Zita Puskár

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.

&NA; GABA and glycine are inhibitory neurotransmitters used by many neurons in the spinal dorsal horn, and intrathecal administration of GABAA 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 naïve 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 naïve 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.

A quantitative and morphological study of projection neurons in lamina I of the rat lumbar spinal cord

In the rat lumbar spinal cord the major supraspinal targets for lamina I projection neurons are the caudal ventrolateral medulla (CVLM), lateral parabrachial area (LPb) and periaqueductal grey matter (PAG). In this study we have estimated the number of lamina I neurons retrogradely labelled from each of these sites in the L4 segment, as well as the proportion that can be labelled by injecting different tracers into two separate sites. Our results suggest that this segment contains approximately 400 lamina I projection neurons on each side, and that approximately 85% of these can be labelled from either the CVLM or the LPb on the contralateral side. Around 120 lamina I cells in L4 project to the PAG, and over 90% of these cells can also be labelled from the CVLM or LPb. Most lamina I neurons projecting to CVLM or LPb are located in the contralateral dorsal horn, but in each case some cells were found to have bilateral projections. We also examined horizontal sections to investigate morphology and the expression of the neurokinin 1 (NK1) receptor in cells labelled from CVLM, LPb or PAG. There were no consistent morphological differences between these groups, however, while cells with strong or moderate NK1 receptor‐immunostaining were labelled from LPb or CVLM, they seldom projected to the PAG. These results suggest that many lamina I cells project to more than one site in the brain and that those projecting to PAG may represent a distinct subclass of lamina I projection neuron.

MOR-1-immunoreactive neurons in the dorsal horn of the rat spinal cord: evidence for nonsynaptic innervation by substance P-containing primary afferents and for selective activation by noxious thermal stimuli

A direct action of μ‐opioid agonists on neurons in the spinal dorsal horn is thought to contribute to opiate‐induced analgesia. In this study we have investigated neurons that express the μ‐opioid receptor MOR‐1 in rat spinal cord to provide further evidence about their role in nociceptive processing. MOR‐1‐immunoreactive cells were largely restricted to lamina II, where they comprised approximately 10% of the neuronal population. The cells received few contacts from nonpeptidergic unmyelinated afferents, but many from substance P‐containing afferents. However, electron microscopy revealed that most of these contacts were not associated with synapses. None of the MOR‐1 cells in lamina II expressed the neurokinin 1 receptor; however, the μ‐selective opioid peptide endomorphin‐2 was present in the majority (62–82%) of substance P axons that contacted them.

A population of large lamina I projection neurons with selective inhibitory input in rat spinal cord

Lamina I of the spinal dorsal horn contains a diverse mixture of neurons. Among these, a group of giant neurons (Waldeyer cells) has long been recognized. In this study we have used immunocytochemistry to characterize a population of Waldeyer cells which were identified by the presence of high levels of the glycine receptor-associated protein gephyrin on their cell bodies and proximal dendrites. Most of these cells (27/29) were retrogradely labelled after injection of cholera toxin B subunit into the parabrachial area, and the majority (26/30) expressed the protein product of immediate-early gene c-fos, Fos, following noxious stimulation. Unlike many lamina I projection neurons, these cells either lacked the neurokinin 1 receptor, or expressed it at a very low level. Most of the gephyrin puncta on the cells were adjacent to axons that contained glutamate decarboxylase (and were therefore presumably GABAergic), which suggests that the cells are under powerful inhibitory control. Only around 35% of the puncta were associated with axons that expressed the glycine transporter GLYT2 (a marker for glycinergic axons); however, the glycine receptor alpha1 subunit was present at all of the gephyrin puncta on these cells. The cells received synapses from axons that contained nitric oxide synthase, most of which were also GABAergic, and in some cases this input was so dense that it outlined the cell bodies and dendrites. The innervation by nitric oxide synthase-containing axons was selective for these cells, compared to other neurons in the dorsal horn. From the results of this study we suggest that the gephyrin-rich cells form a specific population of lamina I projection neurons which convey noxious information to the brain. These cells are under powerful inhibitory control, and the study provides further evidence that inhibitory circuits in the dorsal horn are organized in a specific manner.

