A model of organotypic rat spinal slice culture and biolistic transfection to elucidate factors that drive the preprotachykinin-A promoter

Brain Research Reviews 46 (2004) 191 – 203 www.elsevier.com/locate/brainresrev

Review

A model of organotypic rat spinal slice culture and biolistic transfection to elucidate factors that drive the preprotachykinin-A promoter Kathryn J. Hilton, Alan N. Bateson, Anne E. King* School of Biomedical Sciences, University of Leeds, Clarendon Way, Leeds, LS2 9JT, UK Accepted 23 July 2004 Available online 22 September 2004

Abstract The tachykinin substance P (SP) is a neuropeptide that is expressed in some nociceptive primary sensory afferents and in discrete populations of spinal cord neurons. Expression of spinal SP and the preprotachykinin-A (PPT-A) gene that encodes SP exhibits plasticity in response to conditions such as peripheral inflammation but the mechanisms that regulate expression are poorly understood. We have developed a spinal cord organotypic culture system that is suitable for the analysis of PPT-A gene promoter activity following biolistic transfection of recombinant DNA constructs. Spinal cord organotypic slices showed good viability over a 7-day culture period. Immunostaining for phenotypic markers such as NeuN and h-III tubulin demonstrated preservation of neurons and their structure, although there was evidence of axotomy-induced down-regulation of NeuN in certain neuronal populations. Neurokinin-1 receptor (NK-1R) immunostaining in laminae I and III was similar to that seen in acute slices. Biolistic transfection was used to introduce DNA constructs into neurons of these organotypic cultures. Following transfection with a construct in which expression of enhanced green fluorescent protein (EGFP) is controlled by the PPT-A promoter, we showed that induction of neuronal activity by administration of a forskolin analogue/high K+ (10 AM/10 mM) for 24 h resulted in a fourfold increase in the number of EGFP-positive cells. Similarly, a twofold increase was obtained after treatment with the NK-1R-specific agonist [Sar9,Met (O2)11]-substance P (10 AM). These data demonstrate the usefulness of this model to study physiological and pharmacological factors relevant to nociceptive processing that can modulate PPT-A promoter activity. D 2004 Elsevier B.V. All rights reserved. Theme: Sensory systems Topic: Spinal cord Keywords: Biolistic transfection; Substance P; Preprotachykinin-A; Organotypic culture; Spinal slice

Contents 1.

2.

Introduction. . . . . . . . . . . . . . . . . . . . . . 1.1. Substance P as a spinal neuromodulator . . . 1.2. Regulation of PPT-A gene expression . . . . 1.3. Gene transfer into cultured organotypic tissue Methods . . . . . . . . . . . . . . . . . . . . . . . 2.1. Interface culture of spinal cord . . . . . . . . 2.2. Immunohistochemistry . . . . . . . . . . . . 2.3. Biolistic transfection protocol . . . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

* Corresponding author. Tel.: +44 113 34 34243; fax: +44 113 34 34228. E-mail address: [email protected] (A.E. King). 0165-0173/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.brainresrev.2004.07.016

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

192 192 192 193 193 193 193 194

192

K.J. Hilton et al. / Brain Research Reviews 46 (2004) 191–203

3.

Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Organotypic spinal cord cultures . . . . . . . . . . . . . 3.2. Biolistic DNA transfection protocols . . . . . . . . . . . 3.3. Activation of the PPT-A promoter . . . . . . . . . . . . 4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Biolistic transfection of organotypic spinal cord cultures . 4.2. Plasticity of PPT-A and tachykinin peptide expression . . 4.3. Physiological activation of the PPT-A promoter . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction 1.1. Substance P as a spinal neuromodulator In spinal cord, the tachykinin peptide substance P (SP) performs a number of significant physiological functions including modulation of ventral horn motor pathways [72], regulation of autonomic outflow from sympathetic preganglionic neurons [73] and transmission of nociceptive sensory afferent inputs [71]. Data for the latter establish a link to neural mechanisms that underlie both acute responsiveness to peripheral noxious stimuli [14,65] and central sensitisation phenomena implicated in hyperalgesia or pathological pain [38,66]. Following peripheral nociceptor activation, SP is released from capsaicin-sensitive smallcaliber sensory afferents [34,86] and induces a slow membrane depolarisation of postsynaptic target dorsal horn neurons through neurokinin-1 receptors (NK-1R) [39,79] localised to lamina I, III and IV [48, 89]. Spino-parabrachial and spino-thalamic pathways that subserve ascending nociceptive neurotransmission are derived, at least partly, from NK-1R expressing dorsal horn neurons [51,88,89]. In considering the origins of spinal SP, it is clear that the majority is derived from extrinsic primary afferents innervating peripheral nociceptors [13] and supraspinal sources such as medullary or bulbo-spinal descending pathways [4,29,52]. However, the fact that neither deafferentation nor supraspinal transection can eliminate SP immunoreactivity suggests other intrinsic sources of SP [25,63]. Similarly, the presence of preprotachykinin-A (PPT-A) mRNA in dorsal spinal cord indicates the capacity for SP biosynthesis within intrinsic neuronal structures [53,69,74]. In fact, a number of spinal structures that constitutively express SP have been identified including neurons localised to the dorsal horn [31,43,45,64,82,93], some of which project to parabrachial nucleus [8]. SP-immunoreactive cell bodies that may contribute to a network of proprio-spinal longitudinal fibres [11] and SP-immunoreactive fibre tracts that interconnect dorsal and ventral regions have been described [23]. No physiological function is ascribed to these intrinsic SP networks although a role for SP in ascending nociceptive processing or in local nociceptive or motor circuitry may be inferred.

