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Habituation Training Improves Locomotor Performance in a Forced Running Wheel System in Rats

  • University of Murcia / University of Granada

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Increasing evidence supports that physical activity promotes mental health; and regular exercise may confer positive effects in neurological disorders. There is growing number of reports that requires the analysis of the impact of physical activity in animal models. Exercise in rodents can be performed under voluntary or forced conditions. The former presents the disadvantage that the volume and intensity of exercise varies from subject to subject. On the other hand, a major challenge of the forced training protocol is the low level of performance typically achieved within a given session. Thus, the aim of the present study was to evaluate the effectiveness of gradual increasing of the volumen and intensity (training habituation protocol) to improve the locomotor performance in a forced running-wheel system in rats. Sprague Dawley rats were randomly assigned to either a group that received an exercise training habituation protocol, or a control group. The locomotor performance during forced running was assessed by an incremental exercise test. The experimental results reveals that the total running time and the distance covered by habituated rats was significantly higher than in control ones. We conclude that the exercise habituation protocol improves the locomotor performance in forced running wheels.
Content may be subject to copyright.
published: 08 March 2017
doi: 10.3389/fnbeh.2017.00042
Habituation Training Improves
Locomotor Performance in a Forced
Running Wheel System in Rats
Angel Toval1,2,Raúl Baños 1,2 ,Ernesto De la Cruz3,Nicanor Morales-Delgado1,2,
Jesús G. Pallarés4,Abdelmalik Ayad1,2,Kuei Y. Tseng 5and Jose L. Ferran 1,2 *
1Department of Human Anatomy and Psychobiology, School of Medicine, University of Murcia, Murcia, Spain, 2Institute of
Biomedical Research of Murcia (IMIB), Virgen de la Arrixaca University Hospital, University of Murcia, Murcia, Spain,
3Department of Physical Activity and Sport, Faculty of Sport Science, University of Murcia, Murcia, Spain, 4Human
Performance and Sports Science Laboratory, University of Murcia, Murcia, Spain, 5Department of Cellular and Molecular
Pharmacology, The Chicago Medical School at Rosalind Franklin University, North Chicago, IL, USA
Edited by:
Nuno Sousa,
University of Minho, Portugal
Reviewed by:
Francoise Schenk,
University of Lausanne, Switzerland
Vikas Mishra,
Louisiana State University Health
Sciences Center, USA
Jose L. Ferran
Received: 01 November 2016
Accepted: 24 February 2017
Published: 08 March 2017
Toval A, Baños R, De la Cruz E,
Morales-Delgado N, Pallarés JG,
Ayad A, Tseng KY and Ferran JL
(2017) Habituation Training Improves
Locomotor Performance in a Forced
Running Wheel System in Rats.
Front. Behav. Neurosci. 11:42.
doi: 10.3389/fnbeh.2017.00042
Increasing evidence supports that physical activity promotes mental health; and regular
exercise may confer positive effects in neurological disorders. There is growing number
of reports that requires the analysis of the impact of physical activity in animal models.
Exercise in rodents can be performed under voluntary or forced conditions. The former
presents the disadvantage that the volume and intensity of exercise varies from subject
to subject. On the other hand, a major challenge of the forced training protocol is the
low level of performance typically achieved within a given session. Thus, the aim of the
present study was to evaluate the effectiveness of gradual increasing of the volume and
intensity (training habituation protocol) to improve the locomotor performance in a forced
running-wheel system in rats. Sprague-Dawley rats were randomly assigned to either
a group that received an exercise training habituation protocol, or a control group. The
locomotor performance during forced running was assessed by an incremental exercise
test. The experimental results reveal that the total running time and the distance covered
by habituated rats was significantly higher than in control ones. We conclude that the
exercise habituation protocol improves the locomotor performance in forced running
Keywords: physical activity, exercise, rodents, familiarization protocols, acclimation protocols
According to the World Health Organization (2010), the lack of physical activity is the fourth
leading risk factor for global human mortality. For instance, reduced physical activity is
associated with higher risks of developing obesity, type 2 diabetes, osteoporosis, depression
and cardiovascular diseases (World Health Organization, 2010; Dishman et al., 2013; Mora-
Rodriguez et al., 2016). On the other hand, there is growing evidence supporting a positive
impact of increasing regular levels of physical activity on public health (Dishman et al., 2006;
Hillman et al., 2008; van Praag, 2009; Vivar et al., 2012). These studies suggest that motor
skill training and regular exercise are beneficial to sustaining proper executive functions of
cognition and learning (e.g., motor learning in the spinal cord; Edgerton et al., 2004; Hillman
et al., 2008), and in some cases it may confer protective effects against the onset of neurological
disorders including Parkinson’s disease (Smith and Zigmond, 2003), Alzheimer’s dementia
(Cotman and Berchtold, 2002) and stroke (Stummer et al., 1994). However, the neurobiological
Frontiers in Behavioral Neuroscience | 1March 2017 | Volume 11 | Article 42
Toval et al. Habituation Effects in Forced Wheel
mechanisms associated with physical activity are not entirely
known, partly due to a lack of uniformity and parameterization
in experimental protocols employed to assess the impact
of exercise in animal models. For example, rodent studies
using running-wheels often employ protocols that allow
ad libitum access to the wheel. While such an approach
has its advantages, both the intensity and volume varies
significantly from subject to subject due to the ‘‘voluntary’’
nature of the experimental design (van Praag et al., 2005;
Kregel et al., 2006; Leasure and Jones, 2008; Creer et al.,
2010; Kobilo et al., 2011; Marlatt et al., 2012). One way
to overcome these challenges is to implement a forced
running-wheel protocol in which the same training load is
applied to all subjects (Auriat et al., 2006; Shimizu and
Yamanouchi, 2011; Wang et al., 2013; Chen et al., 2014).
It becomes clear from these studies that the inclusion
of a pretraining stage of habituation prior to the testing
phase is a crucial step to achieving better performances in
response to increasing running demands (Dick, 2007). Thus,
the aim of the present study is to develop and evaluate a
protocol of habituation training to enhance the locomotor
performance of young adult rats subjected to a progressive
incremental running load test in a forced running-wheel system
(Bentley et al., 2007).
