Download Olfactory tract transection in neonatal rats: Evidence for Mitral cell

Indian Journal of Experimental Biology
Vol. 50, November 2012, pp. 755-764
Olfactory tract transection in neonatal rats: Evidence for Mitral cell regeneration
and restoration of functional connectivity with its targets
S P Anil1, T R Laxmi2, Bindu M Kutty & T R Raju*
Department of Neurophysiology, National Institute of Mental Health and Neurosciences (NIMHANS),
Hosur Road, PB No. 2900, Bangalore 560 029, India
Received 14 May 2012; revised 9 August 2012
Central Nervous System (CNS) regeneration and repair mechanism are two important aspects of functional recovery in
the adult central nervous system following brain and spinal cord injury. Following olfactory tract transection in neonatal
rats, functional connectivity between the olfactory bulb and the piriform cortex gets re-established by 120 days. The
recovery of the dendritic morphology was associated with the synchronized oscillatory activity between olfactory bulb and
piriform cortex. Mitral cells which were regenerated after the transection showed profuse branching, indicative of their
undifferentiated state. However, normal dendritic morphology could be seen by 120 days after olfactory tract transection.
These results thus provide a supportive evidence for the restoration of the functional connections between the olfactory bulb
and the piriform cortex at 120 days.
Keywords: Local field potentials, Mitral cells, Olfactory tract transection, Piriform cortex, Regeneration
The mammalian olfactory system has a remarkable
capacity to continuously form new interneurons in
adulthood1 under normal conditions as well as
following an insult. The importance of host
regeneration as the possible mechanism leading to the
functional recovery following nerve transection has
been reported2. Munirathinam et al3. have
demonstrated the axonal regeneration following
olfactory tract transection. However, the study did not
provide any evidence for the functional restoration in
terms of integrated electrical activity between the bulb
and the piriform cortex following olfactory tract
transection.
The Mitral/tufted cells of the olfactory bulb (OB)
are extensively connected with piriform cortex4-7
and various higher cortical regions of the brain8,9.
Owing to these extensive connections with both
neocortical and subcortical structures, including the
hippocampus10, olfactory system is involved in
multiple behaviours such as detection of odours,
food-seeking behaviour, spatial memory and
various maternal behaviours11-13.
______________________
*
Correspondent author
Telephone: +91-80-2699 5178
Fax: +91-80-2656 4830
E-mail: [email protected];
[email protected]
1,2
SP Anil and TR Laxmi have contributed equally
The olfactory system undergoes both functional
and anatomical changes during the first few weeks of
postnatal life14-17. Olfactory tract transection (OTT) at
this stage causes a wide range of functional and
cytoarchitectural changes. However, the tract is
capable of undergoing regeneration and thus sparing
the behavioural abnormality as shown in male
hamsters18,19. The reinnervation of the olfactory bulb
with its target is specific to the regenerative capability
of Mitral cells of the olfactory bulb which is aided by
the olfactory stimulation12,20-22. After 45 days
post-transection, the olfactory tract fibers start
regenerating and by 240 days the fiber density was
near to normal3. It perhaps could be due to the unique
properties of neural progenitor cells of the
subventricular zone which migrate to the olfactory
bulb23 where they differentiate into interneurons, like
granule and periglomerular cells in adults24-27. The
migrating subventricular cells are capable of forming
Mitral cells in the transected rats28.
One of the effective strategies to probe how the
new connections are formed during regenerative
process29 is through investigating the morphological
changes in the Mitral cells following OTT. Analysis
of local field potentials (LFPs) combined with
dendritic morphology of Mitral cells could
substantiate the establishment of functional
connectivity. This is important because synchronized
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INDIAN J EXP BIOL, NOVEMBER 2012
oscillatory activity at theta frequency (4-12Hz) is
thought to be important for information processing in
any sensory systems30-36.
Accordingly, the present study reports a detailed
morphological assessment of regenerating Mitral cells
following OTT in Albino rat pups. The restoration of
functional integrity between the olfactory bulb and its
targets has been evaluated electrophysiologically by
studying the LFPs from olfactory bulb and piriform
cortex.
Materials and Methods
Animals—Albino rat pups (P0) of Wistar strain
were bred and raised in the Central Animal Research
Facility (CARF) of the Institute. Rats housed in
polypropylene cages were maintained on a 12 hour
light/ dark cycle and given food and water ad libitum.
