Identification of somatostatin sst2(a) receptor expressing neurones in central regions involved in nociception

Somatostatin is a neuromodulator and neurotransmitter in the central nervous system. Administration of somatostatin to the spinal cord or brain areas involved in nociception has been shown to result in analgesia. Little information is available about the somatostatin receptor types which may be involved in mediating the neuromodulatory and analgesic effects of the peptide. To define the neuronal systems expressing the sst_2(a) receptor in brain areas associated with analgesia, immunohistochemical co-localisation studies were carried out in the periaqueductal grey (PAG) and spinal cord using an antibody specific for the sst_2(a) receptor. To further define sst_2(a) receptor expressing neurones, sst_2(a) receptor immunohistochemistry was combined with retrograde tracing using fluorogold. In the PAG, sst_2(a) receptor expressing neurones were found to co-express calbindin D28k (36%), the glutamate transporter EAAC-1 (25%), and GABA transporter GAT-1 (10%). A total of 65% of sst_2(a) positive neurones projected to the thalamus. In the spinal cord, the sst_2(a) receptor shows cellular co-localisation with EAAC-1 and GAT-1. Immunohistochemistry and receptor autoradiography using [¹²⁵I]BIM 23027 after dorsal rhizotomy of the lumbar dorsal roots, L4 and L5, suggests that the somatostatin sst_2(a) receptor is not present on primary afferent neurones. Dorsal hemisections of the mid thoracic cord did not alter the immunohistochemical signal for the somatostatin sst_2(a) receptor, providing further evidence for an intrinsic localisation of the receptor protein in the dorsal horn of the spinal cord. These data show that the somatostatin sst_2(a) receptor exists on morphologically and neurochemically heterogenous neurones and is closely associated with brain areas involved in analgesia and the modulation of nociception.     

Selective distribution of GABA(A) receptor subtypes in mouse spinal dorsal horn neurons and primary afferents

In the spinal cord dorsal horn, presynaptic GABA_A receptors (GABA_ARs) in the terminals of nociceptors as well as postsynaptic receptors in spinal neurons regulate the transmission of nociceptive and somatosensory signals from the periphery. GABA_ARs are heterogeneous and distinguished functionally and pharmacologically by the type of α subunit variant they contain. This heterogeneity raises the possibility that GABA_AR subtypes differentially regulate specific pain modalities. Here, we characterized the subcellular distribution of GABA_AR subtypes in nociceptive circuits by using immunohistochemistry with subunit‐specific antibodies combined with markers of primary afferents and dorsal horn neurons. Confocal laser scanning microscopy analysis revealed a distinct, partially overlapping laminar distribution of α1–3 and α5 subunit immunoreactivity in laminae I–V. Likewise, a layer‐specific pattern was evident for their distribution among glutamatergic, γ‐aminobutyric acid (GABA)ergic, and glycinergic neurons (detected in transgenic mice expressing vesicular glutamate transporter 2–enhanced green fluorescent protein [vGluT2–eGFP], glutamic acid decarboxylase [GAD]67–eGFP, and glycine transporter 2 (GlyT2)–eGFP, respectively). Finally, all four subunits could be detected within primary afferent terminals. C‐fibers predominantly contained either α2 or α3 subunit immunoreactivity; terminals from myelinated (Aβ/Aδ) fibers were colabeled in roughly equal proportion with each subunit. The presence of axoaxonic GABAergic synapses was determined by costaining with gephyrin and vesicular inhibitory amino acid transporter to label GABAergic postsynaptic densities and terminals, respectively. Colocalization of the α2 or α3 subunit with these markers was observed in a subset of C‐fiber synapses. Furthermore, gephyrin mRNA and protein expression was detected in dorsal root ganglia. Collectively, these results show that differential GABA_AR distribution in primary afferent terminals and dorsal horn neurons allows for multiple, circuit‐specific modes of regulation of nociceptive circuits. J. Comp. Neurol. 520:3895–3911, 2012. © 2012 Wiley Periodicals, Inc.     

