# Effect of Prenatal Protein Malnutrition on Long-Term Potentiation and BDNF Protein Expression in the Rat Entorhinal Cortex after Neocortical and Hippocampal Tetanization

Authors: Alejandro Hernández; Héctor Burgos; Mauricio Mondaca; Rafael Barra; Héctor Núñez; Hernán Pérez; Rubén Soto-Moyano; Walter Sierralta; Victor Fernández; Ricardo Olivares; Luis Valladares
Journal: Neural Plasticity
 (2008)

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## Abstract

Reduction of the protein content from 25 to 8% casein in the diet of pregnant rats results in impaired neocortical long-term potentiation (LTP) of the offspring together with lower visuospatial memory performance. The present study was aimed to investigate whether this type of maternal malnutrition could result in
modification of plastic capabilities of the entorhinal cortex (EC) in the adult progeny. Unlike
normal eutrophic controls, 55–60-day-old prenatally malnourished rats were unable to
develop LTP in the medial EC to tetanizing stimulation delivered to either the ipsilateral
occipital cortex or the CA1 hippocampal region. Tetanizing stimulation of CA1 also failed
to increase the concentration of brain-derived neurotrophic factor (BDNF) in the EC of
malnourished rats. Impaired capacity of the EC of prenatally malnourished rats to develop
LTP and to increase BDNF levels during adulthood may be an important factor contributing
to deficits in learning performance having adult prenatally malnourished animals.


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## Body

## 1. Introduction

Both human and animal studies indicate that maternal
protein malnutrition alters various maturational events in the brain resulting
in behavioral abnormalities, altered cognitive functioning, and disturbances in
learning and memory (for review, see [1]). Alterations extend
into the postnatal period and continue into adulthood. For example, on reaching
adulthood prenatally malnourished rats on a 6% prenatal/25% postnatal casein
diet exhibit learning disturbances, such as deficits in execution of spatial alternation tasks [2] as well as impaired visual
discrimination learning [3]. In addition, on reaching
adulthood, rats born from 8% case in-restricted mothers showed
decreased visuo-spatial memory performance [4].One of the principal hypotheses in the malnutrition
field relates to the issue that decreases in synaptic plasticity may be a
critical brain mechanism underlying learning deficits observed as a result of
nutritional insults to the developing brain. In this regard, it has been shown
that it is difficult to induce and maintain hippocampal [5, 6] and neocortical [4]
long-term potentiation (LTP) in brains of prenatally malnourished rats. Whether prenatal
malnutrition could affect synaptic plasticity in brain regions
other than the hippocampus and cerebral neocortex is unknown at
present. The entorhinal
cortex (EC) is well situated to play a key role in the bidirectional
interactions between the neocortex and the hippocampus, and is thought to be
critically involved in the formation of declarative (or explicit) memory—the ability to
recollect everyday events and factual knowledge. Potentially, prenatal
malnutrition could alter at this level the bidirectional communication between the neocortex and the hippocampus, thereby disturbing some types of
cognitive processes. However, the
effects of prenatal malnutrition on EC neuroplastic mechanisms have not still
been explored. The superficial layers (I–III) of the EC are
regarded as ‘‘input layers’’ since terminations of the projections from
perirhinal and parahippocampal cortices—the recipients of cortical association areas—occur primarily in these layers [7–11]. Information
processed in the hippocampus and subiculum is then returned to the deep layers
(V-VI) of the EC via
projections from the CA1 field and subiculum [11–14], and these layers in turn
project to forebrain structures [15–17]. Accordingly, the deep layers are
regarded as ‘‘output layers,’’ and therefore functionally segregated from the
superficial layers. Similar
to other brain regions, the EC has been shown to express N-methyl-D-aspartate (NMDA)
receptor-dependent LTP [18–20] and long-term depression [20–24], both in in vitro and in vivo experimental paradigms.The present
study was aimed to investigate whether mild reduction of the protein content of the diet of
pregnant rats can modify plastic capabilities of the EC in vivo. In contrast to severe forms of maternal malnutrition,
mild reduction of the casein content in the diet of pregnant rats from 25 to 8%,
calorically compensated for by carbohydrates, results in apparently normal in
utero development of fetuses, as assessed by normal maternal weight gain during
pregnancy and normal body and brain weights of pups at birth [25]. However,
this insidious form of protein maternal malnutrition, so-called ‘‘hidden’’
prenatal malnutrition [25], results in altered noradrenergic function in the
neocortex of the offspring together with impaired neocortical LTP and lower
visuospatial memory performance [4, 26–28]. The present results provide evidence
that mild prenatal malnutrition in rats leads to impaired long-term synaptic
potentiation together with decreased expression of brain-derived neurotrophic
factor (BDNF) in the EC of adult animals; a neurotropin plays a major role in
regulating induction and maintenance of LTP [29, 30].


## 2. Materials and Methods

### 2.1. Animals and Diets

The
experimental protocol and animal management were in accordance with the NIH
Guide for the Care and Use of Laboratory Animals [31], and was approved by the
Committee for the Ethical Use of Experimental Animals, INTA, University of Chile. Female Sprague-Dawley rats were fed isocaloric purified diets containing either
normal (25% casein, providing 22.5% protein) or low (8% casein, providing 7.2%
protein) amounts of protein. The other components of the purified diets were as
follows. (i) Normal diet: carbohydrate, 50.2%; fat, 15.0%; vitamin mix, 1.0%;
salt mix, 4.7%; water, 1.7%; cellulose, 4.2%; L-methionine, 0.4%. (ii) Low protein
diet: carbohydrate, 66.5%; fat, 15.0%; vitamin mix, 1.0%; salt mix, 4.7%;
water, 1.0%; cellulose, 4.2%; L-methionine, 0.4%. Both diets provide about 4.3 Kcal/g. The dietary paradigm was started 1 day after mating and continued
throughout pregnancy. The body weight gain of the pregnant mothers was
controlled daily. At birth, all pups were weighed and litters were culled to 8
pups (4 males, 4 females). Afterwards, pups born from mothers fed the 7.2%
protein diet were fostered to well-nourished dams (22.5% protein diet) giving
birth on that day. Pups born from mothers receiving the 22.5% protein diet were also fostered to well-nourished dams in
order to equalize among groups other factors that may depend on the rearing
conditions (i.e., stress due to cross-fostering). After weaning, at 22 days of age,
all pups were fed a standard laboratory diet providing 22.5% protein.


