D I Hughes

Publications of D I Hughes

• P boutons in lamina IX of the rodent spinal cord express high levels of glutamic acid decarboxylase-65 and originate from cells in deep medial dorsal horn.

  Authors: D I Hughes, M Mackie, G G Nagy, J S Riddell, D J Maxwell, G Szabó, F Erdélyi, G Veress, P Szucs, M Antal, A J Todd

  Proceedings of the National Academy of Sciences of the United States of America. 07/2005; 102(25):9038-43.

  Presynaptic inhibition of primary muscle spindle (group Ia) afferent terminals in motor nuclei of the spinal cord plays an important role in regulating motor output and is produced by a population ofPresynaptic inhibition of primary muscle spindle (group Ia) afferent terminals in motor nuclei of the spinal cord plays an important role in regulating motor output and is produced by a population of GABAergic axon terminals known as P boutons. Despite extensive investigation, the cells that mediate this control have not yet been identified. In this work, we use immunocytochemistry with confocal microscopy and EM to demonstrate that P boutons can be distinguished from other GABAergic terminals in the ventral horn of rat and mouse spinal cord by their high level of the glutamic acid decarboxylase (GAD) 65 isoform of GAD. By carrying out retrograde labeling from lamina IX in mice that express green fluorescent protein under the control of the GAD65 promoter, we provide evidence that the cells of origin of the P boutons are located in the medial part of laminae V and VI. Our results suggest that P boutons represent the major output of these cells within the ventral horn and are consistent with the view that presynaptic inhibition of proprioceptive afferents is mediated by specific populations of interneurons. They also provide a means of identifying P boutons that will be important in studies of the organization of presynaptic control of Ia afferents.

• Selective loss of spinal GABAergic or glycinergic neurons is not necessary for development of thermal hyperalgesia in the chronic constriction injury model of neuropathic pain.

  Authors: E Polgár, D I Hughes, J S Riddell, D J Maxwell, Z Puskár, A J Todd

  Pain. 08/2003; 104(1-2):229-39.

  GABA and glycine are inhibitory neurotransmitters used by many neurons in the spinal dorsal horn, and intrathecal administration of GABA(A) and glycine receptor antagonists produces behavioural signsGABA and glycine are inhibitory neurotransmitters used by many neurons in the spinal dorsal horn, and intrathecal administration of GABA(A) and glycine receptor antagonists produces behavioural signs of allodynia, suggesting that these transmitters have an important role in spinal pain mechanisms. Several studies have described a substantial loss of GABA-immunoreactive neurons from the dorsal horn in nerve injury models, and it has been suggested that this may be associated with a loss of inhibition, which contributes to the behavioural signs of neuropathic pain. We have carried out a quantitative stereological analysis of the proportions of neurons in laminae I, II and III of the rat dorsal horn that show GABA- and/or glycine-immunoreactivity 2 weeks after nerve ligation in the chronic constriction injury (CCI) model, as well as in sham-operated and nai;ve animals. At this time, rats that had undergone CCI showed a significant reduction in the latency of withdrawal of the ipsilateral hindpaw to a radiant heat stimulus, suggesting that thermal hyperalgesia had developed. However, we did not observe any change in the proportion of neurons in laminae I-III of the ipsilateral dorsal horn that showed GABA- or glycine-immunoreactivity compared to the contralateral side in these animals, and these proportions did not differ significantly from those seen in sham-operated or nai;ve animals. In addition, we did not see any evidence for alterations of GABA- or glycine-immunostaining in the neuropil of laminae I-III in the animals that had undergone CCI. Our results suggest that significant loss of GABAergic or glycinergic neurons is not necessary for the development of thermal hyperalgesia in the CCI model of neuropathic pain.

• Modulation of inhibitory autapses and synapses on rat CA1 interneurones by GABA(A) receptor ligands.

  Authors: H Pawelzik, D I Hughes, A M Thomson

  The Journal of physiology. 03/2003; 546(Pt 3):701-16.

