Articles in PresS. Am J Physiol Regul Integr Comp Physiol (October 8, 2014). doi:10.1152/ajpregu.00329.2014
1 2 3 4
Leptin receptor signaling in the lateral parabrachial nucleus contributes to
5
the control of food intake
6 7
Running title: lPBN LepRb signaling contributes to food intake control
8 9 10 11
Amber L. Alhadeff1, Matthew R. Hayes2, Harvey J. Grill1 1Department
of Psychology, 2Department of Psychiatry, University of Pennsylvania
12
Philadelphia, PA
13 14 15 16 17 18 19
Address correspondence to:
20 21 22 23 24 25
Amber L. Alhadeff, M.A. [email protected] Address: University of Pennsylvania 3720 Walnut Street D25 Philadelphia, PA 19104
26
Key Words: leptin, PBN, food intake, obesity, meal size
Copyright © 2014 by the American Physiological Society.
27 28
Abstract Pontine parabrachial nucleus (PBN) neurons integrate visceral, oral, and
29
other sensory information playing an integral role in the neural control of feeding.
30
Current experiments probed whether lateral PBN (lPBN) leptin receptor (LepRb)
31
signaling contributes to this function. Intra-lPBN leptin microinjection significantly
32
reduced cumulative chow intake, average meal size, and body weight in rats,
33
independent of effects on locomotor activity or gastric emptying. In contrast to the
34
effects observed following LepRb activation in other nuclei, lPBN LepRb stimulation
35
did not affect progressive ratio responding for sucrose reward or conditioned place
36
preference for a palatable food. Collectively, results suggest that lPBN LepRb
37
activation reduces food intake by modulating the neural processing of meal
38
size/satiation signaling, and highlight the lPBN as a novel site of action for leptin-
39
mediated food intake control.
40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
60 61 62
Introduction
63
inputs from a variety of brain regions involved in the control of food intake and
64
energy balance. Oral and viscerosensory information ascends from the nucleus
65
tractus solitarius (NTS) (2, 26) to the lPBN, which in turn sends and receives
66
projections from several hypothalamic (2, 27) and amygdala nuclei (2, 27), the
67
ventral tegmental area (VTA) (25), and the nucleus accumbens (NAc) (23). Previous
68
studies show that a variety of neurochemical signals, including endocannabinoids
69
(7, 8), opioids (32, 33), glutamate (34, 35), and glucagon-like peptide-1 (GLP-1) (1)
70
act directly in the lPBN to affect food intake and reward behavior. Collectively, these
71
data highlight the involvement of lPBN signaling in the control of food intake and
72
encourage further investigations of lPBN neuroendocrine signaling in the control of
73
energy balance. To this end, it is worth noting that the adipose tissue-derived
74
hormone leptin acts on receptors distributed throughout the brain to control for
75
energy balance (13), and that leptin receptors (LepRb) are expressed in lPBN (14,
76
17). A direct analysis of the role of lPBN LepRb signaling in the control of food
77
intake, however, remains unexplored.
Neurons of the lateral parabrachial nucleus (lPBN) receive and integrate
78
The current experiments utilize a combination of behavioral and
79
pharmacological techniques to test the hypothesis that lPBN LepRb signaling is
80
involved in the neural control of food intake. Data show that lPBN leptin
81
microinjection reduces body weight and chow intake via a reduction in meal size. In
82
contrast to effects of LepRb signaling in other CNS nuclei that reduce food reward
83
and motivation, present data show that lPBN LepRb signaling reduces food intake
84
without affecting motivation to feed. Results indicate that lPBN LepRb signaling
85
contributes to food intake control through effects on meal size/satiation signaling
86
and highlight the lPBN as a brain region involved in leptin-mediated food intake
87
control.
