Brain Research Bulletin, Vol. 25, pp. 797-802. Q Pergamon Press plc, 1990. Printed in the U.S.A
0361-9230190 $3.00 + .OO
Serotonin Release in Lateral and Medial Hypothalamus During Feeding and Its Anticipation DAVID H. SCHWARTZ, Department
LUIS HERNANDEZ
of Psychology,
Princeton
Received
AND BARTLEY
University, Princeton,
8 August
G. HOEBEL’
NJ 08544-1010
1990
SCHWARTZ, D. H., L. HERNANDEZ AND B. G. HOEBEL. Serotonin release in lateral and medial hypothalamus during feeding and its anticipation. BRAIN RES BULL 25(6) 797-802, 1990.-In the present experiments we extend previous findings that established a relationship between feeding behavior and hypothalamic serotonin as measured by in vivo microdialysis. The new re+ sult is hypothalamic release of serotonin in anticipation of eating when the animal sees and smells food. We have now verified brain serotonin peaks in four different ways: 1) a serotonergic reuptake blocker (fluoxetine 1 or 10 FM) in the perfusion medium raised basal levels of serotonin, 2) every sample was oxidized at two potentials using a dual potentiostat to confirm the voltage characteristics of each peak, 3) serotonin peaks were reduced by the selective serotonin cell body agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), thus helping confm that most of the serotonin observed in these experiments was neuronal in origin, and 4) lateral and medial hypothalamic microdialysis probes were used simultaneously to monitor the degree of diffusion from one to the other. The results show that extracellular serotonin increases at both sites during preingestive events as well as during eating, but not afterwards. Microdialysis
Feeding
Satiety
Serotonin
5-HIAA
injection of serotonin or its agonists such as fenfluramine inhibit feeding behavior (20, 21, 26, 32). Serotonin injections have some effect in all the medial hypothalamic (MH) regions, and the anorectic effect of fenfluramine is most potent in the paraventricular nucleus (PVN) compared to other hypothalamic regions (29). Conversely, lesions of the PVN attenuate fenfluramine-induced anorexia (2 1,3 1). These results are all consistent with a role for serotonin in an MH satiety system. The role of serotonin in the lateral hypothalamus (LH) is not as clear. Unlike the MH, lesions of the LH enhance fenfluramine-induced anorexia (6,7). In the LH, serotonin iontophoresis inhibits glucose-sensitive neurons but excites others (18). There is some segregation of projections to the medial and lateral hypothalamus from different serotonin cell groups (3,30). Electrophysiological studies of the raphe show that as a role serotonin cell activity varies with behavioral arousal level and does not change in relation to specific environmental or physiological stimuli (10,17). The exception to this rule is a group of serotonin cells that fii during chewing and grooming movements (23). Release of serotonin at hypothalamic terminals may depend heavily on presynaptic influences and therefore could become relatively independent of raphe cell body firing rates. From these pharmacological, anatomical and electrophysiological data, it is not clear what to predict about the relationship between natural
Extracellular
serotonin release and feeding, but in vivo techniques can now directly examine changes in extracellular serotonin in different hypothalamic regions. Microdialysis showed that in the medial region, including the PVN, the serotonin metabolite, 5-HIAA, peaked in the early part of the dark cycle if there was food to eat (28). This suggested that serotonin may be released in association with food intake. We have previously shown that extracellular serotonin in the LH increases during a large daytime meal (24). This left open the issues of dark-phase vs. light-phase eating, site specificity and whether local serotonin reuptake by the terminals was preventing us from detecting subtle changes in release. The present experiments compare light and dark phase eating, and compare the LH and MH using two microdialysis probes for each rat, and use a serotonin uptake blocker to amplify extracellular serotonin. In addition, a number of procedures were instituted to cross-validate the identification of serotonin and its local origin.
HYPOTHALAMIC
‘Requests for reprints should be addressed
Monoamine
METHOD
Subjects and Surgery Thirteen male Sprague-Dawley rats, weighing between 350 and 425 g were individually housed with food and water available ad lib on‘a light cycle of 12 h on/12 h off. Animals were anesthetized with a combination of pentobarbital (20 mg/kg) and ket-
to Bartley G. Hoebel, Department
797
of Psychology,
Princeton University,
Princeton,
NJ 08544-1010.
