Anaesthesia for microlaryngeal and laser laryngeal surgery: impact of subglottic jet ventilation.

OBJECTIVE: Over the past 20 years, jet ventilation techniques have been developed to enable safe and controlled microlaryngoscopy and the accurate treatment of laryngeal pathology. This study examined how advances in jet ventilation tube design have facilitated safe endolaryngeal surgery. STUDY DESIGN: The study documented the development and use of the Jockjet subglottic jet ventilation tube system at the Prince of Wales Hospital, Sydney. The new system consisted of two components: a Teflon tube with an outer diameter of 4 mm at the larynx, and a companion ventilator. The facility for end-tidal carbon dioxide and distal airways pressure monitoring was incorporated via dedicated channels. The Venturi jet was produced via a covered tip to prevent trauma to the tracheal mucosa. SETTING: The Prince of Wales and Sydney Children's Hospitals, incorporated with The University of New South Wales. PATIENTS: From June 2002 to March 2008 inclusive, 1000 consecutive patients underwent microlaryngeal surgery at this institution. Subglottic jet ventilation, via the Jockjet tube, was employed for 332 patients. MAIN OUTCOME MEASURES: Anaesthetic safety and intra-operative surgical access. RESULTS: In all the 332 patients observed, surgical access was optimised and no adverse anaesthetic outcomes were encountered. CONCLUSION: Subglottic jet ventilation facilitates safe airway management during microlaryngeal and laser laryngeal surgery.

Peripheral nerve injury induces cannabinoid receptor 2 protein expression in rat sensory neurons.

We have localized cannabinoid receptor 2 protein in rat and mouse somatic sensory nervous system, using an antibody that recognizes mouse cannabinoid receptor 2. Little or no cannabinoid receptor 2 immunoreactivity was found in sections of naive rat or mouse dorsal root ganglia or spinal cord. This was in accord with the lack of detectable cannabinoid receptor 2 mRNA in (dorsal root ganglion) neurons by in situ hybridization experiments described in the literature. However, we could detect cannabinoid receptor 2 immunoreactivity following unilateral nerve damage-either by sciatic nerve section, or by spinal nerve ligation. It was localized to the superficial laminae of the dorsal horn of the spinal cord, ipsilateral to the nerve damage, coincident with the area of termination of damaged afferents which was marked by loss of isolectin B4 binding. This upregulation was not seen in cannabinoid receptor 2 null mice. The cannabinoid receptor 2 protein in spinal cord appeared to be expressed on sensory neuron afferent terminals as it colocalized with two markers of damaged afferents, namely growth associated protein-43 and the neuropeptide galanin. Moreover, it did not colocalize with markers of activated microglial cells (OX-42) or astroglial cells (glial fibrillary acidic protein) in rat spinal cord. In the peripheral nerve, accumulation of cannabinoid receptor 2 immunoreactivity was seen in nerve sections proximal, but not distal, to the ligation site, suggesting transport down the nerve from the cell bodies. Although convincing cannabinoid receptor 2 immunoreactivity was seen in neither uninjured nor injured dorsal root ganglion neuron cell bodies in tissue sections, expression was detectable in isolated, cultured neurons that had received a prior axotomy in vivo. This clear demonstration of CB(2) receptors on sensory neurons suggests an additional cellular target for CB(2) agonist induced analgesia, at least in neuropathic models.

Regulation and function of spinal and peripheral neuronal B1 bradykinin receptors in inflammatory mechanical hyperalgesia.

