Understanding LTP in pain pathways

"Central sensitisation" is used in the literature in at least two mutually exclusive definitions. Some of the authors use "central sensitisation" as an umbrella term for all forms of changes within the central nervous system which ultimately lead to enhanced pain perception. If using this definition of "central sensitisation" one should keep in mind that none of the presently known phenomena in the central nervous system (CNS) which can be observed in experimental or clinical models of hyperalgesia or allodynia has a proven, causative role for the perception of pain. Thus, all presently proposed mechanisms of "central sensitisation" in this definition would have the status of hypotheses. This includes but is not be limited to the topic of the present review, the LTP at C-fibre synapses in the superficial spinal dorsal horn.

The task force for taxonomy of the International Association for the Study of Pain (IASP) and many other authors define "central sensitisation" as "an enhanced responsiveness of nociceptive neurons in the CNS to their normal afferent input". This is a very clear-cut definition with little error-proneness. The mechanisms underlying "central sensitisation" in this definition can be well studied experimentally. Nociceptive neurons in the CNS may, however, serve very distinct and also antagonistic functions not all of which are related to the perception of pain. Some nociceptive neurons are excitatory, others are inhibitory. Spinal nociceptive neurons may project to different areas in the brain, with a wide spectrum of functions. Other nociceptive spinal neurons may project segmentally to motoneurons and still other may be interneurons with no known function at all. A remarkable exception are those nociceptive neurons which are located in lamina I of the spinal dorsal horn and which express the NK1 receptor for substance P, many of which project supraspinally. If these neurons are destroyed selectively by a toxin conjugated to substance P, then neither inflammation nor neuropathy leads to full expression of hyperalgesia [5, 6]. Interestingly, animal responses to acute noxious stimuli are not altered [5, 6]. Thus, these neurons are indispensable for the development of hyperalgesia but not essential for acute pain. Enhanced responsiveness of different nociceptive neurons in the CNS may thus have distinct and perhaps opposing consequences on pain. Thus, "central sensitisation" defined in this way is fundamentally different from the former definition. Some forms of "central sensitisation" as defined by the IASP task force could contribute to hyperalgesia and/or allodynia while other forms may rather lead to the contrary, i.e. stronger feedback inhibition and endogenous pain control. Many of the presently known forms of "central sensitisation" defined in the latter way would be assigned a yet unknown (if any) role for pain perception.

An utter confusion is created if the term "central sensitisation" is used but not defined or when switching from one definition to the other within the same publication, i.e. if both definitions are not clearly distinguished. This would incorrectly imply that any form of enhanced responsiveness of nociceptive neurons in the CNS to their normal afferent input would lead to an enhanced perception of pain. Before we evaluate the evidence suggesting that a prolonged increase in synaptic strength at C-fibres contributes to hyperalgesia we will first review the fundamental properties of LTP in pain pathways.

LTP is measured as an increase in mono-synaptically-evoked post-synaptic currents or potentials in response to a single pre-synaptic action potential. LTP is often studied in in vitro preparations which allow reliable recordings of synaptic strength. Whole-cell patch-clamp recording is now the most often used technique. It enables some control over the composition of the intracellular fluid of the post-synaptic neurons which may be advantageous to study post-synaptic mechanisms of LTP. If, however a diffusible mediator is involved and dialysis of the post-synaptic neuron has to be avoided, perforated patch-clamp recordings or intracellular recordings with sharp electrodes can be used. To evaluate LTP at the first synapses in nociceptive pathways, transverse slices with long dorsal roots attached can be prepared from lumbar spinal cord of rats or mice to study mono-synaptic, Aδ-fibre or C-fibre evoked excitatory postsynaptic potentials or currents in identified dorsal horn neurons [7, 8].

Some aspects of LTP can only be studied in the entire animal with primary afferent nerve fibres and descending pathways from the brain intact. In vivo C-fibre-evoked field potentials can be measured in superficial spinal dorsal horn e.g. in response to high intensity electrical stimulation of the sciatic nerve for up to 24 h [9]. These extracellulary recorded field potentials reflect summation of post-synaptic, mainly mono-synaptically-evoked currents but not action potential firing [9, 10].

