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1 Departamento de
Neurociencias, The present
study was aimed at characterizing the effects of low-protein
malnutrition (6% casein) on the circadian rhythm of drinking behavior
and on suprachiasmatic nuclei immunohistochemistry in Sprague-Dawley
rats. Recordings were started at 30 days of age under a 12:12-h
light-dark (LD) cycle. At age 150 days, recordings were continued under
constant dim red light, and finally the latency to entrain to complete
and skeleton photoperiods was established. At the end of the recordings
rats were processed for histological analysis. Compared with their
controls, malnournished rats exhibited 1) splitting of
rhythmicity under LD that 2) condensed to one component in
constant dim red light, 3) delayed entrainment to skeleton
photoperiod, and 4) precocious entrainment
under complete photoperiod. Immunohistochemical analysis showed mainly
a decrease in the immunohistochemical detection of vasoactive
intestinal polypeptide and glial fibrillar acid protein cells in
malnourished animals. These results indicate that in malnourished rats
there is a decrease 1) in the
coupling force among the oscillators and 2) in the strength of the phase lock
between the oscillators and the light-dark cycle.
photic entrainment; oscillatory coupling; low-protein malnutrition; hypothalamic immunohistochemistry
CIRCADIAN RHYTHMICITY represents an adaptive mechanism
of organisms to a highly cyclic environment, which enables adequate predictive behavioral and physiological responses from the organism even before the environmental stimuli are present. This process is the
outcome of a genetically coined temporal organization (23). In mammals,
particularly in rodents, the morphological and functional substrates of
circadian rhythmicity have been at least partially elucidated. Nowadays
there is well-founded evidence that the suprachiasmatic nuclei (SCN)
and their afferent and efferent connections are involved in the
generation and regulation of circadian phenomena (see Ref. 17 for a
review).
On the other hand, malnutrition may be viewed as one of the major
adaptive challenges to organism survival. Besides its practical implications for the human population, the experimental paradigms of
malnutrition may be used as tools to understand the adaptive physiological mechanisms of the organism to adverse environmental conditions.
The effect of severe malnutrition on the nervous system has been
studied from different perspectives. At the morphological level its
effects may be summarized as a distortion in neurogenesis due to a
delayed cell division and prolongation of the cell cycle (19), which in
turn affects the neuronal-to-glial ratio (2). Other alterations include
deficient myelination due to a decrease in the number of
oligodendrocytes and changes in myelin metabolism (8) and alterations
in the regulation of several neurochemical systems (see Ref. 21 for a
review). At the behavioral level, long-term changes induced by
malnutrition include locomotor hyperactivity and increased emotional
responses (32) and impairment in some learning and memory tasks (18).
Previous studies on the effects of low-protein malnutrition on the
temporal organization of behavior are very few and most of them are
restricted to the sleep-wake cycle. Forbes et al. (10) found a
redistribution of rapid eye movement (REM) sleep across the diurnal
cycle, characterized by a decrease in the total time of REM sleep
during the light phase and increase during the dark phase; changes were
more evident at the transitions of the light-dark cycle. Such findings
led Forbes et al. to suggest that malnutrition could affect the
circadian system, mainly the phase adjustment to environmental
zeitgebers. Such observations were confirmed by other laboratories with
different nutritional paradigms (3, 27) and were associated with a
redistribution of behavioral patterns, especially grooming (27). With
respect to other behavioral rhythms, Hall et al. (12) reported a phase
delay in the rhythm of feeding behavior and Cipolla-Neto et al. (4)
described a decrease in the amplitude and mesor of different behavioral
patterns as the main effect of malnutrition. In addition, the severity of such changes was related to the age of its onset. Finally, it has
been described that low-protein malnutrition induces a decrease in the
amplitude and a phase delay of plasmatic melatonin rhythm in rats (13).
Such changes in melatonin secretion by the pineal as well as the
behavioral and sleep changes suggest that malnutrition may affect the
expression of circadian rhythmicity in processes such as entrainment to
light-dark cycles, coupling among oscillators, or transmission of
rhythmicity to the effectors.