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

194 194 194 195 196 196 199 200 201 201

It has been apparent for sometime that the expression level of SP in spinal circuitry is not constant but can be either up- or down-regulated after experimental manipulation. One of the best described models for plasticity within spinal peptidergic systems is inflammation which augments expression of SP in dorsal horn and primary sensory neurons [16]. This modified expression of SP is accompanied by increased levels of NK-1R [1] and enhanced spinal release of SP [22]. After inflammation, a dphenotypic switchT triggers transcriptional changes that precipitate de novo expression of SP in large diameter glutamatergic A fibres [67]. Despite the existence of several studies that catalogue spatial and temporal patterns of altered spinal peptide expression in pain models, the mechanisms through which this is achieved are not established. SP, along with neurokinin A (NKA), is derived from the preprotachykininA gene with alternative splicing generating a-, h-, g- and yPPT-A mRNAs [27,41]. Precursor sequences for SP are encoded within all four PPT-A mRNAs whereas NKA is present only within the h- and g-splice variants [27,41]. Electrophysiological and behavioral analyses of mice with a deletion of the PPT-A gene have provided insight into the role of SP in acute nociception, sensitisation and the response to nerve injury [10]. Inflammation or formalininduced up-regulation of PPT-A mRNA in sensory ganglia [26,44] or spinal cord [69,74] is consistent with observed alterations in tachykinin peptide expression. 1.2. Regulation of PPT-A gene expression Inducible expression of the PPT-A gene is regulated in vivo and in vitro by a diversity of factors including nerve growth factor (NGF) [47], steroids [83], cyclic AMP [75], leukaemia inhibitory factors [32] and opioids [54]. The extent to which these or other as yet unidentified factors contribute to transcriptional regulation of SP synthesis in normal and pathological spinal cord is unclear. From a therapeutic perspective, it is possible that transcription factors controlling expression of a specific gene or set of genes may provide suitable targets for direct pharmacological manipulation [6,42,91]. In the central and peripheral nervous systems, neuronal phenotype is specified through development and into adulthood by distinct gene expression

K.J. Hilton et al. / Brain Research Reviews 46 (2004) 191–203

193

profiles. However, it is increasingly clear that post-mitotic neurons display significant plasticity of gene expression in response to physiological effectors [95], pharmacological challenge [5,62] and trauma [80]. Understanding the mechanisms that control normal gene expression and perturbation by biological factors is a central goal of neurobiological research. In spinal cord, this approach could offer a way to limit adverse phenotypic changes, e.g., altered neuropeptide or neurotrophin expression manifest in chronic pain states [36]. To address such issues, diverse novel experimental approaches have been utilised. Data derived from transgenic animals in which the expression of a target gene is either up- or down-regulated have proved useful but may be confounded by parallel compensatory gene expression profiles. More sophisticated transgenic technologies such as conditional knock-outs largely circumvent these problems [24], but do not address the issue of cost, both financially and in terms of the number of animals required.

previously coated with DNA constructs, to high speeds which penetrate cell membranes with minimal damage. Two DNA constructs are used: one that allows the identification of transfected cells and a second that contains part of the PPT-A gene promoter which drives expression of a green fluorescent protein (GFP) variant reporter gene [92]. Promoter activity was determined in response to pharmacological manipulation of the cultured spinal cord slice. There are a number of parameters that affect efficiency of DNA delivery via biolistic gene transfer including the particle material and its size, the DNA concentration and the method used to coat the particles, the delivery pressure and the distance of the sample from the delivery aperture. We utilised known optimal parameters from the literature [2,7,19,49,87] and optimised others for effective delivery into our cultured rat spinal cord slices.

1.3. Gene transfer into cultured organotypic tissue

2.1. Interface culture of spinal cord

An alternative cost-effective approach is to use transient expression of exogenous DNA constructs in ex vivo systems such as primary neuronal or organotypic cultures. The latter represent a useful intermediate between whole animal studies and the reductionist approach of cultured dispersed primary neurons. In contrast to the physical disruption associated with the preparation of cultured primary neurons, organotypic cultures of CNS tissue offer some preservation of cytoarchitecture and neuronal connectivity [21]. However, the introduction of exogenous DNA into primary or organotypic cultured neurons is not without inherent problems, primarily because standard DNA transfection techniques exhibit low efficiency with postmitotic neurons [94]. For example, although very cheap and simple to perform, transfection efficiencies of calcium phosphate-DNA co-precipitates into neurons are below 5% even when using protocols optimised for such purposes [96]. Furthermore, techniques suitable for organotypic cultures are more restricted due to the physical constraints of handling this tissue. Of the current techniques available for the introduction of foreign DNA into neurons in a cultured organotypic brain slice, biolistic gene transfer offers a number of advantages and has proven utility in an increasing number of studies. Biolistic gene transfer uses physical force to introduce DNA-coated biologically inert metal micro-particles into cells [20]. It was originally developed for use in plant cells to overcome cell wall impermeability [40] but subsequently was extended to other tissues including brain in vitro [2,49]. In this paper we describe the first use of the PDS-1000/ He particle delivery system (Bio-Rad) to transfect organotypic rat spinal slices for the analysis of the regulation of PPT-A gene expression. This biolistic system uses helium gas under pressure to accelerate gold micro-particles,

This interface culture method was modified from Stoppini et al. [84]. Female Wistar rats (7–9 days) were anaesthetised with urethane (2 g kg 1, i.p.) for removal of the spinal cord. All procedures were performed under licence and in accordance with relevant UK Home Office legislation. Lumbar spinal slices of 350 Am were cut with a McIlwain tissue chopper and placed onto Millicell organotypic filter inserts. Up to four slices per filter were cultured for the viability experiments, and the characterisation of the cultures and the biolistic protocol. Two slices were cultured per filter for the analysis of transfected PPT-A promoter activity. Filters were placed onto 1 ml of medium in a humidified incubator at 37 8C with a 5% CO2-enriched atmosphere. The culture medium consisted of 50% Neurobasal-A supplemented with B27; 25% heat-inactivated horse serum; 25% Hank’s basic salt solution; 0.25 mM lglutamine; 15 mM HEPES; 15 mM glucose; 25 Ag/ml penicillin/streptomycin (all from Invitrogen Life Technologies). Thrice weekly, 500 Al of the culture medium was removed and replaced with 500 Al of fresh medium.

