Olfactory bulbectomy reduces cerebral glucose utilization: 2-[14C]deoxyglucose autoradiographic study.
ABSTRACT The olfactory bulbectomized (OBX) rat is an extensively investigated animal model of depression. In the present study the effects of olfactory bulbectomy in drug-naive adult male Sprague-Dawley rats (200-240 g) on global (gCGU) and regional cerebral glucose (rCGU) utilization was evaluated. Two weeks following surgery, the autoradiographic measurement of CGU using [14C]-2-deoxyglucose was employed. The levels of CGU in the OBX and sham-operated rats were compared in 40 brain regions. Statistical methods indicate significantly lower levels of global (overall) CGU in the OBX group than in the sham group. Discriminant analysis was done on the z-scores to remove animal to animal variability. The following thirteen regions were identified by the stepwise discriminant analysis of the z-scores as significantly contributing to the differences between the sham and OBX: amygdala, cingulate cortex, caudate putamen at the level of globus pallidus, caudate putamen-lateral part, dorsal subiculum, dorsal thalamus, hypothalamus, median raphe, somatosensory cortex, substantia nigra, ventral hippocampus, ventral tegmental area and the ventral thalamus. The pattern of changes in the rCGU following OBX does not completely correlate with the pattern of connectivity of the olfactory bulbs, however, many regions with direct connection to the olfactory bulbs (e.g., amygdala, hypothalamus, ventral hippocampus, and ventral tegmental area) were found to be important for differentiation. No left to right asymmetries in the rCGU were found. The data suggest that there are very important regional differences in glucose utilization between the OBX and sham operated rats, which points to the need to study antidepressants in an animal model of depression rather than in normal animals.
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ABSTRACT: Local cerebral glucose utilization was measured by the [14C]-deoxyglucose method in five near-term fetal sheep in whom bilateral ablation of the cochleae had been accomplished aseptically 5 to 8 days earlier. The tympanic membrane and ossicles were removed and all turns of each cochlea were unroofed with destruction carried to the modiolus. Mean local cerebral glucose utilization of 33 of 34 gray matter structures and four of four white matter structures in operated animals were significantly lower (p less than 0.05) than that in unoperated control fetuses. The depression in local cerebral glucose utilization was greatest (p less than 0.002) in brain stem auditory nuclei, in which the mean rate of glucose utilization was approximately 25% of the levels in unoperated fetuses. The pattern of glucose utilization in these structures was clearly altered, with a reversal of the normal distribution in density of the inferior colliculus. Tonotopic bands of high local cerebral glucose utilization frequently seen in autoradiographs of inferior colliculus in unoperated fetuses were not observed in operated fetuses. These results show that the glucose utilization of the brain, and by implication the normal growth and maturation of the brain, depends on an intact auditory system during prenatal life.American Journal of Obstetrics and Gynecology 01/1988; 157(6):1438-42. · 3.88 Impact Factor
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ABSTRACT: (1) Bilateral olfactory bulbectomy (OB) produces a series of behavioural changes in the rat, characterised by hyperactivity and hyper-reactivity. It also produces an elevation of plasma 11-hydroxycorticosterone (11-OHCS). (2) Chronic treatment (10–14 days) of OB rats with anti-depressant drugs (amitriptyline, mianserin, viloxazine, nomifensine and iprindole) normalises the behavioural change and the 11-OHCS elevation. The mono-amine oxidase inhibitor, tranylcypromine, does not normalise these parameters. (3) Using two behavioural parameters (step-down avoidance testing and irritability), plus 11-OHCS measurements, it has proved possible to differentiate between anti-depressants, anti-anxiety agents, tranquilisers and central stimulants. OB therefore has potential as a screening method for differentiating between various classes of psychotropic drugs. (4) The quadracyclic anti-depressant, mianserin, proved negative in standard anti-depressant screening and a new derivative, GB-6582 proved positive in the OB model. GB-6582 is an efficient inhibitor of 5-hydroxytryptamine (5-HT) uptake. (5) The neurotoxin, 5–6 DHT, will mimic the behavioural and physiological effects of bulbectomy as will 5–7 DHT with prior treatment of the rat with desmethyl-imipramine, when these neurotoxins are injected directly into the olfactory bulb. The neurotoxin, 6-OHDA, injected into the bulb does not mimic the effects of OB. (6) GB-6582 when injected into the bulb prior to intra-bulbar 5–6 DHT, prevents the appearance of the behavioural and physiological effects of bulbectomy. (7) All the anti-depressants which normalise surgical bulbectomy, show the same effect with chemical bulbectomy induced by 5–6 DHT. (8) Following bulbectomy, neuroanatomical studies demonstrated degenerating fibres in: (a) the anterior hippocampus; (b) the corticomedial nucleii of the amygdala; (c) the bed nucleus of the stria terminalis and (d) the pre-optic area of the hypothalamus. (9) These brain areas are concerned with behavioural arousal, hypothalamic-adrenohypophyseal modulation and sexual behaviour. All these are adversely affected by bulbectomy. (10) The significance of these observations is discussed in terms of 5-HT-catecholamine interaction in the development of diseases of affect.Psychoneuroendocrinology 08/1979; 4(3):253-72. · 5.14 Impact Factor
