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).