2. Methods

2.2. Immunohistochemistry Acute slices were prepared by transcardiac perfusion of 8-day-old Wistar rats with artificial cerebrospinal fluid, removal of the spinal cord, post-fixation in 4% paraformaldehyde (PFA), cryoprotection in 30% sucrose and cutting 50 Am slices on a freezing stage microtome. Organotypic slices were fixed in 4% PFA and removed from the filter. Acute and organotypic slices were washed with 10 mM PBS, incubated in 1% H2O2 for 15 min (DAB slices only), permeabilised in 50% ethanol for 30 min and incubated in a blocking solution of 0.3% Triton X-100/PBS for 1 h. PBS washes of at least 215 min were carried out between each

194

K.J. Hilton et al. / Brain Research Reviews 46 (2004) 191–203

step. Sections were incubated in anti-NK-1R (Sigma) 1:10,000, anti-SP (Sigma) 1:1000, anti-NeuN (Chemicon) 1:1000 or anti-h-III tubulin (Chemicon) 1:100 for 48–72 h. Slices were thoroughly washed in 10 mM PBS. To visualise NK-1R and SP staining, slices were incubated in biotinylated secondary antibody for a minimum of 2 h, avidin biotinylated horseradish peroxidase for 1 h followed by incubation in 3V,3V-diaminobenzidine until the brown precipitate formed. NeuN staining was revealed by incubating slices with Cy3-conjugated secondary antibody (Jackson Immunochemicals) 1:1000 for 2 h. For h-III tubulin immunostaining, slices were incubated with biotinylated secondary antibody for a minimum of 2 h and visualised by incubation with streptavidin Cy3-conjugated secondary antibody (Jackson Immunochemicals) 1:1000 for 2 h. 2.3. Biolistic transfection protocol Slices were transfected on the fifth day of culture using a PDS-1000/He particle delivery system (Bio-Rad). The parameters are detailed in Fig. 5. For the characterisation of transfection, organotypic slices were biolistically transfected with a cytomegalovirus (CMV) promoter-enhanced green fluorescent protein (EGFP) construct and cultured for a further 2 days. Slices were fixed in 4% PFA and processed for NeuN, h-III tubulin and glial acidic fibrillary protein (GFAP) immunostaining. For the latter, slices were incubated in GFAP (Chemicon) 1:1000 for 48–72 h. After washing they were incubated with Cy3-conjugated secondary antibody for 2 h. Two filters of slices were biolistically transfected at one time with PPT-A promoter-EGFP and CMV promoter-DsRed constructs after 5 days in culture. Twenty four hours later, one filter from each pair was treated with 10 AM 7-Deacetyl-7h-(g-N-methylpiperazino) butyrylforskolind 2HCld H2O (L 858051, water-soluble forskolin analogue) and 10 mM K+ or 10 AM [Sar9,Met(O2)11]substance P and cultured for a further 24 h. Slices were fixed and the fluorescent cells counted.

on separate preparations prepared over a 15-week period. This test showed that after 5 or 7 days in culture, the slices had good viability shown by extensive green calcein fluorescence, indicative of live cells (data not shown). On occasion slices had a small number of dead or dying cells, illustrated by cells with intensely fluorescent red nuclei caused by ethidium homodimer-1 bound to the nuclear DNA. Such cells were scattered throughout the slice in small numbers and there was no indication of any specific cell population that was vulnerable in this culture system. To confirm preservation of structure and to appraise further the usefulness of this model, immunostaining was carried out for NK-1R (Fig. 2A,B), SP (Fig. 2C,D), NeuN (Fig. 3) and h-III tubulin (Fig. 4) on slices cultured for 5 days and compared to perfused tissue from comparative aged rats. Immunostaining for NK-1R revealed preservation of the NK-1R immunopositive neurons located in laminae I and III and excellent organotypic structure (Fig. 2A compared to B). Immunostaining for SP revealed a degradation of staining in the superficial dorsal horn concurrent to degeneration of the primary afferent or descending fibre terminals during culture (Fig. 2C compared to D). Immunostaining with the marker NeuN established that a significant proportion of the viable cells were neuronal and that there was acceptable preservation of neurons in the dorsal horn, especially the superficial area (Fig. 3A compared to B). In the ventral horn, however, there was an absence of staining particularly pertaining to the somatic motoneurons (Fig. 3C vs. area indicated on A). This result was inconsistent with that obtained using a LIVE/ DEADR viability/cytotoxicity assay (Molecular Probes) which indicated the presence of viable cells of a size and location consistent with the somatic motoneuron population (data not shown). Further analysis was performed with an antibody to the h-III isoform of tubulin, which is neuronalspecific. Immunostaining for h-III tubulin in the cultured sections revealed that somatic motoneurons are present in these cultures (Fig. 4F). As h-III tubulin is a major component of the neuronal cytoskeleton, more of the morphology of the neurons present in these cultures was revealed (Fig. 4B,D,E).

