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Rom J Morphol Embryol
2011, 52(3):879–886
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Enhanced dendritic arborization of hippocampal
CA3 neurons by Bacopa monniera extract
treatment in adult rats
VENKATA RAMANA VOLLALA1), SUBRAMANYA UPADHYA2), SATHEESHA NAYAK3)
1)
Department of Anatomy,
Rajiv Gandhi Institute of Medical Sciences (RIMS), Adilabad, India
2)
Department of Physiology,
St. George’s University School of Medicine, Grenada, West Indies
3)
Department of Anatomy,
Melaka Manipal Medical College (Manipal Campus), Manipal University, India
Abstract
Objective: Bacopa monniera (BM), a traditional Ayurvedic medicine has been used in treatment for a number of disorders, particularly
those involving anxiety, intellect and poor memory. The current study examined the effects of standardized extract of Bacopa monniera
on the dendritic morphology in adult rats of hippocampal CA3 neurons, one of the regions concerned with learning and memory. Materials
and Methods: Adult Wistar (2.5-month-old) rats were designated into 2-, 4- and 6-week treatment groups. Rats in each of these groups
were divided into 20 mg/kg, 40 mg/kg and 80 mg/kg dose groups (n=8 for each dose). These rats along with age-matched control rats
were then subjected to spatial learning (T-maze) and passive avoidance tests. Subsequent to the T-maze and passive avoidance tests,
these rats were killed by decapitation, brains were removed and hippocampal neurons were impregnated with silver nitrate (Golgi staining).
Hippocampal CA3 neurons were traced using camera lucida. Dendritic branching points (a measure of dendritic arborization) and dendritic
intersections (a measure of dendritic length) were quantified. These data were compared with control rats. Results and Conclusions:
The results showed improvement in spatial learning performance and enhanced memory retention in rats treated with BM extract. There
was a significant increase in the dendritic intersections and dendritic branching points along the length of both apical and basal dendrites in
rats treated with BM extract for four and six weeks. However, the rats treated with BM extract for two weeks did not show any significant
change in hippocampal CA3 neuronal dendritic arborization. We conclude that constituents present in BM extract have neuronal dendritic
growth stimulating properties.
Keywords: Bacopa monniera, spatial learning, passive avoidance, hippocampal CA3 neurons, dendritic arborization, memory.
Introduction
Herbs, a principal form of medicine in developing
countries, are becoming popular throughout the
developing and developed world. In western societies,
there is increasing interest in herbal medicines, which
are often perceived as a more ‘natural’ and ‘soft’
treatment compared to synthetic drugs [1]. Before the
development of modern medicine, people relied on a
large arsenal of natural remedies for the treatment of
central nervous system (CNS) related maladies. The
medicinal plants and/or their constituents which have
been widely used for their reputed effectiveness in
functions of CNS are as follows: Acorus calamus,
Bacopa monniera, Celastrus paniculatus, Centella
asiatica, Clitoria ternatea, Convolvulus pluricaulis,
Ginkgo biloba, Nardostachys jatamansi, Scutellaria
baicalensis, Withania somnifera.
Bacopa monniera (Linn.), syn. Herpestis monniera
(Linn.), a member of the Scrophulariaceae family, is a
small, creeping herb with numerous branches, small
oblong leaves, and light purple flowers. The genus
Bacopa includes over 100 species of aquatic herbs.
It commonly grows in wet, marshy areas throughout
India, Nepal, Sri Lanka, China, Taiwan, and Vietnam;
and is also found in Florida and other southern states
of the USA. The herb can be found at elevations from
sea level to altitudes of 4400 feet [2–6]. Flowers and
fruits appear in summer and the entire plant is used
medicinally [7].
This medicinal plant is popularly known as Brahmi.
The name Brahmi is derived from the word “Brahma”,
the mythical “creator” in the Hindu pantheon. Because
the brain is the centre for creative activity, any
compound that improves brain health is called Brahmi.
The plant has been used in Indian folklore as a nerve
tonic [8].