All experimental procedures were approved by the University of
Murcia’s animal care and use committee according to the Spanish
regulation (Royal Decree 1201/2005) and European Union
Directive 2003/65/EC of the European Parliament (Amending
Council Directive 86/609/EEC) guide for care and use of
laboratory animals.
Animals and Experimental Groups
Young adult male Sprague-Dawley rats (Laboratory Animals
Facilities at the University of Murcia) were group housed
(2–3 rats/cage) and kept in a 12:12 h light/dark cycle room
(dark period from 8 AM to 8 PM) at 21–23C and 55 ±5%
of relative humidity, with food and water available ad libitum.
Rats were randomly assigned to receive either the protocol
of habituation training (habituated) or not (non-habituated)
for eight consecutive days. The locomotor performance to
incremental intensities of forced running was assessed at 1, 3,
31 and 33 days post-last habituation session.
Running-Wheel Training
Six polycarbonate motor running-wheels were purchased
from Lafayette-Campdem system (80805A model, dimensions
129.54 ×45.47 ×42.93 cm). The internal surface of the
running-wheel was covered with custom-made denim fabric
to provide a smooth flatten running surface (Figures 1A–C).
The habituation phase is comprised of 10 sessions distributed
across 8 days of training. During these sessions, both intensity
(speed) and volume (time) were increased following an upward
progressive pattern as summarized in Figures 1D,F. Once the
habituation phase is completed, rats were subjected to the
first incremental exercise test 24 h after the last habituation
session. In order to determine whether the habituation sessions
exert an enduring impact on forced running performance,
rats were tested again at 3, 31 and 33 days from the last
session of habituation training. During the testing phase of
incremental forced running, a defined speed of 9 m/min is
introduced at the beginning of each test followed by increments
of 0.9 m/min every 5 min until a failure to maintain a
running pattern becomes apparent (Figure 1E). Criteria to
stop the incremental test include jumping, crawling and/or
rolling within the running-wheel. Typically, rats are removed
from the running-wheel and the test stopped if two or more
consecutive uncontrolled laps are detected. The time spent in the
running wheel was determined from the beginning of the test
to its termination when the animal fails to maintain a running
Statistical Analysis
All data were presented as mean ±standard error of the
mean. A two-tailed student’s t-test was used for two-group
comparison involving a single continuous variable and a one-way
repeated measures analysis of variance (ANOVA) test was used
for intra-subjects multiple comparison. Differences between the
experimental groups were considered statistically significant at
P<0.05 (StatSoft, Tulsa, OK, USA).
Comercially available running-wheels were modified to
include a custom-made denim fabric into the internal
surface of the wheel to provide a smooth flatten running
surface (Figures 1A–C). In order to assess the impact of
habituation training, a cohort of young adult male Sprague-
Dawley rats were subjected to a protocol of habituation
comprised of 10 sessions in 8 days during which the
intensity (speed) and the volume (time) of the training
sessions were increased following an upward progressive
pattern (Figures 1D–F). We observed a 100% success rate
response, that is, all animals (n= 23) tested with this tranining
protocol managed to complete the entire 8 days of habituation
We next developed a progressive incremental running load
test (Figure 1E) to assess the impact of habituation training
on locomotor performance. Relative to the non-habituated
group (n= 12), rats that underwent the habituation training
phase exhibited a six fold increase in performance to the
incremental running load test as revealed by the time and
distance spent in the wheel (Figure 2). While most of the
animals from the non-habituated group failed to pass the
first 5 min step of the incremental test, habituated rats
run an average of 32 min covering a mean distance of
370 m when tested 24 h after the last habituation session
(Figures 2A,B).
Finally, the incremental test was repeated to evaluate long
term effects of the habituation. The locomotor performance at
3 days remains similar; however it is significantly decreased
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Toval et al. Habituation Effects in Forced Wheel
FIGURE 1 | (A) Forced motor wheel with aluminum bars in the running surface. (B) Running surface of the wheel covered by denim fabric. (C) Forced motor wheel
system during a running session. (D) The schedule shows the exercise program developed during the habituation exercise protocol. The speed, time and number of
sessions by day are described. The incremental test is developed after 24 h finished the habituation, and repeated at 3, 31 and 33 days later. (E) This graphic is a
representation of time variation (Xaxis) in relation to the speed variation (Yaxis) during the development of the incremental exercise test. Notice that every 5 min the
speed changes increasing in 0.9 m/m giving the aspect of steps. (F) Bars and lines graph which represents the training load in each day (Xaxis) of the habituation
protocol. Bars indicate time of running (Yaxis, left). Line indicates speed (Yaxis, right). The training load, speed and time followed an upward progressive pattern.
WU, warming up phase.
both at 31 and 33 days (habituated rats run an average
of 16/18 min covering a mean distance of 162/194 m;
respectively; Figures 3A,B).
These results support that the habituation period improves
the locomotor performance in forced running wheels; but
this effect is progressively drecreased without a permanent
The present study was developed to evaluate the impact of
an habituation training in the locomotor performance, using a
forced running wheel system in young adult rats. It is known
that wild rats cover long distances during the night, running
bursts of short periods at high speeds (Tchernichovski and
Benjamini, 1998). A similar pattern of running is detected
in voluntary exercise laboratory paradigm, that allow rats to
reach higher intensities and cover longer distances than in
forced exercise; but restricting the posibility to manage intensity
and volume of running (training load; Leasure and Jones,
2008). This situation highlights the difficulty of the voluntary
running to do precise correlations between training load and
the observed effects (van Praag et al., 2005; Leasure and Jones,
2008; Creer et al., 2010; Kobilo et al., 2011; Marlatt et al.,
2012). On the other hand, in forced conditions, rats run during
larger periods of time but at lower speed than observed during
voluntary running (Narath et al., 2001; Leasure and Jones, 2008).