Activation of either B1 or B2 bradykinin receptors by kinins released from damaged tissues contributes to the development and maintenance of inflammatory hyperalgesia. Whereas B2 agonists activate sensory neurones directly, B1 agonists were thought only to have indirect actions on sensory neurones. The recent discovery of constitutive B1 receptor expression in the rat nervous system lead us to re-investigate the role of neuronal B1 receptors in inflammatory hyperalgesia. Therefore we have examined B1 bradykinin receptor regulation in rat dorsal root ganglia in a model of inflammatory hyperalgesia, and correlated it with hyperalgesic behaviour. Twenty-four hours after injection of Freund's complete adjuvant into one hindpaw, there was a significant increase in B1 protein expression (measured by immunohistochemistry) in both ipsilateral and contralateral dorsal root ganglion neurones, whereas axotomy resulted in reduction of B1 protein in ipsilateral dorsal root ganglia. In behavioural experiments, the B1 antagonist desArg10HOE140, administered by either intrathecal or systemic routes, attenuated Freund's complete adjuvant-induced mechanical hyperalgesia in the inflamed paw, but did not affect mechanical allodynia. The B1 agonist, desArg9BK, did not affect paw withdrawal thresholds in nai;ve rats following intraplantar administration into the paw, whilst intrathecal administration elicited mechanical hyperalgesia. However, after Freund's complete adjuvant-induced inflammation, desArg9BK caused a marked mechanical hyperalgesia, by either route, of the contralateral, uninflamed hindpaw, correlating with the observed contralateral and ipsilateral increases in receptor levels. Our results suggest a functional role for B1 receptors expressed both in the periphery and in the spinal cord, in mechanical hyperalgesia during inflammation.

Un modelo de dolor asociado a cáncer de huesos en la rata / A rat model of bone cancer pain

En este artículo se describe el primer modelo conocido de dolor por cáncer de huesos en la rata. Ratas SpragueDawley recibieron inyecciones intratibiales de células M R M T-1 singénicas de carcinoma de glándula mamaria de rata y exhibieron conductas indicativas de dolor, como: alodinia mecánica, diferencia en el peso apoyado en las patas traseras e hiperalgesia mecánica. La aparición del tumor óseo y el daño estructural del hueso se vigilaron mediante análisis radiológico, medidas cuantitativas del contenido mineral e histología. Las inyecciones intratibiales de 3 x 103 ó 3 x 104 células M R M T-1 singénicas produjeron un tumor que se propagó rápidamente dentro de la tibia, causando una intensa remodelación del hueso. Las radiografías mostraron importantes daños en el hueso cortical y las trabéculas entre 10 y 14 días después de la inoculación de 3 x 103 células MRMT-1 y, al cabo de 20 días, los daños amenazaban la integridad de la tibia. Mientras que el contenido mineral y la densidad mineral disminuyeron significativamente en hueso afectado por el cáncer, el número de osteoclastos en el hueso compacto peritumoral no experimentó cambios. Sin embargo, la tinción con fosfatasa ácida resistente al tartarato puso de manifiesto la existencia de un gran número de células policariotas parecidas a los osteoclastos presentes dentro del tumor. No se observó crecimiento tumoral después de la inyección de células MRMT-1 destruidas con calor. Las inyecciones intratibiales de 3 x 103 ó 3 x 104 de células MRMT-1, células destruidas con calor o excipiente no p ro d u j e ron cambios en el peso corporal ni en la temperatura interna en los 19-20 días siguientes. La actividad general en los animales que recibieron una inyección de células MRMT-1 vivas o destruidas con calor fue mayor que en los animales de control, pero la actividad del grupo tratado con células MRMT-1 disminuyó al avanzar la enfermedad. En las ratas que recibieron inyecciones intratibiales de células MRMT-1 se produjo la aparición gradual de alodinia mécanica e hiperalgesia mecánica/disminución del peso apoyado en el miembro afectado, a partir de 12-14 ó 1012 días después de la inyección de 3 x 103 ó 3 x 104 células, respectivamente. Estos síntomas no se observaron en las ratas que recibieron células destruidas con calor o excipiente. Los datos sobre la conducta de los animales sugieren un intervalo razonable para la evaluación de los agentes antinociceptivos entre el día 14 y el día 20 después de la inoculación de las células cancerosas en este modelo. El tratamiento agudo con morfina (1-3 mg.kg- 1, por vía subcutánea (s.c.)) consiguió una disminución proporcional a la dosis de la frecuencia de respuesta de retirada de la pata trasera frente a estimulación con filamentos de von Fre y 17 ó 19 días después de la inyección de 3 x 103 células M R M T-1. Se observó también una reducción significativa de la diferencia en el peso apoyado en las patas traseras. El tratamiento agudo con celebrex (10-30 mg.kg- 1 s.c.) no influyó en la alodinia mecánica ni en el diferente peso apoyado en las patas traseras de las ratas 20 días después del tratamiento con 3 x 103 células MRMT- 1 .Aunque la fisiopatología del dolor oncológico no se conoce bien, el aumento significativo de la tinción con proteína acídica fibrilar glial (GFAP) en los correspondientes segmentos de la médula espinal ipsilateral sugiere una posible participación de los astrocitos. En resumen, la inducción de cáncer de huesos en la rata con la línea de células MRMT-1 singénicas de cáncer mamario constituye un modelo preclínico válido del dolor asociado a metástasis óseas. Al progresar el tumor en la cavidad de la médula ósea parece una marcada hiperalgesia mecánica y alodinia, aunque el estado general del animal sigue siendo satisfactorio. El tratamiento agudo con morfina tiene cierto efecto analgésico en el peso apoyado en las patas traseras, pero celebrex, un inhibidor selectivo de COX-2, no influye en los cambios de conducta relacionados con el dolor en este modelo. (AU)