In the ancient Indian system of medicine, viz.,
“Ayurveda”, Bacopa monniera (BM) has been classified
under “Medhya rasayana”, i.e., medicinal plants
rejuvenating intellect and memory. The ancient classical
Ayurvedic treatises, viz., “Charak samhita”, “Susruta
samhita”, and “Astanga hridaya”, have prescribed BM
for the promotion of memory, intelligence, and general
performance. Therefore, this plant has been investigated
in several laboratories in India for its neuropharmaco-
logical effect [9–11]. Its traditional memory-enhancing
claim has been established experimentally in several
animal experimental models of learning [12–15]. There
R J M
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Romanian Jo urnal of
Morphology & Embryology
http://www.rjme.ro/
Venkata Ramana Vollala et al.
880
is no evidence which shows the effect of this plant
extract on the brain regions involved in learning and
memory, namely the hippocampus [16–18], amygdala
and limbic cortex. The cornu ammonis (CA) region,
particularly the CA3 sub region of the hippocampus, is
the key structure of the brain involved in learning and
memory [19–22]. The current study examined the
effects of standardized extract of BM on the dendritic
morphology of hippocampal CA3 neurons in adult rats.
Materials and Methods
Animals and experimental groups
Wistar albino rats of random sex, approximately
2.5-month-old and weighing about 150–200 g were
obtained from “the central animal house”, Manipal
University, Manipal, India. The total number of animals
used for the study was 120 (72 experimental rats and 48
control rats). The experimental protocol was approved
by the institutional animal ethical committee for
experimental clearance IAEC/KMC/02/2005–2006. The
rats were fed “Amrut rat and mice pellet” manufactured
by Pranav Agro Industries Ltd., E/5–6, M.I.D.C.,
Kupwad Block, SANGLI – 416436 (Maharashtra), India.
Four rats were housed in each polypropylene cage and
maintained in a 12:12 hour cycle of dark and light.
Three time intervals (two, four or six weeks) were
used and each time group was divided into dose groups
(n=8 for each dose). Each dose group was fed 20 mg/kg,
40 mg/kg and 80 mg/kg of standardized extract of BM
daily for two, four or six weeks. Age-matched normal
control group (NC) and a gum acacia vehicle control
group (GAC) (n=8 in both groups) were also maintained
for each time period.
Extraction and administration of BM
Standardized plant extract of BM was supplied by
the herbal manufacturer, M/s. Natural Remedies Private
Limited, Bangalore, India. The shelf life of this extract
is 2 years.
The first step was extraction of the botanically
identified plant material with alcohol. The alcoholic
extract was then re-extracted with water and the water-
soluble matter was retained. The final re-extract was
concentrated and dried to make a powder. Phytochemical
analysis revealed that the final extract contained
approximately 10% w/w (10% of the total mass of the
extract) of the active ingredients (Bacosides A and B)
by high-performance liquid chromatography (HPLC)
and high-performance thin layer chromatography
(HPTLC).
The BM extract was administered orally along with
5% gum acacia, using an oral feeding tube and syringe.
Behavioral tests
Following treatment, all groups (NC, GAC, and
BM) were subjected to behavioral tests during the night
(starting at 7 PM). The behavioral tests consisted of a
spatial learning (T-maze) test and a passive avoidance
test and were done as detailed earlier [23].
Rapid Golgi staining procedure
Subsequent to the behavioral tests, these rats were
anesthetized with ether, sacrificed by cervical dislocation
and the brains were removed. Each cerebral hemisphere
was cut coronally to two equal pieces, cortex was
removed from the posterior part and hippocampi were
dissected (but were not compared) and fixed in rapid
Golgi fixative. Tissue was processed for rapid Golgi
staining as detailed previously [24]. Briefly, tissues
were fixed for 5 days in Golgi fixative, and impregnated
with 1.5% aqueous silver nitrate solution for 48 hours.
Sledge microtome sections of 120 µm thicknesses were
cut (as many serial sections as possible in the tissue),
dehydrated, cleared and mounted with Distrin plasticizer
xylene mounting media.
Camera lucida tracing
8–10 hippocampal CA3 neurons of each rat were
traced using camera lucida from the slide, and their
dendritic branching points and dendritic intersections
were quantified. Both right and left side hippocampal
CA3 neurons were used. Neurons with minimal overlap
of dendrites, heavily impregnated with silver nitrate and
without truncate dendrites, were selected for tracing.
