 2843-2850. 5 Boulant, J.A. and Demieville, H.N.. Responses of thermosensitive preoptic and septal neurons to hippocampal and brain stem stimulation, J. Neurophysiol., 40 (1977) 1356-1368. 6 Boulant, J.A. and Hardy, J.D., The effect of spinal and skin temperatures on the firing rate and thermosensitivity of preoptic neurones, J. Physiol. (London), 240 (1974) 639-660. 7 Bullock, T.H., Cooling and integration in receptors and central afferent systems. In M.S. Laverack and D.J. Cosens (Eds.), Sense Organs, Blackie and Son, Glasgow, 1981, pp. 366-380. 8 Carlisle, H.J. and Ingram, D.L., The effects of heating and cooling the spinal cord and hypothalamus on thermoregulatory behavior in the pig, J. Physiol. (London), 231 (1973) 353-364. 9 Carpenter, D.O., Ionic and metabolic bases of neural thermosensitivity, Fed. Proc. Fed. Am. Soc. Exp. Biol., 40 (1981) 2808-2813. 10 Chu, N.-S. and Keenan, C.L., Effects of ethanol (E) on
mechanisms underlying thermosensitivity in D R neurons. Intracellular recordings using current- and voltage-clamp techniques may yield m o r e definitive results on the intrinsic nature of central thermosensitive neurons 33, including those in the raphe nuclei. The involvement of 5-HT either as the transmitter of the D R thermosensitive neurons or as a m o d u l a t o r of the discharge frequencies of these cells will have to be investigated pharmacologically. Presently, the in vivo w o r k of Cronin and B a k e r tl and Bligh 3 coupled with our in vitro study suggest that the D R m a y represent part of a 5-HT midbrain t h e r m o r e g u l a t o r y system serving to influence behavioral and physiological modifications necessary for maintaining thermostasis.
This work was s u p p o r t e d in part by an R C D A to N.-S.C., AA00049, an.d N I A A A Grant R03AA0681801 to C . L . K .
spontaneous activity of dorsal raphe (DR) neurons in vitro, Soc. Neurosci. Abstr., 9 (1983) 1232. 11 Cronin, M.J. and Baker, M.A.. Heat-sensitive midbrain raphe neurons in the anaesthetized cat, Brain Research, 110(1976) 175-181. 12 Dickenson, A.H., Specific responses of rat raphe neurones to skin temperatures, J. Physiol. (London), 173 (1977) 277-293. 13 Eisenman. J.S., Unit activity studies of thermoresponsive neurons. In J. Bligh and R.E. Moore (Eds.), Essays on Temperature Regulation, Elsevier, New York, 1972, pp. 55-69. 14 Hardy, J.D., Models of temperature-regulation - - a review. In J. Bligh and R.E. Moore (Eds.), Essays on Temperature Regulation, Elsevier, New York, 1972, pp. 163-186. 15 Hellon, R.F., Neurophysiology of temperature regulation: problems and perspectives, Fed. Proc. Fed. Am. Soc. Exp. Biol., 40 (1981) 2804-2807. 16 Hensel, H., Therrnoreception and Temperature Regulation, Academic, New York, 1981, pp. 33-79. 17 Hori, T. and Harada, Y., Responses of midbrain raphe neurons to local temperature, Pfluegers Arch., 364 (1976) 205-207. 18 Hori, T., Nakashima, T., Hori, N. and Kiyohara, T., Thermosensitive neurons in hypothalamic tissue slices in vitro, Brain Research, 186 (1980) 203-207. I9 Hori, T., Nakashima, T., Kiyohara, T., Shibata, M. and Hori, N., Effects of calcium removal on thermosensitivity of preoptic neurons in hypothalamic slices, Neurosci. Lett., 20 (1980) 171-175. 20 Jacob, J.J. and Girault, .1.M.T., 5-Hydroxytryptamine. In
