Neuroscience 259 (2014) 84–93
NEONATAL OLFACTORY BULBECTOMY ENHANCES LOCOMOTOR
ACTIVITY, EXPLORATORY BEHAVIOR AND BINDING OF NMDA
RECEPTORS IN PRE-PUBERTAL RATS
G. FLORES, a,b* O. IBAÑEZ-SANDOVAL, a
A. B. SILVA-GÓMEZ, a I. CAMACHO-ABREGO, a
A. RODRÍGUEZ-MORENO c AND
J. C. MORALES-MEDINA b
a
Laboratorio de Neuropsiquiatrı´a, Instituto de Fisiologı´a, Universidad
Autónoma de Puebla, 14 Sur 6301, San Manuel, 72570
Puebla, Mexico
b
Douglas Mental Health University Institute, Department of
Psychiatry, McGill University, Montréal, QC H4H 1R3, Canada
c
Laboratorio de Neurociencia Celular y Plasticidad,
Universidad Pablo de Olavide, Sevilla, Spain
Abstract—In this study, we investigated the effect of neona-
tal olfactory bulbectomy (nOBX) on behavioral paradigms
related to olfaction such as exploratory behavior, locomotor
activity in a novel environment and social interaction. We
also studied the effect of nOBX on the activity of the
N-methyl-D-aspartate (NMDA) subtype of glutamate recep-
tors during development. The behavioral effects of nOBX
(postnatal day 7, PD7) were investigated in pre- (PD30) and
post-pubertal (PD60) Wistar rats. NMDA receptor activity
was measured with [125I]MK-801 in the brain regions associ-
ated with the olfactory circuitry. A significant increase in the
novelty-induced locomotion was seen in the pre-pubertal
nOBX rats. Although the locomotor effect was less marked
than in pre-pubertal rats, the nOBX rats tested post-pubertal-
ly failed to habituate to the novel situation as quickly as the
sham- and normal- controls. Pre-pubertally, the head-dip-
ping behavior was enhanced in nOBX rats compared with
sham-operated and normal controls, while normal explor-
atory behavior was observed between groups in adulthood.
In contrast, social interaction was increased in post-pubertal
animals that underwent nOBX. Both pre- and post-pubertal
nOBX rats recovered olfaction. Interestingly, pre-pubertal
rats showed a significant increase in the [125I]MK-801 binding
in the piriform cortex, dorsal hippocampus, inner and outer
layers of the frontal cortex and outer layer of the cingulate
cortex. At post-pubertal age, no significant differences in
*Correspondence to: G. Flores, Laboratorio de Neuropsiquiatrı́a,
Instituto de Fisiologı́a, Universidad Autónoma de Puebla, 14 Sur
6301, CP 72570 Puebla, Mexico. Tel: +52-2-2295500x7322; fax:
+52-2-2295500x7301.
E-mail addresses: gonzaloflores56@gmail.com, gonzalo.flores@
correo.buap.mx (G. Flores).
Abbreviations: ANOVA, analysis of variance; BL, basolateral; Cg,
cingulate cortex; DG, dentate gyrus; dPFC, dorsal part of the prefrontal
cortex; EDTA, ethylenediaminetetraacetic acid; HBT, hole board test;
MK-801, dizocilpine; NAcc, nucleus accumbens; NMDA, N-methyl-D-
aspartate; nOBX, neonatal bilateral ablation of the olfactory bulbs;
OBX, ablation of the olfactory bulbs; OF, open field; OT, olfactory
tubercle; PBS, phosphate saline solution; PD, postnatal day; PFC,
prefrontal cortex; RMS, rostral migratory stream; SI, social interaction;
SVZ, subventricular zone.
0306-4522/13 $36.00 Ó 2013 IBRO. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.neuroscience.2013.11.047
84
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[125I]MK-801 binding were observed between groups at
any of the brain regions analyzed. These results suggest that
nOBX produces pre-pubertal behavioral disturbances
and NMDA receptor changes that are transitory with recovery
of olfaction early in adulthood. Ó 2013 IBRO. Published by
Elsevier Ltd. All rights reserved.
Key words: neonatal ablation of the olfactory bulbs,
locomotion, social interaction, NMDA receptor, limbic system,
olfactory bulbectomy and depression.
INTRODUCTION
Bilateral ablation of the olfactory bulbs (OBX) in adult rats
leads to a variety of behavioral, neurochemical,
endocrine, and immunological changes that parallel
those seen in depressed subjects (Willner, 1990; Song
and Leonard, 2005). The behavioral changes include
increased locomotor activity in novel environment,
passive avoidance learning deficits, enhanced
vulnerability to stressors, sleep disturbances and altered
food intake. Interestingly, many of the components of
the OBX syndrome are also present in major depression
(DSM-IV), and chronic antidepressant treatment
alleviates many of the behavioral deficits in OBX rats as
observed in the human condition (Kelly et al., 1997).
Such similarities between the OBX syndrome and major
depression had led to be one of the most studied animal
models of depression-related behavior.
The behavioral and neurochemical similarities
observed in depressed patients and in adult OBX rats
are related to disturbances in the organization of the
limbic system (van Riezen and Leonard, 1990;
Richardson, 1991; Morales-Medina et al., 2013b). In this
regard, most of the targets of antidepressant binding in
the brain are structures in the limbic system such as the
prefrontal cortex (PFC), septal nucleus, subiculum, and
hippocampus (Kaulen et al., 1989; Duman and
Aghajanian, 2012). The PFC is of particular interest in
depression because of its importance in emotions,
judgment, planning and cognitive functions (Kolb, 1990).
