Locust primary neuronal culture for the study of synaptic transmission

Abstract

We have designed a cell culture system for thoracic neurons of adult Locusta migratoria that enables the establishment of functional synapses in vitro. Patch-clamp recordings revealed three different neuron classes. About half of the neurons (47%) had unexcitable somata with outward and no inward conductance. The other half generated either single (37%) or multiple action potentials (18%) and differed mainly in lower outward conductance. Selectively stained motor neurons were analyzed to demonstrate varied physiological properties due to culture conditions. Using paired patch clamp recordings we demonstrate directly synaptic transmission in morphologically connected neurons in vitro. Presynaptic stimulation resulted in postsynaptic potentials in 42 pairs of neurons tested, independent of the type of neuron. According to pharmacological experiments most of these synapses were either glutamatergic or GABAergic. In addition to these chemical synapses, electrical synapses were found. With the demonstration of synapse formation in cell culture of adult locust neurons, this study provides the basis for the future analysis of more defined insect neuronal circuits in culture.

Isolamento e cultivo de neurônios e neuroesferas de córtex cerebral aviar

“…É comum visualizar o comprimento dos neuritos entre duas a cinco vezes o diâmetro do soma (Weigel et al 2012) conforme pode ser visualizado nas Figuras 3 a 5. Weigel et al (2012) relatam a existência de distintos tipos morfológicos de neurônios, classificados em unipolares e bipolares.…”

“…É comum visualizar o comprimento dos neuritos entre duas a cinco vezes o diâmetro do soma (Weigel et al 2012) conforme pode ser visualizado nas Figuras 3 a 5. Weigel et al (2012) relatam a existência de distintos tipos morfológicos de neurônios, classificados em unipolares e bipolares. Neurônios unipolares apresentam um úni-co neurito longo primário (Fig.3), enquanto os neurônios bipolares (Fig.4) tipicamente possuem dois neuritos quando comparados aos neurônios unipolares.…”

The role of gap junction proteins in the development of neural network functional topology

“…When comparing the expression pattern of SYT‐1 to that of the innexins, we saw that GJs appeared early on in development, before active chemical synapses. The presence of functional active chemical synapses in insect neural cell cultures was recently demonstrated in locust cell cultures using immunohistochemical (Pfahlert & Lakes‐Harlan, ) and patch clamp studies (Weigel et al ., ). Hence, GJs may play a role in early developmental processes, including the formation of chemical synapses.…”

“…The cultured neurones are fully differentiated adult neurones that have lost their original dendrites and axon, leaving only the soma; during clearly identifiable stages of their in vitro development, neurones regenerate and connect to neighbouring cells, migrate and aggregate to form stable, fully clustered neural networks. The presence of GJs and functional electrical synapses has been also established in similar cultures of locust neurones (Weigel et al ., ), as well as in in vitro networks from other insects (eg Oh et al ., ).…”

Culture of neural cells of the eyestalk of a mangrove crab is optimized on poly-l-ornithine substrate

“…The culture medium for maintaining the cultures of neurons and glial cells was chosen from literature sources: L-15 medium with L-glutamine has been the most widely used for maintaining invertebrate neurons and glial cells and/or brain ganglia in culture (Pearce et al 2003;Sashikumar and Desai 2008;Weigel et al 2012;Zanetti et al 2007). However, the base medium, comprising inorganic salts, amino acids and vitamins, was not sufficient to maintain cell viability; it had to be supplemented with other factors (Valk et al 2010).…”