798
alar (40 mgikg) for implantation of 21-ga guide cannulas aimed at the perifomical LH and MH according to the atlas of Paxinos and Watson (22) at the following stereotaxic coordinates: LH guide cannulas, 6.5 mm anterior to the interaural line, 1.4 mm lateral to the midsagittal sinus, and 4 mm ventral to the level skull surface; MH guide cannulas: A 6.5 mm, L 0.4 mm, and V 4 mm. Probes would later extend 5 mm below the guide cannulas in order to pass into the target structure. Animals recovered for at least 1 week following surgery before a probe was inserted for an experimental session. During this recovery period an obdurator (26 ga, 10.5 mm) in the guide cannula prevented cannula blockage. Dialysis Procedure
Dialysis probes were constructed as previously described (13). Briefly, a 26-ga stainless steel tube was fitted with a porous (6000 molecular weight cutoff) cellulose fiber tip 0.2 mm outer-diameter and 3 mm long. A smaller 36-ga tube was fitted inside the 26-ga tube and served as an outlet. The probes had a relative recovery between 6% and 10% depending on the neurochemical (13). A syringe pump with a 2.5-ml gas-tight syringe perfused a high calcium Ringer’s solution through the probe at a flow rate of 1 pl/min (189 mM NaCl, 3.9 mM KCl, 3.37 mM CaCl, in Procedures 1 and 2, and 2.4 mM CaCl, in Procedure 3: pH 5.6). Fluoxetine was added to this Ringer as specified below. The dialysate exited the outlet tube into PE-10 tubing leading to a 400~1 polyethylene vial clipped to the rat’s headpiece. A double fluid swivel with a counterbalance arm ensured that the tubing did not get tangled as the rat moved around the cage. HPLC Analysis
Dialysates were analyzed by reverse phase high performance liquid chromatography with electrochemical detection (HPLCEC). Samples from the LH and MH were collected by removing the vials from the rat’s headpiece and injecting the contents directly into a Rheodyne valve with a 20-~1 sample loop. The sample was separated on a Brownlee IO-cm column with 3.2-mm bore and 3 PM, C-18 packing. The mobile phase contained 116.8 mM NaOH, 144.7 mM monochloroacetic acid, 1.38 mM l-octane sulfonic acid, 1.38 mM EDTA, and 5% V/V acetonitrile at pH 3.1. 5-HIAA and serotonin were eluted in that order and oxidized on two glassy carbon electrodes at 0.59 V and 0.68 V (Model 400 electrochemical detector with dual potentiostat, Princeton Applied Research Co.) at retention times of approximately 3 and 6 min respectively. The output was then relayed to a 2-pen chart recorder (Linseis Model L-6512). Serotonin peak heights were measured at both potentials and the ratio between the two potentials was computed. Experimental Procedure 1: Lateral and Medial Hypothalamic Serotonin During Light-Phase Feeding
Rats were housed in a room with the lights on at 0900 h and out at 2100 h. They were placed in the experimental chambers in the same room for at least three days and given free access to food and water to accustom them to the setting. On a subsequent day, food was removed at 2300 h and the following afternoon (1300 h) animals were offered access to a palatable mash of sweetened condensed milk and powdered Purina rat chow for two hours to familiarize them with deprivation and a novel, palatable food. On the day before the experimental session (between 1500 and 1800 h), the-obdurators were removed and dialysis probes were
SCHWARTZ,
HERNANDEZ
AND HOEBEL
inserted into the LH and MH guide shafts allowing simultaneous collection of samples. Ringer’s solution plus 10 pM fluoxetine flowed through the probe at 0.5 pJmin overnight to ensure steady baseline levels during the experiment on the following day. The flow rate was increased to 1 pl/min in the morning (0800 h) and the first sample was collected between 0900 and 0930 h. All samples were 30 min in duration. At least three baseline samples were collected, then the palatable mash was presented to the animal under the cage floor out of its reach for one hour sometime between 1100 and 1400. Next, animals were given the mash to eat for one hour during which time food intake and hypothalamic serotonin were measured each 30 min. Three more samples were collected after the food was removed. Each animal was then injected with 8-OH-DPAT (250 pg/kg SC) and two final samples were collected. Serotonin and 5-HIAA peaks were converted to pg amounts by comparison with known standards and then expressed as percent of first baseline sample. Data were analyzed by a two-way analysis of variance (brain site X time) followed by post hoc ttests when justified. Experimental Procedure Serotonin Diffusion
2: Lateral/Medial
Hypothalamic