All of these are affected in both depressed subjects and
the OBX rat.
In the PFC, antidepressant binding sites correlate with
the distribution of glutamate receptor sites, and chronic
antidepressant treatment affects the ligand-binding
properties of N-methyl-D-aspartate (NMDA) receptors
(Nowak et al., 1993). Furthermore, NMDA receptors in
 G. Flores et al. / Neuroscience 259 (2014) 84–93
the frontal lobe may also play an important role in learning
and depression, based on the fact that acquisition of
intracranial self-stimulation at sites in the frontal lobe is
blocked by pretreating rats with ketamine, an NMDA
antagonist (Corbett, 1990). Our group has also found a
significant elevation of NMDA receptors in the medial
PFC after OBX (Webster et al., 2000). In addition to
these early findings, ketamine has recently been shown
as an efficacious acute antidepressant in humans as
well as in animal models of depression-related behavior
(Zarate et al., 2006; Liebrenz et al., 2009; Li et al.,
2011). All together, these findings support a key role of
the glutamatergic system in depression.
The olfactory bulbs (OB) are a unique, dynamic
system that retains the ability to acquire new neurons
throughout life (Altman, 1969). Cells migrate from the
subventricular zone (SVZ) along the rostral migratory
stream (RMS) into the OB (Altman, 1969; Kato et al.,
2012). Interestingly, most neurons are added in the first
days after birth. However, little attention has been given
to alterations in behavior and physiology following
neonatal bilateral ablation of the OB (nOBX) in contrast
to numerous studies of bilateral OBX in the adult rat
(Kelly et al., 1997). Previously, unilateral nOBX between
postnatal days (PDs) 1–6 produced retrograde
degeneration at PD8 and new sensory functional
neurons were reconstituted by PD30 (Hendricks et al.,
1994a,b). Additionally, unilateral nOBX at PD1 showed
a limited recovery in olfaction in early adulthood
(Hendricks et al., 1994a,b). However, to the best of our
knowledge, bilateral nOBX has yet to be compared to
the neurochemical and behavioral effects of the removal
of OB in the adult rat.
OBX produces a constellation of behavioral deficits in
emotion-related paradigms and disrupts the NMDA
receptor activity. The integration of new born cells in the
OB is particularly high early postnatally (Tropepe et al.,
1997). Therefore, we hypothesized that the removal of
OB early in development could produce long-lasting
behavioral disturbances and deregulated NMDA
receptor expression. The aim of the present study was
to assess whether nOBX produces changes in
locomotor activity in novel environment, head-dipping
behavior, social interaction (SI), olfaction and NMDA
receptor activity in the limbic brain regions. To
determine the duration of these possible changes, we
evaluate all the behavioral and neurochemical studies
pre- and post-pubertally.
EXPERIMENTAL PROCEDURES
Animals
Pregnant Wistar rats, bred in our facilities at the
Universidad Autónoma de Puebla, were selected at
gestational days 14–17. Animals were individually
housed in a temperature and humidity-controlled
environment on a 12–12-h light–dark cycle with free
access to standard laboratory chow food and tap water
until the time of delivery. After the day of birth, litters of
6–8 male pups were formed, and on the PD7
(corresponding to a body weight of 12–14 g) pups were
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85
assigned to either normal, sham or nOBX groups. All
experimental procedures were approved by the IF
Animal Care Committee and met governmental
guidelines (Mexican Council for Animal Care, Norma
Oficial Mexicana NOM-062-ZOO-1999) and by the
National Institutes of Health Guide for the Care and Use
of Laboratory Animals. All efforts were made to
minimize animal suffering and to reduce the number of
animals used.
Surgery
Pups were anesthetized by putting them on wet ice for
15–20 min until they reach hypothermia (Solis et al.,
2009). Under aseptic conditions and full anesthesia, a
bilateral craniotomy was performed 6 mm anterior to
bregma in the frontal bone to expose the OB. The OB
stalks were severed with a microknife and the bulbs
were aspirated with a blunt needle connected to a
vacuum pump. Sham-operated rats underwent the same
surgical treatment as OBX rats, except for the actual
ablation of the OB. Control animals were normal,
undisturbed animals. After this procedure, the pups
were placed under a warming lamp for recovery and
then returned to their mothers. At PD21, animals were
weaned and grouped as two or three animals per cage.
nOBX animals shared a cage with a sham, a normal
control or both.
Behavioral testing
Four (PD30, n = 10 per group) or 7 weeks (PD60, n = 10
per group) after surgeries, the behavioral paradigms were
assessed under four testing conditions:
Locomotor activity. Locomotion in a novel
environment was assessed in all groups by placing each
of the animals alone in an activity box to which they had
not been exposed previously. The activity box was
equipped with 8-photocells connected to a counter
(Tecnologı́a Digital, Mexico). The assessment lasted for
six 10-min periods with 1-min long intervals between
testing periods.
