 |
 Previous Article | Next Article 
The Journal of Neuroscience, April 1, 1998, 18(7):2646-2652
The Second Messenger cAMP Elicits Eating by an Anatomically
Specific Action in the Perifornical Hypothalamus
Elizabeth R.
Gillard1,
Arshad M.
Khan2,
Rickinder
S.
Grewal1,
Bara
Mouradi1,
Stefany D.
Wolfsohn1, and
B. Glenn
Stanley1
1 Departments of Neuroscience and Psychology and
2 Division of Biomedical Sciences and Department of
Neuroscience, University of California at Riverside, Riverside,
California 92521
 |
ABSTRACT |
We have previously shown that a membrane-permeant analog of cAMP,
8-bromo-cAMP (8-br-cAMP), elicits a vigorous eating response when
microinjected into the perifornical hypothalamus (PFH) or lateral
hypothalamus (LH) of satiated rats, suggesting that increases in cAMP
in these areas may be important in the neural control of eating. To
determine the locus of this effect, we compared the ability of
8-br-cAMP (1-100 nmol/0.3 µl) to elicit eating after microinjection
into the PFH, LH, or the following bracketing areas: the anterior and
posterior LH, paraventricular nucleus of the hypothalamus, thalamus,
and amygdala. 8-br-cAMP at 50 nmol elicited eating ( 3.4 gm in 2 hr)
exclusively in the PFH and LH. At 100 nmol, 8-br-cAMP elicited a larger
response in these areas and elicited a smaller, more variable response
in the thalamus. We similarly mapped the feeding-stimulatory effects of
compounds that increase endogenous cellular cAMP in naive rats.
Combined microinjection of matched doses (300 nmol) of
3-isobutyl-1-methylxanthine and
7-deacetyl-7-O-(N-methylpiperazino)- -butyryl-forskolin
was effective exclusively in the PFH, eliciting an average 2 hr food intake of 8.4 ± 2.0 gm. Collectively, these results suggest that increases in cellular cAMP within a specific brain site, the PFH, may
play a role in the neural stimulation of eating.
Key words:
perifornical hypothalamus; hypothalamus; cAMP; feeding; second messengers; eating; rat
| |
INTRODUCTION |
Several lines of evidence suggest
that the lateral hypothalamus (LH) and adjacent perifornical
hypothalamus (PFH) are intimately involved in the neural control of
food intake. Specifically, neurotoxic and electrical lesions of the LH
and PFH disrupt eating (Anand and Brobeck, 1951 ; Winn et al., 1984),
whereas electrical stimulation in these areas elicits eating in
satiated animals (Murzi et al., 1991). Similarly, neurochemical
stimulation of LH neurons by glutamate and its receptor agonists
elicits intense eating in satiated rats, and glutamate and its
receptors have been suggested to play a physiological role in the
control of eating and body weight (Stanley et al., 1996). Likewise, the
PFH is most sensitive to both the eating-stimulatory effects of
neuropeptide Y (Stanley et al., 1993) and the feeding-suppressive
effects of catecholamine neurotransmitters, which may participate in
the physiological control of eating (Leibowitz and Stanley, 1986).
Neurons in the LH and PFH also respond to internal signals related to
the nutritive state of the animal, such as changes in circulating
levels of the metabolic fuel glucose (Himmi et al., 1988). More
strikingly, LH and PFH neurons respond with changes in firing rate to
the sight, smell, and taste of food that both precede and predict
feeding in the hungry monkey (Rolls et al., 1986). These neurons also
acquire responses to non-food objects paired repeatedly with food, in
parallel with the learning of behavioral responses to the objects (Mora
et al., 1976).
These data suggest that neuronal elements in the LH and PFH are
responsive to multiple neurotransmitter inputs relaying information about the internal and external environment, and that integration of
this information by LH and PFH neurons may be important in the neural
control of eating. The activity of individual neurons in these areas is
modulated in vitro (Cheng et al., 1988) by several neurotransmitters that affect eating, suggesting that individual cells
may integrate the effects of a variety of feeding-related neurochemicals. Recent evidence suggests that the cAMP-synthesizing enzyme adenylyl cyclase (AC) may function as a coincidence detector for
activation of a variety of metabotropic receptors (Lustig et al.,
1993), and we have shown that PFH and LH microinjection of the cAMP
analog 8-bromo-cAMP (8-br-cAMP) or drugs that increase levels of
endogenous cAMP elicits a robust eating response in satiated animals
(Gillard et al., 1997a). These findings may suggest that cAMP in a
population of LH and/or PFH neurons might participate in integrating
the feeding-stimulatory and feeding-inhibitory effects of some
neurotransmitters. However, because diffusion of centrally injected
substances is common (Myers et al., 1971), it is also possible that the
eating-stimulatory effects we observed were consequent to diffusion
away from the injection site and actions outside of the LH and PFH. To
resolve this issue, in the current study, we compared the ability of
8-br-cAMP and agents that increase cAMP levels to stimulate eating when
injected into the PFH and LH with their effectiveness when injected
into sites bracketing these areas.
Portions of this study have been presented in preliminary form (Gillard
et al., 1994, 1997b).
| |
MATERIALS AND METHODS |
Subjects and surgery. A total of 145 adult male
Sprague Dawley rats obtained from Charles River Laboratories
(Wilmington, MA) were individually housed in a vivarium at 21°C with
a 12 hr light/dark cycle and maintained on Purina Rat Chow pellets and water ad libitum until 3 d after surgery, when the
pellets were removed and permanently replaced with a sweetened
milk-mash diet (46% Purina Rat Chow powder, 37% sucrose, and 17%
Carnation evaporated milk) to which they had free access for the
duration of the experiment.