  To determine whether autaptic inhibition plays a functional role in the adult hippocampus, the action potential afterhyperpolarisations (spike AHPs) of CA1 interneurones were investigated in 25To determine whether autaptic inhibition plays a functional role in the adult hippocampus, the action potential afterhyperpolarisations (spike AHPs) of CA1 interneurones were investigated in 25 basket, three bistratified and eight axo-axonic cells. The spike AHPs showed two minima in all regular-spiking (5), burst-firing (3) and in many fast-spiking cells (17:28). The fast component had a time-to-peak (TTP) of 1.2 +/- 0.5 ms, the slower TTP was very variable (range of 3.3-103 ms). The AHP width at half-amplitude (HW) was 12.5 +/- 5.7 ms in fast-spiking, 29.3 +/- 18 ms in regular-spiking and 99.7 +/- 42 ms in burst-firing cells. Axo-axonic cells never establish autapses, and the fast-spiking variety showed narrow (HW: 3.9 +/- 0.7 ms) spike AHPs with only one AHP minimum (TTP: 0.9 +/- 0.1 ms). When challenged with GABA(A) receptor modulators, spike AHPs in basket and bistratified cells were enhanced by zolpidem (HW by 18.4 +/- 6.2 % in 10:15 cells tested), diazepam (45.2 +/- 0.5 %, 6:7), etomidate (43.9 +/- 36 %, 6:8) and pentobarbitone sodium (41 %, 1:1), and were depressed by bicuculline (-41 +/- 5.7 %, 5:8) and picrotoxin (-54 %, 1:1), and the enhancement produced by zolpidem was reduced by flumazenil (-31 +/- 13 %, relative to the AHP HW during exposure to zolpidem, 3:4). Neuronal excitability was modulated in parallel. The spike AHPs of three axo-axonic cells tested showed no sensitivity to etomidate, pentobarbitone or diazepam. Interneurone-to-interneurone inhibitory postsynaptic potentials (IPSPs), studied with dual intracellular recordings, had time courses resembling those of the spike AHPs. The IPSP HW was 13.4 +/- 2.8 ms in fast-spiking (n = 16) and 28.7 +/- 5.8 ms in regular-spiking/burst-firing cells (n = 6), and the benzodiazepine1-selective modulator zolpidem strongly enhanced these IPSPs (45 +/- 28 %, n = 5). Interneurones with spike AHPs affected by the GABA(A) receptor ligands exhibited 3.8 +/- 1.9 close autaptic appositions. In three basket cells studied at the ultrastructural level 6 of 6, 1 of 2 and 1 of 2 close appositions were confirmed as autapses. Therefore, in the hippocampus autaptic connections contribute to spike AHPs in many interneurones. These autapses influence neuronal firing and responses to GABA(A) receptor ligands.

• Distribution and colocalisation of glutamate decarboxylase isoforms in the rat spinal cord.

  Authors: M Mackie, D I Hughes, D J Maxwell, N J K Tillakaratne, A J Todd

  Neuroscience. 02/2003; 119(2):461-72.

  The inhibitory neurotransmitter GABA is synthesized by glutamic acid decarboxylase (GAD), and two isoforms of this enzyme exist: GAD65 and GAD67. Immunocytochemical studies of the spinal cord haveThe inhibitory neurotransmitter GABA is synthesized by glutamic acid decarboxylase (GAD), and two isoforms of this enzyme exist: GAD65 and GAD67. Immunocytochemical studies of the spinal cord have shown that whilst both are present in the dorsal horn, GAD67 is the predominant form in the ventral horn. The present study was carried out to determine the pattern of coexistence of the two GAD isoforms in axonal boutons in different laminae of the cord, and also to examine the relation of the GADs to the glycine transporter GLYT2 (a marker for glycinergic axons), since many spinal neurons are thought to use GABA and glycine as co-transmitters.Virtually all GAD-immunoreactive boutons throughout the spinal grey matter were labelled by both GAD65 and GAD67 antibodies; however, the relative intensity of staining with the two antibodies varied considerably. In the ventral horn, most immunoreactive boutons showed much stronger labelling with the GAD67 antibody, and many of these were also GLYT2 immunoreactive. However, clusters of boutons with high levels of GAD65 immunoreactivity were observed in the motor nuclei, and these were not labelled with the GLYT2 antibody. In the dorsal horn, some GAD-immunoreactive boutons had relatively high levels of labelling with either GAD65 or GAD67 antibody, whilst others showed a similar degree of labelling with both antibodies. GLYT2 immunoreactivity was associated with many GAD-immunoreactive boutons; however, this did not appear to be related to the pattern of GAD expression.It has recently been reported that there is selective depletion of GAD65, accompanied by a loss of GABAergic inhibition, in the ipsilateral dorsal horn in rats that have undergone peripheral nerve injuries [J Neurosci 22 (2002) 6724]. Our finding that some boutons in the superficial laminae showed relatively high levels of GAD65 and low levels of GAD67 immunoreactivity is therefore significant, since a reduction in GABA synthesis in these axons may contribute to neuropathic pain.

• The expression of vesicular glutamate transporters VGLUT1 and VGLUT2 in neurochemically defined axonal populations in the rat spinal cord with emphasis on the dorsal horn.

  Authors: A J Todd, D I Hughes, E Polgár, G G Nagy, M Mackie, O P Ottersen, D J Maxwell

  The European journal of neuroscience. 02/2003; 17(1):13-27.