Quantification of dendritic branching points
and dendritic intersections
The concentric circle method of Sholl DA (1956)
was used for dendritic quantification [25]. Five
concentric circles on a transparent sheet with a radial
distance of 20 µm between them were used for dendritic
quantification (dendritic branching points and inter-
sections). The sheet was placed on a neuron tracing such
that the center of the cell body of the neuron coincided
with the center of the concentric circles. The number of
branching points between the two concentric circles, i.e.
within each successive 20 µm concentric zone (ring), was
counted. The dendritic intersection is the point where a
dendrite intersects the given concentric circle (Figure 1).
Figure 1 – Diagram showing a hippocampal CA3
neuron and the scheme of dendritic quantification.
A – Apical dendrites, B – Basal dendrites, DBP –
Dendritic branching points, DI – Dendritic inter-
sections, S – Soma, CC – Concentric circles, and 1 to
5 – Concentric zones.
The dendritic intersections at each concentric circle
were counted. Both branching points and intersections
were counted up to a radial distance of 100 µm from the
Enhanced dendritic arborization of hippocampal CA3 neurons by
Bacopa monniera
extract treatment in adult rats
881
center of the soma. Mean number of dendritic branching
points in each concentric zone and number of dendritic
intersections at each concentric circle were calculated.
This method of scoring was applied for both apical and
basal dendritic quantification. The apical dendrite of
pyramidal neuron in CA3 region is a single pole
extending from the apex of soma. It divides into 2–3
main branches from which the secondary and tertiary
branches arise. The basal dendrites of pyramidal
neurons arise from several places along the base of the
soma and these repeatedly branch producing a dense tuft
(Figure 1).
Statistical analysis
Data was analyzed using analysis of variance
(ANOVA) followed by Bonferroni’s test (post hoc) using
GraphPad Prism, version 2.01.
Results
The rats treated with all doses of BM showed
improvement in spatial learning performance and
enhanced memory retention compared to normal control
rats [23]. Briefly, when treated for a longer duration
(four and six weeks), rats showed significant
improvement in their learning behavior in all (20, 40,
and 80 mg/kg) dose groups. During spatial learning
T-maze tests, they showed an increased number of
alternations and decreased percentage bias during
spontaneous alternation test and increased percentage
of correct responses during rewarded alternation test.
In the passive avoidance tests, there was no significant
change in behavior during exploration. However, during
the retention test, rats treated for four and six weeks at
all three doses (20, 40, and 80 mg/kg) spent less time in
the smaller compartment, suggesting improved memory
retention [23].
Hippocampal CA3 neuronal dendritic
quantification
Hippocampal CA3 neuronal dendritic analyses in
BM extract (40 and 80 mg/kg) treated rats (four and six
weeks) showed a significant increase in dendritic length
and branching both in the apical and basal dendrites
(Figures 2–11).
Figure 2 – Photomicrographs (A1, B1, C1, D1) and
camera lucida tracings (A2, B2, C2, D2) of Golgi-
stained hippocampal CA3 neurons from control rats
(A1, A2) and rats treated with BM for four weeks at
doses of 20 mg/kg (B1, B2), 40 mg/kg (C1, C2), and
80 mg/kg (D1, D2). A significant increase in dendritic
arborization in BM 40 and 80 mg/kg groups is
demonstrated.
Figure 3 – Apical dendritic intersections of hippo-
campal CA3 neurons in rats treated with BM for
four weeks, control and gum acacia rats. NC vs.
BM 40 mg/kg: *** p<0.001; NC vs. BM 80 mg/kg:
$$ p<0.01, $$$ p<0.001.
Figure 4 – Apical dendritic branching points of
hippocampal CA3 neurons in rats treated with BM
for four weeks, control and gum acacia rats at
different concentric zones (CZ) and total number of
branching points. Each value represents the mean +
standard deviation of 8–10 neurons from each rat.
NC vs. BM 40 mg/kg: ** p<0.01, *** p<0.001; NC
vs. BM 80 mg/kg: $ p<0.05, $$ p<0.01, $$$ p<0.001.