The cellular localization of 5-HT2A receptors in the spinal cord and spinal ganglia of the adult rat

The localization of serotonin_2A (5-HT_2A) receptors in the adult rat spinal cord and dorsal root ganglia was examined by using a polyclonal antibody that recognizes the C-terminus peptides of the mouse 5-HT_2A receptor. Positive cell bodies of 5-HT_2A receptor were found in several regions of the spinal cord. Generally, large-to-intermediate sized neuronal cell bodies were intensely immunolabeled. Motoneurons in the ventral horn were the most intensely labeled. Dot-like immunoreactive profiles were located beneath the cell membrane of motoneurons. Neuronal somata in the intermediolateral nucleus of the thoracic spinal cord were moderately labeled. The immunoreactivity in the dorsal horn was weak. A considerable number of glial cell bodies in the white matter were immunostained. The majority of both small and large sized neurons were 5-HT_2A immunopositive in the dorsal root ganglion.     

Hyperpolarization-activated and cyclic nucleotide-gated cation channel subunit 2 ion channels modulate synaptic transmission from nociceptive primary afferents containing substance P to secondary sensory neurons in laminae I-IIo of the rodent spinal dorsal horn

We have previously demonstrated that hyperpolarization-activated and cyclic nucleotide-gated cation channel subunit 2 (HCN2) is expressed by terminals of peptidergic nociceptive primary afferents in laminae I-IIo of the rat spinal dorsal horn. In this study, we investigated the possible neurotransmitters and postsynaptic targets of these HCN2-expressing primary afferent terminals in the superficial spinal dorsal horn by using immunocytochemical methods. We demonstrated that HCN2 widely colocalizes with substance P (SP), and that HCN2-positive terminals that are also immunoreactive for SP form serial close appositions with dendrites and perikarya of neurokinin 1 receptor-immunoreactive neurons. It was also found that HCN2-immunoreactive terminals are frequently apposed to neurons that are immunoreactive for calbindin, micro-opioid receptor and the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor subunit GluR2, markers for excitatory interneurons. Investigating HCN2 immunoreactivity in glutamic acid decarboxylase 65-green fluorescent protein transgenic mice, we found that HCN2-positive terminals occasionally also contact cells that contain an isoform of glutamic acid decarboxylase (glutamic acid decarboxylase 65), a marker for GABAergic inhibitory neurons. Application of ZD7288, an antagonist of HCN channels, onto neurons that were recorded in spinal cord slices with whole-cell patch-clamp electrodes reduced the number of monosynaptic excitatory postsynaptic potentials evoked by electrical stimulation of primary afferents at nociceptive intensities. The results suggest that HCN2 may contribute to the modulation of membrane excitability of SP-containing nociceptive primary afferent terminals, may increase the reliability of synaptic transmission from primary afferents to secondary sensory neurons and thus may play a role in the fine-tuning of pain transmission from nociceptive primary afferents to neurons in the spinal dorsal horn.     

Time-dependent influence of the somatostatin analogue octreotide on the proliferation of rat astrocytes and glioma cells

The somatostatin receptor subtype sst₂ was visualized by immunostaining on cultivated rat astrocytes and C6 rat glioma cells. Octreotide, a metabolically stable sst₂ agonist reduced [³H]thymidine incorporation into DNA of both cell types dose-dependently only after short-time application (2–5 h), after prolonged incubation (>12 h) no antiproliferative effect was measurable. We conclude that sst₂ receptors may be desensitized. Thus, desensitization might hinder application of octreotide to reduce glial tumour growth.     

Characterization of forms of immunoreactive somatostatin in sensory neuron and normal and deafferented spinal cord

