Illinois Wesleyan University
Digital Commons @ IWU
Honors Projects
Biology
1990
The Effects of Intracerebral Injections of Substance
P, and a Substance P Fragment (5-11) into the
Medial Preoptic Nucleus on Lordosis Behavior in
the Female Rat
Kellie S. Jones '90
Illinois Wesleyan University
Recommended Citation
Jones '90, Kellie S., "The Effects of Intracerebral Injections of Substance P, and a Substance P Fragment (5-11) into the
Medial Preoptic Nucleus on Lordosis Behavior in the Female Rat" (1990). Honors Projects. Paper 39.
http://digitalcommons.iwu.edu/bio_honproj/39
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THE EFFECTS OF INTRACEREBRAL INJECTIONS OF SUBSTANCE P, AND
A SUBSTANCE P FRAGMENT (5-11) INTO THE MEDIAL PREOPTIC NUCLEUS ON
LORDOSIS BEHAVIOR IN THE FEMALE RAT
Kellie S. Jones
Department of Biology
ILLINOIS WESLEYAN UNIVERSITY, Bloomington, II., 61701
ll.pril 24, 1990
ACKNOWLEDGEMENTS
First and most importantly, I would like to thank Dr. Dornan
t.he chance t.o work
in his lab
were successful endeavors).
wi t.h "Thorn I
and get on his
I
would
began working with
nerves (bot.h of
also like to
in the lab
for
which
thank the
people
as colleagues who
bec."llne
true friends: Leslie Matuszewich, Melissa Peterson, Chris Ballack, and
Pet_er Malan.
The members
of my
commit.tee, Dr.
Thomas Griffiths,
Dr.
Bruce
Crilev, and Dr. John Clavedetcher, spent a great deal of personal time
reviewing and
edi t.ing
my
honors
paper.
I
t.ha.nk
them
for
their
generosity and consideration.
Finally, I wish
from t.he start.
fa~t
to thank
my fiance, Michael
hmV' important. this
work was t.o
that this project placed constraints
urged me to continue the research.
has been the most supportive and
he had
had
previously
little
Of
Monahan, who
knew
He accepted
me.
t.he
upon my time, and still
he
all the people in my life,
he
understanding of a field with
experience.
He
will
always
which
be
my
greatest strength.
This research
was supported
by
an Illinois
Wesleyan
Research
Grant awarded to Dr. Wayne Dornan and a grant awarded to Kellie
by Beta Beta Beta, the
Universit.y.
biological honor society of Illinois
Jones
Wesleyan
•
ABSTRACT
In this
study either
substance
res is tant to enzymatic degradat.ion,
bilaterally into
the
The lordosis
(LIS) were
analogue
or acidified saline was
injected
in ovariectomized
quotient (LQ)
recorded
using a
(MPN) of
nucleus
the
medial
(ovx) estrogen-primed
female
and the
steroid
increase of sexual receptivity.
SP 5-11,
an
medial preoptic
preoptic area (MPOA)
rats.
P (SP),
lordosis intensity
regime that
produced
scale
a
slow
In experiment 1, when receptivity was
low, bilateral injections of 2 different doses of SP (100 ng,1000
cannula), had no effect
injected with
on lordosis behavior
acidified
saline.
receptivity was maximal, SP 5-11
decrease in
lordosis
effects
bilateral
of
responding.
injections
In
when compared to
experiment
')
t... ,
of
results
SP
on
indicate
lordosis
when
marked
t.hat
behavior
dependent on the level of sexual receptivity, and therefore,
levels.
those
however,
(1000 ng!cannulal produced a
The
ng!
the
is
estrogen
•
INTRODUCTION
It has been
the female rat
egt~rogen
demonstrated that sexual
i;:;;
dependent on circulating levels of ovarian
and progesterone (28).
lordosis behavior in
in-tegrity of
specif ic neural
been
rat is dependent
pathways between
nucleus of the
repeatedly found
to
on the
areas of
central gray (MeG), also facilitatory,
of
functional
the
brain,
hypothalamus (VMH), an
be facilitatory,
in
hormone~:;,
Moreover, the hormonal induction
the female
notably the ventromedial
which has
receptivity (lordosis)
the
area
midbrain
and the medial preoptic
area,
an area which is involved in the inhibition of lordosis behavior
(see
28 for revie r..,).
also
been shn"m
Implantation
to
have
a
of estrogen into
dramatic
effect
on
these areas has
:;:"exual
receptivity
in
ovariectomized (ovx) rats (5,18,31,35).
