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The chronic effects of desipramine and sertraline on platelet and synaptosomal 5HT uptake in olfactory bulbectomised rats
Prog. ~eu~Psyc~op~~rmac~1. E?BioI. Psychiat. Printed in Great Britain. All rights reserved
1988, Vol. 12, pp. 585694 Copyright
0
027~5~El88 $0.00 + SO 1988 Pergamon Press pfc
THE CHRONIC EFFECTS OF DESIPRAMINE AND SERTRALINE ON PLATELET AND SYNAPTOSOMAL 5HT UPTAKE IN OLFACTORY BULBECTOMISED RATS JACKIE
BUTLER,
Department
MIRIAM
TANNIAN
and BRIAN
of Pharmacology, University Republic of Ireland
(Final
form, December
E.LEONARD.
College,
Galway,
1987)
Abstract Butler, Jackie, Miriam Tannian and Brian E. Leonard: The chronic effects of desipramine and sertraline on platelet 5HT uptake in olfactory bulbectomised rats. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 1988, 12:585-594 r Depressed patients show a characteristic decrease in the rate of uptake of serotonin into their platelets, which is normalised only by clinically effective antidepressant treatment. 2. We also find a decrease in platelet 5HT uptake rates in the olfactory bulbectomised (OB) rat model of depression, which return to normal following three weeks of treatment with desipramine or sertraline. 3. Synaptosomal 5HT uptake appears to consist of a low affinity, high capacity component and a high affinity, low capacity component, both of which are increased in the OB rat, compared to its sham operated control, and normalised by chronic antidepressant treatment. 4. The low affinity uptake of serotonin is not inhibited by in vitro incubation with sertraline, which suggests that the low affinity system may be associated with a non-specific uptake of 5HT into noradrenergic or dopaminergic nerve endings. Keywords : depression, synaptosome.
olfactory
Abbreviations : olfactory me-
bulbectomy,
bulbectomy
platelet,
COB), serotonin
serotonin
uptake,
(5HT).
Introduction A decreased probable 1979)
state
being
treatment
Leonard,
rate
(Coppen
only by clinically
has also been
effect
uptake
of depression
et al, 1985).
of depressed
immediate
Treatment
found
patients,
to raise
on recovery,
is now recognised
with
et al,
antidepressant
the novel
the platelet to control
of this drug is to inhibit
; Toumisto
et al, 1978
effective
as a
antidepressant,
serotonin levels,
5HT uptake
uptake
although
(Butler
the
and
in preparation).
The olfactory antidepressant After
marker
serotonin
normalised
(Healy
sertraline, rates
platelet
removal
hyperactivity
bulbectomised activity
rat has been used
(vanRiezen
of the olfactory in a stressful
for putative
et al, 1977 ; Cairncross
bulbs, novel
to test
the animals
enviroment,
585
show
which
et al, 1978).
a characteristic
is reversed
only
by
J. Butler et al.
586
chron
ic antidepressant
In the present rats,
before
determine
study,
and after
were
As the platelet tissue
investigation
from the same
the 5HT uptake
comparable
into platelets or sertraline,
to those
seen
we also measured chronic
neurotransmission 5HT uptake treatment
rates
of OB to
in depressed
to be an accessible
1968) and the use of an animal
in brain
rats after
& Tu ite, 1981).
with desipramine
is considered
(Pletscher,
of changes
simultaneously,
(Leonard
we examined treatment
if the changes
patients. neuronal
treatment
model model
of
allows
to be determined into the synaptosomes
with
desipramine
used
in these
or sertraline.
Methods Animals Male were
Sprague-Dawley
housed
light-dark
4/cage
rats
with with
cycle,
(250-28Og)
free access lights
were
to food and water,
experiments.
with
They
a 12-hr
on at 8 am.
Drugs Sertraline Ciba-Geigy,
was obtained UK.
Radiochemicals Surgical
as a gift
The tritiated
from
UK and desipramine
from
from Amersham
Ltd. UK.
Procedure
Bilateral
olfactory
anaesthesia
bulbectomy
was performed
1.0 ml/lOOg
(2.5% w/v;
Following
(1977).
exposure
of the skull,
7 mm anterior
to bregma
points
corresponding
to the posterior
olfactory
bulbs
haemostatic
removed
by surgical
but although
left intact.
they were
handled
margin
by suction
were
daily
were
drilled
was then applied
the bulbs
et al at
side of the mid-line
of the orbit
Sham operated
allowed
tribromoethanol by Cairncross
animals
was cut,
to recover
by the experimenter
at
of the eye.
and the burr holes
powder
clips.
the dura above
The animals
burr holes
and 2 mm either
Tetracycline
sponge.
the skin closed treated,
were
under
i.p) as described
points
which
Pfizer,
5HT was purchased
filled
to the wound were
to reduce
and
similarily
the bulbs
for 14 days,
The
with
were
during
the potential
for aggressiveness. Treatment Following groups
recovery,the
(a,b,cl
i.p. injection "b" was treated
with
08 animals
randomly
10 rats in each group.
for 21 days; Group with
were
desipramine
The animals
"a" received (fO/mg/kg)
divided
a saline
and Group
into three
received injection,
a daily Group
"c" was treated
with
587
Platelet and synaptosomal uptakeinOB rats
sertraline
(IO/m&kg).
Behavioural
The sham operated
The animals
from above measured
were
all animals placed
by a 100 watt
surrounding
aluminium visually
Biochemical
into tubes
the animals
containing
of the method
5HT uptake.
The uptake
using
10 cm squares
crossed
which
and the of the rats was
in 3 min.
sacrified
blood EDTA
by decapitation
flow).