194
21
22
23
24
25
26
27 28
29
30
P. Lomax and E. Schonbaum (Eds.), Body Temperature, Dekker, New York, 1979, pp. 183-230. Jahns, R., Different projections of cutaneous thermal inputs to single units of the midbrain raphe muscle, Brain Research, 101 (1976) 355-361. Kelso, S.R. and Boulant, J.A., Effects of synaptic blockade on thermosensitive neurons in hypothalamic tissue slices, Am. J. Physiol., 243 (1982) R480-R490. Kelso, S.R., Perlmutter, M.N. and Boulant, J.A., Thermosensitive single-unit activity of in vitro hypothalamic slices, Am. J. PhysioL, 242 (1982) R77-R84. Lomax, E., Historical development of concepts of thermoregulation. In P. Lomax and E. Schonbaum (Eds.), Body Temperature, Dekker, New York, 1979, pp. 1-23. Moore, R.Y., The anatomy of central serotonin neuron system in the rat brain. In B.L. Jacobs and A. Gelperin (Eds.), Serotonin Neurotraasmission and Behavior, MIT Press, Cambridge, MA, 1981, pp. 35-70. Mosko, S.S. and Jacobs, B.L., Midbrain raphe neurons: spontaneous activity and response to light, Physiol. Behav., 13 (1974) 589-593. Mosko, S.S. and Jacobs, B.L., Recording of dorsal raphe unit activity in vitro, Neurosci. Lett., 2 (1976) 195-200. Myers, R.D., The role of hypothalamic serotonin in thermoregulation. In J. Barchus and E. Usdin (Eds.), Serotonin and Behavior, Academic, New York, 1973, pp. 293-302. Myers, R.D., Impairment of thermoregulation, food, and water intakes in the rat after hypothalamic injections of 5,6dihydroxytryptamine, Brain Research, 94 (1975) 491-506. Myers, R.D. and Waller, M.B., Thermoregulation and se-
31
32
33
34
35 36
37
38
rotonin. In W.B. Essman (Ed.), Serotonin in Health and Disease, Vol. H, Spectrum Publications, New York, 1978, pp. 1-67. Nakayama, T. and Hardy, J.D., Unit responses in the rabbit's brain stem to changes in brain and cutaneous temperature, J. Appl. Physiol., 27 (1969) 848-857. Nakayama, T. and Hori, T., Effects of anaesthetic and pyrogen on thermal sensitive neurons in the brain stem, J. Appl. Physiol., 34 (1973) 351-355. Nelson, D.O. and Prosser, C.L., Intracellular recordings from thermosensitive preoptic neurons, Science, 213 (1981) 787-789. Reaves, T.A. and Hayward, J.N., Hypothalamic and extrahypothalamic thermoregutatory centers. In P. Lomax and E. Schonbaum (Eds.), Body Temperature, Dekker, New York, 1979, pp. 39-70. Satinoff, E., Neural organization and evolution of thermal regulation in mammals, Science, 201 (1978) 16-22. Steinbusch, H.W.M. and Nieuwenhuys, R., Localization of serotonin-like immunoreactivity in the central nervous system and pituitary of the rat, with special references to the innervation of the hypothalamus. In B. Haber (Ed.), Advances in Experimental Medical Biology, Vol. 133, Plenum, New York, 1981, pp. 7-35. Weiss, B.L. and Aghajanian, G.K., Activation of brain serotonin metabolism by heat: role of midbrain raphe neurons, Brain Research, 26 (1971) 37-48. Whittow, G.C., Evolution of thermoregulation. In G.C. Whittow (Ed.), Comparative Physiology of Thermoregulation, Vol. III, Academic, New York, 1973, pp. 201-258.