Social interaction. This paradigm has been broadly
validated for measuring anxiety in rats (File, 1980; File
and Seth, 2003). In this study, a modified version of the
original model was used one day after the locomotor test
(Silva-Gomez et al., 2003a). A pair of rats was placed in
opposing corners of the open field (OF) apparatus and
was allowed to explore the arena for 10 min. We took
care that a member of a pair do not differ more than
20 g. All of the animals were tested only once under
high-light and unfamiliar conditions. Each pair of rats
was randomly selected within the same test group
(normal–normal, sham–sham, nOBX–nOBX) and placed
into an acrylic cage (80  80  40 cm). The following
behaviors were considered as active SI: sniffing,
following, grooming, mounting, wrestling, jumping on and
crawling under or over the partner (Morales-Medina
et al., 2012a,b). The experiment was videotaped and
scored by an investigator blind to the surgical status of
86 G. Flores et al. / Neuroscience 259 (2014) 84–93
the rat using a computer software developed by the
University of Puebla. Data are expressed as total
number of interactions as well as the time spent in the
active behaviors.
Exploratory behavior. Defined as head-dipping activity
as previously described (Crawley, 1985; Kamei et al.,
2007). Rats were measured individually in an unfamiliar,
black, acrylic box (30  30  20 cm) with a hole (2 cm in
diameter), which was located in one of the walls 2 cm
above the floor level and with a pair of photocells
connected to a computer counter (Tecnologı́a Digital,
México). Every time the rat head-dips the infrared light
of the photocells was blocked and the counter exhibited
the number of head-dips and the total time spent on this
behavior. The number of head-dips and accumulated
time of the head-dipping activity was measured for
10 min.
Olfactory test. Food retrieval tests are commonly used
to measure olfactory ability (Amemori et al., 1989;
Hendricks et al., 1994a,b). After 24 h of food restriction,
individual rats were allowed to search for hidden
chocolate chip cookies (Nabisco, Chewy Chips Ahoy,
Mexico city, DF, Mexico, 07820). The olfactory test for
each rat was conducted in the home cage. The cookies
were hidden in random locations under 2 inches of
cedar bedding on the floor of the box. We measured the
latency for finding a 10-g piece of cookie in the box.
Four consecutive trials were conducted per rat during
the same session.
Histology
Immediately after the last behavioral testing, rats
were deeply anesthetized with sodium pentobarbital
(200 mg/kg, i.p.) and perfused intracardially with 4%
paraformaldehyde. The brains were quickly removed
from the skull and stored in paraformaldehyde at 4 °C
until used. The size of the OB in normal, sham and
nOBX was verified visually using a light microscope.
The rat brains were sectioned in the parasagittal plane
by a Cadwell Vibrotome at 80-lm thickness. The
sections were collected on pre-cleaned, gelatin-coated,
microscope slides (three sections/slide) and stained with
Cresyl Violet.
Ligand autoradiography
Another cohort of rats (which were also from the same
condition), 4 (PD30, n = 6 per group) or 7 weeks
(PD60, n = 6 per group) after nOBx, were used for the
NMDA receptor binding study. The rats were
decapitated with a laboratory guillotine and their brains
were quickly removed from the skull, frozen in
isopentane at 40 °C, and stored at 80 °C until used.
The size of the OB in normal, sham and nOBX was
verified visually. The frozen rat brains were sectioned in
the coronal plane at 20 °C on a Leica (CM1100)
cryostat at 16-lm thickness. The sections were
collected on precleaned, gelatin-coated microscope
slides (3, 4 sections/slide), thaw-mounted, desiccated
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under vacuum at 4 °C overnight, and then stored at
80 °C until the day of the experiment. Brain sections
taken at level of the olfactory tubercle (OT) were also
stained with Cresyl Violet.
For the NMDA receptor binding study, brain sections
from the PFC, striatum, nucleus accumbens (NAcc) and
hippocampus were taken using a previously described
protocol (Webster et al., 2000). Slides were incubated for
60 min at room temperature with 200 pM (+)-3-[125I]Iodo-
MK-801, 50 mM sodium phosphate/0.9% saline solution
(PBS) pH 7.4. Non-specific binding was defined in
adjacent sections by the addition of 5 lM MK-801 to the
incubation buffer. Following incubation, all the slides were
rinsed in ice-cold PBS and then washed twice for 15 min
in ice-cold PBS. After a final brief dipping in ice-cold
distilled water, slides were dried at room temperature and
apposed to [125I]-Hyperfilm (Amersham, Toronto, Ontario,
Canada) with control standards for 8 h.
Autoradiographic films were analyzed with a
computerized image analysis system (MCID-4, Imaging
Reseach, Ste-Catherine, Ontario, Canada). Brain
sections were divided into sub-regions according to the
Paxinos and Watson (1986).
Materials
MK-801 were purchased from RBI (Sigma, St. Louis, MO,
USA). [125I]MK-801 (2200 Ci/mmol) was obtained from
NEN (Boston, MA, USA). Isopentane was obtained from
BDH (Montreal, QC, Canada), EDTA was purchased
from Boehringer-Mannheim (Laval, QC, Canada),
gelatin was obtained from Fisher (Montreal, QC,
Canada) and bovine serum albumin from Calbiochem
(La Jolla, CA, USA). All others chemicals used were of
analytical reagent quality.
Statistical analysis
Behavioral and autoradiographic results were analyzed
with two-way analysis of variance (ANOVA) tests.
