F30 Grant Outline

Specific Aim 1: To test the role of renin-angiotensin system in supporting the chronic activation of vasopressin
neurons in the SON or neurons that project to the SON of bile duct ligated rats.
Hypothesis: Activation of the renin-angiotensin system mediates increased vasopressin release and fluid
retention associated with BDL.
These experiments will involve chronic inhibition of angiotensin converting enzyme !"#$ and central !T%& to
determine the role of these systems in the activation of the SON' fluid retention' and vasopressin release in ()*
rats. &ats and mice will be tested in metabolism cages to measure water inta+e and urine output. &adio
telemetry will be used to monitor blood pressure and heart rate in rats.
1) EFFECTS OF CHRONIC ACE INHIBITION ON FLUID RETENTION AND CHRONIC CNS ACTIVATION IN
BDL RATS.
a) House in metabolism cages for measuring daily food and water intake, and urine output.
b) Measure BP and HR.
c) 4 Treatment Groups:
i$ Sham ligation and tap water
ii$ BDL and tap water
iii$ Sham ligation and enalapril !"# mg$L),
iv$ BDL and enalapril.
d) Tract Tracing it! "os and "osB#$"osB %mmuno!istoc!emica& staining in B'( rats.
i$ One set of rats from each treatment group will receive injection unilaterally in the SON with ,-- nl
of "T.-( /eneral 0ethods 1$.
ii$ Following the injection, the rats will receive either bile duct ligation or sham ligation surgery
(General Methods 2).
iii$ Rats from each group will be perfused at 2, 4, and 6 weeks after surgery.
%$ Ta+e truncal blood measurements pre-perfusion for plasma osmolarity' protein and hct.
iv$ Tract Tracing %mmuno!istoc!emistr)
%$ %os and %osB$∆%osB pero&idase detection General Methods 9).
,$ %orebrain sections also will be processed for 'asopressin immunofluorescence.
2$ Brainstem sections for DBH immunofluorescence General Methods 9).
3$ (he number of c)%os positi'e cells will be determined in the following regions* +(S, ,-, ./L,
0/L, -B+, par'ocellular and magnocellular -/+, S1+, 2n-1, S%1 and 1/L( General
Method !).
4$ Samples will be analy3ed to determine the number of %os and %osB$∆%osB positi'e cells in the
+(S, ./L, 2n-1, 1/L( and S%1 that are retrogradely labeled from the S1+.
5$ ,n additional set of forebrain sections will be processed for %os, (0-/1, and (0-/4.
e) %mmuno!istoc!emistr)*S+, and P-, .non/Tract Tracing Rats).
i$ 67os(87os( peroxidase$
%$ Test the effects of !"#i on SON.
ii$ T&9:3 "y,$
iii$ :asopressin "y2$
f) P&asma Measurements and RT/P0R .non/Tract tracing Rats).
i$ measure plasma 'asopressin, and -0, 5eneral 2ethods 11).
ii$ Section brains for laser capture microscopy /eneral 0ethods %2$.
iii$ "ollect and process samples of SON' 9:N' S7O' O:*T and 0n9O
%$ SON and 9:N ;estern and &T-9"& for< General Methods ").
a$ !T%&
b$ T&9:3
,$ *amina terminalis will be analyzed for 6s in !T%&.
g) E##e$ts o# $hron%$ ACE %nh%&%t%on on the a$t%'%t( o# 'aso)ress%n ne*rons %n '%tro +%th )at$h,
$la-) re$ord%n.s.
i$ 4 groups of rats* Sham ligation and tap water, BDL and tap water, sham ligation and enalapril, and
BDL and enalapril.
ii$ 6sing in vitro patch clamp electrophysiological recording from S1+ neurons we will test changes in
(0-/4 and ,ng77 channel properties resulting from BDL in the presence and absence of
peripheral ,ng77.
