Ruth Ruscheweyh

Lamina-specific membrane and discharge properties of rat spinal dorsal horn neurones in vitro.

Membrane and discharge properties determine the input‐output relationship of neurones and are therefore of paramount importance for the functions of neural circuits. Here, we have tested the hypothesis that neurones in different laminae of the spinal dorsal horn differ in their electrophysiological properties. Whole‐cell patch‐clamp recordings from dorsal horn neurones in a rat transverse spinal cord slice preparation were used to record active and passive membrane properties. Neurones from superficial dorsal horn laminae had higher membrane resistances and broader action potentials than deep dorsal horn neurones. Action potential thresholds were highest in lamina II neurones, representing low membrane excitability. Five types of firing patterns were identified in response to depolarising current injections. Tonic‐firing neurones discharged action potentials at regular intervals throughout the current pulse. Delayed‐firing neurones showed a delayed onset of firing in response to current injections that was due to activation of a transient voltage‐dependent outward current, presumably an A‐current. Another group of neurones fired a short initial burst of action potentials. Single‐spiking neurones discharged only one action potential at the onset of a depolarising pulse. Phasic‐bursting neurones showed irregular bursts of action potentials. Firing patterns were unequally distributed among laminae. Tonic‐firing neurones were numerous in lamina I and deeper laminae but were not found in lamina II. Delayed‐firing neurones were encountered in laminae I and II but not in deeper laminae. Most of the neurones showing an initial burst were found in lamina II. These differences in membrane and discharge properties probably contribute to lamina‐specific processing of sensory, including nociceptive, information.

Modification of classical neurochemical markers in identified primary afferent neurons with Aβ-, Aδ-, and C-fibers after chronic constriction injury in mice

It is functionally important to differentiate between primary afferent neurons with A‐fibers, which are nociceptive or nonnociceptive, and C‐fibers, which are mainly nociceptive. Neurochemical markers such as neurofilament 200 (NF200), substance P (SP), and isolectin B4 (IB4) have been useful to distinguish between A‐ and C‐fiber neurons. However, the expression patterns of these markers change after peripheral nerve injury, so that it is not clear whether they still distinguish between fiber types in models of neuropathic pain. We identified neurons with Aβ‐, Aδ‐, and C‐fibers by their conduction velocity (corrected for utilization time) in dorsal root ganglia taken from mice after a chronic constriction injury (CCI) of the sciatic nerve and control mice, and later stained them for IB4, SP, calcitonin gene‐related peptide (CGRP), NF200, and neuropeptide Y (NPY). NF200 remained a good marker for A‐fiber neurons, and IB4 and SP remained good markers for C‐fiber neurons after CCI. NPY was absent in controls but was expressed in A‐fiber neurons after CCI. After CCI, a group of C‐fiber neurons emerged that expressed none of the tested markers. The size distribution of the markers was investigated in larger samples of unidentified dorsal root ganglion neurons and, together with the results from the identified neurons, provided only limited evidence for the expression of SP in Aβ‐fiber neurons after CCI. The extent of up‐regulation of NPY showed a strong inverse correlation with the degree of heat hyperalgesia. J. Comp. Neurol. 502:325–336, 2007.

Distinctive membrane and discharge properties of rat spinal lamina I projection neurones in vitro

Most lamina I neurones with a projection to the brainstem express the neurokinin 1 receptor and thus belong to a small subgroup of lamina I neurones that are necessary for the development of hyperalgesia in rat models of persisting pain. These neurones are prone to synaptic plasticity following primary afferent stimulation in the noxious range while other nociceptive lamina I neurones are not. Here, we used whole‐cell patch‐clamp recordings from lamina I neurones in young rat spinal cord transverse slices to test if projection neurones possess membrane properties that set them apart from other lamina I neurones. Neurones with a projection to the parabrachial area or the periaqueductal grey (PAG) were identified by retrograde labelling with the fluorescent tracer DiI. The properties of lamina I projection neurones were found to be fundamentally different from those of unidentified, presumably propriospinal lamina I neurones. Two firing patterns, the gap and the bursting firing pattern, occurred almost exclusively in projection neurones. Most spino‐parabrachial neurones showed the gap firing pattern while the bursting firing pattern was characteristic of spino‐PAG neurones. The underlying membrane currents had the properties of an A‐type K+ current and a Ca2+ curent with a low activation threshold, respectively. Projection neurones, especially those of the burst firing type, were more easily excitable than unidentified neurones and received a larger proportion of monosynaptic input from primary afferent C‐fibres. Intracellular labelling with Lucifer yellow showed that projection neurones had larger somata than unidentified neurones and many had a considerable extension in the mediolateral plane.