Monitoring presynaptic activity at synapses of primary afferent nerve fibres is technically quite demanding. In an attempt to monitor pre-synaptic activity in primary afferents optical recordings techniques have been utilised. Some voltage-sensitive dyes can be anterogradely transported in primary afferents to the central terminals mainly in lamina I [11] and may serve as an indicator for pre-synaptic electrical activity but not for transmitter release.

LTP can not be directly investigated by recording action potential discharges of post-synaptic neurons, as action potential firing not only depends upon synaptic strength but also on membrane excitability and the balance between excitatory and inhibitory input to the neuron. For the same reasons poly-synaptically-evoked responses can generally not be used to study synaptic strength and changes thereof.

A-fibre-evoked spinal field potentials are depressed by conditioning 50 Hz stimulation of sciatic nerve fibres. After GABA_A receptor antagonist bicuculline (1 mg/kg i.p.) the same conditioning stimulus now produces LTP rather than LTD [25]. Similarly, 50 Hz conditioning stimulation produces short lasting potentiation followed by LTD in control animals but LTP in animals with a CCI of sciatic nerve [26]. Topical application of muscimol (10 μg), a GABA_A receptor agonist to spinal cord prevents tetanus-induced LTP of A-fibre-evoked field potentials in animals with a CCI [27]. This again suggests that the polarity of synaptic plasticity is context-sensitive and not solely dominated by the type of afferent input.

In principle, LTP can be induced and/or expressed by pre-synaptic [28] or by post-synaptic [29, 30] mechanisms or by any combination thereof. At present, there is clear evidence for a post-synaptic, Ca²⁺-dependent form of LTP induction in spinal cord lamina I neurons. Induction of LTP at C-fibre-synapses requires co-activation of NK1 and NK2 receptors [9], opening of ionotropic glutamate receptors of the NMDA type [8, 12, 13], opening of T-type voltage-gated calcium channels [8, 12], and activation of group I but not group II or III metabotropic glutamate receptors [31]. Activation of NK1 receptors by substance P may directly enhance single NMDA channel opening [32] and NMDA receptor mediated currents in lamina I neurons [8] and all this may lead to substantial rise in post-synaptic [Ca²⁺]_i (Fig. 2). It is presently unknown if Ca²⁺ influx through Ca²⁺-permeable a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors is required for LTP induction in pain pathways. Some indirect evidence suggests, however, that this might be the case [21, 33].

In any case, a rise in post-synaptic [Ca²⁺]_i is essential for LTP induction and the magnitude in [Ca²⁺]_i rise is linearly correlated with the magnitude of LTP in vitro [8]. Recent data demonstrate that LTP-inducing stimuli cause substantial rise in [Ca²⁺]_i in lamina I neurons not only in slice preparations, but also in intact animals [12]. Not surprisingly therefore signal transduction involves Ca²⁺-dependent pathways including activation of protein kinase C, calcium-calmodulin-dependent protein kinase II (CaMKII), protein kinase A (PKA) phospholipase C (PLC), inositoltriphosphate-3 (IP₃) receptors, nitric oxide synthase (NOS) and members of the mitogen-activated protein kinase family (MAPK), including extracellular signal-regulated kinase (ERK) (Fig. 2) [8, 12, 18, 34‐36].

When assessed with voltage-sensitive dyes the pre-synaptic facilitation of electrical activity in primary afferents after LTP-inducing stimuli is partially sensitive to iNOS inhibitor (AMT), a blocker of glial cell metabolisms (MFA), and an mGluR group I antagonist (LY367385) [11].

Inhibition of protein synthesis in spinal cord by either cycloheximide or anisomycin selectively inhibits the maintenance of the late-phase of spinal LTP but does not affect either LTP induction or baseline responses of C-fibre evoked field potentials [37].

Importantly, the very same signal transduction pathways are required for full expression of hyperalgesia in animal models of inflammatory and neuropathic pain e.g. [38‐41].