Previous studies, although suggestive, are limited by the length of
recordings and time sampling in allowing conclusions to be drawn
regarding the circadian organization of the malnourished rats. The
present study was aimed at characterizing the circadian organization
(estimation of endogenous period, phase angle to the light-dark cycle,
latency to entrainment to complete and skeleton photoperiods) of
chronic malnourished rats using long-term recordings of drinking
behavior under different lighting conditions. Recording of drinking
behavior was used because it is a simple and very reliable marker of
activity rhythms in rats. Finally, to establish whether low-protein
malnutrition affects cell groups and fibers in the SCN that underlie
the generation and regulation of circadian rhythmicity, the number of
cells and/or fibers expressing immunohistochemical staining to
vasopressin (VP+), vasoactive intestinal polypeptide (VIP+),
glial fibrillar acidic protein (GFAP+), neuropeptide Y (NPY+),
and serotonin (5-HT+) was compared between control and malnourished
rats.
Compared with their controls, malnourished rats exhibited bimodal
symmetric pattern of drinking under a light-dark cycle, two
free-running activity components when transferred to constant dim red
light that converged to form a single one with an endogenous period
similar to control animals, and delayed entrainment to skeleton
photoperiod and early entrainment or masking of rhythmicity under
complete photoperiods. The main findings of immunohistochemical analysis in malnourished animals were a ~25% decrease in the number and density of VIP+ and GFAP+ cells. These results indicate that in
malnourished rats there is a decrease in
1) the coupling force among the
oscillators and 2) the strength of
the phase lock between these and one of its zeitgebers, the light-dark
cycle.
Animals and General Procedure
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
Adult females to be used as breeders were randomly assigned either to a control or an experimental group. Those in the latter group were fed an isocaloric low-protein diet of 6% casein complemented with methionine (Teklad Mills Labs) 5 wk before mating. The same diet was maintained during pregnancy and lactation. After weaning, the diet was provided to the pups, which were used as the experimental subjects, and was maintained throughout the duration of the study. Control and 6% casein diets contain the same proportion of kilocalories per gram, fat, vitamin mix, minerals, and nonnutritive fillers and only differ in the protein content. Low-protein diet was supplemented with L-methionine (0.4%) because casein lacks this essential amino acid. This procedure causes severe and reproducible low-protein malnutrition characterized by a decreased body and brain weight at birth and a slower body and brain growth rate with respect to control animals (26). Animals from the control group were treated similarly but fed a regular laboratory diet containing 25% casein (Purina Chow). In both groups, the reproductive units consisted of four females and one male. One week before mating, males for the malnourished group were also fed the same low-protein diet. Vaginal smears were inspected for sperm positiveness every morning starting the day after placement of the male in the unit, and sperm-positive females were removed from the unit and housed individually. On the day of delivery, litters were standardized to eight pups regardless of their original size (9-14 pups/litter for control and 6-11 pups/litter for malnourished rats).
On the day of weaning (postnatal day 21), the pups were sorted by gender and housed collectively for 8 to 9 days before the beginning of the behavioral recordings. At 30 days of age the rats from both nutritional conditions were individually placed in the recording cages, and their drinking behavior was continuously monitored for the next 6-10 mo under different lighting schedules. At the end of the recording period the animals were euthanized, and their brains were processed for immunohistochemical analysis of the SCN.