88 89
Methods
90
Subjects and Drugs
91
Male Sprague-Dawley rats (250-300g upon arrival; Charles River
92
Laboratories, Wilmington, MA) were individually housed in hanging metal cages on
93
a 12h light/12h dark cycle with ad libitum access to pelleted chow (Purina Rodent
94
Chow, 5001; 28.5% (kcal) protein, 13.5% fat, 58.0% carbohydrate, 3.3kcal/g) and
95
water except when otherwise noted. All procedures conformed to and received
96
approval from the institutional standards of the University of Pennsylvania animal
97
care and use committee.
98 99 100
Leptin was purchased from Harbor University of California, Los Angeles Research and Education Institute, Torrance, CA, and was dissolved in sodium bicarbonate.
101 102 103
Surgery Rats received intramuscular ketamine (90mg/kg; Butler Animal Health
104
Supply, Dublin, OH), xylazine (2.7mg/kg; Anased, Shenandoah, IA) and
105
acepromazine (0.64mg/kg; Bitler Animal Health Supply) anesthesia and
106
subcutaneous analgesia (2.0mg/kg Metacam; Boehringer Ingelheim Vetmedica, St.
107
Joseph, MO) for all surgeries.
108
Unilateral 26-gauge guide cannulae (Plastics One; Roanoke, VA) were
109
stereotaxically implanted in the lPBN or the cerebral aqueduct according to the
110
following coordinates. lPBN guide cannulae were positioned ±2.0mm lateral from
111
midline, 0.6mm anterior to lambda, and 5.7mm ventral from skull surface using a
112
20° angle (negative slope in anterior to posterior direction) with the injector aimed
113
2.0mm below the end of the guide cannula. Cannula placements were histologically
114
confirmed postmortem: Chicago sky blue ink (100nl) was injected in the lPBN after
115
animals were euthanized; brains were removed and post-fixed in formalin, cut on a
116
cryostat, mounted on glass slides, and analyzed for placement. A representative
117
image of the injection site, as well as a schematic diagram representing injection
118
placements for a cohort of rats, is depicted in Figure 1. Rats with injection sites that
119
were not within the lPBN were excluded from analyses. Aqueduct guide cannulae
120
were positioned anterior to the PBN, ±2.0mm medial from midline, 8.2mm caudal
121
anterior from bregma, and 3.85mm ventral from skull using a 20° angle (negative
122
slope in the lateral to medial direction). Cannula placements were functionally
123
confirmed via measurement of the sympathoadrenal-mediated glycemic response to
124
5-thio-D-glucose (210μg/2μL in artificial cerebrospinal fluid, aCSF) injected into the
125
aqueduct as previously described (28). A post-injection increase in blood glucose
126
level of at least 100% from baseline was necessary for subject inclusion.
127 128
Experimental Procedures
129
Experiment 1: Leptin effects in the cerebral aqueduct – parenchymal dose selection
130
To select leptin doses for lPBN experiments that were subthreshold for effect
131
when delivered into the cerebroventricular system, rats (n=9) that were habituated
132
to experimental procedures received a 100nl unilateral injection of leptin (0.6, 0.3,
133
or 0.1μg) or vehicle via an automated syringe pump (PHD Ultra; Harvard Apparatus;
134
Holliston, MA) in the aqueduct in a within-subjects, counterbalanced experimental
135
design immediately before the onset of the dark cycle. Chow intake was measured
136
at 1h, 3h, 6h, and 24h accounting for spillage. Body weight and water intake was
137
measured 24h post-injection. At least 48h elapsed in between drug injection
138
conditions.
139 140
Experiment 2: lPBN leptin effects on chow intake and meal patterns
141
Rats (n=18) were housed in a custom, automated feedometer consisting of
142
hanging wire cages with a small access hole to a food cup resting on an electronic
143
scale. The associated software (LabView) records the weight of food cups every 10s.
144
Following habituation to the cages and powdered standard chow for 5 days, rats
145
received a 100nl unilateral injection of leptin (0.1, 0.3, or 0.6μg) or vehicle into the
146
lPBN in a within-subjects, counterbalanced experimental design immediately before
147
the onset of the dark cycle. As noted the doses selected were first determined to be
148
subthreshold for effect when delivered to the ventricular system (Experiment 1).
