Elsevier

Neuroscience

Volume 515, 1 April 2023, Pages 25-36
Neuroscience

Research Article
Astrocyte-mediated Transduction of Muscle Fiber Contractions Synchronizes Hippocampal Neuronal Network Development

https://doi.org/10.1016/j.neuroscience.2023.01.028Get rights and content

Highlights

  • Contracting muscle media enhances hippocampal neuronal activity.

  • Contracting muscle media expedites synaptic maturation.

  • Contracting muscle media accelerates accumulation of filamentous actin at synapses.

  • Contracting muscle media induces significant astrocyte and neuron proliferation.

  • Astrocytes release factors that inhibit muscle media-induced neuronal activity.

Abstract

Exercise supports brain health in part by enhancing hippocampal function. The leading hypothesis is that muscles release factors when they contract (e.g., lactate, myokines, growth factors) that enter circulation and reach the brain where they enhance plasticity (e.g., increase neurogenesis and synaptogenesis). However, it remains unknown how the muscle signals are transduced by the hippocampal cells to modulate network activity and synaptic development. Thus, we established an in vitro model in which the media from contracting primary muscle cells (CM) is applied to developing primary hippocampal cell cultures on a microelectrode array. We found that the hippocampal neuronal network matures more rapidly (as indicated by synapse development and synchronous neuronal activity) when exposed to CM than regular media (RM). This was accompanied by a 4.4- and 1.4-fold increase in the proliferation of astrocytes and neurons, respectively. Further, experiments established that factors released by astrocytes inhibit neuronal hyper-excitability induced by muscle media, and facilitate network development. Results provide new insight into how exercise may support hippocampal function by regulating astrocyte proliferation and subsequent taming of neuronal activity into an integrated network.

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).

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