Behavioral Recordings and Analysis
Drinking behavior was continuously monitored by a computerized system described elsewhere (1). Briefly, each time the animal touched the water spout it generated an electric pulse; these events were computed at 15-min intervals and stored in magnetic media for later analysis. Animals from both nutritional conditions (22 control, 37 malnourished) were monitored under the same lighting conditions. The behavior of 16 animals (4-6 controls and 10-12 malnourished) was simultaneously recorded for ~300 days under different lighting conditions: 12:12-h light-dark regimen (LD), constant dim red light (50 lx, DD), or skeleton photoperiod of two light pulses at 12-h intervals (30 min, 400 lx). The experiments were conducted from spring 1991 to spring 1994 in four successive series. Graphic display and analysis of the data were made by the Digital Analysis System Applied to Chronobiology (DISPAC) developed and validated in our laboratory (Instituto de Fisiología Celular-Universidad Nacional Autónoma de México). Other statistical analyses were made using the SPSS-PC software.Histological Procedures
At the end of the recordings all animals received a lethal dose of pentobarbital sodium (100 mg/kg body wt) and were perfused transcardially with 500 ml of 0.9% saline solution followed by 300 ml of fixative (4% paraformaldehyde, 15% saturated picric acid solution in 0.1 M phosphate buffer, pH 7.2). This procedure was accomplished between 1200 and 1400 in pairs of control and experimental animals simultaneously. Brains were removed and postfixed for 1 h at 4°C, then transferred successively to 10, 20, and 30% sucrose solutions in 0.1 M phosphate buffer (pH 7.2, 4 C) until they sank. The anterior hypothalamus was then cut in the coronal plane in 40-µm sections with a cryostat. Sections were serially collected in phosphate buffer (0.1 M, pH 7.6) in four sets and processed for immunohistochemistry according to the avidin-biotin method. Primary antibodies (all from Incstar) against VP, VIP, GFAP, 5-HT, and NPY were diluted 1:1,000 in 0.1 M phosphate buffer containing 1% normal goat serum and 0.3% Triton X-100. Immunoreactive material was designated VP+, VIP+, GFAP+, 5-HT+, and NPY+, respectively. These procedures were made simultaneously in pairs of control and experimental tissue. Each brain was processed at random with four primary antibodies, and a minimum of six animals from each group was studied with each antibody.Experimental Design
Experiment 1. Development of rhythmicity under entrained conditions. This protocol studied the development of rhythmicity from 30 to 130 days of age in rats kept in a LD cycle (12:12 h). Forty-three animals of both genders (22 males, 21 females) and nutritional status (14 controls, 29 malnourished) were studied in a mixed factorial design considering age (3 levels, repeated measures) and nutritional status (2 levels, randomized). Because no differences due to gender were found in a preliminary analysis of the data, this factor was not included in the design. At 30 days of age the animals were housed individually, and the recording of drinking behavior was obtained continuously for the next 150 days as previously described, with the exception of 14 subjects (7 control, 7 malnourished) that were recorded only for 110 days and then were processed for histological analysis.The data were double plotted in an array of 48 h per line. Actograms
thus obtained were visually inspected, and segments of data at 30, 90, and 120 days of age were selected for further analysis. These segments
consisted of 16 days of recording without missing data >24 h or any
other type of system malfunction. A margin of up to 2 wk was allowed to
select the segment in the case of missing data. To determine the rhythm
architecture, the following parameters were considered:
1) duration of activity (
),
2) duration of rest (
),
3) period length (
), and
4) phase relation of activity onset
to lights-off (
). Mean values from 16 consecutive cycles were
computed for each subject and used as an estimator of the parameter
under study, whereas the standard deviation was used as an estimator of
its lability (24). Mathematical estimation of period was accomplished
on data from 16-day segments using the
2 periodogram (28) and the
spectral density analysis (SDA) based on the Fourier transform (31).
Statistical analysis was accomplished by a two-way analysis of variance
(ANOVA). The -level in all the experiments was 0.05 unless otherwise
stated.
Experiment 2. Endogenous period and entrainment to complete photoperiod. This protocol studied rhythmicity of adult rats in free-running conditions and its entrainment to a LD cycle. The remaining 29 rats from the previous experiment (7 controls and 22 malnourished) were used in this study. The experiment started when the previous illumination conditions (LD) were switched to constant dim red light (50 lx, DD), the recordings were continued for 60 days. Then the animals were exposed again to the LD cycle and the recordings continued for 30 days. At the end of the recordings the animals were processed for histological analysis.
From the DD recordings, the endogenous period () was measured by a
graphic procedure and the 2
periodogram. The graphic procedure consisted of a linear regression on
the activity onset of 10 consecutive days; the period was estimated from the slope of the fitted line (1). To describe the time course of
entrainment to the complete photoperiod, the number of days to entrain
to a LD cycle from DD were computed. Entrainment was considered to
occur on the first day when the activity showed a period of 24 h, with
a constant phase angle between dark and activity onset maintained for
at least the following 3 consecutive days. Because the phase angle
between activity onset and the LD cycle may influence the time course
of entraining, the time difference between the activity onset and
turning off the lights on the first day in LD was computed and
expressed in degrees (1 h = 15°). Statistical analysis of these
data was accomplished using the Student's
t-test for independent measures.
