Department of Neuroscience,
University of Florida, Gainesville,
U.S.A.
Summary
ACTH 1-24, ACTH 4-10, ACTH 4-10(D-phe),
lysine vasopressin (LVP) or an amino acid
mixture were administered to mice using
bilateral intraventricular injections (5x1O9
moles per mouse). Behavioral observations were
made for the subsequent 85 minutes, and the
incorporation of subcutaneously injected
[3H]lysine into brain proteins assayed
for the last 10 minutes of this period. Mice
injected with ACTH1-24, showed the previously
reported stretching and yawning syndrome, an
effect also observed with ACTH 1-24(D-phe) but
less often. These same peptides also induced a
pronounced increase in the proportion of time
mice spent grooming. LVP caused a dramatic
hyperactivity; mice so injected moved
continuously about the cage occasionally eating
or grooming, but were never still. Injection
with ACT H 1-24, or ACTR 4-10(D-phe), but not
ACTB 4-10 or LVP, caused significant increases
in the incorporation of [3H]lysine into
brain protein.
The elegant work of De Wied and his
coworkers has established that administration of
ACTH, vasopressin or their analogs can influence
the learning and retention of behavioral tasks
in rata (1). Current indications are that these
effects reflect normal physiological processes
and that the peptides act directly on the brain
(2).
We previously observed that avoidance
training and related stimulation increased the
uptake and incorporation of (HJ lysine into
proteins of mouse brain and liver (3). The
effects seemed to be stress-related, rather than
specifically training-related, and occurred in
response to footshocks, loud buzzer
presentations and handling, as well as to
training. These effects diminished or
disappeared with repeated exposure to the
stimuli over a few days (3). Mediation by
stress-related hormones was suspected, but
adrenalectomized mice showed similar biochemical
responses (4). This suggested that perhaps ACTH
itself might be involved, especially since ACTH
had previously been shown to stimulate the
incorporation of amino acids into brain proteins
of mice (5, 6). We also found that ACTH
administration could mimic the observed E
3H] lyaine incorporation changes (7).
Corticosterone administration to
adrsnalectomired mice was not effective (Bees
and Dunn, submitted for publication). Further
evidence that the response to stimulation is
caused by ACTH is that the effect of footshock
on brain [3H]lysine incorporation was
suppressed by doses of dexemethaaone that
inhibited the secretion of corticoaterone and
presumably therefore of ACTH (Bees and Dunn,
submitted for publication). We have further
observed that whereas ACTH1_21f increased
[3E] lysine incorporation in both brain
and liver, ACTH 4-10 was effective only in the
brain (Dunn, Rees, and luvone, submitted for
publication). However, the response to
subcutaneously injected ACTH 1-24 or ACTH 4-10
was smaller and less consistent than that to
footshock. Allen et al. (8) have provided
evidence that ACTH might he secreted directly
into the cerebral ventricles. Wetherefore
studied the effects of intraventricular
administration of the peptides on
[3H]lysine incorporation. Also, since
ACTH and its analogs have been reported to
produce a behavioral response known as the
stretching and yawning syndrome (9,10), and an
enhancement of grooming behavior in rats (11),
we observed the behavior of the mice for 85
minutas prior to sacrifice.
Discussion
The observation of synchronized stretching
and yawning behavior following intraventricular
injection of ACTH replicates the observations of
Ferrari et al. (9). We observed such effects
most intensely with ACTH 1-24confirming earlier
observations(9,l0,ll). Unlike Ferrari et al.
(9), but in agreement with Baldwin et al. (10)
and Cispen et al. (11) we observed very little
effect with ACTH 4-10. However, ACTH 4-10(D-phe)
was much more potent than ACTH 4-10, although
less potent than ACTH 1-24, as also found by
Cispen et al. (11). This sane order of potencies
of the peptides was also observed with grooming
behavior; ACTH 1-24 being most potent and ACTH
4-10(D-phe) less so. These results in mice are
thus in excellent agreement with those of Cispen
et al. (11) in rats. ACTH 4-10 may not be
without effect since stretching behavior was
often enhanced by this peptide. These behavioral
effects of the peptides are not exclusive to
C57B1/6J mice since we have observed very
similar stretching and yawning, and grooming
responses in CD-1 male mice.