Accumulat.ing evidence has
neuropeptides
present
in
shown that the
the
central
concentration of
nervous
system
dependent on the presence of estrogen (13.17,23,24).
t-hen, there
have been
behavior in
the
female
bilateral injections
Caldwell found
a
a
female rats treated
number of
rat
of the
~Tith
days (7). Dornan at al.
0.5
For
of
following
into the
for three
injections of
MPOA,
in
lordosis
cholecystokinin (CeK)
the media.l preoptic nucleus (MPN) of the MPOA.
ovx
consecutive
(10) also reported a facilitation of
behavior following bilateral
sexual
too
lordosis behavior
ug of estradiol
are
Not surprisingly
example,
neuropeptide, oxytocin,
marked facilitation
(CNS)
neuropeptides linked
(4:,7,11,33).
:c;ome
ln
•
Recently, based
presence of estrogen
studie~')
using
on
the
receptors in
t.he technique
CNS
which
t~he
VMH
one popular
expression of
certain
hypothesis
are released
to
modulate
neurons controlling the
(2) and
which
the
report
genes
concerning
the
the female rat is that
genes, estrogen
precursor proteins for
These peptides are t.hen
(6).
the
expression of certain neuropeptide
(30,32,34),
levels for specific
demonstrated
hypot.halamic neurons
neural regulation of sexual receptivity in
aLter ing
has
of i1l.-..-situ hybridizat.ion
that estrogen regulates the
"Ti thin the
evidence
regulates
by
mRNA
neuropeptides within
t.he
transported to the dMCG where
descending circuits
display of
that
they
regulate
lordosis behavior
motor
in the
female
rat..
One of these peptides is
~ell
bodies and
~;ubstance
fibers have been
found in areas
recef>ti vi ty in t.he female rat (e, 19 ).
concent.rat:e estrogen
fc,und alse' t.o make SP
wi thin it.s
gene
for
relevant to
cells; these
that neurons
preprotachykinins
( 29) ,
SP
sexual
The VMH has been documented
same cells
have
Although in situ hybridizat..:.ion
(2.3) .
have subsequently reported
Indeed, numerous
P (Sp).
within the
information
tel
been
st-.udies
VMH express
';-Thet.her
concerning
estrogen treat.ment. alters preprot.achykinin gene expression within
hypot.halamus has been conflicting (6,29).
has SP
receptors
changes in the
as well
nUlnber of
as
the
t.he
Nonetheless, the MCG, which
cell bodies
18,19,21).
SP immunoreactive fibers
shows
over the
marked
estrous
cycle (13).
Taken t.ogether,
these
results
suggest
important. role in t.he
neural regulation of
female rat:.
Dornan et
Indeed,
al.
that
SP
may
play
lordosis behavior in
(11), reported
that
an
t.he
bilateral
•
injections of three different doses of SP int.o t.he dMCG produced rapid
and
(3
sLlstained
h)
facilitation
B~sed
est.rogen-primed female rats.
of
lordosis
behavior
on these results, they
in
ovx
postulated
that SP prcduced within t.he cell bc/dies of the VMH was transported
t.he MCG
where
it
carried
est:::-ogen suggesting
behaviorally-relevant
the importance
of a
regulation of sexual receptivity in
~:p
immun(=1)~'eactivity
exist.ed
projected to
the dMCG,
l..1beling"
use·)
Wi;.1S
a
inject.ing
retrograde
tracer,
Cells that
Hi thin
double labeling
same cells
at ,::1.1.
