David Andrew

Spinothalamic lamina I neurons selectively sensitive to histamine: a central neural pathway for itch

We found a class of lamina I spinothalamic tract (STT) neurons selectively excited by iontophoretic histamine. The responses of this class of neurons parallel the pure itching sensation this stimulus elicits in humans, and match the responses of peripheral C-fibers that have similar selectivity. These neurons have distinct central conduction velocities and thalamic projections, indicating that they constitute a unique subset of STT neurons. These findings can explain why a lesion of the lateral STT disrupts itch along with pain and temperature sensations. Our findings provide strong evidence that itch is subserved by specific neural elements both peripherally and centrally.

Widespread Expression of the AMPA Receptor GluR2 Subunit at Glutamatergic Synapses in the Rat Spinal Cord and Phosphorylation of GluR1 in Response to Noxious Stimulation Revealed with an Antigen-Unmasking Method

Glutamate, the principal excitatory neurotransmitter in the spinal cord, acts primarily through AMPA receptors. Although all four AMPA subunits are expressed by spinal neurons, we know little about their distribution at glutamatergic synapses. We used an antigen-unmasking technique to reveal the synaptic distribution of glutamate receptor (GluR) 1-4 subunits with confocal microscopy. After pepsin treatment, punctate staining was seen with antibodies against each subunit: GluR2-immunoreactive puncta were distributed throughout the gray matter, whereas GluR1-immunoreactive puncta were restricted to the dorsal horn and were most numerous in laminas I-II. Punctate staining for GluR3 and GluR4 was found in all laminas but was weak in superficial dorsal horn. Colocalization studies showed that GluR2 was present at virtually all (98%) puncta that were GluR1, GluR3, or GluR4 immunoreactive and that most (>90%) immunoreactive puncta in laminas IV, V, and IX showed GluR2, GluR3, and GluR4 immunoreactivity. Evidence that these puncta represented synaptic receptors was obtained with electron microscopy and by examining the association of GluR2- and GluR1-immunoreactive puncta with glutamatergic boutons (identified with vesicular glutamate transporters or markers for unmyelinated afferents). The great majority (96%) of these boutons were associated with GluR2-immunoreactive puncta. Our findings suggest that GluR2 is almost universally present at AMPA-containing synapses, whereas GluR1 is preferentially associated with primary afferent terminals. We also found a substantial, rapid increase in staining for synaptic GluR1 subunits phosphorylated on the S845 residue in the ipsilateral dorsal horn after peripheral noxious stimulation. This finding demonstrates plastic changes, presumably contributing to central sensitization, at the synaptic level.

Spinothalamic lamina I neurones selectively responsive to cutaneous warming in cats

1 In order to further characterize the role of lamina I as the source of central ascending neural pathways for thermoreception and thermoregulation, experiments were performed on anaesthetized cats to determine the quantitative response characteristics of warming‐specific lumbosacral spinothalamic lamina I neurones. 2 We identified 10 neurones out of 474 that were selectively excited by cutaneous warming (Warm cells). Their thresholds were all in the range 35‐37 °C at a baseline of 34.5 °C, and their discharge linearly encoded the temperature of graded, innocuous warming stimuli with a sensitivity of 2.1 Hz °C−1. 3 The stimulus‐response function of the Warm cells plateaued at temperatures that were in the noxious heat range. 4 The Warm cells were distinguished from other classes of spinothalamic lamina I neurones by their peripheral inputs, central conduction velocities and level of ongoing activity. 5 The discharge of Warm cells compares well with the known human psychophysics of warm sensibility, and these neurones are likely to be crucial to discriminative thermoreception. Additionally, a role in thermoregulation, a defining feature of mammalian homeostasis, is suggested.

Quantitative characterization of low‐threshold mechanoreceptor inputs to lamina I spinoparabrachial neurons in the rat

It has been suggested that primary afferent C‐fibres that respond to innocuous tactile stimuli are important in the sensation of pleasurable touch. Although it is known that C‐tactile fibres terminate in the substantia gelatinosa (lamina II) of the spinal cord, virtually all of the neurons in this region are interneurons, and currently it is not known how impulses in C‐mechanoreceptors are transmitted to higher centres. In the current study, I have tested the quantitative response properties of ‘wide dynamic range’ projection neurons in lamina I of the spinal cord to graded velocity brushing stimuli to identify whether low‐threshold mechanoreceptor input to these neurons arises from myelinated or umyelinated nerve fibres. Graded velocity brushing stimuli (6.6–126 cm s−1) were used to characterize the mechanoreceptor inputs to ‘wide dynamic range’ neurons in lamina I of the dorsal horn that had axons that projected to the contralateral parabrachial nucleus. The most effective tactile stimuli for activation of ‘wide dynamic range’ lamina I spinoparabrachial neurons were low velocity brush strokes: peak discharge occurred at a mean velocity of 9.2 cm s−1 (range 6.6–20.4 cm s−1, s.d. 5.0 cm s−1), and declined exponentially as brush velocity increased. The data indicate that C‐fibres, but not A‐fibres, conveyed low‐threshold mechanoreceptor inputs to lamina I projection neurons.