Figure 5 – Basal dendritic intersections of hippo-
campal CA3 neurons in rats treated with BM for
four weeks, control and gum acacia rats. NC vs. BM
40 mg/kg: * p<0.05, ** p<0.01, *** p<0.001; NC vs.
BM 80 mg/kg: $ p<0.05, $$ p<0.01, $$$ p<0.001.
Figure 6 – Basal dendritic branching points of
hippocampal CA3 neurons in rats treated with BM
for four weeks, control and gum acacia rats at
different concentric zones (CZ) and total number
of branching points. Each value represents the mean
+ standard deviation of 8–10 neurons from each
rat. NC vs. BM 40 mg/kg: *** p<0.001; NC vs. BM
80 mg/kg: $$$ p<0.001.
Venkata Ramana Vollala et al.
882
Figure 7 – Photomicrographs (A1, B1, C1, D1) and
camera lucida tracings (A2, B2, C2, D2) of Golgi-
stained hippocampal CA3 neurons from control rats
(A1, A2) and rats treated with BM for six weeks at
doses of 20 mg/kg (B1, B2), 40 mg/kg (C1, C2), and
80 mg/kg (D1, D2). A significant increase in dendritic
arborization in BM 20, 40 and 80 mg/kg groups is
demonstrated.
Figure 8 – Apical dendritic intersections of hippo-
campal CA3 neurons in rats treated with BM for
six weeks, control and gum acacia rats. NC vs. BM
20 mg/kg: ## p<0.01; NC vs. BM 40 mg/kg: ***
p<0.001; NC vs. BM 80 mg/kg: $ p<0.05, $$$ p<0.001.
Figure 9 – Apical dendritic branching points of
hippocampal CA3 neurons in rats treated with BM
for six weeks, control and gum acacia rats at
different concentric zones (CZ) and total number of
branching points. Each value represents the mean +
standard deviation of 8–10 neurons from each rat.
NC vs. BM 20 mg/kg: # p<0.05, ## p<0.01; NC vs. BM
40 mg/kg: * p<0.05, ** p<0.01, *** p<0.001; NC vs.
BM 80 mg/kg: $ p<0.05, $$ p<0.01, $$$ p<0.001.
Treatment with all doses (20, 40 and 80 mg/kg) for
two weeks did not alter the dendritic arborization. There
was no difference in dendritic length and branching
pattern between control and gum acacia treated rats,
suggesting that daily handling of the rats (handling
stress and vehicle) itself did not alter dendritic pattern.
Since there was no significant difference in the dendritic
length and branching between the control and vehicle
groups, only comparisons between the control and
experimental groups are detailed.
Figure 10 – Basal dendritic intersections of hippo-
campal CA3 neurons in rats treated with BM for
six weeks, control and gum acacia rats. NC vs. BM
20 mg/kg: ## p<0.01; NC vs. BM 40 mg/kg: ** p<0.01,
*** p<0.001; NC vs. BM 80 mg/kg: $$ p<0.01, $$$
p<0.001.
Figure 11 – Basal dendritic branching points of
hippocampal CA3 neurons in rats treated with BM
for six weeks, control and gum acacia rats at
different concentric zones (CZ) and total number of
branching points. Each value represents the mean +
standard deviation of 8–10 neurons from each rat.
NC vs. BM 20 mg/kg: ## p<0.01, ### p<0.001; NC vs.
BM 40 mg/kg: ** p<0.01, *** p<0.001; NC vs. BM
80 mg/kg: $ p<0.05, $$ p<0.01, $$$ p<0.001.
Four weeks treatment
Apical dendritic intersections (Figure 3)
No significant change in dendritic intersections/
concentric circle was noted in BM 20 mg/kg group when
compared to normal control group. Both BM 40 and
80 mg/kg groups showed a significant increase in
dendritic intersections at 60, 80 and 100 µm concentric
circles (60 µm concentric circle: 2.41±0.39 in normal
control group vs. 3.76±0.51 in 40 mg/kg group, p<0.001
and 3.59±0.88 in 80 mg/kg group, p<0.01, 80 µm
concentric circle: 3.28±0.60 in normal control group vs.