Briefly,
into
there£,)re
projected from to the dMCG.
anti~;erum
the female rat.
t,he
Fluorogold
and
~Jathway
SP VMH-MCG
the
follows
indicat.e a
study (9),
in
of
t,he
VMH
termed
the
jf
which
"double
this technique
dMCG.
the
first
Fluorogold
patri
which
a
f ir,ers
Brain sections are then incubated with
are doubly labeled
the VMli
about
To determine
neuroanatomical technique
by Dornan
c.?.lL" fen-
SP antisenlffi.
in
information
to
with Fluorogold ..:.md
VMH-dMCG SP
however,
pathway.
although doubly
In
labeled
exist. throughout the VMH, the dense populat.ion that would be
a
SP
this
cells
expected
if a major SP pat.hway existed from the VJII'J-I to t.hs MCG, was absent..
It
has
been
documented
inmunoreactive fibers
aV'idence from
that.
and receptors
AJ.;:esson et
al.
s~me
which
recently reported
111
to the MPN,
produce SP is as
that
the
lesic)ns of
important role in
does
contain
rat (19,21).
t.he
VMH produced
SP
There
but whether these
of yet, unknown.
reduction of SP immunc·reactivi t.y 1n the MPOA.
is not inconceivable that the SP
MPOA
that estrogen-concentrating
(3)
1;vithin the hypot,halamlJs project
are the
the
cells
cells
Malsbury
a
1S;
(20)
significant
Based on the above,
it
innervation of the MPOA may play
an
the neuroregulation
of sexual
receptivity in
the
female rat.
Presently, nothing is
SP innervation of
behavior in
the
determine if SP
the MPN
female
known about the importance of
in regard
rat.
to the
The purpose
injected directly
regulation of
of
into the MPN
this
study
the
lordosis
was
would affect
to
sexual
receptivity in estrogen-primed rats.
GENERAL METHODS
Animals and Surgery
Adult Long-Evans female rats weighing between 210g and 250g
housed. in groups in
21C,
~Tith
steel mesh cages in
a controlled environment
an extended light cycle (on at 0700, off at 2100).
ovariectomy, each animal
sodium pentobarbital at a
at
Food and
Animals were ovariectomized under ~ther.
water were available ad lib.
One week after
were
dosage of 50 mg/kg
was anaesthetized
with
and recAived a pair
of
stereotaxically implant.ed 22-gauge stainless steel guide cannulae wit],
inner
~3tylets
(28 -gauge) aimt:::-d 1rnm above the MPN using the coordinat.es
that were empirically derived from the Paxinos and Watson atlas
AP +2.2, ML
-0.5, DV -5.3
from bregma.
into place with cranioplastic.
The
cannulae were
(25);
cemented
Intracerebral Injections
Each 28-gauge
syringe by
a 12"
lnner cannula
plastic tube.
injector was
connected to
Distilled water
a 2
was drawn
up
tubing until 1 ul of water could be drawn and expelled reliably.
ul
the
Air
was t.hen dra"lD int.o t.he inner cannula at a vol urne of 0.5 ul, "Thereupon
the inner cannula
peptide was
was placed in
drawn.
Movement of
the peptide solution
the
air bubble
ensured actuEtl peptide inj ection into t.he brain.
were 1
mm longer
amount. of
received
than the
lesioning "Then
no
ird eci:ion.
guide cannula
the guide
anaesthesia,
but
during
injections
to decrease
restrained
the
original
placed.
Animals
manually
during
Aliquots of all peptide solutions were prepared on the day
of behavioral testing and stored in small plastic centrifuge tubes
a freezer until
used.
Substance
acid, 0.9% saline (pH 4.7).
glass, and may make
P was
the peptide more stable.
receiving acidified SP as
saline media (15).
(each
dissolved in
were stronger in those
were injected manually
period of 60
place 30-60 s before withdrawal.
SP in a
to
and
animals
normal
at a volume
s. The cannula
in
acetic
In addition, Hall
opposed to those receiving
Solutions
side) over a
0.01 N
Acidified saline prevents adsorption
Stewart found that behavioral effects
0.5 ul
of
The injector canrrnla
cannulae were
were
and 1.5 ul
was left
of
in
•
Behavioral Testing
Mating tests were conducted
in a dimly
lit room in
rectangular
test boxes containing wood chip bedding after animals had been allowed
to recover
one
week
const.ructed with
mea3~re
sexual
from
t.hree
stereotaxic
wooden
receptivity,
5
ides and
the
intensity of the
These responses
vigorous male
rated
on
lordosis; 1 js a mild response
used.