### 2.2. LTP Determinations in the Medial EC

Experiments were
carried out in 16 normal and 16 malnourished rats of 55–60 days of age.
Rats were weighed, anesthetized with 1.5 g/kg i.p. urethane, and placed in a
stereotaxic apparatus under artificial ventilation. Reinforcement of anesthesia
during the experiments was not necessary since surgical procedures and
recordings lasted no longer than 3 hours and, in our experience, 1.5 g/kg i.p.
urethane induces profound anesthesia lasting more than 6 hours. Animals never
regained consciousness and no changes in heart rate in response to stimulation
were detected throughout the experiments.Field responses were evoked in the left medial EC by
electrical stimulation of either the ipsilateral occipital cortex or the ipsilateral
ventral CA1 hippocampal region, in an alternated fashion. After exposure of the
left occipital lobe, electrical stimulation of the occipital cortex and the ventral
CA1 hippocampal region was carried out by means of two independent bipolar side-by-side
electrodes composed each by two glued, parylen-insulated, 50-μm-diameter tungsten wires with a 0.8-mm tip
separation. One stimulating electrode was positioned in the left occipital
cortex at coordinates A=−5.8, L=−3.5, in mm, in such a way that the longer
tip penetrated the cortex by 1.0 mm. The other stimulating electrode was advanced to the ventral
CA1 region at coordinates A=−5.5, L=−5.0,
V=7.0, in mm. As has pointed out recently, progressively
more ventral CA1 regions innervate progressively more medial regions of the
medial entorhinal areas [32],
which in turn receive more strong visual input through the parahippocampal cortex
(postrhinal cortex in the rat [7]).Field responses
were recorded from the left medial EC with another bipolar side-by-side
electrode (two glued, parylen-insulated, 50-μm-diameter tungsten wires with a 0.8-mm tip
separation) positioned at coordinates A=−7.5, L=−5.0, V=6.5, the longer
tip being 0.1-0.2 mm above the
ventral brain surface. Configuration and positioning of the recording electrode
pair into the EC allowed one tip of the bipolar electrode was into layer II-III
and the other tip near layer V. Although bipolar electrode arrangement does not
allow performing laminar analysis of potential reversal, it maximizes field recordings
corresponding to depolarization of neurons (active inward current or sink) situated
near to one electrode tip, while minimizing those produced in distant current
generators affecting rather similarly the two electrode tips. Thus, bipolar
electrodes seem especially appropriate for focalized recording from laminar
cortical structures such as the EC, where differential activation of neurons of
layers II-III by neocortical-EC pathways, or layer V by CA1-EC afferents, will
create, respectively, superficial and deep current sinks. Rostrocaudal (A) and
lateral (L) coordinates were relative to bregma, while vertical (V) coordinates
were relative to the cortical surface, all taken from Paxinos and Watson [33].
Figure 1 shows a scheme of two coronal planes of the rat brain illustrating the
positions of the stimulating electrodes in the occipital cortex and ventral CA1
region of the left hemisphere, as well as the location of the recording electrode
in the ipsilateral EC. Test stimuli, alternately applied to either the
occipital cortex (during 2.5 minutes) or the ventral CA1 region (during 2.5 minutes),
consisted of 100 microseconds duration square-wave pulses at 0.2 Hz generated
by means of a Grass S11 stimulator in conjunction with a Grass SIU-5 stimulus
isolation unit and a Grass CCU 1A constant current unit (Astro-Med Inc., West Warwick, RI, USA). Bipolar recording of EC field
responses to occipital cortex stimulation consisted of a bigger upward negative
wave followed by a downward positive component. Surface negative responses have
been already recorded in vivo from
the EC of rats during stimulation of the piriform cortex [34]. In turn, EC
field responses to CA1 stimulation begin with a marked downward surface
positive deflection followed by a late upward surface negative wave of smaller
amplitude. In vivo recording of
surface positive field responses from the EC of rats during CA1 stimulation has
recently been reported [35–37]. Thus, only
the negative first-wave of occipital cortex-EC responses and the positive
first-wave of CA1-EC responses were measured in the present experiments. Before
beginning each experiment, two full input-output series, one for the occipital
cortex and the other for CA1, were performed at stimulus intensities of 200–1200 μA. Test
stimuli with a stimulation intensity yielding EC responses with first-wave peak
amplitude of 50% of the maximum were used for the remainder of the experiment. Thus,
test stimuli applied to the occipital cortex were similar in frequency and
duration to those applied to CA1, but rather different in intensity. EC
responses evoked from the occipital cortex and from the CA1 region were also subjected
to a 10-pulse, 30 Hz stimulus in order to test the ability of the response to
follow repetitive stimulation. As showed elsewhere [34, 38], polysynaptic
components usually fail at frequencies <40–50 Hz, whereas
monosynaptic components should follow frequencies near 100 Hz. After a 30-minute
stabilization period of alternated occipital cortex and CA1 stimulation with
test stimuli, a 2.5-minute control period of EC basal responses (30 averaged
responses) evoked from the occipital cortex was recorded, followed by another
2.5-minute control period of EC basal responses (30 averaged responses) evoked
from the CA1 region. Thereafter, LTP was induced in the medial EC by applying tetanizing
stimulation either to the occipital cortex (8 normal and 8 malnourished rats) or
to the ventral CA1 region (8 additional normal and 8 additional malnourished
rats). The tetanizing stimulus consisted of three high-frequency trains (100 microseconds
square-wave pulses at 312 Hz) of 500 milliseconds duration each, applied every
30 seconds with intensity 50% higher than the respective test stimuli. Such a stimulating
protocol has been shown to induce saturating LTP in the EC, at least when
activating the EC from the piriform cortex in awake rats, meaning that
subsequent application of additional trains fails to induce further increments of
field responses [34]. A closely similar stimulating protocol (3 trains of
stimuli for 200 milliseconds at 250 Hz with intertrain interval of 30 seconds) applied
to the CA1 hippocampal region has been reported to produce reliable LTP
induction in the EC of uretanized rats [37].(a) Scheme of two coronal planes of the rat brain
illustrating the positions of the stimulating electrodes in the occipital
cortex (OC) and ventral CA1 region of the left hemisphere, as well as the location
of the recording electrode in the ipsilateral entorhinal cortex EC. In the upper part are shown representative examples of the average of 30
successive responses evoked in the medial EC of one rat by ipsilateral
stimulation of the occipital cortex (A and B) or the ventral CA1 hippocampal region (C and D) at 0.2 Hz, obtained before (A and C) and after (B and D) tetanization. Calibration bars are
indicated. Upward potential deflection is negative. First and second arrows indicate, respectively, the beginning and the peak of the early negative (A) or early positive (C) wave, which served to calculate peak
amplitude or slope (amplitude/time
ratio on the nearest sample to the 10% and the 90% level) of the early component. (b) Onset and peak latencies
(values are means ± SEM, in millisecond) of the early component of EC
responses to test stimulus applied to either the OC or CA1 region before tetanization.
(a)(b)Recordings were amplified by a Grass P-511
preamplifier (0.8–1000 Hz bandwidth), and displayed and averaged on a Philips
PM 3365A digital oscilloscope. They were also digitized at a rate of 10000/second
by an A/D converter interfaced to an Acer PC, and stored for retrieval and
offline analysis. In all experiments, body temperature and expired CO2 were monitored and remained within normal limits. Peak latency and peak amplitude
of the early component of averaged field responses were measured using time and
voltage cursors provided in the digital oscilloscope. Slope was determined as
the amplitude/time ratio on the nearest sample to the 10% and the 90% level
between cursors set on the beginning and the peak of the early negative or positive
wave (see Figure 1, first and second arrow, resp., in recordings (A and C)).
The efficacy of the tetanizing train to potentiate cortical evoked responses
was evaluated by measuring both the peak amplitude and the maximal slope
increases. The results were similar but the former procedure led to lower
variability of means (as revealed by statistical variance), so amplitudes were
used for analyses of the experiments.Two hours after occipital cortex or CA1 tetanization,
once the electrophysiological experiments were finished, the animals were
sacrificed by decapitation, the brain rapidly removed and weighed, and the left
and right ECs dissected out. The average weight of dissected entorhinal area (averaged
without taking into consideration left or right hemisphere origin) was5.77±0.61
for normal rats and 5.30±0.50 for malnourished animals (mean ± SEM). These
samples were stored at −80°C before use. Afterwards, the tissues were
examined for expression of BDNF protein level by ELISA.


