Differential effects of GABA in modulating nociceptive vs. non-nociceptive synapses

Abstract

GABA (γ-amino-butyric acid) -mediated signaling is normally associated with synaptic inhibition due to ionotropic GABA receptors that gate an inward Cl(-) current, hyperpolarizing the membrane potential. However, there are also situations where ionotropic GABA receptors trigger a Cl(-) efflux that results in depolarization. The well-characterized central nervous system of the medicinal leech was used to study the functional significance of opposing effects of GABA at the synaptic circuit level. Specifically, we focused on synapses made by the nociceptive N cell and the non-nociceptive P (pressure) cell that converge onto a common postsynaptic target. It is already known that GABA hyperpolarizes the P cell, but depolarizes the N cell and that inhibition of ionotropic GABA receptors by bicuculline (BIC) has opposing effects on the synapses made by these two inputs; enhancing P cell synaptic transmission, but depressing N cell synapses. The goal of the present study was to determine whether the opposing effects of GABA were due to differences in Cl(-) homeostasis between the two presynaptic neurons. VU 0240551 (VU), an inhibitor of the Cl(-) exporter K-Cl co-transporter isoform 2 (KCC2), attenuated GABA-mediated hyperpolarization of the non-nociceptive afferent while bumetanide (BUM), an inhibitor of the Cl(-) importer Na-K-Cl co-transporter isoform 1 (NKCC1), reduced GABA-mediated depolarization of the nociceptive neuron. VU treatment also enhanced P cell synaptic signaling, similar to the previously observed effects of BIC and consistent with the idea that GABA inhibits synaptic signaling at the presynaptic level. BUM treatment depressed N cell synapses, again similar to what is observed following BIC treatment and suggests that GABA has an excitatory effect on these synapses. The opposing effects of GABA could also be observed at the behavioral level with BIC and VU increasing responsiveness to non-nociceptive stimulation while BIC and BUM decreased responsiveness to nociceptive stimulation. These findings demonstrate that distinct synaptic inputs within a shared neural circuit can be differentially modulated by GABA in a functionally relevant manner.

Endocannabinoids Have Opposing Effects On Behavioral Responses To Nociceptive And Non-nociceptive Stimuli

“…Mechanical non-nociceptive stimuli were delivered via von Frey fibers that apply force sufficient to activate the non-nociceptive sensory neurons in Hirudo (0.008 g–2.0 g), but were below the mechanical threshold for activating mechano-nociceptive cells (7.0 g) 21 . Chemical nociceptive stimuli were delivered via the application of 250 μM AITC (800 μL) to the posterior sucker as carried out in previous experiments with Hirudo 25, 36 . The von Frey fibers elicit a localized shortening responses, whereas AITC elicits whole body shortening in which the animal picks up its sucker to withdraw it from the site of AITC application.…”

“…1A) 32, 33 . Nociceptive synapses are “protected” from this disinhibition because they are depolarized by GABA due to elevated levels of intracellular Cl − 33, 36 . At this time the GABAergic neurons in Hirudo that undergo this suggested endocannabinoid-mediated depression have not been identified.…”

“…The techniques for testing Hirudo responses to nociceptive and non-nociceptive stimuli are based on previously published protocols 25, 36 . Individual Hirudo were placed in a testing arena consisting of a plastic petri dish (145 mm diameter) lined with filter paper that had been saturated with pond water and maintained at room temperature.…”

“…For vehicle control experiments, 100 μL of 0.01% DMSO were injected. As previously reported 36 , injections were made just anterior of the posterior sucker, a region where the dorsal and ventral sinuses that are part of the leech vascular system converge 19 . The leech CNS is contained within the ventral sinus so this method of injection is likely to be effective in delivering drugs to the CNS.…”

“…Endocannabinoids potentiate the non-nociceptive synapse (P) via an indirect mechanism in which endocannabinoids depress inhibitory input from an unknown GABAergic interneuron (this is also TRPV-mediated). In N cells the Cl − gradient is dominated by the Cl − importer (NKCC1) while the P cell gradient is dominated by the Cl − exporter (KCC1) 36 . As a result of an elevated intracellular Cl − concentration, N cells are depolarized by GABA and consequently “protected” from disinhibition.…”