A quantitative study of neuronal nitric oxide synthase expression in laminae I-III of the rat spinal dorsal horn.

Nitric oxide produced by neuronal nitric oxide synthase (nNOS) in the spinal cord is required for development of hyperalgesia in inflammatory and neuropathic pain states. nNOS is expressed by some dorsal horn neurons, and an early study that used a histochemical method to identify these cells suggested that they were mainly inhibitory interneurons. We have carried out a quantitative analysis of nNOS-immunoreactivity in laminae I-III of the rat dorsal horn, to determine the proportion of inhibitory and excitatory neurons and axonal boutons that express the protein. nNOS was present in ∼5% of neurons in laminae I and III, and 18% of those in lamina II. Although most cells with strong nNOS immunostaining were GABA-immunoreactive, two-thirds of the nNOS-positive cells in lamina II and half of those in lamina III were not GABAergic, and some of these expressed protein kinase Cγ (PKCγ). We estimate that nNOS is present in 17-19% of the inhibitory interneurons in laminae I-II, and 6% of those in lamina III. However, our results suggest that nNOS is also expressed at a relatively low level by a significant proportion (∼17%) of excitatory interneurons in lamina II. nNOS was seldom seen in boutons that contained vesicular glutamate transporter 2, which is expressed by excitatory interneurons, but was co-localised with the vesicular GABA transporter (VGAT, a marker for GABAergic and glycinergic axons). nNOS was detected in 13% of VGAT boutons in lamina I and in 7-8% of those in laminae II-III. However, it was only found in 2-4% of the VGAT boutons that were presynaptic to PKCγ-expressing interneurons in this region. These results indicate that nNOS is more widely expressed than previously thought, being present in both inhibitory and excitatory neurons. They provide further evidence that axons of neurochemically defined populations of inhibitory interneuron are selective in their post-synaptic targets.

Two populations of neurokinin 1 receptor-expressing projection neurons in lamina I of the rat spinal cord that differ in AMPA receptor subunit composition and density of excitatory synaptic input.

Lamina I of the spinal cord contains many projection neurons that express the neurokinin 1 receptor (NK1r). It has been reported that these cells can undergo long-term potentiation (LTP), which may result from insertion of AMPA-type glutamate receptors (AMPArs) containing GluA1 or GluA4 subunits. We therefore investigated synaptic AMPAr expression on these cells with immunocytochemistry following antigen-retrieval. We also examined their density of glutamatergic input (by analysing AMPAr synaptic puncta and contacts from glutamatergic boutons), and phosphorylation of extracellular signal-regulated kinases (pERKs) following noxious stimulation. Our results indicate that there are two populations of NK1r-expressing projection neurons: large GluA4(+)/GluA1(-) cells with a high density of glutamatergic input and small GluA1(+)/GluA4(-) cells with a much lower input density. Results from pERK experiments suggested that the two groups may not differ in the types of noxious stimulus that activate them. Glutamatergic synapses on distal dendrites of the large cells were significantly longer than those on proximal dendrites, which presumably compensates for the greater attenuation of distally-generated excitatory postsynaptic currents (EPSCs). Both types of cell received contacts from peptidergic primary afferents, however, on the large cells these appeared to constitute over half of the glutamatergic synapses, and were often associated with elongated AMPAr puncta. This suggests that these afferents, which probably contain substance P, provide a powerful, secure synaptic input to large NK1r-expressing projection neurons. These results demonstrate the importance of GluA4-containing AMPArs in nociceptive transmission and raise the possibility that different forms of LTP in lamina I projection neurons may be related to differential expression of GluA1/GluA4.

Soma size distinguishes projection neurons from neurokinin 1 receptor-expressing interneurons in lamina I of the rat lumbar spinal dorsal horn.