Animals weighing 350-450 gm were anesthetized by Metofane inhalation
and implanted stereotaxically with a chronic unilateral 26 gauge
stainless steel guide cannula targeted at the LH, the medial PFH, the
thalamus (THAL) dorsal to these sites, the paraventricular nucleus
(PVN) medial to these sites, the amygdala (AMY) lateral to these sites,
the anterior lateral hypothalamus (ALH), or the posterior lateral
hypothalamus (PLH). For rats weighing 400-425 gm, with the incisor bar
at  3.3 mm, coordinates (in mm) were 6.3 anterior to the interaural
line (A), 2.0 lateral to the midsagittal sinus (L), and 8.0 ventral to
the surface of the skull (V) for the LH; 6.3 A, 1.0 L, and 8.0 V for
PFH; 6.3 A, 1.8 L, and 5.4 V for THAL; 6.8 A, 0.4 L, and 7.4 V for PVN;
6.3 A, 3.5 L, and 8.2 V for AMY; 7.7 A, 1.8 L, and 8.2 V for ALH; and
4.6 A, 1.6 L, and 7.7 V for PLH. For rats weighing >425 gm, 0.1 mm was
added in the anterior direction. The cannula was permanently anchored to the skull with dental acrylic and stainless steel screws, and a
plastic guard was placed around the exposed portion of the cannula. To
maintain patency, a 33 gauge stainless steel obturator was inserted
into the lumen of the cannula. Animals were allowed at least 1 week of
postoperative recovery, during which they were repeatedly handled and
mock-injected to accustom them to the testing procedure.
General test procedure. Tests were conducted during
the early portion of the light phase with freshly prepared mash diet
provided to each subject at least 1.5 hr before testing to maximize
satiety. Central injections were administered in a volume of 0.3 µl
through a 33 gauge injector terminating in the brain 1 mm below the tip of the guide cannula. Food intake was measured 1, 2, and 4 hr after the
final injection. For each experiment, all treatments were administered
in counterbalanced order in tests separated by at least 48 hr.
Experiments were completed within 5 weeks after surgery.
Pharmacological agents. 8-br-cAMP (sodium salt) and
3-isobutyl-1-methylxanthine (IBMX) were obtained from Sigma (St. Louis, MO). A water soluble analog of forskolin,
7-deacetyl-7-O-(N-methylpiperazino)--butyryl-forskolin dihydrochloride (MPB forskolin), was obtained from Calbiochem (San
Diego, CA). IBMX was dissolved in dimethyl sulfoxide (DMSO). All other
agents were dissolved in artificial CSF (aCSF) composed of (in
mM): Na+ 147, Cl
154, K+ 3, Ca2+ 1.2, and
Mg2+ 0.9, pH 7.4.
Experiment 1. To determine the locus of 8-br-cAMP
feeding stimulation, the membrane-permeant cAMP analog 8-br-cAMP (1, 10, and 100 nmol) or its aCSF vehicle was injected into the PFH
(n = 8), LH (n = 10), THAL
(n = 6), PVN (n = 9), AMY
(n = 6), ALH (n = 8), or PLH
(n = 6) (total n = 53).
Experiment 2. To determine the consistency in the
magnitude of the observed eating responses and to better localize the
effect of 8-br-cAMP, a naive group of animals (n = 33)
implanted with PFH (n = 12), LH (n = 11), or THAL (n = 10) cannulas was injected with 100 nmol of 8-br-cAMP or aCSF. Data for each subject were averaged over two
trials of the counterbalanced series.
Experiment 3. To examine the anatomical specificity
of eating elicited by compounds that increase endogenous cellular cAMP, a separate group of animals (total n = 59) received
central injections of IBMX (30, 150, or 300 nmol) followed 10 min later
by a matched dose of MPB forskolin or injection of DMSO vehicle
followed by aCSF vehicle. Animals with cannulas targeted at the PFH
(n = 14), LH (n = 9), THAL
(n = 11), PVN (n = 6), AMY
(n = 6), ALH (n = 7), and PLH
(n = 6) received the resulting four treatments in counterbalanced order. To determine whether IBMX alone or MPB forskolin
alone could elicit eating, after completing this first block of tests
the subjects remaining with intact headpieces and cannulas
(n = 52) were injected in counterbalanced order with DMSO followed 10 min later by aCSF, 300 nmol of IBMX followed by aCSF,
or DMSO followed by 300 nmol of MPB forskolin.
Histological verification of cannula placements and data
analysis. On completion of testing, subjects were killed by
CO2 inhalation and perfused transcardially with 10%
Formalin. Brains were removed and post-fixed for at least 24 hr in
Formalin before sectioning on a cryotome. Coronal sections (100 µm)
were cut through the extent of the visible cannula track and stained
with cresyl violet. The injection sites were then localized by tracing
the image onto size-matched figures adapted from the atlas of Paxinos
and Watson (1986). Food intake data were averaged and analyzed for
effects of drug and injection site using ANOVA, with multiple
comparisons (Duncan's multiple range test) performed at an  of
0.05.
| |
RESULTS |
Histology
The injection sites for 140 of the 145 subjects were localized
histologically; all of these were located in their intended targets.
Photomicrographs of representative injection sites within the seven
brain sites tested are shown in Figure
1.
View larger version (91K):
[in this window]
[in a new window]
|
Figure 1.
