sodium ibotenic acid: fluorogold: introduction a · cec cem cem cel cel cel cec cem cem bla bla bla...
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Novel Instrum
ent for Electrophysiological G
uidance of Nanoliter Injections in the B
rain.R
obert Lundy1, Y
i Kang
1, Chuck Locke
2, and Ralph N
orgren1 * P
enn State C
ollege of Medicine * D
ept. of Neural and B
ehavioral Sciences * H
ershey, PA
1 and Drum
mond S
cientific, Broom
all, PA
2.
Iontophoresis, air pressure, or manual ejection of neuronal tracer
and excitotoxin are techniques often used to study connectivity andfunction of specific brain nuclei. T
he interpretation of data obtainedfrom
such studies depend on two m
ajor parameters -- electro-
physiological identification of the target site and injection volume.
Although not routine, recording extracellular neural activity can be
used to center injections in functionally identified areas. It remains
a challenge, however, to place sm
all injections (<100nl) reliably in
the brain. This often m
akes interpretation difficult especially forsm
all subcortical structures where large injections usually extend
well beyond the target site.
To more reliably place sm
all injections, we m
odified acom
mercialy available device (N
anoject II, Drum
mond S
cientific),to record extracellular neural activity. T
he Nanoject II uses a direct
drive, a stainless steel piston inside a fluid filled pipette, and caninject as little as 2.3nl w
ith each activation. The present experi-
ments tested this new
instrument for electrophysiological localiza-
tion of the gustatory responsive area of the parabrachial nucleus(P
BN
) and injection of small volum
es (<60nl) of the tracer
Fluorogold or the excitotoxin ibotenic acid.
Fluorogold: E
lectrophysiologyIntroduction
PB
N: 23nl Injection
The response of a P
BN
taste cell to 0.1M N
aCl (black bar, 7s) recorded w
ith a pipetteattached to the new
Nanoject II. O
nce gustatory responsive cells were localized, the
retrograde neuronal tracer Fluorogold w
as ejected from the pipette at a rate of 2.3nl/step.
Su
mm
ary
Retrograde Transport: A
mygdala
Sodium
Appetite
Ibotenic Acid Lesions: P
BN
Learned Taste Aversion
The procedures for electrophysiological identification of the gustatory P
BN
were identical to
that for Fluorogold. D
otted area for 11.5 and 23.0nl is the approximate size of excitotoxin
lesion.
PB
N: 59.8nl Injection
Sodium
appetite tests 1, 2, & 3 occured 24hr follow
ing furosemide-
induced sodium loss. T
hree of the 5 rats in the 23nl PB
Nx group
failed to express a sodium appetite. T
he remaining 2 anim
als inthis group and all 4 of those w
ith 11.5nl IBO
injections displayed anorm
al sodium appetite.
All anim
als learned to avoid drinking 0.2M sucrose follow
ingT
rial 1 pairing with LiC
l.
23nl In
jection
Ce, central nucleus of the am
ygdala (M, m
edial; L, lateral; C, capsular); B
LA,
basolateral nucleus of the amygdala. C
olumn 1, approx. -3.14m
m posterior to
bregma; C
olumn 2 approx.-2.8m
m; C
olumn 3 approx.-2.3m
m.
59.8nl In
jection
NS
C
11.5nl
23.0nl
-9.8mm
-9.7mm
-9.6mm
-10mm
-9.8mm
-9.2mm
-10
mm
-9.8
mm
-9.6
5m
m-9
.3m
m
mP
BN
lPB
N
mP
BN
lPB
N
mP
BN
lPB
N
Ce
L
BL
AB
LA
BL
A
Ce
LC
eL
Ce
C
Ce
MC
eM
Ce
LC
eL
Ce
LC
eC
Ce
MC
eM
BL
AB
LA
BL
A
The N
anoject II was successfully m
odified torecord extracellular neural activity.
Sm
all nanoliter injections were consistently
placed in the gustatory parabrachial nucleus.
The effects of sm
all excitotoxic lesions suggesta functional topography w
ithin the gustatoryparabrachial nucleus.
This research w
as supported by NIH
grants DC
005156, DC
006698,D
C005435, and D
C00240.
0 5
10
15
20
Pre
-W
ate
rTria
l 1Tria
l 2T
rial 3
Test
Contro
l
PB
Nx 1
1.5
nl
PB
Nx 2
3nl
0 5
10
15
20
Contro
lP
BN
x 11.5n
lP
BN
x 23nl
Wate
rS
ucro
se
Single-Bottle Intake
Two-B
ottle Intake
Mean Intake (ml/15min)
*
*
**
*
0 5
10
15
20
25
30
35
0.2
50.5
12
24
Cumulative 0.51M NaCl Intake(ml)
Tim
e (hr)
0 5
10
15
20
25
30
35
0.2
50.5
12
24
Con
trolP
BN
11.5
nl
PB
N 2
3nl
PB
NX
23nl
0 5
10
15
20
25
30
35
0.2
50.5
12
24
0 5
10
15
20
25
30
35
0.2
50.5
12
24
Ap
petite Test 1
Ap
petite Test 2
Ap
petite Test 3
Salin
e Co
ntro
l
**
**
**
**
**
**
*
General M
ethodsS
ub
jec
ts: E
igh
tee
n m
ale
Sp
rag
ue
-Da
wle
y rats w
eig
hin
g b
etw
ee
n 3
40
-48
5g
ram
s. An
ima
ls ha
d fre
e a
ccess to
no
rma
l rat ch
ow
an
d d
istilled
wa
ter u
nle
ssotherw
ise noted. In all surgical procedures the rats were anesthetized w
ith a 50m
g/kg
inje
ction
of N
em
bu
tal.