Lamina I of the spinal dorsal horn contains neurons that project to various brain regions, and approximately 80% of these projection cells express the neurokinin 1 receptor (NK1r), the main receptor for substance P. Two populations of NK1r-immunoreactive neurons have been identified in lamina I: small weakly immunoreactive cells and large cells with strong immunolabelling [Cheunsuang O and Morris R (2000) Neuroscience 97:335-345]. The main aim of this study was to test the hypothesis that the large cells are projection neurons and that the small cells are interneurons. Projection neurons were identified by injection of tracers into the caudal ventrolateral medulla and lateral parabrachial area, and this was combined with immunostaining for NK1r. We found a bimodal size distribution for NK1r-immunoreactive neurons. The small cells (with somatic cross-sectional areas <200 microm(2)) showed weak immunoreactivity, while immunostaining intensity was variable among the large cells. Virtually all (99%) of the immunoreactive cells with soma areas >200 microm(2) were retrogradely labelled, while only 10% of retrogradely labelled cells were smaller than this. Soma sizes of retrogradely labelled neurons that lacked NK1r did not differ from those of NK1r-expressing projection neurons. It has been suggested that a population of small pyramidal projection neurons that lack NK1r may correspond to cells activated by innocuous cooling, and we therefore assessed the morphology of retrogradely labelled cells that were not NK1r-immunoreactive. Fifteen percent of these were pyramidal, but these did not differ in size from pyramidal NK1r-immunoreactive projection neurons. These results confirm that large NK1r-immunoreactive lamina I neurons are projection cells, and suggest that the small cells are interneurons. Since almost all of the NK1r-immunoreactive cells with soma size >200 microm(2) were retrogradely labelled, cells of this type can be identified as projection cells in anatomical studies.

Tactile allodynia can occur in the spared nerve injury model in the rat without selective loss of GABA or GABA(A) receptors from synapses in laminae I-II of the ipsilateral spinal dorsal horn.

Although there is evidence that reduced inhibition in the spinal dorsal horn contributes to neuropathic pain, the mechanisms that underlie this are poorly understood. We have previously demonstrated that there is no loss of neurons from laminae I-III in the spared nerve injury (SNI) model [Polgár E, Hughes DI, Arham AZ, Todd AJ (2005) Loss of neurons from laminas I-III of the spinal dorsal horn is not required for development of tactile allodynia in the SNI model of neuropathic pain. J Neurosci 25:6658-6666]. In this study we investigated whether there was a difference between ipsilateral and contralateral sides in the levels of GABA, the vesicular GABA transporter (VGAT), or the beta3 subunit of the GABA(A) receptor at synapses in the medial part of the superficial dorsal horn in this model. Tissue from rats that had undergone SNI 4 weeks previously was examined with an electron microscopic immunogold method to reveal GABA, following pre-embedding detection of GABA(A) beta3 to allow identification of GABAergic terminals. Assessment of labeling for the GABA(A) beta3 subunit and VGAT was performed by using immunofluorescence and confocal microscopy. We found no difference in the intensity of immunolabeling for any of these markers on the two sides of the superficial dorsal horn. These results suggest that there is no significant loss of GABAergic boutons from the denervated area after SNI (which is consistent with the finding that neuronal death does not occur in this model) and that there is no depletion of GABA or GABA(A) receptors at GABAergic synapses within this region. An alternative explanation for disinhibition after nerve injury is that it results from reduced excitatory drive to GABAergic dorsal horn neurons following loss of primary afferent input to these cells.

Characterization of neurons that express preprotachykinin B in the dorsal horn of the rat spinal cord.

