Selection of postsynaptic serotonin receptors during reinnervation of an identified leech neuron in culture

Abstract

Serotoninergic Retzius neurons reform an inhibitory synapse onto pressure-sensitive mechanosensory (P) neurons when the cells are removed from the nervous system of the leech and are juxtaposed in tissue culture. The somas of P cells in situ and single (uninnervated) P cells in culture have both a depolarizing and Cl-dependent hyperpolarizing response to application of the transmitter serotonin (5-HT). Synaptic release of 5-HT by a Retzius cell in situ and in culture evokes a Cl-dependent postsynaptic response but does not appear to activate the depolarizing receptors. We have characterized the ionic currents induced by synaptically released and applied 5-HT in voltage-clamped P cells in culture in order to determine the responses to transmitter and their modifications following innervation. When 5-HT was applied to single P cells, 2 types of channels were activated by 5-HT and could be distinguished by changing the ionic composition of the superfusion solution. In an impermeant cation (TrisCl) solution, a 5-HT-dependent Cl current was activated. When single P cells were superfused with a Cl-free solution (Cl replaced by impermeant SO4), 5-HT activated a monovalent cation current. Following innervation of a P cell by a cocultured Retzius cell, the reversal potential of the peak postsynaptic current depended on the Cl gradient and the synaptic response was blocked by the Cl channel blocker 9-anthracenecarboxylic acid. Thus, synaptic release of 5-HT activated solely the Cl channels and not the cationic channels. Pipette application of 5-HT onto innervated P cells activated a Cl conductance comparable in magnitude to the synaptic response. In contrast, the cationic conductance was reduced roughly 5-fold. It is concluded that innervation of a P cell by a Retzius cell resulted in clustering of the synaptic 5-HT receptors, which activate Cl channels and reduction of the nonsynaptic, cationic response. The result is a selection of receptors in the cultured P cell that mimics the pattern observed in vivo.

Non-Associative Learning and Serotonin Induce Similar Bi-Directional Changes in Excitability of a Neuron Critical for Learning in the Medicinal Leech

“…That 5HT both excited and inhibited the S-cell indicates that at least two different 5HT receptors may be present, as in single hippocampal and prefrontal cortex neurons (Andrade and Nicoll, 1987;Colino and Halliwell, 1987;Araneda and Andrade, 1991), spinal motor neurons (White, 1985;Skydsgaard and Hounsgaard, 1996), and leech mechanosensory P cells (Henderson, 1983;Drapeau and Sanchez-Armass, 1988), for example. In the leech P-cells, application of low levels of 5HT onto the soma produces hyperpolarization of the membrane potential that may be mediated by a 5HT 3 receptor (Ali et al, 1998), whereas more 5HT induces depolarization resulting from the activation of a 5HT 2 -like receptor (Catarsi and Drapeau, 1997).…”

Inhibition of Transmitter Release Correlates with the Proteolytic Activity of Tetanus Toxin and Botulinus Toxin A in Individual Cultured Synapses ofHirudo medicinalis

“…8 B,D, top panels). In postsynaptic P-cells, activation of the 5-HT-induced chloride conductance (Drapeau and Sanchez-Armass, 1988) was recorded as a depolarization of the membrane potential, because the postsynaptic microelectrode contained a high Cl Ϫ concentration solution (Fig. 8 B,D, bottom panels).…”

Tyrosine phosphorylation during synapse formation between identified leech neurons.

“…In some experiments in which cell pairs were later separated (by repeated passage through the aspiration pipette), we used R cells mildly fixed for 30 min in 0-02 % (w/v) paraformaldehyde and 0-03 % (w/v) glutaraldehyde (Sigma) in phosphate-buffered saline (PBS); the fixed R cells were transferred at 10 min intervals into three serial droplets of L-15 medium in order to remove the aldehydes before being paired with untreated P cells (Drapeau et al 1989). (Drapeau, 1990 Instruments, Burlingame, CA, USA), as described previously (Drapeau & Sanchez-Armass, 1988). Cell-attached patches were formed on P cells, as described previously (Drapeau, 1990), using 5-10 M1Q electrodes that were coated to within 50 ,sm of the tip with dental wax to reduce the capacitance.…”

“…At different times following the juxtaposition of R and P cell bodies, the P cells were recorded intracellularly in a Cl--free solution (see Methods) while applying 100 /M 5-HT from a pressure pipette. At a holding potential of -50 mV (near the normal resting potential), the only detectable response to 5-HT is due to the activation of a monovalent cation conductance (Drapeau & Sanchez-Armass, 1988). Both single P cells plated for 3 days ( Fig.…”

“…Although the Clcurrent is activated by 5-HT application even after application of tyrosine kinase inhibitors, the lack of Cl--dependent synaptic responses under this condition suggests that the assembly of a functional (inhibitory) synapse is similarly dependent on tyrosine kinase activity. The contact-dependent selection of 5-HT responses would limit the inhibitory, synaptic actions of 5-HT to sites of contact in the neuropil; on cell bodies, chronic exposure to 5-HT desensitizes the Cl--dependent response and thus activates only the depolarizing response (Henderson et al 1983;Drapeau & Sanchez-Armass, 1988).…”