A rat model of bone cancer pain.

This study describes the first known model of bone cancer pain in the rat. Sprague-Dawley rats receiving intra-tibial injections of syngeneic MRMT-1 rat mammary gland carcinoma cells developed behavioural signs indicative of pain, including: mechanical allodynia, difference of weight bearing between hind paws and mechanical hyperalgesia. The development of the bone tumour and structural damage to the bone was monitored by radiological analysis, quantitative measurement of mineral content and histology. Intra-tibial injections of 3 x 10(3) or 3 x 10(4) syngeneic MRMT-1 cells produced a rapidly expanding tumour within the boundaries of the tibia, causing severe remodelling of the bone. Radiographs showed extensive damage to the cortical bone and the trabeculae by day 10-14 after inoculation of 3 x 10(3) MRMT-1 cells, and by day 20, the damage was threatening the integrity of the tibial bone. While both mineral content and mineral density decreased significantly in the cancerous bone, osteoclast numbers in the peritumoural compact bone remained unchanged. However, tartarate-resistant acid phosphatase staining revealed a large number of polykariotic cells, resembling those of osteoclasts within the tumour. No tumour growth was observed after the injection of heat-killed MRMT-1 cells. Intra-tibial injections of 3 x 10(3) or 3 x 10(4) MRMT-1 cells, heat-killed cells or vehicle did not show changes in body weight and core temperature over 19-20 days. The general activity of animals after injection with live or heat-killed MRMT-1 cells was higher than that of the control group, however, the activity of the MRMT-1 treated group declined during the progress of the disease. Rats receiving intra-tibial injections of MRMT-1 cells displayed the gradual development of mechanical allodynia and mechanical hyperalgesia/reduced weight bearing on the affected limb, beginning on day 12-14 or 10-12 following injection of 3 x 10(3) or 3 x 10(4) cells, respectively. These symptoms were not observed in rats receiving heat-killed cells or vehicle. Behavioural data suggest a reasonable time window for evaluation of anti-nociceptive agents between day 14 and 20 after cancer cell inoculation in this model. Acute treatment with morphine (1-3mg/kg, subcutanously (s.c.)) produced a dose-dependent reduction in the response frequency of hind paw withdrawal to von Frey filament stimulation 17 or 19 days following intra-tibial injections of 3 x 10(3) MRMT-1 cells. A significant reduction in the difference in hind limb weight bearing was also observed. Acute treatment with celebrex (10-30 mg/kg, s.c.) did not affect mechanical allodynia or difference in weight bearing in rats 20 days following treatment with 3 x 10(3) MRMT-1 cells. Although the pathophysiology of cancer pain is largely unknown, significant enhancement of glial fibrillary acidic protein (GFAP) staining in the corresponding segments of the ipsilateral spinal cord highlights the possible involvement of astrocytes. In summary, the induction of bone cancer in the rat by the syngeneic MRMT-1 mammary tumour cell line provides a valid pre-clinical model for pain associated with bone metastases. Significant mechanical hyperalgesia and allodynia develops in association with the progression of the tumour in the bone marrow cavity, while the general condition of the animal remains satisfactory. While acute treatment with morphine has some analgesic effect on hind limb sparing the selective COX-2 inhibitor, celebrex, has no influence on the pain-related behavioural changes in this model.

