Conference Agenda

I will discuss how GABAA/glycine-mediated inhibition shapes many aspects of information processing, from segregation of inputs to short and long-term plasticity. Yet, the strength of this form of inhibition depends on intracellular Cl- concentration, which is mainly regulated by the K-Cl-cotransporter KCC2 in the adult central nervous system. We have found a heterogeneity in Cl- homeostasis among neurons, which represents a substrate of metaplasticity as well as determining the level of crosstalk between sensory modalities. I will show that, in turn, the regulator is itself regulated: heterogeneous Cl- homeostasis result from modulation of KCC2 expression and function via both inter- and intra-cellular signaling mechanisms, yielding multiple forms and spatiotemporal scales of plasticity and helps explain several unintuitive aspects of the complex experience of normal and abnormal (pathological) pain, with applications to other brain disorders, including drug dependence and neurodegenerative diseases.

The neurotrophin BDNF, not only plays a critical role in the maturation of the nervous system, but also acts as a neuromodulator. In spinal nociceptive pathways, BDNF and its preferred receptor TrkB have been shown to underlie several forms of maladaptive plasticity leading to pathological pain. One key mechanism through which BDNF/TrkB signaling alters nociceptive transmission is by affecting spinal KCC2, the main regulator of Cl- homeostasis in the CNS, thus leading to disinhibition and spinal hyperexcitability. However, little is known so far whether BDNF/TrkB also modulates spinal inhibition under normal conditions and how this shapes sensory transmission

In this lecture, I’ll show recent data showing that the distribution and function of TrkB receptors is uneven in the spinal dorsal horn and differentially affects KCC2 functions. Indeed, TrkB is more active in the spinal dorsal horn laminae (lamina I and II outer) that mainly process thermal input, leading to lower KCC2 and weaker inhibition in these areas. On the other hand, TrkB signaling is weaker and inhibition stronger in lamina II inner, where mechanical input is mainly processed.

Central circuits with weaker inhibition are likely to undergo to unconstrained forms of plasticity which favor sensitization. By using optogenetics in combination with molecular targets of either thermal- and mechanical-transmitting afferent fibers, the thermal circuits in the dorsal horn were shown to undergo sensitization in response to repetitive stimulation more readily than mechanical circuits.

Interestingly, mechanical allodynia is a hallmark of different forms of neuropathic pain, suggesting that mechanical circuits, that normally display high inhibition and little tendency to sensitization, may switch to a highly disinhibited and plastic configuration.

Thus, the way BDNF/TrKB/KCC2 shapes sensory modalities in normal animals may inform the way the system is altered in pathological conditions.

Serotonergic modulation of pain transmission in the spinal cord is mediated by several types of receptors (5-HT1-7). 5-HT7 receptors are the most recently identified and have been involved in nociceptive modulation at several levels along the pain axis. By activating various intracellular pathways (including the cAMP signaling), these receptors induce neuronal depolarization, regulate neurotransmitter release, and modulate synaptic plasticity.

In the nociceptive system, 5-HT7 receptors are expressed both peripherally (on the primary nociceptors) and centrally, in the spinal cord and supraspinal centers. Behavioral studies indicate that these receptors play a prevalent pronociceptive role when activated peripherally, while they seem to exert an antinociceptive action in the central nervous system. Although the role of 5-HT7 receptors in pain modulation has been extensively investigated, little is known so far about the neural circuits and the cellular mechanisms involved.

In this presentation, I will show data obtained in mouse spinal cord dorsal horn about the effects of 5-HT7 receptors on synaptic transmission and neuronal excitability. Following the application of the selective 5-HT7 agonist LP-211, an enhancement of both glutamatergic and GABA/glycinergic transmission was observed, by recording synaptic responses spontaneously generated or evoked by dorsal root stimulation. The impact of 5-HT7 receptors was stronger on inhibitory transmission. Accordingly, in the presence of LP-211, synaptic responses evoked by repetitive stimulation of the dorsal root were more likely to evoke action potentials in tonic firing neurons, mainly corresponding to inhibitory interneurons. Recent data, obtained through the optogenetic stimulation of GABAergic interneurons, confirm a potentiating effect of 5-HT7 receptors on the inhibitory transmission in the spinal cord dorsal horn.

These results suggest that 5-HT7 receptors exert a differential modulation of excitatory and inhibitory circuits in the spinal cord dorsal horn and are consistent with a prevalent central antinociceptive effect.

Perception of pain derives from the integration and elaboration of sensory stimuli conveyed to different areas of the central nervous system (CNS) through nociceptive pathways. At the spinal cord level, the superficial dorsal horn (SDH) is the nociceptive-specific termination site of primary afferent fibers, where modality-specific circuits are activated for the elaboration of pain. A complex network of excitatory and inhibitory interneurons exists, and their balance shapes the way nociceptive signals reach the brain. At the cortical level, multiple areas participate in pain processing.

To assess the hypothesis that inflammatory pain activates specific inhibitory circuits in the CNS, adult (>45d) male and female CD1 mice were used to generate a model of inflammation induced by hindpaw intraplantar injection of zymosan. Behavioral tests to verify the presence of mechanical allodynia were performed 4 hours after injection. Through immunofluorescence, activation of inhibitory neurons was detected by the expression of a common marker of neuronal activation (Fos) with phenotypic markers of inhibitory neurons (Pax2 in SDH, calbindin/parvalbumin/calretinin in the primary somatosensory cortex). The colocalization between markers was ascertained by confocal microscopy.

The von Frey test showed mechanical allodynia both in male and female. Male mice showed a significant ipsilateral increase in Fos expression at SDH and contralateral at the cortical level. However, a significant increase in the activation of inhibitory neurons was only observed in SDH nociceptive-specific areas, while no changes were detected in the somatosensory cortex. Preliminary data on female mice showed related results in the SDH, while a different activation of inhibitory neurons occurred in the somatosensory cortex.

These results confirm the pivotal role of inhibition in the SDH in constraining the spread of nociceptive information in inflammatory pain, with differences related to sex. Thus, local inhibitory circuits in this area could represent a potential target for controlling chronic pain.