### 2.3. Determination of BDNF in the EC

Whole samples of EC were homogenized in ice-cold lysis
buffer, containing 137 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1%
Triton X-100, 10% glycerol, and 2μL/mL protease inhibitor cocktail P8340 (Sigma-Aldrich, St. Louis, MO, USA). The tissue homogenate solutions were centrifuged at 14000 x g for 5 minutes at 4°C. The supernatants
were collected and diluted 1/5 in buffer DPBS and then acidified in 1 N HCl. They
were then incubated for 15 minutes at room temperature and neutralized with 1 N NaOH until pH 7.6. BDNF was assessed using the E-Max ImmunoAssay system ELISA
kit (Promega, Co., Madison, Wis, USA). Briefly, standard 96-well
flat-bottom NUNC-immuno maxisorp ELISA plates were incubated overnight at 4°C with a monoclonal
anti-BDNF antibody. The plates were blocked by incubation for 1 hour at
room temperature (RT) with a 1x block and sample buffer. Serial dilutions of
known amounts of BDNF ranging from 0 to 500 pg/mL were performed in
duplicate for standard curve determination. Wells containing the standard
curves and supernatants of brain tissue homogenates were incubated at room
temperature for 2 hours, as specified by the protocol. They were then
incubated with a secondary antihuman BDNF polyclonal antibody for 2 hours
at room temperature, as specified by the protocol. A species-specific antibody
conjugated to horseradish peroxidase was used for tertiary reaction for
1 hour at room temperature following this incubation step. TMB one
solution was used to develop color in the wells. This reaction was terminated
with 1 N hydrochloric acid at a specific time (10 minutes) at room
temperature, and absorbance was then recorded at 450 nm in a microplate
reader within 40 minutes of stopping the reaction. The neurotrophin values
were determined by comparison with the regression line for BDNF and expressed
as pg BDNF/mg wet weight. Using this kit, BDNF can be quantified in the range
of 7.8–500 pg/mL.


### 2.4. Statistical Analyses

All statistical analyses were performed with
GraphPad Instat version 3.00 (GraphPad Software, Inc., San Diego, Calif, USA).
For the effect of dietary treatments on body and brain weights, intergroup
comparisons were made using unpaired Student’st-test. For the analysis of the time-course in LTP studies, a one-way
ANOVA was performed for intragroup comparisons followed by Dunnett’s multiple comparisons
post-hoc test. For analyzing results of BDNF protein expression, intergroup
comparisons between normal and malnourished groups were made using unpaired
Student’s t-test, while the effect of
tetanization was assessed using nonparametric ANOVA (Kruskal-Wallis test)
followed by Dunn’s multiple comparisons post-hoc test.


## 2.1. Animals and Diets

The
experimental protocol and animal management were in accordance with the NIH
Guide for the Care and Use of Laboratory Animals [31], and was approved by the
Committee for the Ethical Use of Experimental Animals, INTA, University of Chile. Female Sprague-Dawley rats were fed isocaloric purified diets containing either
normal (25% casein, providing 22.5% protein) or low (8% casein, providing 7.2%
protein) amounts of protein. The other components of the purified diets were as
follows. (i) Normal diet: carbohydrate, 50.2%; fat, 15.0%; vitamin mix, 1.0%;
salt mix, 4.7%; water, 1.7%; cellulose, 4.2%; L-methionine, 0.4%. (ii) Low protein
diet: carbohydrate, 66.5%; fat, 15.0%; vitamin mix, 1.0%; salt mix, 4.7%;
water, 1.0%; cellulose, 4.2%; L-methionine, 0.4%. Both diets provide about 4.3 Kcal/g. The dietary paradigm was started 1 day after mating and continued
throughout pregnancy. The body weight gain of the pregnant mothers was
controlled daily. At birth, all pups were weighed and litters were culled to 8
pups (4 males, 4 females). Afterwards, pups born from mothers fed the 7.2%
protein diet were fostered to well-nourished dams (22.5% protein diet) giving
birth on that day. Pups born from mothers receiving the 22.5% protein diet were also fostered to well-nourished dams in
order to equalize among groups other factors that may depend on the rearing
conditions (i.e., stress due to cross-fostering). After weaning, at 22 days of age,
all pups were fed a standard laboratory diet providing 22.5% protein.