Lesion condition and age were considered as
independent factors. For behavioral studies, time-of-
testing was also an independent factor. Newman–Keuls
and Mann–Whitney tests were used as appropriate for
post hoc comparisons with p < 0.05 considered as
significant.
RESULTS
nOBX: Bulb size
OBX results in permanent damage of the OB without
regrowth (Kelly et al., 1997; Song and Leonard, 2005).
With this in mind, we evaluated the effects of nOBX in
the size of OB pre- and post-pubertally. The nOBX
animals showed bilateral reduction in the size with
regrown of OB pre- and post-pubertally as observed in
Fig. 1. Cresyl Violet-stained sections obtained from
nOBX animals, at both pre- and post-pubertal age,
revealed smaller size of OB compared to sham and
control animals. Additionally, the general
cytoarchitecture of the regrown bulbs showed
deformation of the OB of nOBX animals (Fig. 1).
 G. Flores et al. / Neuroscience 259 (2014) 84–93
Fig. 1. Neonatal olfactory bulbectomy (nOBX) showed regrowth of
olfactory bulbs pre-pubertally. Left, nOBX animals showed bilateral
reduction in the size of the olfactory bulbs pre-pubertally. Right, nissl
staining sagittal section of the OB showed reduction in the size and
deformation of the structures in the nOBX animals.
nOBX: Behavioral testing
We used the locomotor activity test to determine whether
nOBX induced hyperlocomotion pre- and post-pubertally.
All groups showed active exploratory behavior as initial
response of rats placed in a novel environment. As
shown in Fig. 2, nOBX increased locomotor activity
observed pre-pubertally relative to sham and normal
animals of the same age (p < 0.05).
Two-way ANOVA revealed that locomotion was
significantly affected by the nOBX (F2,54 = 5.81,
p = 0.005), but not by age (F1,54 = 0.98, p = 0.3) or by
lesion  age interactions (F2,54 = 1.78, p = 0.1). Post
hoc tests showed that there was a significant increase
in locomotor activity observed at PD35 in the nOBX
group relative to sham and normal animals of the same
age (p < 0.05). No difference in locomotor activity in
novel environment was observed between sham, normal
and OBX groups post-pubertally. In addition, when the
data were analyzed in separate 10-min blocks, it was
evident that pre-pubertal rats that underwent nOBX
were more active than shams and control animals
during the period between 30 and 60 min of the test.
Post-pubertal rats were habituated to the novel
environment at a slower rate than non-lesioned animals.
OBX produced hyperlocomotion in novel environment;
however, this behavior was not seen in other models of
depression-related behavior (Kelly et al., 1997; Morales-
Medina et al., 2013b). For this reason, we used the hole
board test (HBT) to measure additional exploratory
behavior after nOBX pre- and post-pubertally.
Fig. 3A, B shows the effect of nOBX on exploratory
behavior at both ages. ANOVA reveals that the total
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87
Fig. 2. Neonatal olfactory bulbectomy induces hyperlocomotion pre-
pubertally. Temporal profile of locomotor activity (mean number of
beam interruptions per 10 min ± SEM) in pre- (A) and post-pubertal
(B) animals. Analysis of total activity scores (mean number of beam
interruptions per 60 min ± SEM) of pre- and post-pubertal animals
(C), ⁄p < 0.05.
time spent as exploratory behavior was significantly
affected by nOBX (F2,54 = 3.3, p = 0.04) and age
(F1,54 = 82.9, p < 0.001) but not by nOBX  age
interaction (F2,54 = 0.10, p = 0.9). Similarly, the number
of episodes of dipping-head shows significant effects of
nOBX (F2,54 = 4.9, p = 0.01) and age (F1,54 = 82.1,
p < 0.001) but not nOBX  age interaction
(F2,54 = 0.24, p = 0.7). Post hoc test only showed that
nOBX animals at PD30 exhibited an increase in the
number and time of episodes of dipping-head compared
to sham and normal animals (p < 0.05).
OBX animals are widely known to display
hyperlocomotion (Kelly et al., 1997; Song and Leonard,
2005); however, effects of OBX on SI were only recently
established. These results suggest that OBX causes
deficits in SI in rat and mice (Wang et al., 2007;
Morales-Medina et al., 2012a,b). Fig. 3C, D shows the
effect of nOBX on social behavior at both ages. The
number of episodes of social encounters was affected
88 G. Flores et al. / Neuroscience 259 (2014) 84–93

Fig. 3. Effect of neonatal bulbectomy in the hole board test and social interaction assessed. Neonatal bulbectomy increases the number (A) as well
as the time of (B) head-dipping behavior pre-pubertally. Post-pubertally nOBX does not modify either the number (A) or the time (B) of head-dipping
behavior. Each column represents the mean ± SEM, ⁄p < 0.05. In addition, neonatal olfactory bulbectomy increases active social contacts in the
post-pubertal rat. The nOBX increases number of encounters (C) or the time (D) of social contacts post-pubertally, while nOBX has no effect of the
number of encounters (C) or time (D) of social contacts pre-pubertally. Each column represents the mean ± SEM, ap < 0.01 vs normal; bp < 0.05
vs sham; cp < 0.01 vs normal.

by nOBX (F2,54 = 0.04, p = 0.83), with trend to age
(F1,54 = 3.33, p = 0.07) and without effects of
nOBX  age interactions (F2,54 = 2.22, p = 0.11).
