Dynamic balance of metabotropic inputs causes dorsal horn neurons to switch functional states Dominique Derjean1, 3, Sandrine Bertrand1, 3, Gwendal Le Masson1, Marc Landry1, Valérie Morisset2 & Frédéric Nagy1 Nature Neuroscience 6, 274 - 281 (2003) Introduction • The dorsal horn of the spinal cord is the first central relay for inputs from primary nocieptor sensory fibers. Relay deep dorsal horn neurons (DHNs) integrate both innocuous and nociceptive inputs and are of fundamental importance for plasticity in information processing and transfer. • Individual neurons have characteristic and different intrinsic membrane properties that affect how they respond to same sensory drive. • “Intrinsic membrane properties” refer to a neuron’s electrical properties in isolation, in the absence of synaptic inputs. • In normal state, the deep DHNs show three kinds of intrinsic membrane properties: (a) Tonically firing neurons (b) Plateau neurons (c) Oscillatory neurons • Intrinsic membrane properties are not immutable. Rather, neuromodulatory substances released from neighboring neurons that act via second messenger systems often alter one or more of the voltage and time-dependant currents in a neuron and change a neuron’s intrinsic membrane properties for seconds, minutes or hours. • Derjean et al. in this issue show that the spinal cord neurons that receive information from the sensory neurons activated by painful stimuli are not simple faithful followers of the train of action potentials fired by the sensory neurons. Instead, the spinal cord neurons display complex membrane properties that transform the sensory signal. As a consequence, short-term modulation of these membrane properties could contribute to modifications of pain sensitivity. Result Three firing modes: a balance of 1. Activation of group-I metabotropic glutamate receptors (mGluRs) by the metabotropic controls agonists ACPD .(Fig1b) 2. applied the mGluR1 antagonist 4-CPG on those neurons with spontaneous plateau properties. (Fig1g) *This indicates that the active properties of the deep DHNs are under a sustained basal glutamatergic modulatory control. 3. activating GABAB receptors with the agonist baclofen .(Fig1C) 4. superfusion of the GABAB antagonist CGP55845 .(Fig1E) *This shows that the metabotropic GABAB system exerts a tonic inhibitory control over the active properties of deep DHNs. Evidently, it is the balance of permissive and suppressive modulatory systems that controls the active properties of deep DHNs. • To test whether these antagonistic modulatory pathways share a common cellular mediator, Authors targeted a family of channels linked to a variety of metabotropic receptors: the G-protein dependent potassium channel Kir3. Membrane currents modified by bath-application of GABAB receptors or mGluR agonists were analyzed using a voltage ramp and a subtraction procedure and on synaptically isolated DHNs. • Current–voltage curves were determined using voltage ramps from -55mV to -155 mV, Antagonistic regulation of a Kir3 current GABAB receptors agonists mGluR agonists Erev, -99.4 ± 2.2 mV Outward current Erev, -95.3 ± 2.2 mV K+ equilibrium potential (-95.8 mV) Inward current The chord conductance was The chord measured at two different conductance of the potentials that were equidistant DHPG-suppressed from Erev (Fig. 2b). currents was higher for hyperpolarized potentials (Fig. 2h) In all cells tested, the conductance was significantly higher for the most negative potential (Fig. 2d). the suppressive effect of DHPG was The effect of baclofen was abolished in a voltage-independent manner in the presence of Ba2+ (Fig. 2c and d) and blocked by the superfusion of Ba2+ at low in a voltage-dependent manner in the presence of Cs+ (Fig. 2d), which is characteristic of Kir currents. concentrations (Fig. 2g–h). • Together, these results indicate that activation of GABAB and mGlu receptors exert antagonistic regulation— enhancement and inhibition, respectively—on a Kir current in deep DHNs. The morphological evidence showing that a single DHN received both glutamatergic and GABAergic synaptic contacts and expressed Kir channel Triple immunostaining showed a dendrite of a plateau- generating DHN injected with biocytin (blue) receives both VgluT-a marker of GABAergic fibers - (green, open arrowhead) and GAD65-a marker of glutamatergic fiber - (red, filled arrowhead) containing fibers. Kir3.1 immunoreactivity reveals that Kir3 channels (green, arrows) are expressed in the soma of a biocytin- injected DHN (blue) • These observations suggest that the Kir current that is described in electrophysiological experiments and modulated by both mGlu and GABAB receptors is of the Kir3 