VR1 protein expression increases in undamaged DRG neurons after partial nerve injury.

Changes in phenotype or connectivity of primary afferent neurons following peripheral nerve injury may contribute to the hyperalgesia and allodynia associated with neuropathic pain conditions. Although earlier studies using partial nerve injury models have focused on the role of damaged fibres in the generation of ectopic discharges and pain, it is now thought that remaining undamaged fibres may be equally important. We have examined the expression of the sensory neuron-specific cation channel Vanilloid Receptor 1 (VR1), an important transducer of noxious stimuli, in three models of nerve injury in the rat, using anatomical separation or fluorescent retrograde tracers to identify damaged or undamaged sensory neurons. After total or partial sciatic nerve transection, or spinal nerve ligation, VR1-immunoreactivity (IR) was significantly reduced in the somata of all damaged dorsal root ganglion (DRG) neuronal profiles, compared to controls. However, after partial transection or spinal nerve ligation, VR1 expression was greater in the undamaged DRG somata than in controls. Unexpectedly, after L5 spinal nerve ligation, VR1-IR of the A-fibre somata increased approximately 3-fold in the uninjured L4 DRG compared to controls; a much greater increase than seen in the somata with C-fibres. Furthermore, we found that VR1-IR persisted in the transected sciatic nerve proximal to the lesion, despite its down-regulation in the damaged neuronal somata. This persistence in the nerve proximal to the lesion after nerve section, together with increased VR1 in DRG neurons left undamaged after partial nerve injury, may be crucial to the development or maintenance of neuropathic pain.

mGlu5 receptors and nociceptive function II. mGlu5 receptors functionally expressed on peripheral sensory neurones mediate inflammatory hyperalgesia.

Previous studies have demonstrated that the metabotropic glutamate receptor subtype 5 (mGlu5 receptor) is expressed in the cell bodies of rat primary afferent neurones. We have further investigated the function and expression of mGlu5 receptors in primary afferent neurones, and their role in inflammatory nociception. Freund's complete adjuvant-induced inflammatory hyperalgesia of the rat hind paw was significantly reduced by intraplantar, but not by intracerebroventricular or intrathecal microinjection of the selective mGlu5 receptor antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP). Pharmacological comparison in vivo of the nociceptive effects of glutamate, and ionotropic and metabotropic glutamate (mGlu) receptor agonists applied to the rat hind paw, indicated that group I mGlu receptor agonists induce a dose-dependent decrease in paw withdrawal threshold (mechanical hyperalgesia). Group I mGlu agonist-induced hyperalgesia was inhibited by co-microinjection of MPEP, but not by the mGlu1 receptor antagonist (S)-4-carboxy-phenylglycine (4-CPG). Carrageenan-induced inflammatory hyperalgesia was inhibited by pre-treatment of the inflamed hind paw with MPEP, but not following MPEP injection into the contralateral hind paw. Dorsal horn neurones receiving peripheral nociceptive and non-nociceptive afferent input were recorded in anaesthetized rats following microinjection of CHPG into their peripheral receptive fields. CHPG significantly increased the frequency and duration of firing of dorsal horn wide dynamic range (WDR) neurones and this activity was prevented by co-administration of CHPG and MPEP into their receptive fields. Immunohistochemical experiments revealed the co-expression of mGlu5 receptor protein and betaIII tubulin in skin from naive rats, indicating the constitutive expression of mGlu5 receptors on peripheral neurones. Double-labelling of adult rat DRG cells with mGlu5 receptor and vanilloid receptor subtype 1 antisera also supports the expression of mGlu5 receptors on peripheral nociceptive afferents. These results suggest that mGlu5 receptors expressed on the peripheral terminals of sensory neurones are involved in nociceptive processes and contribute to the hyperalgesia associated with inflammation.