## 2.2. LTP Determinations in the Medial EC

Experiments were
carried out in 16 normal and 16 malnourished rats of 55–60 days of age.
Rats were weighed, anesthetized with 1.5 g/kg i.p. urethane, and placed in a
stereotaxic apparatus under artificial ventilation. Reinforcement of anesthesia
during the experiments was not necessary since surgical procedures and
recordings lasted no longer than 3 hours and, in our experience, 1.5 g/kg i.p.
urethane induces profound anesthesia lasting more than 6 hours. Animals never
regained consciousness and no changes in heart rate in response to stimulation
were detected throughout the experiments.Field responses were evoked in the left medial EC by
electrical stimulation of either the ipsilateral occipital cortex or the ipsilateral
ventral CA1 hippocampal region, in an alternated fashion. After exposure of the
left occipital lobe, electrical stimulation of the occipital cortex and the ventral
CA1 hippocampal region was carried out by means of two independent bipolar side-by-side
electrodes composed each by two glued, parylen-insulated, 50-μm-diameter tungsten wires with a 0.8-mm tip
separation. One stimulating electrode was positioned in the left occipital
cortex at coordinates A=−5.8, L=−3.5, in mm, in such a way that the longer
tip penetrated the cortex by 1.0 mm. The other stimulating electrode was advanced to the ventral
CA1 region at coordinates A=−5.5, L=−5.0,
V=7.0, in mm. As has pointed out recently, progressively
more ventral CA1 regions innervate progressively more medial regions of the
medial entorhinal areas [32],
which in turn receive more strong visual input through the parahippocampal cortex
(postrhinal cortex in the rat [7]).Field responses
were recorded from the left medial EC with another bipolar side-by-side
electrode (two glued, parylen-insulated, 50-μm-diameter tungsten wires with a 0.8-mm tip
separation) positioned at coordinates A=−7.5, L=−5.0, V=6.5, the longer
tip being 0.1-0.2 mm above the
ventral brain surface. Configuration and positioning of the recording electrode
pair into the EC allowed one tip of the bipolar electrode was into layer II-III
and the other tip near layer V. Although bipolar electrode arrangement does not
allow performing laminar analysis of potential reversal, it maximizes field recordings
corresponding to depolarization of neurons (active inward current or sink) situated
near to one electrode tip, while minimizing those produced in distant current
generators affecting rather similarly the two electrode tips. Thus, bipolar
electrodes seem especially appropriate for focalized recording from laminar
cortical structures such as the EC, where differential activation of neurons of
layers II-III by neocortical-EC pathways, or layer V by CA1-EC afferents, will
create, respectively, superficial and deep current sinks. Rostrocaudal (A) and
lateral (L) coordinates were relative to bregma, while vertical (V) coordinates
were relative to the cortical surface, all taken from Paxinos and Watson [33].
Figure 1 shows a scheme of two coronal planes of the rat brain illustrating the
positions of the stimulating electrodes in the occipital cortex and ventral CA1
region of the left hemisphere, as well as the location of the recording electrode
in the ipsilateral EC. Test stimuli, alternately applied to either the
occipital cortex (during 2.5 minutes) or the ventral CA1 region (during 2.5 minutes),
consisted of 100 microseconds duration square-wave pulses at 0.2 Hz generated
by means of a Grass S11 stimulator in conjunction with a Grass SIU-5 stimulus
isolation unit and a Grass CCU 1A constant current unit (Astro-Med Inc., West Warwick, RI, USA). Bipolar recording of EC field
responses to occipital cortex stimulation consisted of a bigger upward negative
wave followed by a downward positive component. Surface negative responses have
been already recorded in vivo from
the EC of rats during stimulation of the piriform cortex [34]. In turn, EC
field responses to CA1 stimulation begin with a marked downward surface
positive deflection followed by a late upward surface negative wave of smaller
amplitude. In vivo recording of
surface positive field responses from the EC of rats during CA1 stimulation has
recently been reported [35–37]. Thus, only
the negative first-wave of occipital cortex-EC responses and the positive
first-wave of CA1-EC responses were measured in the present experiments. Before
beginning each experiment, two full input-output series, one for the occipital
cortex and the other for CA1, were performed at stimulus intensities of 200–1200 μA. Test
stimuli with a stimulation intensity yielding EC responses with first-wave peak
amplitude of 50% of the maximum were used for the remainder of the experiment. Thus,
test stimuli applied to the occipital cortex were similar in frequency and
duration to those applied to CA1, but rather different in intensity. EC
responses evoked from the occipital cortex and from the CA1 region were also subjected
to a 10-pulse, 30 Hz stimulus in order to test the ability of the response to
follow repetitive stimulation. As showed elsewhere [34, 38], polysynaptic
components usually fail at frequencies <40–50 Hz, whereas
monosynaptic components should follow frequencies near 100 Hz. After a 30-minute
stabilization period of alternated occipital cortex and CA1 stimulation with
test stimuli, a 2.5-minute control period of EC basal responses (30 averaged
responses) evoked from the occipital cortex was recorded, followed by another
2.5-minute control period of EC basal responses (30 averaged responses) evoked
from the CA1 region. Thereafter, LTP was induced in the medial EC by applying tetanizing
stimulation either to the occipital cortex (8 normal and 8 malnourished rats) or
to the ventral CA1 region (8 additional normal and 8 additional malnourished
rats). The tetanizing stimulus consisted of three high-frequency trains (100 microseconds
square-wave pulses at 312 Hz) of 500 milliseconds duration each, applied every
30 seconds with intensity 50% higher than the respective test stimuli. Such a stimulating
protocol has been shown to induce saturating LTP in the EC, at least when
activating the EC from the piriform cortex in awake rats, meaning that
subsequent application of additional trains fails to induce further increments of
field responses [34]. A closely similar stimulating protocol (3 trains of
stimuli for 200 milliseconds at 250 Hz with intertrain interval of 30 seconds) applied
to the CA1 hippocampal region has been reported to produce reliable LTP
induction in the EC of uretanized rats [37].(a) Scheme of two coronal planes of the rat brain
illustrating the positions of the stimulating electrodes in the occipital
cortex (OC) and ventral CA1 region of the left hemisphere, as well as the location
of the recording electrode in the ipsilateral entorhinal cortex EC. In the upper part are shown representative examples of the average of 30
successive responses evoked in the medial EC of one rat by ipsilateral
stimulation of the occipital cortex (A and B) or the ventral CA1 hippocampal region (C and D) at 0.2 Hz, obtained before (A and C) and after (B and D) tetanization. Calibration bars are
indicated. Upward potential deflection is negative. First and second arrows indicate, respectively, the beginning and the peak of the early negative (A) or early positive (C) wave, which served to calculate peak
amplitude or slope (amplitude/time
ratio on the nearest sample to the 10% and the 90% level) of the early component. (b) Onset and peak latencies
(values are means ± SEM, in millisecond) of the early component of EC
responses to test stimulus applied to either the OC or CA1 region before tetanization.
(a)(b)Recordings were amplified by a Grass P-511
preamplifier (0.8–1000 Hz bandwidth), and displayed and averaged on a Philips
PM 3365A digital oscilloscope. They were also digitized at a rate of 10000/second
by an A/D converter interfaced to an Acer PC, and stored for retrieval and
offline analysis. In all experiments, body temperature and expired CO2 were monitored and remained within normal limits. Peak latency and peak amplitude
of the early component of averaged field responses were measured using time and
voltage cursors provided in the digital oscilloscope. Slope was determined as
the amplitude/time ratio on the nearest sample to the 10% and the 90% level
between cursors set on the beginning and the peak of the early negative or positive
wave (see Figure 1, first and second arrow, resp., in recordings (A and C)).
The efficacy of the tetanizing train to potentiate cortical evoked responses
was evaluated by measuring both the peak amplitude and the maximal slope
increases. The results were similar but the former procedure led to lower
variability of means (as revealed by statistical variance), so amplitudes were
used for analyses of the experiments.Two hours after occipital cortex or CA1 tetanization,
once the electrophysiological experiments were finished, the animals were
sacrificed by decapitation, the brain rapidly removed and weighed, and the left
and right ECs dissected out. The average weight of dissected entorhinal area (averaged
without taking into consideration left or right hemisphere origin) was5.77±0.61
for normal rats and 5.30±0.50 for malnourished animals (mean ± SEM). These
samples were stored at −80°C before use. Afterwards, the tissues were
examined for expression of BDNF protein level by ELISA.