Experiment 3. Entrainment to skeleton photoperiod. To discriminate between the masking effect due to the complete photoperiod and actual entrainment of rhythmicity, the latency of entrainment to a skeleton photoperiod was measured in an additional eight animals from each group. To do so, 120-day-old rats were recorded in the following lighting conditions: 1) LD cycle for 20 days, 2) DD for 45 days, and 3) skeleton photoperiod for 45 days. The skeleton photoperiod consisted of 30 min of light each 12 h (lights on at 0800 and 2000, 400 lx). The period in DD, the latency of entrainment to the skeleton photoperiod as well as the phase angle between activity onset and the next light pulse given at 2000 of the skeleton photoperiod were computed as described in experiment 2. Statistical comparison between both nutritional groups was made using the Student's t-test for independent measures.
Experiment 4. SCN cytoarchitecture. At the end of the recordings animals were transferred to an LD room for 2 to 3 wk and then processed for histological analysis. To control for the variability inherent to immunohistochemical procedures on the measurements, particular care was taken to simultaneously incubate pairs of control and experimental sections throughout the entire procedure.
Manual morphometric analysis was made with the aid of the
MCID imaging analysis system as follows: for each pair of
control and experimental tissue equivalent sections from the middle
level of the SCN were identified, corresponding approximately to
anterioposterior (AP) 
1.3 mm from bregma (22). A representative
section for each subject was selected for analysis. The
cytoarchitectonic borders of the SCN and the regions showing dense and
sparse cellular or fiber distributions were delimited with
phase-contrast microscopy. The area for each of those regions, which
correspond to the dorsomedial and ventrolateral divisions of the SCN,
was measured. Cell counts for VP+ and VIP+ and fiber counts for 5-HT+
and NPY+ were made at ×600 in each of the regions previously
described. GFAP+ cells, which exhibit a less-regionalized distribution,
were counted in the total SCN. Fiber counting was accomplished by using
a reticule made of parallel sinusoidal waves 60 µm apart and with a
length of 60 µm between crests (modified from Ref. 30). The criterion to manually select the targets to be counted was a minimum ratio for
background to immunoreactivity of 1:3 in relative optic density. Whenever overlapping of targets in the
z-axis was apparent, this was verified
by adjusting the focus plane in the microscope, and in such cases the
targets were counted individually. Both cell and fiber densities
(expressed in 100 µm2) were
estimated from the number of targets counted and the area of the region
from which they were collected. Statistical comparisons between groups
were made from six subjects with the use of a one-way ANOVA. The
-level was set at 0.05.
To establish whether histological changes induced by malnutrition were
specific to the SCN, similar measurements were performed in other brain
areas that express the studied peptides as follows: the magnocellular
division of the paraventricular hypothalamic nuclei (PVN) for VP+
cells, the frontoparietal cortex from the rhinal fissure to the dorsal
edge of the caudate putamen for VIP+ cells, and the parietal cortex
immediately dorsal to the rhinal fissure (from 16) for GFAP+ cells.
Cortical VIP+ and GFAP+ cells were counted from the same section used
for SCN. VP+ cells were counted from equivalent sections of the PVN
corresponding approximately to AP 1.8 mm from bregma (22).
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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At birth, pups from females fed the 6% protein diet weighed significantly less (5.2 ± 0.2 g) than pups from mothers fed with 25% casein diet (6.08 ± 0.15 g). At 30 days of age, when rats were placed in the behavioral recording cages, malnourished animals weighed only 21 ± 1.34 g, whereas control rats weighed 65.8 ± 2.3 g, which means a deficit of 66% of body weight. It was evident throughout the study that malnourished rats maintained a decreased body size, although to avoid perturbations in the behavioral recording, no further weight measures were performed. Data obtained in other studies in our laboratory indicate that the weight deficit in malnourished animals persists up to 220 days of life (198 ± 30 g in malnourished and 627 ± 191 in control rats).