3. Results 3.2. Biolistic DNA transfection protocols 3.1. Organotypic spinal cord cultures Organotypic spinal slice cultures were successfully set up (Fig. 1) using the interface method developed by Stoppini et al. [84] with culture medium modifications that increase viability in the spinal cord. Spinal cord cultures prepared in this way maintain an excellent gross morphology with the dorsal and ventral horns distinguishable from each other, facilitating the identification of neuronal populations. Slices spontaneously attach to the filter supports and flatten to less than 100 Am in thickness from the original starting thickness of 350 Am. Viability testing was carried out using the LIVE/ DEADR viability/cytotoxicity assay kit (Molecular Probes)

The cultured spinal cord slices were biolistically transfected with DNA constructs using a PDS-1000/He particle delivery system as shown schematically in Fig. 5. The rupture pressure and the distance between the target shelf and the launch assembly were optimised and the filters were placed on an agar plate and covered with a nylon baffle to protect the slices from trauma caused by the helium blast. During optimisation trials, transfected cells were identified by the expression of EGFP from a CMV promoter construct. Transfected cells were present throughout all areas of the slices, and transfection was especially successful where the slice had flattened and was thinnest at the edges. There were

K.J. Hilton et al. / Brain Research Reviews 46 (2004) 191–203

195

Fig. 1. Summary schematic of the interface culture method.

a number of different cell morphologies transfected and immunostaining with NeuN (Fig. 6A) and h-III tubulin (Fig. 6C,D) revealed co-localisation with transfected cells. However, a large proportion of cells did not co-localise with these markers. There were also a minimal number of cells co-localised with the astrocytic marker GFAP (Fig. 6B), though in this model no anti-mitotic protocol is followed to restrict proliferation of this cell type. 3.3. Activation of the PPT-A promoter Organotypic slices cultured for 5 days were biolistically co-transfected with PPT-A-EGFP and CMV promoter-DsRed (CMV-DsRed) with an efficiency of 41F4.6

transfected cells/slice (n=38) in control cultures. Two filters were transfected at one time and after a further 24h incubation, either a forskolin analogue (L 858051)/high potassium (10 AM and 10 mM, respectively) or [Sar9,Met(O2)11]-substance P (10 AM) was added to the medium for one filter of each pair for 24 h. After the application of forskolin analogue and high potassium (Fig. 7A), there was a significant fourfold increase in PPT-A promoter activity. This was shown by an increase in the proportion of DsRed-positive transfected cells that coexpress EGFP. An example of the EGFP-positive cells is shown for forskolin analogue/high potassium-treated slices (Fig. 7B, left panel) and control slices (Fig. 7C, left panel). Co-expressed in these cells was DsRed (Fig. 7B

Fig. 2. Immunostaining for NK-1R (A, acute; B, organotypic) and SP (C, acute; D, organotypic) in the superficial dorsal horn of acute and organotypic spinal slices. Scale bars=100 Am.

196

K.J. Hilton et al. / Brain Research Reviews 46 (2004) 191–203

4. Discussion 4.1. Biolistic transfection of organotypic spinal cord cultures

Fig. 3. Immunostaining for NeuN in acute (A) and organotypic spinal slices (B, superficial dorsal horn; C, ventral horn, area pertaining to that marked in A by the dashed box). (A) Scale bar=70 Am; (B) scale bar=200 Am; (C) scale bar=50 Am.

and C, right panel). Similarly, there was a significant twofold increase in the number of EGFP-positive cells after the application of [Sar9,Met(O2)11]-substance P (Fig. 8A) when compared to controls. Examples of EGFP- and DsRed-positive cells for drug-treated (Fig. 8B, left and right panels) and control (Fig. 8C, left and right panels) are shown.

In this study, we report for the first time the successful biolistic transfection of neurons in spinal cord organotypic cultures and the use of this model system to study factors that modulate PPT-A promoter activity. Our organotypic spinal cord slices are viable, with minimal cell death. Further, they retain their morphology and the expression of certain neurochemical markers through the culture period. We also provide evidence to suggest that the commonly used neuronal-specific immunochemical marker NeuN may not be suitable for all preparations as its expression appears to be down-regulated over time in certain neurons. Two methods of organotypic slice culture have emerged for long-term studies of neuronal tissue. These are the roller-tube method, which has been characterised in depth for certain neuronal tissues by Gahwiler et al. [21], and the interface method developed by Stoppini et al. [84]. We chose the interface method for this study because following adherence to the filter, the cultured slices are easy to handle and readily accessible for biolistic transfection. We found that the gross morphology of our spinal interface cultures compared well with that reported [15] for the roller-tube method. Further, the morphological distortion that sometimes results from tissue rotation in the rollertube method [15] does not occur with the interface method. In a modification of Stoppini’s interface protocol, we substituted the minimum essential medium component with Neurobasal A supplemented with B27 (D. Marsh, The John P. Robarts Research Institute, Canada, personal communication). Visual inspection with differential interference contrast microscopy of cultures maintained in this

Fig. 4. Immunostaining for h-III tubulin in acute (A) and organotypic spinal slices (B, lamina I; (C, lamina II; D, lamina III; E, lamina IV; F, ventral horn. (A) Scale bar=400 Am; (B) scale bar=45 Am; (C) scale bar=100 Am; (D and E), scale bar=45 Am; (F) scale bar=200 Am.