In order to determine the contribution made by primary sensory afferents and supraspinal projections to the immunoreactive somatostatin (IRS) content of the spinal cord, measurements were made of the concentration of IRS in the dorsal and ventral halves of the cord in cats subjected to unilateral lumbosacral dorsal rhizotomy (L1-S3) alone or combined with spinal cord transection. The molecular forms of IRS (characterized by gel chromatography) in L7 lumbar spinal cord, L6-S1 dorsal roots, ventral roots and dorsal root ganglia, and sciatic nerve were also determined. S14 was the predominant form in all tissues examined, but two additional molecular forms corresponding to S28 and S11.5 kdalton were present in dorsal root ganglia and spinal cord; S28 but not S11.5 kdalton was detected in both dorsal roots and sciatic nerves. These results indicate that S14 and S28 and S28 are transported along the central and peripheral processes of dorsal root ganglia, but that spinal cord S11.5 kdalton originates in the central nervous system. IRS in the dorsal horn was reduced by ca. 40% following dorsal root section. Neither disruption of descending pathways by spinal transection nor surgical isolation of the lumbar segements lowered cord somatostatin content below that produced by dorsal root section, indicating that most of the somatostatin within the cord arises from the dorsal root and from neurons in local spinal segments. Although the total content of IRS in the dorsal horn was reduced by ca. 40% following dorsal rhizotomy, the pattern of molecular forms was not changed accordingly. Since S14 and S28 but not S11.5 kdalton are transported via the dorsal root, the dorsal root section would be predicted to produce a relatively greater decrease in S14 and S28 than in S11.5 kdalton. Therefore, failure to find a selective loss of S14 and S28 suggests that dorsal rhizotomy affects dorsal horn IRS content not only by removing afferent input but possibly also by modifyinh the processing of IRS by the remaining somatostatinergic neurons.     

Expression of hyperpolarization-activated and cyclic nucleotide-gated cation channel subunit 2 in axon terminals of peptidergic nociceptive primary sensory neurons in the superficial spinal dorsal horn of rats

Hyperpolarization-activated cyclic nucleotide-gated cation channel proteins (HCN1-4), which are potentially able to modulate membrane excitability, are abundantly expressed by neurons in spinal dorsal root ganglia (DRG). In the present experiment, we investigated whether HCN2 protein is confined exclusively to the perikarya of DRG neurons or is transported from the somata to the central axons of DRG neurons that terminate in the spinal dorsal horn. Using immunohistochemical methods, we have demonstrated that laminae I-IIo of the superficial spinal dorsal horn of the adult rat spinal cord show a strong punctate immunoreactivity for HCN2. Dorsal rhizotomy resulted in a complete loss of immunostaining in the dorsal horn, suggesting that HCN2 is confined to axon terminals of primary afferents. In double labelling immunohistochemical studies, we have also shown that HCN2 widely co-localizes with calcitonin gene-related peptide, but is almost completely segregated from isolectin-B4 binding, indicating that HCN2 is primarily expressed in peptidergic nociceptive primary afferents. The expression of HCN2 in central terminals of peptidergic primary afferents was also verified with electron microscopy. Utilizing the pre-embedding nanogold method, we found that HCN2 is largely confined to axon terminals with dense-core vesicles. Within these terminals, some of the silver grains marking the accurate location of HCN2 molecules were associated with the cell membrane, and others were scattered in the axoplasm. Within the cell membrane, HCN2 was found almost exclusively in extrasynaptic locations. The results suggest that HCN2 may contribute to the modulation of membrane excitability of nociceptive primary afferent terminals in the spinal dorsal horn.     

CSF somatostatin is elevated in patients with postzoster neuralgia

We evaluated the concentration of the neuropeptide somatostatin (SOM) in the CSF of patients with several neurologic diseases. Since SOM is localized in high concentrations in primary sensory pathways, such as the dorsal root ganglia and dorsal horn of the spinal cord, it might be involved in conditions of chronic pain due to functional alterations of nociceptive neurons, such as postinfectious zoster neuralgia. Our study indicated a marked elevation of SOM in patients suffering from postzoster neuralgia compared with controls. Comparison with other neurologic diseases revealed decreased CSF SOM levels in Parkinson's and Alzheimer's disease, unchanged values in patients with amyotrophic lateral sclerosis, and increased concentrations in patients with brain tumors. In neurodegenerative disorders, SOM levels in CSF seemed to reflect the anatomic distribution as well as a reduction or preservation of the peptide in certain brain areas affected by the disease process. In postzoster patients, postinfectious degeneration of dorsal root ganglia cells might cause deafferentation of dorsal horn neurons and activation of SOM-containing systems with increased release either locally from neurons in the dorsal horn of the spinal cord or from descending fiber projections. The results suggested that SOM may take part in the modulation of nociceptive responses.     