Although forced models are consistent with the uniformity of
the physical activity carried out for the group of rats, running
on a treadmill or a motorized wheel can be a challenge for
the animals. In fact, as many as 10% of the rats refuse to walk
or run on a treadmill; and these animals must be removed
from exercise studies (Jasperse and Laughlin, 1999; Koch and
Britton, 2001; Kregel et al., 2006). Our results support that
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Toval et al. Habituation Effects in Forced Wheel
FIGURE 2 | (A) The graph represents the total time of running endured during
the incremental test comparing non-habituated (blue) and habituated (orange)
young adult rats. Individual measures are indicated by diamonds and the
mean comparison by bars (non habituated: X= 5.42 ±0.5 min; habituated:
X= 31.70 ±1.8 min). (B) The graph represents the total distance covered
during the incremental test comparing non-habituated (blue) and habituated
(orange) rats. Individual measures are indicated by diamonds and the mean
comparison by bars (non habituated: X= 49.57 ±4.87 m; habituated:
X= 368.29 ±25.49 m). ∗∗∗p<0.0001; two-tailed student’s t-test.
habituation exercise training are key to get better locomotor
performances to develop succesful training programs in forced
Only a few of the current works in forced running paradigm
consider the implementation of an habituation phase as a good
strategy to reduce the number of rats classified as nonrunners
(Kregel et al., 2006; O’Dell et al., 2007; Chen et al., 2014).
For this aim the animals are introduced in treadmills or
motor wheels with a gradual increase in training load. This
FIGURE 3 | (A) The graph represents the total time of running of each
individual (gray lines) and the mean of time (orange line) during the four
incremental tests carried out at 1 day after the completion of the habituation
phase and repeated after 3 (X= 27.9 ±1.74 min), 31 (X= 15.81 ±1.46 min)
and 33 (X= 18.58 ±1.54 min) days. (B) The graph represents the total
distance covered during the four incremental tests for each individual (gray
lines) and the mean of distance (orange line) of habituated rats after 1, 3
(X= 314.99 ±24.2 m), 31 (X= 162.27 ±17.29 m) and 33
(X= 194.95 ±18.53 m) days. A comparison between tests at 1 and 3 day vs.
performed at 31 and 33 day show statystically significant differences
(p<0.002; one-way repeated measures analysis of variance (ANOVA)).
condition may be relevant to improve locomotor performance
and minimize potential injures that can occur in this new
environment for the rodents. In our study a 100% of rats
finished the whole running habituation period. However, the
habituation protocol is absent in some studies using forced
motor wheel; which maintain a constant intensity and volume
from the beginning and throughout all the sessions during
their programs (Clement et al., 1993; Ji et al., 2014). The
intensity of the exercise developed in these works during the
training protocol was between 1.22 m/min and 12 m/min;
that is lower in comparison with other forced protocols that
includes a progressive increment in the training load, and
reaches a maximum speed of 30 m/min (Clement et al.,
1993; Leasure and Jones, 2008; Chen et al., 2014; Ji et al.,
2014). Some works developed habituation-like protocols, that
consist in an increase of training load pattern during the
entire training period. Under this conditions the highest speed
reached during the training protocol was between 12 m/min
and 14 m/min (Sandrow-Feinberg et al., 2009; Caton et al.,
2012; Griesbach et al., 2013). However, others works that
implement pretraining sessions do not report the training load
details of the protocol employed, such us duration, speed
or number of sessions; but reported a maximum speed of
21 m/min (O’Dell et al., 2007; Hu et al., 2010;Kennard and
Woodruff-Pak, 2012). Finally, only few studies indicate all
the detailed features of the habituation protocol developed;
reaching the highest speed (30 m/min; Auriat et al., 2006;
Shimizu and Yamanouchi, 2011; Wang et al., 2013; Chen et al.,
2014). According to Chen et al. (2014), running behavior
in motor wheels for rodents is more laborious than the
linear motion of the treadmill, in particular in the absence
of an adaptive learning stage. Our results demonstrated that
habituated rats can sustain a higher forced running speed
when compared to the non-habituated group. Interestingly,
such locomotor improvement is transient as the forced running
speed decreases progressively over time within a period of
30 days.
Several mechanisms could contribute to sustaining a higher
running speed following a progressive habituation protocol
to the running wheel. It is well known that physical activity
produces biochemical changes that improves the muscular
erobic metabolism (Terjung and Hood, 1986). While data on
how a forced running wheel affects muscular endurance are
lacking, evidences from a number of studies using treadmill
suggest that a 60 min/day/1 week of running at 25 m/min
is needed to induce muscular endurance as determined by
changes in cytochrome c, citrate synthase, 3-Ketaocid-CoA
transferase (Booth and Holloszy, 1977;Terjung, 1979; Dudley
et al., 1982), and myoglobin concentration (Lawrie, 1953;
Pattengale and Holloszy, 1967; Terjung and Hood, 1986).
In this regard, it is unlikely that 7 days of progressive
augmentation in duration and intensity reaching a maximal
speed of 9 m/min for 1 h during the last day is sufficient
to elicit adaptive changes in muscular endurance. Another
contributing factor is stress. Stress hormones are known
to increase during forced exercise (Saito and Soya, 2004;
Chen et al., 2016), which in turn are increased during
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Toval et al. Habituation Effects in Forced Wheel
acute running according to exercise intensity and duration
(Chennaoui et al., 2002; Kawashima et al., 2004; Saito and
Soya, 2004; Chen et al., 2016). Acute stress conditions promote
fight-fight responses producing elevation of peripheral blood
pressure and heart rate; and facilitates energy utilization (Maier
et al., 1998; Moraska et al., 2000; Norris and Carr, 2013).
However, our habituation protocol reaches a maximal speed
of 9 m/min only during the last day for 1 h; and the
evidence from a number of studies using treadmill suggest
that a running speed of at least 25 m/min (just above the
lactate threshold) is required to induce changes in blood lactate,
plasma ACTH, plasma glucose and adrenaline (Timofeeva
et al., 2003; Saito and Soya, 2004; Soya et al., 2007; Chen
et al., 2016). One study showed that activation of CRH
neurons in the paraventricular nucleus emerges following
1 h of acute forced running wheel (Yanagita et al., 2007),
yet data showing changes in stress hormones are currently
Based on currently available literature, it is possible that a
change in the dopaminergic system may contribute to enhance
the forced running-wheel performance observed following the
habituation training. It has been shown that pharmacological
activation of the dopaminergic system is sufficient to improve
motor coordination and its endurance (Tamasy et al., 1981;
Freed and Yamamoto, 1985; Boldry et al., 1991; Meeusen and
De Meirleir, 1995; Sutoo and Akiyama, 1996; Chen et al.,
2016). Future studies are warranted to determine the role
of dopamine and related catecholamines in sustaining better
locomotor performance in a forced running-wheel system.