Synaptic mechanisms of hyperalgesia

Publisher Summary Hyperalgesia and allodynia often aggravate pain for variable periods after trauma, surgery, and inflammation. Pain that is induced by normally nonpainful stimuli (allodynia) or abnormally intense pain elicited by noxious stimuli (hyperalgesia) may be the consequence of an increased sensitivity of nociceptors (peripheral sensitization) or may be due to an increased responsiveness of neurons in the central nervous system (central sensitization). The sensitization of nociceptors is typically limited in time to the period of primary injury. This chapter discusses the neurobiological mechanisms underlying persistent pain and hyperalgesia. The peripheral mechanisms of hyperalgesia are discussed. Four principle mechanisms of afferent-induced central sensitization are synaptic mechanisms, membrane excitability, phenotypical changes, and morphological reorganization. The chapter focuses on synaptic mechanisms in superficial spinal dorsal horn that may contribute to some forms of hyperalgesia. Afferent-induced hyperalgesia may last for minutes to months and may include mechanisms such as longterm potentiation (LTP) of synaptic strength or impairment of pre- or postsynaptic inhibition at the first central synapse. It is likely that similar synaptic changes also occur at later stages of nociception. To assess changes in synaptic strength it is essential to record monosynaptically evoked postsynaptic currents or potentials, either with intracellular single cell recordings or as extracellular field potentials.

Role of kainate receptors in nociception.

Nociceptive nerve fibers use L-glutamate as a fast excitatory neurotransmitter and it is therefore not surprising that both, ionotropic and metabotropic glutamate receptors play pivotal roles for transmission of nociceptive information in spinal cord. A subtype of ionotropic glutamate receptors, the kainate receptor, is present in spinal dorsal horn. However, its role has remained obscure as specific antagonists and agonists have become available only recently. Kainate receptors are present on small, including nociceptive, dorsal root ganglion cells and on intrinsic dorsal horn neurons, and those two locations can be targeted separately by appropriate agonists and antagonists. Postsynaptic kainate receptors on spinal dorsal horn neurons are activated by high intensity electrical stimulation of the dorsal root entry zone that activates nociceptive primary afferent fibers. In contrast, low intensity stimulation that activates only non-nociceptive fibers is ineffective. Selective blockade of kainate receptors may produce analgesia. Here, we review what is known about localization of kainate receptors in dorsal root ganglia and spinal dorsal horn and their physiological and pathophysiological importance with special reference to nociceptive pathways. A short overview on molecular biology and agonist and antagonist pharmacology is included.

Synaptic input of rat spinal lamina I projection and unidentified neurones in vitro

Spinal lamina I projection neurones that transmit nociceptive information to the brain play a pivotal role in hyperalgesia in various animal models of inflammatory and neuropathic pain. Consistently, activity‐dependent long‐term potentiation can be induced at synapses between primary afferent C‐fibres and lamina I projection neurones but not unidentified neurones in lamina I. The specific properties that enable projection neurones to undergo long‐term potentiation and mediate hyperalgesia are not fully understood. Here, we have tested whether lamina I projection neurones differ from unidentified neurones in types or strength of primary afferent input and/or action potential‐independent excitatory and inhibitory input. We used the whole‐cell patch‐clamp technique to record synaptic currents in projection and unidentified lamina I neurones in a transverse lumbar spinal cord slice preparation from rats between postnatal day 18 and 37. Lamina I neurones with a projection to the parabrachial area or the periaqueductal grey were identified by retrograde labelling with a fluorescent tracer. The relative contribution of NMDA receptors versus AMPA/kainate receptors to C‐fibre‐evoked excitatory postsynaptic currents of lamina I neurones significantly decreased with age between postnatal day 18 and 27, but was independent of the supraspinal projection of the neurones. We did not find a significant contribution of kainate receptors to C‐fibre‐evoked excitatory postsynaptic currents. Lamina I projection and unidentified neurones possessed functional GABAA and glycine receptors but received scarce action potential‐independent spontaneous GABAergic and glycinergic inhibitory input as measured by miniature inhibitory postsynaptic currents. The miniature excitatory postsynaptic current frequencies were five times higher in projection than in unidentified neurones. The predominance of excitatory synaptic input to projection neurones, taken together with the previous finding that their membranes are more easily excitable than those of unidentified neurones, may facilitate the induction of synaptic long‐term potentiation.

