Pergamon

0306-4522(94)E0133-0
Neuroscience Vol. 60, No. 4, pp. 837-841, 1994
Elsevier Science Ltd
Copyright 0 1994 IBRO
Printed in Great Britain. All rights reserved
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Letter to ~e~fosciewe
CHRONIC INT~STRIATAL QUINOLINIC ACID
PRODUCES REVERSIBLE CHANGES IN PERIKARYAL
CALBINDIN AND PARVALBUMIN XMMUNOREACTIVITY
T. J. BAZZETT,*~$ J. B. BECKER,P$II R. C. FALIK and R. L. ALBIN*//
*Department of Neurology, @Department of Psychology, TReproductive Sciences Program,
IlNeuroscience Program, The University of Michigan, 1103 E. Huron Street, Ann Arbor,
MI 48104-1687, U.S.A.
We recently reported the use of a chronic dialytic
delivery system for intrastriatal administration of
quinolinic acid in the rat. This system produces
~~~e~ration with some characteristics simihtr to
posf ntortem brain tissue from Hun~ons disease
patients, including reduced cytochrome oxidase stain-
ing, a decreased number of Nissl-stained neurons, and
relative sparing of striatal NADPH-diaphorase con-
taining neurons. The present findings show that
chronic dialytic delivery of q~~g~c acid also pro-
duces a Huntingtons disease-like pattern of reduced
caibindin and parvalbumin perikaryal hnmunoreactiv-
ity that is reversed in rats allowed four to eight weeks
recovery after cessation of quinolinic acid. Further-
more, cytochrome oxidase staining and the number of
Nissl-stained cells were unhand in the region of
transient calbindin and parvalbumiu immunoreactive
perikaryal staining alterations. These results suggest
that changes in calbindin and parvalbumin perikaryal
immunoreactivity provide a relatively sensitive measure
of quinolitic acid induced ~urotoxici~. The revers-
ible nature of reduced perikaryal immu~reactivity
suggests a premorbid state of neurotoxicity, possibly
marked by cellular redistribution of calbindin and
parvalbumin.
Quinolinic acid acts as an agonist at N-methyl-D-
aspartate (NMDA) receptors, causing an increase
in intracellular calcium concentration.20 To maintain
intracellular ionic equilibrium, calcium can be se-
questered by intracellular organelles or buffered by
calcium binding proteins. I3 Excessive calcium influx
through the NMDA receptor complex results in
excitotoxicity. 10*5~2*22 Cells containing the calcium
fTo whom correspondence. should be addressed at the
Department of Ne~ro~o~/Neuro~ience Laboratory
Building.
Abbreviations: HD, Huntingtons disease; NMDA, N-
methyl-o-aspartate.
buffering protein, calbindin, are reduced in post
mortem striatal tissue from all grades of Huntingtons
disease (HD).13,19 Cells containing parvalbumin,
another calcium buffering protein, appear more re-
sistant and are reduced only in tissue from advanced
HDe9
In a previous report, Nissl staining showed that
chronic intrastriatal dialytic delivery of 4 mM quino-
linic acid had no effect on neuronal density, while
15 mM quinolini~ acid produced a decrease in neur-
onal density that was restricted to an area approxi-
mately 400 pm radial to the dialysis probe in the rat.4
In the present experiments, quantitation of Nissl-
stained neuron density revealed findings identical to
the previous study for both 4 and 15 mM concen-
trations of quinolinic acid. However, as shown
in Fig. 1, perikaryal calbindin and parvalbumin
immunoreactivity was significantly reduced through-
out a region 400pm radial to the dialysis probe in
animals receiving 4 mM quinolinic acid (calbindin:
control 170 + 9.55 vs quinolinic acid 73.3 _t 11.19,
P < 0.001; parvalbumin: control 24.25 -+ 2.33 vs
quinolinic acid 14.3 + 2.68, P < 0.005; mean number
of perikarya _+ S.E.M., two-tailed Students paired
t-test). Animals that received 15 mM quinolinic
acid showed reduced calbindin and parvalbumin
perikaryal immunoreacti~ty throughout most of the
striatum (Table 1, Fig. 2).
There was a dose-dependent effect of quinolinic
acid on cytochrome oxidase staining. Analysis of
brain selections containing the dialysis probe tract
revealed that 15 mM quinolinic acid produced a
significantly greater area of decreased staining than
did 4 mM quinolinic acid (Veh 12.8 f 2.53 mm2 vs
4mM 27.1 f l.22mm2, meanfS.E.M., P ~0.001;
4mM 27.1 -+ 1.22mm2 vs 15mM 56.1 j-5.91 mm2,
P < 0.001; Students unpaired t-test). For both doses,
the decrease in perikaryal calbindin and parvalbumin
immunoreactivity was apparent well beyond the area
of decreased cytochrome oxidase staining.
837
838
T.J. BAZZETT et al.