K.J. Hilton et al. / Brain Research Reviews 46 (2004) 191–203

197

Fig. 5. Schematic of the biolistic transfection protocol. Slices were transfected on the fifth day of culture using a PDS-1000/He particle delivery system (BioRad). For the optimisation experiments, only one filter containing up to four slices was used per transfection.

modified medium were less grainy in appearance and exhibited markedly reduced numbers of rounded cells with dark plasma membranes (data not shown). Such cells were

presumed to be functionally compromised and possibly apoptotic. Previous workers have demonstrated significant necrosis in the first 24 h following slice preparation that

Fig. 6. Characterisation of transfected cell type by immunostaining for NeuN (A, deep dorsal horn) GFAP (B, dorsal horn) and h-III tubulin (C, dorsal horn; D, ventral horn). Organotypic slices were biolistically transfected with a CMV promoter-EGFP construct on day 5 and cultured for a further 2 days. (A) Scale bar=10 Am; (B and C) scale bar=20 Am; (D) scale bar=15 Am.

198

K.J. Hilton et al. / Brain Research Reviews 46 (2004) 191–203

and demonstrates maintained elements of dorsal horn nociceptive circuitry in the cultures. Other areas of the spinal slice also showed the expected NK-1R expression pattern (data not shown). The NeuN antibody recognises an unknown antigen in the nuclei and, to a lesser extent, the cytoplasm of neurons [60]. It has been widely used as a neuronal marker for different brain regions in acute CNS slices [85] and cultured cells [30]. In cultured slices, however, NeuN expression was not equivalent to that seen in acute slices as although NeuN immunostaining was preserved in the dorsal horn, in particular the superficial laminae, it was absent in the ventral horn. This might suggest a loss of certain neuronal populations over the culture period but we do not believe that this is the case for a number of reasons. In the viability assay, live cells are detected by their ability to take up calcein-AM (which is not fluorescent and passively crosses intact plasma cell membranes) and convert it to the fluorescent calcein by cell esterases,

Fig. 7. PPT-A promoter activation after treatment with forskolin analogue (L 858051)/high potassium. Histogram showing a fourfold increase in PPT-A promoter activity (A) and individual examples of PPT-A-driven EGFP and CMV-driven DsRed expression in drug treated (B, superficial dorsal horn) and control cultured slices (C, ventral horn). Data are expressed as the mean percentage of red cells that are green (to account for any differences in transfection efficiency)˘S.E.M., n=12 slices per condition, prepared from a total of three rats over two separate experiments. *Pb0.001 vs. control (MannWhitney U-test). (B) Scale bar=45 c¸m; (C) scale bar=20 c¸m. (For interpretation of the references to colour in this figure legend, the readers is referred to the web version of this article).

can take up to 4 days to resolve [68,70]. Using the LIVE/ DEADR stain, we found that from day 5 onwards, little cell death was apparent with the modified culture media. The possibility that certain neuronal populations display differential viability following the trauma of organotypic spinal slice preparation was assessed by the examination of immunohistochemical markers. We found NK-1R-positive neurons in laminae I and III/IV of the dorsal horn, consistent with their localisation in vivo [89]. We did not detect significant levels of SP in this region presumably because axotomy of primary afferents from the dorsal root ganglia and their subsequent degeneration removes a main source of extrinsic SP. Similarly, the degeneration of the terminals derived from descending spinal pathways would eliminate substantive SP expression. Axotomy of sensory afferents does not appear to cause widespread degeneration of target neuronal populations, including those that express NK-1R,

Fig. 8. PPT-A promoter activation after treatment with [Sar9,Met(O2)11]substance P. Histogram showing twofold increase in PPT-A promoter activity (A) and individual examples of PPT-A-driven EGFP expression and CMV-driven DsRed in drug treated (B, ventral horn) and control cultured slices (C, superficial dorsal horn). Data are expressed as the mean percentage of red cells that are green (to account for any differences in transfection efficiency)˘S.E.M., n=26 slices per condition, prepared from a total of eight rats over four separate experiments. * Pb0.05 vs. control (MannWhitney U-test). (B) Scale bar=22 c¸m; (B, inset) scale bar=100 c¸m; (C) scale bar=20 c¸m. (For interpretation of the references to colour in this figure legend, the readers is referred to the web version of this article).

K.J. Hilton et al. / Brain Research Reviews 46 (2004) 191–203

thereby demonstrating preservation of intracellular metabolic function. In the ventral horn of cultured slices, live cells that were morphologically indistinct from motoneurons were easily identified, although these cells did not express NeuN. Detection of dead cells utilises the property of ethidium homodimer-1 to enter only those cells with compromised cell membranes and bind to nuclear DNA: no motoneuron-like cells were detected with ethidium homodimer-1. In addition, immunostaining for the neuronal-specific marker h-III tubulin [17] reveals neurons in ventral horn with extensive processes similar to motoneurons. Finally, h-III tubulin-positive neurons can be biolistically transfected by plasmid CMV promoter constructs driving expression of EGFP or DsRed, as shown by the subsequent de novo production of these fluorescent marker proteins. The ability of these neurons to survive disruption of their membranes in this protocol and subsequently express the protein products of the exogeneous genes thereby introduced is a further indication of their viability. The loss of NeuN immunoreactivity in certain cells may be due to their axotomised state. Indeed, other workers have demonstrated a loss of NeuN expression over time in axotomised facial motoneurons but not in rubrospinal neurons [55]. Thus, the differential NeuN immunostaining we observe is most likely due to the specific downregulation of NeuN expression in subsets of neurons, particularly those present in the ventral horn. Biolistic transfection has been extended from the original use in plant cell transfection [40] to the transfection of neuronal tissue [2,49]. Although there are a number of parameters that require optimisation, once achieved, there is a great potential for this system and significant advantages over viral transfection systems such as the ability to introduce large DNA molecules, the avoidance of virus particle production and absence of a subsequent immune response [9]. One previous report describes an attempt to biolistically transfect interface spinal cultures [50]. These authors failed to transfect neurons, as defined by the lack of co-localisation of transfected cells with NeuN and the restriction of transfected cells to the white matter. They ascribed this to a lack of microcarrier penetration at a blast pressure of 200 psi and, in their hands, higher pressures were reported to dislodge cultured slices from the supporting filter. In our study, we were able to use the recommended rupture pressure of 1100 psi [49] without dislodging the slices or causing significant damage to them due to the use of a nylon mesh between the stopping screen and the tissue sample (modified from Lo et al. [49]). Indeed, we were able to transfect successfully at 1350 psi (data not shown); however, in agreement with other protocols for tissue slices, we found 1100 psi to be optimal. As this culture system potentially maintains all cell types found in spinal tissue, it would be expected that they could all be transfected biolistically as it is a mechanical process. We have shown that neurons can be transfected; however, given the uncertainty regarding the suitability of NeuN as a