Mo
dified
Nan
oject II: T
he Nanoject II uses direct piston displacem
ent. Toe
na
ble
reco
rdin
g o
f extra
cellu
lar n
eu
ral a
ctivity, the
pisto
n/flu
id-fille
d p
ipe
ttew
as e
lectrica
lly isola
ted
from
the
rest o
f the
instru
me
nt.
Gu
statory L
ocalizatio
n: B
y recording through the solution in the Nanoject II
pipette, gustatory neurons were identified by their responsiveness to stim
ulationo
f the
an
terio
r ton
gu
e w
ith 0
.1M
Na
Cl.
Flu
oro
go
ld an
d Ib
oten
ic Acid
: The retrograde neuronal tracer F
luorogold(F
G) w
as used at a concentration of 2% m
ixed with 0.15M
NaC
l. The volum
eo
f FG
inje
cted
wa
s 59
.8n
l (n=
1), 2
3n
l (n=
2), o
r 11.5
nl (n
=3
). Th
e e
xcitoto
xinib
ote
nic a
cid (IB
O) w
as u
sed
at 2
0m
g/m
l mixe
d in
ph
osp
ha
te b
uffe
red
salin
e.
The volum
e of IBO
inje
cted
wa
s 23
nl (n
=5
) or 11
.5n
l (n=
4). T
he control group(n=
4) consisted of two non-surgical rats and tw
o rats in which the gustatory P
BN
wa
s loca
lized
, bu
t IBO
wa
s no
t inje
cted
.
Imm
un
oh
istoch
emistry:
Flu
oro
go
ld: F
ive days post injection, rats were euthanized w
ith a lethal dose ofN
em
bu
tal (1
00
mg
/kg ip
) an
d p
erfu
sed
with
0.9
% h
ep
arin
ized
salin
e, fo
llow
ed
by b
uffe
red
4%
pa
rafo
rma
lde
hyd
e (p
H 7
.4). T
he
bra
in w
as re
mo
ved
, cut a
t5
0µ
m, a
nd
pro
cesse
d fo
r inte
nsifica
tion
of F
luo
rog
old
.Ib
oten
ic Acid
: The procedures w
ere identical to those described above with 2
exce
ptio
ns. F
irst, the
an
ima
ls we
re p
erfu
sed
follo
win
g b
eh
avio
ral te
sting
, wh
ichlasted about 2 m
onths. Second, the tissue sections w
ere stained for the neuronalm
arke
r Ne
uN
(Ch
em
icon
).
So
diu
m A
pp
etite: One w
eek after the LTA experim
ents, animals w
ere housedin w
ire-mesh m
etabolic cages. For 7 days, the anim
als had access to water and
0.5
1M
Na
Cl a
ttach
ed
to th
e fro
nt o
f the
cag
es. O
n d
ay 8
, an
ima
ls we
re m
ad
esodium
deficient by injection of furosemide (6m
g/kg sc). Only w
ater and sodiumd
eficie
nt ch
ow
(Tekla
d) w
ere
ava
ilab
le o
vern
igh
t. Th
e n
ext d
ay w
ate
r an
d N
aC
lsolution w
ere returned to the cages and intake was m
easured at .25, .5, 1, 2, and2
4h
r inte
rvals. W
ate
r an
d N
aC
l rem
ain
ed
on
the
cag
es fo
r 6 m
ore
da
ys. Th
issequence, an injection day w
ith furosemide follow
ed by a test day and a subse-quent 6 day baseline period, w
as repeated a second and third time. F
inally, thissequence w
as repeated a fourth time except that the injection day w
as with an
eq
uiva
len
t volu
me
of 0
.9%
salin
e in
stea
d o
f furo
sem
ide
.
Learn
ed Taste A
version
(LTA): T
wo w
eeks after surgery, animals w
erew
ate
r restricte
d w
ith 1
5m
in a
ccess e
ach
mo
rnin
g a
nd
1h
r ea
ch a
ftern
oo
n. T
he
con
ditio
nin
g p
roce
du
re co
nsiste
d o
f rep
lacin
g m
orn
ing
wa
ter w
ith 0
.2M
sucro
se(C
S) fo
llow
ed
30
min
late
r with
an
inje
ction
of 0
.15
M L
iCl (U
S; 1
.33
ml/1
00
g ip
).W
ater was available for the next tw
o days. This sequence w
as repeated a seconda
nd
third
time
for a
tota
l of 3
CS
-US
pa
iring
s. Fo
llow
ing
two
wa
ter d
ays, th
ea
nim
als a
ga
in h
ad
15
min
acce
ss to th
e C
S, b
ut n
ot su
bse
qu
en
tly inje
cted
with
LiCl (1-bottle test). F
ollowing tw
o more w
ater days, the animals w
ere presentedw
ith water and the C
S (2-bottle test).