Opioids and central sensitisation: II. Induction and reversal of hyperalgesia

Opioids are powerful analgesics when used to treat acute pain and some forms of chronic pain. In addition, opioids can pre‐empt some forms of central sensitization {Sandkühler and Ruscheweyh, Eur. J. Pain, in press, doi:10.1016/j.ejpain.2004.05.012}. Here we review evidence that opioids may also induce and perhaps reverse some forms of central sensitization.

Epileptiform activity in rat spinal dorsal horn in vitro has common features with neuropathic pain

Neuropathic pain and epileptic seizures bear several similarities, among them is the response to anticonvulsant drugs. It has therefore been hypothesized that epileptiform activity of nociceptive spinal dorsal horn neurons may contribute to paroxysmal forms of neuropathic pain. We used patch‐clamp and field potential recordings from young rat spinal cord slices to test if nociceptive dorsal horn structures are indeed able to sustain epileptiform activity. Application of the convulsant 4‐aminopyridine (100 &mgr;M) evoked epileptiform activity that was most pronounced in superficial dorsal horn and involved nociceptive lamina I neurons with a projection to the brain. The epileptiform activity was dependent on fast excitatory and inhibitory synaptic transmission through ionotropic glutamate receptors and GABAA receptors. During epileptiform activity, previously silent polysynaptic pathways from primary afferent C‐fibers to superficial dorsal horn neurons were opened. Stimulation of primary afferents at A&dgr;‐ and C‐fiber intensity interfered with the epileptiform rhythm, suggesting that both affect the same dorsal horn structures. Similar to neuropathic pain, spinal dorsal horn epileptiform activity was much less reduced by classical analgesics than by anticonvulsant agents.

Long‐range oscillatory Ca2+ waves in rat spinal dorsal horn

Synchronous activity of large populations of neurons shapes neuronal networks during development. However, re‐emergence of such activity at later stages of development could severely disrupt the orderly processing of sensory information, e.g. in the spinal dorsal horn. We used Ca2+ imaging in spinal cord slices of neonatal and young rats to assess under which conditions synchronous activity occurs in dorsal horn. No spontaneous synchronous Ca2+ transients were detected. However, increasing neuronal excitability by application of 4‐aminopyridine after pretreatment of the slice with blockers of (RS)‐alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA)/kainate, γ‐aminobutyric acid (GABA)A and glycine receptors evoked repetitive Ca2+ waves in dorsal horn. These waves spread mediolaterally with a speed of 1.0 ± 0.1 mm/s and affected virtually every dorsal horn neuron. The Ca2+ waves were associated with large depolarizing shifts of the membrane potential of participating neurons and were most likely synaptically mediated because they were abolished by blockade of action potentials or N‐methyl‐d‐aspartate (NMDA) receptors. They were most pronounced in the superficial dorsal horn and absent from the ventral horn. A significant proportion of the Ca2+ waves spread to the contralateral dorsal horn. This seemed to be enabled by disinhibition as primary afferent‐induced dorsal horn excitation crossed the midline only when GABAA and glycine receptors were blocked. Interestingly, the Ca2+ waves occurred under conditions where AMPA/kainate receptors were blocked. Thus, superficial dorsal horn NMDA receptors are able to sustain synchronous neuronal excitation in the absence of functional AMPA/kainate receptors.

Bidirectional actions of nociceptin/orphanin FQ on Aδ-fibre-evoked responses in rat superficial spinal dorsal horn in vitro

The present study investigated the modulatory actions of nociceptin/orphanin FQ on excitatory glutamatergic transmission in spinal dorsal horn. In transverse spinal cord slices with an attached dorsal root, mono- and polysynaptic A delta-fibre-evoked extracellular field potentials were recorded from superficial dorsal horn. Nociceptin/orphanin FQ showed bidirectional effects on monosynaptic transmission with a potentiation at lower concentrations (100-300 nM) and a dose-dependent depression at higher concentrations (1-3 microM). The polysynaptic field potential was dose-dependently depressed by nociceptin/orphanin FQ (100 nM-3 microM). None of the actions of nociceptin/orphanin FQ was reversed by the non-specific opioid receptor antagonist naloxone, the N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonovaleric acid or the peptide nocistatin. The bidirectional actions of nociceptin/orphanin FQ on the monosynaptic field potential may provide an in vitro model for the bidirectional actions of nociceptin/orphanin FQ in behavioural studies showing hyperalgesia at low doses of intrathecal nociceptin/orphanin FQ and analgesia at higher doses.