A simplified procedure was used for a study of spread of neurochemical from one site to the other. Dialysis probes were inserted at least one hour before the experimental session and Ringer’s solution was pumped through the probe at a flow rate of 1 pl/min. Following the collection of 20 min baseline samples, a section of PE tubing preloaded with d-fenfluramine (d-FEN; 5 pg/pl) was intercalated into the inlet tubing of the LH probe so that d-FEN was free to diffuse out of the probe and affect the terminals in the region. At least four samples were collected from both probes. Following this, the procedure was reversed so that only the MH probe was exposed to d-FEN while samples were drawn from both. Extracellular serotonin recovered by the two probes was compared by t-test. Experimental Procedure 3: Lateral Hypothalamic During Dark-Phase Feeding
Serotonin
The dialysis procedure was similar to that described in the light-phase feeding study; however, only one probe was used, Ringer’s calcium concentration was lowered to 2.4 mM and fluoxetine to 1 pM. Animals were housed in the dialysis chambers for at least three days prior to the experiment and allowed to feed freely. Probes were implanted at least 24 hours preceding the experiment. The lights were automatically turned on at 0330 h and off at 1530 h. Thirty-min samples were collected starting at 1300 h in order to obtain a lights-on baseline. Food pellets remained freely available to the animals until 2 h before lights-out at which time they were removed from the cage floor to insure that animals would eat during the feeding test. Sample collection continued into the dark period (using red illumination). Ninety min after lights-out, food (sweet mash as in previous experiment) was presented out of reach under the cage floor. Following the inaccessible food test, rats were offered the mash to eat for 60 min during which time intake was measured at 30 min intervals. Three final samples were collected after the food was removed. As in the light-phase experiment, data were expressed as percent of first baseline sample. One-way analysis of variance followed by Fischer’s least significant difference test was used when appropriate. RESULTS
Basal LH Serotonin
Exceeds
MH Serotonin
As shown in Fig. 1, there was a significantly
greater amount
FEEDING INCREASES LH AND MH SEROTO~
799
FOOD ACCESS
FOOD ACCESS
LH*
180. % f
6b
GO
18.0
240
300
MINUTES
FIG. 1. Serotonin during the light-phase before, during and after a meal in 14-h deprived rats. Extracellular serotonin and 5-HL4A were higher in the LH than the MH during baseline, sight/smell of food and during access to food (*p
0361-9230190 $3.00 + .OO
Serotonin Release in Lateral and Medial Hypothalamus During Feeding and Its Anticipation DAVID H. SCHWARTZ, Department
LUIS HERNANDEZ
of Psychology,
Princeton
Received
AND BARTLEY
University, Princeton,
8 August
G. HOEBEL’
NJ 08544-1010
1990
SCHWARTZ, D. H., L. HERNANDEZ AND B. G. HOEBEL. Serotonin release in lateral and medial hypothalamus during feeding and its anticipation. BRAIN RES BULL 25(6) 797-802, 1990.-In the present experiments we extend previous findings that established a relationship between feeding behavior and hypothalamic serotonin as measured by in vivo microdialysis. The new re+ sult is hypothalamic release of serotonin in anticipation of eating when the animal sees and smells food. We have now verified brain serotonin peaks in four different ways: 1) a serotonergic reuptake blocker (fluoxetine 1 or 10 FM) in the perfusion medium raised basal levels of serotonin, 2) every sample was oxidized at two potentials using a dual potentiostat to confirm the voltage characteristics of each peak, 3) serotonin peaks were reduced by the selective serotonin cell body agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), thus helping confm that most of the serotonin observed in these experiments was neuronal in origin, and 4) lateral and medial hypothalamic microdialysis probes were used simultaneously to monitor the degree of diffusion from one to the other. The results show that extracellular serotonin increases at both sites during preingestive events as well as during eating, but not afterwards. Microdialysis
Feeding
Satiety
Serotonin
5-HIAA