Photomicrographs of coronal sections stained with
cresyl violet showing representative injection sites, indicated by
arrows, located in A, G,
lateral hypothalamus (LH); B,
paraventricular nucleus of the hypothalamus
(PVN); C, perifornical
hypothalamus (PFH); D, posterior
lateral hypothalamus (PLH); E,
anterior hypothalamus (ALH); F,
thalamus (THAL); H, amygdala
(AMY). The top row shows 8-br-cAMP
injection sites, and the bottom row shows IBMX-MPB
forskolin injection sites. Scale bar, 1 mm.
|
|
Experiment 1
Seven brain sites within or bracketing the hypothalamus were
tested for sensitivity to the eating-stimulatory effect of 8-br-cAMP (1-100 nmol). As shown in Figure 2, the
membrane-permeant cAMP analog stimulated eating only after injection
into the PFH, LH, and THAL. Statistical analysis revealed significant
effects of both injection site and drug dose 1 hr
(F6,230 = 2.71; p < 0.02; F4,230 = 3.91; p < 0.005, respectively), 2 hr (F6,230 = 2.75; p < 0.02; F4,230 = 5.27;
p < 0.0005, respectively), and 4 hr after injection
(F6,230 = 3.66; p < 0.002;
F4,230 = 6.25; p < 0.0002). The
only significant interaction was between drug dose and injection site
at 4 hr after injection (F24,230 = 1.85;
p < 0.0002). Multiple comparison tests showed that
significant eating was elicited at some postinjection times after
injection of 8-br-cAMP into the LH with doses as low as 1 nmol and the
PFH with doses as low as 10 nmol (p < 0.05),
and reliable eating was obtained with injections of 50 and 100 nmol of
8-br-cAMP in both of these sites. The 50 nmol dose elicited eating of
3.4 gm in 2 hr, and the highest dose of 8-br-cAMP (100 nmol) elicited
food intake of 4.7 ± 1.7 and 5.8 ± 2.0 gm in 2 hr in the LH
and PFH, respectively. Intake 4 hr after injection into the LH and PFH
was 5.7 ± 1.6 and 6.1 ± 2.0 gm, respectively. Although
there was no significant increase in eating during the first hour,
injection of the highest dose of 8-br-cAMP into the THAL dorsal to the
LH and PFH elicited significant eating 2 hr (4.4 ± 1.9 gm) and 4 hr after injection (6.3 ± 2.3 gm) (p < 0.05). The eating response to 8-br-cAMP injected into the THAL was more
variable between individuals and of a longer latency than the response
obtained after injection into the LH or PFH, suggesting that LH and PFH
injections caused eating via actions of 8-br-cAMP on local brain tissue
rather than after diffusion dorsally to the THAL. Injection of
8-br-cAMP into areas bracketing the PFH and LH in all other directions
was ineffective in stimulating eating.
View larger version (24K):
[in this window]
[in a new window]
|
Figure 2.
Cumulative food intake 1, 2, and 4 hr after
injection (mean ± SEM) as a function of injection site
(ALH, anterior hypothalamus; LH, lateral
hypothalamus; PLH, posterior lateral hypothalamus; PFH, perifornical hypothalamus; PVN,
paraventricular nucleus; THAL, thalamus;
AMY, amygdala) and dose of 0-100 nmol of 8-br-cAMP. *p < 0.05, greater than vehicle at the
corresponding time. Downward carats indicate lower than
PFH and LH scores at the corresponding time; p < 0.05, both by Duncan's multiple range test.
|
|
Figure 3A presents food intake
induced by 8-br-cAMP as a function of the mean histologically derived
injection sites for all brain areas tested. As shown here, the LH and
PFH injection sites were bracketed dorsally by the THAL, medially by
the PVN, laterally by the AMY, anteriorly by the ALH, and posteriorly
by the PLH. The PFH, LH, THAL, and AMY injections were located at
approximately the same coronal level: lateral to and just posterior to
the PVN. The PFH and LH injections were separated by an average of 1 mm. Injection sites in the AMY were within the medial nucleus, the basomedial nucleus, and the anterior cortical nucleus. The PVN injections were located an average of 0.8 mm from the PFH injection sites. ALH injections were placed an average of 1.1 mm anterior to the
LH, and the PLH injections were placed an average of 2 mm posterior to
the LH. The THAL injections were placed an average of 1.9 mm dorsal to
the LH in the ventrolateral, ventromedial, and ventral posterolateral
thalamic nuclei. As shown in Figure 3A, injection of 100 nmol of 8-br-cAMP elicited significant eating within the first hour
after injection into the LH and PFH, whereas all other sites, including
the THAL dorsal to the LH and the PVN just 0.8 mm medial to the PFH,
injections were ineffective.
View larger version (57K):
[in this window]
[in a new window]
|
Figure 3.
Brain sites from which eating is elicited by
injection of 8-br-cAMP and agents that increase endogenous cAMP.
Cumulative food intake, rounded to the nearest gram, elicited by 100 nmol of 8-br-cAMP (A) or combined 300 nmol of
IBMX and MPB forskolin (B), 1 (left side of brain) and 4 hr (right side of brain)
after injection into the histologically determined mean sites
represented by the circles (filled
circles represent sites from which eating was elicited;
p < 0.05, Duncan's multiple range test;
open circles represent ineffective sites) on
size-matched schematics (Paxinos and Watson, 1986) in the horizontal
(top) and coronal (bottom) planes. In the
top schematics, sections in the horizontal plane have
been collapsed to depict the anteroposterior and mediolateral relation
of the injection sites to each other, whereas in the bottom planes, anteroposterior sections have been
collapsed to depict the dorsoventral and mediolateral position of the
injection sites. ALH, Anterior hypothalamus;
LH, lateral hypothalamus; PLH, posterior
lateral hypothalamus; PFH, perifornical hypothalamus; PVN, paraventricular nucleus; THAL,
thalamus; AMY, amygdala.
|
|
Experiment 2
As shown in Figure 4 and consistent
with the previous results, in a group of naive animals 100 nmol of
8-br-cAMP elicited marked eating after injection into the LH and PFH
and elicited a smaller eating response after injection into the THAL.