189
BRE 22239
Thermosensitivity of dorsal raphe neurons in vitro C. Larry Keenan and Nai-Shin Chu Department of Neurology, California College of Medicine, University of California at Irvine, lrvine, CA 92717 (U. S. A. ) (Accepted 27 January 1987) Key words: Brainstem; Dorsal raphe; Thermoregulation
Thermosensitivity of raphe neurons was studied in tissue slices of rat brainstems (400-500 !tm). Measurement of activity of single cells in the dorsal raphe region of the slices revealed that the majority of neurons (89%) were sensitive to changes in temperature. Over the range 34 to 42 °C, 3 classes of thermosensitive cells were found: warm (61%), cold (15%) and biphasic type cells (13%). Many dorsal raphe neurons may be intrinsically temperature sensitive and may serve as extrahypothalamic thermodetector components in the integrative process of central thermoregulation. Identification and characterization of central thermoregulatory neurons are important goals in the study of neural substrates underlying thermostasis, a homeostatic function in mammals vital for their survival. Studies over several decades have implied an hierarchical arrangement of parallel centers capable of integrating heat-loss, heat-production and heatconservation mechanisms during the process of thermostasis 2'3'8Aa'15"24'35. The study of these neuronal populations may reveal the processing mechanisms of central neurons that function as integrators or detectors at the interface between peripheral and central thermal inputs to regulate responses of thermoeffectors. The raphe complex in the brainstem is a major extrahypothalamic site proposed to have thermoregulatory functions lt'12A7'21'34'37. Here we report that dorsal raphe (DR) neurons in an in vitro slice preparation 1°'27 retain thermosensitivity in the absence of ascending or descending inputs. A preliminary report appeared in abstract form earlier 1°. Both warm-responding and cold-responding D R cells exhibited linear or non-linear responses to temperature changes. Thermal coefficients and Ql0'S of the cells studied resemble those reported for D R in vivo and hypothalamic temperature-sensitive cells in vivo and in vit-
ro 5'6'1t'18'21'23. To our knowledge, the D R cells are the only other central extrahypothalamic neurons with thermoregulatory functions investigated to date that retain thermosensitivity in vitro. Our findings that the thermosensitivity of these deafferented cells can be manipulated in vitro provide a unique opportunity to further our understanding of the complex, multiple integrating systems involved in thermoregulation in mammals. Although thermosensitivity in a neuron does not necessarily imply that the cell has a role in thermoregulation 9, on the basis of numerous studies, D R neurons are considered candidates for having a thermoregulatory role (for reviews see refs. 3, 15, 28, 35 and 38). Fluorescence histochemistry and selective brain lesion studies have shown that the majority of serotonergic neurons are localized to the brainstem raphe nuclei 16"2°'25'36'37. Lesions of afferent pathways containing axons from spinal and cutaneous thermoreceptors that are presumed to transmit thermosensory information to hypothalamic structures result in degeneration of neurons in the raphe nuclei and the inability of several species to thermoregulate at low ambient temperature ~6. Injection of serotonin (5HT) causes temperature changes, as do several pharmacological manipulations that either enhance or de-
Correspondence: C.L. Keenan. Present address: Beckman Research Institute of the City of Hope, 1450 E. Duarte Rd., Duarte, CA 91010, U.S.A.
I1" A
m
34°C~*--b~
ii '°
42°C
~ Recorders & Monitors
I
o
J--~elethermometer 1 L
lO
B.
Spikes/secI 1.0min bJgll~k= I,
. ~- "t--1-- ........
b--~----~q
| ~ ['"L'= "~"Ld'UL li i e......