5.84±0.69 in 40 mg/kg group, p<0.001 and 5.66±0.73 in
80 mg/kg group, p<0.001, 100 µm concentric circle:
3.59±0.72 in normal control group vs. 6.20±0.76 in
40 mg/kg group, p<0.001 and 5.93±0.68 in 80 mg/kg
group, p<0.001).
Apical dendritic branching points – At different
concentric zones (Figure 4)
No significant change was observed in the dendritic
branching points in any of the concentric zones in BM
20 mg/kg group. However both BM 40 and 80 mg/kg
groups showed significant increase in dendritic branching
points in 40–60, 60–80 and 80–100 µm concentric zones
(40–60 µm concentric zone: 0.87±0.27 in normal control
group vs. 1.81±0.74 in 40 mg/kg group, p<0.001 and
Enhanced dendritic arborization of hippocampal CA3 neurons by
Bacopa monniera
extract treatment in adult rats
883
1.65±0.51 in 80 mg/kg group, p<0.01, 60–80 µm
concentric zone: 1.38±0.33 in normal control group vs.
2.36±0.41 in 40 mg/kg group, p<0.001 and 2.27±0.45 in
80 mg/kg group, p<0.01, 80–100 µm concentric zone:
1.26±0.45 in normal control group vs. 2.17±0.62 in
40 mg/kg group, p<0.01 and 2.03±0.40 in 80 mg/kg
group, p<0.05).
Total number of dendritic branching points
There was no significant change in the total number
of branching points in BM 20 mg/kg group when
compared to normal control group. However, in both
BM 40 and 80 mg/kg groups there was a significant
increase in the total number of branching points
(4.52±0.85 in normal control group vs. 7.71±0.69 in
40 mg/kg group, p<0.001 and 7.21±1.05 in 80 mg/kg
group, p<0.001).
Basal dendritic intersections (Figure 5)
There were no significant changes in the dendritic
intersections at any of the concentric circles in BM
20 mg/kg group when compared with the normal control
group. Both BM 40 and 80 mg/kg groups showed
significant increase in dendritic intersections at 40, 60,
80 and 100 concentric circles (40 µm concentric circle:
5.86±0.81 in normal control group vs. 7.81±1.16 in
40 mg/kg group, p<0.01 and 7.58±1.24 in 80 mg/kg
group, p<0.01, 60 µm concentric circle: 6.63±1.08 in
normal control group vs. 9.67±1.32 in 40 mg/kg group,
p<0.001 and 9.12±1.19 in 80 mg/kg group, p<0.001,
80 µm concentric circle: 4.13±0.72 in normal control
group vs. 8.18±0.76 in 40 mg/kg group, p<0.001 and
7.74±0.83 in 80 mg/kg group, p<0.001, 100 µm
concentric circle: 3.21±0.61 in normal control group vs.
4.43±0.62 in 40 mg/kg group, p<0.05 and 4.32±0.74 in
80 mg/kg group, p<0.05).
Basal dendritic branching points – At different
concentric zones (Figure 6)
No significant changes were observed in the dendritic
branching points at any of the concentric zones in
BM 20 mg/kg group when compared to normal control
group. However, both BM 40 and 80 mg/kg groups
showed a significant increase in dendritic branching
points in concentric zone 20–40 µm (2.63±0.39 in
normal control group vs. 4.17±0.52 in 40 mg/kg group,
p<0.001 and 4.11±0.42 in 80 mg/kg group, p<0.001),
concentric zone 40–60 µm (1.53±0.42 in normal control
group vs. 2.97±0.46 in 40 mg/kg group, p<0.001 and
2.83±0.53 in 80 mg/kg group, p<0.001), concentric zone
60–80 µm (0.53±0.27 in normal control group vs.
1.57±0.50 in 40 mg/kg group, p<0.001 and 1.48±0.39 in
80 mg/kg group, p<0.001).
Total number of branching points
There was no significant change in the total number
of branching points in BM 20 mg/kg group when
compared to normal control group. However, the total
number of branching points was found significantly
increased in BM 40 and 80 mg/kg groups (5.37±0.57 in
normal control group vs. 9.72±0.73 in 40 mg/kg group,
p<0.001 and 9.63±0.55 in 80 mg/kg group, p<0.001).