Statistical analysis revealed significant effects of both drug
treatment and injection site at 1 and 2 hr after injection
(F1,60 = 42.88; p < 0.0001;
F2,60 = 3.67; p < 0.04 at 1 hr;
F1,60 = 47.25; p < 0.0001; F2,60 = 4.10; p < 0.03 at 2 hr), with 8-br-cAMP eliciting significant feeding after injection into
all three sites tested. By 4 hr after injection, the effect of drug
treatment only was significant (F1,60 = 35.94;
p < 0.0001), and 4 hr eating scores after 8-br-cAMP
were significantly greater than vehicle scores in all three sites
tested. Although 8-br-cAMP was effective in stimulating eating when
injected into the LH, PFH, and THAL, injections into the THAL resulted in a significantly smaller feeding response within the 2 hr after injection than that elicited by LH or PFH injections
(p < 0.05). Food intake 2 hr after injection
into the THAL (4.0 ± 1.0 gm) was <48% of that observed after
injection into the PFH (8.6 ± 1.2 gm) and LH (8.5 ± 1.8 gm).
View larger version (24K):
[in this window]
[in a new window]
|
Figure 4.
Cumulative food intake 1, 2, and 4 hr after
injection (mean ± SEM) elicited by 100 nmol of 8-br-cAMP as a
function of injection site (LH, lateral hypothalamus;
PFH, perifornical hypothalamus; THAL,
thalamus). *p < 0.05, greater than vehicle at the
corresponding time; downward carats indicate lower than
PFH and LH scores at the corresponding time; p < 0.05, both by Duncan's multiple range test.
|
|
Experiment 3
As shown in Figure 5, eating was
significantly stimulated by combined injection of the phosphodiesterase
inhibitor IBMX and the adenylyl cyclase activator MPB forskolin only
into the PFH. A significant interaction between injection site and drug
dose was found at 1 hr (F18,208 = 4.54; p < 0.0001), 2 hr (F18,208 = 3.28; p < 0.0001), and 4 hr (F18,208 = 2.43; p < 0.005) after injection. A significant effect of drug dose alone on
eating was found only in the PFH (F3,52 = 11.61;
p < 0.0001 at 1 hr after injection; F3,52 = 9.75; p < 0.001 at 2 hr after injection; and F3,52 = 3.73; p < 0.02 at 4 hr after
injection), with stimulation of eating compared with vehicle scores
occurring after combined injection of the highest doses (300 nmol) of
IBMX and MPB forskolin (p < 0.05 at all time
points). The eating response was robust, with food intake 2 hr after
injection of this dose into the PFH averaging 8.4 ± 2.0 gm,
compared with an average intake of 1.3 ± 0.3 gm 2 hr after
injection of vehicle solution. In contrast, combined injection of IBMX
and MPB forskolin into all other surrounding brain sites failed to
stimulate eating with respect to vehicle scores.
View larger version (21K):
[in this window]
[in a new window]
|
Figure 5.
Cumulative food intake 1, 2, and 4 hr after
injection (mean ± SEM) as a function of injection site
(ALH, anterior hypothalamus; LH, lateral
hypothalamus; PLH, posterior lateral hypothalamus; PFH, perifornical hypothalamus; PVN,
paraventricular nucleus; THAL, thalamus;
AMY, amygdala) and matched doses of 0-300 nmol of IBMX
and MPB forskolin. *p < 0.05, greater than vehicle
at the corresponding time; downward carats indicate
lower than PFH scores at the corresponding time; p < 0.05, both by Duncan's multiple range test.
|
|
A significant effect of injection site alone on eating was found only
at the effective dose (300 nmol) of IBMX and MPB forskolin (F6,52 = 5.74; p < 0.0001 at 1 hr after
injection; F6,52 = 4.81; p < 0.0006 at 2 hr after injection; and F6,52 = 4.36; p < 0.0012 at 4 hr after injection), with eating after injection into the PFH being significantly greater than that resulting from injection into
all other sites (p < 0.05 by Duncan's multiple
range test at all time points).
Figure 3B depicts the histologically determined mean
injection sites for all brain areas examined and shows that the
effective PFH injections were bracketed in all directions by
ineffective sites. The distribution of tested brain sites is similar to
that of Experiment 1. The effective PFH injections were concentrated at
a coronal level ~0.5 mm posterior to the ineffective PVN injections and 0.7 mm medial to the average LH placement. The ALH injections 0.9 mm anterior to the LH and the PLH injections 1.6 mm posterior to the LH
were also ineffective in stimulating eating. Likewise, injections into
the THAL ~1.6 mm dorsal to the LH at the same coronal level as the
PFH and LH failed to stimulate eating, as did injections into the AMY
nuclei slightly anterior to and ~2.3 mm lateral to the PFH. Eating
elicited by combined injection of IBMX and MPB forskolin (300 nmol)
into the PFH was 8.1 ± 2.0 and 8.8 ± 2.0 gm at 1 and 4 hr
after injection, respectively, whereas injection of this dose into
sites other than the PFH never exceeded a mean of 3.8 gm at any time
point measured.