The trunk
disodium
dissected
salt,
of the
cortex
of Slotkin
plasma
0.05 pM tritiated The incubation
The platelets filtration
which
buffered
were
through
isolated Gellman
with
saline,
a
was
five concentrations pH 7.0, containing
to measure
out at OoC to provide
was used
using
and synaptosomes
(25 ~1) was incubated
was also carried
were
(1974).
5HT, for five min at 370C
diffusion,
aspirated.
et al (1978).
of Toumisto
(0.1 pM - 2 pM) in Phosphate
centrifuged
plasma
and amygdala,
under
blood was
on ice and synaptosomes
of 5HT into platelets
the method
(a) Platelet-rich
counted
illuminated
The ambulation
were
0.15%
were
from the P2 pellets
passive
into
of squares
(to facilitate
of the animals
modification
of 5HT
to the 'open field'
apparatus,
at 700g for 10 min and the platelet-rich
The brains
measured
similarily.
The base of the apparatus,
was divided
as the number
anaesthesia
immediately
prepared
treated
Methods
ether
collected
subjected
wall was 45 cm high.
On day 21 of treatment, light
were
in a cylindrical
light-bulb.
90 cm in diameter,
estimated
were
Methods
On day 18 of treatment, test.
animals
as the blank
of
in the calculations.
medium
filters,
uptake.
an estimate
value
from the incubation 0.2 pm cellulose
active
by vacuum
which
were
then
for radioactivity.
(b) The synaptosomes of 5 mg/ml. platelet-
rich plasma.
two-component performed
uptake
using
incubations estimate
resuspended
system
also carried
to the potency
at this concentration be inhibited. by vacuum
for sertraline
scheme
of sertraline,
through
incubation
were
Gellman
proposed
only
pM
to
and/or
is 0.06 PM for 5HT
by Koe
(Koe et al, 1983), (1976).
the serotonergic
isolated 0.45
was
sets of
of 0.25 FM sertraline
of 5HT into noradrenergic
and 1.3 pM for DA uptake
rating
The synaptosomes
filtration
a further
for the
a
from 0.01 PM to 0.5 pM. Both
The IC50
0.54 J.IMfor NA uptake
according
concentration
as described indicated
out in the presence
uptake
synaptosomes.
experiments
in the synaptosomes,
5HT concentrations
were
in PBS to a protein was incubated
As preliminary
the non-specific
dopaminergic uptake,
were
25 pl of this preparation
Therefore,
uptake
should
from the incubation
medium
cellulose
filters
and counted
J.Butleret al.
588
for
radioactivity. The protein
determined also of
concentration using
determined platelets
the to
ensure
(data
Statistical
method
not
of
the
of
Lowry
that
platelet et
protein
and synaptosomal
al
membranes
Platelet
(1951).
concentrations
numbers
reflected
was were
the
number
shown).
Analysis
The platelet weighted
and high-affinity
non-linear
was estimated
and the
by Lineweaver-Burk
The behavioural biochemical
synaptosomal
regression
results
results
was analysed
by
synaptosomal
uptake
analysis.
were
were
uptake
low-affinity
compared
compared
by the
using
the
Mann-Whitney
Students
U-test
t-test
for
and the
unpaired
data.
Results
Behavioural Table
Results
1 illustrates
ambulation
of
characteristic controls
rats
in
effect the
hyperactivity
(p < 0.001).
sertraline effect
the
the
of ‘open
normalised
field’.
in this
Treatment
of
+Saline 155.1 700.0
Platalet serotonin controls sertraline
the
desipramine
while
having
or
no
1 Activity of Apparatus
+Desipramine
2 10.49: _t 5.34
125.3 84.9
OB and Sham
+Sertraline
2 7.59. -+ 8.60
88.9 704.1
Results expressed as % sham + saline (S + Sal.) * p < 0.001 vs. S + Sal.; l p < 0.001 vs. OB + Sal. Ambulation score of sham controls q 76 2 5.82 squares Biochemical
showed
to the sham operated
either
of the OB group,
on the
rats.
Desipramine and Sertraline on the Operated Rats in the ‘Open Field’
Animals OB Sham
The OB rats
test as compared
Table Effect
and sertraline
for 18 days with
the behaviour
on the sham operated
desipramine
crossed
+ 7.778 2 7.87
/ 3 min
Findings
: The maximal
5HT uptake was decreased
(p < 0.01) for three
rate
(Vmaxl
in the OB rat compared
(Table weeks
2).
Treatment
normalised
with
of platelet
uptake
of
to the sham operated either
this uptake
desipramine
rate.
Treatment
or of the
589
Platelet and synaptosomal uptakeinOB rats
sham operated
rats with
There
uptake.
sertraline
the OB and sham operated on Km values resulted (Table
also resulted
was no difference
in either
in a raised
Treatment
groups.
group,
in an increased
in the affinity
(Km) of uptake
with
desipramine
but the three weeks
Km for uptake
sertraline
rate of between
had no effect treatment
in both the OB and sham operated
groups
2). Table
Effect
2
of Desipramine and Sertraline on the Uptake of 3H-5HT Platelets of OB and Sham Operated Rats.