Six weeks treatment
Apical dendritic intersections (Figure 8)
BM 20, 40 and 80 mg/kg groups showed significant
increase in dendritic intersections at 80 and 100 µm
concentric circles (80 µm concentric circle: 3.83±0.46
in normal control vs. 5.01±0.76 in 20 mg/kg group,
p<0.01, 6.38±0.67 in 40 mg/kg group, p<0.001 and
6.05±0.65 in 80 mg/kg group, p<0.001, 100 µm
concentric circle: 4.20±0.47 in normal control vs.
5.55±0.79 in 20 mg/kg group, p<0.01, 6.55±0.64 in
40 mg/kg group, p<0.001 and 6.11±0.68 in 80 mg/kg
group, p<0.001). BM 40 and 80 mg/kg groups also
showed significant increased number of dendritic inter-
sections in 60 µm concentric circle (2.80±0.35 in normal
control vs. 4.12±0.84 in 40 mg/kg group, p<0.001 and
3.73±0.51 in 80 mg/kg group, p<0.05). In addition, BM
40 mg/kg group alone produced significant increased
number of dendritic intersections in 40 µm concentric
circle (1.90±0.16 in normal control vs. 2.32±0.24 in
40 mg/kg group, p<0.001).
Apical dendritic branching points – At different
concentric zones (Figure 9)
All the groups treated with BM showed significant
increase in the dendritic branching points in 60–80 µm
and 80–100 µm concentric zones (60–80 µm concentric
zone: 1.76±0.27 in normal control vs. 2.35±0.36 in
20 mg/kg group, p<0.05, 2.67±0.35 in 40 mg/kg group,
p<0.001 and 2.51±0.39 in 80 mg/kg group, p<0.001,
80–100 µm concentric zone: 1.56±0.31 in normal control
vs. 2.08±0.31 in 20 mg/kg group, p<0.05, 2.35±0.34 in
40 mg/kg group, p<0.001 and 2.18±0.37 in 80 mg/kg
group, p<0.01). In addition, BM 40 and 80 mg/kg groups
also showed significantly increased number of dendritic
branching points in 20–40 µm, 40–60 µm concentric
zones (20–40 µm concentric zone: 0.82±0.28 in normal
control vs. 1.40±0.35 in 40 mg/kg group, p<0.05 and
1.37±0.45 in 80 mg/kg group, p<0.05, 40–60 µm
concentric zone: 1.15±0.30 in normal control vs.
2.03±0.43 in 40 mg/kg group, p<0.01 and 1.87±0.56 in
80 mg/kg group, p<0.05).
Total number of dendritic branching points
Total number of branching points was found
significantly increased in all the three groups treated
with BM (20, 40 and 80 mg/kg) when compared to
normal control group (5.46±0.67 in normal control vs.
7.32±0.74 in 20 mg/kg group, p<0.01, 8.77±0.75 in
40 mg/kg group, p<0.001 and 8.16±1.22 in 80 mg/kg
group, p<0.001).
Basal dendritic intersections (Figure 10)
BM 20, 40 and 80 mg/kg groups showed significant
increase in dendritic intersections at 40, 60 and 80 µm
concentric circles (40 µm concentric circle: 5.91±0.56
in normal control vs. 7.40±1.03 in 20 mg/kg group,
p<0.01, 9.90±0.88 in 40 mg/kg group, p<0.001 and
8.88±0.63 in 80 mg/kg group, p<0.001, 60 µm concentric
circle: 7.15±0.87 in normal control vs. 8.82±0.49 in
20 mg/kg group, p<0.01, 11.08±0.82 in 40 mg/kg group,
p<0.001 and 9.96±1.10 in 80 mg/kg group, p<0.001,
80 µm concentric circle: 5.96±0.78 in normal control vs.
Venkata Ramana Vollala et al.
884
7.88±0.89 in 20 mg/kg group, p<0.01, 10.15±0.52 in
40 mg/kg group, p<0.001 and 8.83±1.49 in 80 mg/kg
group, p<0.001). In 20 and 100 µm concentric circles
both BM 40 and 80 mg/kg groups showed significantly
increased number of dendritic intersections (20 µm
concentric circle: 2.97±0.62 in normal control vs.