Although it is established that neurokinin B is expressed by some neurons in laminae I-III of the rat spinal dorsal horn, little is known about the proportions of cells in these laminae that express neurokinin B, or whether these are excitatory or inhibitory neurons. Neurokinin B is derived from preprotachykinin B, and we have used an antibody against preprotachykinin B to address these issues. We found that preprotachykinin B-immunoreactive neurons were present throughout laminae I-III, constituting 10-11% of the neuronal population in laminae I-II, and 4% of that in lamina III. They formed a prominent band in the ventral half of lamina II (where they made up 16% of the population) and the dorsalmost part of lamina III. The great majority (99%) of preprotachykinin B-immunoreactive axonal boutons contained the vesicular glutamate transporter 2, while none contained glutamic acid decarboxylase. Since most of these boutons are likely to be derived from local preprotachykinin B-expressing cells, these observations suggest that most of the latter are excitatory interneurons. Although 9% of preprotachykinin B-labeled axonal varicosities were substance P-immunoreactive, none contained calcitonin gene-related peptide, which is consistent with reports that neurokinin B is not expressed by primary afferent axons. Many of the preprotachykinin B-immunoreactive cells contained compounds that are present in putative excitatory neurons in laminae I-III: calbindin (84%), protein kinase Cgamma (76%) or somatostatin (31%). However, there was little or no overlap between preprotachykinin B and three other markers associated with excitatory neurons in these laminae: the mu opioid receptor MOR-1, the neurokinin 1 receptor and neurotensin. These results suggest that neurokinin B is expressed by specific populations of excitatory neurons in the superficial dorsal horn. By examining expression of Fos protein in response to intraplantar injection of formaldehyde we provide evidence that many of the preprotachykinin B cells in lamina I and the outer part of lamina II respond to noxious stimulation.

Lack of evidence for significant neuronal loss in laminae I-III of the spinal dorsal horn of the rat in the chronic constriction injury model.

Peripheral nerve injury leads to structural and functional changes in the spinal dorsal horn, and these are thought to be involved in the development of neuropathic pain. In the chronic constriction injury (CCI) model, abnormal 'dark' neurons and apoptotic nuclei have been observed in laminae I-III of the dorsal horn in the territory innervated by the injured sciatic nerve. These findings have been taken as evidence that there is significant neuronal death in this model, and it has been suggested that loss of inhibition resulting from death of GABAergic inhibitory interneurons contributes to the neuropathic pain. However, loss of neurons from the dorsal horn has not been directly demonstrated in neuropathic models, even though this issue is of considerable importance for our understanding of the mechanisms that underlie neuropathic pain. In this study, we have looked for evidence of neuronal death by using a stereological method (the optical disector) with NeuN-immunostaining, and examining spinal cords of naïve rats, and of rats that had undergone CCI or sham operations. All of the CCI animals showed clear signs of thermal hyperalgesia. However, the numbers of neurons in laminae I-III of the ipsilateral dorsal horn in these animals did not differ significantly from those on the contralateral side, nor from those of sham-operated or naïve animals. These results do not, therefore, support the suggestion that there is significant neuronal death in the dorsal horn in this model.

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.

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 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.

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.

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 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.

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.

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.

The types of neuron which contain protein kinase C gamma in rat spinal cord.

Protein kinase C (PKC) is thought to have a role in sensitization of dorsal horn neurons in certain pain states, and a recent study has reported that mice which lack the gamma isoform (PKCgamma) show reduced neuropathic pain after peripheral nerve injury. Although PKCgamma is present at high levels in the ventral part of lamina II we have limited information concerning the types of neuron in which it is located. In this study we have used immunocytochemistry to characterise the neurons which contain PKCgamma. Immunoreactive neurons were concentrated in ventral lamina II, but were also present in lamina III. Some weakly-immunoreactive neurons were located in the dorsal part of lamina II and in lamina I. The great majority (92%) of cells with PKCgamma were not GABA-immunoreactive, and these cells are likely to be excitatory interneurons. Dual-immunofluorescence labelling showed that PKCgamma was not randomly distributed amongst non-GABAergic neurons, since it was present in 76% of cells with neurotensin and 45% of those with somatostatin, but only 5% of those with the mu-opioid receptor (MOR-1). Cells with the neurokinin 1 receptor are found in lamina I and lamina III, and PKCgamma was present in 22% and 37% of these populations, respectively. These results suggest that excitatory interneurons in laminae II and III which lack the micro-opioid receptor may have a significant role in generating neuropathic pain.