into
____________-___-_-_____-_________________________________________________ Animals +Saline +Desipramine +Sertraline ___________________________________________________________~___~___~___~__ 0 Vmax 119.6 -+ 9.58' 96.7 + 6.42 40.9 2 3.97' OB Km 106.7 + 20.25 80.8 + 15.19 521.5 + 140.51*' ________-___-_______~~~~~~~~--~--~~~~~~~-~-~~~--~~~-~~~--~~--~~--~~~~~~~~~ Vmax 100.0 -+ 3.13. 120.2 + 13.64 166.0 -+ 4.041 Sham Km 100.0 + 17.70 96.2 + 20.25 169.6 + 24.05" ____________________--_______--_________-________________________________ Results expressed as % sham + saline * p < 0.01 vs sham + saline; l p < 0.01 vs OB + saline; *o p < 0.01 vs sham + saline, p < 0.01 vs OB + saline Sham control Vmax = 15.1 A 0.47 pmoles/mg protein Sham control Km = 0.5 + 0.08 pM. Synaptosomal which
5HT uptake
was inhibited
by in vitro
(p < 0.001)
and normalised
rats showed
no change
showed
a raised
group,
after
in either
after
sertraline,
in uptake
Vmax after
which
treatment. treatment
There
with
of 5HT (0.01-0.5
was raised
drug
treatment.
desipramine
Sham operated The OB group
in the sham
was no effect
PM),
in the OB group
treatment.
was not apparent
Table Tffect H-5HT
uptake
by the antidepressant
Km of uptake,
sertraline
group
: The high-affinity
operated
on the Km of uptake
( Table 3).
3
of Desipramine and Sertraline on the Hi gh Affinity Uptake of into Synaptosomes of Bulbectomised and Sham Operated Rats
______-_____________~~~~~~~~_~~~-_~~~~--~~~-~~~~~~~~~~~~~~-~~~_~~-_~~~__~Animals +Saline +Desipramine +Sertraline _____-__~___~______~_---__-~-~_~~~_____-~~~~~~~~~~~~~~~~_~~~_~~~-~~__~____-_ Vmax 82.7 ) 7.72' 111.8 -+ 49.25' 259.9 + 47.57" OB Km 89.0 + 30.91 95.5 + 10.91 158.2 + 36.40' -________-_______-__-~-~-~~~~~-~~~~~------~~~~~~~~_~~~~~~~_~~~_~~~__~_~_~_ Vmax 100.0 -+ 25.53' 80.2 ) 5.27 67.9 -+ 28.8" Sham _f__""___________l"~~~_~_~~~~~_______"ll_~~~~~__ Results expressed as % sham + saline * p < 0.001 vs sham + saline; l p < 0.01 vs OB + saline Sham control Vmax = 35.7 _c 9.13 pmoles/mg protein Sham control Km q 0.8 + 0.43 PM.
590
J.Butler etal.
At the higher uptake
concentration
was increased
normalised
by three weeks
did not reach
saturation
not be analysed sertraline
5HT.
(Table
compared with
non-linear
Treatment
either
drug.
range
regression,
'low-affinity'
of animals
for
(p < 0.011,
This uptake,
used and hence
rats with
synaptosomal
by Lineweaver-Burk after
the Vmax
treatment
could by
desipramine
uptake
analysis,
with
and
which
was not inhibited
of the sham operated
the
as estimated
in both groups
PM),
to controls
in the concentration
did not effect
The Km of uptake,
unchanged
of 5HT (0.1-2.0
treatment
by weighted
in vitro.
or sertraline
range
in the OB group
either
of
was drug
4) Table
Effecs
4
of Desipramine and Sertraline on the Low Affinity Uptake H-5HT into Synaptosomes of OB and Sham Operated Rats
of
_____________________~~-~____~~~___~~~~~~~~-~~~~~--~~~~~~-~~~~~-~~~~~--~~~ Animals +Saline +Desipramine +Sertraline _______________________~~____~~~____~-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-~~~ 104.6 2 21.55. 104.9 L 13.78' Vmax 232.0 + 15.51" OB 91.0 2 20.86 110.6 -+ 12.21 Km 89.1 2 13.10 _________________________________________~~-~~-~~~~~~-~~~~~~~~-~~~~~~~~_________ 100.0 -+ 10.73. Vmax 115.0 + 10.28 99.8 -+ 13.81 Sham Km 100.0 2 13.91 82.4 2 9.25 87.5 -+ il.80 ____________________~~~~~~~~~~-~~~~~~-~~----~~-----~~~----~~~---~~~~~~-~~~ Results expressed as % sham + saline * p < 0.01 vs sham + saline; l p < 0.001 vs sham + saline Sham control Vmax q 266 _c 37 pmoles/mg protein Sham control Km = 4.3 5 0.24 PM. Discussion The olfactory
bulbectomised
OB rats have been used (Earley matter
& Leonard,
a reliable,
(Leonard
shown
those
in which
corrects
& Leonard, several
& Leonard,
It was in an attempt
1986).
it was concluded changes
(vanRiezen
1983) chronic,
of these
for new antidepressants
see if there was an abnormality the OB rat and if so, whether
that occur patient.
it is a or merely
of novel
of a short
review
that the physiological, following
Furthermore,
et al, 1977
but not acute,
behavioural
to extend
However,
of depression
test for the detection
in the depressed
here and elsewhere
Janscgr
in the search
this has been the subject
and behavioural seen
of depression
the OB rat is a model
screening
Recently,
et al, 1987)
neurochemical resemble
whether
if tedious,
antidepressants.
as a model
; O'Connor
1985
of conjecture
rat as a model
; Cairncross
bulbectomy as has been et al, 1979
antidepressant
treatment
deficits.
our previous
studies
in the transport this could
that we decided
of 5HT into platelets
be normalised
by chronic
to of
;
Platelet and synaptosomal uptakeinOB rats
antidepressant (Healy
It has been established
treatment.