Test
quotient
scale
were
front..
To
and
the
(LO)
The LIS was determined
and recording
following ten mounts
d
boxes
Plexiglass
with the female
lordosis reflex
were
a
lordo.3is
lordosis intensity scale (LIS) were
pairing a sexually
surgery.
from
by the
0-3: where
0
the
male.
is
consisting of very little arch of
back; 2 equals the normal lordosis response--back arching and
evaluat.ic>ll of
t.he
lordosis
lordos L:; rf,sponses
absence of
of the nose
to t.hs
re:.o;ponse
nunlber of
proceptive behaviors
SP
injectio~s
assessed on a
following
paradigm which
repeat.ed
estrogen
behavioral paradigm
( Yo •
10 ) .
content.
Lordosis
produces a
was scored
ratios
the
male mounts.
of
The presence
or
and
no a priori indication
and
slow increase
without
of SP
mating
knowledge of
the
of
was
injections
tests.
in sexual
ear
v.rhet.her
facilitate lordosis, the behavior
treatment.
of
number
hopping, darting,
recorded the effects
the
none
t.he test. sessio:)n as indication
Since there was
would inhibit or
The LQ
which
such as
wiggling were also recorded during
overall receptivity.
( 16 ) .
no
tipping
of head back; 3 being an exaggerated response with extreme flexion
the back and elevation
by
This
receptivity
injection's
Histological Analysis
After all data had been collected, all animals were anaesthetized
with
an
oVE."rdose
of
sodium
pentobarbital.
Brains
;3ubsequently and 46 um sections were taken (see fig 1).
were stained with cresyl
violet and the
were
The
removed
sections
cannulae placements in
each
brain were located using microscopy.
Statistical Analysis
A one way analysis of variance with repeated measures was applied
to the data.
In experiment
2, a two-sample
independent t-·test
was
used to determine the effect of the SP analogue on lordosis responses.
EXPERIMENT 1
Each female
received a
estradiol benzoate (EBl in
were
divided
randomly
subcutaneous (s.c.)
sesame oil 53
into
3
groups
injection of
h before testing.
which
received
5
Females
bilateral
injections into the MPN of 100 ng SP, 1000 ng gp, or acidified
in a
latinized, repeated
injections on sexual
measures
design.
behavior were assessed
The
effects of
saline
the
by placing females
sexually vigorous males 30 minutes following injection.
ug
SP
with
•
FIGURE 1
Illust.ration of bilat.eral cannula placement.s in nine females
which received injections of SP
experiments 1 and 2.
Klippel (196:-»
let~ters
the
Numbers
corpus callosum;
In
The numbers identify the figure in the Konig and
MPOA
are
approximations
following the
animals with this placement.
CC,
or near t.he MPN
atlas from Ylhich t.he figures v.Tere redrawn.
wi-thin
pI acements .
and SP 5-11 into
lett.ers
of
actual
indicate the
Abbreviations: ac, anterior
CL, claustrum; CP,
Small typed
cannulae
number
of
commissure;
caudate putamen; F,
fornix;
LV, lateral ventricle; MAH, medial anterior hypothalamus; MPOA, medial
preopt-i(~
area;
oc, optic
chiasm; pvm,
periventricular nucleus;
stria medullaris thalamus; st, st.ria terminalis.
SM,
!
•
/
CD
3
'8
20
22
F
24
MAH
21
?,
.
RESULTS
As can
be seen
in figures
revealed that at low levels of
2 and
3, the
analysis of
variance
receptivity both doses of SP (100
ng,
1000 ngl injected into the MPN had no statistically significant effect
on the
lordosis
behavior
F(2,16l=1.89 P>O.051.
response (LQ,
F(2,16)=2.68
Interestingly, there
was
P)0.05;
a trend
LIS,
towards
a
facilitation of sexual receptivity with the largest dosage of SP, 1000
ng (see fig. 2 dnd fig. 3, test 11.