149
Automated food measurements were made for 24h post-injection; body weight was
150
recorded manually 24h post-injection. In a subset of the total group of rats (n=8),
151
24h water intake was recorded manually. Meal patterns were subsequently
152
analyzed for all rats, with a meal defined as any intake ≥0.25g; ≥10min must elapse
153
for feeding bouts to be considered two separate meals. At least 48h elapsed in
154
between drug injection conditions.
155 156 157
Experiment 3: lPBN leptin effects on high-fat diet intake and meal patterns Rats (n=10) were subjected to the same procedures as in Experiment 2,
158
except that they were maintained on powdered high-fat diet (HFD; Research Diets,
159
New Brunswick, NJ; 20% (kcal) protein, 45% fat, 35% carbohydrate, 3.73 kcal/g)
160
throughout the duration of the experiment.
161 162 163
Experiment 4: lPBN leptin effects on progressive ratio responding Rats (n=11) maintained ad libitum on standard chow were habituated to
164
45mg sucrose pellets (Bio-Serv; Frenchtown, NJ) in their home cage and trained as
165
we have previously described (19) to press a lever for pellets at a fixed ratio (FR)-3
166
schedule of reinforcement (three lever presses required to receive one pellet). For
167
all training sessions, the right lever was active and an inactive left lever served as a
168
control for non-conditioned changes in operant responding.
169
Rats were given three tests in a within-subjects design using a progressive
170
ratio (PR) schedule of reinforcement. A 100nl unilateral lPBN injection of leptin (0.3
171
or 0.6μg) or vehicle was delivered 3h prior to each PR test session in a within-
172
subjects, counterbalanced experimental design. Animals were returned to their
173
home cage for the 3h between injection and test session and food was withheld.
174
During the PR test, the effort required to obtain each pellet increased exponentially
175
throughout the session as we have previously described (1, 19), using the formula
176
F(i)=5e0.2i-5, where F(i) is the number of lever presses required to obtain the next
177
pellet at i=pellet number. The PR session ended when a 20min period elapsed
178
without the rat earning a pellet.
179 180 181
Experiment 5: lPBN leptin effects on food-conditioned place preference expression Rats (n=21) maintained ad libitum on standard chow were trained for CPP as
182
we have previously described (19). All CPP training and testing sessions were
183
performed in a dimly lit room. Animals were trained in an apparatus consisting of
184
two identical plexiglass compartments (74cm long, 57.4cm wide, and 24.7cm high)
185
separated by a divider wall with a door that was closed during training but open
186
during habituation and testing. The two environments within the CPP box were
187
made distinguishable by different wall color and design and floor texture. Rats were
188
habituated to the CPP chamber (with access to both environments) for one 15min
189
video-recorded session. The differential time spent in each of the two environments
190
was analyzed via ANY-Maze software (Stoelting Co., Wood Dale, IL) and a baseline
191
environment preference was determined. For each rat, the environment that was
192
least preferred during habituation was subsequently paired with the palatable food
193
for all training, whereas the preferred side was never paired with palatable food.
194
CPP training consisted of 16 consecutive days of training (15min sessions): 8 days of
195
training in a food-paired environment, where 5g of a high-fat diet (60%kcal/fat,
196
Research Diets, New Brunswick, NJ) was divided into 10 aliquots and scattered
197
throughout the environment, alternating with 8 days of training in the other
198
environment without food.
199
CPP testing commenced the day after the training was completed using a
200
between-subjects design. Rats were matched for baseline preference (n=11 vehicle,
201
n=10 leptin) and given a unilateral injection of leptin (0.6μg) or vehicle 3h prior to
202
CPP test. The animals were returned to their home cages for the 3 intervening
203
hours and food was withheld. With the divider door open and no food present, the
204
rats were videotaped for the 15-min CPP test to determine the total time spent in
205
the environments previously associated with palatable food or without food. The
206
time spent in each environment was analyzed via ANY-maze software, and the
207
percentage shift in preference (from habituation baseline) for the food-paired side
208
was calculated. As a control for potential leptin-induced effects on locomotor
209
activity parameters, total time active and total distance traveled were also analyzed
210
via ANY-maze software.