Differentiation of lamina I spinomedullary and spinothalamic neurons in the cat.

We characterized spinomedullary neurons that project to the ventrolateral portion of the medulla that receives lamina I terminations in two sets of experiments in the cat. First, their distribution was examined using single unilateral iontophoretic injections of cholera toxin subunit B. The injection sites were characterized by microelectrode recordings from nociceptive‐ and thermoreceptive‐specific units, indicative of lamina I input. The spinomedullary neurons were symmetrically distributed bilaterally, predominantly (63–69%) in lamina I but also in laminae V–VIII and the thoracic lateral horn (intermediolateral cell column). In horizontal sections, spinomedullary lamina I neurons included all three main morphological types described earlier. Second, spinomedullary and spinothalamic neurons were compared in retrograde double‐labeling experiments. Different combinations of tracers were injected in the right thalamus and the left or right ventrolateral medulla (guided by recordings). The numbers of spinomedullary and spinothalamic neurons on the left side were comparable, and the segmental and laminar distributions were similar, except that a greater proportion of spinomedullary neurons originated from thoracic segments. However, the proportion of double‐labeled neurons was consistently ∼1%, indicating that spinomedullary and spinothalamic pathways arise from separate subpopulations. Spinomedullary neurons were more ventrally located within lamina I than spinothalamic neurons. A significantly greater proportion of spinomedullary neurons had fusiform somata (49% vs. 36%). These observations indicate that lamina I is the major source of spinal input to this portion of the ventrolateral medulla, that the projection includes several morphological types of inputs, and that this projection is distinct from the spinothalamic projection. These findings are consistent with the concept that lamina I projections constitute an ascending homeostatic afferent pathway relating the physiological condition of the body. J. Comp. Neurol. 458:257–271, 2003.

Quantitative responses of spinothalamic lamina i neurones to graded mechanical stimulation in the cat

Nociceptive spinothalamic tract (STT) neurones in lamina I of the lumbosacral spinal cord of anaesthetized cats were characterized by recording their responses to graded mechanical stimulation with controlled forces of 10‐120 g and probes of 5.0, 0.5 and 0.1 mm2 contact area. Neurones were identified by antidromic activation from the contralateral thalamus, and cells that responded to noxious stimulation were categorized as either nociceptive specific (NS, n= 20) or as polymodal nociceptive (HPC, responsive to heat, pinch and cold, n= 19) based on their responses to quantitative thermal stimuli. The mean responses of the 39 units increased linearly as stimulus intensity increased, and the population stimulus‐response curves evoked by each of the three probes were all significantly different from each other. Thresholds were 45 g for the 5.0 mm2 probe, 30 g for the 0.5 mm2 probe and 20 g for the 0.1 mm2 probe. Further analysis showed that the NS neurones encoded both stimulus intensity and area (probe size) significantly better than HPC neurones in terms of their thresholds to individual probes, their peak discharge rates, their suprathreshold responsiveness and their ability to discriminate the three different probe sizes. These differences are consistent with the known differences between the mechanical encoding properties of A‐fibre nociceptors, which provide the dominant inputs to NS neurones, and C‐fibre nociceptors, which are the dominant inputs to HPC cells. Comparison of the stimulus‐response curves of NS and HPC neurones indicated that the discharge of NS neurones better match the psychophysics of mechanical pain sensations in humans than the discharge of the HPC neurones do. Our findings support the view that NS neurones have a prominent role in mechanical pain and sharpness, and they corroborate the concept that the lamina I STT projection comprises several discrete channels that are integrated in the forebrain to generate qualitatively distinct sensations.

Sensitization of lamina I spinoparabrachial neurons parallels heat hyperalgesia in the chronic constriction injury model of neuropathic pain.

