Physiology & Behavior, Vol. 20, pp. 117-120. Pergamon Press and Brain Research Publ., 1978. Printed in the U.S.A.
Diurnal Changes in Serotonin Content of Frontal Pole and Pain Sensitivity in the R a t I ARTHUR J. SCHLOSBERG 2 AND JOHN A . HARVEY
Department o f Psychology, University o f Iowa, Iowa City, IA 52242 (Received 18 December 1976) SCHLOSBERG, A. J. AND J. A. HARVEY. Diurnal changes in serotonin content or frontal pole and pain sensitivity in the rat. PHYSIOL. BEHAV. 20(2) 117-120, 1978. - This study examined the relationship between diurnal variations in pain sensitivity, and serotonin content of frontal pole, hippocampus and amygdala in the rat. Rats demonstrated significantly longer paw-lick latencies in response to a painful thermal stimulus during light hours as compared with dark hours. Similarly, all three brain regions demonstrated a significantly higher serotonin content during light as compared with dark hours. There was a significant correlation (0.66) between paw-lick latencies and serotonin content of frontal pole but not with serotonin content of hippocampus or amygdala. The occurrence, in a normal animal, of a heightened sensitivity to a painful stimulus during dark hours when serotonin content of brain is also lowest is consistent with studies demonstrating that lowering of serotonin content by means of lesions, drugs or tryptophan deficient diets also produces an increased sensitivity to painful stimuli. Diurnal rhythm
Serotonin
Pain sensitivity
Frontal pole
SEROTONIN content exhibits a systematic diurnal variation in whole brain [3] as well as in discrete regions such as frontal pole, frontal cortex, hippocampus, caudate, amygdala, hypothalamus and brain stem [5, 8, 11, 17, 18, 19]. In all of these brain regions, serotonin content is highest during light hours, the period of inactivity and sleep for the rat, and lowest during dark hours, the period of maximum activity in the rat [17]. The largest diurnal fluctuations in serotonin content occur in regions receiving serotonergic inputs (i.e., terminal rich areas) while the region of the brain stem containing the cell bodies of the serotonin neurons demonstrates the least fluctuation [11,18]. Although all regions of brain listed above demonstrate the greatest content of serotonin during light hours, these rhythmic fluctuations are not completely in phase with each other [ 11,18]. At least part of the diurnal fluctuations in serotonin content appears to be related to changes in the synthesis of serotonin with synthesis being highest during light hours and decreasing by approximately 50% during dark hours [ 11 ]. It has been previously demonstrated that decreases in serotonin content of brain result in an increased sensitivity to painful stimuli regardless of whether the decrease in serotonin is produced by: (1) lesions which interrupt the ascending serotonergic pathways [2, 6, 9, 12, 15]; (2) p-chlorophenylalanine [7,20] ;or (3) a tryptophan deficient diet [141. The lesion studies suggest that the decreases in serotonin content responsible for the increased pain sensitivity occur in regions rostral to the septal region [9]. In particular, septal lesions produce significant decreases in serotonin
Hippocampus
Amygdala
content of frontal poles and hippocampus but not of amygdala [10]. If the proposed relationship between serotonin content and pain sensitivity suggested by these studies is correct then an animal's pain sensitivity should be greatest at night when serotonin content in rostral telencephalic regions such as frontal poles and hippocampus is lowest. It was found that normal rats exhibited an increased pain sensitivity during dark as compared with light hours, as measured by a 55% decrease in paw-lick latencies during dark hours [8]. A separate group of rats was assayed for serotonin content of frontal poles, hippocampus and amygdala during the light and dark hours at which animals had been tested for pain thresholds. In agreement with previous findings, the serotonin content of all 3 regions was significantly lower at night than during the day [8]. The present experiment was directed at a further examination of the relationship between diurnal variations in pain sensitivity and serotonin content of frontal poles, hippocampus and amygdala in the same animal in order to more clearly specify those areas of brain which may demonstrate a high degree of correlation between serotonin content and pain sensitivity. METHOD
Animals Animals were 17 male albino rats, 6 0 - 6 5 days of age, from Carworth Farms, NJ. The animals were housed one per cage in a constant temperature (23°C) colony room and were given free access to Purina Lab Chow and water throughout the duration of the experiment. The animal
This research was supported by U.S.P.H.S. Grant No. MH 16841 (J.A.H.), and by U.S.P.H.S. predoctoral fellowship MH 08333 to A.J.S. We thank Irwin Lucki and Kenny Simansky for their generous assistance during portions of this research. 2Present address: Department of Nutrition and Food Sciences, M.I.T., Cambridge, MA 02139. 117
IIS colony was maintained cycle with lights on at care of the animals was hr on alternating days. rats were unhandled and
SCHLOSBER(; ANI) ttARVt:5 on a 12-hr alternating light-dark 0800 hr. General maintenance and scheduled between 0800 and 0830 Except for behavioral testing, the left undisturbed.
