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Department of Psychology, University of Pennsylvania, Philadelphia, Pennsylvania 19104
Address all correspondence and requests for reprints to: Diana L. Williams, Department of Psychology, University of Pennsylvania, 3815 Walnut Street, Philadelphia, Pennsylvania 19104. E-mail: dianaw{at}psych.upenn.edu
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Abstract |
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80%)
to the same extent in vagotomized and surgical control rats. We
conclude that stimulation or antagonism of MC3/4-Rs in the dorsal vagal
complex yields effects on food intake that do not require an intact
vagus nerve. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The majority of studies on the MC system have focused on the
hypothalamus (4, 6, 10). Anatomical evidence, however, clearly
indicates that potentially relevant components of the MC system are
present in the hindbrain. POMC, the precursor to the endogenous MC4-R
agonist, 
MSH, is produced in only two locations in the brain: the
arcuate nucleus of the hypothalamus and the commissural nucleus of the
solitary tract (cNTS) (11). Importantly, immunoreactivity for MSH
itself has been demonstrated in the cNTS (12). In addition, MC3/4-Rs in
the cNTS and in the dorsal motor nucleus of the vagus nerve (DMX) were
demonstrated in a binding study using the high affinity ligand,
Nle4,D-Phe7-MSH
(13). The DMX, in fact, expresses the highest density of MC4-R
messenger RNA (mRNA) in the brain, whereas weak expression is found in
the medial NTS (14). The functional relevance of hindbrain MC3/4-Rs to
feeding and body weight control was suggested in a recent report in
which dose-dependent decreases or increases in feeding and body weight
were obtained upon fourth intracerbroventricular (4th-icv)
administration of MTII or SHU9119, respectively (5). The response to
4th-icv delivery of these ligands was no weaker than that obtained upon
lateral icv delivery.
The high MC4-R mRNA density in the DMX and the proximity of this nucleus to the fourth ventricle are consistent with the suggestion that receptors within the DMX, or in the dorsal vagal complex (DVC; including the DMX and NTS) more generally, at least partially mediate the effects of 4th-icv MTII and SHU9119. One aim of the present study was to explore this suggestion by delivery of MTII and SHU9119 unilaterally into the DVC at doses subthreshold for 4th-icv response.
The second aim of the present work is to investigate the possibility that an intact vagus nerve is required for the anorexic effect of 4th-icv MTII. One would expect that vagotomized (VX) rats will not show a 4th-icv MTII effect if, in fact, the receptors on DMX motor neurons represent the substrate for the response. Indeed, the efferent branch of the vagus nerve is the only known output pathway for motor neurons in that nucleus. A reversal of the 4th-icv effect with vagotomy would be consistent with but would not prove that DMX mediates MTII effects on intake. The integrity of vagus nerve may also be necessary for feeding effects obtained upon stimulation of receptors located some distance from the vagal complex. An appropriate example is the reversal by vagotomy of the hyperphagia obtained when norepinephrine is delivered lateral-icv or to the paraventricular nucleus (15, 16). In this report, we evaluate the effects of subdiaphragmatic vagotomy on the intake and body weight effects obtained independently from 4th- and lateral-icv injection of MTII. Vagal mediation of the effects resulting from stimulation of different MC4-R populations would be supported if vagotomy blocks responses evoked from both ventricles. Alternately, vagotomy could block responses from one ventricle but not affect the response of the other. The suggestion that the DMX itself mediates icv MTII effects would be challenged if vagotomy had no effect on the response from either ventricle.