In contrast to the effectiveness of combined injection of IBMX and MPB
forskolin (300 nmol) in the PFH, neither compound alone stimulated
eating in any brain site tested (p > 0.20 by
ANOVA) (data not shown).
| |
DISCUSSION |
The present study demonstrates that functionally increasing cAMP
concentrations by means of 8-br-cAMP (Greengard, 1978) injection into
the PFH and LH elicits eating, whereas injections into other sites are
either less effective or ineffective. The effective sites in the PFH
and LH were bracketed anteriorly, posteriorly, medially, and laterally
at average distances of only 0.8-2.0 mm by ineffective sites, and the
only other brain site into which injection of 100 nmol of 8-br-cAMP
stimulated eating was the THAL 1.9 mm dorsal to the LH. One question is
to what extent 8-br-cAMP injected into the PFH and LH stimulated
feeding by a local action in these sites, or by diffusion to dorsal
brain structures, such as the THAL. Eating consequent to diffusion from
the LH and PFH seems unlikely, however, because THAL injections were
effective only at the highest dose of 8-br-cAMP tested (100 nmol), and
even this dose did not consistently stimulate eating within the first hour after injection into the THAL, in contrast to the consistent stimulation at this time after PFH and LH injections (Figs. 2, 4). When
eating was elicited during the first hour after THAL injections, 1 and
2 hr food intake was <48% of the food intake of PFH- and LH-injected
subjects (Fig. 4). Additionally, the eating elicited by THAL injections
might be mediated by biochemical mechanisms distinct from those that
produce eating after PFH and LH injections. This was suggested by our
previous evidence that the cGMP analog 8-br-cGMP was completely
ineffective in eliciting eating after injection into the PFH or LH but
did elicit a feeding response after THAL injection that was equivalent
to that elicited by 8-br-cAMP injected into that site (Gillard et al.,
1997a). Further supporting local, rather than thalamic, actions of PFH
injections is the current finding that agents that increase endogenous
cAMP (IBMX and MPB forskolin) stimulated eating after injection into
the PFH but not the THAL. Although diffusion and reflux of centrally injected chemicals up the cannula track into the cerebroventricles can
occur and may mediate behavioral effects (Myers et al., 1971; Johnson
and Epstein, 1975), the smaller effects observed with THAL injections
close to the lateral ventricles and the ineffectiveness of injections
into the PVN just lateral to the third ventricle argue against
mediation of eating by diffusion or reflux of 8-br-cAMP into the
ventricular system. Collectively, these data argue that 8-br-cAMP
elicited feeding after injection into the LH and PFH via an action on a
cellular cAMP effector in these locations rather than after diffusion
or reflux to the THAL or the cerebroventricles, because if the
8-br-cAMP was acting after diffusion to these sites, then the THAL
injections would be expected to yield greater responses than LH and PFH
injections.
It is ultimately of interest to determine whether the eating observed
after introduction of exogenous 8-br-cAMP reflects cellular processes
within the PFH that are relevant to the physiological control of food
intake. The present study confirms that injection of drugs that
increase endogenous cAMP can stimulate intense eating (Gillard et al.,
1997a), and it is the first to show that this effect is attributable to
actions specifically in the PFH. We have shown previously that 100 nmol
of cAMP itself, which does not cross cell membranes appreciably, does
not stimulate eating in the PFH and LH (Gillard et al., 1997a),
suggesting that the membrane-permeant cAMP analog 8-br-cAMP stimulates
eating via an intracellular action on local cell bodies or processes
rather than via osmotic effects or activation of adenosine receptors. Consistent with this, we demonstrated in the present study that combined PFH injection of the phosphodiesterase inhibitor IBMX, which
reduces the breakdown of cAMP, and the AC stimulator MPB forskolin,
which stimulates cAMP production, significantly stimulates eating of up
to 8.8 ± 2.0 gm (Fig. 5). Several findings argue that combined
IBMX-MPB forskolin injection elicited eating via actions on a common
system, the cAMP system. Although IBMX can have
phosphodiesterase-independent effects (Schwabe et al., 1978), and
unmodified forskolin is known to have cAMP-independent effects (McHugh
and McGee, 1986; Laurenza et al., 1989; Baxter and Byrne, 1990), the
failure of either compound alone to elicit eating when injected
unilaterally argues against mediation of eating by the different
nonspecific effects of either compound. In addition, preliminary
studies have shown that whereas bilateral PFH injection of 300 nmol of
MPB forskolin alone elicited eating, a forskolin analog having the same
non-cAMP-dependent effects as forskolin but lacking cAMP-related
activity (Laurenza et al., 1989) did not (Gillard et al., 1997b). These
results collectively argue against mediation of eating by either
osmotic effects or non-cAMP-dependent effects and, when combined with
the ineffectiveness of IBMX-MPB forskolin in the other sites tested
(particularly the LH and THAL), argue for the PFH as the most likely
locus of feeding stimulation by endogenous intracellular cAMP.
Several findings also suggest that the effects of 8-br-cAMP and agents
that increase endogenous cAMP are attributable to actions in the PFH
rather than in the nearby ventromedial hypothalamic nucleus (VMH),
which has also been implicated in eating control. Specifically, in
contrast to the effectiveness of PFH injections of 8-br-cAMP and agents
that increase cAMP, injections into the PVN ~0.8 mm medial to the PFH
were ineffective despite the proximity of these injections to the VMH,
also located medially to the PFH. In addition, separate experiments in
our laboratory have shown that VMH injections of 8-br-cAMP and
IBMX-MPB forskolin at the same doses used in the present study do not
elicit significant increases in eating with respect to vehicle
injections (p > 0.2) (E. R. Gillard, A. M. Khan, B. G. Stanley, unpublished observations).