; Butler
et al, 1983,1985
the platelets effective
et al, 1980)
; Coppen
et al, 1979
of depressed
that the defective
patients
is corrected
5HT uptake
The results particular
of this study
that at least
antidepressants
associated
with
a return
normalises
It is of interest
patients
et al, 1985) appears
specificity
uptake
The increased
sites
Km of uptake
competitive
seen with
the noradrenaline
of 5HT into
Synaptosomal
5HT uptake
The nature
but the platelet
synaptosome
of the same
increased contrast
antidepressants Two components It is also synaptosomes
rate being
system.
to synaptosomal
sites
(Kimelberg,
study
has indicated
inhibited
in vitro
found
which
is not
those
depression
attention
into both suggest
occuring
because
and
that changes
of 5HT
in central
were
of
In the present
the platelet
in the synaptosomal
rates
is
neurons,
fractions
In both normalised
an
in
platelets by
deficit.
5HT uptake that
the transport
on both a low and a high have reported
the transport
that
treatment
by sertraline,
et al, 1968).
the behavioural
1985) while
19861,
sertraline
rate in the platelets.
investigations
may involve
(eg.
is as yet unclear.
desipramine.
The results
from this study
is dependent
& Singhal,
of the
or noradrenergic
considerable
the 5HT uptake
that corrected
Previous
(Ahluwalia
uptake
however,
apparent
was
of the presumed
underlying
in 5HT transport
animals.
to a decreased
and synaptosomes,
chronic
(Pletscher
do not reflect
5HT uptake
the effect
mechanism
lesion(s)
neurons
changes
into platelets
; this effect
in both OB rats and depressed
inhibitor,
has received
to central
we examined
two
of the OB rats to
(eg. sertraline)
with
the
with
in the OB rat
of the neurochemical
its similarity
Vmax
of 5HT uptake
uptake
to this
treatment
to be independent
associated
inhibition
regard
rate
activity
and the precise
indicates
unclear,
5HT uptake
of the drug on the 5HT
desipramine)
the uptake
to find that
on platelet
(Healy
with
that chronic
of the locomotor
antidepressants
systems
transport
by therapeutically
the OB rat resembles
function,
We have also shown
patient.
values.
show
of platelet
dissimilar
uptake
(Toumisto
in the OB rat
'marker'
depressed
study,
laboratories
and elsewhere
treatment.
Platelet
control
in these
1986)
& Leonard,
591
others
for this
the low affinity
evidence
for such a system that one of these
is
(i.e. glial)
slight.
5HT uptake
5HT uptake
transport
suggested
of 5HT into non-neuronal
but evidence
by the specific
have
of 5HT into
affinity
system
inhibitor,
The present is not
sertraline.
This
J.Butler etal.
592
suggests
that the low affinity
aminergic activity
neurons. of both
the OB rat than sites
the low and high affinity
of other
control
by antidepressant
to studying
transport
be associated
it is clear
its sham operated
is normalised
devoted
site might
Nevertheless,
the effects biogenic
transport
site
that the
is greater
and that the activity
treatment.
of various
amine
with catechol-
from this study
Further
studies
antidepressants
neurotransmitters
in
of both will
be
on the
into synaptosomes
of
OB rats.
Conclusions It may be concluded depressant
treatment
depressed
patients
as a model showing
that the similarity on defective
and OB rats
of depression.
a biochemical
profile
is interesting
in this study
antidepressant
treatment,
putative
appear
evidence
is the first
similar
report
in favour
could
anti-
be used
model
patients.
of normal as a model
of
of the OB rat
of an animal
to that of depressed
is the restoration
which
of chronic
of 5HT into the platelets
function
What
with
for screening
antidepressants.
Despite neurone
is further
This
in the effect
transport
the well
regarding that there
the nature
established the nature
similarities of receptors,
are also marked
of the 5HT transport
between
the platelet
transport
differences
between
sites
and the
etc.,
them with
it would regard
to
sites.
Acknowledgement J.B.
is a post-doctoral
Ireland. used
The authors
in this study
fellow
thank
sponsored
Pfizer
by the Health
UK Ltd. for supplying
and for financial
assistance
towards
Research
Board
of
the sertraline the cost of the
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LOWRY, O.H., ROSENBROUGH, N.J., FARR, A.L. & RANDALL, R.J. (1951) measurement with the Folin phenol reagent. J. Biol. Chem. 193,
Protein 265-275.
O'CONNOR, W.T. & LEONARD, B.E. (1986) Effect of chronic administration of the 6-aza analogue of mianserin (Org. 37701 and its enantiomers on behaviour and changes in noradrenaline metabolism of olfactory bulbectomised rats in the 'open field' apparatus. Neuropharmacol. ;t5, 267-270.
J.Butler et al.
594
PLETSCHER, A. (19681 Metabolism, transfer platelets. Br. J. Pharmacol. 32, 1-16.
and storage
of 5HT in blood
SLOTKIN, T.A., SEIDLER, F.J., WHITMORE, W.L., SALVAGGIO, M. & LAU, C. Ionic and nucleotide cofactor requirements for uptake of $1978) H-norepinephrine by rat brain synaptic vesicle preparations. Mol. Pharmacol. 14, 868-878. TOUMISTO, J. (1974) A new modification for studying 5HT uptake by blood platelets : A re-evaluation of tricyclic antidepressants as uptake inhibitors. J. Pharm. Pharmacol. 6, 92-100. TOUMISTO, J., TUKIANEN, E. & AHLFORS, U.G. (1979) Decreased uptake of 5-hydroxytryptamine in blood platelets from patients with endogenous depression. Psychopharmacol. 65, 141-147. VANRIEZEN, H., SCHNIEDEN, H. & WREN, A.F. (1977) Behavioural changes following olfactory bulbectomy in rats : A possible model for the detection of antidepressant drugs. Br. J. Pharmacol. 21, 426p-427~.