Bradykinin B1 receptor is constitutively expressed in the rat sensory nervous system.

Using immunocytochemistry with an antibody raised to a specific rat bradykinin B1 receptor sequence, we showed that the B1 receptor was expressed in the naive rat sensory nervous system. B1 immunoreactivity was seen in laminae 1 and 2 of the dorsal horn of the spinal cord, where primary afferents terminate, and on peripheral nerve terminals in the bladder. B1 receptor was co-expressed preferentially with IB4 positive, but not calcitonin gene-related peptide (CGRP) containing C-cell bodies in the dorsal root ganglion. B1 activation has an important role in the hyperalgesia associated with inflammation, but the site of action of B1 antagonists has generally been believed to be on peripheral, non-neuronal cells. The striking distribution of B1 receptors on sensory neurones suggests that a direct action of B1 activators on the nervous system may also contribute to hyperalgesia.

Expression of the 5-HT1B receptor by subtypes of rat trigeminal ganglion cells.

The type of trigeminal ganglion cells that express 5-HT1B receptors has not been well characterized, despite the fact that these receptors are important targets for anti-migraine drugs. We have therefore used combined in situ hybridization and immunofluorescence to examine the expression of 5-HT1B receptor messenger RNA in identified subpopulations of rat trigeminal ganglion cells. 5-HT1B-expressing cells accounted for 15% of all trigeminal ganglion cells, were medium sized, and showed immunoreactivity for either 200,000 mol. wt neurofilament, calcitonin gene-related peptide, or nerve growth factor receptor (trkA). In contrast few 5-HT1B cells showed immunoreactivity for substance P or binding of the lectin Griffonia simplicifolia IB4. Our results are consistent with 5-HT1B receptors acting to control the release of calcitonin gene-related peptide from trigeminal neurons with finely myelinated axons. 5-HT1B receptor agonists may reduce neurogenic vasodilation by activating such receptors. However many nociceptive trigeminal neurons, including the substance P and IB4-binding populations, do not express the 5-HT1B receptor.

Antibodies against the extracellular domain of the 5-HT3 receptor label both native and recombinant receptors.

We have developed polyclonal antibodies (pAb120) against a peptide corresponding to a region within the extracellular domain of the 5-hydroxytryptamine3 (5-HT3) receptor subunit, thus permitting, for the first time, localization of 5-HT3 receptors at the cell surface in intact (non-permeabilized) systems. The antibodies are both specific and sensitive: pAb120 recognized as little as 63 ng of protein from HEK293 cells expressing recombinant 5-HT3 receptors, whilst Western blots of recombinant 5-HT3 receptors purified from Sf9 cells revealed two bands at 48 and 54 kDa, and native 5-HT3 receptors from N1E-115 cell membranes produced a broad band at 50-54 kDa with a smaller band at 35 kDa. These bands were also labelled by antibodies against the intracellular loop of the 5-HT3 receptor. Immunofluorescent labelling revealed a ring of intense fluorescence in the plasma membrane of non-permeabilized HEK293 cells expressing recombinant 5-HT3 receptors. Studies on native 5-HT3 receptors revealed that pAb120 could recognize 5-HT3 receptors on presynaptic terminals isolated from rat striatum, and immunohistochemical studies in rat brain sections revealed labelling of cell bodies, dendrites and varicose axons in hippocampus, cortex and lateral hypothalamus; all of these areas have been reported to express 5-HT3 receptors. We conclude that pAb120 is a highly specific and sensitive antiserum that will assist in clarifying fundamental questions about 5-HT3 receptor neurobiology.

Intraregional variation in expression of serotonin transporter messenger RNA by 5-hydroxytryptamine neurons.