Why did application of agents that typically increase endogenous cAMP
elicit eating with much greater anatomical specificity than did
microinjection of 8-br-cAMP? The disparity may simply reflect
differences in the pattern and extent of diffusion in brain tissue
among these chemically diverse compounds. However, the major functional
difference among these compounds is their mode of action. Whereas
8-br-cAMP penetrates cells indiscriminately, the ability of both IBMX
and MPB forskolin to increase cAMP should be dependent on cellular
phosphodiesterase and adenylyl cyclase, with IBMX-MPB forskolin
treatment being most effective in cells having the highest levels of
phosphodiesterase and adenylyl cyclase. Consistent with this idea is
evidence that the abundance and types of adenylyl cyclase vary markedly
across different brain sites (Williams et al., 1969; Worley et al.,
1986; Xia et al., 1992; Mons et al., 1993; Cali et al., 1994; Mons and
Cooper, 1994). Such a difference in sensitivity between cells in
different areas, coupled with the requirement for relatively high doses
to be effective in stimulating eating, might account for the finding
that IBMX-MPB forskolin elicited eating only when applied in the PFH
and not in the LH, although neurons in both of these areas have
demonstrated roles in orchestrating eating behavior, and both are
responsive to the eating-stimulatory effect of 8-br-cAMP.
Although further study is required to resolve the identity of the
cellular elements on which 8-br-cAMP and IBMX-MPB forskolin act in the
PFH, that injections into other sites were ineffective suggests that
stimulation of cell bodies or processes in the PFH rather than of
fibers of passage elicited eating. Injections into areas bracketing the
PFH and LH, particularly the ALH and PLH, would be expected to
intersect the course of major fiber systems coursing through the LH and
PFH. It is unknown which cell types in the PFH are affected by local
increases in cAMP resulting from application of 8-br-cAMP or agents
that increase endogenous cAMP. However, the abundance of evidence
implicating PFH neurons in natural eating, as well as the particular
effectiveness of PFH electrical stimulation in eliciting eating
(Leibowitz and Stanley, 1986; Murzi et al., 1991), suggests that these
agents may act on neurons intrinsic to this area and/or on the local
processes of extrinsic neurons to stimulate eating.
The results of the present study suggest that cAMP may be an important
second messenger in PFH neurons participating in a neural circuit
concerned specifically with eating regulation. In this regard, it is
interesting that the hypothalamus has a high rate of cAMP production
(Williams et al., 1969). Because its activity is subject to regulation
by inhibitory and stimulatory G-proteins as well as components of other
signaling systems, the cAMP-synthesizing enzyme AC has the potential to
act as a coincidence detector for the activation of a variety of
metabotropic neurotransmitter receptors (Bourne and Nicoll, 1993;
Lustig et al., 1993). The cAMP second messenger system may be a
powerful means of integrating the actions of multiple neurotransmitters
that affect eating through actions in the PFH. Although it is unknown
which neurotransmitters might act directly or indirectly on the cAMP
system to stimulate food intake, the PFH is most sensitive to the
eating-suppressive effects of dopamine and epinephrine and the
eating-stimulatory effect of neuropeptide Y (NPY) (Stanley et al.,
1993), suggesting that PFH cAMP may contribute to the effects of these
neurochemicals. However, all of the known NPY receptor subtypes couple
to decreases in cAMP, suggesting that increased PFH cAMP is unlikely to
elicit eating by acting exclusively on PFH cells directly responsive to
NPY. Rather, increases in cAMP may act on a variety of cell types, with
the effects on neurons downstream of NPY-responsive neurons being
critical for eating stimulation. That PFH NPY may ultimately induce
increases in cAMP in neurons downstream of those bearing NPY receptors
is consistent with recent findings that both PFH injection of NPY and
food deprivation increased cAMP response element binding to rat
hypothalamic nuclear extracts (Sheriff et al., 1997). Although this
explanation appears to be the most parsimonious, it may be premature to
dismiss the possibility that increased cAMP within NPY-responsive
neurons might contribute to the observed stimulation of eating, given
that it has not been clearly established that NPY elicits eating
exclusively by decreasing cAMP in the eating-relevant neurons bearing
NPY receptors. Specifically, (1) eating-relevant NPY receptors may
couple to other second messenger systems, such as increases in
intracellular Ca2+ (Herzog et al., 1992); (2)
pertussis toxin, which blocks both eating and inhibition of cAMP
production in response to NPY (Chance et al., 1989), also blocks
increases in intracellular Ca2+ mediated by the
Go protein; and (3) PFH NPY has been shown to result in
increased activity of hypothalamic calcium- and calmodulin-dependent protein kinase II (Sheriff et al., 1997). These findings may suggest that the effects of PFH NPY and cAMP on eating might not be mutually exclusive. Consistent with this, our preliminary studies indicate that
PFH administration of 8-br-cAMP does not reduce eating elicited by NPY
(E. R. Gillard, A. M. Khan, A. U. Haq, B. G. Stanley, unpublished observations).
The dramatic stimulation of eating by increases in PFH cAMP suggests
that this second messenger may play a role in a neural circuit(s)
controlling eating. In addition to integrating the effects of multiple
neurotransmitters in the PFH, cAMP may also contribute to the
plasticity evidenced by PFH neurons during food-related learning (Mora
et al., 1976).
| |
FOOTNOTES |
Received Aug. 12, 1997; revised Dec. 29, 1997; accepted January 8, 1998.