Further studies using metabolic chambers or implanted chip
systems will be essential to develop the best training condition
and standardized exercise training programs in rodents. A
rigorous analysis of physiological variations such us VO2max,
lactate threshold, heart rate or body composition, a set of data
that is currently used in elite sports training in humans, is
necessary to adapt the training program to the specific features
of each experimental subject (Copp et al., 2009; Zhou et al.,
AT and JLF: study conception and design, data collection and
analysis, interpretation, drafting and revising the manuscript.
RB: data collection and analysis, interpretation, drafting and
revising the manuscript. EDC: study conception and design,
data collection and analysis, interpretation, and revising the
manuscript. NM-D: data collection and analysis, revision
of the manuscript. JGP: study conception and design, data
collection and analysis, revision of the manuscript. AA: data
analysis, interpretation, revision of the manuscript. KYT: study
conception and design, data analysis, interpretation, revision of
the manuscript. All authors have approved the final manuscript
Granted by MAPFRE Foundation (2014); and the Spanish
Ministry of Science and Technology (MEC) and European
Regional Development Fund (FEDER; BFU2014-57516P to JLF).
The authors gratefully acknowledge helpful comments and
laboratory support from Luis Puelles.
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Frontiers in Behavioral Neuroscience | 6March 2017 | Volume 11 | Article 42
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2017 Toval, Baños, De la Cruz, Morales-Delgado, Pallarés, Ayad, Tseng
and Ferran. This is an open-access article distributed under the terms of the Creative
Commons Attribution License (CC BY). The use, distribution and reproduction in
other forums is permitted, provided the original author(s) or licensor are credited
and that the original publication in this journal is cited, in accordance with accepted
academic practice. No use, distribution or reproduction is permitted which does not
comply with these terms.
Frontiers in Behavioral Neuroscience | 7March 2017 | Volume 11 | Article 42
... Animal research with voluntary or forced exercise reporting the positive impact of physical activity in health is skyrocketing (13)(14)(15)(16)(17). Forced treadmill and wheel exercise, but not voluntary, are the preferred modalities to develop the same training in all animals. Specifically, forced wheel is an emergent modality that should ensure the exercise load reproducibility and avoid non-specific stress-related responses (18,19), but methods provided in most of these works do not seem to be properly detailed. In this sense, current guidelines do not guarantee the reproducibility of the training programs in forced wheel or any other forced modality (6). ...
... Particularly in exercise, variables such as habituation, duration of the session, speed, frequency and duration of the exercise program are determinant to guarantee the reproducibility of the forced wheel training (18,19,31). Finally, reporting the time elapsed between the last session of exercise and the test or biological analysis should avoid circadian and metabolic misinterpretations (32)(33)(34). ...
... Supplementary File 2 describes a summary of the rodent model and the exercise program used in each study included in this review. Rats were the rodents used in 75.9% (41/54) of the studies (18,, and mice in 24.1% (13/54) (81)(82)(83)(84)(85)(86)(87)(88)(89)(90)(91)(92)(93). In rats, the most frequent strain was Sprague-Dawley (22/54) (18, 43, 46-49, 51-55, 61, 64-67, 70, 71, 76, 77, 79, 80), followed by Wistar (10/54) (44, 45, 57-60, 63, 68, 69, 73). ...
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A well-documented method and experimental design are essential to ensure the reproducibility and reliability in animal research. Experimental studies using exercise programs in animal models have experienced an exponential increase in the last decades. Complete reporting of forced wheel and treadmill exercise protocols would help to ensure the reproducibility of training programs. However, forced exercise programs are characterized by a poorly detailed methodology. Also, current guidelines do not cover the minimum data that must be included in published works to reproduce training programs. For this reason, we have carried out a systematic review to determine the reproducibility of training programs and experimental designs of published research in rodents using a forced wheel system. Having determined that most of the studies were not detailed enough to be reproducible, we have suggested guidelines for animal research using FORCED exercise wheels, which could also be applicable to any form of forced exercise.
... However, little is known about why it has such a profound effect on the brain. Neurobiological mechanisms associated with physical activity are not entirely known, which is partly due to a lack of uniformity and parameterization in experimental protocols employed to assess the impact of exercise in animal models [15]. Therefore, in the present study, we have used a forced wheel-running protocol in which the same training load is applied to all the subjects [15,16]. ...
... Neurobiological mechanisms associated with physical activity are not entirely known, which is partly due to a lack of uniformity and parameterization in experimental protocols employed to assess the impact of exercise in animal models [15]. Therefore, in the present study, we have used a forced wheel-running protocol in which the same training load is applied to all the subjects [15,16]. ...
... In the present study, six custom-made polycarbonate motor running wheels were used. The advantage of this system is the possibility of controlling the training; all animals run at the same speed for the same period of time, in contrast to voluntary activity where the level of activity depends on the animal's preferences [15]. The rats were exercised 5 days/week for 6 weeks, of which the first week was an introduction week (the habituation phase). ...
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Background: In the pathogenesis of central nervous system disorders (e.g., neurodegenerative), an important role is attributed to an unhealthy lifestyle affecting brain energy metabolism. Physical activity in the prevention and treatment of lifestyle-related diseases is getting increasing attention. Methods: We performed a series of assessments in adult female Long Evans rats subjected to 6 weeks of Western diet feeding and wheel-running training. A control group of lean rats was fed with a standard diet. In all experimental groups, we measured physiological parameters (animal weights, body composition, serum metabolic parameters). We assessed the impact of simultaneous exposure to a Western diet and wheel-running on the cerebrocortical protein expression (global proteomic profiling), and in the second part of the experiment, we measured the cortical levels of protein related to brain metabolism (Western blot). Results: Western diet led to an obese phenotype and induced changes in the serum metabolic parameters. Wheel-running did not reduce animal weights or fat mass but significantly decreased serum glucose level. The global proteome analysis revealed that the altered proteins were functionally annotated as they were involved mostly in metabolic pathways. Western blot analysis showed the downregulation of the mitochondrial protein-Acyl-CoA dehydrogenase family member 9, hexokinase 1 (HK1)-enzyme involved in principal glucose metabolism pathways and monocarboxylate transporter 2 (MCT2). Wheel-running reversed this decline in the cortical levels of HK1 and MCT2. Conclusion: The cerebrocortical proteome is affected by a combination of physical activity and Western diet in female rats. An analysis of the cortical proteins involved in brain energy metabolism provides a valuable basis for the deeper investigation of changes in the brain structure and function induced by simultaneous exposure to a Western diet and physical activity.