Behavioral Recordings
Experiment 1. Most of the subjects showed clear circadian rhythms from the beginning of the experiment at 30 days of age. Some others presented an ultradian pattern of drinking behavior, which became clearly circadian within the next 2 wk. Such patterns were similar for both groups and remained about the same for the first 2 mo of recording (Fig. 1). However, a difference between groups was seen in the persistence of variability in the phase of activity onset in the malnourished animals. Such variability was present in both groups at 30 days of age but became less evident in the control animals as they grew older, reaching a minimum at ~90 days of age. With regard to the number of water spout contacts, control animals showed 6,961 ± 2,074 spout contacts per day (251 ± 101 per 15-min bin, mean ± SD) whereas malnourished animals showed 2,520 ± 1,239 contacts per day (96 ± 48 per 15-min bin). These values were similar for the entire study.
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The rhythm architecture at 30, 90, and 120 days of age is shown in
Table 1. For both experimental groups, at
30 days of age the rest-to-activity ratio was smaller and was
larger than at 90 and 120 days of age. The mean period was 24 h as expected for the entrained condition. The most
striking difference between the two groups was found at ~120 days of
age, the time at which most of the malnourished subjects (70%) showed
a change in the pattern of activity that was characterized by bouts of
drinking at 12-h intervals. In some animals such change occurred
abruptly within 3-4 days (Fig.
1B), but in others the change
developed gradually as two components of activity moved out of phase
(Fig. 1C), therefore with two
simultaneous periods of activity. During the time when this was
observed rats were maintained under a LD regimen. Such a pattern was
not observed in the animals from the control group. The results of the
ANOVA are summarized in Table 1; most parameters were affected by both
the age and the nutritional condition and all showed significant
interactions between these factors.
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According to the spectral density analysis and
2 periodogram, control animals
showed a main component of 24 h, which increased in density as the rats
grew older. Malnourished animals showed a similar pattern at 30 and 90 days of age, although at 120 days 70% of the malnourished animals also
showed an increase in amplitude of the 12-h component (Fig.
2).
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Experiment
2. Control animals showed free running
of rhythmicity with a stable period of 24:25 ± 0:14 (mean ± SD;
h:min) (Fig. 3A). On
the other hand, malnourished animals showed free running of both
activity components found under LD, with the values for both short and
long periods being 23:48 ± 0:37 and 24:20 ± 0:12, respectively.
Both components gradually converged to form a single component after
~30 days under DD (Fig. 3, B and
C). In this latter condition
malnourished animals showed a comparable to control animals (24:34 ± 00:16), very similar to the data from the
2 periodogram, which showed for
controls and malnourished subjects periods of 24:24 ± 0:24 and
24:36 ± 0:54, respectively.
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At the end of the recording in DD, animals were returned to the LD cycle (lights on at 0800, 400 lx). The mean angle to lights off for the first day on LD was 140 ± 51° for control and 217 ± 101° for malnourished rats (Fig. 4). Malnourished animals appeared to entrain almost immediately after the onset of the LD cycle (Fig. 5). To quantify this phenomenon, the latency for entrainment to the complete photoperiod was estimated by computing the days taken to obtain a constant phase relation between lights off and activity onset for at least 3 consecutive days, with a period equal to 24 h. The mean latencies for control and malnourished animals were 6.9 ± 2.6 and 2.6 ± 1.3 days, respectively. Such differences were statistically significant [F = 10.79, t = 6.5, degrees of freedom (df) 20, P < 0.05].
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Because the mean phase angles between both groups were significantly different, it is possible that this may explain the differences in the number of transients found between control and malnourished animals. To contrast this hypothesis, eight animals from each group were paired according to their phase angle to the entraining stimulus, and then the endogenous period and the number of transients to reentrainment were compared by a Student's t-test (Table 2). In this condition the difference in the number of transients persists (control 5.75 ± 1.58; malnourished 2.12 ± 0.35; t = 5.9, df 14, P < 0.05) but no difference was found in the endogenous period, which indicates that this effect depends neither on the phase angle nor the endogenous period.
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Experiment
3. To discriminate between a possible
masking effect due to the complete photoperiod and actual entrainment
of rhythmicity, the latency of entrainment to a skeleton photoperiod was measured in eight additional animals from each group. To do so, the
animals were held in DD for 45 days and then exposed to a skeleton
photoperiod (lights on at 0800 and 2000, 400 lx, Fig. 6). The free-running periods in control and
malnourished animals were similar to those reported in
experiment
2. The phase angle to the light pulse
given at 2000 on the first day of skeleton photoperiod was 227 ± 101° for control and 191 ± 99° for malnourished rats (Fig.