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ABSTRACT: Following an olfactory bulb lesion in guinea pig (2 to 3 days), neuronal degeneration occurs in several olfactory-bulb-related areas, primarily in the piriform cortex. The degenerating neurons, which are argyrophilic, are also found in the posterolateral cortical amygdaloid nucleus and the ventrolateral entorhinal cortex. It is suggested that the neurons degenerate because of a transneuronal effect due to a sudden loss of afferent input from the olfactory bulb, although a retrograde effect acting in concert with transneuronal factors cannot be excluded. Terminal degeneration can be identified in several areas outside the olfactory bulb projection area, and is interpreted as degeneration in the axons of the degenerating cortical neurons. Such terminal degeneration, which is best seen 3 to 4 days postoperatively, has been identified in part of the basolateral amygdaloid complex, in the basomedial amygdaloid nucleus, and in the temporal parts of the fascia dentata of the hippocampal formation. Terminal degeneration has also been observed in the deep layers of the anterior olfactory nucleus, the olfactory tubercle, the nucleus of the lateral olfactory tract, and the anterior amygdaloid area. All these projections, apparently, represent the second link in two-neuron pathways, where mitral or tufted cells in the olfactory bulb make up the first neuron. This interpretation was confirmed in control experiments in which areas of argyrophilic neurons coincided with the location of retrogradely labeled neurons following injection of fluorescent substances into several of the above-mentioned areas of terminal degeneration.The Journal of Comparative Neurology 07/1982; 208(2):196-208. · 3.66 Impact Factor
Brain Research Bulletin 76 (2008) 485–492
Olfactory bulbectomy reduces cerebral glucose utilization:
2-[14C]deoxyglucose autoradiographic study?
Ivan Skelina, Hiroki Satoa,1, Mirko Diksica,b,∗
aCone Neurological Research laboratory, Department of Neurology and Neurosurgery, McGill University, Montreal, QC, Canada
bFaculty of Medicine, The J.J. Strossmayer University, Osijek, Croatia
Received 27 November 2007; received in revised form 18 January 2008; accepted 22 January 2008
Available online 20 February 2008
The olfactory bulbectomized (OBX) rat is an extensively investigated animal model of depression. In the present study the effects of olfactory
bulbectomy in drug-naive adult male Sprague–Dawley rats (200–240g) on global (gCGU) and regional cerebral glucose (rCGU) utilization was
evaluated. Two weeks following surgery, the autoradiographic measurement of CGU using [14C]-2-deoxyglucose was employed. The levels of
CGU in the OBX and sham-operated rats were compared in 40 brain regions. Statistical methods indicate significantly lower levels of global
(overall) CGU in the OBX group than in the sham group. Discriminant analysis was done on the z-scores to remove animal to animal variability.
The following thirteen regions were identified by the stepwise discriminant analysis of the z-scores as significantly contributing to the differences
between the sham and OBX: amygdala, cingulate cortex, caudate putamen at the level of globus pallidus, caudate putamen-lateral part, dorsal
subiculum, dorsal thalamus, hypothalamus, median raphe, somatosensory cortex, substantia nigra, ventral hippocampus, ventral tegmental area
and the ventral thalamus. The pattern of changes in the rCGU following OBX does not completely correlate with the pattern of connectivity of
the olfactory bulbs, however, many regions with direct connection to the olfactory bulbs (e.g., amygdala, hypothalamus, ventral hippocampus, and
ventral tegmental area) were found to be important for differentiation. No left to right asymmetries in the rCGU were found. The data suggest that
there are very important regional differences in glucose utilization between the OBX and sham operated rats, which points to the need to study
antidepressants in an animal model of depression rather than in normal animals.
© 2008 Elsevier Inc. All rights reserved.
Keywords: Model of depression; Olfactory bulbectomy; Cerebral glucose utilization; Autoradiography; [14C]-2-deoxyglucose
The olfactory bulbectomized (OBX) rat is an animal model
of depression induced by the removal of the olfactory bulbs
Abbreviations: OBX, olfactory bulbectomy; OB, olfactory bulbs; 2DG,
2-deoxyglucose; rCGU, regional cerebral glucose utilization; PET, positron
emission tomography; 5-HT, serotonin; 5-HIAA, 5-hydroxyindolacetic
acid; 5-HTT, serotonin transporter; 5,7-DHT, 5,7-dihydroxytryptamine; NA,
noradrenaline; DA, dopamine; NMDA, N-methyl-d-aspartic acid; GABA, ?-
?This work was presented, in part, at the 2006 Society for Neuroscience
Annual Meeting in Atlanta, GA Oct 13–17 2006.