211 212 213
Experiment 6: lPBN leptin effects on gastric emptying Rats (n=25) were overnight (12h) food deprived and given an injection of
214
leptin (0.3 or 0.6μg) or vehicle 3.5h prior to access to a 6g meal of chow. 90min
215
post-refeed, animals were euthanized via C02 asphyxiation and their stomachs were
216
rapidly removed and stomach contents collected. Stomach contents were baked
217
overnight at 80°C and the dry weight of the stomach contents was compared to the
218
size of the dry weight of the refeed meal (accounting for spillage) to calculate the
219
percentage of the meal that was emptied from the stomach prior to euthanasia
220
which occurred 5h post-injection of leptin or vehicle.
221
222
Statistical Analyses
223
Data for each experiment were analyzed separately using Statistica (version
224
7; StatSoft Inc., Tulsa, OK) and expressed as mean ± SEM. For all behavioral
225
experiments, repeated measures or one-way ANOVA and post hoc Neumann-Keuls
226
comparisons were made. Alpha levels were set to α=0.05 for all analyses.
227 228
Results
229
Experiment 1: Evaluation of lPBN effects in the cerebral aqueduct
230
Cerebral aqueduct delivery of leptin did not significantly affect cumulative
231
chow intake at 1h [F(3,24)=1.69], 3h [F(3,24)=0.84], 6h [F(3,24)=0.89], or 24h
232
[F(3,24)=1.46] compared to vehicle treatment (Figure 2). Additionally, post-hoc
233
comparisons revealed no significant drug effects for any doses or time points.
234 235
Experiment 2: lPBN leptin significantly reduces chow intake via a reduction in meal
236
size
237
There was a significant main effect of lPBN leptin (0.1, 0.3, 0.6μg) on
238
cumulative food intake at 5h [F(3,51)=3.80, p
1 2 3 4
Leptin receptor signaling in the lateral parabrachial nucleus contributes to
5
the control of food intake
6 7
Running title: lPBN LepRb signaling contributes to food intake control
8 9 10 11
Amber L. Alhadeff1, Matthew R. Hayes2, Harvey J. Grill1 1Department
of Psychology, 2Department of Psychiatry, University of Pennsylvania
12
Philadelphia, PA
13 14 15 16 17 18 19
Address correspondence to:
20 21 22 23 24 25
Amber L. Alhadeff, M.A. [email protected] Address: University of Pennsylvania 3720 Walnut Street D25 Philadelphia, PA 19104
26
Key Words: leptin, PBN, food intake, obesity, meal size
Copyright © 2014 by the American Physiological Society.
27 28
Abstract Pontine parabrachial nucleus (PBN) neurons integrate visceral, oral, and
29
other sensory information playing an integral role in the neural control of feeding.
30
Current experiments probed whether lateral PBN (lPBN) leptin receptor (LepRb)
31
signaling contributes to this function. Intra-lPBN leptin microinjection significantly
32
reduced cumulative chow intake, average meal size, and body weight in rats,
33
independent of effects on locomotor activity or gastric emptying. In contrast to the
34
effects observed following LepRb activation in other nuclei, lPBN LepRb stimulation
35
did not affect progressive ratio responding for sucrose reward or conditioned place
36
preference for a palatable food. Collectively, results suggest that lPBN LepRb
37
activation reduces food intake by modulating the neural processing of meal
38
size/satiation signaling, and highlight the lPBN as a novel site of action for leptin-
39
mediated food intake control.