4). The latency of entrainment was computed as described for the
complete photoperiod. In these conditions, control animals showed very
similar values for this parameter to those found in animals entrained
to the complete photoperiod, 6.1 ± 3.1 days; in
contrast, malnourished subjects showed a mean latency of 12.3 ± 6.0 days (F = 7.44, t = 2.41, df 14, P < 0.05). No statistical differences were found in the endogenous period and the phase angle to the reentrainment stimulus between control and malnourished rats (Table 2).
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SCN Cytoarchitecture
Experiment 4. No clear differences in SCN morphology between nutritional groups were found with visual inspection of immunostained material under bright-field microscopy. The middle level of the SCN stained for VP+, VIP+, GFAP+, NPY+, and 5-HT+ in control and malnourished animals is shown in Fig. 7. It can be observed that characteristic SCN cellular groups maintained their anatomic distribution. Both VP+ and VIP+ neurons and their dense fiber plexes are located in the dorsomedial and ventrolateral regions, respectively, whereas sparse fibers extend to the complementary portion of the nuclei. GFAP+ cells are distributed throughout the extent of the nuclei. NPY+- and 5-HT+-dense fiber plexes were found in the ventral region, although sparse fibers extended to the remaining SCN.
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The morphometric analysis of cells and fibers of SCN and control regions is shown in Table 3. In the SCN and cortex of malnourished animals a significant decrease in cell number and density for VIP+ and GFAP+ was found. In contrast, VP+ cell number and density were decreased in the dorsomedial and total SCN but not in the ventrolateral region, whereas in PVN only a decrease in the cell number but not in the cellular density was found. Afferent fibers to the SCN were less affected by malnutrition; 5-HT+ fibers were only reduced in number in the dorsomedial and total SCN. No changes were found for NPY+ fibers.
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The low body weight observed at birth and 30 days of age, as well as the reduced body size observed throughout the study in chronic 6% casein-fed rats, is similar to values reported in previous studies (26) and confirm that this diet is effective in producing malnutrition in rats.
Behavioral Aspects
During the first 2 mo of recording, control animals exhibited an ontogenic pattern characterized by 1) condensation of the,
2) increase of the rest-to-activity
ratio, 3) improvement in the phase
adjustment to the entrainment signal, and
4) tuning of the period to that of
the zeitgeber. These findings are similar to previous reports (6).
The total number of water spout contacts during 24 h was reduced in
malnourished rats to 64% of their controls. Because malnourished animals showed a similar decrement in body weight (66%), it seems that
the difference in the frequency of drinking behavior may be related to
the different corporal mass between the two groups. Despite such
differences, malnourished animals exhibited a similar pattern of
drinking behavior to their controls until ~90 days of age, while
showing a poor phase adjustment to the LD cycle manifested in the
lability of (Table 1). Previous studies addressing the effect of
malnutrition on the sleep-wake cycle indicate a decrease in the total
time of REM sleep during the light phase and increase during the dark
phase, which may reflect an alteration in the phase adjustment to the
light-dark cycle (3, 10, 27). The results from the present study extend
such observations and indicate that phase changes in the sleep-wake
cycle may be due to alterations in the entrainment of malnourished
animals to the light-dark cycle.
At ~120 days of age, malnourished animals showed two components of
activity at ~12-h intervals. The spectral density analysis and the
2 periodogram indicated an
increase of the 12-h component (Fig. 2). When animals were recorded in
constant darkness, the two components of activity exhibited different
endogenous periods and afterward converged to form a single component
with an endogenous period similar to the controls (Fig. 3). Such a
pattern of activity resembles the splitting of rhythmicity found in
some nocturnal rodent species held in constant bright light (24). In
such cases splitting has been considered to reflect the drifting apart
of two oscillators due to a decrease in the coupling force between
them. Present results reflect uncoupling between two groups of
oscillators. Thus malnutrition may induce a decrease in the coupling
force among oscillators of the system. Alternatively, malnutrition may affect the endogenous period of the oscillators, which in turn would
make it difficult for them to attain a stable coupling state. The
finding of a similar period in control and malnourished animals after
some weeks in constant dim red light suggests that this is not the case
because malnutrition does not affect the endogenous period of the
oscillators.