199

marker, we cannot be certain that we have identified all neurons present in these cultures. We also observed transfection of astrocytes, as judged by co-localisation with GFAP, but there remained a substantial proportion of transfected cells that we could not positively identify. One way to circumvent this problem is to transfect with constructs that drive marker expression from cell-type specific promoters. The PPT-A promoter construct used in this study exhibits neuronal specific expression as a consequence of a neuron restrictive silencer element motif close to the transcriptional start site [78]. Activity of this promoter has also been shown to be modulated by physiological and pharmacological manipulations in isolated dorsal root ganglia [28]. No studies to date have examined whether such factors can modulate PPT-A promoter activity in spinal cord cultures, although the promoter construct has been successfully expressed in cultured cortical slices [92]. We co-transfected a PPT-A promoter construct that expresses EGFP with a CMV promoter construct that expresses DsRed to enable identification of all transfected cells (DsRed) and PPT-A promoter-expressing neurons (EGFP). This allowed normalisation of the number of EGFP-positive cells, thereby correcting for variations in transfection efficiency between slices. Application of a forskolin analogue/high potassium or [Sar9,Met(O2) 11]-substance P caused a significant increase in the number of cells expressing the PPT-A promoter construct when compared to control slices. EGFPpositive cells were located in the superficial laminae, an area previously found to contain SP immunoreactive neurons [31]. Our data demonstrate that pharmacological manipulation of promoter activity is possible in physiologically relevant areas. A surprising observation was that [Sar9,Met(O2)11]substance P stimulated spinal slices exhibited a small number of EGFP-positive neurons in the ventral horn. Their size, morphology and location in lamina IX fulfil previous criteria set for the identification of motoneurons in organotypic spinal slices [15]. Whilst SP immunoreactive tracts from extrinsic sources are associated in vivo with the ventral horn and motoneurons [23], motoneurons themselves do not normally express SP. However, the up-regulation of PPT-A mRNA levels in spinal cord motoneurons in response to axotomy suggests a reactive expression that could be a mechanism of neuropeptide support for the damaged neurons [97]. 4.2. Plasticity of PPT-A and tachykinin peptide expression From a functional perspective, it is clear that there is a potential for considerable plasticity within the expression profile of the PPT-A gene in spinal neurons. Similarly, there is a plethora of data indicating experimentally induced alterations in tachykinin peptide content in both spinal cord and sensory ganglia. For example, after the induction of peripheral inflammation, both PPT-A gene

200

K.J. Hilton et al. / Brain Research Reviews 46 (2004) 191–203

expression and the protein product SP are up-regulated. Whilst a significant proportion of enhanced spinal SP expression presumably reflects NGF-dependent elevated peptide content of sensory ganglia [16], reported increases in spinal PPT-A mRNA suggest enhanced biosynthesis of SP in intrinsic peptidergic spinal neurons [58,69,74]. Modified expression of tachykinin peptides is apparently not restricted to cells with a clearly established tachykinin peptidergic phenotype, thus a population of non-peptidergic sensory afferents display de novo expression of SP after inflammation [67]. In motoneurons, peptides such as SP that are not detectable under normal condition are transiently expressed after axotomy [97]. In several brain regions, increased PPT-A expression has been observed in reactive astrocytes following tissue insult [46,90]. These studies on plasticity within the tachykinin system may provide a physiological context for the current finding of enhanced PPT-A promoter activity in biolistically transfected organotypic spinal cord cultures. Here, the expression of EGFP under the control of the PPT-A promoter was enhanced by prolonged exposure of the tissue to either forskolin analogue/high K+or [Sar9,Met(O2)11]-substance P. Both of these pharmacological strategies are presumed to induce sustained excitation of intrinsic spinal neurons and therefore may mimic, albeit crudely, ongoing activation of spinal neurons by sensitised primary afferents that release SP and glutamate [81,95]. This in vitro spinal cord model, therefore, could provide a useful means to investigate mechanisms that underpin activity-dependent modification of PPT-A gene transcription. If activation of the PPT-A promoter results in augmented SP and de novo spinal release from intrinsic neurons, then this will impact on spinal neurotransmission and may contribute to abnormal somatosensory phenomena that characterise chronic pain. 4.3. Physiological activation of the PPT-A promoter Activity-dependent activation of transcription factors and the consequential alteration of gene expression drive phenotypic changes in individual neurons that will radically alter the physiological status of central circuitry. For example, in spinal cord nociceptive circuitry, a temporal sequence of post-translational and transcriptional modifications is implicated in the generation of central hypersensitivity and inflammatory pain [95]. Distinct patterns of gene expression in central neurons are controlled by both negative and positive regulatory elements localised to neuronal-specific promoters [18]. External factors that initiate intracellular signal-transduction pathways and the activation or expression of transcription factors associated with these promoter response elements will, in turn, influence promoter activity. In this study using organotypic spinal cord slices maintained in culture for several days, we revealed a link between activation of the PPT-A promoter and putative neuronal excitation. In the first protocol, 24-h