LTP induction can be prevented by blockade of any of the above mentioned essential elements of signal transduction for LTP. In mature rats deep (surgical) level of anaesthesia with either urethane, isoflurane or sevoflurane is, however, insufficient to pre-empt LTP induction of C-fibre-evoked field potentials [42]. LTP is prevented by low dose intravenous infusion of μ-opioid receptor agonist fentanyl [42]. Similarly, LTP of spinal field potentials elicited by stimulation in the tract of Lissauer in spinal cord slices is blocked by DAMGO, a more specific agonist at these receptors [19]. Activation of spinal a₂-adrenoreceptors by clonidine [43] or spinal application of the benzodiazepine diazepam [44] also prevents LTP induction in vivo.

Functional blockade of glial cells by i.t. administration of fluorocitrate changes the polarity of HFS induced synaptic plasticity. When HFS is given 1 h, but not 3 h after fluorocitrate LTD but no LTP of C-fibre-evoked field potentials is induced [14].

LTP of C-fibre-evoked field potentials can be reversed by brief, high frequency conditioning electrical stimulation of sciatic nerve fibres at Aδ-fibre intensity [15]. Reversal of LTP by Aδ-fibre stimulation is time-dependent and effective only when applied 15 or 60 min but not 3 h after LTP induction [45].

Spinal application of either, NK1 or NK2 receptor antagonists one to three hours after HFS, i.e., after LTP is established, does not affect maintenance of LTP [9], suggesting that activation of these receptors, which are required for the induction of LTP are not essential for its maintenance.

Modulation of synaptic strength is a powerful mechanism to control signal flow in selected pathways. A typical consequence of LTP at excitatory synapses would be an increase in action potential firing of the same and perhaps also of downstream neurons in response to a given stimulus. And indeed, LTP-inducing conditioning stimuli have been found to facilitate action potential firing of multireceptive neurons in deep dorsal horn e.g. [46‐48]. This is likely due to LTP at the first synapse in the nociceptive pathway but other mechanisms of facilitation should not be excluded. Action potential firing would also be enhanced if membrane excitability is increased, i.e. the thresholds for action potential firing are lowered, if inhibition is less effective or if inhibition is even reversed and becomes excitatory e.g. due to a reversal of the anion gradient in the post-synaptic neuron [49, 50].

HFS of sciatic nerve fibres which induces LTP at synapses of C-fibres in spinal cord has behavioural consequences in rats and causes thermal hyperalgesia at the ipsilateral hind paw for six days [34]. This suggests that LTP at C-fibre synapses has an impact on nociceptive behaviour.

An indispensable proof for any proposed mechanism of hyperalgesia is an appropriate correlate in the human. And indeed, conditioning HFS of cutaneous peptidergic afferents in humans cause increased pain perception in response to electrical test stimuli applied through the same stimulation electrode [51]. Noxious stimulation with punctate mechanical probes in skin adjacent to the HFS conditioning skin site uncovers a marked (2–3 fold) increase in pain sensitivity, i.e. secondary hyperalgesia [51]. Touching the skin around the conditioning stimulation electrode with a soft cotton wisp evokes pain only after HFS. Thus, HFS also induces secondary mechanical allodynia. Hyperalgesia at the conditioned site but not secondary hyperalgesia or allodynia at adjacent skin areas is prevented by pre-treatment with ketamine [52], a clinically used substance which, among other effects, also blocks NMDA receptors.

Interestingly, all thermal modalities comprising cold and warm detection thresholds, cold and heat pain thresholds as well as pain summation (perceptual "wind-up") remain unaltered after conditioning HFS of peptidergic skin nerve fibres [53].

When verbal pain descriptors are used to evaluate pain in addition to its perceived intensity after HFS, a significant long-term increase in scores for sensory but not for affective descriptors of pain is detected [54]. Within the sensory descriptors, those describing superficial pain, those for heat pain and those for sharp mechanical pain are all potentiated. The authors conclude that brief painful stimuli rarely have a strong affective component and that perceived pain after HFS exhibits predominantly a potentiation of the C-fibre-mediated percepts hot and burning [54].

In humans subjects conditioning LFS causes also an increased pain sensitivity in the area around the LFS conditioned skin site but a depression of pain evoked by stimulation through the same electrode [51].