GABAergic neurons that contain neuropeptide Y selectively target cells with the neurokinin 1 receptor in laminae III and IV of the rat spinal cord.

Neuropeptide Y (NPY) is contained in a population of GABAergic interneurons in the spinal dorsal horn and, when administered intrathecally, can produce analgesia. We previously identified a strong monosynaptic link between substance P-containing primary afferents and cells in lamina III or IV with the neurokinin 1 (NK1) receptor. Because some of these cells belong to the spinothalamic tract, they are likely to have an important role in pain mechanisms. In this study, we used confocal microscopy to examine the input to lamina III/IV NK1 receptor-immunoreactive neurons from NPY-containing axons. All of the cells studied received a dense innervation from NPY-immunoreactive axons, and electron microscopy revealed that synapses were often present at points of contact. Most NPY-immunoreactive boutons were also GABAergic, which supports the suggestion that they are derived from local neurons. The association between NPY-containing axons and NK1 receptor-immunoreactive neurons was specific, because postsynaptic dorsal column neurons (which were located in laminae III-V but did not possess NK1 receptors) and lamina I neurons with the NK1 receptor received significantly fewer contacts from NPY-immunoreactive axons. In addition, the NK1 receptor-immunoreactive lamina III/IV cells received few contacts from nitric oxide synthase-containing axons (which belong to a different population of GABAergic dorsal horn neurons). The NPY-containing axons appeared to be targeted to the NK1 receptor-immunoreactive neurons themselves rather than to their associated substance P-immunoreactive inputs. The dense innervation of these cells by NPY-containing axons suggests that they may possess receptors for NPY and that activation of these receptors may contribute to NPY-mediated analgesia.

Immunohistochemical localization of neurokinin-l receptor in the lumbar spinal cord of young rats: morphology and distribution.

The type and distribution of neurokinin-1 (NK-1) receptor-expressing neurones were studied in young (14-day-old) rats' lumbar spinal cord using pre-embedding immunohistochemistry. The heaviest immunoreactivity was observed in the middle part and lateral fourth of lamina I where the great majority of immunoreactive perikarya represented fusiform and multipolar cells. In lamina II the middle and medial part showed moderate immunoreactivity, most of the cells resembled stalked cells. In lamina III the labelled perikarya were evenly distributed, while those in lamina IV accumulated mainly in the lateral part. In both laminae most of the labelled neurones represented central cells, the rest of them belonged to the antenna-type cells with long dorsally directed dendrites penetrating the superficial laminae. The immunoreactivity in laminae V-VII was uniform and relatively weak. In lamina VIII the immunopositive perikarya were encountered only rarely while in lamina IX virtually all motoneurones showed weak immunoreactivity. Lamina X contained small, multipolar and fusiform labelled perikarya. In conclusion, we found that the general appearance of the NK-1 receptor immunostaining and the major type of NK-I receptor-expressing neurones were similar to that found previously in adult spinal cord. Using the same method as Brown and colleagues the number of labelled NK- 1 receptor immunoreactive cells was similar in young and adult animals except lamina I where the number of immunoreactive neurones was twice that in adults.

Neurokinin-1 receptor expression in dorsal root ganglion neurons of young rats.