Inspection of Fig. 4 and Table 2 shows that, in both experimental
groups (control and malnourished), the individual phase angles between
activity onset and the entraining stimulus (either complete or skeleton
photoperiod) were scattered throughout the entire 24-h cycle. In
control animals mean values of phase angles were significantly
different (t = 2.33, df = 14, P < 0.05) between both entraining
conditions and, even so, no difference was found in the number of
transients to entrain either to a complete or to a skeleton
photoperiod. In contrast, in malnourished animals no difference in the
mean values of phase angle to each entrainment condition were found
(t = 1.14, df = 14); even so,
entrainment to the complete photoperiod was found to occur in about
one-half the time required for control rats and it took about twice the time needed by controls to entrain to a skeleton photoperiod. Altogether, the finding of a similar endogenous period for control and
malnourished rats and the fact that the phase angle did not contribute
to the differences in entrainment latency suggest that the precocious
entrainment induced by complete photoperiod and the late entrainment to
skeleton photoperiod of the malnourished animals maybe due to a
decrease in the responsiveness of the circadian system to light as a
zeitgeber, rather than to an increased responsiveness to the LD cycle
(masking-like effect).
Precocious entrainment of rhythmicity to the complete photoperiod may be due to alterations in visual afferents to the SCN. It has been reported that in some experimental conditions, light may drive overt rhythmicity through sprouting of retinal fibers into the anterior hypothalamus (15). In early stages of development of normal rats, the retinohypothalamic tract (RHT), the main visual pathway for light entrainment (15), projects to the SCN, adjacent hypothalamic areas, and the lateral hypothalamus; briefly after parturition a regression of projections takes place, leading to the adult pattern in which most fibers terminate in the SCN and only sparse fibers are in the adjacent and lateral hypothalamus (29). During development, malnutrition induces permanent alterations in the growth of neuronal processes and connectivity in different brain areas (7, 9). It is thus conceivable that malnutrition also induces an abnormal development of the RHT. Tract tracing studies in malnourished rats remain to be done to directly test such a hypothesis. Nevertheless, preliminary data from our laboratory indicate that in control animals 13 of 180 neurons (7%) recorded in the hypothalamus adjacent to the SCN responded to brief pulses of bright light (5 ms, 600 lx). In contrast, in malnourished rats there was an increase in the number of neurons (17 of 131, 13%) responding to light pulses outside the SCN (11). These results are consistent with the previous hypothesis that malnutrition may induce an abnormal development of the RHT.
SCN Cytoarchitecture
Low-protein malnutrition induced a long-term decrease in VIP+ and GFAP+ cells within the SCN in both the cell number and cellular density of ~25% compared with control animals. VP+ cells were also reduced in the total cell number and density by ~15% with respect to controls, but not in the ventrolateral SCN region. The deleterious effect of malnutrition on these cell groups was not specific to the SCN because similar effects were observed in other brain areas analyzed (decrease of VIP+ 21%, GFAP+ 23%, and VP+ 11% with respect to controls). The reduction in the cell number may reflect lower intracellular concentration of the peptides and protein localized in the cells, which would render them undetectable with this immunohistochemical procedure, as a consequence of changes in the metabolism in neurons and glia. Further studies combining stereological assessment of the SCN cell number and direct estimation of the concentration of peptides or its mRNA are needed to address this issue.Present results are consistent with previous studies showing that glial cells are particularly vulnerable to malnutrition (5). On the other hand, we have not found previous studies on the effects of malnutrition on specific peptidergic neuronal populations. Our results indicate a higher vulnerability to malnutrition for VIP+ and GFAP+ with respect to VP+ cells.
Afferent fibers to the SCN were less affected by malnutrition; 5-HT+ total fiber number showed only an 8% reduction, whereas NPY fibers showed no differences between the two nutritional groups. These results are consistent with the hypothesis that sprouting of terminal fields may compensate cellular loss induced by malnutrition, as has been suggested by previous studies in developmental brain plasticity (2, 21).