## 2.3. Determination of BDNF in the EC

Whole samples of EC were homogenized in ice-cold lysis
buffer, containing 137 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1%
Triton X-100, 10% glycerol, and 2μL/mL protease inhibitor cocktail P8340 (Sigma-Aldrich, St. Louis, MO, USA). The tissue homogenate solutions were centrifuged at 14000 x g for 5 minutes at 4°C. The supernatants
were collected and diluted 1/5 in buffer DPBS and then acidified in 1 N HCl. They
were then incubated for 15 minutes at room temperature and neutralized with 1 N NaOH until pH 7.6. BDNF was assessed using the E-Max ImmunoAssay system ELISA
kit (Promega, Co., Madison, Wis, USA). Briefly, standard 96-well
flat-bottom NUNC-immuno maxisorp ELISA plates were incubated overnight at 4°C with a monoclonal
anti-BDNF antibody. The plates were blocked by incubation for 1 hour at
room temperature (RT) with a 1x block and sample buffer. Serial dilutions of
known amounts of BDNF ranging from 0 to 500 pg/mL were performed in
duplicate for standard curve determination. Wells containing the standard
curves and supernatants of brain tissue homogenates were incubated at room
temperature for 2 hours, as specified by the protocol. They were then
incubated with a secondary antihuman BDNF polyclonal antibody for 2 hours
at room temperature, as specified by the protocol. A species-specific antibody
conjugated to horseradish peroxidase was used for tertiary reaction for
1 hour at room temperature following this incubation step. TMB one
solution was used to develop color in the wells. This reaction was terminated
with 1 N hydrochloric acid at a specific time (10 minutes) at room
temperature, and absorbance was then recorded at 450 nm in a microplate
reader within 40 minutes of stopping the reaction. The neurotrophin values
were determined by comparison with the regression line for BDNF and expressed
as pg BDNF/mg wet weight. Using this kit, BDNF can be quantified in the range
of 7.8–500 pg/mL.


## 2.4. Statistical Analyses

All statistical analyses were performed with
GraphPad Instat version 3.00 (GraphPad Software, Inc., San Diego, Calif, USA).
For the effect of dietary treatments on body and brain weights, intergroup
comparisons were made using unpaired Student’st-test. For the analysis of the time-course in LTP studies, a one-way
ANOVA was performed for intragroup comparisons followed by Dunnett’s multiple comparisons
post-hoc test. For analyzing results of BDNF protein expression, intergroup
comparisons between normal and malnourished groups were made using unpaired
Student’s t-test, while the effect of
tetanization was assessed using nonparametric ANOVA (Kruskal-Wallis test)
followed by Dunn’s multiple comparisons post-hoc test.


## 3. Results

### 3.1. Effect of Dietary Treatment on Body and Brain Weights

Body and brain weights measurements
revealed that there were no significant differences in body weight gain of
pregnant mothers receiving 7.2% or 22.5% protein diet (data not shown). Full
data on the effects of this dietary treatment on maternal weight gain during
the first, second, and third weeks of pregnancy was published elsewhere [26]. At
days 1, 8, and 55–60 of postnatal
life, no significant differences in body and brain weights were found between
rats born from mothers receiving 7.2% or 22.5% protein diet (Table 1).Table 1
Body and brain weights of normal
and prenatally malnourished rats. Values
are means ± SEM.N=16
rats in each group. No statistically significant differences (NS) were found
when comparing body and brain weights of normal and malnourished groups of same
ages (unpaired Student’s t-test).
Body weight (g)Brain weight (mg)AgeDay 1Day 8Day 55Day 55–60Normal7.3±0.0718.9±0.37235±101322.0±16.1Malnourished7.2±0.0919.1±0.45229±121318.6±15.7PNSNSNSNS


### 3.2. LTP In Vivo in the Medial EC

In rats of 55–60 days of age, bipolar
recording of basal EC field responses to occipital cortex stimulation consisted
of a prominent upward negative wave followed by a positive component. This is
consistent with the arrangement of the side-by-side bipolar electrode located
into the EC, where the longer tip is expected to be recording an early superficial
sink generated by depolarization of stellate and pyramidal principal neurons within
input layers II-III (in relation to rather silent deep layers). In contrast, basal
EC responses to CA1 stimulation began with a marked downward surface positive
deflection followed by a late surface negative wave, which is consistent with
the recording through the shorter tip of an early deep sink resulting from depolarization
of pyramidal cells within output layer V. Figure1(a) shows typical recordings
of basal (A and C) and potentiated (B and D) averaged EC field responses evoked
by stimulation of the occipital cortex (A and B) or the CA1 region (C and D). In
normal eutrophic rats, the onset and peak latencies of the early negative
component of basal EC responses evoked from the occipital cortex were 18.7±2.2 and 30.6±1.8 milliseconds, respectively, while the onset and peak latencies
of the early positive wave in basal responses evoked from CA1 were 9.6±0.7
and 17.9±0.8 milliseconds (Figure 1(b)). For both type of responses, the
differences in latency before and after potentiation were not statistically
significant (paired Student’s t-test,
N=8). Shape, latencies, and wave amplitudes of basal field responses evoked
in the EC of prenatally malnourished rats, either from the occipital cortex or
the CA1 area, did not differ from those of normal eutrophic rats (unpaired
Student’s t-test, N=8 in each group). Frequency testing
showed that the early component of the EC
potential evoked from the occipital cortex declined rapidly with a stimulus frequency of 30 Hz, thus suggesting a polysynaptic nature of the
response. In contrast, the early component of the EC response elicited from CA1
was able to follow 30 Hz stimulation
frequency with decreasing amplitude of less than 20%, which is characteristic
of monosynaptic responses.In normal animals, tetanizing stimulation applied
to either the occipital cortex or the CA1 hippocampal region produced a
significant increase in peak amplitude of the early component
evoked in the ipsilateral
medial EC, which remained unchanged throughout the
recording period (Figure2). After tetanizing the occipital cortex, the early
negative wave to occipital cortex test stimuli was potentiated to neocortical test
stimuli in all blocks over the time-course (ranging from 107 to 136%, Dunnett’s
multiple comparisons test) excepting for block 2.5–5 minutes, while no
significant potentiation to CA1 stimuli was observed in the early component of EC
responses (Dunnett’s multiple comparisons test); however, a transient but
complete inhibition was early observed on block 0–2.5 minutes. After
tetanizing the ventral CA1, the early positive wave of EC responses to CA1 test
stimuli was potentiated in all blocks over the time-course (ranging from 59 to 72%,
Dunnett’s multiple comparisons test) excepting for block 2.5–5 minutes, while
no significant potentiation to occipital cortex stimulation was observed.Time-course of LTP induced in the medial
entorhinal cortex of 55–60-day-old normal
eutrophic rats by applying tetanizing stimulation to the occipital cortex (a) or
to the ventral CA1 hippocampal region (b). The arrow indicates time of
application of the tetanizing stimulus.N=8 rats in all groups. Values are
means ± SEM of peak-to-peak amplitudes, 30 responses averaged per rat. Note the
occurrence of homosynaptic, but not heterosynaptic potentiation. One-way ANOVA followed
by Dunnett’s multiple comparisons test indicated significant intragroup differences
in peak-to-peak amplitudes (P*<.05, P**<.01) when comparing
post-tetanizing values with the last pretetanizing basal point (at 0 minute),
excepting for block 2.5–5 minutes (a),
where significant inhibition (P**<.01) occurred.
(a)(b)In contrast to that
occurred in normal controls, no significant increase of the early component of
EC field responses evoked from the occipital cortex or from the CA1 region (P>.05
for all blocks, Dunnett’s multiple comparisons test) was observed in
malnourished animals after applying neocortical or hippocampal tetanizing
stimulation (Figure 3).Failure of tetanizing stimulation applied to
the occipital cortex (a) or to the ventral CA1 hippocampal region (b) to induce
LTP in the medial entorhinal cortex of 55–60-day-old prenatally
malnourished rats. The arrow indicates time of application of the tetanizing
stimulus.N=8 rats in all groups. Values are means ± SEM of peak-to-peak
amplitudes, 30 responses averaged per rat. It can be noted that neither
homosynaptic nor heterosynaptic potentiation occurred in the EC of malnourished
animals. One-way ANOVA followed by Dunnett’s multiple comparisons test
indicated no significant intragroup differences in peak-to-peak amplitudes when
comparing posttetanizing values with the last pretetanizing basal point (at 0
minute), excepting for block 2.5–5 minutes (a) where
significant inhibition (P**<.01) occurred.
(a)(b)