40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
60 61 62
Introduction
63
inputs from a variety of brain regions involved in the control of food intake and
64
energy balance. Oral and viscerosensory information ascends from the nucleus
65
tractus solitarius (NTS) (2, 26) to the lPBN, which in turn sends and receives
66
projections from several hypothalamic (2, 27) and amygdala nuclei (2, 27), the
67
ventral tegmental area (VTA) (25), and the nucleus accumbens (NAc) (23). Previous
68
studies show that a variety of neurochemical signals, including endocannabinoids
69
(7, 8), opioids (32, 33), glutamate (34, 35), and glucagon-like peptide-1 (GLP-1) (1)
70
act directly in the lPBN to affect food intake and reward behavior. Collectively, these
71
data highlight the involvement of lPBN signaling in the control of food intake and
72
encourage further investigations of lPBN neuroendocrine signaling in the control of
73
energy balance. To this end, it is worth noting that the adipose tissue-derived
74
hormone leptin acts on receptors distributed throughout the brain to control for
75
energy balance (13), and that leptin receptors (LepRb) are expressed in lPBN (14,
76
17). A direct analysis of the role of lPBN LepRb signaling in the control of food
77
intake, however, remains unexplored.
Neurons of the lateral parabrachial nucleus (lPBN) receive and integrate
78
The current experiments utilize a combination of behavioral and
79
pharmacological techniques to test the hypothesis that lPBN LepRb signaling is
80
involved in the neural control of food intake. Data show that lPBN leptin
81
microinjection reduces body weight and chow intake via a reduction in meal size. In
82
contrast to effects of LepRb signaling in other CNS nuclei that reduce food reward
83
and motivation, present data show that lPBN LepRb signaling reduces food intake
84
without affecting motivation to feed. Results indicate that lPBN LepRb signaling
85
contributes to food intake control through effects on meal size/satiation signaling
86
and highlight the lPBN as a brain region involved in leptin-mediated food intake
87
control.
88 89
Methods
90
Subjects and Drugs
91
Male Sprague-Dawley rats (250-300g upon arrival; Charles River
92
Laboratories, Wilmington, MA) were individually housed in hanging metal cages on
93
a 12h light/12h dark cycle with ad libitum access to pelleted chow (Purina Rodent
94
Chow, 5001; 28.5% (kcal) protein, 13.5% fat, 58.0% carbohydrate, 3.3kcal/g) and
95
water except when otherwise noted. All procedures conformed to and received
96
approval from the institutional standards of the University of Pennsylvania animal
97
care and use committee.
98 99 100
Leptin was purchased from Harbor University of California, Los Angeles Research and Education Institute, Torrance, CA, and was dissolved in sodium bicarbonate.
101 102 103
Surgery Rats received intramuscular ketamine (90mg/kg; Butler Animal Health
104
Supply, Dublin, OH), xylazine (2.7mg/kg; Anased, Shenandoah, IA) and
105
acepromazine (0.64mg/kg; Bitler Animal Health Supply) anesthesia and
106
subcutaneous analgesia (2.0mg/kg Metacam; Boehringer Ingelheim Vetmedica, St.
107
Joseph, MO) for all surgeries.
108
Unilateral 26-gauge guide cannulae (Plastics One; Roanoke, VA) were
109
stereotaxically implanted in the lPBN or the cerebral aqueduct according to the
110
following coordinates. lPBN guide cannulae were positioned ±2.0mm lateral from
111
midline, 0.6mm anterior to lambda, and 5.7mm ventral from skull surface using a
112
20° angle (negative slope in anterior to posterior direction) with the injector aimed
113
2.0mm below the end of the guide cannula. Cannula placements were histologically
114
confirmed postmortem: Chicago sky blue ink (100nl) was injected in the lPBN after
115
animals were euthanized; brains were removed and post-fixed in formalin, cut on a
116
cryostat, mounted on glass slides, and analyzed for placement. A representative
117
image of the injection site, as well as a schematic diagram representing injection
118
placements for a cohort of rats, is depicted in Figure 1. Rats with injection sites that
119
were not within the lPBN were excluded from analyses. Aqueduct guide cannulae
120
were positioned anterior to the PBN, ±2.0mm medial from midline, 8.2mm caudal
121
anterior from bregma, and 3.85mm ventral from skull using a 20° angle (negative
122
slope in the lateral to medial direction). Cannula placements were functionally
123
confirmed via measurement of the sympathoadrenal-mediated glycemic response to
124
5-thio-D-glucose (210μg/2μL in artificial cerebrospinal fluid, aCSF) injected into the
125
aqueduct as previously described (28). A post-injection increase in blood glucose
126
level of at least 100% from baseline was necessary for subject inclusion.