The distribution of the messenger RNA encoding the 5-hydroxytryptamine transporter was investigated in rat brain. 5-Hydroxytryptamine transporter messenger RNA was found exclusively in the B1-B9 cell groups containing the cell bodies of 5-hydroxytryptamine neurons. Combined in situ hybridization and 5-hydroxytryptamine immunocytochemistry demonstrated 5-hydroxytryptamine transporter gene expression in the majority of and exclusively in 5-hydroxytryptamine neurons. Cells differed in their levels of expression of 5-hydroxytryptamine transporter messenger RNA and 5-hydroxytryptamine immunofluorescence, but with a tight correlation between the two parameters. Image analysis of cells from B7, the dorsal raphe nucleus, and B8, the median raphe nucleus, revealed significant differences between groups in the mean cellular level of 5-hydroxytryptamine transporter gene expression. Cells in the ventromedial subdivision of B7 displayed higher levels of expression than cells in B8 or cells in the lateral wings of B7. There was also heterogeneity in the distribution of the cellular levels of expression for two other genes expressed by 5-hydroxytryptamine neurons: l-aromatic amino acid decarboxylase messenger RNA and tryptophan hydroxylase messenger RNA. However, the relative levels of expression of these two genes within the four regions studied differed from that of 5-hydroxytryptamine transporter messenger RNA. These results indicate intraregional differences between 5-hydroxytryptamine neurons with respect to 5-hydroxytryptamine transporter messenger RNA levels. Such differences may account for the differential sensitivity of 5-hydroxytryptamine neurons to cytotoxins.

Constitutive expression of calmodulin-binding phosphoprotein GAP-43 in rat serotonergic and noradrenergic cell groups which project to the spinal cord.

In situ hybridization was combined with serotonin (5-hydroxytryptamine, 5-HT) or tyrosine hydroxylase immunocytochemistry and with Fluoro-Gold retrograde labeling of bulbo-spinal pathways in order to investigate the expression of GAP-43 mRNA in monoamine cell groups of the adult rat brain stem. Consistent with previous reports, GAP-43 mRNA was observed in serotonin and dopamine cell groups in the pons. In addition, GAP-43 expressing cells were observed in all the major monoamine cell groups in the medulla. Thus the B1, B2 and B3 serotonin cell groups all showed high GAP-43 expression in all contained many GAP-43 expressing serotonin cells with spinal cord projections. The A1, A2, A5 and A6 noradrenaline cell groups also showed high GAP-43 expression, although cells with spinal cord projections were largely restricted to the A5 group and A6 subcoeruleus region. In all areas, GAP-43 expressing cells with spinal cord projections were also observed which were not serotonergic or noradrenergic.

Co-expression of GAP-43 with dopamine-beta-hydroxylase in adult rat spinal cord.

Dual colour immunofluorescence was used to compare the distribution of dopamine-beta-hydroxylase (DBH) and GAP-43 in the spinal cord of the adult rat. GAP-43 immunostaining was observed in all spinal cord regions containing DBH immunoreactivity. DBH and GAP-43 double-labelled fibres and varicosities were present in the lateral and ventral funiculi of the white matter and in the ventral horn where they were most prominent around motoneurones. In the dorsal horn and around the central canal, GAP-43 immunoreactivity was too abundant to determine whether DBH immunoreactive fibres were double labelled. We conclude that the noradrenergic system is one of a small number of spinal cord systems that express high basal levels of GAP-43 in the adult.

Chronic D-fenfluramine decreases serotonin transporter messenger RNA expression in dorsal raphe nucleus.

In situ hybridization was used to measure the effects of chronic fenfluramine administration on serotonin transporter messenger RNA expression in cells of the dorsal raphe nucleus complex. Fenfluramine produced a significant, but transient, down-regulation of serotonin transporter mRNA in cells which lie in the ventral portion of the dorsal raphe nucleus, but not in the dorsal part of the dorsal raphe nucleus. Our findings suggest that cells which lie in the ventral part of the dorsal raphe nucleus are more sensitive to the effects of chronic fenfluramine administration, but that fenfluramine does not cause long-term changes in gene expression in serotonin cell bodies.

