Transmission efficacy and plasticity in glutamatergic synapses formed by excitatory interneurons of the substantia gelatinosa in the rat spinal cord.

BACKGROUND: Substantia gelatinosa (SG, lamina II) is a spinal cord region where most unmyelinated primary afferents terminate and the central nociceptive processing begins. The glutamatergic excitatory interneurons (EINs) form the majority of the SG neuron population, but little is known about the mechanisms of signal processing in their synapses. METHODOLOGY: To describe the functional organization and properties of excitatory synapses formed by SG EINs, we did non-invasive recordings from 183 pairs of monosynaptically connected neurons. An intact presynaptic SG EIN was specifically stimulated through the cell-attached pipette while the evoked EPSCs/EPSPs were recorded through perforated-patch from a postsynaptic neuron (laminae I-III). PRINCIPAL FINDINGS: We found that the axon of an SG EIN forms multiple functional synapses on the dendrites of a postsynaptic neuron. In many cases, EPSPs evoked by stimulating an SG EIN were sufficient to elicit spikes in a postsynaptic neuron. EPSCs were carried through both Ca(2+)-permeable (CP) and Ca(2+)-impermeable (CI) AMPA receptors (AMPARs) and showed diverse forms of functional plasticity. The synaptic efficacy could be enhanced through both activation of silent synapses and strengthening of already active synapses. We have also found that a high input resistance (R(IN), >0.5 GOmega) of the postsynaptic neuron is necessary for resolving distal dendritic EPSCs/EPSPs and correct estimation of their efficacy. CONCLUSIONS/SIGNIFICANCE: We conclude that the multiple synapses formed by an SG EIN on a postsynaptic neuron increase synaptic excitation and provide basis for diverse forms of plasticity. This functional organization can be important for sensory, i.e. nociceptive, processing in the spinal cord.

Excitatory interneurons dominate sensory processing in the spinal substantia gelatinosa of rat.

Substantia gelatinosa (SG, lamina II) is a spinal cord region where most unmyelinated primary afferents terminate and the central nociceptive processing begins. It is formed by several distinct groups of interneurons whose functional properties and synaptic connections are poorly understood, in part, because recordings from synaptically coupled pairs of SG neurons are quite challenging due to a very low probability of finding connected cells. Here, we describe an efficient method for identifying synaptically coupled interneurons in rat spinal cord slices and characterizing their excitatory or inhibitory function. Using tight-seal whole-cell recordings and a cell-attached stimulation technique, we routinely tested about 1500 SG interneurons, classifying 102 of them as monosynaptically connected to neurons in lamina I-III. Surprisingly, the vast majority of SG interneurons (n = 87) were excitatory and glutamatergic, while only 15 neurons were inhibitory. According to their intrinsic firing properties, these 102 SG neurons were also classified as tonic (n = 49), adapting (n = 17) or delayed-firing neurons (n = 36). All but two tonic neurons and all adapting neurons were excitatory interneurons. Of 36 delayed-firing neurons, 23 were excitatory and 13 were inhibitory. We conclude that sensory integration in the intrinsic SG neuronal network is dominated by excitatory interneurons. Such organization of neuronal circuitries in the spinal SG can be important for nociceptive encoding.

Selective postsynaptic inhibition of tonic-firing neurons in substantia gelatinosa by mu-opioid agonist.