However, the total time spent in SIs was only
significantly affected by age (F1,54 = 37.2, p < 0.001),
without effect of nOBX (F2,54 = 0.63, p = 0.5) and
nOBX  age interactions (F2,54 = 1.55, p = 0.2). Post
hoc test shows that nOBX animals at P60 exhibited
higher number of episodes of social encounters
compared with the sham and normal animals (p < 0.05
and p < 0.01, respectively), with more time spent in
social contact (p < 0.01).
OBX results in loss of olfaction. We next evaluated the
effects of nOBX in olfactory behavior pre- and post-
pubertally (Fig. 4A, B). As illustrated in the Fig. 4C,
ANOVA reveals that after 24 h of food restriction,
normal, sham and nOBX animals at both ages searched
and found hidden pieces of chocolate chip cookies.
nOBX: NMDA receptor expression
Previous research described a long-lasting increase in
NMDA receptor expression after OBX in the PFC
(Webster et al., 2000) as well as decreased protein
expression of NMDA receptor subunit 1 (NR1) in the
PFC, hippocampus and amygdala (Wang et al., 2007).
In the last set of experiments, we examined the
expression of NMDA receptors in cortical and
subcortical regions (PFC, NAcc, Cpu, TO, thalamus,
basolateral (BL)-amygdala and hippocampus) at pre-
and post-pubertal age following bilateral nOBX. The
results showed a significant increase in the level of
[125I]MK-801 binding to the NMDA receptor in the inner
and outer parts of the dorsal PFC (dPFC-outer), outer
part of the cingulate cortex (Cg-outer), piriform cortex
and CA1, CA3 and dentate gyrus (DG) of the
hippocampus of the neonatal OBX rats in comparison
with the sham-operated rats (Table 1, Figs. 5–7). The
increase in the level of NMDA receptor in the piriform
cortex (+43%); in the outer and inner part of the dPFC
(+36%, +28%, respectively), and CA3 of the
hippocampus (+32%) of nOBX animals compared to
sham rats (Table 1; Figs. 5–7). The changes in NMDA
receptor binding in the inner part of the PFC was less
pronounced (+32.1%) than in the outer cortex, but was
nevertheless, statistically significant (p < 0.05). Other
brain areas showed a moderate, but significant,
increase in the level of NMDA receptors, outer part of
the Cg (+20%), caudate–putamen (+19%), stratum
oriens, pyramidal and molecular layers of the CA1 of the
hippocampus (+20%, +22% and +16%) and
molecular layer of the DG of the hippocampus (+17%)
(Table 1, Figs. 5–7).
DISCUSSION
The present study demonstrates that nOBX in rats
increases locomotion and attentional behavior
(exploration) in pre- but not post-pubertal age. The pre-
pubertal abnormal behaviors were accompanied with
NMDA-enhanced activity in several brain regions
including the PirC, hippocampus and PFC. In addition,
nOBX animals showed an increase in SI at post-
pubertal age. In the olfactory test, there was no

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 G. Flores et al. / Neuroscience 259 (2014) 84–93
Fig. 4. Latency for food finding across testing. Neonatal olfactory
bulbectomy does not modify the latency to find the hidden food either
pre- (A) or post-pubertally (B). C shows the cumulative data of the
4 days evaluated with no significant difference between groups. Each
column represents the mean ± SEM.
difference between lesioned and control animals at either
pre- or post-pubertal age, and the size of the regrown
bulbs were similar in all groups. Altogether, these data
suggest that the OB suffers strong compensatory plastic
changes after early postnatal ablation.
nOBX induces hyperlocomotion and head-dipping
behavior only pre-pubertally
In the present study, we observed for the first time that
nOBX produces hyperlocomotion in a novel environment
pre-pubertally. In contrast, post-pubertal nOBX animals
displayed a similar level of locomotion compared to
control animals. Previously, cumulative evidence has
shown that OBX produces hyperlocomotion in adult rats
and mice (Kelly et al., 1997; Wang et al., 2007).
Hyperlocomotion is associated with a failure to adapt to
a novel environment, as a trait of depression-like
behavior in this animal model (Kelly et al., 1997;
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89
Morales-Medina et al., 2013b). Several other neonatal
manipulations produce hyperlocomotion in a novel
environment post-pubertally. Such manipulations include
lesions of the ventral hippocampus (Flores et al., 2005),
amygdala (Solis et al., 2009), early social isolation
(Silva-Gomez et al., 2003b), blockade of nitric oxide
early in development (Morales-Medina et al., 2008) as
well as in adult offspring of dams with prenatal
inflammation (Aguilar-Valles et al., 2010). In the later
models, hyperlocomotion has been interpreted as a
schizophrenia-related behavior. In the OBX model as
well as those neonatal manipulations, hyperlocomotion
have been associated with maladaptations of the limbic
system.
The HBT has been used as a tool to measure anxiety,
emotionality and responses to stress and novel
environment (Crawley, 1985; Casarrubea et al., 2009).
We observed an increase in the number and time of
head-dipping behavior in nOBX rats pre-pubertally.