Repeated administration of MDMA down-regulates preprocholecystokinin mRNA expression but not tyrosine hydroxylase mRNA expression in neurones of the rat substantia nigra.

The effect of repeated administration of 3,4-methylenedioxymethamphetamine (MDMA) on the expression of tyrosine hydroxylase and preprocholecystokinin (CCK) messenger RNAs in substantia nigra was examined by in situ hybridisation histochemistry. Sections hybridised with 35S-labelled oligonucleotides were subjected to computerised image analysis to determine the density of silver grains above positively labelled cells as an index of steady state mRNA levels. In the substantia nigra pars compacta, CCK mRNA levels were significantly reduced in drug-treated animals 24 h and at 2 weeks after the last dose of MDMA (10 mg/kg i.p., twice daily for 4 days). In the same animals, MDMA caused no change in the level of tyrosine hydroxylase mRNA in this brain region. The results show that MDMA can produce changes in dopamine neurones. Furthermore, since tyrosine hydroxylase and cholecystokinin are co-expressed in substantia nigra pars compacta, these results suggest that the expression of the tyrosine hydroxylase and CCK genes are regulated independently.

Serotonin and NADPH-diaphorase in the dorsal raphe nucleus of the adult rat.

NADPH-diaphorase histochemistry, employed as a marker for nitric oxide synthase (NOS), was combined with serotonin (5-HT) immunofluorescence to investigate the relationship between NOS and 5-HT in the rat dorsal raphe nucleus. Many NADPH-diaphorase labelled cells and varicose axons were observed in the nucleus. Coexistence between NADPH-diaphorase and 5-HT occurs in cells of the dorsomedial and ventromedial subgroups but not in the lateral subgroups. Coexistence was not observed in axons, but NADPH-diaphorase labelled axons contact 5-HT/NADPH-diaphorase containing cell bodies. These findings have implications for the role of nitric oxide in 5-HT pathways and for the mechanism of action of 5-HT neurotoxins.

Serotonergic terminals express a growth associated protein (GAP-43) in the adult rat spinal cord.

Dual colour immunofluorescence has been used to compare the distribution of serotonin (5-hydroxytryptamine, 5-HT) and GAP-43 in the adult rat. GAP-43 immunostaining was observed in all spinal cord regions containing 5-HT immunoreactivity. 5-HT and GAP-43 double labelled fibres and varicosities were present and were most evident around motoneurones, in lamina X, and in the intermediolateral cell column. Single labelled GAP-43 fibres and varicosities were also observed and were the dominant population in the dorsal horn and in certain fibre tracts. We conclude that the 5-HT system is one of a small number of spinal cord systems that express high levels of GAP-43 in the adult.

A combined in situ hybridization and immunofluorescence procedure allowing visualisation of peptide mRNA and serotonin in single sections.

We describe a novel procedure for combining immunocytochemistry with in situ hybridisation. In contrast to previously published procedures, the technique involves immunofluorescence followed by in situ hybridization and is particularly suitable for antigens which are labile or sensitive to in situ hybridization processing. We have evaluated the technique using 5-hydroxytryptamine (5-HT, serotonin) immunofluorescence and neuropeptide in situ hybridization employing 35S-labelled oligonucleotide probes. Successful double labelling was obtained and showed that galanin messenger RNA (mRNA) is expressed by 5-HT immunoreactive cells in the dorsal raphe nucleus of the rat. In contrast, somatostatin mRNA in the same region is expressed by a separate non-serotonergic cell population. Double-labelled preparations produced using this technique can be conveniently viewed using epipolarised combined with epifluorescent illumination. Careful analysis of procedural variables revealed that it is not possible to carry out high-sensitivity 5-HT immunocytochemistry following in situ hybridization. The immunostaining is much poorer on slide-mounted sections than on free-floating sections, and 5-HT appears to be lost during the in situ hybridization steps of dehydration/delipidation and incubation in hybridization buffer. The procedure we describe avoids these problems but with a slight loss of in situ hybridization sensitivity.