All the experimental work was carried out with due
clearance from the Institutional animal ethics
committee. It was ensured that the room was properly
ventilated and the ambient temperature was
maintained.
Experimental groups— A total 144 male rats were
used. All rat pups were randomly divided into three
groups, lateral olfactory tract transected (OTT), sham
control (Sham) and normal control (NC). OTT was
achieved by lateral olfactory tract (LOT) transection
at postnatal day 4 (P4). Sham animals received the
same treatment, except that the LOT was not
transected. Normal control animals were not exposed
to any surgical procedures.
Surgery—Olfactory tract transection procedure was
carried out under halothane anesthesia as reported
earlier with a few modifications3,37. The olfactory
tract was accessed by unilateral incision on the skull,
posterior to the olfactory bulb. A sterile surgical blade
(no.11) was inserted along the orbital surface of
frontal bones and gently drawn back and forth to
ensure complete bilateral transection of the tract. The
wound was cleaned with antiseptic and sealed with
Healex spray. Following surgery the pups were
warmed and returned to their mothers or colony.
Same day old littermates were used as experimental
controls.
Tissue preparation for the morphological studies—
After completion of experimental procedures, rats
(n=8-10 per group) from all groups (NC, SC and
OTT) were sacrificed by decapitation at two time
points: 70 and 120 days following transection. The
cerebrum and olfactory bulbs were removed together,
carefully avoiding any damage to the bulbs. The
tissues including the frontal cortex were processed for
rapid Golgi impregnation38-40. After impregnation, the
olfactory bulbs were carefully removed and
embedded in hot wax. The sagittal sections of
140-160 µ thickness were obtained using rotary
microtome. Camera lucida neuronal tracing of Mitral
cells (with minimal damage, high quality of
impregnation) was done under 625x magnification
using a Leica microscope (Leica, Germany). The
criteria for selection of Mitral cells for drawing were:
(i) neurons must be confined to the Mitral Cell Layer,
(ii) they must be darkly stained, and (iii) neuron
should not have any truncation of branches. Neurons
were chosen from identical regions of olfactory bulbs.
A total 60-80 neurons (Mitral cells per group) were
used for quantitative analysis of dendritic
morphology.
To study the changes in the dendritic arborization,
the dendritic intersections of the Mitral cells were
taken with 20 µ increment using Sholl’s analysis41. In
brief, concentric circles were drawn in a transparent
sheet of 20 µ increments using a stage micrometer
scale at the same magnification at which the neurons
were drawn. Using the center of the cell as a reference
point, intersections and branching points were
measured from the cell body in each successive circle.
The number of dendritic branching points within two
adjacent concentric circles was counted as described
earlier38-40. The dendritic branching points and
intersections were studied up to a distance of 260 µm
from the soma.
Local field potential (LFP) recordings—A total 35
rat pups (n=8-10 per group) were used for the
electrophysiological study. The rats were randomly
divided into following four groups; normal control
(NC) for 70 days (n=8), NC for 120 days (n=7), OTT
for 70 days (n=10) and OTT for 120 days (n=10).
Implantation procedure—Rats were anesthetized
with a combination of Ketamine (80 mg/kg body
weight) and Xylazine (10 mg/kg body weight). In
addition, xylocaine (2%) was injected subcutaneously
before the surgery. The depth electrodes were
carefully implanted stereotaxically on bilateral sides
into the olfactory bulb and Piriform Cortex (PCx) as
per the dorso-ventral (DV) co-ordinates adapted from
Paxinos and Watson42. The insulated electrodes were
implanted stereotaxically into the olfactory bulb
(Mitral cell layer (MCL) (anterior-posterior (AP):
+6.7 mm, medio-lateral (ML): 2.0 mm, dorsoventral
(DV): 2.5 mm) and layer 3 PCx (AP: +1.5 mm, ML:
ANIL et al.: OLFACTORY TRACT TRANSECTION AND MITRAL CELL REGENERATION
3.9 mm, DV: 7.4 mm). Grounding electrode was
placed in the right parietal cortex. All these electrodes
were soldered and assembled into a 5-pin integrated
circuit socket and the connector was fixed to the skull
using the dental acrylic cement (Dental Products of
India Ltd., Mumbai, India) and was allowed to
recover from surgery. Healex was applied to all the
wound edges and the rat was finally placed back in its
home cage for 5 days for the recovery from the
surgical effects. Following surgical recovery, LFPS
recordings were carried out in their familiar home
cage concurrent with the spontaneous behaviour.