  Two vesicular glutamate transporters, VGLUT1 and VGLUT2, have recently been identified, and it has been reported that they are expressed by largely nonoverlapping populations of glutamatergic neuronsTwo vesicular glutamate transporters, VGLUT1 and VGLUT2, have recently been identified, and it has been reported that they are expressed by largely nonoverlapping populations of glutamatergic neurons in the brain. We have used immunocytochemistry with antibodies against both transporters, together with markers for various populations of spinal neurons, in an attempt to identify glutamatergic interneurons in the dorsal horn of the mid-lumbar spinal cord of the rat. The great majority (94-100%) of nonprimary axonal boutons that contained somatostatin, substance P or neurotensin, as well as 85% of those that contained enkephalin, were VGLUT2-immunoreactive, which suggests that most dorsal horn neurons that synthesize these peptides are glutamatergic. In support of this, we found that most somatostatin- and enkephalin-containing boutons (including somatostatin-immunoreactive boutons that lacked calcitonin gene-related peptide and were therefore probably derived from local interneurons) formed synapses at which AMPA receptors were present. We also investigated VGLUT expression in central terminals of primary afferents. Myelinated afferents were identified with cholera toxin B subunit; most of those in lamina I were VGLUT2-immunoreactive, whereas all those in deeper laminae were VGLUT1-immunoreactive, and some (in laminae III-VI) appeared to contain both transporters. However, peptidergic primary afferents that contained substance P or somatostatin (most of which are unmyelinated), as well as nonpeptidergic C fibres (identified with Bandeiraea simplicifolia isolectin B4) showed low levels of VGLUT2-immunoreactivity, or were not immunoreactive with either VGLUT antibody. As all primary afferents are thought to be glutamatergic, this raises the possibility that unmyelinated afferents, most of which are nociceptors, express a different vesicular glutamate transporter.

• Gating of vagal inputs by sciatic afferents in nonspinally projecting neurons in the rat rostral ventrolateral medulla oblongata.

  Authors: A Zagon, D I Hughes

  The European journal of neuroscience. 03/2001; 13(4):781-92.

  Integration and coordination of somato-visceral sensory information is crucial to achieve adaptive behavioural responses. We have recently shown that sensory vagal and somato-sensory (sciatic nerve)Integration and coordination of somato-visceral sensory information is crucial to achieve adaptive behavioural responses. We have recently shown that sensory vagal and somato-sensory (sciatic nerve) inputs converge in neurons of the rostral ventrolateral medulla oblongata, which was implicated in adjusting visceral activities to changing somatic performances. In the present study, the neuronal mechanism of interaction between sciatic and vagal sensory inputs was examined in the rostral ventrolateral medulla oblongata using in vivo intracellular recording and labelling. Conditioning stimulation of the contralateral sciatic nerve (2 V) led to a time-dependent inhibition of responses to vagal stimulation (100 microA) in each RVLM neuron that received convergent sciatic and vagal sensory inputs (n = 50). None of these neurons had direct spinal projections, and only 8% of them exhibited a visible response to stimulation of the aortic depressor nerve. A significant attenuation of the amplitude of vagal test responses was present for up to 800 ms of conditioning delay, although the duration of this sciatico-vagal inhibition was greatly dependent on the intensity of both stimuli. The electrophysiological data indicated that sciatico-vagal inhibition is mediated presynaptically, via activation of GABAB receptors. Morphological evidence of axo-axonic interactions that may underlie sciatico-vagal inhibition was subsequently found in the electron microscope. It is suggested that during movements of the hindleg, activation of sciatic sensory fibres leads to re-patterning of neuronal activity in RVLM neurons via inhibition of visceral sensory inputs. Sciatico-vagal inhibition is likely to affect the activity of those RVLM neurons that modulate higher neuronal activities via ascending projections.

• Double immunofluorescence, peroxidase labelling and ultrastructural analysis of interneurones following prolonged electrophysiological recordings in vitro.

  Authors: D I Hughes, A P Bannister, H Pawelzik, A M Thomson

  Journal of neuroscience methods. 10/2000; 101(2):107-16.