EXPERIMENT 2
Because
animals
which
received
bilateral
injections
of
the
largest dose of SP 11000 ng) exhibited a trend toward an inhibition in
test 2, in
experiment 2
random groups and placed
s.c. tor
j
all remaining
animals were
on a high-dose estrogen
consecutive days
to ensure maximum
divided into
regime of 10 ug
receptivity.
On
2
EB
the
fourth day all animals received
bilat.eral injections into the MPN
of
1000 ng SP 5-11 (more resistant.
to enzymatic degradation than SP)
or
saline and were paired with sexually vigorous males.
•
FIGURE 2.
Effects of SP (lOOng,
1000 ng) injections into the MPN
lordosis behavior during 3 mating tests (means ±S.E.M.).
on
-
3
saline
c::=J 100 ng
~ 1000 ng
w
-.J
«
0
(f)
~
2
(f)
z
w
~
Z
(f)
-
(f)
0
1
0
0:::
0
-.J
a
1
2
TEST
3
FIGURE 3.
Effect"s of SP (100 ng,
1000 ng) injections into the MPN
lordosis intensity during 3 mating tests (means ±S.E.M.).
on
/
-
100
I­
z
saline
CJ 100 ng
~ 1000 ng
75
w
I­
0
:J
a
(f)
50
(f)
0
0
0:::
0
--.J
25
O..L.---­
1
2
TEST
3
~
~ ~~~---------~
-------------a
RESULTS
As illustrated in fig.
into the
MPN
compared to
produced
saline
4, bilateral injections of 1000 ng SP 5-11
a significant
controls
inhibition
reflected in
both
of
the
lordosis
LQ
\vhen
(t(8)~3.26
P<O.Ol) and the LIS (t(8)=4.56 P<O.Ol).
DISCUSSION
In experiment. 1, bilateral inj ect.ions
of two different doses
of
SP into the MPN produced no appreciable effect on lordosis behavior in
ovx
estrogen-primed
acidified saline.
female
rats
In experiment
1000 ng of the SP fragment,
when
compared
to
injections
of
2, however, bilateral injections
of
SP 5-11 produced a significant
reduction
in bot.h the LO' sand t.he LIS's when compared to animals which received
saline injections.
Using a
resul ts have
number of
st.rongly
different kinds
implicated
the
of experimental
MPOA (in
which
proceduref:,
t.he
MPN
is
cent.rally locat.ed) and the VMH as important areas in the regulation of
lordosis behavior.
It has been
the VMH stimulates
lordosis, while stimulation
lordosis
expression
(27).
found that electrical stimulation
One
explanation
in the MPOA
for
the
of
inhibits
results
of
experiment 2 then is that the inhibition of lordosis behavior produced
by an excitatory neuropeptide such
,jf neun:>n5 intrinsic t,) the MPN.
as SP resulted from an
activation
•
FIGURE
Effects of
~.
behavior (A)
and on
(means ±S.E.M.).
~ontrols
(p<O.Ol).
SP 5-11
(1000 ng)
lordosis intensity
Asterisk
indicates
into the
(B) during
significantly
MPN on
lordosis
one mating
test
different
froln
100
-
SALINE
~ Substance P (5-11)
1000 ng
I­
z
75
w
I-
a
::::J
a
(f)
50
(f)
Q
0
0:::
a-l
25
0.1....--------­
**
This explanation,
however,
does
lordosis scores seen in experiment 1.
not explain
the
increase
Alternatively, since rats
that
received only 5 ug EB did not respond to the doses of SP and only
highest dose
ng)
(1000
in
inhibition, something very
test two
produced
similar to
1.S
yet
unknown, it
trend
the action
into the MPN may be occurring in the SP circuit.
mechanism
a
seems that an
specific
sensiti vi ty
occurs in the CCK circuit, producing effects parallel to the
in LQ with repeated estrogen doses (10).
ylay as
CCK, only
during high
concent-rat.ions are
at. their
become sensitized so that
increase
If SP acts much in the
levels of
recept.ivity, when
highest, does
an effect.
same
estrogen
t.he MPOA-VMH-MCG
the SP can produce
circuit
This
could
have very profound effects on the lordosis regulatory circuit.
