Cell culture of mechanoreceptor neurons innervating proleg sensory hairs in Manduca sexta larvae, and co-culture with target motoneurons.
The tip of each proleg in Manduca sexta larvae bears a dense array of mechanosensory hairs termed planta hairs (PHs), each innervated by a single sensory neuron (termed a PH-SN) located in the underlying epidermis. In the CNS, axon terminals of PH-SNs make direct, excitatory, nicotinic cholinergic synapses with proleg retractor motoneurons including the accessory planta retractor (APR). These synapses mediate a proleg withdrawal reflex, exhibit multiple forms of activity-dependent plasticity and weaken during the prepupal peak of ecdysteroids. In the present study we developed methods to dissociate PH-SNs from the epidermis and culture them alone or with APRs. The PH-SNs were fluorescently labeled in situ by introducing dye through the cut hair shaft or by retrograde axonal staining. Alternatively, unlabeled PH-SNs were utilized. The epidermis beneath the planta hair array was separated from the cuticle, enzymatically treated and mechanically dissociated into single cells. PH-SNs were cultured on glass coverslips coated with concanavalin A and laminin, in modified Leibovitz's IL-15 medium. Supplementation with medium conditioned by an insect cell line produced the best results. Dissociated PH-SNs had somatic diameters of ~10 micro m and typically bore a stout dendrite consisting of the inner and, occasionally, the outer dendritic segment. An axonal stump was sometimes retained. Viable PH-SNs typically extended new processes and often survived for 2-4 weeks. When co-cultured, PH-SNs and APRs exhibited robust growth and made close anatomical appositions. This culture system provides convenient experimental access to PH-SNs and may potentially permit sensorimotor synapses to be investigated in vitro.
Melville
J M
JM
Institute of Neuroscience, University of Oregon, Eugene, OR 97403-1254, USA.
Hoffman
K L
KL
Jarrard
H E
HE
Weeks
J C
JC
D000818:Animals; D002455:Cell Division; D048429:Cell Size; D002478:Cells, Cultured; D018920:Coculture Techniques; D005456:Fluorescent Dyes; D018402:Manduca; D008465:Mechanoreceptors / Q000166:cytology*; D009046:Motor Neurons / Q000166:cytology*; D009473:Neuronal Plasticity / Q000502:physiology; D009475:Neurons, Afferent / Q000166:cytology*; D013569:Synapses / Q000502:physiology
D005456:Fluorescent Dyes