Behavioral Methods and Procedure Pain sensitivity was measured by a modification of the hot-plate method of Eddy and Leimbach [4]. A Plexiglas cylinder, 35.5 cm high and 18.8 cm inner diameter, was used to restrict rats to the heated surface of a copper plate, 30.0 cm long and 25.0 cm wide. The copper plate was attached to the bottom of a piece of Plexiglas (1.2 cm thick) with a hole 20.0 cm in diameter; this assemblage was secured in an insulated tank with a capacity of 58 1 of distilled water. The top of the tank was enclosed leaving exposed the copper plate (18.8 cm in diameter). The copper plate, submerged 0.6 cm below the surface of the water, was heated to a temperature of 51.5 ° -+ 0.5°C employing a thermoregulated water circulating pump (Bench Scale Equipment Co., Dayton, OH). Temperature of the surface of the hot-plate as well as the tank water was monitored by a Tel-Tru thermometer (Tel-Tru Manufacturing Co., Rochester, NY) and bulb thermometer, respectively. At the time of testing, animals were individually transported in a cage other than the home cage to the testing room. Simultaneous with placement of an animal on the hot-plate, a 1/100th sec timer was hand-activated. The first lick of a fore- or hind-paw was used to determine response latency (recorded in 0.10 sec) to the heat stimulus. Therefore, paw-lick latencies were defined as the time in seconds between placement of the animal on the hot-plate and the initial appearance of a,paw-lick response. In the absence of this response, a 45.0 sec cut-off was employed, and the animal was removed from the plate. Four animals demonstrated 45 sec latencies and in each case this occurred during testing in the light cycle. Testing during the animals' dark cycle was conducted under red-light conditions. A red flashlight was used to transport animals from the colony to the test room which was illuminated by two red 75 W light bulbs. Except for this illumination, all other lights between the colony and test rooms were extinguished. Employing a counter-balanced design, ten animals were first tested during their light (day) cycle (between 1230 and 1600 hr) and then during their dark (night) cycle between 2300 and 0400 hr with approximately 5 8 - 6 0 hr intervening between these two tests. Seven animals were tested in the reverse order. Testing began 3 weeks after arrival of animals in the colony. Brain Dissection and Serotonin Assay Immediately after each animal completed its second hot-plate test during light or dark conditions, it was transported to an adjacent room for decapitation (under red light during dark periods). Upon decapitation, the brain was rapidly removed and placed on a Plexiglas platform over ice. Three brain areas were dissected for later determination of 5-HT content: frontal pole, hippocampus, and amygdala. To obtain the frontal poles, the olfactory bulbs were removed from all brains and a coronal section was made at an acute angle of approximately 5 4 - 5 6 ° and 3.0 mm caudal to the tips of the frontal poles. To obtain
the hippocampus the occipital and temporal regions were peeled forward and the telencephalon was separated from the brainstem using the optic chiasm and tile base of the' coronal radiations as dissecting landmarks. Both hippo-campi were then removed by peeling each back from the ventral surface of the posterior telencephalon and section.ing at the base of the lateral ventricles. Lastly. the amygdala was removed from each hemisphere by cutting along the rhinal fissure. This tissue was bound rostrally by the lateral olfactory tract, and extended caudally to include periamygdaloid cortex, amygdaloid complex, and entorhinal cortex. Upon its removal, each tissue sample was weighed, wrapped in aluminum foil, frozen, and stored on dry-ice. All tissue samples taken from animals given their second hot-plate test under light or dark periods were assayed together under the same extraction conditions within 24 hr of decapitation. Mean tissue weights (N = 17) were (rag -+SEM): frontal pole, 53.5 + t.8; hippocampus, 112.9 ± 3.1 ; and amygdala, 103.9 -+ 2.5. Serotonin Assay Serotonin content was determined according to the o-pthalaldehyde (OPT) method [ 13] after butanol extraction [1]. The fluorescence of samples and internal standards (serotonin creatinine sulfate, Regis Chemical Co.) were read in an Aminco-Bowman spectrophotoflourometer equipped with a Xenon lamp. Uncorrected activation and emission wave-lengths were 360 and 484 nm, respectively. Emission spectra were obtained for each sample and these were always found to be the same as that of authentic serotonin. Serotonin content was expressed as /.