### 3.3. BDNF Expression in the EC

Serial dilutions of
known amounts of BDNF ranging from 0 to 500 pg/mL allowed to determine a
standard curve demonstrating a
direct relationship between optical density and BDNF concentration (r2=0.9106).Table2 shows that
application of tetanizing stimulation to the left CA1 region in normal rats
resulted in a significant increase of BDNF concentration in the ipsilateral EC
(P<.05, Dunn’s multiple comparisons test) two hours after
tetanization, while application of tetanizing stimuli to the left occipital
cortex did not significantly modify the BDNF level in the ipsilateral EC. In
contrast, tetanizing stimulation applied to either the occipital cortex or the
CA1 hippocampal region in malnourished rats was ineffective in modifying the
BDNF concentration in the ipsilateral EC. Table 2 also shows that on days 55–60 of postnatal
life, malnourished rats exhibited significant lower concentration of BDNF
protein in the right medial EC (corresponding to the nonstimulated cerebral
hemisphere) than that observed in normal animals of same ages (P<.05,
unpaired Student’s t-test).Table 2
Changes in BDNF
expression (pg/mg wet tissue) in the left entorhinal cortex (EC) of 55–60-day-old normal
and prenatally malnourished rats two hours after applying ipsilateral
tetanizing stimulation to the occipital cortex (OC) or the CA1 hippocampal
region, as compared to BDNF levels in the right EC. Values are means ± SEM. The
number of samples in each group is shown in parentheses. BDNF concentrations in
right EC samples after tetanizing the left OC or left CA1 did not significantly
differ between them, and were therefore pooled. Comparisons of BDNF levels
between normal and malnourished groups were made using unpaired Student’st-test, and PNC is the probability level for comparisons related to
the nutritional condition (NS = not significant). Comparisons between basal
BDNF levels (right EC) with those obtained after OC or CA1 tetanization (left
EC) were made using nonparametric ANOVA (Kruskal-Wallis test) followed by
Dunn’s multiple comparisons post-hoc test, and PT is the probability level for comparisons between
right and left EC samples (different superscripts indicate a significant
difference, P<.05; NS = not
significant).
Pooled OC + CA1 tetanizationOC tetanizationCA1 tetanizationPT(right EC)(left EC)(left EC)Normal15.7±3.5a(8)14.4±2.6a(4)25.1±2.2b(4)<0.05Malnourished9.5±0.82 (8)11.4±2.0 (4)8.1±1.6 (4)NSPNC<0.05NS<0.001


## 3.1. Effect of Dietary Treatment on Body and Brain Weights

Body and brain weights measurements
revealed that there were no significant differences in body weight gain of
pregnant mothers receiving 7.2% or 22.5% protein diet (data not shown). Full
data on the effects of this dietary treatment on maternal weight gain during
the first, second, and third weeks of pregnancy was published elsewhere [26]. At
days 1, 8, and 55–60 of postnatal
life, no significant differences in body and brain weights were found between
rats born from mothers receiving 7.2% or 22.5% protein diet (Table 1).Table 1
Body and brain weights of normal
and prenatally malnourished rats. Values
are means ± SEM.N=16
rats in each group. No statistically significant differences (NS) were found
when comparing body and brain weights of normal and malnourished groups of same
ages (unpaired Student’s t-test).
Body weight (g)Brain weight (mg)AgeDay 1Day 8Day 55Day 55–60Normal7.3±0.0718.9±0.37235±101322.0±16.1Malnourished7.2±0.0919.1±0.45229±121318.6±15.7PNSNSNSNS