After 4-6 days of post surgical recovery, the rats
were connected to the 8-channel LFPS Polygraph
(Grass Model 78D Polygraph) via a flexible, shielded
cable, which did not restrain or cause discomfort to
the animal. The rat was provided with food and water
ad libitum during the recording session. The rats were
kept in the home cage with the electrodes connected
to the socket for the exploration of the environment,
prior to the recording session. The recording session
lasted for 10 min and the spontaneous behaviour of
the rat such as exploratory locomotion, grooming,
rearing and in the stationary phase (quiet) was
monitored throughout the recording session and
marked.
Offline analysis—All the spontaneous behaviours
exhibited by the adult transected and normal control
rats were quantified offline using spike 2 software.
The total percentage of different spontaneous
behaviour was taken. Spontaneous changes in the
behavioural pattern of rats in their home cage
following OTT and behavioural recovery of rats after
70 and 120 days post-transection were compared with
the data obtained from normal control rats. The
changes in behavioural patterns correlated with the
Mitral cell regeneration in rats. The analysis was done
in both normal control and olfactory tract transected
rats. For each time point, data from 10 rats per group
were used for both NC and OTT.
Power spectral analysis—The power spectral
analysis was carried out using fast fourier
transformation (FFT) to compare the synchronized
rhythmic features of olfactory oscillations following
OTT. The LFPs signals were amplified, band pass
filtered (0.3 Hz-0.3 KHz) and sampled at the rate of
1000 Hertz, digitized by a CED A/D converter (CED,
1401 A/D converter, Cambridge, UK), and offline
analysis was carried out using the Spike 2 software
(Cambridge, UK).
757
Histological verification of placement of
electrodes—After the completion of each set of
experiments, the rats were sacrificed to identify the
electrode placement sites, by taking coronal brain
sections and stained with cresyl violet as described43.
Statistical analysis—All statistical analyses were
conducted using the SPSS Software. One/Two-way
analysis of variance (ANOVA) procedures were used
in the data analysis. After obtaining significant
ANOVAs, post-hoc comparisons of means were
carried out with Bonferroni/Tukey’s multiple
comparison tests. In addition, Student’s t-test was also
used to compare between groups following OTT to
assess the spontaneous behaviours expressed by both
NC and OTT rats.
Results
Body weight changes due to OTT—The body
weight was evaluated every week after surgery and
the increase in weight was compared with that of
control rats. The mean body weight gain of OTT rats
did not differ significantly from control rats (Fig. 1).
Morphology of the olfactory bulb—Rapid Golgi
silver impregnation staining techniques were employed
in all groups of rats to assess the cytoarchitecture of
Mitral cells as well as structural changes in the
dendritic arborization due to OTT. The olfactory tract
appeared to establish an anatomical continuity with
their target by 70 days post transection. Gradual
recovery in the size of olfactory bulb in 70 days post
transection and with a complete recovery in the bulb
size by 120 days was also observed.
Morphological
assessment
of
dendritic
arborization—A representative example of camera
lucida tracings and photomicrographs of rapid Golgi
Fig. 1—Effect of olfactory tract transection (OTT) in rats on body
weight mass. [Data were subjected to two way ANOVA
indicating no significant differences between groups across the
age (n=15/group). Values are mean ± SE].
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INDIAN J EXP BIOL, NOVEMBER 2012
stained olfactory bulb neurons from NC and OTT are
given in Figs. 2a and b as well as 3a and b.
To investigate the impact of OTT on dendritic
arborization in a greater detail, a segmental analysis
was performed using the two-way ANOVA to track
the changes in dendritic length and branch points as a
function of radial distance from the cell soma. The
statistical analysis suggest that OTT-induces
significant changes in the dendritic arborization of
Mitral cells with a significant increase in the total
number of dendritic branches (F22,248=2.263, P<0.001)
and dendritic intersection (F18,208=3.782, P<0.0001).