It has been proposed that spinal lamina I neurons with ascending axons that project to the midbrain play a crucial role in hyperalgesia. To test this hypothesis the quantitative properties of lamina I spinoparabrachial neurons in the chronic constriction injury (CCI) model of neuropathic pain were compared to those of unoperated and sham‐operated controls. Behavioural testing showed that animals with a CCI exhibited heat hyperalgesia within 4 days of the injury, and this hyperalgesia persisted throughout the 14‐day post‐operative testing period. In the CCI, nociceptive lamina I spinoparabrachial neurons had heat thresholds that were significantly lower than controls (43.0 ± 2.8°C vs. 46.7 ± 2.6°C; P < 10−4, ANOVA). Nociceptive lamina I spinoparabrachial neurons were also significantly more responsive to graded heat stimuli in the CCI, compared to controls (P < 0.02, 2‐factor repeated‐measures ANOVA), and increased after‐discharges were also observed. Furthermore, the heat‐evoked stimulus–response functions of lamina I spinoparabrachial neurons in CCI animals co‐varied significantly (P < 0.03, ANCOVA) with the amplitude of heat hyperalgesia determined behaviourally. Taken together these results are consistent with the hypothesis that lamina I spinoparabrachial neurons have an important mechanistic role in the pathophysiology of neuropathic pain.

Cone beam computed tomography for imaging orbital trauma—image quality and radiation dose compared with conventional multislice computed tomography

We compared the image quality and radiation dose to the lens of the eye in patients with suspected orbital fractures who were imaged using cone beam computed tomography (CBCT) or conventional multislice computed tomography (CT). Although CBCT has a lower radiation dose than conventional CT, it is not known whether the image quality is comparable for diagnostic purposes. We identified fractures of the orbit (floor or roof, or both) in 6/10 patients who were scanned using CBCT and in 5/10 patients who were scanned using multislice CT (orbital floor and medial wall). Impingement of the rectus muscle on fracture lines was identified with both techniques, but retro-orbital haemorrhage was detected only on multislice CT. The mean radiation dose to the lens of the eye was 42% lower (range 23-53, SD 10) for CBCT than for multislice CT (p<0.001), and the effective dose (a measure of the risk of developing a radiation-induced cancer) was also significantly lower. CBCT can therefore be used to diagnose orbital fractures, and is associated with a significantly lower radiation dose than multislice CT.

Effects of Src-kinase inhibition in cancer-induced bone pain

Background Bone metastases occur frequently in advanced breast, lung, and prostate cancer, with approximately 70% of patients affected. Pain is a major symptom of bone metastases, and current treatments may be inadequate or have unacceptable side effects. The mechanisms that drive cancer-induced bone pain are not fully understood; however, it is known that there is sensitization of both peripheral bone afferents and central spinal circuits. It is well established that the N-methyl-D-aspartate receptor plays a major role in the pathophysiology of pain hypersensitivity. Inhibition of the non-receptor tyrosine kinase Src controls N-methyl-D-aspartate receptor activity and inhibiting Src reduces the hypersensitivity associated with neuropathic and inflammatory pains. As Src is also implicated in osteoclastic bone resorption, we have investigated if inhibiting Src ameliorates cancer-induced bone pain. We have tested this hypothesis using an orally bioavailable Src inhibitor (saracatinib) in a rat model of cancer-induced bone pain. Results Intra-tibial injection of rat mammary cancer cells (Mammary rat metastasis tumor cells -1), but not vehicle, in rats produced hindpaw hypersensitivity to thermal and mechanical stimuli that was maximal after six days and persisted for at least 13 days postinjection. Daily oral gavage with saracatinib (20 mg/kg) beginning seven days after intra-tibial injection reversed the thermal hyperalgesia but not the mechanical allodynia. The analgesic mechanisms of saracatinib appear to be due to an effect on the nervous system as immunoblotting of L2-5 spinal segments showed that mammary rat metastasis tumor cells-1 injection induced phosphorylation of the GluN1 subunit of the N-methyl-D-aspartate receptor, indicative of receptor activation, and this was reduced by saracatinib. Additionally, histology showed no anti-tumor effect of saracatinib at any dose and no significant effect on bone preservation. Conclusions This is the first demonstration that Src plays a role in the development of cancer-induced bone pain and that Src inhibition represents a possible new analgesic strategy for patients with bone metastases.

Processing of C-Tactile Information in the Spinal Cord

Functional brain imaging places C-tactile (CT) fibres within an interoceptive network that is important for the sensation of pleasurable touch and emotional well-being, but the circuits that relay this information from periphery to forebrain are not well understood. In vivo single unit recordings from projection neurons in lamina I of the rat spinal cord showed that the activity of CT fibres is integrated with that of nociceptors before being relayed to the brainstem parabrachial nucleus. There is at least one interneuron in the pathway that relays CT activity from mechanoreceptor to lamina I output neuron, with preliminary data suggesting that such interneurons express the γ isoform of protein kinase C and receive inputs from vesicular glutamate transporter three-labelled primary afferents. A role for CT fibres in mechanical allodynia is also suggested, as pilot data shows that these fibres can provide low-threshold inputs to sensitized nociceptive-specific lamina I projection neurons. Thus, the homeostatic signal provided via CT fibres can be directly modulated by pain, placing the CT system firmly within an interoceptive network that regulates the body’s internal environment.