## 3.2. LTP In Vivo in the Medial EC

In rats of 55–60 days of age, bipolar
recording of basal EC field responses to occipital cortex stimulation consisted
of a prominent upward negative wave followed by a positive component. This is
consistent with the arrangement of the side-by-side bipolar electrode located
into the EC, where the longer tip is expected to be recording an early superficial
sink generated by depolarization of stellate and pyramidal principal neurons within
input layers II-III (in relation to rather silent deep layers). In contrast, basal
EC responses to CA1 stimulation began with a marked downward surface positive
deflection followed by a late surface negative wave, which is consistent with
the recording through the shorter tip of an early deep sink resulting from depolarization
of pyramidal cells within output layer V. Figure1(a) shows typical recordings
of basal (A and C) and potentiated (B and D) averaged EC field responses evoked
by stimulation of the occipital cortex (A and B) or the CA1 region (C and D). In
normal eutrophic rats, the onset and peak latencies of the early negative
component of basal EC responses evoked from the occipital cortex were 18.7±2.2 and 30.6±1.8 milliseconds, respectively, while the onset and peak latencies
of the early positive wave in basal responses evoked from CA1 were 9.6±0.7
and 17.9±0.8 milliseconds (Figure 1(b)). For both type of responses, the
differences in latency before and after potentiation were not statistically
significant (paired Student’s t-test,
N=8). Shape, latencies, and wave amplitudes of basal field responses evoked
in the EC of prenatally malnourished rats, either from the occipital cortex or
the CA1 area, did not differ from those of normal eutrophic rats (unpaired
Student’s t-test, N=8 in each group). Frequency testing
showed that the early component of the EC
potential evoked from the occipital cortex declined rapidly with a stimulus frequency of 30 Hz, thus suggesting a polysynaptic nature of the
response. In contrast, the early component of the EC response elicited from CA1
was able to follow 30 Hz stimulation
frequency with decreasing amplitude of less than 20%, which is characteristic
of monosynaptic responses.In normal animals, tetanizing stimulation applied
to either the occipital cortex or the CA1 hippocampal region produced a
significant increase in peak amplitude of the early component
evoked in the ipsilateral
medial EC, which remained unchanged throughout the
recording period (Figure2). After tetanizing the occipital cortex, the early
negative wave to occipital cortex test stimuli was potentiated to neocortical test
stimuli in all blocks over the time-course (ranging from 107 to 136%, Dunnett’s
multiple comparisons test) excepting for block 2.5–5 minutes, while no
significant potentiation to CA1 stimuli was observed in the early component of EC
responses (Dunnett’s multiple comparisons test); however, a transient but
complete inhibition was early observed on block 0–2.5 minutes. After
tetanizing the ventral CA1, the early positive wave of EC responses to CA1 test
stimuli was potentiated in all blocks over the time-course (ranging from 59 to 72%,
Dunnett’s multiple comparisons test) excepting for block 2.5–5 minutes, while
no significant potentiation to occipital cortex stimulation was observed.Time-course of LTP induced in the medial
entorhinal cortex of 55–60-day-old normal
eutrophic rats by applying tetanizing stimulation to the occipital cortex (a) or
to the ventral CA1 hippocampal region (b). The arrow indicates time of
application of the tetanizing stimulus.N=8 rats in all groups. Values are
means ± SEM of peak-to-peak amplitudes, 30 responses averaged per rat. Note the
occurrence of homosynaptic, but not heterosynaptic potentiation. One-way ANOVA followed
by Dunnett’s multiple comparisons test indicated significant intragroup differences
in peak-to-peak amplitudes (P*<.05, P**<.01) when comparing
post-tetanizing values with the last pretetanizing basal point (at 0 minute),
excepting for block 2.5–5 minutes (a),
where significant inhibition (P**<.01) occurred.
(a)(b)In contrast to that
occurred in normal controls, no significant increase of the early component of
EC field responses evoked from the occipital cortex or from the CA1 region (P>.05
for all blocks, Dunnett’s multiple comparisons test) was observed in
malnourished animals after applying neocortical or hippocampal tetanizing
stimulation (Figure 3).Failure of tetanizing stimulation applied to
the occipital cortex (a) or to the ventral CA1 hippocampal region (b) to induce
LTP in the medial entorhinal cortex of 55–60-day-old prenatally
malnourished rats. The arrow indicates time of application of the tetanizing
stimulus.N=8 rats in all groups. Values are means ± SEM of peak-to-peak
amplitudes, 30 responses averaged per rat. It can be noted that neither
homosynaptic nor heterosynaptic potentiation occurred in the EC of malnourished
animals. One-way ANOVA followed by Dunnett’s multiple comparisons test
indicated no significant intragroup differences in peak-to-peak amplitudes when
comparing posttetanizing values with the last pretetanizing basal point (at 0
minute), excepting for block 2.5–5 minutes (a) where
significant inhibition (P**<.01) occurred.
(a)(b)


## 3.3. BDNF Expression in the EC

Serial dilutions of
known amounts of BDNF ranging from 0 to 500 pg/mL allowed to determine a
standard curve demonstrating a
direct relationship between optical density and BDNF concentration (r2=0.9106).Table2 shows that
application of tetanizing stimulation to the left CA1 region in normal rats
resulted in a significant increase of BDNF concentration in the ipsilateral EC
(P<.05, Dunn’s multiple comparisons test) two hours after
tetanization, while application of tetanizing stimuli to the left occipital
cortex did not significantly modify the BDNF level in the ipsilateral EC. In
contrast, tetanizing stimulation applied to either the occipital cortex or the
CA1 hippocampal region in malnourished rats was ineffective in modifying the
BDNF concentration in the ipsilateral EC. Table 2 also shows that on days 55–60 of postnatal
life, malnourished rats exhibited significant lower concentration of BDNF
protein in the right medial EC (corresponding to the nonstimulated cerebral
hemisphere) than that observed in normal animals of same ages (P<.05,
unpaired Student’s t-test).Table 2
Changes in BDNF
expression (pg/mg wet tissue) in the left entorhinal cortex (EC) of 55–60-day-old normal
and prenatally malnourished rats two hours after applying ipsilateral
tetanizing stimulation to the occipital cortex (OC) or the CA1 hippocampal
region, as compared to BDNF levels in the right EC. Values are means ± SEM. The
number of samples in each group is shown in parentheses. BDNF concentrations in
right EC samples after tetanizing the left OC or left CA1 did not significantly
differ between them, and were therefore pooled. Comparisons of BDNF levels
between normal and malnourished groups were made using unpaired Student’st-test, and PNC is the probability level for comparisons related to
the nutritional condition (NS = not significant). Comparisons between basal
BDNF levels (right EC) with those obtained after OC or CA1 tetanization (left
EC) were made using nonparametric ANOVA (Kruskal-Wallis test) followed by
Dunn’s multiple comparisons post-hoc test, and PT is the probability level for comparisons between
right and left EC samples (different superscripts indicate a significant
difference, P<.05; NS = not
significant).
Pooled OC + CA1 tetanizationOC tetanizationCA1 tetanizationPT(right EC)(left EC)(left EC)Normal15.7±3.5a(8)14.4±2.6a(4)25.1±2.2b(4)<0.05Malnourished9.5±0.82 (8)11.4±2.0 (4)8.1±1.6 (4)NSPNC<0.05NS<0.001