127 128
Experimental Procedures
129
Experiment 1: Leptin effects in the cerebral aqueduct – parenchymal dose selection
130
To select leptin doses for lPBN experiments that were subthreshold for effect
131
when delivered into the cerebroventricular system, rats (n=9) that were habituated
132
to experimental procedures received a 100nl unilateral injection of leptin (0.6, 0.3,
133
or 0.1μg) or vehicle via an automated syringe pump (PHD Ultra; Harvard Apparatus;
134
Holliston, MA) in the aqueduct in a within-subjects, counterbalanced experimental
135
design immediately before the onset of the dark cycle. Chow intake was measured
136
at 1h, 3h, 6h, and 24h accounting for spillage. Body weight and water intake was
137
measured 24h post-injection. At least 48h elapsed in between drug injection
138
conditions.
139 140
Experiment 2: lPBN leptin effects on chow intake and meal patterns
141
Rats (n=18) were housed in a custom, automated feedometer consisting of
142
hanging wire cages with a small access hole to a food cup resting on an electronic
143
scale. The associated software (LabView) records the weight of food cups every 10s.
144
Following habituation to the cages and powdered standard chow for 5 days, rats
145
received a 100nl unilateral injection of leptin (0.1, 0.3, or 0.6μg) or vehicle into the
146
lPBN in a within-subjects, counterbalanced experimental design immediately before
147
the onset of the dark cycle. As noted the doses selected were first determined to be
148
subthreshold for effect when delivered to the ventricular system (Experiment 1).
149
Automated food measurements were made for 24h post-injection; body weight was
150
recorded manually 24h post-injection. In a subset of the total group of rats (n=8),
151
24h water intake was recorded manually. Meal patterns were subsequently
152
analyzed for all rats, with a meal defined as any intake ≥0.25g; ≥10min must elapse
153
for feeding bouts to be considered two separate meals. At least 48h elapsed in
154
between drug injection conditions.
155 156 157
Experiment 3: lPBN leptin effects on high-fat diet intake and meal patterns Rats (n=10) were subjected to the same procedures as in Experiment 2,
158
except that they were maintained on powdered high-fat diet (HFD; Research Diets,
159
New Brunswick, NJ; 20% (kcal) protein, 45% fat, 35% carbohydrate, 3.73 kcal/g)
160
throughout the duration of the experiment.
161 162 163
Experiment 4: lPBN leptin effects on progressive ratio responding Rats (n=11) maintained ad libitum on standard chow were habituated to
164
45mg sucrose pellets (Bio-Serv; Frenchtown, NJ) in their home cage and trained as
165
we have previously described (19) to press a lever for pellets at a fixed ratio (FR)-3
166
schedule of reinforcement (three lever presses required to receive one pellet). For
167
all training sessions, the right lever was active and an inactive left lever served as a
168
control for non-conditioned changes in operant responding.
169
Rats were given three tests in a within-subjects design using a progressive
170
ratio (PR) schedule of reinforcement. A 100nl unilateral lPBN injection of leptin (0.3
171
or 0.6μg) or vehicle was delivered 3h prior to each PR test session in a within-
172
subjects, counterbalanced experimental design. Animals were returned to their
173
home cage for the 3h between injection and test session and food was withheld.
174
During the PR test, the effort required to obtain each pellet increased exponentially
175
throughout the session as we have previously described (1, 19), using the formula
176
F(i)=5e0.2i-5, where F(i) is the number of lever presses required to obtain the next
177
pellet at i=pellet number. The PR session ended when a 20min period elapsed
178
without the rat earning a pellet.
