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F30 Grant Outline

Specific Aim 1:To test the role of renin-angiotensin system in supporting the chronic activation of vasopressinneurons 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) EFFECTSOFCHRONICACE INHIBITIONONFLUIDRETENTIONANDCHRONICCNS ACTIVATIONINBDL 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 waterii$ BDL and tap wateriii$ 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 -/+, S+, 2n-, S% and /L( GeneralMethod !).

4$ Samples will be analy3ed to determine the number of %os and %osB$%osB positi'e cells in the+(S, /L, 2n-, /L( and S% that are retrogradely labeled from the S+.

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%&.

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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%ns.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 vitropatch clamp electrophysiological recording from S+ neurons we will test changes in

(0-/4 and ng77 channel properties resulting from BDL in the presence and absence ofperipheralng77.

,$ ""0TS +"0,TRA(AT1R B(+02A'%,B(' RATS. %S+(ATPR%PHRA(""0TS)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,+Se3pression that occur in the "NS of ()* rats.

b) Treatment Groups: Genera& Met!ods )

%$ 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 -/+, S+, 2n-, S% and /L( GeneralMethod !).

4$ Samples will be analy3ed to determine the number of %os and %osB$%osB positi'e cells in the+(S, /L, 2n-, /L( and S% that are retrogradely labeled from the S+.

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$.

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e) ffects 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 vitropatch clamp electrophysiological recording from S+ neurons, we will test changes in

(0-/4 and ng77 channel properties resulting from BDL in the presence and absence of centralng77.

Specifc Aim 2: Test the hypothesis that increasedexpression o FosB in the SON produces cellularadaptations that contribute to increased excitability in theSONHypothesis: 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 enhancedexcitability of the vasopressin system associated with ()*.

9PR%M,TA: AA-/M'%AT'%,H%B%T%+,+"$"+SB A,'"(;%'RT,T%+,A,'-AS+PRSS%,

R(AS';R%,GB'( 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

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c. laser capture micro-dissection of the SON followed by &T-9"& analyses for T&9:3' and n,+S

Genera& Met!od 1).

9PR%M,T0: TH""0TS+"%,H%B%T%+,+"$"+SB +,THA0T%-%T+"-AS+PRSS%,,;R+,S

%,B'( RATSI!"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 vitroelectrophysiological whole cell patch clamp recording from S+ neurons.

"igure 1=: ?n experiment %! enalapril will be used

to bloc+ converting enzyme and prevent theformation of angiotensin in ()* rats.

"igure 1: ?n these experiments !T%& +noc+out

mice will be used to determine if these receptors areimportant for vasopressin release associated with

()*

F%*re /0 7n e&periment 1, central infusionsof lorsartan will be used to block central (1receptors in rats with established BDL tore'erse the effects of 'asopressin release onwater 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 vasopressinrelease associated with ()* in the rat.