After 70 days of OTT, there was a pronounced
decrease in the number of branching points of Mitralneurons within a distance of 120-160 µm from the
soma as revealed by Bonferroni’s post-hoc test
(Fig. 2c). In addition, an increased number of
branches within a distance of 40-80 µm from the
soma were observed in 70 days of the OTT group
(Fig. 2d). However, the numbers were comparable to
that of control rats by 120 days (F18,210=1.559,
P<0.07) (Fig. 3d). Bonferroni’s multiple comparison
test further revealed that dendritic hypertrophy
induced by OTT was gradually ameliorated from
70 days (Fig. 2c) to 120 days (Fig. 3c) after OTT with
a significant reduction in the number of intersections
at 100-140 µ segment from the soma respectively.
The differences in both dendritic intersections and in
dendritic branches were not prominent between the
control and sham control groups at any time points
(Figs 2 and 3).
In addition to dendritic intersections and dendritic
branch points, we have also observed several
structural differences in Mitral cells between NC and
OTT rats. Firstly, the olfactory tract transection
induced distortion in the Mitral cell layer (MCL) of
the 70 days OTT group (Fig. 4). Secondly, non-spiny
Mitre-shaped cells were also observed in the central
core of the olfactory bulb near to the rostral migratory
stream (RMS). Thirdly, compared with normal
olfactory bulb, olfactory bulb of OTT group showed
increased vascularity characteristic feature of
regenerating tissue during establishment of its
connections with its target44,45. Finally, more studded
spinier granule cells were specifically detected in the
granule cell layer of OTT rats indicating neural
reaction to olfactory tract transection.
Histological confirmation of the electrode
placement—Histological analysis with Cresyl
violet stained sections confirmed that all bipolar
Fig. 2—Olfactory tract transection after 70 days increases dendritic arborization in short shaft Mitral neurons of olfactory bulb compared
with NC and Sham. (A) representative Mitral cell tracing from 70 days OTT group; (B) example of Golgi stained Mitral cell; (C)
dendritic intersections; (D) total number of dendritic branches of Mitral cells at various distance from soma showing nerve sprouting
following 70 days post-transection (n=10/group and 10 neurons/rat). NC=normal control, Sham=sham control, OTT=olfactory tract
transected. Values are mean ± SE. P values: ** P value: <0.01, *** P value: <0.001 between NC and OTT rats.
ANIL et al.: OLFACTORY TRACT TRANSECTION AND MITRAL CELL REGENERATION
759
Fig. 3—Olfactory tract transection after 120 days did not show any significant differences in dendritic arborization along a wide range of
dendritic segment in comparison with NC and sham group. (A) representative Mitral cells tracing showing no detectable nerve sprouting
after 120 days OTT; (B) example of Golgi stained Mitral cell; (C) dendritic intersections and (D) total number of dendritic branches of
Mitral cells at various distance from soma; (n=10rats/group and 10 neurons/rat). NC=normal control, Sham=sham control,
OTT=olfactory tract transected. Values are mean + SE *P<0.05 between NC and OTT rats.
Fig. 4—Mitral cell layer in (A) normal control (NC), and (B) olfactory tract transected (OTT) group of rat. White arrow indicates Mitral
cell layer (MCL). Note disrupted MCL in OTT olfactory bulb. Black arrow indicates Mitral –like cell in central core of the olfactory bulb
(Rostral Migratory Stream) of OTT rat.
electrodes were indeed implanted into the MCL and
PCx (layer 3) (Fig. 5).
Spontaneous exploratory behaviour of the rats in
home cage—The home cage behaviour of the rat was
compared between intact (NC and sham) and rats with
bilateral OTT. After 70 days of olfactory tract
transection, OTT rats reared more frequently than NC
rats (P<0.001) (Fig. 6). However, 120 days OTT rats
exhibited increased exploratory locomotion (t-test,
P<0.05) and with normal spontaneous behaviours.
Associative changes in the neuronal activity of
MCL and PCx during spontaneous exploratory
locomotion behaviour in the home cage—Since the
exploratory locomotor activity in the home cage has
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INDIAN J EXP BIOL, NOVEMBER 2012
to be considered in evaluating motivation-induced
responses following transection, a detailed spectral
analysis of the neuronal activities recorded from
olfactory bulb and Piriform Cortex during this
behaviour was carried out.