  Inhibitory hippocampal and neocortical interneurones comprise a physiologically, morphologically and neurochemically heterogenous cell population. To identify the roles each class of interneuroneInhibitory hippocampal and neocortical interneurones comprise a physiologically, morphologically and neurochemically heterogenous cell population. To identify the roles each class of interneurone plays within a given circuit it is necessary to correlate the electrophysiological properties of individual cells with their neurochemistry and morphology at both the light and electron microscopic level. However, the optimal conditions required for any one part of the protocol typically compromise the results from another. We have developed a protocol which allows the neurochemical content, gross morphology and ultrastructure details of biocytin-filled neurones to be recovered following long, dual intracellular recordings in thick mature slices maintained in an interface recording chamber, helping define sub-populations which could not otherwise be determined. Dual immunofluorescence is performed by incubating the tissue in monoclonal and polyclonal antibodies simultaneously, prior to visualization of biocytin-labelling with precipitation of a peroxidase reaction product. By using a biotinylated anti-avidin D antibody (Vector Laboratories), the intensity of this precipitation can be enhanced further where necessary. It is envisaged that this protocol can not only help determine the neurochemical content of cells recorded in similar in vivo studies, but that the ability to amplify peroxidase labelling in poorly filled cells is also of interest.

• Differential sensitivity to Zolpidem of IPSPs activated by morphologically identified CA1 interneurons in slices of rat hippocampus.

  Authors: A M Thomson, A P Bannister, D I Hughes, H Pawelzik

  The European journal of neuroscience. 03/2000; 12(2):425-36.

  Hippocampal pyramidal cells express several alpha-subunits, which determine the affinity of GABAA (gamma-aminobutyric acid) receptors for benzodiazepine site ligands. This study asked whetherHippocampal pyramidal cells express several alpha-subunits, which determine the affinity of GABAA (gamma-aminobutyric acid) receptors for benzodiazepine site ligands. This study asked whether inhibitory postsynaptic potentials (IPSPs) elicited by specific interneuronal subclasses were differentially sensitive to the alpha1-preferring agonist Zolpidem, i.e. whether different receptors mediate different inhibitory connections. Paired intracellular recordings in which the presynaptic cell was an interneuron and the postsynaptic cell a CA1 pyramid were performed in slices of adult rat hippocampus. Resultant IPSPs were challenged with Zolpidem, cells filled with biocytin and identified morphologically. IPSPs elicited by fast spiking (FS) basket cells (n = 9) were enhanced more than IPSPs elicited by regular spiking (RS) basket cells (n = 10). At FS basket cell synapses the efficacy of Zolpidem was equivalent to that of Diazepam, while RS basket cell IPSPs are enhanced 50% less by Zolpidem than by Diazepam. Thus, while alpha1 subunits may dominate at synapses supplied by FS basket cells, RS basket cell synapses also involve alpha2/3 subunits. Two bistratified cell IPSPs tested with Zolpidem did not increase in amplitude, despite powerful enhancements of bistratified cell IPSPs by Diazepam, consistent with previous indications that these synapses utilize alpha5-containing receptors. Enhancements of basket cell IPSPs by Zolpidem and Diazepam were bi- or triphasic with steep amplitude increases separated by plateaux, occurring 10-15, 25-30 and 45-55 min after adding the drug to the bath. The entire enhancement was, however, blocked by the antagonist Flumazenil (n = 7). Flumazenil, either alone (n = 3), or after Zolpidem, reduced IPSP amplitude to approximately 90% of control, suggesting that alpha4-containing receptors were not involved.

• Up-regulation of substance P in low-threshold myelinated afferents is not required for tactile allodynia in the chronic constriction injury and spinal nerve ligation models

  Authors: D I Hughes, D.T. Scott, J S Riddell, A J Todd

  It has been proposed that substance P and calcitonin gene-related peptide (CGRP) are up-regulated in low-threshold myelinated primary afferents following certain types of nerve injury, and thatIt has been proposed that substance P and calcitonin gene-related peptide (CGRP) are up-regulated in low-threshold myelinated primary afferents following certain types of nerve injury, and that release of substance P from these afferents contributes to the resulting tactile allodynia. To test this hypothesis, we looked for neuropeptides in Aβ primary afferent terminals in the ipsilateral gracile nucleus and spinal dorsal horn in three nerve injury models: sciatic nerve transection (SNT), spinal nerve ligation (SNL) and chronic constriction injury (CCI). We also looked for evidence of neurokinin 1 (NK1) receptor internalisation in the dorsal horn following electrical stimulation of Aβ afferents. We found no evidence of either substance P or CGRP expression in injured Aβ terminals in the spinal cord in any of the models. Although substance P was not detected in terminals of injured afferents in the gracile nucleus, CGRP was expressed in between 32 and 68% of these terminals, with a significantly higher proportion in the SNL and CCI models, compared to SNT. In addition, we did not detect any Aβ-evoked NK1 receptor internalisation in neurons from laminae I, III or IV of the dorsal horn in the CCI or SNL models. These results do not support the proposal that substance P is present at significant levels in the terminals of injured Aβ primary afferents in neuropathic models. They also suggest that any release of substance P from injured Aβ afferents is unlikely to activate NK1 receptors in the dorsal horn or contribute to neuropathic pain.