... The aim of the present study is to determine whether the dopamine system modulates exercise capacity during adolescence using a rodent model of forced running wheel we recently implemented. Briefly, rats were exposed to an 8day period of habituation in the running wheel before assessing their locomotor performance in response to an incremental load of forced running [42,43]. Both systemic and striatal administration of dopamine D1 (SCH23390) and D2 (raclopride) receptor antagonists were delivered 15-30 min prior to the onset of the incremental test, and changes in the level of locomotor performance were compared. ...
... An 8-day exercise habituation protocol was implemented using a forced running wheel system (Lafayette-Campden, model 8085A). Speed and time of running were progressively increased throughout the sessions of the protocol, from 5 at 0 m/min the first day to 30 min running at 9 m/min the last day, as described in Toval et al. (2017Toval et al. ( , 2020 [42,43]. Nonhabituated rats remained in locked wheels, without any exercise stimulus, for the same time as the habituated groups. ...
... An 8-day exercise habituation protocol was implemented using a forced running wheel system (Lafayette-Campden, model 8085A). Speed and time of running were progressively increased throughout the sessions of the protocol, from 5 at 0 m/min the first day to 30 min running at 9 m/min the last day, as described in Toval et al. (2017Toval et al. ( , 2020 [42,43]. Nonhabituated rats remained in locked wheels, without any exercise stimulus, for the same time as the habituated groups. ...
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Improving exercise capacity during adolescence impacts positively on cognitive and motor functions. However, the neural mechanisms contributing to enhance physical performance during this sensitive period remain poorly understood. Such knowledge could help to optimize exercise programs and promote a healthy physical and cognitive development in youth athletes. The central dopamine system is of great interest because of its role in regulating motor behavior through the activation of D1 and D2 receptors. Thus, the aim of the present study is to determine whether D1 or D2 receptor signaling contributes to modulate the exercise capacity during adolescence and if this modulation takes place through the striatum. To test this, we used a rodent model of forced running wheel that we implemented recently to assess the exercise capacity. Briefly, rats were exposed to an 8-day period of habituation in the running wheel before assessing their locomotor performance in response to an incremental exercise test, in which the speed was gradually increased until exhaustion. We found that systemic administration of D1-like (SCH23390) and/or D2-like (raclopride) receptor antagonists prior to the incremental test reduced the duration of forced running in a dose-dependent manner. Similarly, locomotor activity in the open field was decreased by the dopamine antagonists. Interestingly, this was not the case following intrastriatal infusion of an effective dose of SCH23390, which decreased motor performance during the incremental test without disrupting the behavioral response in the open field. Surprisingly, intrastriatal delivery of raclopride failed to impact the duration of forced running. Altogether, these results indicate that the level of locomotor response to incremental loads of forced running in adolescent rats is dopamine dependent and mechanistically linked to the activation of striatal D1 and extra-striatal D2 receptors.
... Increasing evidence has shown strong correlations between physical activity and mental health, facilitating neuroplasticity, improving brain function, and preventing mental disorders such as depression, Parkinson or Alzheimer disease (Cotman and Berchtold, 2002;Smith and Zigmond, 2003;Dishman et al., 2006;Hotting and Roder, 2013;Cooney et al., 2014). However, only a reduced number of studies in animal models were dealing with the causal mechanisms by which physical activity produces health benefits (Toval et al., 2017). ...
... Reproducible conditions are essential in order to understand the neurobiological mechanisms of physical activity in rodent models. Animals are required to undergo similar loads of exercise, avoiding irregular motor performance and non-specific stress responses (Kregel et al., 2006;Leasure and Jones, 2008;Toval et al., 2017;Rudeck et al., 2020). Nevertheless, voluntary running is the most used exercise model in rodents; and the animals are rarely exposed to the equal intensities and volumes of running (Van Praag et al., 2005;Kregel et al., 2006;Leasure and Jones, 2008;Creer et al., 2010;Marlatt et al., 2012). ...
... These individual differences can be avoided using forced models such as treadmill or motorized wheels. However, around 10% of the rodents reject running in forced models; a situation that can be surpassed by applying an exercise habituation (HAB) protocol (Jasperse and Laughlin, 1999;Koch and Britton, 2001;Kregel et al., 2006;Toval et al., 2017). HAB to exercise is an adaptive period preceding the main training phase, highlighted by a progressive increase of speed and time of running. ...
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It has been demonstrated that physical activity contributes to a healthier life. However, there is a knowledge gap regarding the neural mechanisms producing these effects. One of the keystones to deal with this problem is to use training programs with equal loads of physical activity. However, irregular motor and stress responses have been found in murine exercise models. Habituation to forced exercise facilitates a complete response to a training program in all rodents, reaching the same load of physical activity among animals. Here, it was evaluated if glucose and lactate – which are stress biomarkers – are increased during the habituation to exercise. Sprague-Dawley rats received an 8-days habituation protocol with progressive increments of time and speed of running. Then, experimental and control (non-habituated) rats were subjected to an incremental test. Blood samples were obtained to determine plasmatic glucose and lactate levels before, immediately after and 30 min after each session of training. Crh and Avp mRNA expression was determined by two-step qPCR. Our results revealed that glucose and lactate levels are not increased during the habituation period and tend to decrease toward the end of the protocol. Also, Crh and Avp were not chronically activated by the habituation program. Lactate and glucose, determined after the incremental test, were higher in control rats without previous contact with the wheel, compared with habituated and wheel control rats. These results suggest that the implementation of an adaptive phase prior to forced exercise programs might avoid non-specific stress responses.
... In addition, this period is characterized by a larger gain in weight and adipose tissue content compared to the young adult stage . Accurate characterization of the distribution of TH-positive cells in the hypothalamic region during the adolescent period is important for experimental designs aiming to determine their action during modulation of motor and metabolic responses in this period of life (Caballero and Tseng, 2016;Toval et al., 2017Toval et al., , 2020Toval et al., , 2021Kutsenko et al., 2021). The aim of the present study was to map more precisely the distribution of THpositive neurons in specific nuclei or strata of the hypothalamic region in adolescent rats following the updated area map provided by the prosomeric model (Puelles et al., 2012a;Ferran et al., 2015a;. ...