179 180 181
Experiment 5: lPBN leptin effects on food-conditioned place preference expression Rats (n=21) maintained ad libitum on standard chow were trained for CPP as
182
we have previously described (19). All CPP training and testing sessions were
183
performed in a dimly lit room. Animals were trained in an apparatus consisting of
184
two identical plexiglass compartments (74cm long, 57.4cm wide, and 24.7cm high)
185
separated by a divider wall with a door that was closed during training but open
186
during habituation and testing. The two environments within the CPP box were
187
made distinguishable by different wall color and design and floor texture. Rats were
188
habituated to the CPP chamber (with access to both environments) for one 15min
189
video-recorded session. The differential time spent in each of the two environments
190
was analyzed via ANY-Maze software (Stoelting Co., Wood Dale, IL) and a baseline
191
environment preference was determined. For each rat, the environment that was
192
least preferred during habituation was subsequently paired with the palatable food
193
for all training, whereas the preferred side was never paired with palatable food.
194
CPP training consisted of 16 consecutive days of training (15min sessions): 8 days of
195
training in a food-paired environment, where 5g of a high-fat diet (60%kcal/fat,
196
Research Diets, New Brunswick, NJ) was divided into 10 aliquots and scattered
197
throughout the environment, alternating with 8 days of training in the other
198
environment without food.
199
CPP testing commenced the day after the training was completed using a
200
between-subjects design. Rats were matched for baseline preference (n=11 vehicle,
201
n=10 leptin) and given a unilateral injection of leptin (0.6μg) or vehicle 3h prior to
202
CPP test. The animals were returned to their home cages for the 3 intervening
203
hours and food was withheld. With the divider door open and no food present, the
204
rats were videotaped for the 15-min CPP test to determine the total time spent in
205
the environments previously associated with palatable food or without food. The
206
time spent in each environment was analyzed via ANY-maze software, and the
207
percentage shift in preference (from habituation baseline) for the food-paired side
208
was calculated. As a control for potential leptin-induced effects on locomotor
209
activity parameters, total time active and total distance traveled were also analyzed
210
via ANY-maze software.
211 212 213
Experiment 6: lPBN leptin effects on gastric emptying Rats (n=25) were overnight (12h) food deprived and given an injection of
214
leptin (0.3 or 0.6μg) or vehicle 3.5h prior to access to a 6g meal of chow. 90min
215
post-refeed, animals were euthanized via C02 asphyxiation and their stomachs were
216
rapidly removed and stomach contents collected. Stomach contents were baked
217
overnight at 80°C and the dry weight of the stomach contents was compared to the
218
size of the dry weight of the refeed meal (accounting for spillage) to calculate the
219
percentage of the meal that was emptied from the stomach prior to euthanasia
220
which occurred 5h post-injection of leptin or vehicle.
221
222
Statistical Analyses
223
Data for each experiment were analyzed separately using Statistica (version
224
7; StatSoft Inc., Tulsa, OK) and expressed as mean ± SEM. For all behavioral
225
experiments, repeated measures or one-way ANOVA and post hoc Neumann-Keuls
226
comparisons were made. Alpha levels were set to α=0.05 for all analyses.
227 228
Results
229
Experiment 1: Evaluation of lPBN effects in the cerebral aqueduct
230
Cerebral aqueduct delivery of leptin did not significantly affect cumulative
231
chow intake at 1h [F(3,24)=1.69], 3h [F(3,24)=0.84], 6h [F(3,24)=0.89], or 24h
232
[F(3,24)=1.46] compared to vehicle treatment (Figure 2). Additionally, post-hoc
233
comparisons revealed no significant drug effects for any doses or time points.
234 235
Experiment 2: lPBN leptin significantly reduces chow intake via a reduction in meal
236
size
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There was a significant main effect of lPBN leptin (0.1, 0.3, 0.6μg) on
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cumulative food intake at 5h [F(3,51)=3.80, p