i %,k~lmBlllllll~PdA4~,-
---'~
,,14/Jlf
[lo s,ik,s
__
37 °C 32
Fig. 1. A: schematic drawing of experimental recording set-up. A, the microelectrode and micromanipulator assembly used to record the single unit activity. The tissue slices (B) are supported by a nylon netting and submerged in ACSF. The left inset is a drawing of a transversely cut brain slice at the level of the inferior colliculus (IC) approximately 600/~m anterior to the locus coeruleus. Electrode placement into the dorsal raphe (DR) nucleus is easily accomplished by visual guidance using a dissecting microscope. Bar = 2.0 mm. The heating element (C) heats the ACSF (D) in the outer chamber (E). A needle valve (F) controls the flow rate of the ACSF which is saturated with 95% 0 2 - 5 % CO 2 (G). The inset to the right is an example of the extracellular action potential recorded from a warmsensitive DR neuron and the characteristic waveform differences (indicated by the arrows and the duration of the a and b components of the potentials) that accompany changes in firing rate (FR) when temperature is changed. B: polygraph display of FR responses of a warm-sensitive DR neuron to temperature changes. The action potentials were amplified (xl00) with frequency filter cut-offs at 100 Hz and 10 kHz and recorded on magnetic tape while the unit activity was monitored on a storage oscilloscope. Simultaneously the spikes were converted to pulses of constant size and duration by a window discriminator (WPI). The output of the discriminator was counted linearly by a ratemeter and recycled every second. The output of the ratemeter was displayed by a vertical deflection on a polygraph (Beckman Dynograph R612) as shown in the top trace of the example. Measurements and analyses were made during or after the experiments from such polygraph records which displayed the FR (spikes/s) obtained, a slope integration of FR obtained via the output of the window discriminator and resetting integrator (Beckman 9837B) (middle trace), and a calibrated DC voltage output (bottom trace) from the bath thermistor (Yellowspring Telethermometer). For each neuron recorded, temperature was varied at least twice in both the ascending and descending directions over the range of 34 to 42 °C. In the example shown, the discontinuity on the descending portion of the temperature change consists of 6.8 min. Rate of temperature change never exceeded 0.02 °C/s, so that the temperature effects were assumed primarily to reflect static responses of thermodetectors rather than dynamic responses, although the latter cannot be completely ruled out and may contribute to the effects.
191 c r e a s e b r a i n 5 - H T levels 2°29'3°. Single unit studies in
g u l a t i o n and a direct i n f l u e n c e of t h e r m o s e n s i t i v e
v i v o h a v e s h o w n that e i t h e r p e r i p h e r a l o r local t e m -
D R n e u r o n s on the t h e r m o r e g u l a t o r y cells of t h e hy-
p e r a t u r e c h a n g e s alter firing rates of d o r s a l and o t h e r r a p h e n e u r o n s 1U2'15'17'21. L o c a l t h e r m a l s t i m u l a t i o n
pothalamus.
of dorsal r a p h e cells in v i v o alters firing rates of ther-
t h e r m o s e n s i t i v e , the effect of t e m p e r a t u r e on the fir-
m o s e n s i t i v e units in the h y p o t h a l a m i c r e g i o n s k n o w n
ing rates of 57 D R cells w e r e s t u d i e d e l e c t r o p h y s i o -
to be i n v o l v e d in t h e r m o r e g u l a t i o n while the o p p o -
logically in b r a i n s t e m slices o b t a i n e d f r o m adult rats.
site e x p e r i m e n t has no effect o n r a p h e t h e r m o s e n s i tive n e u r o n s 1t'13'31'32. T h e s e results suggest the par-
T h e slices w e r e p r e p a r e d using p r e v i o u s l y d e s c r i b e d
ticipation of r a p h e s e r o t o n e r g i c n e u r o n s in t h e r m o r e -
uously p e r f u s e d with artificial c e r e b r o s p i n a l
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T o i n v e s t i g a t e w h e t h e r D R cells are intrinsically
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Fig. 2. A: responses of thermosensitive DR neurons to temperature changes. Top: thermosensitivities of two cells with bell-shaped curves. Middle: thermosensitive responses of two DR cells illustrate the increase in firing rate as temperature was increased. Bottom: responses of two neurons which increased firing rates as temperature was lowered. Mean Frs were calculated for each degree Celsius (Tx) over the temperature range from TX- 0.5 °C to Tx + 0.5 °C from polygraph records as in Fig. 1B and the data plotted as a function of temperature. B: FR/temperature functions of warm-sensitive cells (top) and cold-sensitive cells (bottom). Mean FRs as functions of temperature (T i, the initial, lowest temperature used; Tx, any temperature in the arithmetic progression Ti, Ti÷ 1, Ti+2 . . . Ti÷(,_l )) have been plotted as individual points and then fitted by least-squares regression analysis using a microcomputer and a curve-fitting program (solid lines). The majority of warm-sensitive neurons respond to temperature as illustrated by the curves in the top graph obtained from 3 cells. The curves are exponentials of the form FR = Aekrwith A = slope, k = constant, and T = (T x- Ti) x 10-1 (°C). The majority of the cold-sensitive neurons have sigmoid functions as illustrated by 3 cell responses to temperature in the bottom graph. The functions are described by the equation FR = A x (1 + BkT)-1. The value B = expected y-intercept. Other values have the same meanings as in the equation for the warm-responding cells.
