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. 2023 Apr 1:515:25-36.
doi: 10.1016/j.neuroscience.2023.01.028. Epub 2023 Feb 2.

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

Ki Yun Lee  1 , Justin S Rhodes  2 , M Taher A Saif  3
Affiliations

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

Ki Yun Lee et al. Neuroscience. .

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.

Keywords: Exercise; astrogliogenesis; hippocampus; in vitro model; muscle; neural network; neurogenesis; neuronal maturation; synaptogenesis.

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Conflict of interest statement

Declaration of interest

All authors declare no conflicts of interest in this work.

Figures

Fig 1.
Fig 1.. Muscle contraction-conditioned media increases hippocampal neuronal activity
(A) Primary mouse hippocampal cells were cultured on a multi-electrode array (MEA) for 9 days with the following treatments: regular media (RM), muscle contraction-conditioned media (CM), RM with N-benzyl-p-toluene sulphonamide (BTS-RM), CM with BTS (BTS-CM). Average number of spikes per second (± SEM) across all electrodes normalized to the average value for RM on day 2 are shown. (B) Average number of bursts per minute normalized to the average value for RM on day 2. (C) Average synchrony index (n = 12 for control and 6 for BTS cultures). Different lowercase letters above bar plots indicate significant differences among treatments within each day (p < 0.05). (D) Average muscle contraction amplitudes between control and 10 μM BTS (left). Skeletal muscle contraction patterns in the presence of 0 (top right) and 10 μM BTS (bottom right). Scale bar in x, y: 1 s, 1 μm. (E) Peak fluorescent changes between control and 10 μM BTS (n = 3). Traces of calcium dynamics in the presence of 0 (top right) and 10 μM BTS (bottom right). Scale bar in x, y: 2 s, 1 ΔF⁄F0. Different lowercase letters above box plots indicate significant differences between treatments (p < 0.05). (F) Raster plots representing spike trains from nine electrodes from RM (top left), RM with BTS (top right), CM (bottom left), and CM with BTS (bottom right) on day 9. Each line and the red box represent a single firing, and synchronous burst, respectively. Scale bar: 2 s.
Fig 2.
Fig 2.. Muscle contraction-conditioned media accelerates hippocampal neuron synapse development
(A) Primary mouse hippocampal cells were cultured for 9 days with regular media (RM) or muscle contraction-conditioned media (CM). Average number of synapses (± SEM), as measured by immunohistochemical detection of pre and postsynaptic markers, are shown for RM and CM on days 2 and 9. (B) Average vesicle accumulation per synapse on days 2 and 9. Data are shown normalized to the average value for RM on day 2. (C) Average F-actin intensity on day 9 (F-actin levels were below the limit of detection on day 2). Data are shown normalized to the average value for RM. (D) Average number of astrocytes on days 2 and 9. The inset shows RM on day 2 on a smaller scale (n = 6 per group). (E) Images of astrocytes in the cultures on day 2 in RM (left) and CM (right) (S100β, green; DAPI, blue). Scale bar: 50 μm. (F) Average number of neurons on day 3 and 9 (n = 6 for day 3 and n = 8 for day 9). (G) Images of neurons and astrocytes in the cultures on day 9 in RM (left) and CM (right) (NeuN, red; S100β, green; DAPI, blue). Scale bar: 100 μm. Different lowercase letters above box plots indicate significant differences between treatments and days (p < 0.05).
Fig 3.
Fig 3.. Astrocytes release factors that tame muscle-media-induced neuronal activity
(A) Average number of astrocytes in control and glia-reduced group on day 4 (n = 8). (B) Images of the control and glia-reduced cultures on day 4 (S100β, green; DAPI, blue). Scale bar: 100 μm. (C) Average number of spikes per second (± SEM) normalized to RM on day 2 in the presence and absence of astrocytes (n = 18 for control and 6 for glia-reduced cultures). Different lowercase letters above bar plots indicate significant differences among treatments and cultures within each day (p < 0.05). Results for burst rate and synchrony index are shown in supplementary materials (Fig S4 and Appendix S1).
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