BACKGROUND: Spinal substantia gelatinosa (SG) is a site of action of administered and endogenous opioid agonists and is an important element in the system of antinociception. However, little is known about the types of neurons serving as specific postsynaptic targets for opioid action within the SG. To study the spinal mechanisms of opioidergic analgesia, the authors compared the action of mu-opioid agonist [D-Ala, N-Me-Phe, Gly-ol]-enkephalin (DAMGO) on SG neurons with different intrinsic firing properties. METHODS: Whole cell patch clamp recordings from spinal cord slices of Wistar rats were used to study the sensitivity of SG neurons to DAMGO. RESULTS: Three groups of neurons with distinct distributions in SG were classified: tonic-, adapting-, and delayed-firing neurons. DAMGO at 1 microm concentration selectively hyperpolarized all tonic-firing neurons tested, whereas none of the adapting- or delayed-firing neurons were affected. The effect of DAMGO on tonic-firing neurons was due to activation of G protein-coupled inward-rectifier K conductance, which could be blocked by 500 microm Ba and 500 microm Cs but increased by 50 microm baclofen. As a functional consequence of DAMGO action, a majority of tonic-firing neurons changed their pattern of intrinsic firing from tonic to adapting. CONCLUSIONS: It is suggested that tonic-firing neurons, presumably functioning as excitatory interneurons, are primary postsynaptic targets for administered and endogenous opioid agonists in spinal SG. Functional transition of cells in this group from tonic to adapting firing mode may represent an important mechanism facilitating opioidergic analgesia.

Mechanism of spike frequency adaptation in substantia gelatinosa neurones of rat.

Using tight-seal recordings from rat spinal cord slices, intracellular labelling and computer simulation, we analysed the mechanisms of spike frequency adaptation in substantia gelatinosa (SG) neurones. Adapting-firing neurones (AFNs) generated short bursts of spikes during sustained depolarization and were mostly found in lateral SG. The firing pattern and the shape of single spikes did not change after substitution of Ca2+ with Co2+, Mg2+ or Cd2+ indicating that Ca2+-dependent conductances do not contribute to adapting firing. Transient KA current was small and completely inactivated at resting potential suggesting that adapting firing was mainly generated by voltage-gated Na+ and delayed-rectifier K+ (KDR) currents. Although these currents were similar to those previously described in tonic-firing neurones (TFNs), we found that Na+ and KDR currents were smaller in AFNs. Discharge pattern in TFNs could be reversibly converted into that typical of AFNs in the presence of tetrodotoxin but not tetraethylammonium, suggesting that lower Na+ conductance is more critical for the appearance of firing adaptation. Intracellularly labelled AFNs showed specific morphological features and preserved long extensively branching axons, indicating that smaller Na+ conductance could not result from the axon cut. Computer simulation has further revealed that down-regulation of Na+ conductance represents an effective mechanism for the induction of firing adaptation. It is suggested that the cell-specific regulation of Na+ channel expression can be an important factor underlying the diversity of firing patterns in SG neurones.

Ionic basis of tonic firing in spinal substantia gelatinosa neurons of rat.

Ionic conductances underlying excitability in tonically firing neurons (TFNs) from substantia gelatinosa (SG) were studied by the patch-clamp method in rat spinal cord slices. Ca(2+)-dependent K(+) (K(CA)) conductance sensitive to apamin was found to prolong the interspike intervals and stabilize firing evoked by a sustained membrane depolarization. Suppression of Ca(2+) and K(CA) currents, however, did not abolish the basic pattern of tonic firing, indicating that it was generated by voltage-gated Na(+) and K(+) currents. Na(+) and K(+) channels were further analyzed in somatic nucleated patches. Na(+) channels exhibited fast activation and inactivation kinetics and followed two-exponential time course of recovery from inactivation. The major K(+) current was carried through tetraethylammonium (TEA)-sensitive rapidly activating delayed-rectifier (K(DR)) channels with a slow inactivation. The TEA-insensitive transient A-type K(+) (K(A)) current was very small in patches and was strongly inactivated at resting potential. Block of K(DR) rather than K(A) conductance by 1 mM TEA lowered the frequency and stability of firing. Intracellular staining with biocytin revealed at least three morphological groups of TFNs. Finally, on the basis of present data, we created a model of TFN and showed that Na(+) and K(DR) currents are sufficient to generate a basic pattern of tonic firing. It is concluded that the balanced contribution of all ionic conductances described here is important for generation and modulation of tonic firing in SG neurons.