Research ArticleAstrocyte-mediated Transduction of Muscle Fiber Contractions Synchronizes Hippocampal Neuronal Network Development
Introduction
Exercise is a highly effective strategy for maintaining cognitive health throughout life, even when initiated at late stages in life (Churchill et al., 2002, Erickson and Kramer, 2009, Erickson et al., 2011). Many studies have shown robust long-term changes in the hippocampus from increased physical activity, such as increased adult hippocampal neurogenesis, synaptogenesis, and enlarged hippocampal volume which likely support enhanced cognition (van Praag et al., 1999, Redila and Christie, 2006, Clark et al., 2009, Clark et al., 2011, Erickson et al., 2011). However, the mechanisms by which exercise produces such dramatic changes in the hippocampus remain elusive. Uncovering the mechanisms that are responsible for enlarging the hippocampus and enhancing its function could be used to reverse-engineer treatments for cognitive pathologies that result in a diminished size and function of the hippocampus, such as Alzheimer’s disease, stress, depression, anxiety, PTSD, Cushing’s disease, epilepsy, and normal aging (Dhikav and Anand, 2007).
Cumulative research over the past few decades has suggested that factors released from contracting muscles such as lactate (el Hayek et al., 2019), growth factors (Trejo et al., 2001, Fabel et al., 2003), trophic factors (Church et al., 2016), and myokines (Wrann et al., 2013, Moon et al., 2016) provide crucial signals that support enhanced plasticity (Delezie and Handschin, 2018). However, how muscle factors affect hippocampal cells is still being worked out. Recently, we found that repeated electrical contractions of the hindlimb muscles of anesthetized mice in a pattern that produced endurance adaptations in the muscles (40 reps, twice a week for 8 weeks) caused increased numbers of new astrocytes in the hippocampus and enlarged the volume of the dentate gyrus by approximately 10% (Gardner et al., 2020). This suggests astrocytes are sensitive to muscle factors and proliferate when they detect muscle factors in the blood. Given the role that astrocytes play in forming the blood–brain barrier, they are well situated to transduce signals from the blood into the brain.
One way to study the interactions between contracting muscle cells and hippocampal cells including neurons and astrocytes is to isolate the cells and perform experiments in vitro. For example, previous in vitro studies found that muscle-conditioned media attracted neurites of spinal cord motor neurons to form neuro-muscular junctions (McCaig, 1986). Along this line, our lab has been examining cross-talk between muscles and neurons in vitro. We recently found that when media from contracting muscle fibers derived from a C2C12 mouse myoblast cell line is applied to neuronal cultures derived from a mouse embryonic stem cell line plated on a micro-electrode array, it enhanced overall neural firing rates of the neurons (Aydin et al., 2020).
To further explore how factors from contracting muscles might influence hippocampal cells, we developed an in vitro preparation in which primary mouse skeletal muscle cells are plated on a functionalized substrate. The myoblasts develop bundles of myotubes and begin to contract spontaneously. We then take the media surrounding the contracting muscles (conditioned media, CM) and apply that media to in vitro primary hippocampal cell cultures that include neurons and astrocytes. The objectives of this study were to determine whether CM influences the function and maturation of hippocampal neuronal networks, and to investigate the role of astrocytes in the process of transduction of muscle contractions to the activity of hippocampal neuronal networks in vitro.