These results differ from the effects of
ACTH peptides on avoidance behavior, in which
ACTH 4-10 and ACTH 1-24 were equipotent in
delaying extinction, whereas ACTH14...10(D-phe)
accelerated extinction (1). Apparently this
effect of ACTH-10 (D-phe) was not caused simply
by competition with endogenous ACTH since ACTH
4-10(Dphe) was effective in hypophysectomized
rats. However, ACTH 4-10(Dphe) was as potent as
ACTR 4-10 in facilitating passive avoidance
behavior (15). These data suggest then that
there may be separate receptors for ACTH 1-24
and ACTH 4-10(D-phe).
That ACTH peptides should affect the
incorporation of amino acids irto protein is
consistent with earlier data. Semiginovsky and
Jakoubek (5) showed that ACTH increased the
incorporation of [U-14C]Ieucine into
mouse brain protein. kudman et al. (6) also
showed that ACTH or B-MSH increased the
incorporation of [U-14C]valine,
[UC]tyrosine, [U-14C]Ieucine and
[U-1C]lysine into mouse brain protein.
Using ACTH 4-10, Reading (16) observed an
increased incorporation of [11C]leucine
into rat brain protein and we have observed a
similar effect with [311]lysine in mice
(Dunn, Rees and luvone, submitted for
publication). In hypophysectomized rats there is
a decreased rate of cerebral protein synthesis
indicated by a diminished aggregation of
ribosomes and polyribosomes (17) and a decreased
in vitro protein synthesis (18). In these
animals ACTH stimulated the incorporation of
[3H]leucine into protein (19).
Furthermore, ACTH 11D in vitro stimulated
[3H]leucine incorporation into brain
stem slices from hypophysectomized rats
(20,21).
The effects of ACTH 4-10 D (D-phe) obtained
here are inconsistent with the observations of
Schotman et al. (19) who found that this peptide
decreased the [3H]leucine incorporation
of hypophysectomized rats and with those of
Reith et al. (21) who found no effect on the
incorporation by brain stem slices from
hypophysectonized rats.
The dramatic behavioral effects of
intraventricular LVP have not been previously
reported and are worthy of further study. It is
interesting to speculate to what extent they
might be related to the effects of adrenergic
compounds (22). The lack of effect of LVP on
[3R]lysine incorporation into brain
protein is also consistent with the lack of
effect of subcutaneously injected LVP (Dunn,
Rees and luvone, submitted for
publication).
The results of the present study suggest
that the effect of ACTH on brain protein
synthesis might be mediated centrally as also
indicated by the results of Reith et al. (20,21)
mentioned above. The observations further
indicate that the intraventricular route of
administration is effective. This is consistent
with the behavioral data of de Wiedts group (see
for example Ref. 23) and may also explain why in
our studies the effects of stimulation (electric
footshock) are larger end more consistent then
those of peripheral administration of ACTH
(Dunn, Race and luvone, submitted for
publication). That ACTH is secreted into the
ventricles is indicated by its presence in human
CSF and the lack of correlation between plasma
and CSF concentrations of peripherally
administered ACTH (8).
The dosage of ACTH 1-24 used in these
studies (about 15 pg or 1500 nU per mouse) is
above the physiological rangs, but is similar to
the dosages used by several other investigators
(9,10,11). Increased grooming behavior has been
reported following intraventricular injection of
as little as 0.01 pg of ACTH 1-24 in rats
(11).
Our results indicate a correlation between
the activity of ACTH peptides in eliciting
grooming, stretching and yawning behavior, and
their effects on [3H]lysine
incorporation. The correlation between grooming
behavior and [3H]lysine incorporation
into cerebrum protein was significant
(coefficient of linear regression r-0.38, df-22,
p<O.O5) when the results of all treatments
were combined. Whether or not these behavioral
activities bear any relation to the effects on
leaning remains to be seen. However, the
observations are consistent with a central
action of the peptides and a possible role in
leaning mediated by changes in cerebral protein
synthesis.