The expression of neurokinin-1 receptors was studied in the fourth lumbar dorsal root ganglia of young rats using immunohistochemical and electrophysiological techniques. Use of a specific immunoserum raised against the C-terminal fragment of rat neurokinin-1 receptor revealed immunoreactivity in 32 +/- 1.5% of dorsal root ganglion neurons. The diameter of the majority of the neurokinin-1 receptor immunostained neurons was smaller than 30 microm. Double immunohistochemical labelling using neurokinin-1 receptor and substance P antibodies revealed that about 1/3 of the neurokinin-1 receptor expressing neuron contains substance P. Likewise, about 1/3 of the substance P producing DRG cells expressed the neurokinin-1 receptor. Superfusion of substance P (1 microM) to an in vitro preparation of the fourth lumbar dorsal root ganglion induced a reversible long-lasting depolarization as measured by extracellular suction electrodes attached to the dorsal roots. This response to substance P was only partially antagonized by the selective neurokinin-1 receptor antagonist RP 67580 (1 microM). Intracellular recordings distinguished between Aalpha/beta-, Adelta- and C-sub-types of ganglion neurons. Superfusion of substance P (1 microM) evoked excitatory responses in Adelta- and C-type neurons. These results demonstrate the expression of functional neurokinin-1 receptors on a subpopulation of Adelta- and C-type sensory ganglion neurons. Our data suggest the possible physiological importance of peripheral neurokinin-1 receptors located on dorsal root ganglion neurons.

Alterations of substance P immunoreactivity in lumbar and thoracic segments of rat spinal cord in ultraviolet irradiation induced hyperalgesia of the hindpaw.

Substance P immunostaining was quantified on sections from the 4th-5th lumbar and midthoracic spinal segments of rats at the peak of hyperalgesia following ultraviolet irradiation-induced inflammation of one hindpaw. The area of the immunostaining in the lumbar dorsal horn was significantly decreased on both sides by 50%, while in the thoracic spinal cord, it was increased by 18% on the contralateral and stayed unchanged on the ipsilateral side.

A quantitative study of neurons which express neurokinin-1 or somatostatin sst2a receptor in rat spinal dorsal horn.

The neurokinin-1 and somatostatin sst2a receptors have both been identified on spinal cord neurons. In this study we have estimated the proportions of neurons in different parts of the spinal cord which express these receptors, by using a monoclonal antibody against a neuronal nuclear protein named NeuN and combining the optical disector method with confocal microscopy. The NeuN antibody was initially tested on over 3200 neurons identified with antisera against a variety of compounds, including neuropeptides, enzymes and receptors, and also on astrocytes and oligodendrocytes. All of the neurons, but none of the glial cells that were examined possessed NeuN-immunoreactivity, which suggests that NeuN is a reliable marker for all spinal cord neurons. We found that approximately 45% of neurons in lamina I, 23-29% of those in laminae IV-VI and 18% in lamina X possessed the neurokinin-1 receptor, while the receptor was present on a smaller proportion of neurons in laminae II and III (6% and 11%, respectively). Thirteen percent of lamina I neurons and 15% of those in lamina II expressed the sst2a receptor. To provide further information about the types of neuron which possess the sst2a receptor, we searched for possible co-existence with the neurokinin-1 receptor as well as with GABA and glycine. sst2a and neurokinin-1 receptors were not co-localized on neurons in laminae I and II. All of the sst2a-immunoreactive neurons examined were also GABA-immunoreactive, and 83.5% were glycine-immunoreactive, indicating that the receptor is located on inhibitory neurons in the superficial dorsal horn. These results demonstrate the proportions of neurons in each region of the spinal cord which can be directly activated by substance P or somatostatin acting through these receptors. Levels of receptors can change in pathological states, and this method could be used to determine whether or not these changes involve alterations in the number of neurons which express receptors. In addition, the method can be used to estimate the sizes of neurochemically-defined populations of spinal cord neurons.

Spinal neurokinin NK1 receptor down-regulation and antinociception: effects of spinal NK1 receptor antisense oligonucleotides and NK1 receptor occupancy.