5.12±0.88 in 40 mg/kg group, p<0.001 and 4.75±0.44
in 80 mg/kg group, p<0.001, 100 µm concentric circle:
3.83±1.02 in normal control vs. 6.03±1.12 in 40 mg/kg
group, p<0.01 and 5.70±1.08 in 80 mg/kg group,
p<0.01).
Basal dendritic branching points – At different
concentric zones (Figure 11)
All the groups treated with BM showed a significant
increase in dendritic branching points in 20–40 µm and
40–60 µm concentric zones (20–40 µm concentric zone:
2.78±0.45 in normal control vs. 3.93±0.41 in 20 mg/kg
group, p<0.01, 4.81±0.74 in 40 mg/kg group, p<0.001
and 4.36±0.64 in 80 mg/kg group, p<0.001, 40–60 µm
concentric zone: 1.77±0.35 in normal control vs.
2.67±0.19 in 20 mg/kg group, p<0.01, 3.67±0.46 in
40 mg/kg group, p<0.001 and 3.28±0.70 in 80 mg/kg
group, p<0.001). In addition, BM 40 and 80 mg/kg
groups also showed significantly increased number
of dendritic branching points in 0–20, 60–80 and
80–100 µm concentric zones (0–20 µm concentric zone:
0.62±0.23 in normal control vs. 1.45±0.38 in 40 mg/kg
group, p<0.001 and 1.28±0.39 in 80 mg/kg group,
p<0.01, 60–80 µm concentric zone: 0.76±0.24 in normal
control vs. 1.76±0.36 in 40 mg/kg group, p<0.001 and
1.55±0.33 in 80 mg/kg group, p<0.001, 80–100 µm
concentric zone: 0.22±0.16 in normal control vs.
0.76±0.33 in 40 mg/kg group, p<0.01 and 0.66±0.41 in
80 mg/kg group, p<0.05).
Total number of branching points
Total number of branching points was found
significantly increased in all the three groups treated
with BM (20, 40 and 80 mg/kg) when compared to
normal control group (6.13±0.53 in normal control vs.
9.21±0.43 in 20 mg/kg group, p<0.001, 12.46±1.47 in
40 mg/kg group, p<0.001 and 11.26±1.14 in 80 mg/kg
group, p<0.001).
Discussion
Advanced neuroscience research has shown that the
learning process is associated with alterations in the
dendritic morphology of the hippocampal [24, 26–28]
and amygdaloid [29–32] neurons. Rao BS et al. (1993)
showed that self-stimulation rewarding experience
promotes structural changes in pyramidal neurons of the
CA3 region of the hippocampus in adult Wistar rats
[24]. An operant conditioning study carried out by
Mahajan DA and Desiraju T (1988) on Wistar rat pups
of brain-growth-spurt age to assess the plasticity of
apical dendritic branching of CA3 pyramidal neurons of
the hippocampus revealed a significant increase in the
number of branching points of the dendrites of the
learning group compared with the other groups [26].
Environmental enrichment promotes structural and
behavioral plasticity in the adult brain. Short-term
exposure to an enriched environment enhances dendritic
branching points in the hippocampus of rats [27]. There
are few reports in the literature correlating enhanced
learning and memory with histological changes in brain.
Clitoria ternatea (commonly called Shankapushpi)
aqueous root extract treatment in rats has shown: (i)
improved learning and memory (including passive
avoidance learning) [33], (ii) increase in dendritic
arborization of CA3 neurons of the hippocampus [34],
(iii) an increase in the acetylcholine content of the
hippocampus [35], and (iv) increase in dendritic
arborization of amygdala neurons [31].
A few other herbal extracts have been reported to
show similar effects. Fresh leaf extracts of medicinal
herbs (reported in Ayurveda to have ‘Medhya’
properties) such as, Centella asiatica and Ocimum
sanctum have been shown to improve learning and
memory, correlated with an increase in dendritic
arborization of amygdaloid neurons and hippocampal
neurons with the former [36], and alterations in
cytoarchitecture of neurons of the hippocampus and
substantia nigra with the latter [37]. These studies
suggest that dendritic arborizations are highly plastic in
regions such as the hippocampus and amygdala and that
they tend to bring about changes in learning and
memory.