Somatostatin 2a receptors are not expressed on functionally identified respiratory neurons in the ventral respiratory column of the rat

Microinjection of somatostatin (SST) causes site‐specific effects on respiratory phase transition, frequency, and amplitude when microinjected into the ventrolateral medulla (VLM) of the anesthetized rat, suggesting selective expression of SST receptors on different functional classes of respiratory neurons. Of the six subtypes of SST receptor, somatostatin 2a (sst_2a) is the most prevalent in the VLM, and other investigators have suggested that glutamatergic neurons in the preBötzinger Complex (preBötC) that coexpress neurokinin‐1 receptor (NK1R), SST, and sst_2a are critical for the generation of respiratory rhythm. However, quantitative data describing the distribution of sst_2a in respiratory compartments other than preBötC, or on functionally identified respiratory neurons, is absent. Here we examine the medullary expression of sst_2a with particular reference to glycinergic/expiratory neurons in the Bötzinger Complex (BötC) and NK1R‐immunoreactive/inspiratory neurons in the preBötC. We found robust sst_2a expression at all rostrocaudal levels of the VLM, including a large proportion of catecholaminergic neurons, but no colocalization of sst_2a and glycine transporter 2 mRNA in the BötC. In the preBötC 54% of sst_2a‐immunoreactive neurons were also positive for NK1R. sst_2a was not observed in any of 52 dye‐labeled respiratory interneurons, including seven BötC expiratory‐decrementing and 11 preBötC preinspiratory neurons. We conclude that sst_2a is not expressed on BötC respiratory neurons and that phasic respiratory activity is a poor predictor of sst_2a expression in the preBötC. Therefore, sst_2a is unlikely to underlie responses to BötC SST injection, and is sparse or absent on respiratory neurons identified by classical functional criteria. J. Comp. Neurol. 524:1384–1398, 2016. © 2015 Wiley Periodicals, Inc.     

Neuronal and glial localization of the cannabinoid-1 receptor in the superficial spinal dorsal horn of the rodent spinal cord

A long line of experimental evidence indicates that endogenous cannabinoid mechanisms play important roles in nociceptive information processing in various areas of the nervous system including the spinal cord. Although it is extensively documented that the cannabinoid‐1 receptor (CB₁‐R) is strongly expressed in the superficial spinal dorsal horn, its cellular distribution is poorly defined, hampering our interpretation of the effect of cannabinoids on pain processing spinal neural circuits. Thus, we investigated the cellular distribution of CB₁‐Rs in laminae I and II of the rodent spinal dorsal horn with immunocytochemical methods. Axonal varicosities revealed a strong immunoreactivity for CB₁‐R, but no CB₁‐R expression was observed on dendrites and perikarya of neurons. Investigating the co‐localization of CB₁‐R with markers of peptidergic and non‐peptidergic primary afferents, and axon terminals of putative glutamatergic and GABAergic spinal neurons we found that nearly half of the peptidergic (immunoreactive for calcitonin gene‐related peptide) and more than 20% of the non‐peptidergic (binding isolectin B4) nociceptive primary afferents, more than one‐third and approximately 20% of the axon terminals of putative glutamatergic (immunoreactive for vesicular glutamate transporter 2) and GABAergic (immunoreactive for glutamic acid decarboxylase; GAD65 and/or GAD67) spinal interneurons, respectively, were positively stained for CB₁‐R. In addition to axon terminals, almost half of the astrocytic (immunoreactive for glial fibrillary acidic protein) and nearly 80% of microglial (immunoreactive for CD11b) profiles were also immunolabeled for CB₁‐R. The findings suggest that the activity‐dependent release of endogenous cannabinoids activates a complex signaling mechanism in pain processing spinal neural circuits into which both neurons and glial cells may contribute.     