Reprint
requests
should
Dr. J. Butler Department of Pharmacology University College Galway Republic of Ireland.
be addressed
to :
1988, Vol. 12, pp. 585694 Copyright
0
027~5~El88 $0.00 + SO 1988 Pergamon Press pfc
THE CHRONIC EFFECTS OF DESIPRAMINE AND SERTRALINE ON PLATELET AND SYNAPTOSOMAL 5HT UPTAKE IN OLFACTORY BULBECTOMISED RATS JACKIE
BUTLER,
Department
MIRIAM
TANNIAN
and BRIAN
of Pharmacology, University Republic of Ireland
(Final
form, December
E.LEONARD.
College,
Galway,
1987)
Abstract Butler, Jackie, Miriam Tannian and Brian E. Leonard: The chronic effects of desipramine and sertraline on platelet 5HT uptake in olfactory bulbectomised rats. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 1988, 12:585-594 r Depressed patients show a characteristic decrease in the rate of uptake of serotonin into their platelets, which is normalised only by clinically effective antidepressant treatment. 2. We also find a decrease in platelet 5HT uptake rates in the olfactory bulbectomised (OB) rat model of depression, which return to normal following three weeks of treatment with desipramine or sertraline. 3. Synaptosomal 5HT uptake appears to consist of a low affinity, high capacity component and a high affinity, low capacity component, both of which are increased in the OB rat, compared to its sham operated control, and normalised by chronic antidepressant treatment. 4. The low affinity uptake of serotonin is not inhibited by in vitro incubation with sertraline, which suggests that the low affinity system may be associated with a non-specific uptake of 5HT into noradrenergic or dopaminergic nerve endings. Keywords : depression, synaptosome.
olfactory
Abbreviations : olfactory me-
bulbectomy,
bulbectomy
platelet,
COB), serotonin
serotonin
uptake,
(5HT).
Introduction A decreased probable 1979)
state
being
treatment
Leonard,
rate
(Coppen
only by clinically
has also been
effect
uptake
of depression
et al, 1985).
of depressed
immediate
Treatment
found
patients,
to raise
on recovery,
is now recognised
with
et al,
antidepressant
the novel
the platelet to control
of this drug is to inhibit
; Toumisto
et al, 1978
effective
as a
antidepressant,
serotonin levels,
5HT uptake
uptake
although
(Butler
the
and
in preparation).
The olfactory antidepressant After
marker
serotonin
normalised
(Healy
sertraline, rates
platelet
removal
hyperactivity
bulbectomised activity
rat has been used
(vanRiezen
of the olfactory in a stressful
for putative
et al, 1977 ; Cairncross
bulbs, novel
to test
the animals
enviroment,
585
show
which
et al, 1978).
a characteristic
is reversed
only
by
J. Butler et al.
586
chron
ic antidepressant
In the present rats,
before
determine
study,
and after
were
As the platelet tissue
investigation
from the same
the 5HT uptake
comparable
into platelets or sertraline,
to those
seen
we also measured chronic
neurotransmission 5HT uptake treatment
rates
of OB to
in depressed
to be an accessible
1968) and the use of an animal
in brain
rats after
& Tu ite, 1981).
with desipramine
is considered
(Pletscher,
of changes
simultaneously,
(Leonard
we examined treatment
if the changes
patients. neuronal
treatment
model model
of
allows
to be determined into the synaptosomes
with
desipramine
used
in these
or sertraline.
Methods Animals Male were
Sprague-Dawley
housed
light-dark
4/cage
rats
with with
cycle,
(250-28Og)
free access lights
were
to food and water,
experiments.
with
They
a 12-hr
on at 8 am.
Drugs Sertraline Ciba-Geigy,
was obtained UK.
Radiochemicals Surgical
as a gift
The tritiated
from
UK and desipramine
from
from Amersham
Ltd. UK.
Procedure
Bilateral
olfactory
anaesthesia
bulbectomy
was performed
1.0 ml/lOOg
(2.5% w/v;
Following
(1977).
exposure
of the skull,
7 mm anterior
to bregma
points
corresponding
to the posterior
olfactory
bulbs
haemostatic
removed
by surgical
but although
left intact.
they were
handled
margin
by suction
were
daily
were
drilled
was then applied
the bulbs
et al at
side of the mid-line
of the orbit
Sham operated
allowed
tribromoethanol by Cairncross
animals
was cut,
to recover
by the experimenter
at
of the eye.
and the burr holes
powder
clips.
the dura above
The animals
burr holes
and 2 mm either
Tetracycline
sponge.
the skin closed treated,
were
under
i.p) as described
points
which
Pfizer,
5HT was purchased
filled
to the wound were
to reduce
and
similarily
the bulbs
for 14 days,
The
with
were
during
the potential
for aggressiveness. Treatment Following groups
recovery,the
(a,b,cl
i.p. injection "b" was treated
with
08 animals
randomly
10 rats in each group.
for 21 days; Group with
were
desipramine
The animals
"a" received (fO/mg/kg)
divided
a saline
and Group
into three
received injection,
a daily Group
"c" was treated
with
587
Platelet and synaptosomal uptakeinOB rats
sertraline
(IO/m&kg).
Behavioural
The sham operated
The animals
from above measured
were
all animals placed
by a 100 watt
surrounding
aluminium visually
Biochemical
into tubes
the animals
containing
of the method
5HT uptake.