In the present study, treatment with all doses (BM
20, 40 and 80 mg/kg) for two weeks did not alter the
dendritic arborization. However, treatment with BM 40
and 80 mg/kg for four weeks and with BM extract 20,
40 and 80 mg/kg for six weeks resulted in a significant
increase in the dendritic length (dendritic intersections)
and branching points. This may be the key neural basis
for improved learning and memory in these rats.
Thus, dendritic structural reorganization is the key
feature in learning and memory. Such structural alteration
is induced by Bacopa monniera and a few other herbal
extracts: (a) root powder extract of Withania somnifera
has been shown to protect the hippocampal (CA2 and
CA3) neurons undergoing neurodegeneration (due to
stress exposure) to 80% [38, 39]; (b) methanol extracts
from the dried roots of Scutellana baicalensis,
administered intraperitoneally significantly protected
CA1 neurons against 10 mm transient forebrain
ischemia [40]; (c) Semecarpus anacardium protects
the hippocampal neurons from stress induced neuro-
degeneration [38]; (d) Oren-gedoku-to a traditional
Chinese drug shown to work against impairment of
learning and memory induced by transient cerebral
ischemia [41]; (e) Biota, a traditional Chinese herbal
medicine affects learning and memory process in the
central nervous system and improves the impairment
of memory, acquisition and retention disturbances
produced by basal forebrain lesions [42]; (f) DX-9386,
a traditional Chinese medicine consisting of Ginseng,
Polygala, Acorus and Hoelen in the ratio of 1:1:25:50
potentiates LTP formation in the hippocampus and is
useful in amelioration of learning deficits [43]; (g)
Clitoria ternatea and jatamansi have also been reported
to be excellent memory enhancers [33, 44].
Our findings with BM extract also suggest similar
morphological changes in hippocampus. With BM
Enhanced dendritic arborization of hippocampal CA3 neurons by
Bacopa monniera
extract treatment in adult rats
885
dendritic length was significantly increased in the
hippocampus beyond 40 µm from the cell body both in
apical and basal dendrites in the rats which received 40
and 80 mg/kg of BM daily for four and six weeks.
However, no significant increase in dendritic length
was observed in 20 mg/kg/day group at any time. This
suggests that these doses of plant extract were adequate
to induce structural changes in these neurons. Naturally,
such changes will have a profound effect on the
behavior [23] because of the additional dendrites, which
are available on these neurons for the formation of new
synapses [45]. From the results it can be noted that a
significant number of additional dendritic branches are
formed in CA3 neurons of BM extract treated rats. This
result in more rapid and effective conduction of impulses,
which may be one of the reasons for enhanced learning
and memory in these rats, reported earlier [23].
Various molecular mechanisms have been proposed
for the dendritic enhancement in different parts of the
brain in vivo and in vitro [46–49]. Though we have
not tested such a role of BM, it may possess such a
stimulative property.
The beneficial effects of BM have been attributed to
the active constituent saponin, as Bacoside A [50] and
Bacoside B [51]. Bacosides A and B were found to
facilitate the capacity for mental retention in rats and
were active in both positive and negative reinforcement
experiments [13]. The mixture of saponins Bacosides A
and B in clinical trials showed facilitatory effect on both
memory and learning [15]. The exact mechanism of
action of BM could be attributed to a combination of
cholinergic modulation [52] and antioxidant effects
[53].
Conclusions
The results of our experiment suggest that Bacopa
monniera extract treatment in rats with higher doses for
longer periods induce structural changes in hippocampal
CA3 neurons, which improve their learning and memory.
Acknowledgements
The authors sincerely thank M/s. Natural Remedies
Private Limited for supplying the BM extract and
Manipal University (MU) for providing experimental
facilities to carry out this work.
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Corresponding author
Venkata Ramana Vollala, Assistant Professor of Anatomy, MSc, PhD, Rajiv Gandhi Institute of Medical Sciences
(RIMS), Adilabad, Andhra Pradesh, India; Phone +91 9494306083, e-mail: ramana.anat@gmail.com
Received: November 19th, 2010
Accepted: August 2nd, 2011