To define the effects of antisense oligonucleotides on spinal neurokinin 1 (NK1) receptor function in nociceptive processing, several antisense oligonucleotides directed against the NK1 receptor mRNA were intrathecally injected into rats via an implanted catheter, and their effect on the behavioural response to formalin injected into the paw was assessed. We observed that there was no significant reduction of pain behaviour or immunostaining of spinal NK1 receptors after repeated daily intrathecal treatment with an antisense oligonucleotide. However, spinal application of substance P (SP) in the antisense oligonucleotide-treated animals resulted in a profound and long-lasting reduction in the behavioural response to formalin injection, and a parallel reduction in the NK1 receptor immunoreactivity normally observed in spinal dorsal horn. Intrathecal SP in the control groups, i.e., rats treated with an oligonucleotide containing four mismatched bases, the corresponding sense oligonucleotide, a mixture of the sense and the antisense oligonucleotides, in each case had no effect. The effects of SP were blocked by NK1 receptor antagonists and were not mimicked by NMDA. The mechanism underlying these effects is not clear. It may be due to partial degradation of the internalised receptors, which cannot be replaced by newly synthesised receptors because of the action of the NK1 antisense oligonucleotide.

Direct evidence of an extensive GABAergic innervation of the spinal dorsal horn by fibres descending from the rostral ventromedial medulla.

A long line of studies emphasizes the contribution of serotonergic fibres descending from the rostral ventromedial medulla in the control of spinal nociceptive information processing. A growing body of evidence, however, suggests that the relative contribution of serotonin to the mediation of spinal neuronal activity from the rostral ventromedial medulla may require re-evaluation. It has recently been substantiated that, in addition to the serotonergic fibres, the spinal dorsal horn receives an abundant non-serotonergic projection from the rostral ventromedial medulla. Furthermore, stimulation in the rostral ventromedial medulla could result in a powerful inhibition of nociceptive spinothalamic tract cells without any detectable serotonin release in the dorsal horn. After labelling raphe-spinal axons and axon terminals in the rat by iontophoretic injections of the anterograde axonal tracer Phaseolus vulgaris leucoagglutinin into the central region of the rostral ventromedial medulla (nucleus raphe magnus) and revealing GABA and glycine immunoreactivities of the labelled raphe-spinal terminals and their postsynaptic targets by postembedding immunocytochemical methods, here we demonstrate an extensive GABAergic projection from the rostral ventromedial medulla to the spinal dorsal horn. We show that the majority of the labelled raphe-spinal terminals in laminae I-IIo and IV-V contain GABA and some of the GABA-immunoreactive terminals are also immunoreactive for glycine. We also disclose that GABA-immunoreactive raphe-spinal terminals establish synaptic contacts primarily with GABA- and glycine-negative, presumably excitatory, spinal neurons, including Calbindin-D28k- as well as parvalbumin-immunoreactive cells in both laminae I-IIo and IV-V. The results suggest that volleys in fibres descending from the rostral ventromedial medulla may evoke GABA release from raphe-spinal terminals, and the released GABA, in some cases probably acting together with glycine, might play a crucial, as yet mostly unidentified, role in the inhibition of nociceptive information processing in the dorsal horn of the spinal cord.

Sub-population of capsaicin sensitive primary afferent neurons in thoracic, lumbar and sacral dorsal root ganglion in young rats revealed by stimulated cobalt uptake.

The number of dorsal root ganglion (DRG) neurons, the relative number of capsaicin sensitive DRG cells and the diameter distribution of these neurons were investigated in the thoracic 11, lumbar 5 and sacral 1 DRG ganglia in young rats. The capsaicin sensitivity of DRG cells was shown by the stimulated cobalt uptake technique in in vitro conditions. Cobalt labelled and non-labelled neurons were counted using the dissector method. Our results show that the total number of DRG cells in the Th11, L5 and S1 segments (4200-6500 per ganglion) were not significantly different from each other and about 8% of these cells were capsaicin sensitive in the segments studied. These findings also show that the capsaicin sensitive DRG cells belong to the small diameter DRG cell population. Control experiments on neonatally capsaicin injected animals indicate that the capsaicin stimulated cobalt uptake provides selective and specific staining of capsaicin sensitive DRG neurons.