,$ 1""10TS +" 01,TRA( AT1R B(+02A'1 %, B(' RATS. .%S+(AT1 P1R%PH1RA( 1""10TS)
a) This experiment will use chronic icv infusions of the !T% receptor antagonist losartan to test the role of these
receptors in chronic changes in urine output and vasopressin release associated with ()*. This experiment will
evaluate the role of !T%&s in changes in T&9:3' and n,+S e3pression that occur in the "NS of ()* rats.
b) 4 Treatment Groups: .Genera& Met!ods 4)
%$ sham ligation and icv vehicle sterile physiological saline$'
,$ sham ligation and icv losartan , ug8ml in sterile saline$'
2$ ()* and icv vehicle'
3$ ()* and icv losartan'
4$ sham ligation and sc vehicle'
5$ sham ligation and sc losartan'
=$ ()* and sc vehicle'
1$ ()* and sc lorsartan.
c) Tract Tracing it! "os and "osB#$"osB %mmuno!istoc!emica& staining in B'( rats.
i$ One set of rats from treatment groups 2>5 will receive injection unilaterally in the SON with ,-- nl
of "T.-( /eneral 0ethods 1$.
ii$ Following the injection, the rats will receive either bile duct ligation or sham ligation surgery
according to their treatment groups (General Methods 2).
iii$ Rats from each group will be perfused at 2, 4, and 6 weeks after surgery.
%$ Ta+e truncal blood measurements pre-perfusion for plasma osmolarity' protein and hct.
iv$ Tract Tracing %mmuno!istoc!emistr)
%$ %os and %osB$∆%osB pero&idase detection General Methods 9).
,$ %orebrain sections also will be processed for 'asopressin immunofluorescence.
2$ Brainstem sections for DBH immunofluorescence General Methods 9).
3$ (he number of c)%os positi'e cells will be determined in the following regions* +(S, ,-, ./L,
0/L, -B+, par'ocellular and magnocellular -/+, S1+, 2n-1, S%1 and 1/L( General
Method !).
4$ Samples will be analy3ed to determine the number of %os and %osB$∆%osB positi'e cells in the
+(S, ./L, 2n-1, 1/L( and S%1 that are retrogradely labeled from the S1+.
5$ ,n additional set of forebrain sections will be processed for %os, (0-/1, and (0-/4.
d) Si3 rats from eac! group i&& be instrumented it! radio te&emetr) transmitters !en t!e)
recei5ed B'( or s!am &igation surger).
i$ &ats with &adio telemetry Transmitters will be decapitated 5 wee+s after ()* surgery and their
tissues harvested for ;estern blot and &T-9"& analysis.
%$ Trun+ blood will be collected into a heparized centrifuge tube for measuring plasma vasopressin'
?*-5' and 9&! Genera& Met!ods 11$.
,$ The SON and 9:N samples will be processed for ;estern blot and &T-9"& analysis for !T%&s'
and T&9:36 Genera& Met!ods 7$.
2$ Samples from the lamina terminalis will be analyzed for changes in !T%&.
ii$ The remaining rats from each groups will be anesthetized %-- mg8+g inactin ip$ and perfused for
immunohistochemistry.
%$ SON and 9:N will be processed for 67os( peroxidase$' T&9:3 "y,$' and vasopressin "y2$
staining.
,$ SON and 9:N will be processed for 67os( peroxidase$ and vasopressin "y2$.
e) 1ffects of c!ronic AT1R b&oc8ers on t!e acti5it) of 5asopressin neurons in 5itro it! !o&e ce&&
patc!/c&amp recordings.
i$ 4 groups of rats* Sham ligation and tap water, BDL and tap water, sham ligation and enalapril, and
BDL and enalapril.
ii$ 6sing in vitro patch clamp electrophysiological recording from S1+ neurons, we will test changes in
(0-/4 and ,ng77 channel properties resulting from BDL in the presence and absence of central
,ng77.
Specifc Aim 2: Test the hypothesis that increased
expression of ΔFosB in the SON produces cellular
adaptations that contribute to increased excitability in the
SON
Hypothesis: Increased expression of 6osB mediates cellular adaptations that support inappropriate vasopressin release
associated with hepatic cirrhosis.