192 (ACSF) (Fig. 1). Conventional electrophysiological methods were used to record single unit activity (details are given in Fig. 1B). Extracellular action potentials from the neurons were recorded a minimal 10 min at 37 °C to assure stability of each cell and to obtain a baseline firing rate (FR). The majority (74.3%) of DR cells studied in vitro had spontaneous FRs of <6 spikes/s at 37 °C. This compares favorably with discharge rates recorded in vivo and in vitro IA°'26'27. Three types of temperature-sensitive cells were found in the DR (Fig. 2A). Biphasic responses in firing rates were observed in 13% of the cells (Fig. 2A, top). These cells had temperature-response curves that were bell-shaped. In 61% of the D R cells studied, FRs increased as temperature was raised from 34 to 42 °C (Fig. 2A, middle). Increasing discharges were observed in 15% of the cells in response to temperature decreases over the same range (Fig. 2A, bottom). Both warm- and cold-sensitive cells exhibited temperature-dependent changes in action potential waveform. An example of changes recorded from a warm-sensitive cell is shown in the right inset of Fig. 1A. Only 11% of the D R cells studied were considered temperature insensitive because they did not respond to temperature changes or responded with Q10's ~< 2. The low rate of firing in vivo and in vitro was the principle reason for using the al0 effect as the criterion for determining thermosensitivity of a cell. It is for this reason that we report a higher percentage of temperature-sensitive DR neurons (89%) than that reported for preoptic/anterior hypothalamic cells (40-50%) 4'5"32. Thermal coefficients and Ql0's were obtained over the temperature ranges to which the cells were most thermosensitive. Thermal coefficients ranged from 0.1 to 1.0 impulses/s-°C for warm-sensitive cells, and from -0.1 t o - 0 . 8 impulses/s.°C for cold-sensitive cells. Ql0'S ranged from 2 to >100 for warm-respond-
ing cells and 0.006 to 0.5 for cold-responding cells when extrapolated from the thermosensitive ranges of the cells (see Table I for summary). Warm-responding neurons were generally most responsive at temperatures >39 °C and cold-responding cells were most sensitive to temperatures <37 °C. Analysis of the FR functions of the thermosensitive cells using a microprocessor and a curve-fitting program (Jawston, 1982, unpublished) revealed that the majority of the warm-sensitive neurons (28/35) had temperature-dependent FRs best fit by single exponentials of the form FR = Ae kT (Fig. 2B, top). Temperature-dependent FRs of the majority of coldsensitive cells (5/9) had functional relationships best described by sigmoid curves of the form FR = A x (1 + Bkr) -1 (Fig. 2B, bottom). Two cold-sensitive cells had discharge functions that 'decayed exponentially as temperature was increased, while several warmand cold-sensitive cells had linear discharge functions (correlation coefficient > 0.95). The significance of the different discharge functions of the warm- and cold-sensitive cells is not clear. Input-output curves of first and higher order sensory cells have been used in the past as criteria by which to judge whether the cells were primary (involved in the transduction of stimulus intensity) or secondary t6. Although there are many examples of neuronal responses coding stimulus intensity as some power function, there are also sufficient examples of stimulus intensity-frequency functions of sensory cells that are linear, logarithmic, or some higher order polynomial depending on the range of the stimulus or the responsiveness and type of the sensory receptors involved. At present one must conclude that there are no general input-output functions applying to all sensory cells 7. Nevertheless, the similarities between the temperature-dependent discharge functions of DR cells and thermosensitive neurons in the anterior
TABLE I Classification of DR neurons according to FR responses to temperature increases Cell type