injection of serotonin or its agonists such as fenfluramine inhibit feeding behavior (20, 21, 26, 32). Serotonin injections have some effect in all the medial hypothalamic (MH) regions, and the anorectic effect of fenfluramine is most potent in the paraventricular nucleus (PVN) compared to other hypothalamic regions (29). Conversely, lesions of the PVN attenuate fenfluramine-induced anorexia (2 1,3 1). These results are all consistent with a role for serotonin in an MH satiety system. The role of serotonin in the lateral hypothalamus (LH) is not as clear. Unlike the MH, lesions of the LH enhance fenfluramine-induced anorexia (6,7). In the LH, serotonin iontophoresis inhibits glucose-sensitive neurons but excites others (18). There is some segregation of projections to the medial and lateral hypothalamus from different serotonin cell groups (3,30). Electrophysiological studies of the raphe show that as a role serotonin cell activity varies with behavioral arousal level and does not change in relation to specific environmental or physiological stimuli (10,17). The exception to this rule is a group of serotonin cells that fii during chewing and grooming movements (23). Release of serotonin at hypothalamic terminals may depend heavily on presynaptic influences and therefore could become relatively independent of raphe cell body firing rates. From these pharmacological, anatomical and electrophysiological data, it is not clear what to predict about the relationship between natural
Extracellular
serotonin release and feeding, but in vivo techniques can now directly examine changes in extracellular serotonin in different hypothalamic regions. Microdialysis showed that in the medial region, including the PVN, the serotonin metabolite, 5-HIAA, peaked in the early part of the dark cycle if there was food to eat (28). This suggested that serotonin may be released in association with food intake. We have previously shown that extracellular serotonin in the LH increases during a large daytime meal (24). This left open the issues of dark-phase vs. light-phase eating, site specificity and whether local serotonin reuptake by the terminals was preventing us from detecting subtle changes in release. The present experiments compare light and dark phase eating, and compare the LH and MH using two microdialysis probes for each rat, and use a serotonin uptake blocker to amplify extracellular serotonin. In addition, a number of procedures were instituted to cross-validate the identification of serotonin and its local origin.
HYPOTHALAMIC
‘Requests for reprints should be addressed
Monoamine
METHOD
Subjects and Surgery Thirteen male Sprague-Dawley rats, weighing between 350 and 425 g were individually housed with food and water available ad lib on‘a light cycle of 12 h on/12 h off. Animals were anesthetized with a combination of pentobarbital (20 mg/kg) and ket-
to Bartley G. Hoebel, Department
797
of Psychology,
Princeton University,
Princeton,
NJ 08544-1010.
798
alar (40 mgikg) for implantation of 21-ga guide cannulas aimed at the perifomical LH and MH according to the atlas of Paxinos and Watson (22) at the following stereotaxic coordinates: LH guide cannulas, 6.5 mm anterior to the interaural line, 1.4 mm lateral to the midsagittal sinus, and 4 mm ventral to the level skull surface; MH guide cannulas: A 6.5 mm, L 0.4 mm, and V 4 mm. Probes would later extend 5 mm below the guide cannulas in order to pass into the target structure. Animals recovered for at least 1 week following surgery before a probe was inserted for an experimental session. During this recovery period an obdurator (26 ga, 10.5 mm) in the guide cannula prevented cannula blockage. Dialysis Procedure
Dialysis probes were constructed as previously described (13). Briefly, a 26-ga stainless steel tube was fitted with a porous (6000 molecular weight cutoff) cellulose fiber tip 0.2 mm outer-diameter and 3 mm long. A smaller 36-ga tube was fitted inside the 26-ga tube and served as an outlet. The probes had a relative recovery between 6% and 10% depending on the neurochemical (13). A syringe pump with a 2.5-ml gas-tight syringe perfused a high calcium Ringer’s solution through the probe at a flow rate of 1 pl/min (189 mM NaCl, 3.9 mM KCl, 3.37 mM CaCl, in Procedures 1 and 2, and 2.4 mM CaCl, in Procedure 3: pH 5.6). Fluoxetine was added to this Ringer as specified below. The dialysate exited the outlet tube into PE-10 tubing leading to a 400~1 polyethylene vial clipped to the rat’s headpiece. A double fluid swivel with a counterbalance arm ensured that the tubing did not get tangled as the rat moved around the cage. HPLC Analysis
Dialysates were analyzed by reverse phase high performance liquid chromatography with electrochemical detection (HPLCEC). Samples from the LH and MH were collected by removing the vials from the rat’s headpiece and injecting the contents directly into a Rheodyne valve with a 20-~1 sample loop. The sample was separated on a Brownlee IO-cm column with 3.2-mm bore and 3 PM, C-18 packing. The mobile phase contained 116.8 mM NaOH, 144.7 mM monochloroacetic acid, 1.38 mM l-octane sulfonic acid, 1.38 mM EDTA, and 5% V/V acetonitrile at pH 3.1. 5-HIAA and serotonin were eluted in that order and oxidized on two glassy carbon electrodes at 0.59 V and 0.68 V (Model 400 electrochemical detector with dual potentiostat, Princeton Applied Research Co.) at retention times of approximately 3 and 6 min respectively. The output was then relayed to a 2-pen chart recorder (Linseis Model L-6512). Serotonin peak heights were measured at both potentials and the ratio between the two potentials was computed. Experimental Procedure 1: Lateral and Medial Hypothalamic Serotonin During Light-Phase Feeding