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Most of the studies on neurochemical mapping, connectivity, and physiology in the hypothalamic region were carried out in rats and under the columnar morphologic paradigm. According to the columnar model, the entire hypothalamic region lies ventrally within the diencephalon, which includes preoptic, anterior, tuberal, and mamillary anteroposterior regions, and sometimes identifying dorsal, intermediate, and ventral hypothalamic partitions. This model is weak in providing little or no experimentally corroborated causal explanation of such subdivisions. In contrast, the modern prosomeric model uses different axial assumptions based on the parallel courses of the brain floor, alar-basal boundary, and brain roof (all causally explained). This model also postulates that the hypothalamus and telencephalon jointly form the secondary prosencephalon, separately from and rostral to the diencephalon proper. The hypothalamus is divided into two neuromeric (transverse) parts called peduncular and terminal hypothalamus (PHy and THy). The classic anteroposterior (AP) divisions of the columnar hypothalamus are rather seen as dorsoventral subdivisions of the hypothalamic alar and basal plates. In this study, we offered a prosomeric immunohistochemical mapping in the rat of hypothalamic cells expressing tyrosine hydroxylase (TH), which is the enzyme that catalyzes the conversion of L-tyrosine to levodopa (L-DOPA) and a precursor of dopamine. This mapping was also combined with markers for diverse hypothalamic nuclei [agouti-related peptide ( Agrp ), arginine vasopressin ( Avp ), cocaine and amphetamine-regulated transcript ( Cart ), corticotropin releasing Hormone ( Crh ), melanin concentrating hormone ( Mch ), neuropeptide Y ( Npy ), oxytocin/neurophysin I ( Oxt ), proopiomelanocortin ( Pomc ), somatostatin ( Sst ), tyrosine hidroxilase ( Th ), and thyrotropin releasing hormone ( Trh )]. TH-positive cells are particularly abundant within the periventricular stratum of the paraventricular and subparaventricular alar domains. In the tuberal region, most labeled cells are found in the acroterminal arcuate nucleus and in the terminal periventricular stratum. The dorsal retrotuberal region (PHy) contains the A13 cell group of TH-positive cells. In addition, some TH cells appear in the perimamillary and retromamillary regions. The prosomeric model proved useful for determining the precise location of TH-positive cells relative to possible origins of morphogenetic signals, thus aiding potential causal explanation of position-related specification of this hypothalamic cell type.
... At 24 h, before tests, rats were placed in the OFT arena for 5 min of habituation. The habituation to the open field has been shown to reduce data variability while improving performance on the behavioral tests (Tatem et al., 2014;Toval et al., 2017). Rats were tested in a square arena with 100cm × 100cm × 50cm under uniform 25 lux intensity infrared light. ...
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Major Depressive Disorder (MDD) with Peripartum Onset was classified in 2013 by the Diagnostic and Statistical Manual, Fifth Edition (DMS-5) and approved in 2019 by the World Health Organization (WHO). These diagnostic revisions call for the development of new animal models of maternal depression, emphasizing the pregnancy period. We have recently described a novel rat model of maternal MDD with a Peripartum Onset. Exposure to pre-gestational chronic mild stress (CMS) with repeated restrain resulted in maternal depressive-like behavior and impacted offspring's neurodevelopment. The present study examined gender differences in short- vs. long-term neurodevelopmental impact of pre-gestational maternal stress. Stress response was assessed in Sprague Dawley CMS-exposed dams (n = 7) by metabolic, hormonal, and behavioral changes and compared to controls dams (n = 7). Short-term impact of maternal stress on offspring was examined in terms of metabolic, neurodevelopmental, and behavioral tests in male (n = 40) and female (n = 35) adolescent offspring on a postnatal day (PD) 48; the long-term impact was assessed in adult male (n = 13) and female (n = 12) offspring on PD 225. Brain tissue was collected from adolescent and adult offspring for biochemical analysis. Maternal stress was associated with decreased body weight and increased urinary corticosterone during the pre-pregnancy period, but depressive-like behavior was delayed until later in pregnancy. No significant neurodevelopmental changes in suckling male or female offspring derived from the stress-exposed dams were observed. However, adolescent male and female offspring of stress-exposed dams displayed an increased depressive-like behavior and gender-dependent increase in anxiety-like behavior in female offspring. These changes were associated with a brain-region-specific increase in brain-derived neurotrophic factor (BDNF) protein and BDNF receptor (TrkB) mRNA in males. Behavioral changes observed in the adolescents receded in adult male and female offspring. However, plasma BDNF was elevated in stress-exposed adult female offspring. These results suggest that pre-gestational maternal stress is associated with gender-dependent short- vs. long-term neurodevelopmental impact in the offspring. Presented data are of significant public health relevance, and there is an urgent need for further research to confirm these findings and probe the underlying mechanisms.
... Moderate aerobic exercise involved physical activity at 40-59% maximum volume of oxygen [VO 2 in mL] in healthy individuals (Perk et al., 2012). Mice were randomly assigned to two groups, sedentary (S) and exercised (Ex) mice, and they had an adaptation period of exercise for two weeks with a gradual increase in speed and exercise time until reaching 10 m/min for 30 min/day (Toval et al., 2017). ...
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Exercise encourages active and healthy aging, maintaining functional and physical capabilities. This study aimed to assess the effects of a long-term moderate aerobic exercise protocol on bone microarchitecture and fragility associated with chronic inflammation and oxidative stress in aging. Male BALB/c mice (n = 10 per group) underwent a moderate exercise protocol from 13 weeks to 27 (adulthood age) or 108 weeks of age (elderly age) and were then sacrificed. Age-match sedentary mice were included as a control group. Serum cortisol concentrations were determined by chemiluminescent immunoassay, C-reactive protein (CRP) by a turbidimetric assay, advanced glycation end-products (AGEs) and malondialdehyde (MDA) by fluorescent spectroscopy, and total glutathione (GSH) by colorimetric method. The right femur was dissected for morphometric and densitometric analysis by computerized microtomography (µCT), and biomechanical properties were assessed using a three-point bending device. Muscle from the same extremity was obtained to determine relative mRNA expression of pro-inflammatory cytokines (TNF-α and IL-6) by RT-qPCR. Statistical differences were evaluated by two-way ANOVA and Holm-Sidak method post hoc with P<0.05. In elderly mice, moderate exercise increased glutathione levels and microarchitecture complexity but decreased bone fragility and oxidative stress markers, cortisol, and pro-inflammatory cytokines. In conclusion, these results suggest a strong link between a pro-inflammatory state and age-conditioned oxidative stress on bone quality. Thus, on a human scale, moderate aerobic exercise may improve bone quality during aging.