~g/g of tissue, fresh weight. RESULTS All rats irrespective of order of testing demonstrated lower paw-lick latencies during dark as compared with light hours. A two factor (lighting condition by test order) mixed analysis of variance, with repeated measures on one factor (test order) was performed on paw-lick latency. The only significant effect was found for lighting condition (light vs dark), F( I ,1 5 ) = 27.23, p0.10, or for the main effect of test order, F(1,15) = 0.14, p>0.20. The data for light and dark periods, irrespective of test order, are presented in Table 1. The pawqick latencies during the light period were 74.7% longer than during the dark period of testing indicating a higher pain threshold during light as compared with dark hours. The behavioral and neurochemical data for the 10 TABLE 1 PAW-LICK LATENCIES AS A FUNCTION OF TESTING DURING DARK OR LIGHT HOURS
Paw-lick Latencies (see) Mean -+ SEM
Percent change in latency from Dark to Light
N
Dark Hours
Light Hours
Mean _+ SEM
17
16.34 _+_ 1.28
27.77 __+2.67
74.7 _+ 14.4"
*Percent change is significant at p
Diurnal Changes in Serotonin Content of Frontal Pole and Pain Sensitivity in the R a t I ARTHUR J. SCHLOSBERG 2 AND JOHN A . HARVEY
Department o f Psychology, University o f Iowa, Iowa City, IA 52242 (Received 18 December 1976) SCHLOSBERG, A. J. AND J. A. HARVEY. Diurnal changes in serotonin content or frontal pole and pain sensitivity in the rat. PHYSIOL. BEHAV. 20(2) 117-120, 1978. - This study examined the relationship between diurnal variations in pain sensitivity, and serotonin content of frontal pole, hippocampus and amygdala in the rat. Rats demonstrated significantly longer paw-lick latencies in response to a painful thermal stimulus during light hours as compared with dark hours. Similarly, all three brain regions demonstrated a significantly higher serotonin content during light as compared with dark hours. There was a significant correlation (0.66) between paw-lick latencies and serotonin content of frontal pole but not with serotonin content of hippocampus or amygdala. The occurrence, in a normal animal, of a heightened sensitivity to a painful stimulus during dark hours when serotonin content of brain is also lowest is consistent with studies demonstrating that lowering of serotonin content by means of lesions, drugs or tryptophan deficient diets also produces an increased sensitivity to painful stimuli. Diurnal rhythm
Serotonin
Pain sensitivity
Frontal pole
SEROTONIN content exhibits a systematic diurnal variation in whole brain [3] as well as in discrete regions such as frontal pole, frontal cortex, hippocampus, caudate, amygdala, hypothalamus and brain stem [5, 8, 11, 17, 18, 19]. In all of these brain regions, serotonin content is highest during light hours, the period of inactivity and sleep for the rat, and lowest during dark hours, the period of maximum activity in the rat [17]. The largest diurnal fluctuations in serotonin content occur in regions receiving serotonergic inputs (i.e., terminal rich areas) while the region of the brain stem containing the cell bodies of the serotonin neurons demonstrates the least fluctuation [11,18]. Although all regions of brain listed above demonstrate the greatest content of serotonin during light hours, these rhythmic fluctuations are not completely in phase with each other [ 11,18]. At least part of the diurnal fluctuations in serotonin content appears to be related to changes in the synthesis of serotonin with synthesis being highest during light hours and decreasing by approximately 50% during dark hours [ 11 ]. It has been previously demonstrated that decreases in serotonin content of brain result in an increased sensitivity to painful stimuli regardless of whether the decrease in serotonin is produced by: (1) lesions which interrupt the ascending serotonergic pathways [2, 6, 9, 12, 15]; (2) p-chlorophenylalanine [7,20] ;or (3) a tryptophan deficient diet [141. The lesion studies suggest that the decreases in serotonin content responsible for the increased pain sensitivity occur in regions rostral to the septal region [9]. In particular, septal lesions produce significant decreases in serotonin