A typical example showing the local field
potentials recorded bilaterally from the MCL and PCx
when the rat was exploring the home cage is shown in
Fig. 5—Cresyl violet stained brain sections showing the electrode
placement sites (arrow) in the (i) Mitral cell layer (MCL) of the
olfactory bulb and (ii) layer 3 of the piriform cortex.
Fig. 6—Comparisons of the spontaneous behaviours exhibited by
the rat in their home cage. (A) 70 days (n=10 for NC and n=12 for
OTT), and (B) 120 days (n=11 for NC and n=10 for OTT) rats.
Values are mean + SE, *P<0.05 comparisons were made between
normal control (NC) and olfactory tract post-transected (OTT)
group.
Fig. 7. Averaged data showing power spectra at theta
range (4-12 Hz range) calculated for the periods of
exploratory locomotor activity from MCL and PCx in
70 and 120 days NC and OTT rats is given in Fig. 8.
During the exploratory locomotion, there was a peak,
evident at 4-12 Hz in the PCx (bilaterally) of the NC
rats at all age groups. Clear peaks at 12-20 Hz were
also present in PCx and MCL (right hemisphere)
during spontaneous exploratory locomotion. A
significant increase in the relative power at theta
range was seen in PCx of 70 days OTT rats when
compared to NC (t1,16=2.137; P<0.05) (Fig. 8c). The
difference in the theta peaks in MCL or PCx was not
obvious between NC and OTT rats of 120 days group
(Fig. 8 a-d). In addition, it was also observed that
when the rat was exhibiting exploratory walking in
the home cage, oscillatory activity at theta range was
visible in both PCx, as well as in the MCL of
olfactory bulb. However, the synchronized theta
oscillation was more pronounced in the PCx than in
the olfactory bulb of all the age groups. The
synchronization was seen only when the rat was
exploring the home cage, but did not occur when the
animal was immobile.
Further to quantify the reinnervation of the Mitral
cells with their target (Piriform cortex), coherence
analysis was carried out. There was a significant
decrease in theta coherence between the right
Fig. 7—Representative example showing continuous recording of
extracellular local field potentials from Mitral cell layer (MCL) of
olfactory bulb (OB) and layer 3 of piriform cortex. (A) 70 days;
(B) 120 days rats of NC and OTT groups. Normal control (NC)
and olfactory tract transected (OTT). Amplitude 100µV; Time
scale: 1 second.
ANIL et al.: OLFACTORY TRACT TRANSECTION AND MITRAL CELL REGENERATION
olfactory bulb and the right piriform cortex of 70 days
OTT rats at 4-12 Hz (t1,16= 2.226; P<0.04) (Fig. 9).
Reduction in the coherence was not evident in
120 days-OTT rats (P>0.05).
Discussion
The present findings extend our understanding of
regeneration in the central nervous system and further
throw light on the lesion induced genesis of new
Fig. 8—The effect of olfactory tract transection on theta
frequency in the olfactory bulb and piriform cortex. (a) The
average histogram showing the power spectral density (4-12Hz)
from left olfactory bulb in the Mitral cell layer (MCL); (b) right
olfactory bulb; (c) left piriform cortex and (d) right piriform
cortex. Filled bars is from 70days; hashed bars is from 120 days
group. NC (n=8 for 70 day and n=7 for 120 day) and OTT (n=10).
Value are mean+SE, *P<0.05.
Fig. 9—Changes in LFP theta (4-12Hz) coherence from NC and
OTT rats during exploratory behavior. (a) average LFP coherence
between right MCL and right PCx at 70 days (b) 120 days
(c) average coherence between left MCL and left PCx at 70 days
and (d) 120 days group of rats. Data were compared between NC
(filled box) and OTT (empty box) groups of rats. MCL, Mitral
cell layer of olfactory bulb; PCx, piriform cortex; NC, normal
control (n=8); OTT, olfactory tract
761
Mitral cells and their connectivity with the target. The
electrophysiological data revealed that neurons in
Mitral cell layer of the olfactory bulb developed
synchronized firing with piriform cortex after
120 days of olfactory tract transection. The present
results also demonstrate the structural and functional
restoration of the Mitral cells following early
postnatal olfactory tract transection, although
produced an initial degeneration of Mitral cells.