... The handling procedure consisted of picking up one rat at a time and petting it for 2 min. From postnatal day 26, female and male animals of the experimental groups were exposed to an 8-days exercise habituation protocol in a forced running wheel system (Campden Instruments, 80805A) as described previously (Fig. 1) 52 . The training program consisted of two sessions per day, on ZT14 and ZT20. ...
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Determining the body composition during adolescence can predict diseases such as obesity, diabetes, and metabolic syndromes later in life; and physical activity became an effective way to restore changes in body composition. However, current available literature assessing the body composition before, during and after adolescence in female and male rodents by in vivo techniques is scarce. Thus, by using computerized tomography, we aimed to define the baseline of the weight and body composition during the adolescence and young adulthood of female and male Sprague–Dawley rats (on P30, P60 and P90) under standard diet. Then, we determined the effect of 18 days of forced exercise on the body weight and composition during the early adolescence (P27-45). The highest percentual increments in weight, body volume and relative adipose contents occurred during the female and male adolescence. Forced running during the early adolescence decreased weight, body volume and relative adipose delta and increment values in males only. The adolescence of rats is a period of drastic body composition changes, where exercise interventions have sex-dependent effects. These results support a model that could open new research windows in the field of adolescent obesity.
... Moreover, in order to challenge the cognition and creativity of rats, new protocols could be established in which the pattern of stair intervals after a short period of time would be altered. Also, in order to determine the level of exercise necessary to obtain the observed responses it would be crucial to explore forced exercises (treadmill or motorized wheels), because forced exercise ensures the control of exercise load that will determine the level of physical activity necessary for the observed and induced changes (Toval et al., 2017). Furthermore, it would be interesting to perform the protocols on animals of different ages, to investigate any changes that could be associated with different periods of life. ...
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Purpose One of the most frequent complications associated with diabetes mellitus is apoptosis within the brain which can lead to cognitive disorders. Exercise is considered the best non‐pharmacological approach to reduce the severity and extent of cell death through poorly‐understood mechanisms. The aim of this study was to investigate the effects of voluntary complex and regular wheel running on the levels of 8‐oxoguanine DNA glycosylase (OGG1), semaphorin 3B (sema3B), hydrogen peroxide (H2O2), and apoptosis in the hippocampus of diabetic rats. Methods 48 Wistar male rats were randomly divided into 6 groups: healthy control (C), diabetes control (D), regular wheel running + diabetes (RWD), complex wheel running + diabetes (CWD), healthy regular wheel running (RW), and healthy complex wheel running (CW). The diabetic rat model was produced by intraperitoneal injection of streptozotocin (STZ). The protocol encompassed a 4‐week voluntary running training regimen on regular and complex wheel running apparatus. The rats were sacrificed 48 hr after the last training session. To measure the protein concentrations within the hippocampus, ELISA has been utilized. One‐way ANOVA was used to compare the groups. Results There were no significant differences in OGG1 protein levels between the groups. H2O2 level in the D group was significantly higher than the C group (p = .002), while this in RWD and CWD groups was considerably lower than the D group (p = .002 and p = .003, respectively). In the D group, the levels of apoptosis and Sema3B were significantly (p = .001 and p = .007, respectively) higher than C, RWD (p = .001, p = .0001, respectively), and CWD groups (p = .001, p = .006, respectively). Nevertheless, there were not any significant differences between RWD and CWD groups. Conclusion The increased levels of Sema3B, H2O2, and apoptosis within the hippocampus associated with diabetes could be noticeably restored by both types of voluntary wheel running protocols.
Running wheel exercise training (RWE) and skilled reaching training (SRT) are physical training approaches with positive effects on cognitive function. However, few studies have compared the different effects of these exercises on long-term memory, and their mechanism remains unknown. This study investigated the effects of SRT and RWE, at the recovery stage, on the cognitive function of transient middle cerebral artery occlusion (tMCAO) rats and explored their association with NgR1/Rho-A/ROCK/LOTUS/LGI1 signaling. Adult Sprague-Dawley rats (n = 55) were divided into four groups after pretraining: SRT, RWE, tMCAO, and Sham. Rats were subjected to modified neurological severity score (mNSS) measurements and forelimb grip strength and the Morris water maze tests. Using immunofluorescence and western blotting, we evaluated axonal growth inhibitor expression in the peri-infarct cortex on days 28 and 56 after tMCAO. Results showed the mNSS reduced, whereas the grip strengths improved in RWE and SRT groups. The escape latency in the Morris water maze test was shorter, whereas the number of times of crossing the platform was higher in both the SRT and RWE groups than in the tMCAO group on day 56; furthermore, the parameters in the SRT group improved compared to those in the RWE group. Physical exercise training could improve cognitive functions by reducing the expression of the NgR1/RhoA/ROCK axon growth inhibitors and increasing the expression of the endogenous antagonists LOTUS/LGI1. Exercise training beginning at the recovery stage could improve the cognitive function in tMCAO rats through a mechanism probably associated with the axonal growth inhibitor pathway.
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Exercise is known to have beneficial effects on cognition, mood, and the brain. However, exercise also activates the hypothalamic–pituitary–adrenal axis and increases levels of the glucocorticoid cortisol (CORT). CORT, also known as the “stress hormone,” is considered a mediator between chronic stress and depression and to link various cognitive deficits. Here, we review the evidence that shows that while both chronic stress and exercise elevate basal CORT levels leading to increased secretion of CORT, the former is detrimental to cognition/memory, mood/stress coping, and brain plasticity, while the latter is beneficial. We propose three preliminary answers to the exercise-CORT paradox. Importantly, the elevated CORT, through glucocorticoid receptors, functions to elevate dopamine in the medial prefrontal cortex under chronic exercise but not chronic stress, and the medial prefrontal dopamine is essential for active coping. Future inquiries may provide further insights to promote our understanding of this paradox.