Hippocampus
Amygdala
content of frontal poles and hippocampus but not of amygdala [10]. If the proposed relationship between serotonin content and pain sensitivity suggested by these studies is correct then an animal's pain sensitivity should be greatest at night when serotonin content in rostral telencephalic regions such as frontal poles and hippocampus is lowest. It was found that normal rats exhibited an increased pain sensitivity during dark as compared with light hours, as measured by a 55% decrease in paw-lick latencies during dark hours [8]. A separate group of rats was assayed for serotonin content of frontal poles, hippocampus and amygdala during the light and dark hours at which animals had been tested for pain thresholds. In agreement with previous findings, the serotonin content of all 3 regions was significantly lower at night than during the day [8]. The present experiment was directed at a further examination of the relationship between diurnal variations in pain sensitivity and serotonin content of frontal poles, hippocampus and amygdala in the same animal in order to more clearly specify those areas of brain which may demonstrate a high degree of correlation between serotonin content and pain sensitivity. METHOD
Animals Animals were 17 male albino rats, 6 0 - 6 5 days of age, from Carworth Farms, NJ. The animals were housed one per cage in a constant temperature (23°C) colony room and were given free access to Purina Lab Chow and water throughout the duration of the experiment. The animal
This research was supported by U.S.P.H.S. Grant No. MH 16841 (J.A.H.), and by U.S.P.H.S. predoctoral fellowship MH 08333 to A.J.S. We thank Irwin Lucki and Kenny Simansky for their generous assistance during portions of this research. 2Present address: Department of Nutrition and Food Sciences, M.I.T., Cambridge, MA 02139. 117
IIS colony was maintained cycle with lights on at care of the animals was hr on alternating days. rats were unhandled and
SCHLOSBER(; ANI) ttARVt:5 on a 12-hr alternating light-dark 0800 hr. General maintenance and scheduled between 0800 and 0830 Except for behavioral testing, the left undisturbed.
Behavioral Methods and Procedure Pain sensitivity was measured by a modification of the hot-plate method of Eddy and Leimbach [4]. A Plexiglas cylinder, 35.5 cm high and 18.8 cm inner diameter, was used to restrict rats to the heated surface of a copper plate, 30.0 cm long and 25.0 cm wide. The copper plate was attached to the bottom of a piece of Plexiglas (1.2 cm thick) with a hole 20.0 cm in diameter; this assemblage was secured in an insulated tank with a capacity of 58 1 of distilled water. The top of the tank was enclosed leaving exposed the copper plate (18.8 cm in diameter). The copper plate, submerged 0.6 cm below the surface of the water, was heated to a temperature of 51.5 ° -+ 0.5°C employing a thermoregulated water circulating pump (Bench Scale Equipment Co., Dayton, OH). Temperature of the surface of the hot-plate as well as the tank water was monitored by a Tel-Tru thermometer (Tel-Tru Manufacturing Co., Rochester, NY) and bulb thermometer, respectively. At the time of testing, animals were individually transported in a cage other than the home cage to the testing room. Simultaneous with placement of an animal on the hot-plate, a 1/100th sec timer was hand-activated. The first lick of a fore- or hind-paw was used to determine response latency (recorded in 0.10 sec) to the heat stimulus. Therefore, paw-lick latencies were defined as the time in seconds between placement of the animal on the hot-plate and the initial appearance of a,paw-lick response. In the absence of this response, a 45.0 sec cut-off was employed, and