Fig. 1. Data from Experiment 1 showed that 21 days of 4 mM quinolinic acid produced a significant
decrease in cdlbindin and parvalbumin immunoreactive perikarya compared to control striata. Detailed
methods for the dialytic delivery system are described elsewhere. Perikarya were analysed in a
I .02 x 10 - mm volume of striatal tissue 400 pm radial from the necrotic core or a homologous region
in the contralateral (control) striatum of t 2 SpragueDawley rats (Harlan Sprague-Dawiey, Inc.
Indianapolis, IN). In the control striatum calbindin (A) and parvalbum~n (C) immunoreactive staining
is normal. Exposure to 4 mM quinolinic acid for 21 days produced a significant decrease in calbindin (El)
and parvalbumin (D) immunoreactive perikarya and an increase in neuropil staining. Calbindin and
parvalbumin antibodies (Sigma) were diluted 1: 2000 and immunohistochemical assays performed using
Vectastain mouse IgG Elite ARC kit (Vector Labs, Inc.) according to manufacturers instructions. There
was no significant difference in the number of nissl-stained cells (not shown) in control (186.5 + 10.46)
vs quinolinic acid-exposed (160.3 f 14.82) striata. Scale bar = IOOpm.
Table 1, Combined data from Experiment 2 showing a relatively rapid and sustained loss, and
Experiment 3 showing the subsequent recovery, of calbindin and parvalbumin immunoreactive
perikaryal staining in response to 15 mM quinolinic acid
Calbindin Parvalbumin
Quinolinic acid Control Qu~noli~c acid Control
Time course
5 days (n = 6) 84.0 f 16.8** 246.0 + 15.4 9.2 + 2.4** 25.8 + 2.1
10 days (n = 4) 48.0 k 24.4* 212.1 + 26.1 14.4 f. 5.5 25.0 + 2.5
15 days (n = 4) 73.8 & 19.1* 251.0 +44.4 15.7 + 0.9* 29.3 + 2.4
21 days (n = 7) 65.4 F 23.3** 246.6 & 24.6 II.5 * 2.0* 19.0 + 0.8
Recovered
4 weeks (n = 5) 162.4 f 20.1 209.6 k 30.5 15.8 + 1.2 16.2 + 1.8
8 weeks (n = 4) 118.3 k 33.9 193.3 + 36.3 18.0 + 1.8 19.8 & 3.3
Perikarya were analysed in a 1.02 x 10 _I mm3 volume of tissue from the ventral lateral
striatum approximately 1 mm caudal from the necrotic core. Two-tailed paired Students
t-tests were used to determine significant changes in the number of calbindin and
parvaibu~n ~mmunor~c~ve perikarya between control and quinoIinic acid exposed striata
(*P < 0.05 **P < 0.005).
Cresyl Violet stained sections were also analysed in animals receiving 21 days in = 7)
administration. There was no significant difference in the number of Nissl-stained CdlS
in control (110.4 + 4.58) vs quinolinic acid exposed (98.3 & 10.45) striata.
Fig. 2. Representative sections
._ __ . . ._ . _.
compare posterior lateral striatal tissue after: 21 days vehicle, 2f days
I5 mM qumolmic actd, and 21 days IS mM quinolinic acid plus four weeks recovery. Cresyl Violet
staining was similar across the three groups. Calbindin and parvalbumin immunoreactive perikarya
decreased after 21 days 21 mM quinolinic acid, but not different after 15 mM quinotinic acid plus four
weeks recovery, when compared to vehicle. Cresyl Violet staining after 21 days vehicle (A), 21 days
quinoiin~c acid (B), 21 days quinolinjc acid + four weeks of recovery (C). Calbindin immunoreactive
staining 21 days vehicle (D), 21 days quinolinic acid (E), 21 days quinolinic acid + four weeks recovery
(F). Parvalbumin immunoreactive staining 21 days vehicle (G), 21 days quinolinic acid (H) 21 days
quinohnic acid f four weeks recovery (I). cc, Corpus cailosum. *Blood vessel provides a landmark
for comparison of consecutive 40-ym sections from a brain exposed to 21 days quinolinic acid.
Chronic intrastriatal quinolinic acid
839
Scale bar = 100 urn.
A time course evaluation of animals receiving and is sustained throughout the 21 day course of
15 mM quinolinic acid showed that the decrease quinolinic acid delivery (Table 1).
in calbindin and parvalbumin perikaryal immuno- The ventrolateral region of the caudal striatum
reactivity occurs relatively rapidly (within five days), showed no change in the density of Nissl-stained cells
840 T. J. BAZZETT et al.
but decreased perikaryal calbindin and parvalbumin
immunoreactivity after 21 days of 15 mM quinolinic
acid compared to vehicle administration. If animals
were allowed to survive either four or eight weeks
after cessation of quinolinic acid administration, the
number of immunoreactive calbindin and parvalbu-
min perikarya in this brain region was not different
from the vehicle-treated striatum (Table 1, Fig. 2).