incubation in a medium containing an analogue of the cAMP activator forskolin and elevated K+ induced a significant enhancement of PPT-A promoter-driven EGFPexpressing cells compared to untreated controls. These data are consistent with increased activity of the PPT-A promoter in transfected cortical neurons [92] or the endogenous PPTA gene in dorsal root ganglion neurons [59] after exposure to forskolin/high K+. In a separate study, inclusion of the selective NK-1R agonist [Sar9,Met(O2)11]-substance P in the extracellular medium for 24 h induced a twofold elevation of EGFP-expressing cells. The signal-transduction cascade that links neuronal excitation induced by forskolin/high K + or [Sar 9,Met(O2)11]-substance P to activation of the PPT-A promoter in organotypic spinal cord slices is unknown, but insight into putative mechanisms may be gained from other published studies investigating neuronal-specific transcriptional control of the PPT-A gene and peptide expression [18,28,75,77,78]. These studies have identified multiple regulatory domains with enhancer and silencer elements localised to the PPT-A promoter that are targets for multiple transcription factors. Activator regions within the PPT-A promoter include AP-1 and cAMP-responsive element (CRE) consensus binding sequences that bind jun/ fos and cAMP-responsive element binding protein (CREB), respectively [75,77]. The AP-1 complex is particularly interesting in the context of the present study because augmented spinal neuron expression of the immediate early genes c-fos and c-jun that interact with the AP-1 site after noxious stimulation or inflammation is well documented [61]. Similarly, the mitogen-activated protein (MAP) kinase cascade and CREB phosphorylation has been linked to Fos expression in tissue injury-induced inflammation [35]. In dorsal horn neurons, SP is a potent activator of Fos [3] although glutamate acting through the NMDA ion-channel complex [12] is also effective. Interestingly, complete Freund’s adjuvant up-regulation of SP encoding mRNA persists in mice that lack NK-1R suggesting that SP-dependent excitation is not prerequisite for up-regulation of intrinsic neuronal peptide expression [74]. It is plausible that other released sensory afferent neurotransmitters such as glutamate acting through metabotropic receptors, pro-nociceptive modulators such as brain-derived neurotrophic factor interacting with spinal TrkB receptors [37,56] or neuronal survival factors such as leukaemia inhibitory factor [32] may contribute to modulation of spinal PPT-A gene expression. NGF, a major driver of SP expression in sensory ganglia, may also be a candidate since TrkA-containing neurons are localised to the spinal cord [57]. Interestingly, TrkA immunoreactivity specifically associated with a population of spinoreticular neurons is enhanced by adjuvant-induced arthritis [76]. In models of persistent pain, a number of intracellular signalling molecules are simultaneously up-regulated in spinal cord [33]. The fact that PPT-A gene expression is subject to control by multiple positive and negative

K.J. Hilton et al. / Brain Research Reviews 46 (2004) 191–203

regulatory elements [18] suggests putative convergence of more than one of these signal-transduction cascades onto the promoter. Further studies using this organotypic spinal cord model will be required to clarify these and other issues such as the identity of intracellular signalling molecules downstream from the activated membrane receptors.

Acknowledgements The authors would like to thank Dr. M. Ackley for assistance with the DIC microscopy. The PPT-A-EGFP plasmid was kindly supplied by Prof. J.P. Quinn, University of Liverpool. K.J.H. is in receipt of a BBSRC Committee studentship.

References [1] C. Abbadie, J.L. Brown, P.W. Mantyh, A.I. Basbaum, Spinal cord substance P receptor immunoreactivity increases in both inflammatory and nerve injury models of persistent pain, Neuroscience 70 (1996) 201 – 209. [2] D. Arnold, L. Feng, J. Kim, N. Heintz, A strategy for the analysis of gene expression during neural development, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 9970 – 9974. [3] H. Badie-Mahdavi, M.A. Worsley, M.A. Ackley, A.U. Asghar, J.R. Slack, A.E. King, A role for protein kinase intracellular messengers in substance P- and nociceptor afferent-mediated excitation and expression of the transcription factor Fos in rat dorsal horn neurons in vitro, Eur. J. Neurosci. 14 (2001) 426 – 434. [4] R.P. Barber, J.E. Vaughn, J.R. Slemmon, P.M. Salvaterra, E. Roberts, S.E. Leeman, The origin, distribution and synaptic relationships of substance P axons in rat spinal cord, J. Comp. Neurol. 184 (1979) 331 – 351. [5] A.N. Bateson, Basic pharmacologic mechanisms involved in benzodiazepine tolerance and withdrawal, Curr. Pharm. Des. 8 (2002) 5 – 21. [6] K. Befort, M. Costigan, C.J. Woolf, Differential gene expression— how to find new analgesic targets, Curr. Opin. Investig. Drugs 2 (2001) 396 – 398. [7] J.E. Biewenga, O.H. Destree, L.H. Schrama, Plasmid-mediated gene transfer in neurons using the biolistics technique, J. Neurosci. Methods 71 (1997) 67 – 75. [8] A. Blomqvist, L. Mackerlova, Spinal projections to the parabrachial nucleus are substance P-immunoreactive, NeuroReport 6 (1995) 605 – 608. [9] A.P. Byrnes, J.E. Rusby, M.J. Wood, H.M. Charlton, Adenovirus gene transfer causes inflammation in the brain, Neuroscience 66 (1995) 1015 – 1024. [10] Y.Q. Cao, P.W. Mantyh, E.J. Carlson, A.M. Gillespie, C.J.H. Epstein, A.I. Basbaum, Primary afferent tachykinins are required to experience moderate to intense pain, Nature 392 (1998) 390 – 394. [11] Z. Changgeng, W. Zenker, M. Celio, Substance P-positive structures in rat spinal cord—a longitudinal bundle ventral to the central canal, Anatomischer Anzeiger 154 (1983) 193 – 203. [12] V. Chapman, J. Buritova, P. Honore, J.M. Besson, Physiological contributions of neurokinin 1 receptor activation, and interactions with NMDA receptors, to inflammatory-evoked spinal c-Fos expression, J. Neurophysiol. 76 (1996) 1817 – 1827. [13] S. De Biasi, A. Rustioni, Glutamate and substance P coexist in primary afferent terminals in the superficial laminae of spinal cord, Proc. Natl. Acad. Sci. U. S. A. 85 (1988) 7820 – 7824.