Despite the large body of literature observing
hyperlocomotion in the open field in OBX animals, little
attention has been given to the effect of removal of OB
in similar behaviors. Kamei’s group observed that OBX
increases head-dipping behaviors and two different
anxiolytic drugs normalize this abnormal behavior in the
OBX rat (Saitoh et al., 2006; Kamei et al., 2007). This
group interpreted such behavior as an impulsive-like
behavior. Thus, nOBX increases impulsive-like behavior
pre-pubertally as observed in the hyperlocomotion in
novel environment. Given the plasticity of the OB and
that the OB have only a secondary association with the
limbic system through connections to the hippocampus
and amygdala, these results suggest that nOBX results
in transient disrupted exploratory behaviors.
nOBX increases SI post-pubertally
The SI test has been validated as an experimental
paradigm to measure anxiolytic as well as anxiogenic
compounds or phenotypes (File and Seth, 2003; Li
et al., 2011). We observed that nOBX increases SI in
the rat post-pubertally. In contrast, our group and others
have found that OBX decreases active social contacts
in rats and mice (Wang et al., 2007; Morales-Medina
et al., 2012a). Wang et al. (2007) also observed deficits
in the elevated plus maze after OBX. These results
suggest that the age of lesion certainly account for this
phenomenon, and nOBX modify circuits in the brain that
modulate anxiety-like behavior in an opposite manner to
the adult lesion.
nOBX produces regrowth of OB
The OBs are a unique brain region since, along the
hippocampus, they receive newly born neurons
throughout the life (Altman, 1969; Weiss et al., 1996).
The SVZ stem cells give rise to neuroblasts that migrate
along the RMS to the OB, once there these cells migrate
radially and complete their differentiation into neurons
(Carlen et al., 2002; Ninkovic et al., 2007). With this in
mind, histological evaluation of nOBX bulbs showed
regrowth, but smaller than those in control animals.
90 G. Flores et al. / Neuroscience 259 (2014) 84–93

Table 1. Neonatal olfactory bulbectomy modify NMDA binding pre-pubertally

Region P30 P60

Normal Sham nOBx Normal Sham nOBx
⁄#
dPFC-inner 4.9 ± 0.23 5.0 ± 0.36 6.4 ± 0.42 4.5 ± 0.3 4.6 ± 0.15 4.9 ± 0.37
dPFC-outer 7.5 ± 0.31 6.9 ± 0.53 9.4 ± 0.64⁄⁄# 6.7 ± 0.28 7.1 ± 0.34 7.1 ± 0.33
Cg-inner 5.1 ± 0.32 5.4 ± 0.45 6.3 ± 0.37 4.6 ± 0.36 4.7 ± 0.13 5.1 ± 0.25
Cg-outer 7.0 ± 0.4 6.9 ± 0.3 8.3 ± 0.4⁄# 6.3 ± 0.35 6.5 ± 0.16 7.0 ± 0.23
Piriform 5.2 ± 0.37 4.7 ± 0.49 6.7 ± 0.2⁄⁄# 4.7 ± 0.41 5.0 ± 0.39 6.0 ± 0.32
Cpu 4.1 ± 0.22 4.2 ± 0.3 5.0 ± 0.2⁄# 3.9 ± 0.27 3.9 ± 0.21 4.1 ± 0.23
NAcc-core 4.1 ± 0.3 4.5 ± 0.32 5.0 ± 0.26 3.9 ± 0.2 4.4 ± 0.3 4.5 ± 0.23
NAcc-shell 4.1 ± 0.29 4.4 ± 0.33 5.2 ± 0.27# 3.9 ± 0.25 4.4 ± 0.31 4.5 ± 0.23
OT 5.4 ± 0.39 5.3 ± 0.31 6.5 ± 0.36 5.1 ± 0.54 5.6 ± 0.44 5.7 ± 0.4
CA1 striatu oriense 8.8 ± 0.76 9.7 ± 0.26 11.6 ± 0.28⁄## 8.0 ± 0.6 8.7 ± 0.50 9.9 ± 0.46
CA1 pyramidal cell 6.2 ± 0.63 6.8 ± 0.17 8.3 ± 0.47⁄# 6.3 ± 0.62 6.4 ± 0.61 8.2 ± 0.52
CA1 striatum reticulatum 10.5 ± 0.73 11.7 ± 0.28 12.9 ± 0.15 9.8 ± 0.53 10.4 ± 0.56 11.9 ± 0.38
CA1 molecular cell 8.1 ± 0.58 9.8 ± 0.27 11.4 ± 0.41⁄## 7.9 ± 0.43 8.7 ± 0.43 10.4 ± 0.56
CA2 8.6 ± 0.9 10.6 ± 0.4 11.4 ± 0.43# 8.5 ± 0.30 9.6 ± 0.47 10.0 ± 0.9
CA3 7.3 ± 0.7 8.2 ± 0.21 9.7 ± 0.43⁄# 6.7 ± 0.33 7.1 ± 0.29 7.8 ± 0.4
DG pyramidal cell 4.7 ± 0.92 4.5 ± 0.19 4.9 ± 0.2 3.8 ± 0.31 3.9 ± 0.42 5.0 ± 0.28
DG polymorphic cell 7.7 ± 0.64 8.2 ± 0.19 9.1 ± 0.41 6.6 ± 0.28 7.1 ± 0.36 7.7 ± 0.69
DG molecular cell 8.6 ± 0.23 9.3 ± 0.33 10.9 ± 0.28⁄⁄## 8.3 ± 0.41 9.8 ± 0.47 10.8 ± 0.51
AI-inner 9.3 ± 0.75 10.3 ± 0.83 10.1 ± 0.91 7.7 ± 0.64 8.8 ± 0.41 9.1 ± 0.43
AI-outer 11.9 ± 1.11 11.9 ± 0.89 12.5 ± 1.2 9.4 ± 0.91 10.3 ± 0.18 11.3 ± 0.71
LO-inner 8.9 ± 0.78 9.4 ± 0.67 9.4 ± 1.27 7.4 ± 0.59 8.4 ± 0.40 8.3 ± 0.45
LO-outer 11.5 ± 1.19 12.0 ± 0.76 12.3 ± 1.03 9.5 ± 0.89 10.7 ± 0.40 11.0 ± 0.68