The number of calbindin and parvalbumin immuno-
reactive perikarya were not different between four-
and eight-week animals (Table 1).
The present results show that chronic dialytic
delivery of quinolinic acid can decrease the number of
calbindin and parvalbumin immunoreactive peri-
karya. The reversible nature of this effect suggests a
change in production or distribution of these intra-
cellular proteins. This hypothesis is further supported
by the findings that there is no decrease in cyto-
chrome oxidase staining or in the number of Nissl-
stained cells in regions exhibiting transient changes in
calcium binding proteins.
Analysis of striata exposed to chronic dialytic
delivery of quinolinic acid reveals three primary
regions of specific neuronal reactions. The first re-
gion, a central necrotic core directly adjacent to the
probe tract, is marked by inflammatory infiltrate and
a lack of neurons. The second region, previously
referred to as the transition zone,4s5 is marked by an
absence of perikaryal staining for parvalbumin and
calbindin, a reduction in the number of Nissl-stained
cells and a decrease in cytochrome oxidase staining.
Though many Nissl-stained neurons in the transition
zone appear structurally intact, a decrease in cyto-
chrome oxidase staining suggests metabolic impair-
ment. Beyond the transition zone is the third region,
marked only by a decrease in perikaryal calbindin
and parvalbumin immunoreactivity. However, unlike
the quinolinic acid-induced decrease in cytochrome
oxidase and Nissl cell staining in the transition
zone, decreased calbindin and parvalbumin staining
in the region outside the transition zone is in part
reversible.
There was also an increase in calbindin and parv-
albumin neuropil staining throughout the striatum
with both doses of quinolinic acid, and at all time
points after 15 mM quinolinic acid delivery. This
increase was also apparent after acute quinolinic acid
injections,23 and may have resulted from immuno-
histochemical neuropil staining caused by immuno-
globulin extravasation.
There are at least two explanations for the transient
decrease in perikaryal calbindin and parvalbumin
immunoreactivity following quinolinic acid adminis-
tration. First, it is possible that some cells were
excluded from analysis because they were not
distinguished from increased neuropil staining.
However, it is unlikely that a significant number of
cells were excluded since the increase in neuropil
staining at no time exceeded the intensity of staining
seen in the vast majority of perikarya from the
homologous region of the control striatum; and
some immunoreactive neurons could be clearly seen
against the increased background on the lesion side
(Figs 1, 2).
Alternatively, the transient decrease in perikaryal
calbindin and parvalbumin immunoreactivity may be
caused by a change in distribution of these proteins
within neurons. When stimulated, NMDA receptors
allow an influx of calcium into dendritic processes
where they are concentrated.6s22 Dendritic processes,
which are capable of independent regulation of
changes in intracellular calcium,8~14 could recruit
calcium buffering proteins. Calbindin exhibits charac-
teristics of a fast mobile calcium buffering protein,
capable of diffusing from surrounding cytoplasm to
local sites of calcium influx. Ferrante and col-
leagues found the intensity of calbindin immuno-
reactivity shifts to distal dendritic arbors in HD
striatal tissue. A similar calbindin and parvalbumin
shift in response to quinolinic acid could explain the
observations of decreased perikaryal staining and
increased neuropil staining.
It is also interesting to note that two weeks
after acute intrastriatal quinolinic acid injection,
levels of somatostatin and the glutamic acid decar-
boxylase isoforms GAD65 and GAD67 were de-
creased,16 whereas in a similar paradigm calbindin
levels were unchanged. It is possible that exposure
to quinolinic acid may produce long-term effects on
production of some cellular components, while pro-
ducing only temporary changes in production or
distribution of others.
Calbindin-containing neurons appear less resist-
ant than parvalbumin neurons to the neurodegen-
erative effects of HD.9s9 The present results reveal
that chronic quinolinic acid administration also re-
duces a greater percentage of calbindin- compared
to parvalbumin-immunoreactive perikarya (Table 1).
These results strengthen the contention that the pat-
tern of neuronal vulnerability in HD is shared by stri-
atal tissue exposed to chronic NMDA agonist
administration.
The importance of assessing physiological neur-
onal states that precede cell death associated with
neurodegenerative disorders has recently been dis-
cussed.* In the present report, reversible quinolinic
acid induced changes in calbindin and parvalbumin
immunoreactivity may represent one such premorbid
neuronal state. Further assessment of these changes
may offer new insight into the neurodegenerative
processes of HD.
Acknowledgements-This work was supported by NSO0130,
NS19613 and NS22157. Dr Bazzett was supported by
training arants from The UM Reproductive Sciences
Program -(HDO7048) and The UM Department of
Neuroloev (NS07222-12). The authors would like to thank
Kevin <;a&, Brady Bustany and Elizabeth Vitarbo for
their contributions to this project. We thank one of the
anonymous reviewers for constructive criticism.
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Chronic intrastriatal quinolinic acid
841
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(Accepted 7 March 1994)