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Materials and Methods |
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Intraparenchymal and icv cannulation
Guide cannulas (Plastics One, Roanoke, VA) were implanted
2.0 mm above injection sites of naive rats under ketamine (90 mg/kg)
and xylazine (15 mg/kg im) anesthesia. For Exp 1, intraparenchymal
cannulas (26 gauge) were placed above the right DVC, 13.8 mm posterior
to bregma, 0.8 mm lateral to the midline, and 3.7 mm dorsal to the
interaural line. For Exp 2, icv cannulas (22 gauge) were implanted
above the lateral and fourth ventricles. Placement for the lateral
ventricle cannula was 0.9 mm posterior to bregma, 1.5 mm lateral to the
midline, and 2.0 mm below the dura; fourth ventricle cannula placement
was 2.5 mm anterior to the occipital suture, on the midline, and 4.5 mm
below the dura. Cannulas were cemented to four jewelers screws attached
to the skull and were closed with obturators. The rats recovered in
their home cages for at least 5 days while daily food intake and body
weight were recorded.
Injections
For intraparenchymal injection, MTII and SHU9119 (Phoenix,
Belmont, CA) were dissolved in artificial cerebrospinal fluid
(aCSF). A 33-gauge injector 2 mm longer than the guide cannula was
inserted, and a 0.5-µl volume of MTII, SHU9119, or aCSF was infused
over 5 min using a microsyringe pump (Harvard Apparatus, Natick, MA).
The injector was removed after another minute. For icv injection, MTII
was prepared by dissolving the peptide in deoxygenated distilled water.
A 23-gauge injector 2 mm longer than the guide cannula was inserted,
and a 3-µl volume of MTII or saline was injected over 3 min using a
Hamilton microsyringe (Reno, NV). The injector was left in place for
another minute before removal. All injections took place an average of
30 min before the beginning of the dark cycle.
Verification of cannula placement
Parenchymal placements were verified by injecting 0.5 µl India
ink 1.5 mm below the drug injection site just before transcardial
perfusion with saline followed by 10% formalin. Brains were then
removed and postfixed in a 10% sucrose-formalin solution. Fifty-micron
sections were cut in the coronal plane and then stained with cresyl
violet. Where possible, the presence of gliosis was used to reconstruct
injector placement. In some cases, glial damage was not apparent or not
determined; in these cases injector placement was assessed in relation
to the dye reference (see Fig. 1
). Only
those data for animals that were verifiably injected into the DVC are
discussed here. The icv cannula placement was evaluated after recovery
from surgery by measuring a sympathetically mediated increase in plasma
glucose after icv injection of 210 µg 5-thio-D-glucose in
3 µl saline (18). Only rats that showed at least a doubling of plasma
glucose level in response to this treatment were used in
experiments.
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Statistical analysis. Data were analyzed with Student’s t tests, comparing drug and vehicle values for feeding, drinking, and body weight. Significance at the 0.05 level was evaluated with one-tailed tests given the direction of effects reported in the literature for MTII and SHU9119.
Exp 2: fourth icv MTII effects in intact and VX animals
Vagotomy. After cannula placement verification, six rats
were anesthetized with ketamine-xylazine, and the trunks of the
subdiaphragmatic vagus were transected. A midline abdominal incision
was made, and the dorsal and ventral branches of the vagus nerve were
dissected from the esophagus. Each branch of the nerve was tied with
surgical suture at two points separated by approximately 1 cm, and then
cauterized between the sutures. Sham surgeries were also
performed (n = 5), in which each trunk of the nerve was exposed,
but not tied or cauterized. The incision was then closed, and rats were
allowed to recover for at least 1 week until liquid diet intake
stabilized. After the experiment, vagotomies were functionally verified
with a test of short term liquid diet intake after cholecystokinin
octapeptide (CCK8; Bachem, Torrance, CA) treatment. Intake
suppression after CCK8 requires an intact vagus nerve (19). For this
test, rats were restricted to 15 ml liquid diet overnight (30% of
the average 24-h intake), and then injected with CCK8 (4 µg/kg, ip)
or saline in counterbalanced order. Liquid diet was made available 5
min after injections, and intake was recorded at 15 and 30 min. A lack
of response to CCK8 by VX rats was taken as verification of
subdiaphragmatic vagotomies.