Indirect Effects of Malnutrition on Circadian Rhythmicity
It is conceivable that, although nonspecific to the SCN, alterations induced by malnutrition on its neurochemical organization may be related, at least to some extent, to the differences in the expression of circadian rhythmicity previously reported. On the other hand, malnutrition may indirectly induce such differences by either altering the metabolism of other neuroactive substances and hormones or even the induction of a general catabolic state.It has been previously described that malnutrition induces a decrease in the amplitude of plasma melatonin rhythm in rats (13). Also, it has been suggested that the pineal gland, the main source of melatonin in mammals, may function as a phase integrator in rodents because pinealectomy increases uncoupling among circadian oscillators in subjects exposed to constant bright light (1) and facilitates entrainment to reverse photoperiods, decreasing the number of transients needed to reach a stable phase relation to the LD cycle (25). Taken together, these observations suggest that the differences in circadian rhythmicity induced by malnutrition with respect to controls, that is splitting of rhythmicity under LD, precocious entrainment to complete photoperiod, and late entrainment to skeleton photoperiod, may be partially due to the decrease in melatonin secretion from the pineal.
There is evidence of a circadian oscillator entrainable to food availability (FEO), which is independent of the SCN. Food anticipatory activity, as well as entrainment of corticosterone rhythms, has been observed in response to daily meals under circadian food access schedules, even after complete SCN lesions (20). It also has been shown that a catabolic state is necessary to the expression of the FEO, because the duration of the interval of fasting has to be long enough to induce a catabolic state to entrain corticosterone rhythms to food pulses (14). Furthermore, free running of food anticipatory activity occurs in fasting but not ad libitum food conditions (20). Considering that chronic low-protein malnutrition is known to induce a general catabolic state in the organisms, an alternative explanation to the effects of malnutrition on the expression of circadian rhythms is that such a catabolic state would enable the expression of FEO.
In conclusion, chronic malnourished animals were capable of exhibiting remarkably normal circadian rhythmicity, which indicates the strength of the circadian organization to persist despite such a gross insult. Although alterations in the neurochemical organization of the SCN were found, these were not specific to these nuclei, confirming previous observations regarding the vulnerability of brain organization to chronic low-protein malnutrition. Thus the different organization of circadian rhythms in malnourished animals in relation to controls may be due not only to the alterations in the SCN but also to other influences of this insult on the organism, such as alterations in the metabolism of neuroactive substances and hormones, or even the induction of a general catabolic state that may expose other circadian oscillators.
Perspectives
Although malnutrition affects the circadian system at different levels, such as biochemical (13), morphological, and behavioral organization (present study), the persistence itself of circadian rhythmicity indicates the robustness and relevance of this process for animal survival and adaptation to the environment. It is clear that malnourished animals are able to exhibit circadian rhythmicity, although with a different organization with respect to control animals. We may speculate that changes in malnourished rats reflect the development of an alternative temporal pattern of behavior, which in natural conditions enables the animals to increase their foraging activity under conditions of food shortage. The development of such alternative strategies would depend on both the multioscillatory nature of the circadian system and the responsiveness of the system to entraining signals other than the LD cycle, such as food availability. The challenge of malnutrition may release such mechanisms, which under other conditions would be of secondary relevance to the animal, to generate adequate predictive behavioral and physiological patterns. Previous hypotheses may be addressed at the behavioral level by studying the relative potency of different entraining signals in relation to LD cycles and at the morphological and functional levels by studying the RHT and the response of SCN neurons to light pulses and other stimuli in malnourished animals.| |
ACKNOWLEDGEMENTS |
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We thank Dr. Robyn Hudson (University of Munich) for valuable comments and enriching discussion of the manuscript.
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FOOTNOTES |
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This work was supported by DGAPA Grants IN200794 and IN202891.
Address for reprint requests: R. Aguilar-Roblero, Neurociencias, IFC, UNAM, Apdo. Postal 70-253,, Mexico DF 04510, Mexico.
Received 13 September 1996; accepted in final form 26 June 1997.
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REFERENCES |
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Abstract
Introduction
Methods
Results
Discussion
References
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) and to 20-h light pulse for
skeleton photoperiod (
) in control
(A) and malnourished
(B) rats. Arrows indicate mean value
for each group under each lighting condition (continuous line, complete
photoperiod; broken line, skeleton photoperiod), and 0° represents
phase of reference of the entraining cycle. 
, Mean
value for complete photoperiod; 
, mean value for skeleton
photoperiod.