∗Corresponding author at: Montreal Neurological Institute, McGill Univer-
sity, 3801 University Street, Montreal, QC H3A2B4, Canada.
Tel.: +1 514 398 8526; fax: +1 514 398 8195.
E-mail address: Mirko.firstname.lastname@example.org (M. Diksic).
1Permanent address: Department of Neurosurgery, University of Yamanashi,
1110 Shimokato Tamaho-cho, Nakakoma-gun, Yamanashi 409-3898, Japan.
. It has been observed that the OBX syndrome develops
2 weeks post-surgery. Neurochemical disturbances in several
neurotransmitter systems have been found in OBX rats, such as
decreased noradrenaline (NA) brain levels , altered tissue
concentration of the 5-HT (5-hydroxytriptamine), 5-HIAA (5-
in density of the GABA-A and GABA-B receptors , and
decreased density of the muscarinic receptors . At the behav-
ioral level, its main features are hyperactivity in the open field
(OF) test, irritability, aggression, decreased habituation, and
deficiencies in spatial memory and aversive learning (review
). Apart from chronic mild stress , OBX is the only
animal model of depression whose pathological characteristics
are normalized following chronic, but not acute, antidepressant
therapy. Also, many psychotropic drugs, other than antidepres-
sants, have no effect on the normalization of OBX-induced
changes . Animal models of MDD (major depressive dis-
order) should have three different distinct values : (1) face
0361-9230/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
I. Skelin et al. / Brain Research Bulletin 76 (2008) 485–492
validity (how closely the model resembles the psychiatric con-
dition); (2) construct validity (consistency of the model with the
action of the drugs in the model resemble the actions of these
drugs in the human disease). The OBX rat model of depression
possesses all of these three values , with a relatively large
number of similarities to the human disease. However, we still
do not have a good understanding of the relationship between
model and human depression.
since temporary peripheral anosmia did not result in hyperac-
tivity . It is likely that the OBX syndrome is a consequence
of anterograde and retrograde neuronal degeneration, as well as
the non-physiological circuitry that follows the removal of the
Autoradiographic measurement of cerebral glucose utiliza-
tion (CGU) using [14C]-labeled 2-deoxyglucose (2DG) has a
relatively good resolution (about 0.1mm). CGU represents
total energy use by the brain, but the elevation in electrical
activity of a cell results in a larger increase of metabolism in
the terminals than in the cell itself . As the brain neuronal
transmission, in addition to other processes, require energy (and
therefore require glucose), and because systematic studies of
glucose utilization in the OBX rat model of depression have
not been performed, it was deemed important to investigate this
aspect of the brain metabolic activity.
it is still not clear which brain regions or pathways are critical
for the development of OBX syndrome and how their dysfunc-
tion in OBX parallels the dysfunction of analogue structures in
human depression. There is also no clear understanding which
of the regional metabolic changes occur in the brain of OBX
rats during the development of the OBX syndrome. Therefore,
cose utilization (rCGU) in OBX rats and compare it with that
in sham-operated rats. This is the first in vivo study of rCGU
in OBX rats using a relatively good anatomical resolution (with
autoradiography) in which measurements were done bilaterally
in forty brain regions.
2. Materials and methods
Sprague–Dawley male rats (Charles River, St. Constant, Quebec, Canada),
and Sham). Under 2% isoflurane anesthesia, a hole was made in the frontal
bone, with a posterior edge of 5.2mm from the Bregma. Olfactory bulbs were
cut using the microscissors and aspirated by the vacuum pump, taking care not
to damage the frontal cortex. The sham procedure was identical except that the
was introduced to the dead space to contain the blood loss. As an analgesic, the
animals were given 0.03mg/kg of buprenorphine s.c., and the 2%-xylocaine
gel was applied to the suture on the skin. Following the brain extraction, the
specimens were checked for a complete removal of the olfactory bulbs and for
any possible damage to the frontal cortex. If the bulbs were not completely
removed or if there was damage to the frontal cortex, the brains were not further
processed. From extensive experience, this generally occurs in less than 2% of
the animals. The animals were kept for 2 weeks to recover from the surgery and
to develop the OBX syndrome. They were housed two per cage and monitored
closely for any sign of distress. All of these procedures were approved by the
Animal Care and Use Committee of the Montreal Neurological Institute and
McGill University and were done according to the procedures of the Canadian
Council on Animal Care.