Methods. After recovery from surgery, animals were given icv saline vehicle (3 µl) to habituate them to the injection procedure. The experiment began when liquid diet intake was stable. Four injection conditions, 1 nmol MTII [a dose that substantially affects intake and body weight (5)] and vehicle injections in the fourth and lateral ventricles, were separated by 72 h and counterbalanced across rats. After injections, at the onset of the dark cycle, rats were given ad libitum access to liquid diet in graduated tubes hung on the cage. The initial volume was recorded, and intake at 2, 4, and 24 h postinjection was determined by reading the volume in the tube at that time and subtracting that from the initial volume. Rats were given fresh liquid diet daily, and intakes at 48 and 72 h postinjection were determined in the same way. Twenty-four-, 48-, and 72-h water intakes were also measured by weighing the bottles each day and subtracting their weights from their initial weights. Animals were weighed daily. Forty-eight and 72 h data for two rats (one VX and one sham-operated) were not available.
Statistical analyses. Data were analyzed with three-way ANOVA, including VX as a between-subjects factor and injection site (lateral or fourth ventricle) and drug as repeated measures factors. Post-hoc comparisons were made using least significant difference tests.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) are included in these analyses. Data from 3 rats were
excluded based on cannula placements outside of the DVC.
As shown in Fig. 2, food intake after
unilateral MTII injection was significantly decreased at 2 h
[t(9) = 2.232; P < 0.05], 4 h
[t(9) = 3.255; P < 0.01], and
24 h [t(9) = 3.90; P < 0.005]
compared with that after vehicle treatment. Food intakes at 48 and
72 h after MTII were similar to those after aCSF. Twenty-four
hours after MTII injection, rats lost more weight than after aCSF
[t(9) = 2.064; P < 0.05]. Water
intake was reduced 2 h after MTII injections [aCSF mean,
5.44 g; MTII mean, 3.52 g; t(9) = 1.982;
P < 0.05], but did not vary across conditions at
subsequent times.
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]. The drug affected
water intake only at 4 h, increasing it slightly [aCSF mean,
10.3 g; SHU9119 mean, 13.04 g; t(6) = -2.717;
P < 0.05]. Body weight did not vary across conditions
at any time.
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and 5]. There was no difference in the
responses of VX and sham-operated rats [24-h intake: F(1, 9) =
1.693; P = NS] and no two-factor interaction. At
48 h, liquid diet intake after MTII was lower than after vehicle
in both groups of animals [F(1, 9) = 17.056; P <
0.005]. VX rats, however, had significantly lower intakes than control
rats [F(1, 7) = 7.860; P < 0.03]. By 72 h,
intakes were not significantly different across conditions. There were
no significant differences between the intake effects of lateral- and
4th-icv delivery of MTII (i.e. no main effects of placement
and no interactions involving this factor).
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, MTII decreased body
weight at 24 h [F(1, 9) = 40.442; P <
0.001] and 48 h [F(1, 7) = 16.555; P <
0.005] relative to vehicle injections for both VX and sham-operated
controls, which did not differ [F(1, 9) = 1.668; P
= NS]. Seventy-two hours after MTII injections, rats still had not
completely returned to their preinjection weights, but this fell short
of statistical significance [F(1, 7) = 4.42; P =
0.07]. There were no differences in body weight change depending on
the ventricle injected.
Water intake. VX rats showed no change in 24-h water intake
after MTII, whereas control animals increased (P =
0.01) water consumption 24 h after the drug injection [VX x
MTII interaction: F(1, 9) = 10.881; P < 0.01]. At
48 h, MTII significantly increased water intake in both VX and
sham-operated rats [F(1, 7) = 19.99; P < 0.003;
see Fig. 6]. Seventy-two hours after
injections, there were no differences in water consumption across
conditions. Water intake did not vary based on injection site under any
condition.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although centered in the DVC, the infusions probably spread beyond its boundaries. It is unlikely, however, that the intake effects of these injections are mediated by structures outside of the DVC. Studies of MC4-R mRNA expression and MC3/4-R binding (13, 14) suggest that receptors are located in the DMX and medial NTS, and not in neighboring structures. Further work is needed to determine whether receptors in the DMX, NTS, or both nuclei mediate the effects of MTII and SHU9119 within the DVC.