Section snippets
Primary mouse skeletal muscle and hippocampus dissection
Muscle tissues were isolated from the hindlimbs of 4-week-old CD1 mice. We used a total of 6 mice and did not differentiate by sex. The muscle tissue was collected and dissociated using a standard protocol (Wang et al., 2017) with slight modifications. Briefly, the tissues were collected in cold PBS (Corning), minced, and digested for 30 minutes in digestion media consisting of DMEM, 2.5% HEPES, 1% GlutaMAX (all from Gibco), and 1% Penicillin-Streptomycin (Lonza) with the addition of 400
Contracting muscle-conditioned media enhances neuronal activity measured by microelectrode arrays
Consistent with our previous MEA study with C2C12 mouse myoblast cell line and mouse embryonic stem cell-derived neuronal culture (Aydin et al., 2020), CM from primary skeletal muscle cells increased spike and burst rates of primary hippocampal neurons across days (Fig. 1A and 1B). The general pattern of development of spike trains over time in RM was consistent with other studies using primary hippocampal cells and primary sensory neurons at a similar cell seeding density (Biffi et al., 2013,
Discussion
Here we establish for the first time an in vitro platform to explore interactions between contracting primary muscle cells and primary hippocampal cells. One of the leading hypothesized mechanisms for pro-cognitive effects of exercise is that muscle contractions release factors that cross into the brain where they directly influence hippocampal cells involved in cognition (van Praag et al., 1999, Trejo et al., 2001, Wrann et al., 2013). This hypothesis is supported by our recent discovery that
Acknowledgements
We are grateful to Dr. Gelson Pagan-Diaz of the University of Texas for discussions of MEA, Jennie Gardner for husbandry of animal subjects and mouse muscle dissections at the early stage of the study, Md Saddam Hossain Joy for discussions of the double-fluorescent label method, Carlos Renteria for discussions of calcium imaging and MATLAB code, Meghan Connolly for discussions of neurogenesis, and Dr. Onur Aydin of the University of Illinois for ideations and overall insightful discussions of
Declaration of interest
All authors declare no conflicts of interest in this work.
Funding
This work was supported by the National Institutes of Health (R21 NS109894), and National Science Foundation (CMMI 1935181).
References (43)
- et al.
Development of 3D neuromuscular bioactuators
APL Bioeng
(2020) - et al.
The influence of neuronal density and maturation on network activity of hippocampal cell cultures: A methodological study
PLoS One
(2013) - et al.
Adult mouse sensory neurons on microelectrode arrays exhibit increased spontaneous and stimulus-evoked activity in the presence of interleukin-6
J Neurophysiol
(2018) - et al.
A small-molecule inhibitor of skeletal muscle myosin II
Nat Cell Biol
(2002) - et al.
Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis
Cell
(2005) - et al.
Astrocytes mediate synapse elimination through MEGF10 and MERTK pathways
Nature
(2013) - et al.
Astrocytes control synapse formation, function, and elimination
Cold Spring Harb Perspect Biol
(2015) - et al.
Comparison of high-intensity vs. high-volume resistance training on the BDNF response to exercise
J Appl Physiol
(2016) - et al.
Exercise, experience and the aging brain
Neurobiol Aging
(2002) - et al.
Functional analysis of neurovascular adaptations to exercise in the dentate gyrus of young adult mice associated with cognitive gain
Hippocampus
(2009)
Adult hippocampal neurogenesis and c-Fos induction during escalation of voluntary wheel running in C57BL/6J mice
Behav Brain Res
Genetic influences on exercise-induced adult hippocampal neurogenesis across 12 divergent mouse strains
Genes Brain Behav
Endocrine crosstalk between Skeletal muscle and the brain
Front Neurol
Is hippocampal atrophy a future drug target?
Med Hypotheses
Astrocyte transforming growth factor beta 1 promotes inhibitory synapse formation via CaM kinase II signaling
Glia
Colocalization of synapse marker proteins evaluated by STED-microscopy reveals patterns of neuronal synapse distribution in vitro
J Neurosci Methods
Lactate mediates the effects of exercise on learning and memory through sirt1-dependent activation of hippocampal brain-derived neurotrophic factor (BDNF)
J Neurosci
Aerobic exercise effects on cognitive and neural plasticity in older adults
Br J Sports Med
Exercise training increases size of hippocampus and improves memory
Proc Natl Acad Sci U S A
VEGF is necessary for exercise-induced adult hippocampal neurogenesis
Eur J Neurosci
The distribution of synapsin I and synaptophysin in hippocampal neurons developing in culture
J Neurosci
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