2.2. Tracer experiment
After the 2-week period, the animals were fasted overnight, but given water
ad libitum. This was done to achieve the stable plasma glucose concentrations
on the day of the tracer experiment. The following day, the animals were anes-
thetized using isoflurane (5% for the induction of anesthesia and 2% for the
tively. A plastic cast was put over the hind limbs to restrain the animals, and
they were allowed to awaken. Arterial oxygen (PaO2), carbon dioxide (PaCO2),
pH, hematocrit and glucose levels were measured immediately prior to, and 10
and 40min following, the tracer injection. The animal’s body temperature was
maintained using a heating lamp. Two hours following the surgery, 20?Ci of
[14C]-labeled 2-DG dissolved in 1ml of saline was injected over 2min and 12
blood samples were collected at progressively longer time periods, up to 45min
following the injection of the tracer. Forty-five minutes following the tracer
injection, the animals were decapitated using a guillotine, and the brains were
at −80◦C until sectioned in the microtome at −20◦C. Slices were contacted
with X-ray films for 5 days along with14C-plastic standards calibrated to the
tissue equivalent. Images were digitized and analyzed with an image analyzer
(MCID, Image Research Co., St. Catherines, Ontario, Canada) and the optic
density converted to the rCGU (?mol/g/min) using the standard set of rate con-
stants, based on a 3-compartment biological model, and a lumped constant of
0.48  in 40 regions of interest. Further details on the method as adapted in
our laboratory can be found in previous publications [38,22]. The rCGU values
were determined in four consecutive slices and on the left and right sides of the
brain. After finding that there were no significant differences between the left
used in the subsequent analysis. The brain regions were identified using the rat
brain atlas . The brain areas (vulumes) used in the quantitation of glucose
utilization are exemplified for several brain regions in Fig. 1.
2.3. Data analysis
The rCGU values were converted to the z-scores (zi; standard normal devi-
ates) and used for the stepwise discriminant analysis. The main objective of the
discriminant analysis is to find a subset of the brain regions, out of the forty
that were measured, which demonstrates the best separation between the OBX
classification was applied). This transformation has been routinely used in sev-
eral positron emission tomography studies because after converting the data to
the normalized z-scores, the data has a minimal dependence on the absolute
values of the glucose utilization in each rat and a further propagation of error
is very small or non-existent . The z-scores were calculated by dividing the
differences of the rCGU (Rgi) and the rat brain mean glucose utilization (Rmean)
with the standard deviation (S.D.) of that mean [14,41] [zi=(Rgi−Rmean)/S.D.].
The Rmeanand S.D. were calculated as the weighted mean whole brain glu-
cose utilization and standard deviation. The regional values were weighed by
the number of pixels (the regional brain volume) used in the calculation of the
individual brain region glucose utilization, and the mean whole brain values and
S.D. were calculated using standard statistical formulas.
All statistical analyses were done with STATISTICA 7 software. A p<0.05
be satisfied for the multivariate statistical evaluations, and the test showed that
the data conforms to the multivariate normality (Mauchley Sphericity Test) and
equality of the variance-covariance matrix. The absolute values of rCGU were
I. Skelin et al. / Brain Research Bulletin 76 (2008) 485–492
Fig. 1. A schematic representation of the areas/volumes used in the quantification of several brain regions. The regions were read on both sides of the brain (see
Section 2). Abreviations are given under Table 2. Distances from the Bregma are marked in the lower right corner. These were adapted from the cross-sections given
in reference [35; with permission].
compared using a multivariate analysis of variance with the global brain glu-
cose utilization as a covariate (MANCOVA) to evaluate the overall differences
between the two groups as well as the influence of the covariate.
Because of the rather large number of brain regions analyzed, an attempt
was made to identify the regions that are the most important for the differ-
ence between the sham and OBX rats using a discriminate analysis . This
approach was deemed necessary because of the large number of brain regions
and it avoids the use of multiple ANOVA and corrections for multiple statistical
tests. A stepwise discriminant analysis, with z-scores, was used to identify the
discriminating) the OBX and sham groups. This stepwise procedure enters one
variable at a time and repeats the group classification procedure utilizing the
“jackknife” procedure until an optimal set of variables is selected. In the pro-
cedure, the values in each rat were removed from the model and the function
was calculated for the remaining (n−1) cases followed by the classification of
the last (left-out) case. Because the case which was being classified was left out
from the function calculation, the classification of that case to the OBX or sham
group represented a less biased estimate of the true one.
cose utilizations between the sham and OBX rats in all of the regions analyzed,
as an attempt to discover a global effect of the OBX on glucose utilization in
the brain. These ratios were compared using a one sample two-tailed t-test to a
reference value of 1 with a standard deviation of zero (1±0). If the OBX does
not produce a global effect, the mean ration should be 1.