VO-inner 9.8 ± 1.24 10.7 ± 0.66 9.5 ± 0.77 7.8 ± 0.60 8.6 ± 0.40 8.1 ± 0.37
VO-outer 11.6 ± 1.27 12.5 ± 0.57 12.4 ± 0.91 10.0 ± 0.92 11.1 ± 0.5 10.8 ± 0.54
Thalamus 3.9 ± 0.26 4.7 ± 0.22 4.9 ± 0.14# 3.5 ± 0.24 3.9 ± 0.28 4.0 ± 0.22
Thalamus-VPL 4.1 ± 0.25 5.0 ± 0.2 5.2 ± 0.12# 3.8 ± 0.27 4.2 ± 0.3 4.4 ± 0.24
Thalamus-VPM 3.7 ± 0.27 4.4 ± 0.28 4.6 ± 0.16 3.3 ± 0.22 3.6 ± 0.26 3.67 ± 0.2
BL-amygdala 3.9 ± 0.32 4.0 ± 0.41 4.5 ± 0.22 3.5 ± 0.25 3.8 ± 0.36 4.0 ± 0.23
PRH 3.6 ± 0.26 3.8 ± 0.52 4.3 ± 0.21 3.3 ± 0.12 3.9 ± 0.37 4.6 ± 0.54
Receptor levels are expressed in fmol/mg of wet tissue and represent the mean ± SEM of pooled values obtained from four sections per animal. Regions are defined
according to Paxinos and Watson Atlas (1986). AI, agranular insular cortex, BL, basolateral; CA, cornu Ammonis; Cg, cingulate cortex; CPu, caudate–putamen; DG, dentate
gyrus; LO, lateral orbital cortex; NAcc, nucleus accumbens; OT, olfactory tubercle; dPFC, dorsal part of the prefrontal cortex; PRH, perirhinal cortex, VPL, ventral
posterolateral; VPM, ventral posteromedial; VO, ventral orbital cortex. ⁄p < 0.05; ⁄⁄p < 0.01 compared with age-matched sham group; #p < 0.05; ##p < 0.01 compared with
age-matched normal group.

reduced size was partially reversed after the naris was

Fig. 5. Photomicrographs of [125I]MK-801 binding sites in the agran-
ular insular and orbital cortex. Abbreviations: dPFC, dorsal part of the
prefrontal cortex; LO, lateral orbital cortex; PrL, prelimbic cortex: VO,
ventral orbital cortex.
Previously, different groups have used sensory
deprivation models, where one or both OBs are deprived
of olfaction by naris closure or plug constriction early
postnatally, resulting in OB reduced size (Fiske and
Brunjes, 2001; McLean et al., 2001). Additionally, this
reopened (Cummings et al., 1997). However, if the naris
closure was performed after PD20, the recovery was
less dramatic (Brunjes, 1994). In this regard, early
postnatally, there is a considerable maturation of cells by
two different mechanisms (Ninkovic et al., 2007) as well
as cell death as part of refining connections in normal
OB. After PD14, new neurons have to integrate into a
preexisting neuronal network reducing the integration
rate (Belluzzi et al., 2003; Carleton et al., 2003).
Neonatal sensory deprivation extended for weeks the
process of cell death (Fiske and Brunjes, 2001),
decreased the expression of interleukin-1 beta (IL-1b)
(Lim and Brunjes, 1999) and microglia activation (Fiske
and Brunjes, 2000). In our model, the only process
affected is the cell proliferation from cells coming from
the RMZ. The long-lasting cell death process observed
after neonatal naris closure occurs is absent in nOBX
animals. In contrast to these early models of olfactory
deprivation where olfaction is recovered, adult OBX
results in long-lasting loss of olfaction (Kelly et al., 1997).
Thus, our data suggest that SVZ postnatal neurogenesis
plays a key role in the increase in size of the OB, and
most of the circuit integrate pre-pubertally after the
lesion, since the OB do not recover post-pubertally.

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 G. Flores et al. / Neuroscience 259 (2014) 84–93
Fig. 6. Photomicrographs of [125I]MK-801 binding sites in dorsal part of the prefrontal cortex (dPFC), cingulate cortex (Cg) and striatum (caudate–
putamen, CPu and nucleus accumbens, NAcc). Neonatal removal of olfactory bulbs produce an increase of NMDA receptor binding in the dPFC, Cg
and CPu compared to sham or control animals at pre-pubertal age.
Fig. 7. Photomicrographs of [125I]MK-801 binding sites in thalamus,
dorsal hippocampus (DH) and amygdala. Neonatal removal of
olfactory bulbs produce an increase of NMDA receptor binding in
the DH compared to sham animals at pre-pubertal age.
nOBX present normal olfaction
Smell has a key role in mammalian evolution, and this
highly complex and dynamic process uses a unique
mechanism of cellular and synaptic plasticity.