The uptake
using
10 cm squares
crossed
which
and the of the rats was
in 3 min.
sacrified
blood EDTA
by decapitation
flow).
The trunk
disodium
dissected
salt,
of the
cortex
of Slotkin
plasma
0.05 pM tritiated The incubation
The platelets filtration
which
buffered
were
through
isolated Gellman
with
saline,
a
was
five concentrations pH 7.0, containing
to measure
out at OoC to provide
was used
using
and synaptosomes
(25 ~1) was incubated
was also carried
were
(1974).
5HT, for five min at 370C
diffusion,
aspirated.
et al (1978).
of Toumisto
(0.1 pM - 2 pM) in Phosphate
centrifuged
plasma
and amygdala,
under
blood was
on ice and synaptosomes
of 5HT into platelets
the method
(a) Platelet-rich
counted
illuminated
The ambulation
were
0.15%
were
from the P2 pellets
passive
into
of squares
(to facilitate
of the animals
modification
of 5HT
to the 'open field'
apparatus,
at 700g for 10 min and the platelet-rich
The brains
measured
similarily.
The base of the apparatus,
was divided
as the number
anaesthesia
immediately
prepared
treated
Methods
ether
collected
subjected
wall was 45 cm high.
On day 21 of treatment, light
were
in a cylindrical
light-bulb.
90 cm in diameter,
estimated
were
Methods
On day 18 of treatment, test.
animals
as the blank
of
in the calculations.
medium
filters,
uptake.
an estimate
value
from the incubation 0.2 pm cellulose
active
by vacuum
which
were
then
for radioactivity.
(b) The synaptosomes of 5 mg/ml. platelet-
rich plasma.
two-component performed
uptake
using
incubations estimate
resuspended
system
also carried
to the potency
at this concentration be inhibited. by vacuum
for sertraline
scheme
of sertraline,
through
incubation
were
Gellman
proposed
only
pM
to
and/or
is 0.06 PM for 5HT
by Koe
(Koe et al, 1983), (1976).
the serotonergic
isolated 0.45
was
sets of
of 0.25 FM sertraline
of 5HT into noradrenergic
and 1.3 pM for DA uptake
rating
The synaptosomes
filtration
a further
for the
a
from 0.01 PM to 0.5 pM. Both
The IC50
0.54 J.IMfor NA uptake
according
concentration
as described indicated
out in the presence
uptake
synaptosomes.
experiments
in the synaptosomes,
5HT concentrations
were
in PBS to a protein was incubated
As preliminary
the non-specific
dopaminergic uptake,
were
25 pl of this preparation
Therefore,
uptake
should
from the incubation
medium
cellulose
filters
and counted
J.Butleret al.
588
for
radioactivity. The protein
determined also of
concentration using
determined platelets
the to
ensure
(data
Statistical
method
not
of
the
of
Lowry
that
platelet et
protein
and synaptosomal
al
membranes
Platelet
(1951).
concentrations
numbers
reflected
was were
the
number
shown).
Analysis
The platelet weighted
and high-affinity
non-linear
was estimated
and the
by Lineweaver-Burk
The behavioural biochemical
synaptosomal
regression
results
results
was analysed
by
synaptosomal
uptake
analysis.
were
were
uptake
low-affinity
compared
compared
by the
using
the
Mann-Whitney
Students
U-test
t-test
for
and the
unpaired
data.
Results
Behavioural Table
Results
1 illustrates
ambulation
of
characteristic controls
rats
in
effect the
hyperactivity
(p < 0.001).
sertraline effect
the
the
of ‘open
normalised
field’.
in this
Treatment
of
+Saline 155.1 700.0
Platalet serotonin controls sertraline
the
desipramine
while
having
or
no
1 Activity of Apparatus
+Desipramine
2 10.49: _t 5.34
125.3 84.9
OB and Sham
+Sertraline
2 7.59. -+ 8.60
88.9 704.1
Results expressed as % sham + saline (S + Sal.) * p < 0.001 vs. S + Sal.; l p < 0.001 vs. OB + Sal. Ambulation score of sham controls q 76 2 5.82 squares Biochemical
showed
to the sham operated
either
of the OB group,
on the
rats.
Desipramine and Sertraline on the Operated Rats in the ‘Open Field’
Animals OB Sham
The OB rats
test as compared
Table Effect
and sertraline
for 18 days with
the behaviour
on the sham operated
desipramine
crossed
+ 7.778 2 7.87
/ 3 min
Findings
: The maximal
5HT uptake was decreased
(p < 0.01) for three
rate
(Vmaxl
in the OB rat compared
(Table weeks
2).
Treatment
normalised
with
of platelet
uptake
of
to the sham operated either
this uptake
desipramine
rate.
Treatment
or of the
589
Platelet and synaptosomal uptakeinOB rats
sham operated
rats with
There
uptake.
sertraline
the OB and sham operated on Km values resulted (Table
also resulted
was no difference
in either
in a raised
Treatment
groups.
group,
in an increased
in the affinity
(Km) of uptake
with
desipramine
but the three weeks
Km for uptake
sertraline
rate of between
had no effect treatment
in both the OB and sham operated
groups
2). Table
Effect
2
of Desipramine and Sertraline on the Uptake of 3H-5HT Platelets of OB and Sham Operated Rats.