## 4. Discussion

Mild reduction of the protein
content of the maternal diet of pregnant rats did not significantly alter body
and brain weights of pups at birth, indicating that protein deficiency in the
7.2% protein group was masked by caloric compensation with carbohydrates,
leading to apparently normal fetal development as assessed by body and brain
weights at birth. A similar result has been reported elsewhere [25, 39]. As
discussed by others [25, 39], fetal growth retardation and reductions in brain
weight after prenatal malnutrition are only produced by severe protein
restriction, that is, reduction of the protein content of the maternal diet to
less than 6%.The foregoing electrophysiological data show that the
medial EC of normal eutrophic rats can develop LTP in vivo to tetanization of both the occipital cortex and the CA1
hippocampal region. This is consistent with previous data showing that the EC could
express LTP to tetanizing stimulation of some cortical and hippocampal regions in
in vivo conditions. For example, Chapman and Racine [34]
have reported a surface negative response that could be evoked in vivo in the EC of rats by
stimulation of the piriform cortex, and that these responses undergo LTP to
high-frequency stimulation of the piriform cortex. However, Ivanco and Racine [35] found that stimulation of the motor
cortex failed to elicit EC responses. On the other hand, surface positive field
responses have been elicited in vivo in
the EC of rats by stimulation of CA1 [35–37]. In all these studies, the early
positive component of the EC response supported LTP to high frequency
stimulation.The present results also suggest that the early negative response evoked in the
entorhinal cortex (EC) by occipital cortex stimulation is apparently
polysynaptic, since it had slow onset and
peak latencies and it was very sensitive to 30 Hz stimuli. As reported
previously, these inputs are
synaptically relayed within the perirhinal
and/or parahippocampal cortices before to reach the superficial layers of the
EC [7–11]. Polysynaptic LTP often involves local circuits within the recipient
brain region, but sometimes is synaptically relayed by brain intermediate
regions that are more distant from the recipient zone thus involving long axonmediated
connections. Regarding the present results, it is not possible to directly
known if potentiation occurred in neurons of the final recipient entorhinal
region or in intermediate perirhinal neurons relaying the response (or in both)
and, therefore, the site of LTP occurrence remains rather unresolved. In
contrast, the early positive
response evoked in the entorhinal cortex (EC) by CA1 stimulation is apparently
monosynaptic since it had shorter onset
and peak latencies and followed a
stimulus frequency of 30 Hz without showing a significant amplitude decrease. As
mentioned previously, direct projections from the CA1 field to the deep V-VI layers of the
EC have already been reported [15–17].The fact that tetanization of the occipital cortex or
the CA1 region only potentiates the responses driven by the tetanized region confirms
that the early component of EC responses to either occipital cortex or CA1
stimulation represents the activation of two distinct set of neurons
(presumably located in superficial layers II-III and deep layer V, resp.).
Nevertheless, prenatally
malnourished adult rats were unable to develop LTP in the medial EC, at least
when submitted to a similar tetanizing stimulation protocol to that applied to
the occipital neocortex or the ventral CA1 region in normal eutrophic animals,
thus suggesting that mild prenatal malnutrition impairs some neural substrate
involved in the generation and/or maintenance of EC plasticity. Previous reports have shown that it
is difficult to induce and maintain hippocampal [5, 6] and neocortical [4] LTP
in brains of prenatally malnourished rats, but the underlying cellular/molecular
mechanisms are still unresolved. Reduced plastic response in the hippocampus of
prenatally malnourished rats seems to be related to significant increases in GABAergic inhibition in the
dentate gyrus [1, 40], while in neocortex it may be correlated with decreased
noradrenaline release due to enhanced α2C adrenoceptor expression [4, 28]. However, the effect of prenatal malnutrition on
neuroplastic mechanisms operating in the EC had not yet been explored.Interestingly, the medial EC of normal eutrophic rats showed
increased BDNF concentration two hours after delivering tetanizing stimulation
to the ipsilateral CA1, whereas the same stimulatory protocol failed in
modifying the BDNF level in the EC of prenatally malnourished rats. As has previously been reported, high-frequency
stimulation inducing LTP evokes significant increases in BDNF mRNA expression [41–44] and BDNF release [45] in the
hippocampus, although changes in hippocampal BDNF protein levels after LTP
induction, have not
still been evaluated. In addition, released BDNF activates distinct mechanisms
to regulate the induction, early maintenance, and late maintenance phases of hippocampal
LTP [29, 30]. Curiously, LTP induced by unilateral perforant
path stimulation seems to produce bilateral induction of BDNF mRNA, although limited to the dentate gyrus [42–46]. A more detailed study addressing this aspect was carried
out by Bramham et al. [47], who demonstrated that unilateral LTP induction in
the dentate gyrus of awake rats led to highly selective ipsilateral (trkB and
NT-3 mRNA) or bilateral (trkC, BDNF, and nerve growth factor mRNA) increases in
gene expression, indicating that LTP triggers an interhemispheric communication
manifested as selective, bilateral increases in gene expression at multiple
sites in the hippocampal network. Whether changes in BDNF concentration
occurred bilaterally in the medial EC after unilateral tetanization of CA1
could not be assessed in the present study, because of the BDNF level in the EC
of the nonstimulated right side served as control for the BDNF value obtained
in the EC of the left stimulated side. Nevertheless, despite the inexistence of
a proper control taken from nontetanized rats, the present data show that there
was a significant difference in BDNF concentration when values from right
(nonstimulated) ECs were compared with those from left (stimulated) ECs in
normal eutrophic rats, whereas such a difference was not present in entorhinal
tissue from malnourished animals. Failure of tetanizing stimulus in modifying BDNF levels in the ipsilateral
EC of prenatally malnourished rats (so-called ‘‘instructive mechanisms’’
[29, 30], those initiated in response
to high-frequency stimulation and required for subsequent development of LTP) clearly match the inability of the
medial EC to induce LTP in malnourished animals, but caution must be exercised regarding
this issue because this observation reveals a correlational but not causal
relationship. Additionally, malnourished animals had significantly lower
concentration of BDNF in the right EC (supposedly ‘‘basal’’ levels in the nonstimulated
side) than normal ones, thus suggesting a possible additional deficit in ‘‘permissive
mechanisms’’ of BDNF (those that make
synapses competent for LTP [29, 30]).Why application of tetanizing stimulation to the occipital
cortex of normal eutrophic rats resulted in potentiation of ipsilateral evoked
EC responses, but not in increased BDNF concentration in the same EC, is
presently unclear. One plausible explanation is that occipital cortex
tetanization really increased BDNF expression but solely in some restricted
layers of the EC and therefore they were not detected by staining the whole EC.
Alternatively, it is possible that this type of polysynaptic LTP did not
actually lead to increased BDNF expression. Then, this negative result in the
occipital cortex-EC pathway should be interpreted with caution as the analysis
performed is quite preliminary. Comparable tetanizing stimulation of the
occipital cortex in malnourished rats did not induce either LTP or BDNF protein
enhancement in the ipsilateral EC. Furthermore, high-frequency stimulation of
the occipital cortex gave rise to a short period (about 5 minutes) of depression
(or even irresponsiveness) of ipsilateral EC neurons to CA1 stimulation, both
in normal and malnourished animals (see Figures2(a) and 3(a)). Whether this
transient presumably intra-EC inhibitory activity resulted from a feedforward
inhibitory mechanism [48] or from a feedback mechanism triggered by the returning
CA1 output into deep layers of the EC (see Craig and Commins [36]) remains to be determined. Also, the possibility that the
complete loss of the CA1-evoked EC response after tetanizing the OC could be
the result of generating local spreading depression cannot be discarded. In
this regard, spreading depression-like episodies that were confined to the
first 5 minutes after tetanizing the perforant path-granule cell pathway have
been reported in anesthetized rats [49].In summary, the present data show that mild prenatal
protein malnutrition resulted in impaired ability of the EC to undergo LTP and
to increase BDNF levels in response to tetanizing stimulation of the
ipsilateral ventral CA1 hippocampal region during postnatal life. On the basis
that EC is part of a circuit underlying networked representations of previous experiences via bidirectional connections between the
neocortex with the hippocampus, impaired EC plasticity may be an important
factor contributing to deficits in explicit learning having adult, prenatally
malnourished animals.



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*Source: 646919-2008-06-30.xml*