Fos induction in lamina I projection neurons in response to noxious thermal stimuli

Lamina I of the spinal cord contains many projection neurons: the majority of these are activated by noxious stimulation, although some respond to other stimuli, such as innocuous cooling. In the rat, approximately 80% of lamina I projection neurons express the neurokinin 1 (NK1) receptor, on which substance P acts. Lamina I neurons can be classified into three main morphological classes: pyramidal, fusiform and multipolar cells. It has been reported that in the cat, pyramidal cells respond to innocuous cooling, and whilst both fusiform and multipolar cells are activated by noxious mechanical and heat stimuli, only cells in the latter group respond to noxious cold [Nat Neurosci 1 (1998) 218]. However, we have previously shown that NK1 receptor-immunoreactive projection neurons belonging to each morphological class are equally likely to up-regulate the transcription factor Fos after noxious chemical stimulation, and that the density of innervation by substance P-containing (nociceptive) afferents is similar for cells of each type [J Neurosci 22 (2002) 4103]. This suggests that the morphological-physiological correlation that has been reported in the cat may not apply in the rat. We have tested this further by examining Fos expression in lamina I spinoparabrachial neurons in the rat after application of noxious heat or noxious cold stimuli under general anesthesia. Following noxious heat, 57-69% of NK1 receptor-immunoreactive spinoparabrachial neurons expressed Fos, and the proportion did not differ significantly between morphological groups. However, after noxious cold stimulation Fos was present in 63% of multipolar neurons, but only 19-26% of fusiform or pyramidal cells. These results suggest that although most NK1 receptor-expressing spinoparabrachial neurons are activated by noxious stimuli, responsiveness to noxious cold is significantly more common in those of the multipolar type. There therefore appears to be a correlation between morphology and function for lamina I projection neurons in the rat.

Selective innervation of lamina I projection neurones that possess the neurokinin 1 receptor by serotonin-containing axons in the rat spinal cord

Axons containing serotonin descend from brainstem to spinal cord and are thought to contribute to stimulation-produced and opioid analgesia, partly by a direct inhibitory action of serotonin on projection neurones. The density of serotoninergic innervation is highest in lamina I, which contains many nociceptive projection neurones. Two sets of anatomical criteria have been used to classify lamina I projection neurones: somatodendritic morphology and presence or absence of the neurokinin 1 receptor. To test whether the strength of serotoninergic innervation of lamina I projection neurones was related to morphology or neurokinin 1 receptor expression, we used confocal microscopy to determine the density of serotoninergic contacts on 60 cells retrogradely labelled from the caudal ventrolateral medulla. The contact density on neurones with the neurokinin 1 receptor was variable, with some cells receiving heavy input and others having few contacts. However, on average they received significantly more contacts (5.64 per 1000 microm(2) plasma membrane +/- 0.47, S.E.M.) than neurones which lacked the receptor (2.49 +/- .36). Among the neurokinin 1 neurones, serotoninergic innervation density was not related to morphology. Since the majority of serotoninergic boutons in lamina I of rat spinal cord do not appear to form synapses, we carried out electron microscopy on three heavily innervated neurokinin 1 receptor-immunoreactive projection neurones. Symmetrical synapses were found at 89% of serotoninergic contacts. These results indicate that serotoninergic innervation of lamina I projection neurones in the rat spinal cord is related to expression of neurokinin 1 receptors, but not to morphology, and that (at least on heavily innervated neurones) most serotonin-containing boutons which are in contact form synapses.

Cocaine- and amphetamine-regulated transcript peptide (CART) is present in peptidergic C primary afferents and axons of excitatory interneurons with a possible role in nociception in the superficial laminae of the rat spinal cord

Cocaine‐ and amphetamine‐regulated transcript peptides (CART) have been implicated in the regulation of several physiological functions, including pain transmission. A dense plexus of CART‐immunoreactive fibres has been described in the superficial laminae of the spinal cord, which are key areas in sensory information and pain processing. In this study, we used antibody against CART peptide, together with markers for various types of primary afferents, interneurons and descending systems to determine the origin of the CART‐immunoreactive axons in the superficial laminae of the rat spinal cord. Calcitonin gene‐related peptide (CGRP), a marker for peptidergic primary afferents in the dorsal horn, was present in 72.6% and 34.8% of CART‐immunoreactive axons in lamina I and II, respectively. The majority of these fibres also contained substance P (SP), while a few were somatostatin (SOM)‐positive. The other subpopulation of CART‐immunoreactive boutons in lamina I and II also expressed SP and/or SOM without CGRP, but contained vesicular glutamate transporter 2, which is present mainly in excitatory interneuronal terminals. Our data demonstrate that the majority of CART‐immunoreactive axons in the spinal dorsal horn originate from peptidergic nociceptive primary afferents, while the rest arise from excitatory interneurons that contain SP or SOM. This strongly suggests that CART peptide can affect glutamatergic neurotransmission as well as the release and effects of SP and SOM in nociception and other sensory processes.