This study was supported by National Institutes of Health Grant
NS24268, Eli Lilly, Sigma Xi, and the University of California, Riverside Department of Neuroscience.
Correspondence should be addressed to Dr. E. R. Gillard,
Department of Psychology, University of California at Riverside, Riverside, CA 92521.
| |
REFERENCES |
-
Anand BK,
Brobeck JR
(1951)
Hypothalamic control of food intake in rats and cats.
Yale J Biol Med
24:123-140[Web of Science][Medline].
-
Baxter DA,
Byrne JH
(1990)
Reduction of voltage-activated K+ currents by forskolin is not mediated via cAMP in pleural sensory neurons of Aplysia.
J Neurophysiol
64:1474-1483[Abstract/Free Full Text].
-
Bourne HR,
Nicoll R
(1993)
Molecular machines integrate coincident synaptic signals.
Cell [Suppl]
72:65-75.
-
Cali JJ,
Zwaagstra JC,
Mons N,
Cooper DMF,
Krupinski J
(1994)
Type VIII adenylyl cyclase.
J Biol Chem
269:12190-12195[Abstract/Free Full Text].
-
Chance WT,
Sheriff S,
Foley-Nelson T,
Fischer JE,
Balasubramaniam A
(1989)
Pertussis toxin inhibits neuropeptide Y-induced feeding in rats.
Peptides
10:1283-1286[Web of Science][Medline].
-
Cheng J-T,
Sessler FM,
Azizi SA,
Chapin JK,
Waterhouse B
(1988)
Electrophysiological actions of norepinephrine in rat lateral hypothalamus. II. An in vitro study of the effects of iontophoretically applied norepinephrine on LH neuronal responses to -aminobutyric acid (GABA).
Brain Res
446:90-105[Web of Science][Medline].
-
Gillard ER,
Khan AM,
Haq AU,
Grewal RS,
Stanley BG
(1994)
Stimulation of feeding behavior in the rat by intrahypothalamic injection of 8-br-cAMP.
Soc Neurosci Abstr
20:1680.
-
Gillard ER,
Khan AM,
Haq AU,
Grewal RS,
Mouradi B,
Stanley BG
(1997a)
Stimulation of eating by the second messenger cAMP in the perifornical and lateral hypothalamus.
Am J Physiol
273:R107-R112[Abstract/Free Full Text].
-
Gillard ER,
Mouradi B,
Khan AM,
Wolfsohn S,
Stanley BG
(1997b)
Eating elicited by elevation of endogenous cAMP in the perifornical hypothalamus (PFH) of the rat is anatomically specific.
Soc Neurosci Abstr
23:577.
-
Greengard P
(1978)
In: Cyclic nucleotides, phosphorylated proteins, and neuronal function. New York: Raven.
-
Herzog H,
Hort YJ,
Ball HJ,
Hayes G,
Shine J,
Selbie LA
(1992)
Cloned human neuropeptide Y receptor couples to two different second messenger systems.
Proc Natl Acad Sci USA
89:5794-5798[Abstract/Free Full Text].
-
Himmi T,
Boyer A,
Orsini JC
(1988)
Changes in lateral hypothalamic neuronal activity accompanying hyper- and hypoglycemias.
Physiol Behav
44:347-354[Medline].
-
Johnson AK,
Epstein AN
(1975)
The cerebral ventricles as the avenue for the dipsogenic action of intracranial angiotensin.
Brain Res
86:399-418[Web of Science][Medline].
-
Laurenza A,
Sutkowski EM,
Seamon KB
(1989)
Forskolin: a specific stimulator of adenylyl cyclase or a diterpene with multiple sites of action?
Trends Pharmacol Sci
10:442-447[Medline].
-
Leibowitz SF,
Stanley BG
(1986)
Neurochemical controls of appetite.
In: Feeding behavior: neural and humoral controls (Ritter RC,
Ritter S,
Barnes CD,
eds), pp 191-234. Orlando, FL: Academic.
-
Lustig KD,
Conklin BR,
Herzmark P,
Taussig R,
Bourne HR
(1993)
Type II adenylylcyclase integrates coincident signals from Gs, Gi, and Gq.
J Biol Chem
268:13900-13905[Abstract/Free Full Text].
-
McHugh EM,
McGee Jr R
(1986)
Direct anesthetic-like effects of forskolin on the nicotinic acetylcholine receptors of PC12 cells.
J Biol Chem
261:3103-3106[Abstract/Free Full Text].
-
Mons N,
Yoshimura M,
Cooper DMF
(1993)
Discrete expression of Ca2+/calmodulin-sensitive and Ca2+-insensitive adenylyl cyclases in the rat brain.
Synapse
14:51-59[Web of Science][Medline].
-
Mons N,
Cooper DMF
(1994)
Selective expression of one Ca2+-inhibitable adenylyl cyclase in dopaminergically innervated rat brain regions.
Mol Brain Res
22:236-244[Medline].
-
Mora F,
Rolls ET,
Burton MJ
(1976)
Modulation during learning of the responses of neurons in the lateral hypothalamus to the sight of food.
Exp Neurol
53:508-519[Web of Science][Medline].
-
Murzi E,
Baptista T,
Hernandez L
(1991)
Hypothalamic sites affecting masticatory neurons in rats.
Brain Res Bull
26:321-325[Web of Science][Medline].
-
Myers RD,
Tytell M,
Kawa A,
Rudy T
(1971)
Micro-injection of 3H-acetylcholine, 14C-serotonin and 3H-norepinephrine into the hypothalamus of the rat: diffusion into tissue and ventricles.