Distribution of RET immunoreactivity in the rodent spinal cord and changes after nerve injury

RET (for “rearranged during transfection”) is a transmembrane tyrosine kinase signaling receptor for members of the glial cell line‐derived neurotrophic factor (GDNF) family of ligands. We used RET immunohistochemistry (IHC), double‐labeling immunofluorescence (IF), and in situ hybridization (ISH) in adult naïve and nerve‐injured rats to study the distribution of RET in the spinal cord. In the dorsal horn, strong RET‐immunoreactive (‐ir) fibers were abundant in lamina II‐inner (II_i), although this labeling was preferentially observed after an antigen‐unmasking procedure. After dorsal rhizotomy, RET‐ir fibers in lamina II_i completely disappeared from the dorsal horn, indicating that they were all primary afferents. After peripheral axotomy, RET‐ir in primary afferents decreased in lamina II_i and appeared to increase slightly in laminae III and IV. RET‐ir was also observed in neurons and dendrites throughout the dorsal horn. Some RET‐ir neurons in lamina I had the morphological appearance of nociceptive projection neurons, which was confirmed by the finding that 53% of RET‐ir neurons in lamina I colocalized with neurokinin‐1. GDNF‐ir terminals were in close proximity to RET‐ir neurons in the superficial dorsal horn. In the ventral horn, RET‐ir was strongly expressed by motoneurons, with the strongest staining in small, presumably γ‐motoneurons. Increased RET expression following peripheral axotomy was most pronounced in α‐motoneurons. The expression and regulation pattern of RET in the spinal cord are in line with its involvement in regenerative processes following nerve injury. The presence of RET in dorsal horn neurons, including nociceptive projection neurons, suggests that RET also has a role in signal transduction at the spinal level. This role may include mediating the effects of GDNF released from nociceptive afferent fibers. J. Comp. Neurol. 500:1136–1153, 2007. © 2006 Wiley‐Liss, Inc.     

Recovery of substance P but not somatostatin in the cat spinal cord after unilateral lumbosacral dorsal rhizotomy: A quantitative study

The dorsal horn of the cat spinal cord contains substance P and somatostatin within nerve endings which arise from cells located in dorsal root ganglia and from cells within the neuraxis. Previous studies from this laboratory have demonstrated that dorsal rhizotomy depletes both peptides from the dorsal horn. However, the changes in the two peptides differ. Substance P is at first severely depleted by dorsal rhizotomy and then recovers in part, whereas somatostatin is diminished less but does not recover. In the present experiments the validity of these conclusions which were based on anatomical observations has been evaluated quantitatively with the use of radioimmunoassay. After a 74% reduction at 10-14 days postoperative, substance P immunoreactivity in the deafferented dorsal horn shows a small, statistically significant recovery by 30 days to 60% of normal values. In contrast, somatostatin is reduced by 46% at 10-14 days but does not return significantly. As previously suggested by immunocytochemistry, dorsal rhizotomy produces no significant decline of either peptide in the ventral horn. The differing response of the two peptides is consistent with the hypothesis that intrinsic spinal substance P-containing neurons increase their projections (or their production of substance P) in the deafferented dorsal horn, but that somatostatin-containing neurons do not. Because synaptic number returns to normal in at least the deafferented lamina II of the cat yet substance P recovers only partially, it is likely that axons which contain transmitters other than substance P or somatostatin also increase the numbers of their terminals in response to dorsal rhizotomy.     

Neuropeptides in dissociated cell cultures of mammalian spinal cord and dorsal root ganglion

Immunohistochemical studies of leucine-enkephalin, somatostatin, vasoactive intestinal polypeptide and neurotensin were carried out in dissociated cell co-cultures of embryonic mouse spinal cord and dorsal root ganglion, using the peroxidase-antiperoxidase technique. Leucine-enkephalin immunoreactivity exceeded that of the other peptides in these coculture preparations. Leucine-enkephalin, substance P and somatostatin were also studied in spinal cord cultures (without dorsal root ganglia) and in dorsal root ganglia cultures (without spinal cord). Each of these peptides was present in only a small percentage (<10%) of perikarya and processes in spinal cord cultures. No leucine-enkephalin immunoreactivity was seen in dorsal root ganglion cultures; a considerable proportion of the processes were immunoreactive for substance P or somatostatin. These observations suggest that co-cultures of spinal cord and dorsal root ganglia can provide a simplified in vitro “model” of the nervous system for the study of peptidergic interactions.     

Role of kainate receptors in nociception

Nociceptive nerve fibers use -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.     