3.1. Physiological data
The physiological data for the OBX and sham rats are pro-
In most of the rats from both groups, the glucose levels had
a tendency of rising in subsequent measurements during the
blood sampling, but this rise was not significant and did not
have any influence on the rCGU. There was no significant dif-
ference in the mean physiological variables assessed during the
experiments between the groups, except the body weight gain
Physiological parameters of OBX and sham rats
Weight gain (g)
61.1 ± 5.8
6.34 ± 0.62
7.43 ± 0.10
37.4 ± 1.3
92.1 ± 4.2
42.2 ± 2.0
82.0 ± 2.5
6.06 ± 0.28
7.46 ± 0.1
36.7 ± 1.4
86.0 ± 3.1
43.1 ± 0.7
aValues are given as the mean±S.E.M.
(Table 1). The repeated measures ANOVA analysis of the body
weight gain in two groups showed a significant statistical inter-
action between the groups and weight at two different times
(prior to, and 14 days following, OBX); F(1,6)=13.2; p<0.02.
A post hoc comparison with Newman–Keuls correction showed
significant differences (p<0.05) in the weight of the rats in the
OBX and sham groups 14 days following the surgery, without a
weight gain: 82.0±2.5g in the sham group and 61.1±5.8g in
the OBX group.
3.2. Global and regional CGU data
A set of autoradiographic images exemplifying the regional
differences in the glucose utilization at different cross-sections
of the rat brains for the OBX and sham operated rats are shown
in Fig. 2. It is evident from an examination of these autoradio-
grams that the rCGU is rather heterogeneous in both the sham
operated and OBX rats, but a small global difference between
the groups is not possible to appreciate from these images. The
values of the rCGU, the mean gCGU and the regional z-scores
of the rCGU are provided in Table 2 as mean±S.E.M.. The one
sample t-test indicated a significant reduction of the gCGU in
the OBX rats relative to that in the sham operated rats [ratio
(mean±S.E.M.)=0.94±0.01; t=−5.39; N=40; p<0.001].
MANCOVA analysis revealed a significant difference in the
rCGU among the brain regions [F(39,507)=3.84; p<0.001], a
global CGU [F(39,507)=9.51; p<0.001; region×global inter-
metries in the rCGU in either of these groups. MANCOVA
also showed a significant influence of gCGU on the analysis
[F(1,13)=2272; p<0.001; covariate significance]. To identify
the brain regions which are responsible for these differences,
the data was analyzed using a stepwise discriminant analysis,
because the identification of the individual brain regions with a
large number of variables is rather difficult when using a post
hoc analysis after MANCOVA.
for an optimal differentiation between the sham and OBX rats.
I. Skelin et al. / Brain Research Bulletin 76 (2008) 485–492
The mean of the rCGU values (?mol/g/min)±S.E.M. and the mean of the z-scores±S.E.M. for the OBX and sham groups
Brain global49.7 ± 5.0
AON 39.6 ± 2.8
Acx57.2 ± 1.4
Amy39.7 ± 1.2
AN 43.9 ± 1.1
Cincx 53.4 ± 1.3
Clu37.8 ± 1.2
CPGP44.1 ± 0.6
CPL 51.4 ± 0.8
CPM 51.1 ± 1.1
DHi36.4 ± 1.1
DS 38.3 ± 2.4
Dth 56.4 ± 1.4
Encx 36.0 ± 1.5
Fcx 48.8 ± 1.3
GP 29.1 ± 0.8
Hyp26.1 ± 1.1
IC caud 84.5 ± 2.9
IC rost 68.6 ± 3.5
LC25.2 ± 1.1
LG42.7 ± 1.5
MFB 47.9 ± 1.6
MG55.8 ± 0.9
Ocx 70.4 ± 1.7
Pcx 46.7 ± 1.2
Pfcx52.4 ± 1.2
SC43.2 ± 0.9
Sch 26.5 ± 1.1
Scx50.3 ± 1.0
SepN30.0 ± 1.0
Smcx 50.7 ± 1.1
SNc37.4 ± 1.2
SNr 37.0 ± 1.4
Vcx47.9 ± 1.4
VHi 32.8 ± 1.2
VTA33.6 ± 1.1
VTh 49.4 ± 1.1
DR43.8 ± 2.0
MR 36.5 ± 2.4
Rpo49.3 ± 2.0
RM33.5 ± 3.5
aThe structure abbreviations are: AON (anterior olfactory nucleus); Acx (auditory cortex); Amy (amygdala); AN (accumbens nucleus); Cincx (cingulate cortex);
Clu (clustrum); CPGP (caudate putamen at the level of globus pallidus); CPL (caudate putamen—lateral part); CPM (caudate putamen—medial part); DHi (dorsal
hippocampus); DS (dorsal subiculum); DTh (dorsal thalamus); Encx (enthorinal cortex); Fcx (frontal cortex); GP (globus pallidus); Hyp (hypothalamus); IC caud
(inferior colliculi—caudal part); IC rost (inferior colliculi—rostral part); LC (locus coeruleus); LG (lateral geniculate); MFB (median forebrain bundle); MG (medial
geniculate); Ocx (orbitofrontal cortex); Pcx (parietal cortex); Pfcx (prefrontal cortex); SC (superior colliculi); Sch (suprachiasmatic nucleus); Scx (sensory cortex);
hippocampus); VTA (ventral tegmental area); VTh (ventral thalamus); DR (dorsal raphe); MR (median raphe); RPo (raphe pontine); RM (raphe magnus).