the animal was removed from the plate. Four animals demonstrated 45 sec latencies and in each case this occurred during testing in the light cycle. Testing during the animals' dark cycle was conducted under red-light conditions. A red flashlight was used to transport animals from the colony to the test room which was illuminated by two red 75 W light bulbs. Except for this illumination, all other lights between the colony and test rooms were extinguished. Employing a counter-balanced design, ten animals were first tested during their light (day) cycle (between 1230 and 1600 hr) and then during their dark (night) cycle between 2300 and 0400 hr with approximately 5 8 - 6 0 hr intervening between these two tests. Seven animals were tested in the reverse order. Testing began 3 weeks after arrival of animals in the colony. Brain Dissection and Serotonin Assay Immediately after each animal completed its second hot-plate test during light or dark conditions, it was transported to an adjacent room for decapitation (under red light during dark periods). Upon decapitation, the brain was rapidly removed and placed on a Plexiglas platform over ice. Three brain areas were dissected for later determination of 5-HT content: frontal pole, hippocampus, and amygdala. To obtain the frontal poles, the olfactory bulbs were removed from all brains and a coronal section was made at an acute angle of approximately 5 4 - 5 6 ° and 3.0 mm caudal to the tips of the frontal poles. To obtain
the hippocampus the occipital and temporal regions were peeled forward and the telencephalon was separated from the brainstem using the optic chiasm and tile base of the' coronal radiations as dissecting landmarks. Both hippo-campi were then removed by peeling each back from the ventral surface of the posterior telencephalon and section.ing at the base of the lateral ventricles. Lastly. the amygdala was removed from each hemisphere by cutting along the rhinal fissure. This tissue was bound rostrally by the lateral olfactory tract, and extended caudally to include periamygdaloid cortex, amygdaloid complex, and entorhinal cortex. Upon its removal, each tissue sample was weighed, wrapped in aluminum foil, frozen, and stored on dry-ice. All tissue samples taken from animals given their second hot-plate test under light or dark periods were assayed together under the same extraction conditions within 24 hr of decapitation. Mean tissue weights (N = 17) were (rag -+SEM): frontal pole, 53.5 + t.8; hippocampus, 112.9 ± 3.1 ; and amygdala, 103.9 -+ 2.5. Serotonin Assay Serotonin content was determined according to the o-pthalaldehyde (OPT) method [ 13] after butanol extraction [1]. The fluorescence of samples and internal standards (serotonin creatinine sulfate, Regis Chemical Co.) were read in an Aminco-Bowman spectrophotoflourometer equipped with a Xenon lamp. Uncorrected activation and emission wave-lengths were 360 and 484 nm, respectively. Emission spectra were obtained for each sample and these were always found to be the same as that of authentic serotonin. Serotonin content was expressed as /.~g/g of tissue, fresh weight. RESULTS All rats irrespective of order of testing demonstrated lower paw-lick latencies during dark as compared with light hours. A two factor (lighting condition by test order) mixed analysis of variance, with repeated measures on one factor (test order) was performed on paw-lick latency. The only significant effect was found for lighting condition (light vs dark), F( I ,1 5 ) = 27.23, p0.10, or for the main effect of test order, F(1,15) = 0.14, p>0.20. The data for light and dark periods, irrespective of test order, are presented in Table 1. The pawqick latencies during the light period were 74.7% longer than during the dark period of testing indicating a higher pain threshold during light as compared with dark hours. The behavioral and neurochemical data for the 10 TABLE 1 PAW-LICK LATENCIES AS A FUNCTION OF TESTING DURING DARK OR LIGHT HOURS
Paw-lick Latencies (see) Mean -+ SEM
Percent change in latency from Dark to Light
N
Dark Hours
Light Hours
Mean _+ SEM
17
16.34 _+_ 1.28
27.77 __+2.67
74.7 _+ 14.4"
*Percent change is significant at p