Subcellular Localization of the GABAA Receptor γ2 Subunit in the Rat Spinal Cord

The fine subcellular organization of the GABA_A receptor complex in the adult rat spinal ventral horn was analysed by immunocytochemistry using a specific polyclonal antiserum raised against the γ subunit. This subunit confers benzodiazepine sensitivity on the chloride channel of the GABA_A receptor. With both fluorescent and peroxidase staining, the immunoreactivity was mainly observed in the grey matter and more specifically in the dorsal and ventral horns on medium and large neurons. A high number of immunostained somata were clustered in regions corresponding to motor nuclei. On the neuronal surface, labelling appeared as fluorescent dots over the more diffuse staining that was present on the soma and proximal part of dendrites. At the ultrastructural level, peroxidase end product was in most cases associated with the internal side of postsynaptic differentiations facing terminal boutons enriched with pleiomorphic small clear vesicles. The positively stained synapses were encountered on proximal dendrites of neurons and throughout the neuropil of the ventral horn (layers VII-IX). An immunoreactivity on the postsynaptic membrane was occasionally found to decorate large pieces of membrane not directly apposed to presynaptic active zones. In addition, presynaptic labelling was observed at axoaxonic contacts and at extrasynaptic sites on membranes within boutons, sometimes themselves apposed to γ2 immunoreactivity. Finally, we also observed γ2 immunoreactivity at the cytosolic face of the plasma membrane of some glial elements. These results give morphological evidence for the involvement of GABA_A receptors in both post- and presynaptic inhibition in the rat spinal ventral horn. The presence of γ2 subunit immunoreactivity at these different synaptic contacts suggests that the two types of inhibition can be modulated by benzodiazepine drugs. The findings also provide anatomical evidence for the possible regulation of GABA release through an autoreceptor, and for GABAergic communication between neuronal and glial components.     

Differential expression patterns of K+/Cl− cotransporter 2 in neurons within the superficial spinal dorsal horn of rats

γ‐Aminobutyric acid (GABA)‐ and glycine‐mediated hyperpolarizing inhibition is associated with a chloride influx that depends on the inwardly directed chloride electrochemical gradient. In neurons, the extrusion of chloride from the cytosol primarily depends on the expression of an isoform of potassium–chloride cotransporters (KCC2s). KCC2 is crucial in the regulation of the inhibitory tone of neural circuits, including pain processing neural assemblies. Thus we investigated the cellular distribution of KCC2 in neurons underlying pain processing in the superficial spinal dorsal horn of rats by using high‐resolution immunocytochemical methods. We demonstrated that perikarya and dendrites widely expressed KCC2, but axon terminals proved to be negative for KCC2. In single ultrathin sections, silver deposits labeling KCC2 molecules showed different densities on the surface of dendritic profiles, some of which were negative for KCC2. In freeze fracture replicas and tissue sections double stained for the β3‐subunit of GABA_A receptors and KCC2, GABA_A receptors were revealed on dendritic segments with high and also with low KCC2 densities. By measuring the distances between spots immunoreactive for gephyrin (a scaffolding protein of GABA_A and glycine receptors) and KCC2 on the surface of neurokinin 1 (NK1) receptor‐immunoreactive dendrites, we found that gephyrin‐immunoreactive spots were located at various distances from KCC2 cotransporters; 5.7 % of them were recovered in the middle of 4–10‐µm‐long dendritic segments that were free of KCC2 immunostaining. The variable local densities of KCC2 may result in variable postsynaptic potentials evoked by the activation of GABA_A and glycine receptors along the dendrites of spinal neurons. J. Comp. Neurol. 523:1967–1983, 2015 © 2015 Wiley Periodicals, Inc.     

Partial sciatic nerve transection causes redistribution of pain-related peptides and lowers withdrawal threshold