into
____________-___-_-_____-_________________________________________________ Animals +Saline +Desipramine +Sertraline ___________________________________________________________~___~___~___~__ 0 Vmax 119.6 -+ 9.58' 96.7 + 6.42 40.9 2 3.97' OB Km 106.7 + 20.25 80.8 + 15.19 521.5 + 140.51*' ________-___-_______~~~~~~~~--~--~~~~~~~-~-~~~--~~~-~~~--~~--~~--~~~~~~~~~ Vmax 100.0 -+ 3.13. 120.2 + 13.64 166.0 -+ 4.041 Sham Km 100.0 + 17.70 96.2 + 20.25 169.6 + 24.05" ____________________--_______--_________-________________________________ Results expressed as % sham + saline * p < 0.01 vs sham + saline; l p < 0.01 vs OB + saline; *o p < 0.01 vs sham + saline, p < 0.01 vs OB + saline Sham control Vmax = 15.1 A 0.47 pmoles/mg protein Sham control Km = 0.5 + 0.08 pM. Synaptosomal which
5HT uptake
was inhibited
by in vitro
(p < 0.001)
and normalised
rats showed
no change
showed
a raised
group,
after
in either
after
sertraline,
in uptake
Vmax after
which
treatment. treatment
There
with
of 5HT (0.01-0.5
was raised
drug
treatment.
desipramine
Sham operated The OB group
in the sham
was no effect
PM),
in the OB group
treatment.
was not apparent
Table Tffect H-5HT
uptake
by the antidepressant
Km of uptake,
sertraline
group
: The high-affinity
operated
on the Km of uptake
( Table 3).
3
of Desipramine and Sertraline on the Hi gh Affinity Uptake of into Synaptosomes of Bulbectomised and Sham Operated Rats
______-_____________~~~~~~~~_~~~-_~~~~--~~~-~~~~~~~~~~~~~~-~~~_~~-_~~~__~Animals +Saline +Desipramine +Sertraline _____-__~___~______~_---__-~-~_~~~_____-~~~~~~~~~~~~~~~~_~~~_~~~-~~__~____-_ Vmax 82.7 ) 7.72' 111.8 -+ 49.25' 259.9 + 47.57" OB Km 89.0 + 30.91 95.5 + 10.91 158.2 + 36.40' -________-_______-__-~-~-~~~~~-~~~~~------~~~~~~~~_~~~~~~~_~~~_~~~__~_~_~_ Vmax 100.0 -+ 25.53' 80.2 ) 5.27 67.9 -+ 28.8" Sham _f__""___________l"~~~_~_~~~~~_______"ll_~~~~~__ Results expressed as % sham + saline * p < 0.001 vs sham + saline; l p < 0.01 vs OB + saline Sham control Vmax = 35.7 _c 9.13 pmoles/mg protein Sham control Km q 0.8 + 0.43 PM.
590
J.Butler etal.
At the higher uptake
concentration
was increased
normalised
by three weeks
did not reach
saturation
not be analysed sertraline
5HT.
(Table
compared with
non-linear
Treatment
either
drug.
range
regression,
'low-affinity'
of animals
for
(p < 0.011,
This uptake,
used and hence
rats with
synaptosomal
by Lineweaver-Burk after
the Vmax
treatment
could by
desipramine
uptake
analysis,
with
and
which
was not inhibited
of the sham operated
the
as estimated
in both groups
PM),
to controls
in the concentration
did not effect
The Km of uptake,
unchanged
of 5HT (0.1-2.0
treatment
by weighted
in vitro.
or sertraline
range
in the OB group
either
of
was drug
4) Table
Effecs
4
of Desipramine and Sertraline on the Low Affinity Uptake H-5HT into Synaptosomes of OB and Sham Operated Rats
of
_____________________~~-~____~~~___~~~~~~~~-~~~~~--~~~~~~-~~~~~-~~~~~--~~~ Animals +Saline +Desipramine +Sertraline _______________________~~____~~~____~-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-~~~ 104.6 2 21.55. 104.9 L 13.78' Vmax 232.0 + 15.51" OB 91.0 2 20.86 110.6 -+ 12.21 Km 89.1 2 13.10 _________________________________________~~-~~-~~~~~~-~~~~~~~~-~~~~~~~~_________ 100.0 -+ 10.73. Vmax 115.0 + 10.28 99.8 -+ 13.81 Sham Km 100.0 2 13.91 82.4 2 9.25 87.5 -+ il.80 ____________________~~~~~~~~~~-~~~~~~-~~----~~-----~~~----~~~---~~~~~~-~~~ Results expressed as % sham + saline * p < 0.01 vs sham + saline; l p < 0.001 vs sham + saline Sham control Vmax q 266 _c 37 pmoles/mg protein Sham control Km = 4.3 5 0.24 PM. Discussion The olfactory
bulbectomised
OB rats have been used (Earley matter
& Leonard,
a reliable,
(Leonard
shown
those
in which
corrects
& Leonard, several
& Leonard,
It was in an attempt
1986).
it was concluded changes
(vanRiezen
1983) chronic,
of these
for new antidepressants
see if there was an abnormality the OB rat and if so, whether
that occur patient.
it is a or merely
of novel
of a short
review
that the physiological, following
Furthermore,
et al, 1977
but not acute,
behavioural
to extend
However,
of depression
test for the detection
in the depressed
here and elsewhere
Janscgr
in the search
this has been the subject
and behavioural seen
of depression
the OB rat is a model
screening
Recently,
et al, 1987)
neurochemical resemble
whether
if tedious,
antidepressants.
as a model
; O'Connor
1985
of conjecture
rat as a model
; Cairncross
bulbectomy as has been et al, 1979
antidepressant
treatment
deficits.
our previous
studies
in the transport this could
that we decided
of 5HT into platelets
be normalised
by chronic
to of
;
Platelet and synaptosomal uptakeinOB rats
antidepressant (Healy
It has been established
treatment.