It has
been thus far a plausible hypothesis that neuropeptide input from
VMH to
t.he dMCG
ul timatel y regulates
related to the expression
demonst~'ated
that
the descending
of sexual receptivity.
the estrogen-concentrat.ing
inherent SP immunoreactivity do not by
motor
the
neurons
Since it has
cells of
an
injections
Although a
increase in
the
toward
of CCK
in
been
t.he VMH
and large ext.end t.o the
with
<..'!MCG,
it can be speculated that regulation produced by SP may originate from
the MPOA and
extend to the
MeG.
estrogen sensitizes the circuit
reach a certain
level, SP is
inhibi tory MPN.
The
lordosis circuit
focus on
to SP.
affects the
and inhibiting
by
the
using a
line of
When estrogen
released from the
MPN t.hen
this hypothesis
Following this
concentrations
\nMH and excites
dMCG by
behavior.
reasoning,
turn ing off
Future
high-dose estrogen
studies
regime
the
the
may
on
female rats, injecting SP into the MPOA to determine if it will have a
similar effect a3 did the SP analogue, SP 5-11.
------_._~~.~---------_._-------
Although the MPOA has been shown to have a net inhibitory
('11
lordo;3is express ion,
it must
be noted that
uniform cell type throughout.
The
with different
of
distributions
this area
MPN itself has three
action
in not
of
subdivisions
steroid-concentrating
cells
( 22 ) .
Integration of these distinct cellular areas requires that signals
of
The
;JP
0ne type be augmented over
others during certain situations.
signal may, therefore, be amplified when estrogen concentratiolls reach
high levels.
It can be also
signal for
the
speculated that SP 5-11
release
of a
classic
1S
acting as a
neurotransmi t.ter
chemical
which
directly produces the inhibitory response in the female rat.s.
et a1.
(10) hypotrlesized that CC'K injected into the nucleus
t.hen
Dornan
accumbens
dcts t.o facilitate lordosis by decreasing the inhibit.cry act.ion of the
dopamine
on
lordosis
behavior
In
(1,12) .
fact,
it
has
demonstrated in other areas such as the substantia nigra and
~hat
SP
induces
Therefore, it is
a
release
newly
of
not inconceivable
MPN-injected SP may
be produced
synthesized
that the
striatum
dopamine
inhibi~ion
by an increase
been
(26).
produced
in dopamine
by
release
within the MPOA.
It is interesting to note, although rejection behaviors were
not
recorded, it seems that the SP analogue-injected females were far more
aggressive towards the males
The test animals
than were the saline-injected
often times turned
and reared at
controls.
the males.
When
females ran from the males, they would often kick t.he male in the face
with their hind legs.
times per mating
These kicks were reported to be as often as
session.
These
females fought
viciously with
pursuing males; males usually being forced onto their backs as a
30
the
sign
of 3ubmission.
In fact, some testing sessions were terminated
before
the prerequisite ten mounts due to the aggressiveness of the female in
preventing male mounts.
In fact, a similar finding has been
by Hall and Stewart (14).
7-2.1,
injected
reported
They found t.hat a SP terminal analogue,
intraperitoneally
(i.p.)
in
rats
significantly
increased the duration of isolation-induced aggression and the
of these
aggressive
attacks.
This
finding
of SP
7-11
number
increasing
aggression correlates with the effect produced by the SP 5-11 used
experiment 1.
A
terminated before
larger percentage
the ten
mounts
of
these mating
t.han occurred
SP
sessions
with the
SP.
in
were
The
inhibition produced by SP 5-11 may, therefore, be more of motivational
then mot.or.
In summary, the res111 ts of experiment 2 support t.he idea that the
SP
innervation
of
the
MPOA
plays
neuroregulation of sexual receptivity
however, furt.her studies ·::tre necessary
within the
understand.
MPOA on
sexual receptivity
an
important
role
in the female rat.
in
Obviously,
before the role of SP
in the
female rat
the
neurons
is
fully
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