47.1 ± 2.6
32.2 ± 2.8
59.0 ± 1.4
37.9 ± 1.2
40.0 ± 1.1
49.0 ± 1.3
36.5 ± 1.2
45.4 ± 0.6
50.0 ± 0.8
47.8 ± 1.1
37.2 ± 1.1
36.5 ± 2.4
52.5 ± 1.4
32.6 ± 1.5
44.2 ± 1.3
30.0 ± 0.8
26.0 ± 1.1
83.9 ± 2.9
68.7 ± 3.5
24.6 ± 1.1
39.4 ± 1.5
46.0 ± 1.6
52.9 ± 0.9
58.7 ± 1.7
47.8 ± 1.2
49.0 ± 1.2
41.5 ± 0.9
26.3 ± 1.1
51.5 ± 1.0
29.6 ± 1.0
46.9 ± 1.1
34.0 ± 1.2
33.5 ± 1.4
46.2 ± 1.4
30.8 ± 1.2
31.4 ± 1.1
45.0 ± 1.1
38.9 ± 2.0
24.7 ± 2.4
43.2 ± 2.0
31.8 ± 3.5
Hyp, MR, Smcx, SNr, VHi, VTA, and VTh. In addition, the Pcx
showed p<0.01 [F(1,1)=5875.8], but including this structure
would not add any significant power to the separation between
these two groups. The canonical variable correlation coefficient
cates that using variables identified by the discriminant analysis
to be important for the differentiation of these two groups are
different at p<0.001 (p value was obtained from χ2approxi-
mation; χ2=84.9; degrees of freedom=13). This value of the
Wilk’s lambda suggests that more than 99% of the variance of
the grouping variable is explained by the predictor variables
provided in Table 3.
The groups described by the above mentioned grouping
variables (Table 3) are significantly different (F(13,2)=103;
the canonical variables are also provided in Table 3. An evalua-
tion of these coefficients suggests a different influence of spe-
cific regions on the group separation. While some brain regions
(caudate putamen – lateral part, substantia nigra – pars reticu-
lata, amygdala, ventral thalamus, median raphe, hypothalamus)
I. Skelin et al. / Brain Research Bulletin 76 (2008) 485–492
Fig. 2. Representative autoradiograms from OBX (left row) and sham (right
row) drug-naive rats. Autoradiographic images of tissue radioactivity at coronal
sections through the brain after 45min of exposure to systemic [14C]-2-
deoxyglucose. Cincx cingulate cortex, AN nucleus accumbens, DHi dorsal
hippocampus, CPM caudate putamen—medial part, Hyp hypothalamus, MG
contribute to the separation in a positive direction, others (cin-
gulated cortex, dorsal thalamus, sensorymotor cortex, ventral
putamen at the level of globus pallidus) make a contribution in
an opposite direction. Note that unstandardized coefficients of
the discriminant function have a negative constant which needs
to be taken into account when separation is calculated. From
the standardized coefficients one can conclude that the glucose
utilization in the caudate putamen at the level of globus pal-
Canonical discriminant function coefficients obtained by forward stepwise dis-
criminant analysis of the z-scores provided in Table 2
lidus, followed by caudate putamen – lateral part, amygdala,
ventral hippocampus, substantia nigra – pars reticulata, ventral
tegmental area, etc. (Table 3) has the most prominent influence
on the differences in the glucose utilization between OBX and
sham operated rats. Similarly superior colliculi have the small-
est influence from the selected variables on the separation of the
OBX and sham rats. The eigenvalue, representing the ratio of
the sum of squares between and within the group was found to
be 1.84×105. This suggests a very large and good separation
of these two groups.
The stepwise discriminant analysis indicates that the mea-
surement in these regions (see Table 3) would be sufficient
to identify differences between these two groups. However, if
one carries out any kind of pharmacological treatment, some
other regions could become important because the treatments
may affect different brain regions in different ways, thus sug-
gesting the need to measure more brain regions for an optimal
differentiation between the experimental groups.