Structural recovery of Mitral cells following
bilateral olfactory tract transaction—The olfactory
bulb has a considerable degree of plasticity46-48 and
has the capability to undergo successful regeneration
following olfactory tract transection3. In the present
study, an initial exuberant growth of dendrites was
observed within a distance of 40-80 µm by 70 days
after the transection which could represent a possible
mechanism involved in the reinnervation of Mitral
cells with piriform cortex49,50. By 120 days a gradual
reduction in the dendritic intersections at distal segments
of mitral cells was observed which was comparable to
that of controls. This pattern of dendritic proliferation
followed by pruning and ending with a single apical
dendrite51 indicates an important phenomenon
associated with the maturation and reinnervation of
Mitral cells with its target. The presence of increased
vascularity with enhanced dendritic sprouting in the bulb
following OTT could also be associated with
regeneration related changes45. Finally, rapid Golgi
stained sections along the line of transection strengthens
an argument for a proper continuity in the central core of
the olfactory tract indicating that rodent olfactory tract is
capable of regenerating spontaneously following
neonatal olfactory tract transection.
Electrophysiological correlates of the home cage
behaviour of the rat to demonstrate the functional
integration of the Mitral cells with Piriform cortex
following OTT—Based on the available literature
from the hippocampal and non-hippocampal studies,
it is suggested that synchronization of the cortical
neurons depends on the periodic activation of
inhibitory
interneurons52
and
information
53
processing . In the present study, decreased
rhythmicity was observed at theta range within and
between the olfactory bulb and piriform cortex after
70 days of OTT. Asynchronous relationship within
and between the bulb and cortex, more predominantly
with the right hemispheres during exploratory activity
reflects the functional decoupling of the anatomical
circuit at the site of the transection of olfactory tract.
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INDIAN J EXP BIOL, NOVEMBER 2012
The possible mechanisms that may be responsible for
the desynchronized pattern of neuronal activity in
both the bulb and the cortex following OTT could be
due to the following reasons: one of the unique
characteristic features of the olfactory cortex is that
all the primary sensory inputs from the olfactory bulb
are processed and filtered at the level of piriform
cortex54 without the involvement of the thalamus as
seen in other sensory systems55. The filtering of the
incoming signals at the cortical neurons during
exploratory behaviour can result in desynchronization
with a decreased rhythmicity that may occur as a
result of less entrainment of principal neurons either
with their afferents or with local interneurons as
indicated with higher theta power in the piriform
cortex. This highlights the significance of filtering of
afferent signals at piriform cortex56, resulting in the
convergence of olfactory inputs, which is necessary
for the discriminatory information processing.
Further a question which may be asked here is
what may be the spontaneous behavioural
consequences of the observed morphological and
electrophysiological changes in olfactory bulb and
piriform cortex. Odour-induced deficits have been
reported in the behaviour after bulbectomy57, and
lateral olfactory tract transection19, suggesting the
lesion-induced memory deficits in tests based on
olfactory cues. The present results also suggest that
transection of olfactory nerve fibers leads to
deafferentation of olfactory bulbs and a loss of
olfactory mediated behaviour58. However, the present
findings show the spontaneous behavioural recovery
of the OTT rats in their home cage by 120 days. In
general, OTT also led to certain behavioural
enhancements, such as increased time spent in
exploring and rearing behaviour in the home cage as
compared to normal control rats.
Consideration of possible lesion-dependent
changes in dendritic sprouting of afferent neurons
and increased asynchronous firing in olfactory bulb
and piriform cortex remains speculative. It is
possible that OTT-induced dendritic re-modeling in
the Mitral cells of the olfactory bulb after OTT could
be one of the triggering factors for the regeneration
of Mitral axons to re-innervate the piriform cortex.
Lower coherence of LFP between MCL and PCx
with recovery of the dendritic sprouting indicates the
bulbocortical coherences probably driven by
excessive excitatory inputs from Mitral axons into
the piriform cortex.
Since the olfactory tract transection was bilateral
and complete leading to total anosmia, an
alternative mechanism exists to compensate for the
functional deficit which is critically required for the
survival of the animal. The present results have
shown that the olfactory tract transected rats
survived the axotomy and reached the experimental
time points without any significant weight loss or
food intake. The present study, thus, provides
several potential evidences to support the
compensatory
replacement
of
the
neural
connections between olfactory bulb and piriform
cortex following olfactory tract transection.
Acknowledgement
The authors are thankful to Department of
Biotechnology (DBT), India for financial assistance
and NIMHANS for the support.
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