Rats were housed in a room with the lights on at 0900 h and out at 2100 h. They were placed in the experimental chambers in the same room for at least three days and given free access to food and water to accustom them to the setting. On a subsequent day, food was removed at 2300 h and the following afternoon (1300 h) animals were offered access to a palatable mash of sweetened condensed milk and powdered Purina rat chow for two hours to familiarize them with deprivation and a novel, palatable food. On the day before the experimental session (between 1500 and 1800 h), the-obdurators were removed and dialysis probes were
SCHWARTZ,
HERNANDEZ
AND HOEBEL
inserted into the LH and MH guide shafts allowing simultaneous collection of samples. Ringer’s solution plus 10 pM fluoxetine flowed through the probe at 0.5 pJmin overnight to ensure steady baseline levels during the experiment on the following day. The flow rate was increased to 1 pl/min in the morning (0800 h) and the first sample was collected between 0900 and 0930 h. All samples were 30 min in duration. At least three baseline samples were collected, then the palatable mash was presented to the animal under the cage floor out of its reach for one hour sometime between 1100 and 1400. Next, animals were given the mash to eat for one hour during which time food intake and hypothalamic serotonin were measured each 30 min. Three more samples were collected after the food was removed. Each animal was then injected with 8-OH-DPAT (250 pg/kg SC) and two final samples were collected. Serotonin and 5-HIAA peaks were converted to pg amounts by comparison with known standards and then expressed as percent of first baseline sample. Data were analyzed by a two-way analysis of variance (brain site X time) followed by post hoc ttests when justified. Experimental Procedure Serotonin Diffusion
2: Lateral/Medial
Hypothalamic
A simplified procedure was used for a study of spread of neurochemical from one site to the other. Dialysis probes were inserted at least one hour before the experimental session and Ringer’s solution was pumped through the probe at a flow rate of 1 pl/min. Following the collection of 20 min baseline samples, a section of PE tubing preloaded with d-fenfluramine (d-FEN; 5 pg/pl) was intercalated into the inlet tubing of the LH probe so that d-FEN was free to diffuse out of the probe and affect the terminals in the region. At least four samples were collected from both probes. Following this, the procedure was reversed so that only the MH probe was exposed to d-FEN while samples were drawn from both. Extracellular serotonin recovered by the two probes was compared by t-test. Experimental Procedure 3: Lateral Hypothalamic During Dark-Phase Feeding
Serotonin
The dialysis procedure was similar to that described in the light-phase feeding study; however, only one probe was used, Ringer’s calcium concentration was lowered to 2.4 mM and fluoxetine to 1 pM. Animals were housed in the dialysis chambers for at least three days prior to the experiment and allowed to feed freely. Probes were implanted at least 24 hours preceding the experiment. The lights were automatically turned on at 0330 h and off at 1530 h. Thirty-min samples were collected starting at 1300 h in order to obtain a lights-on baseline. Food pellets remained freely available to the animals until 2 h before lights-out at which time they were removed from the cage floor to insure that animals would eat during the feeding test. Sample collection continued into the dark period (using red illumination). Ninety min after lights-out, food (sweet mash as in previous experiment) was presented out of reach under the cage floor. Following the inaccessible food test, rats were offered the mash to eat for 60 min during which time intake was measured at 30 min intervals. Three final samples were collected after the food was removed. As in the light-phase experiment, data were expressed as percent of first baseline sample. One-way analysis of variance followed by Fischer’s least significant difference test was used when appropriate. RESULTS
Basal LH Serotonin
Exceeds
MH Serotonin
As shown in Fig. 1, there was a significantly
greater amount
FEEDING INCREASES LH AND MH SEROTO~
799
FOOD ACCESS
FOOD ACCESS
LH*
180. % f
6b
GO
18.0
240
300
MINUTES
FIG. 1. Serotonin during the light-phase before, during and after a meal in 14-h deprived rats. Extracellular serotonin and 5-HL4A were higher in the LH than the MH during baseline, sight/smell of food and during access to food (*p