family. • Finally, the author addressed the question of whether the three firing modes of deep DHNs correspond to different properties of sensory information transfer and thus may define different functional states. The author measured the input–output relationships between primary afferent spiking and the output firing of a single DHN with tonic, plateau or oscillatory firing patterns. To precisely control the afferent spiking and avoid complex polysynaptic effects associated with dorsal root stimulation, the author designed a simple canonical circuit using the hybrid network method. That is, a computer model of primary nociceptor discharge was connected to an intracellularly recorded DHN through an artificial excitatory synapse. State-dependent capabilities of information transfer The modeled nociceptive fiber discharge produced slowly adapting responses to depolarizing stimuli (frequency range, 4–32 Hz). The corresponding cross-correlogram (Fig. 4d) Every spike triggered an artificial synaptic current yielded a broad peak of small amplitude, injected through the recording pipette (Fig. 4a), indicating that the recorded DHN was not mimicking realistic AMPA excitatory postsynaptic responding in a one-to-one manner to every potentials (EPSPs) in the DHN (Fig. 4b). presynaptic spike. Conversely, a plateau-generating neuron The spike transfer was quantified using cross- reacted to the same input with a higher correlation analysis between the afferent frequency, accelerating spike train (Fig. 4e), discharge and the DHN response. and had a more precise correlation as shown by the higher and narrower peak in the cross correlogram (Fig. 4f). The averaged CC (n = 6) was significantly a mean input firing higher (0.26) for plateau-generating neurons (Fig. 4i) compared to tonic neurons (0.08). The frequency of 24 Hz lower CI for plateau neurons (0.1 versus 0.23 in tonic mode) These results suggest that expression of active plateau properties in dorsal horn relay neurons significantly increased the transmission of afferent single spikes. The responses obtained during spontaneous oscillations and rhythmic bursting of DHNs (Fig. 4g) were characterized by both a low CC (Fig. 4h and i, 0.11) and a low CI (Fig. 4j, 0.07), indicating that the oscillatory firing mode was filtering out most of the afferent activity. Analysis of the delay between the occurrence of an input spike and the generation of a correlated output spike (Fig. 4k) revealed a shorter latency for the plateau firing mode compared to the tonic mode. The mean delay in the oscillatory mode was not significantly different from that in the tonic mode, but did show a higher variability (larger s.e.m.). Chord conductance? • For type of channel at a single time, we can define the conductance through the channel as the inverse of the resistance - i.e. g=I/V. However, at a microscopic level channel behave in a stochastic and binary way, and so we cannot predict the behaviour of individual channels in this way. The chord conductance is a measure of the permeability of all of one type of channel for a particular cell, allowing us to predict the actual amount of current that will flow across the whole membrane. • Chord conductance lets us describe the macroscopic behaviour of the channels, giving an indication of the permeability of a class of channels for a given voltage. • Potassium is outward, sodium is inward and Cl- is inward. • Then given that : • gK+ = 0.78 ìsiemens; gNa+ = 0.06 ìsiemens; gCl- = 0.25 ìsiemens • use the chord conductance equation, but need to determine equilibrium potentials for each ion using the Nernst Equation • Ena = ~ +82.5 mV; Ek = ~ -70mV; Ecl = ~-48 mV • Calculate the membrane potential, and Em = ~ -61mV • B. Determine the necessary change in gNa+ if the recorded membrane potential is +34 mV. gNa = ~ 2.0 usiemens • (Assuming all other quantities remain constant.) • C. What is the physiological importance for a membrane that can shift it conductance between the states given in A and B? cross-correlation analysis • Two indexes were computed: (i) the correlation coefficient (CC), which indicates the ratio of input spikes that are transmitted as output spikes in the DHN neuron and thus characterizes the global efficacy of input–output spike transfer . (ii) the contribution index (CI), which quantifies the percentage of output spikes that were precisely correlated with afferent input spikes. It estimates the probability that a DHN spike was triggered by an input spike rather than generated spontaneously Input spike CC : 3/10 = 0.3 Output spikes CI : 3/5 = 0.6 Output spikes were precisely correlated with afferent input spikes.
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