!n adeno-associated viral vector that expresses /79 and a dominant negative construct for 67os( !!:-6@un provided
by the laboratory of )r. #ric Nestler from AT-Southwest 0edical "enter$ will be used to test the role of this transcription
factor in activity dependent changes in gene and protein expression in the SON that are related to water retention and
vasopressin release. These experiments will test the hypothesis that increased 67os( participates in the enhanced
excitability of the vasopressin system associated with ()*.
19P1R%M1,T :A: AA-/M1'%AT1' %,H%B%T%+, +" $"+SB A,' "(;%' R1T1,T%+, A,' -AS+PR1SS%,
R1(1AS1 ';R%,G B'( 0%RRH+S%S.
%. Stereotactically inject in the right and left SON with either !!:-/79 3-- nl$ or !!:-6@un 3-- nlB see Genera&
Met!ods 4$.
,. After one ee8 of reco5er) t!e rats i&& be operated on to perform B'( or s!am &igation .Genera&
Met!ods :).
a. !t this time the rats will also be instrumented with radio telemetry transmitters for continuous recording
of blood pressure and heart rate Genera& Met!ods <$.
2. Si3 Treatment Groups:
%$ sham ligation and !!:-/79'
,$ ()* and !!:-/79'
2$ sham ligation and !!:-6@un'
3$ ()* and !!:-6@un'
4$ un-injected sham ligation'
5$ un-injected ()*.
3. %mmuno!istoc!emistr) at 4 and = ee8s after t!e second surger).
a. Separate sets of sections from each brain will be processed for 67os( immunohistochemistry Genera&
Met!ods >$.
b. Sections that contain the SON and 9:N will also be processed for vasopressin and T&9:3
immunofluorescence.
c. The number of 7os( positive cells that are also positive for vasopressin' T&9:3' will be recorded and
statistical comparisons will be made among the different groups using one-way !NO:!. Genera&
Met!ods 1?$.
d. The sections will also be analyzed for the presence of the /79 reporter in the SON neurons.
19P1R%M1,T :B: AA- M1'%AT1' %,H%B%T%+, +" $"+SB A,' 0HA,G1S %, TRP-46 ,,+S 19PR1SS%+,
%, TH1 S+,.
1. Stereotactic and treatment groups same as #xperiment 2!.
:. @estern B&ot and RT/P0R: :6 46 and = ee8s post surger).
a. 9unches containing the SON will be used for ;estern blot analysis to measure 7os(' T&9:3' and n,+S
following sucrose gradient fractionation as previously described Genera& Met!ods 7$.
b. ;estern blots for the expression of /79 and @un) in order to evaluate the expression of the !!:
constructs.
c. laser capture micro-dissection of the SON followed by &T-9"& analyses for T&9:3' and n,+S
.Genera& Met!od 1:).
19P1R%M1,T :0: TH1 1""10TS +" %,H%B%T%+, +" $"+SB +, TH1 A0T%-%TA +" -AS+PR1SS%, ,1;R+,S
%, B'( RATS I! "I#$% .
• 4 groups ) 11! groups of rats* 1)Sham ligation and ,,/)5%-, !)BDL and ,,/)5%-, 8)sham ligation and
,,/)∆c9un, and 4)BDL and ,,/)∆c9un.
In vitro electrophysiological whole cell patch clamp recording from S1+ neurons.
"igure 1=: ?n experiment %! enalapril will be used
to bloc+ converting enzyme and prevent the
formation of angiotensin in ()* rats.
"igure 1B: ?n these experiments !T%& +noc+out
mice will be used to determine if these receptors are
important for vasopressin release associated with
()*.
F%.*re /0 7n e&periment 1., central infusions
of lorsartan will be used to block central ,(1
receptors in rats with established BDL to
re'erse the effects of 'asopressin release on
water retention.
7igure ,,< these studies will use an !!: vector with
a dominant negative construct against 67os( to test
the hypothesis that changes in gene expression
mediated by 67os( are reCuired for vasopressin
release associated with ()* in the rat.