The method of isolation of the crayfish abdominal stretch receptor maintaining a connection of the sensory neuron to the ventral nerve cord ganglion

Abstract

The crayfish stretch receptor consisting of the single mechanoreceptor neurons enveloped by satellite glial cells is the simplest functioning neuroglial preparation. However, during isolation, its axons are usually transected that eliminates afferent regulation and induces complex axotomy-related signaling responses in neurons and satellite glia. We developed new microsurgical method of crayfish stretch receptor isolation, which preserves connections of sensory neurons to the ventral nerve cord ganglion. The stretch receptor may either remain on the abdominal carapace, or be completely isolated. In both cases, it may be either intact, or axotomized. The integrity of axons was confirmed by firing recording from proximal and distal axon points. Normal, necrotic and apoptotic cells were visualized using double fluorochroming with Hoechst 33342 and propidium iodide. The isolated mechanoreceptor neurons maintain regular firing during 8-10 or more hours. Glial cells surrounding non-axotomized neurons demonstrate lower necrosis and apoptosis levels than the axotomized ones. Unlike the existing method, in which the sensory neurons were axotomized, the present method preserves links between the sensory neurons and the ganglion and makes possible to avoid consequences of axotomy in neurons and satellite glia. The present neuroglial preparation may be used as a simple but informative model object in studies of axotomy-induced degeneration and survival of peripheral neurons, the role of glia in neuron injury, the signaling mechanisms of neuroglial interactions, and the effects of diverse physical and chemical factors on neuronal and glial cells.

Calcium in Neuronal and Glial Response to Axotomy

“…Eight hours of incubation of a sample in saline are sufficient for the appearance of morphological signs (fragmented nuclei) of spontaneous or induced glial apoptosis. The apoptosis did not increase in the following hour of incubation in intact samples (so it is a spontaneous apoptosis), but continued to grow in axotomized cells [113], thus suggesting that AT is a long-lasting damaging factor, although the lumen was instantly sealed mechanically after the transection [19]. Our further analysis showed that AT makes remote glia susceptible to apoptosis promoted by increased extracellular Ca 2+ and impaired the functions of ryanodine receptors and ER Ca 2+ ATPase (SERCA).…”

“…In crayfish stretch receptors, AT not only decreased firing duration (the functional "lifetime") of mechanoreceptor neurons [19], but also promoted necrosis and apoptosis of remote glial cells (RGC) [30,113]. Eight hours of incubation of a sample in saline are sufficient for the appearance of morphological signs (fragmented nuclei) of spontaneous or induced glial apoptosis.…”

E2F1 Expression and Apoptosis Initiation in Crayfish and Rat Peripheral Neurons and Glial Cells after Axonal Injury

“…The structure of the MRN is shown in Figure 1a. Satellite glial cells (SGC) and their interaction with MRN were studied in detail at the optical and ultrastructural levels [12][13][14][15][16]. MRN generates impulses directed to the VNC ganglion with a frequency proportional to the lengthening of the receptor muscle, which is strengthened.…”

“…MRN axotomy was performed by cutting the axon at a distance of 5-8 mm from the cell body. We have developed a technique for isolating control MRNs, the axon of which is not cut and retains a connection with the VNC ganglion [14]. The technique of MRN isolation and axotomy is described in more detail in our previous works [14,54].…”

“…Previously, ultrastructural changes were shown in axotomized hippocampal neurons [6][7][8] and rat dorsal root ganglia [6], as well as in invertebrates, including aplysia [9], earthworm, squid and crayfish [10,11]. In our laboratory, the electrophysiology, morphology, ultrastructure and biochemistry of mechanoreceptor neurons (MRN) of crayfish and surrounding glial cells were previously studied in detail, as well as their responses to various physicochemical effects, inhibitors, or activators of various proteins [12][13][14][15][16]. As a result of these experiments, our team demonstrated the involvement of a number of signaling pathways and transcription factors in the survival and death of neurons and glial cells during axotomy.…”