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Extensive research on humans suggests that exercise could have benefits for overall health and cognitive function, particularly in later life. Recent studies using animal models have been directed towards understanding the neurobiological bases of these benefits. It is now clear that voluntary exercise can increase levels of brain-derived neurotrophic factor (BDNF) and other growth factors, stimulate neurogenesis, increase resistance to brain insult and improve learning and mental performance. Recently, high-density oligonucleotide microarray analysis has demonstrated that, in addition to increasing levels of BDNF, exercise mobilizes gene expression profiles that would be predicted to benefit brain plasticity processes. Thus, exercise could provide a simple means to maintain brain function and promote brain plasticity.
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Physiological variables, such as maximum work rate or maximal oxygen uptake (V̇O2max), together with other submaximal metabolic inflection points (e.g. the lactate threshold [LT], the onset of blood lactate accumulation and the pulmonary ventilation threshold [VT]), are regularly quantified by sports scientists during an incremental exercise test to exhaustion. These variables have been shown to correlate with endurance performance, have been used to prescribe exercise training loads and are useful to monitor adaptation to training. However, an incremental exercise test can be modified in terms of starting and subsequent work rates, increments and duration of each stage. At the same time, the analysis of the blood lactate/ventilatory response to incremental exercise may vary due to the medium of blood analysed and the treatment (or mathematical modelling) of data following the test to model the metabolic inflection points. Modification of the stage duration during an incremental exercise test may influence the submaximal and maximal physiological variables. In particular, the peak power output is reduced in incremental exercise tests that have stages of longer duration. Furthermore, the VT or LT may also occur at higher absolute exercise work rate in incremental tests comprising shorter stages. These effects may influence the relationship of the variables to endurance performance or potentially influence the sensitivity of these results to endurance training. A difference in maximum work rate with modification of incremental exercise test design may change the validity of using these results for predicting performance, and prescribing or monitoring training. Sports scientists and coaches should consider these factors when conducting incremental exercise testing for the purposes of performance diagnostics.
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We developed a forced non-electric-shock running wheel (FNESRW) system that provides rats with high-intensity exercise training using automatic exercise training patterns that are controlled by a microcontroller. The proposed system successfully makes a breakthrough in the traditional motorized running wheel to allow rats to perform high-intensity training and to enable comparisons with the treadmill at the same exercise intensity without any electric shock. A polyvinyl chloride runway with a rough rubber surface was coated on the periphery of the wheel so as to permit automatic acceleration training, and which allowed the rats to run consistently at high speeds (30 m/min for 1 h). An animal ischemic stroke model was used to validate the proposed system. FNESRW, treadmill, control, and sham groups were studied. The FNESRW and treadmill groups underwent 3 weeks of endurance running training. After 3 weeks, the experiments of middle cerebral artery occlusion, the modified neurological severity score (mNSS), an inclined plane test, and triphenyltetrazolium chloride were performed to evaluate the effectiveness of the proposed platform. The proposed platform showed that enhancement of motor function, mNSS, and infarct volumes was significantly stronger in the FNESRW group than the control group (P<0.05) and similar to the treadmill group. The experimental data demonstrated that the proposed platform can be applied to test the benefit of exercise-preconditioning-induced neuroprotection using the animal stroke model. Additional advantages of the FNESRW system include stand-alone capability, independence of subjective human adjustment, and ease of use.
Vertebrate Endocrinology represents more than just a treatment of the endocrine system-it integrates hormones with other chemical bioregulatory agents not classically included with the endocrine system. It provides a complete overview of the endocrine system of vertebrates by first emphasizing the mammalian system as the basis of most terminology and understanding of endocrine mechanisms and then applies that to non-mammals. The serious reader will gain both an understanding of the intricate relationships among all of the body systems and their regulation by hormones and other bioregulators, but also a sense of their development through evolutionary time as well as the roles of hormones at different stages of an animal's life cycle.
Our purpose in this study was to investigate efficient and sustainable combinations of exercise and diet-induced weight loss (DIET), in order to combat obesity in metabolic syndrome (MetS) patients. We examined the impact of aerobic interval training (AIT), followed by or concurrent to a DIET on MetS components. 36 MetS patients (54±9 years old; 33±4 BMI; 27 males and 9 females) underwent 16 weeks of AIT followed by another 16 weeks without exercise from the fall of 2013 to the spring of 2014. Participants were randomized to AIT without DIET (E CON, n=12), AIT followed by DIET (E-then-D, n=12) or AIT concurrent with DIET (E+D, n=12) groups. Body weight decreased below E CON similarly in the E-then-D and E+D groups (~5%). Training improved blood pressure and cardiorespiratory fitness (VO2peak) in all groups with no additional effect of concurrent weight loss. However, E+D improved insulin sensitivity (HOMA) and lowered plasma triglycerides and blood cholesterol below E CON and E-then-D (all P<0.05). Weight loss in E-then-D in the 16 weeks without exercise lowered HOMA to the E+D levels and maintained blood pressure at trained levels. Our data suggest that a new lifestyle combination consisting of aerobic interval training followed by weight loss diet is similar, or even more effective on improving metabolic syndrome factors than concurrent exercise plus diet. © Georg Thieme Verlag KG Stuttgart · New York.
Exercise performance seems to be greatly affected by the chronic level of physical activity experienced by the animal or individual. For example, differences in the capacity for prolonged exercise seem obvious between wild and domesticated animals. This is probably due, in part, to inherent biochemical differences between the muscles of active and less active species (1). Muscles of wild animals appear darker than those of their domesticated counterparts (2). Further, variations in activity patterns due to seasonal change (3) or hibernation (4), are associated with differences in the enzymes related to oxidative metabolism. Thus, in a general sense physical activity seems to be associated with biochemical changes that enhance the muscle's capacity for aerobic metabolism. Muscle Adaptations The specific biochemical changes induced by increased physical activity are well characterized from laboratory studies and have been the subject of a number of excellent reviews (5-9 ). The fundamental change found in