; Butler
et al, 1983,1985
the platelets effective
et al, 1980)
; Coppen
et al, 1979
of depressed
that the defective
patients
is corrected
5HT uptake
The results particular
of this study
that at least
antidepressants
associated
with
a return
normalises
It is of interest
patients
et al, 1985) appears
specificity
uptake
The increased
sites
Km of uptake
competitive
seen with
the noradrenaline
of 5HT into
Synaptosomal
5HT uptake
The nature
but the platelet
synaptosome
of the same
increased contrast
antidepressants Two components It is also synaptosomes
rate being
system.
to synaptosomal
sites
(Kimelberg,
study
has indicated
inhibited
in vitro
found
which
is not
those
depression
attention
into both suggest
occuring
because
and
that changes
of 5HT
in central
were
of
In the present
the platelet
in the synaptosomal
rates
is
neurons,
fractions
In both normalised
an
in
platelets by
deficit.
5HT uptake that
the transport
on both a low and a high have reported
the transport
that
treatment
by sertraline,
et al, 1968).
the behavioural
1985) while
19861,
sertraline
rate in the platelets.
investigations
may involve
(eg.
is as yet unclear.
desipramine.
The results
from this study
is dependent
& Singhal,
of the
or noradrenergic
considerable
the 5HT uptake
that corrected
Previous
(Ahluwalia
uptake
however,
apparent
was
of the presumed
underlying
in 5HT transport
animals.
to a decreased
and synaptosomes,
chronic
(Pletscher
do not reflect
5HT uptake
the effect
mechanism
lesion(s)
neurons
changes
into platelets
; this effect
in both OB rats and depressed
inhibitor,
has received
to central
we examined
two
of the OB rats to
(eg. sertraline)
with
the
with
in the OB rat
of the neurochemical
its similarity
Vmax
of 5HT uptake
uptake
to this
treatment
to be independent
associated
inhibition
regard
rate
activity
and the precise
indicates
unclear,
5HT uptake
of the drug on the 5HT
desipramine)
the uptake
to find that
on platelet
(Healy
with
that chronic
of the locomotor
antidepressants
systems
transport
by therapeutically
the OB rat resembles
function,
We have also shown
patient.
values.
show
of platelet
dissimilar
uptake
(Toumisto
in the OB rat
'marker'
depressed
study,
laboratories
and elsewhere
treatment.
Platelet
control
in these
1986)
& Leonard,
591
others
for this
the low affinity
evidence
for such a system that one of these
is
(i.e. glial)
slight.
5HT uptake
5HT uptake
transport
suggested
of 5HT into non-neuronal
but evidence
by the specific
have
of 5HT into
affinity
system
inhibitor,
The present is not
sertraline.
This
J.Butler etal.
592
suggests
that the low affinity
aminergic activity
neurons. of both
the OB rat than sites
the low and high affinity
of other
control
by antidepressant
to studying
transport
be associated
it is clear
its sham operated
is normalised
devoted
site might
Nevertheless,
the effects biogenic
transport
site
that the
is greater
and that the activity
treatment.
of various
amine
with catechol-
from this study
Further
studies
antidepressants
neurotransmitters
in
of both will
be
on the
into synaptosomes
of
OB rats.
Conclusions It may be concluded depressant
treatment
depressed
patients
as a model showing
that the similarity on defective
and OB rats
of depression.
a biochemical
profile
is interesting
in this study
antidepressant
treatment,
putative
appear
evidence
is the first
similar
report
in favour
could
anti-
be used
model
patients.
of normal as a model
of
of the OB rat
of an animal
to that of depressed
is the restoration
which
of chronic
of 5HT into the platelets
function
What
with
for screening
antidepressants.
Despite neurone
is further
This
in the effect
transport
the well
regarding that there
the nature
established the nature
similarities of receptors,
are also marked
of the 5HT transport
between
the platelet
transport
differences
between
sites
and the
etc.,
them with
it would regard
to
sites.
Acknowledgement J.B.
is a post-doctoral
Ireland. used
The authors
in this study
fellow
thank
sponsored
Pfizer
by the Health
UK Ltd. for supplying
and for financial
assistance
towards
Research
Board
of
the sertraline the cost of the
project. References AHLUWALIA, P. & SINGHAL, R.L. (1985) Kinetics of the uptake into synaptosomes from rat brain : Consequences of lithium withdrawal. Neuropharmacol. 4, 713-720.
of monoamines treatment and
BUTLER, J. & LEONARD, B.E. (1986) Post-partum depression and the effect of nomifensine treatment. Int. Clin. Psychopharmacol. 1, 244-252. CAIRNCROSS, K.D., COX, B., FORSTER, C. & WREN, A.F. (1977) The olfactory bulbectomised rat : A simple model for detecting drugs with antidepressant potential. Br. J. Pharmacol. 61, l-497. CAIRNCROSS, K.D., COX, B., FORSTER, C. & WREN, A.F. (1978) A new model for the detection of antidepressant drugs : Olfactory bulbectomy in the rat compared with existing models. J. Pharmacol. Methods l_, 131-143.
593
Platelet and synaptosomal uptakeinOB rats
CAIRNCROSS, K.D., COX, B., FORSTER, C. & WREN, A.F. (1979) Olfactory Psychoendocrinol. projection system, drugs and behaviour : A review. 253-272. Platelet 5HT accumulation COPPEN, A., SWADE, C. & WOOD, K. (1978) depressive illness. Clin. Chim. Acta, 87, 165-168.
5,
in
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