Given our conclusion that the DMX and/or NTS contributes to the intake suppression of 4th-icv MTII, one might expect subdiaphragmatic vagotomy to reverse these effects at least in part. In fact, vagotomy had no effect on the feeding response to 4th-icv MTII. This result clearly indicates that nonvagal pathways from the DVC mediate these effects. A discussion of such pathways would naturally begin with the NTS. For example, projections from the NTS to centers of sympathetic nervous system control are well known (20, 21), and sympathetic actions of central MC4-R stimulation have been described. Recently, lateral-icv MTII was shown to increase sympathetic nerve activity to the brown adipose tissue (22). It is also possible that MTII effects are mediated by projections from the NTS to other structures of relevance to intake control, including the parabrachial nucleus, amygdala, and hypothalamic nuclei (23, 24).
The failure of vagotomy to reverse 4th-icv MTII effects discounts a role for efferents arising from DMX motor neurons and challenges the hypothesis that MC4-Rs in the DMX are a critical substrate. The DMX cannot be ruled out given potentially relevant nonvagal outputs from this nucleus. It has been known since the time of Cajal that there are at least two kinds of neurons in the DMX, medium (motor neurons) and small (25, 26). The small neurons do not contribute axons to the vagus and survive vagotomy with no evidence of chromatolysis (27, 28). Projections from these neurons to the parabrachial nucleus (29) and as far as the paraventricular nucleus of the hypothalamus (30) have been described. Another nonvagal output of the DMX may arise from the motor neurons, themselves. A substantial portion of them survive subdiaphragmatic vagotomy (31). These cells have long dendrites that extend into the NTS (32). MC4-Rs on DMX neurons could influence NTS projection neurons through dendrodendritic or dendrosomatic synapses, the existence of which has not been confirmed (32). Thus, the DMX, a site containing the highest density of MC4-R mRNA expression and MC3/4-R binding, remains a candidate for the mediation MTII effects in intact as well as VX rats.
Although vagotomy did not block the suppression of liquid diet intake after MTII, other effects of vagotomy were seen in this study. Intact rats increased water intake substantially during each of 2 days after MTII injection. VX rats did not show this response on the first day and exhibited a blunted increase in water intake between 24 and 48 h after injection. This increased water intake in control rats after MTII treatment is in sharp contrast with the decrease in both water and food intake seen in rats maintained ad libitum on solid food. Rats maintained on liquid diet drink very little water, only 10–15% of their total fluid intake. Given that MTII suppressed liquid diet intake by about 80%, it seems appropriate to interpret the increase in water intake as a compensatory response. This effect provides the strongest evidence to date that MTII does not suppress ingestive behavior in general, but, rather, specifically reduces nutrient intake. The delayed response in VX rats, therefore, appears to reflect their inability to adjust water intake in response to a challenge to fluid homeostasis.
The present work clearly demonstrates effects on feeding and body weight after stimulation of caudal brainstem MC3/4-Rs. Unilateral DVC injections of MC3/4-R ligands at doses subthreshold for ventricular response were effective; MTII injection resulted in deep suppression of intake and body weight, and administration of SHU9119 increased intake. These data do not speak to the question of whether forebrain MC3/4-Rs can give rise to similar effects. In fact, forebrain receptor action is suggested by the finding that the dose-response curves for lateral- and 4th-icv administration of MTII are virtually indistinguishable, as are the curves for icv administration of SHU9119 (5). Furthermore, recent work has shown that injections of low doses of MTII or SHU9119 into the paraventricular nucleus of the hypothalamus yield effects on daily intake (33) similar to those obtained here with DVC injections. It seems that similar responses can be elicited by pharmacological stimulation or inhibition of disparate MC4-R subpopulations. The present results suggest that a complete discussion of MC4-R contributions to intake and body weight control must address at least these two sets of receptors.
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Acknowledgments |
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Footnotes |
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Received October 4, 1999.
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