Subpopulation-specific patterns of intrinsic connectivity in mouse superficial dorsal horn as revealed by laser scanning photostimulation

•  Sensory neurons that detect painful and non‐painful stimulation of body tissues have axons that project to the dorsal horn of the spinal cord, where their terminations are partially segregated into superficial (I–II) and deep (III–IV) dorsal horn laminae, respectively. •  The dorsal horn contains many excitatory and inhibitory interneurons whose axons synapse on other dorsal horn neurons to enhance or suppress sensory transmission. •  This study used a localized stimulation technique (laser scanning photostimulation) for high‐resolution mapping of synaptic connections between dorsal horn interneurons, in an in vitro‘slice’ preparation of the mouse lumbar spinal cord. •  Some neurons in superficial layers of the dorsal horn have long dendrites that extend ventrally into deeper layers of the dorsal horn, and these neurons can receive excitatory or inhibitory synaptic input from neurons in the deeper layers. •  These interlaminar connections may be involved in interactions between transmission of signals underlying painful versus non‐painful sensations.

Nonselective innervation of lamina I projection neurons by cocaine- and amphetamine-regulated transcript peptide (CART)-immunoreactive fibres in the rat spinal dorsal horn.

Cocaine‐ and amphetamine‐regulated transcript (CART) peptides have been implicated in spinal pain transmission. A dense plexus of CART‐immunoreactive fibres has been described in the superficial laminae of the spinal cord, which are key areas in sensory information and pain processing. We demonstrated previously that the majority of these fibres originate from nociceptive primary afferents. Using tract tracing, multiple immunofluorescent labelling and electronmicroscopy we determined the proportion of peptidergic primary afferents expressing CART, looked for evidence for coexistence of CART with galanin in these afferents in lamina I and examined their targets. Almost all (97.9%) randomly selected calcitonin gene‐related peptide (CGRP)‐immunoreactive terminals were substance P (SP)‐positive (+) and CART was detected in approximately half (48.6%) of them. Most (81.4%) of the CGRP/SPergic boutons were galanin+ and approximately half (49.0%) of these contained CART. Many (72.9%) of the CARTergic boutons which expressed CGRP were also immunoreactive for galanin, while only 8.6% of the CARTergic terminals were galanin+ without CGRP. Electron microscopy showed that most of the CART terminals formed asymmetrical synapses, mainly with dendrites. All different morphological and neurochemical subtypes of spinoparabrachial projection neurons in the lamina I received contacts from CART‐immunoreactive nociceptive afferents. The innervation density from these boutons did not differ significantly between either the different neurochemical or the morphological subclasses of these cells. This suggests a nonselective innervation of lamina I projection neurons from a subpopulation of CGRP/SP afferents containing CART peptide. These results provide anatomical evidence for involvement of CART peptide in spinal pain transmission.

Development, neurochemical properties, and axonal projections of a population of last-order premotor interneurons in the white matter of the chick lumbosacral spinal cord

There is general agreement that last-order premotor interneurons-a set of neurons that integrate activities generated by the spinal motor apparatus, sensory information and volleys arising from higher motor centres, and transmit the integrated signals to motoneurons through monosynaptic contacts-play crucial roles in the initiation and maintenance of spinal motor activities. Here, we demonstrate the development, neurochemical properties, and axonal projections of a unique group of last-order premotor interneurons within the ventrolateral aspect of the lateral funiculus of the chick lumbosacral spinal cord. Neurons expressing immunoreactivity for neuron-specific enolase were first detected in the ventrolateral white matter at embryonic day 9 (E9). The numbers of immunoreactive neurons were significantly increased at E10-E12, while most of them were gradually concentrated in small segmentally arranged nuclei (referred to as major nuclei of Hofmann) protruding from the white matter in a necklace like fashion dorsal to the ventral roots. The major nuclei of Hofmann became more prominent at E12-E16, but substantial numbers of cells were still located within the ventrolateral white matter (referred to as minor nucleus of Hofmann). The distribution of immunoreactive neurons achieved by E16 was maintained during later developmental stages and was also characteristic of adult animals. After injection of Phaseolus vulgaris-leucoagglutinin unilaterally into the minor nucleus of Hofmann, labeled fibres were detected in the ventrolateral white matter ipsilateral to the injection site. Ascending and descending fibres were revealed throughout the entire rostro-caudal length of the lumbosacral spinal cord. Axon terminals were predominantly found within the lateral motor column and the ventral regions of lamina VII ipsilateral to the injection site. Several axon varicosities made close appositions with somata and dendrites of motoneurons, which were identified as synaptic contacts in a consecutive electron microscopic study. With the postembedding immunogold method, 21 of 97 labeled terminals investigated were immunoreactive for glycine and 2 of them showed immunoreactivity for gamma-aminobutyric acid (GABA). The axon trajectories of neurons within the minor nucleus of Hofmann suggest that some of these cells might represent a population of last-order premotor interneurons. J. Exp. Zool. 286:157-172, 2000.