RESUMEN
Cultured embryonic neurons develop functional networks that transmit synaptic signals over multiple sequentially connected neurons as revealed by multi-electrode arrays (MEAs) embedded within the culture dish. Signal streams of ex vivo networks contain spikes and bursts of varying amplitude and duration. Despite the random interactions inherent in dissociated cultures, neurons are capable of establishing functional ex vivo networks that transmit signals among synaptically connected neurons, undergo developmental maturation, and respond to exogenous stimulation by alterations in signal patterns. These characteristics indicate that a considerable degree of organization is an inherent property of neurons. We demonstrate herein that (1) certain signal types occur more frequently than others, (2) the predominant signal types change during and following maturation, (3) signal predominance is dependent upon inhibitory activity, and (4) certain signals preferentially follow others in a non-reciprocal manner. These findings indicate that the elaboration of complex signal streams comprised of a non-random distribution of signal patterns is an emergent property of ex vivo neuronal networks.
Asunto(s)
Potenciales de Acción/fisiología , Red Nerviosa/fisiología , Neuronas/fisiología , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Animales , Bicuculina/farmacología , Células Cultivadas , Corteza Cerebral/citología , Estimulación Eléctrica , Embrión de Mamíferos , Antagonistas de Receptores de GABA-A/farmacología , Ratones , Ratones Endogámicos C57BL , Red Nerviosa/embriología , Factores de TiempoRESUMEN
Developing myofibers require chemical and electrical stimulation to induce functional muscle tissue. Tissue engineering protocols utilize either or both of these to initiate differentiation ex vivo. Current methodologies typically deliver multi-volt electrical signals, which may be hazardous to developing tissues. In attempts to mimic in vivo muscle development, we stimulated cultured muscle precursor cells with a low-voltage (1 mV) digitized synaptic signal derived from cultured cortical neurons. This synaptic signal induced larger and more adherent myofibers, along with markers of myoblast differentiation, compared to those induced following stimulation with a conventional (28 V) square signal. These findings suggest that stimulation with a digitized synaptic signal may be useful in tissue engineering and physical therapy.
Asunto(s)
Mioblastos Esqueléticos/metabolismo , Neuronas/metabolismo , Células Madre/metabolismo , Sinapsis , Transmisión Sináptica , Animales , Línea Celular , Técnicas de Cocultivo , Ratones , Mioblastos Esqueléticos/citología , Neuronas/citología , Células Madre/citología , Ingeniería de Tejidos/métodosRESUMEN
A number of laboratories have modeled aspects of synaptic plasticity using neuronal networks established on micro-electrode arrays. Such studies demonstrate that external stimulation can increase or hasten maturation of network signaling as evidenced an increase in complex bursts. Herein, we demonstrate that repetitive stimulation with a recorded synaptic signal was capable of increasing overall signaling, including the percentage of bursts, over a 5-day period, but that this increase was completely prevented by the presence of the GABAergic antagonist bicuculline. These findings demonstrate a critical role for inhibitory neurons in signal maturation following stimulation, which supports the purported role for inhibitory neuronal activity in long-term potentiation and learning in situ.
Asunto(s)
Potenciales de Acción/fisiología , Estimulación Eléctrica/métodos , Red Nerviosa/fisiología , Inhibición Neural/fisiología , Plasticidad Neuronal/fisiología , Neuronas/fisiología , Transmisión Sináptica/fisiología , Animales , Células Cultivadas , Ratones , Ratones Endogámicos C57BLRESUMEN
A predominance of excitatory activity, with protracted appearance of inhibitory activity, accompanies cortical neuronal development. It is unclear whether or not inhibitory neuronal activity is solicited exclusively by excitatory neurons or whether the transient excitatory activity displayed by developing GABAergic neurons contributes to an excitatory threshold that fosters their conversion to inhibitory activity. We addressed this possibility by culturing murine embryonic neurons on multi-electrode arrays. A wave of individual 0.2-0.4 mV signals ("spikes") appeared between approx. 20-30 days in culture, then declined. A transient wave of high amplitude (>0.5 mV) epileptiform activity coincided with the developmental decline in spikes. Bursts (clusters of ≥3 low-amplitude spikes within 0.7s prior to returning to baseline) persisted following this decline. Addition of the GABAergic antagonist bicuculline initially had no effect on signaling, consistent with delayed development of GABAergic synapses. This was followed by a period in which bicuculline inhibited overall signaling, confirming that GABAergic neurons initially display excitatory activity in ex vivo networks. Following the transient developmental wave of epileptiform signaling, bicuculline induced a resurgence of epileptiform signaling, indicating that GABAergic neurons at this point displayed inhibitory activity. The appearance of transition after the developmental and decline of epileptiform activity, rather than immediately after the developmental decline in lower-amplitude spikes, suggests that the initial excitatory activity of GABAergic neurons contributes to their transition into inhibitory neurons, and that inhibitory GABAergic activity is essential for network development. Prior studies indicate that a minority (25%) of neurons in these cultures were GABAergic, suggesting that inhibitory neurons regulate multiple excitatory neurons. A similar robust increase in signaling following cessation of inhibitory activity in an artificial neural network containing 20% inhibitory neurons supported this conclusion. Even a minor perturbation in GABAergic function may therefore foster initiation and/or amplification of seizure activity, as well as perturbations in long-term potentiation.
Asunto(s)
Potenciales de Acción , Relojes Biológicos , Epilepsia/embriología , Epilepsia/fisiopatología , Neuronas GABAérgicas , Red Nerviosa/embriología , Red Nerviosa/fisiopatología , Animales , Células Cultivadas , Ratones , Ratones Endogámicos C57BLRESUMEN
Neuronal networks established on micro-electrode arrays provide useful models for synaptic plasticity. Whether or not this represents a facet of learning is debated since ex vivo networks are deprived of organismal interaction with the environment. We compared developmental signaling of such networks with and without stimulation with a prerecorded synaptic signal from another mature culture as a model of sensory input. Unstimulated networks displayed a developmental increase in individual signals that eventually declined, yielding a pattern containing organized bursts of signaling. Minimal stimulation, to model the onset of sensory input hastened the onset of developmental signaling. However, the overall developmental pattern of stimulated networks, including the total number and type of signals as well as the length of this developmental period, was identical to that of unstimulated networks. One interpretation of these findings is that ongoing plasticity may be essential to establish an appropriate platform for learning once sensory input ensues.
Asunto(s)
Aprendizaje/fisiología , Red Nerviosa/crecimiento & desarrollo , Red Nerviosa/fisiología , Plasticidad Neuronal/fisiología , Transducción de Señal/fisiología , Animales , Fenómenos Electrofisiológicos/fisiología , Femenino , Potenciación a Largo Plazo/fisiología , Ratones , Ratones Endogámicos C57BL , EmbarazoRESUMEN
Multielectrode arrays (MEAs) are used for analysis of neuronal activity. Here we report two variations on commonly accepted techniques that increase the precision of extracellular electrical stimulation: (i) the use of a low-amplitude recorded spontaneous synaptic signal as a stimulus waveform and (ii) the use of a specific electrode within the array adjacent to the stimulus electrode as a hard-grounded stimulus signal return path. Both modifications remained compatible with manipulation of neuronal networks. In addition, localized stimulation with the low-amplitude synaptic signal allowed selective stimulation or inhibition of otherwise spontaneous signals. These findings indicate that minimizing the area of the culture impacted by external stimulation allows modulation of signaling patterns within subpopulations of neurons in culture. The simple modifications described herein may be useful for precise monitoring and manipulation of neuronal networks.