Complete nerve transection results in loss of sensation and paralysis of the involved extremity. Such injury drastically reduces content of the nociceptive peptides, substance P, and somatostatin in the dorsal horn of the spinal cord and dorsal root ganglia innervating the limb. Partial nerve injuries occur more commonly in clinical practice, however, and frequently result in the development of chronic neuropathic pain. To investigate mechanisms underlying this pathologic pain syndrome, rats were subjected to partial sciatic nerve transection. Withdrawal thresholds determined with Von Frey hairs dropped dramatically in the operated limb. On postoperative Day 4, thresholds had decreased from 15 g to less than 5 g on the operated side, whereas those on the contralateral (unoperated) side or those from sham-operated rats did not change. Sciatic hemisection had no effect on total content of either substance P or somatostatin in the dorsal spinal cord and lumbar dorsal root ganglia as measured by radioimmunoassay on postoperative Days 4, 7, or 14. However, when examined immunohistochemically, there was a marked redistribution of both peptides associated with partial transection. On the contralateral side or in sham-operated rats, both substance P and somatostatin were confined to the superficial laminae of the dorsal horn. By contrast, on the operated side, content of both peptides was reduced by more than half in the superficial laminae. There was a compensatory increase in content in the deeper laminae where nociceptive peptides are not usually found. Redistribution of substance P and somatostatin may be due to axonal sprouting, increased peptide expression by interneurons, or aberrant expression of nociceptive peptides by neurons normally mediating mechanical sensation. The presence of increased levels of nociceptive peptides in regions of the spinal cord that mediate innocuous sensation may underlie development of allodynia.     

The actions of neuropeptides on dorsal horn neurons in the rat spinal cord slice preparation: an intracellular study

Responses of dorsal horn neurons to bath application of substance P, somatostatin and enkephalin were studied by intracellular recording in the neonatal spinal cord slice preparation. Substance P depolarized dorsal horn neurons and increased their excitability. The depolarization was most commonly associated with an increase in neuronal input resistance. Somatostatin and enkephalin hyperpolarized dorsal horn neurons and caused reduction or abolition of spontaneous firing. While the hyperpolarization produced by enkephalin was always associated with a fall in neuronal input resistance, in the case of somatostatin the similar effect was less consistently observed.     

Cervical primary afferent input to vestibulospinal neurons projecting to the cervical dorsal horn: An anterograde and retrograde tracing study in the cat

Vestibulospinal neurons in the caudal half of the medial and descending vestibular nuclei terminate in the cervical spinal cord, not only in the ventral horn and intermediate zone but also in the dorsal horn. The purpose of the present study was to examine whether the areas containing these vestibulospinal neurons are reached by cervical primary afferents. In one group of experiments, wheat germ agglutinin-horseradish peroxidase conjugate and horseradish peroxidase were pressure injected into spinal ganglia C₂-C₈ and revealed anterogradely labeled fibers and boutons in the caudal part (caudal to the dorsal cochlear nucleus) of the ipsilateral medial and descending vestibular nuclei. This projection was verified in experiments in which wheat germ agglutinin-horseradish peroxidase conjugate was microiontophoretically injected into the caudal half of either the medial or the descending vestibular nuclei and revealed retrogradely labeled cells only in ipsilateral spina ganglia C₂-C₇, with a maximum of cells in C₃. In another group of experiments, after microiontophoretic injections of Phaseolus vulgaris leucoagglutinin or Biocytin into either the medial or the descending vestibular nuclei, anterogradely labeled fibers and boutons were present in the cervical spinal cord, mainly bilaterally in the dorsal horn (laminae I–VI) but also, to a lesser extent, in the ventral horn and intermediate zone. The existence of a loop that relays cervical primary afferent information to vestibulospinal neurons projecting to the cervical spinal cord, in particular the dorsal horn, may have implications for vestibular control over local information processing in the cervical dorsal horn. © 1995 Wiley-Liss, Inc.     

Somatostatin-like neurons are expressed in fetal neocortical homografts in adult rat spinal cord

Fetal central nervous system homografts to adult spinal cord are considered a potential aid for recovery of function after paraplegia. This study utilizes somatostatin (SOM) immunohistochemistry to study the organization of an embryonic day 14 (E14) neocortical homograft into the spinal cord of an adult host over 6 postoperative months. Although the E14 homograft does not contain SOM-positive cells, SOM-reactive neurons are expressed by 30 days postimplantation and are still present in 6-month-old homografts. SOM-immunoreactive neurons are bitufted or multipolar and have dendrites that are confined to the graft. The homograft contains SOM-immunoreactive axons entering and/or exiting from lamina II in the host dorsal horn and SOM-positive homografted neurons send axons into the host ventral columns. These data show that the SOM peptide neocortical phenotype is preserved in homografts to spinal cord but there is anatomical host-homograft integration.