Physiol Behav
7:743-751[Medline].
-
Paxinos G,
Watson C
(1986)
In: The rat brain in stereotaxic coordinates. Orlando, FL: Academic.
-
Rolls ET,
Murzi E,
Yaxley S,
Thorpe SJ,
Simpson SJ
(1986)
Sensory-specific satiety: food-specific reduction in responsiveness of ventral forebrain neurons after feeding in the monkey.
Brain Res
368:79-86[Web of Science][Medline].
-
Schwabe U,
Ohga Y,
Daly JW
(1978)
The role of calcium in the regulation of cyclic nucleotide levels in brain slices of rat and guinea pig.
Naunyn Schmiedebergs Arch Pharmacol
302:141-151[Web of Science][Medline].
-
Sheriff S,
Chance WT,
Fischer JE,
Balasubramaniam A
(1997)
Neuropeptide Y treatment and food deprivation increase cyclic AMP response element-binding in rat hypothalamus.
Mol Pharmacol
51:597-604[Abstract/Free Full Text].
-
Stanley BG,
Magdalin W,
Seirafi A,
Thomas W,
Leibowitz SF
(1993)
The perifornical area: the major focus of (a) patchily distributed hypothalamic neuropeptide Y sensitive feeding system(s).
Brain Res
604:304-317[Web of Science][Medline].
-
Stanley BG,
Willett III VL,
Donias HW,
Dee II MG,
Duva MA
(1996)
Lateral hypothalamic NMDA receptors and glutamate as physiological mediators of eating and weight control.
Am J Physiol
270:R443-R449[Abstract/Free Full Text].
-
Williams RH,
Little SA,
Ensinck JW
(1969)
Adenyl cyclase and phosphodiesterase activities in brain areas of man, monkey, and rat.
Am J Med Sci
258:190-202[Web of Science][Medline].
-
Winn P,
Tarbuck A,
Dunnett SB
(1984)
Ibotenic acid lesions of the lateral hypothalamus: comparison with the electrolytic lesion syndrome.
Neuroscience
12:225-240[Web of Science][Medline].
-
Worley PF,
Baraban JM,
De Souza EB,
Snyder SH
(1986)
Mapping second messenger systems in the brain: differential localizations of adenylate cyclase and protein kinase C.
Proc Natl Acad Sci USA
83:4053-4057[Abstract/Free Full Text].
-
Xia Z,
Choi E-J,
Wang F,
Storm DR
(1992)
The type III calcium/calmodulin-sensitive adenylyl cyclase is not specific to olfactory sensory neurons.
Neurosci Lett
144:169-173[Web of Science][Medline].
Copyright © 1998 Society for Neuroscience 0270-6474/98/1872646-07$05.00/0
This article has been cited by other articles:
|
|
|
|
 
A. M. Khan, T. A. Ponzio, G. Sanchez-Watts, B. G. Stanley, G. I. Hatton, and A. G. Watts
Catecholaminergic Control of Mitogen-Activated Protein Kinase Signaling in Paraventricular Neuroendocrine Neurons In Vivo and In Vitro: A Proposed Role during Glycemic Challenges
J. Neurosci.,
July 4, 2007;
27(27):
7344 - 7360.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Vuilleumier and J. Driver
Modulation of visual processing by attention and emotion: windows on causal interactions between human brain regions
Phil Trans R Soc B,
May 29, 2007;
362(1481):
837 - 855.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Mogi, T. Funabashi, D. Mitsushima, H. Hagiwara, and F. Kimura
Sex Difference in the Response of Melanin-Concentrating Hormone Neurons in the Lateral Hypothalamic Area to Glucose, as Revealed by the Expression of Phosphorylated Cyclic Adenosine 3',5'-Monophosphate Response Element-Binding Protein
Endocrinology,
August 1, 2005;
146(8):
3325 - 3333.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. M. Khan, H. H. Cheung, E. R. Gillard, J. A. Palarca, D. S. Welsbie, J. W. Gurd, and B. G. Stanley
Lateral Hypothalamic Signaling Mechanisms Underlying Feeding Stimulation: Differential Contributions of Src Family Tyrosine Kinases to Feeding Triggered Either by NMDA Injection or by Food Deprivation
J. Neurosci.,
November 24, 2004;
24(47):
10603 - 10615.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Lewis, C. Li, M. H. Perrin, A. Blount, K. Kunitake, C. Donaldson, J. Vaughan, T. M. Reyes, J. Gulyas, W. Fischer, et al.
Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor
PNAS,
June 19, 2001;
98(13):
7570 - 7575.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Fulton, B. Woodside, and P. Shizgal
Modulation of Brain Reward Circuitry by Leptin
Science,
January 7, 2000;
287(5450):
125 - 128.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
A. M. Khan, M. C. Curras, J. Dao, F. A. Jamal, C. A. Turkowski, R. K. Goel, E. R. Gillard, S. D. Wolfsohn, and B. G. Stanley
Lateral hypothalamic NMDA receptor subunits NR2A and/or NR2B mediate eating: immunochemical/behavioral evidence
Am J Physiol Regulatory Integrative Comp Physiol,
March 1, 1999;
276(3):
R880 - R891.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. R. Gillard, A. M. Khan, B. Mouradi, O. Nalamwar, and B. G. Stanley
Eating induced by perifornical cAMP is behaviorally selective and involves protein kinase activity
Am J Physiol Regulatory Integrative Comp Physiol,
August 1, 1998;
275(2):
R647 - R653.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
Top
Abstract
Introduction