4.1. Global rCGU
The overall gCGU was significantly lower in the OBX than
in the sham-operated rats (one sample t-test), MANCOVA and
discriminant analysis (see results). Several mechanisms could
OBX rats  could explain this. The latter hypothesis is based
on the findings of increased levels of the inhibitory amino acid,
glycine , and decreased levels of the excitatory amino acid,
glutamate  and aspartate . These authors suggested that
in GABA turnover, an inhibitory neurotransmitter, in the amyg-
the efferents from the olfactory bulbs (OB) are mostly gluta-
matergic , their loss following the removal of the olfactory
bulbs would also contribute to the excitatory–inhibitory imbal-
ance in their target regions. This predominant loss of the excita-
receptors in the cerebral cortex and amygdalae was found at
approximately the same time-point after surgery (16 days) 
as the present measurements of CGU. With a proposed decrease
in the reactivity of the NMDA receptors in the OBX rats ,
decreased CGU in OBX rats could be through the elevation of
brain 5-HT content . Watanabe et al. [52,53] and Hasegawa
cortical and subcortical regions as assessed by autoradiography
using ?-[14C]methyl-l-tryptophan. Sato et al.  reported a
widespread down-regulation of 5-HT1A receptors in OBX rats,
which suggests increased levels of 5-HT and its interaction with
5-HT receptors, as an indirect consequence of OBX. As the
could also contribute to the lowering of gCGU levels in OBX.
I. Skelin et al. / Brain Research Bulletin 76 (2008) 485–492
Finally, region specific cerebral hypometabolism in OBX
shown by the studies of CGU following several different brain
lesions. Mihara  found a decrease in neocortical CGU 1
ibotenic acid, while Orzi et al.  found an ipsilateral reduc-
tion in rCGU (18%) 2 weeks following the unilateral lesion of
nucleus basalis of Meynert (local kainate injection).
4.2. Regional CGU
Olfactory bulbs project ipsilaterally to the secondary olfac-
tory structures, which include the olfactory tubercle, amygdala,
habenula, enthorhinal and pyriform cortices. They also project
to the nucleus accumbens, hippocampus, hypothalamus, ven-
tral tegmental area and reticular formation . The OB main
afferents are ipsilateral, serotonergic and noradrenergic projec-
tions from the dorsal raphe and locus coeruleus, respectively
. OBX produced changes in rCGU in some (e.g., amygdala,
hypothalamus, ventral hippocampus, ventral tegmental area),
but not all, of the regions directly connected with the olfac-
tory bulbs. Differences were also found in some regions not
directly connected with the olfactory bulbs (cingulate cortex,
caudate putamen – lateral part, caudate putamen at the level of
globus pallidus, dorsal subiculum, median raphe, sensorymotor
cortex, substantia nigra – pars reticulata and ventral thalamus).
The changes in rCGU in the regions directly connected with
OB are likely a consequence of the transneuronal degeneration,
while the changes in the regions not directly connected with
OB may be related to an indirect influence of neurons degen-
erated following olfactory bulbs removal. This hypothesis is in
line with the findings reporting post-OBX neural degeneration
in the areas distant from the OB, such as the amygdala, hip-
pocampus, and dorsal raphe [15,3,31]. The profile of changes in
the rCGU does not completely correspond with the connectivity
of the OB. This discrepancy is similar to those found in other
lesion studies [30,34].
Although OBX has been found to induce changes in many
neurotransmitter systems, the full behavioral profile of OBX
syndrome has been elicited only by injecting the 5-HT neuron-
specific toxin 5,7-dihydroxytryptamine (5,7-DHT) into OB ,
suggesting the critical role of the 5-HT system in the OBX
syndrome. The raphe nuclei are the major source of 5-HT inner-
vations in the brain. The present data suggest the importance of
the median raphe in differentiating between the OBX and sham
groups, while other raphe nuclei (dorsal, pontine and magnus
raphe) are not very important in differentiating between these
two groups. The terminal projections in many forebrain regions
(e.g., caudate putamen, globus pallidus, amygdala) are mostly
from the dorsal and, to a lesser extent (20%), the median raphe
. Some other terminal regions receive predominantly projec-
tions from the median raphe (e.g., nucleus accumbens, ventral
inant analysis as important for differentiation of the sham and
OBX rats (Table 3). Regions that predominantly receive projec-
for the differentiation between the OBX and sham rats, sug-
gesting that it is not just the processes occurring in the cell
two groups. This hypothesis stems from the fact that there is a
receiving projections from it. Nesterova et al.  reported the
neurodegeneration in the dorsal raphe 28 days following bul-
bectomy in mice, suggesting that a reduction in the CGU in
the median raphe observed in the present investigation could be
related to neuronal degeneration.