Response to T (34-42 °C)
No. of cells
%
Qlo (range)
Thermal coef. (To) (range)
Warm Cold Biphasic Insensitive
Increased FR Decreased FR Maxima/minima FR near 38 °C Small to no change in rate
35 9 7 6
61 15 13 I1
5- 180 0.006-0.5 <2
0.1-1.0 (-0.1)-(-0.8) -0.01 < Tc < (0.1)
193 h y p o t h a l a m u s , in vivo and in vitro, and cutaneous t h e r m o r e c e p t o r s are striking 43H6"23. O u r present results suggest that the D R thermosensitive n e u r o n s are intrinsically capable of t h e r m o d e t e c t i o n . W e cannot, however, rule out the possibility that some of the D R cells are driven by interneurons which are true t h e r m o d e t e c t o r s . F u r t h e r studies in which synaptic activity is blocked have to be c o m p l e t e d to assess this possibility. Thermosensitivity of w a r m - r e s p o n d i n g p r e o p t i c / a n t e r i o r h y p o t h a l a m i c cells in tissue slices is u n c h a n g e d when synaptic mechanisms are b l o c k e d by lowering Ca 2÷ concentration and raising Mg 2+ concentration of the external m e d i u m . Kelso and Boulant 22, and H o r i et al. t9 interpret this as evidence that those cells are p r o b a b l y t h e r m o d e t e c t o r s , and thus are intrinsically thermosensitive, even though an equally compelling a r g u m e n t might be that changes induced by Ca 2+ m a n i p u l a t i o n s simply reflect Ca 2+ involvement in transducer mechanisms t6. Similar experiments may permit further insight into
1 Aghajanian, G.K. and Weiss, B.L., Block by LSD of the increase in brain serotonin turnover induced by elevated ambient temperature, Nature (London), 220 (1968) 795-796. 2 Bligh, J., Neuronal models of mammalian temperature regulation. In J. Bligh and R.E. Moore (Eds.), Essays on Temperature Regulation, Elsevier, New York, 1972, pp. IXXIII. 3 Bligh, J., The control neurology of mammalian thermoregulation, Neuroscience, 4 (1979) 1213-1236. 4 Boulant, J.A., Hypothalamic mechanisms in thermoregulation, Fed. Proc. Fed. Am. Soc. Exp. Biol., 40 (1981) 2843-2850. 5 Boulant, J.A. and Demieville, H.N.. Responses of thermosensitive preoptic and septal neurons to hippocampal and brain stem stimulation, J. Neurophysiol., 40 (1977) 1356-1368. 6 Boulant, J.A. and Hardy, J.D., The effect of spinal and skin temperatures on the firing rate and thermosensitivity of preoptic neurones, J. Physiol. (London), 240 (1974) 639-660. 7 Bullock, T.H., Cooling and integration in receptors and central afferent systems. In M.S. Laverack and D.J. Cosens (Eds.), Sense Organs, Blackie and Son, Glasgow, 1981, pp. 366-380. 8 Carlisle, H.J. and Ingram, D.L., The effects of heating and cooling the spinal cord and hypothalamus on thermoregulatory behavior in the pig, J. Physiol. (London), 231 (1973) 353-364. 9 Carpenter, D.O., Ionic and metabolic bases of neural thermosensitivity, Fed. Proc. Fed. Am. Soc. Exp. Biol., 40 (1981) 2808-2813. 10 Chu, N.-S. and Keenan, C.L., Effects of ethanol (E) on
mechanisms underlying thermosensitivity in D R neurons. Intracellular recordings using current- and voltage-clamp techniques may yield m o r e definitive results on the intrinsic nature of central thermosensitive neurons 33, including those in the raphe nuclei. The involvement of 5-HT either as the transmitter of the D R thermosensitive neurons or as a m o d u l a t o r of the discharge frequencies of these cells will have to be investigated pharmacologically. Presently, the in vivo w o r k of Cronin and B a k e r tl and Bligh 3 coupled with our in vitro study suggest that the D R m a y represent part of a 5-HT midbrain t h e r m o r e g u l a t o r y system serving to influence behavioral and physiological modifications necessary for maintaining thermostasis.
This work was s u p p o r t e d in part by an R C D A to N.-S.C., AA00049, an.d N I A A A Grant R03AA0681801 to C . L . K .
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