Cumulative evidence has shown that sensory
deprivation, as well as sensory enrichment, can
suppress or accelerate dendritic morphogenesis of
newborn OB interneurons (Saghatelyan et al., 2005;
Livneh et al., 2009; Kato et al., 2012; Yoshihara et al.,
2012). Therefore, the most exciting finding of our work
was that nOBX animals present normal olfaction at both
ages evaluated, suggesting that postnatal new born
cells integrate in the OB and become functional. Kato
et al. (2012) recently observed that 6 weeks after naris
reopening, animals with olfactory deprivation recover
their functionality. The difference between naris
occlusion and neonatal ablation of the OB is not only
the long-lasting cell death process, which produced
hypotrophy in the naris occlusion, but nOBX animals are
readily exposed to odors right after the surgery while the
OB of occluded animals remained deprived for weeks.
While a considerable number of new OB neurons are
added postnatally, the amount become stable from PD20
until +20 months of age (Tropepe et al., 1997; Enwere
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91
et al., 2004). New postnatal OB neurons integrate and
are functional (Carlen et al., 2002; Belluzzi et al., 2003;
Carleton et al., 2003). However, not all the cells that
integrate survive long. In this regard, Belluzzi et al.
(2003) proposed that these new cells compete for the
synaptic contacts and those that do not receive such
contact eventually die. Thus, the absence of competition
of new postnatal OB cells as well as odor stimulation
may increase the survival rate of these neurons in the
nOBX animals.
nOBX modify the NMDA receptor binding
pre-pubertally
In the present study, nOBX induced a deregulation in the
NMDA receptors pre-pubertally. Postnatal new OB
neurons are primarily GABAergic interneurons, with
unique properties compared to preexisting cells in the
OB. In this regard, Carleton et al. (2003) elegantly
showed the properties of cells that migrate from the
RVZ to the OB at different stages. This group and other
observed that RVZ cells required 2 weeks to migrate
and integrate/become functional in the OB circuit
(Belluzzi et al., 2003). These new cells first express
GABA receptors but once they stop migrating from the
RMS, they move radially and express NMDA-R. In the
next stage of maturation, these cells receive GABAergic
synapses followed by glutamatergic synapses. This
group also suggested that, whereas these glutamatergic
synapses could trigger dendritic growth, these cells only
release glutamate transiently at early stages of the
maturation process. On the other hand, Belluzzi et al.
(2003) observed that these newly generated neurons
present greater excitability. Therefore, this frame of
glutamatergic hyperactivation could account for the
increases in the expression of NMDA receptors
observed in the hippocampus, PFC and particularly high
within the PirC in the nOBX rat pre-pubertally. In this
regard, the OB has strong connections to the PirC
(Morales-Medina et al., 2013a) and the hippocampus
receives afferent projections directly from the OB, as
well as indirectly through the PirC, and has efferent
projections to the OB (de la Rosa-Prieto et al., 2009).
This increased expression of NMDA receptors in these
92 G. Flores et al. / Neuroscience 259 (2014) 84–93
brain regions could be associated with the postnatal
neurogenesis and the possible enhanced integration of
these new cells in the OB after nOBX.
Additionally, our group recently observed deregulated
neurogenesis in the DG of hippocampus in the OBX rat
(Morales-Medina et al., 2013b), and it could be possible
that this effect is also observed in nOBX and thus
explains, at least partially, the behavioral deficits.
CONCLUSION
Locomotion and attentional behavior are two of the most
salient (endurable) features of the adult OBX rat. The
changes between pre- and post-puberty may have been
influenced by several processes operating in OBX
animals. Additionally, the OB have shown a remarkable,
inherent capacity for neural plasticity compared to other
brain regions. Therefore, the clues used for postnatal
neurogenesis and synaptogenesis of the OB could be
useful for the development of new pharmacotherapy for
diseases including depression and schizophrenia.
CONTRIBUTORS
G.F., A.B.S.G., O.I.S. and A.R.M. designed the study and
wrote the protocol. G.F., A.B.S.G., and O.I.S. performed
the experiments. G.F., J.C.M.M., A.B.S.G., O.I.S. and
A.R.M. managed the literature searches and analysis,
G.F., J.C.M.M. and A.R.M. undertook the statistical
analysis and J.C.M.M., G.F. and A.R.M. wrote the first
draft of the manuscript. All contributing authors have
approved the final manuscript.
FUNDING SOURCES
Funding for this study was provided by grants from VIEP-
BUAP Grant (No. FLAG-SAL13-Ind), PROMEP and PIFI
(BUAP-CA-120), and CONACYT Grant (Nos. 129303
and 138663) to G. Flores. None of the funding
institutions had any further role in the study design, the
collection of data, analyses and interpretation of data,
writing of the report or in the decision to submit the
paper for publication.
Acknowledgments—The authors wish to thank Dr. Carlos
Escamilla for his help with the animal care. J.C.M.M. acknowl-
edges the CONACYT for the scholarship. J.C.M.M., O.I.S. and
G.F. acknowledge the National Research System of Mexico for
membership. Thanks are given to Dr. Ashutosh Rastogi and
the Kent State University writing commons for proofreading and
editing of the manuscript.
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