The pattern of rCGU changes observed in rats treated with
ilar to our findings, including the changes in the basal ganglia
sory areas with a high content of dopamine (DA) were found
following the treatment of rats with the 5-HT1Aagonist, 8-OH
the OBX rats have elevated tissue 5-HT, it can stimulate the
release of DA and NA, which could act together with 5-HT on
the regional CGU.
Somewhat discrepant result from those presented here was
glucose uptake in the amygdala slices of OBX rats. They inter-
preted this as an increase in the glucose utilization in the
amygdala, however an increase in the glucose uptake does not
necessarily mean that there has been an increase in glucose uti-
tegmental area and both receive projections from the OB ,
which are cut in the slice preparation producing rather different
situation than that in an intact animal. The ventral tegmen-
tal area is considered to be a part of the reward-circuit and
the dysfunctions of this circuit underlay anhedonia, one of the
core symptoms of depression . Anhedonia has been shown
in OBX rats by decreased sucrose preference . Further,
the ventral tegmental area has mostly reciprocal connections
, with several other regions in which differences in rCGU
have been found (hypothalamus, hippocampus, cingulate cor-
The reciprocal connections of the ventral tegmental area, with
most of the regions found to have changes in the rCGU in OBX,
point to the ventral tegmental area as a potential key node in the
spreading of changed neural activity following OBX. Changes
in OBX, could be implicated in changes in reward sensitivity,
as well as changes in locomotion. Further, this structure is very
important in many affective disorders.
related to changes in meal frequency , fluid intake  and
thermoregulation , all of which have already been demon-
strated to be different in OBX rats when compared to the sham
In the present study, a rCGU difference in the ventral hip-
pocampus was also found between the two groups. This could
be related to deficiencies in habituation to novel environment,
impaired spatial memory and learning found in OBX rats .
OBX produces a decrease in hippocampal neurogenesis 
which is reversed by chronic therapy with citalopram  and
I. Skelin et al. / Brain Research Bulletin 76 (2008) 485–492
imipramine , suggesting the role of adult hippocampal neu-
Further, the present investigation indicates rCGU differences
the structure that has been implicated in the pathogenesis and
therapeutic response to antidepressant regimes of very different
categories, ranging from drugs to electroconvulsive therapy and
deep brain stimulation [27,28]. As mentioned above, this defi-
ciency is likely a result of a different interplay between several
The CGU changes in the sensory and motor structures could
also be explained in the context of different startling sensitiv-
ity and increased locomotion, respectively, in OBX rats. rCGU
changes in OBX brain metabolism at 14 days following surgery
is in accordance with several other lesion studies which also
deprived the animal from one sensory modality. Specifically,
changes in rCGU have been almost abolished 10 days after the
surgical removal of the cochlea , while the eye enucleation
resulted in a rCGU decrease in the denervated superior colli-
culi, which was spontaneously normalized within a few days
Several other studies show that although profound distur-
bance of brain neurotransmission may be present, differences
in rCGU are not detected or are marginal. For example, after
neurotoxic lesions of the 5-HT system using 5,7-DHT, approxi-
substantial decrease in components of brain 5-HT transmission
(89% decrease in (3H)paroxetine binding). However, despite
this large reduction in paroxetine binding, 2 weeks after the
lesion, significant changes in the rCGU were only found in
the hippocampus . In the present study, a relatively large
part of the limbic brain areas (e.g., cingulate cortex, amygdala,
ventral hippocampus, hypothalamus) was found to be impor-
tant in the differentiation between the sham and OBX rats
(see Table 3). However, some important limbic structures (e.g.,
nucleus accumbens, enthorhinal cortex) have not been found
to be significantly important for the differentiation between the
OBX and sham rats, but this does not mean that the rCGU was
not affected in these regions. On the basis of these studies, it
can be suggested that the changes observed between the sham
and OBX groups in glucose utilization represent integral differ-
ences in several neuronal systems as well as the effect of locally
Using [14C]-2-deoxyglucose autoradiography, we have
demonstrated a lower global CGU in OBX rats than sham-
operated rats. Further, 13 brain regions have been identified as
being important when discriminating between OBX and sham
rats. The pattern of change does not strictly correlate with the
connectivity of the OB. These functional changes are present
in the brain regions previously implicated in the pathogenesis
of depression, and are also important in different aspects of
memory, motivation and reward-related behavior. In addition,
differences were found in the regions that are components of
the sensory and motor systems, which could be related to the
hyperactivity of the OBX rats.
The research reported here was supported in part by the
Croatian Ministry of Science, Education and Sports (219-
1081970-2032) and the Canadian Institute for Health Research
(MOP-42438). We would also like to thank Ms. Valerie-Ann
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