201

[14] Y. De Koninck, J.L. Henry, Substance P-mediated slow excitatory postsynaptic potential elicited in dorsal horn neurons in vivo by noxious stimulation, Proc. Natl. Acad. Sci. U. S. A. 88 (1991) 11344 – 11348. [15] J. Delfs, J. Friend, S. Ishimoto, D. Saroff, Ventral and dorsal horn acetylcholinesterase neurons are maintained in organotypic cultures of postnatal rat spinal cord explants, Brain Res. 488 (1989) 31 – 42. [16] J. Donnerer, R. Schuligoi, C. Stein, R. Amann, Upregulation, release and axonal transport of substance P and calcitonin gene-related peptide in adjuvant inflammation and regulatory function of nerve growth factor, Regul. Pept. 46 (1993) 150 – 154. [17] E. Draberova, Z. Lukas, D. Ivanyi, V. Viklicky, P. Draber, Expression of class III beta-tubulin in normal and neoplastic human tissues, Histochem. Cell Biol. 109 (1998) 231 – 239. [18] C. Fiskerstrand, J.P. Quinn, The molecular biology of preprotachykinin-A gene expression, Neuropeptides 30 (1996) 602 – 610. [19] E.M. Fitzgerald, Regulation of voltage-dependent calcium channels in rat sensory neurones involves a Ras-mitogen-activated protein kinase pathway, J. Physiol. 527 (2000) 433 – 444. [20] P.A. Furth, Gene transfer by biolistic process, Mol. Biotechnol. 7 (1997) 139 – 143. [21] B.H. Gahwiler, M. Capogna, D. Debanne, R.A. McKinney, S.M. Thompson, Organotypic slice cultures: a technique has come of age, Trends Neurosci. 20 (1997) 471 – 477. [22] M.G. Garry, K.M. Hargreaves, Enhanced release of immunoreactive CGRP and substance P from spinal dorsal horn slices occurs during carrageenan inflammation, Brain Res. 582 (1992) 139 – 142. [23] S.J. Gibson, S.R. Bloom, J.M. Polak, A novel substance P pathway linking the dorsal and ventral horn in the upper lumbar segments of the rat spinal cord, Brain Res. 301 (1984) 243 – 251. [24] J.R. Gingrich, J. Roder, Inducible gene expression in the nervous system of transgenic mice, Annu. Rev. Neurosci. 21 (1998) 377 – 405. [25] D.L. Hammond, M.A. Ruda, Developmental alterations in nociceptive threshold, immunoreactive calcitonin gene-related peptide and substance P, and fluoride-resistant acid phosphatase in neonatally capsaicin-treated rats, J. Comp. Neurol. 312 (1991) 436 – 450. [26] U. Hanesch, U. Pfrommer, B.D. Grubb, B. Heppelmann, H.G. Schaible, The proportion of CGRP-immunoreactive and SP-mRNA containing dorsal root ganglion cells is increased by a unilateral inflammation of the ankle joint of the rat, Regul. Pept. 46 (1993) 202 – 203. [27] A.J. Harmar, V. Hyde, K. Chapman, Identification and cDNA sequence of delta-preprotachykinin, a fourth splicing variant of the rat substance P precursor, FEBS Lett. 275 (1990) 22 – 24. [28] P.T. Harrison, R.G. Dalziel, N.A. Ditchfield, J.P. Quinn, Neuronalspecific and nerve growth factor-inducible expression directed by the preprotachykinin-A promoter delivered by an adeno-associated virus vector, Neuroscience 94 (1999) 997 – 1003. [29] C.J. Helke, J.J. Neil, V.J. Massari, A.D. Loewy, Substance P neurons project from the ventral medulla to the intermediolateral cell column and ventral horn in the rat, Brain Res. 243 (1982) 147 – 152. [30] T. Hirai, Y. Yoneda, Functional alterations in immature cultured rat hippocampal neurons after sustained exposure to static magnetic fields, J. Neurosci. Res. 75 (2004) 230 – 240. [31] S.P. Hunt, J.S. Kelly, P.C. Emson, J.R. Kimmel, R.J. Miller, J.Y. Wu, An immunohistochemical study of neuronal populations containing neuropeptides or gamma-aminobutyrate within the superficial layers of the rat dorsal horn, Neuroscience 6 (1981) 1883 – 1898. [32] O.J. Igwe, c-Src kinase activation regulates preprotachykinin gene expression and substance P secretion in rat sensory ganglia, Eur. J. Neurosci. 18 (2003) 1719 – 1730. [33] O.J. Igwe, L. Ning, Regulation of the second-messenger systems in the rat spinal cord during prolonged peripheral inflammation, Pain 58 (1994) 63 – 75. [34] T.M. Jessell, Substance P in nociceptive sensory neurons, Ciba Found. Symp. (1982) 225 – 248. [35] R.R. Ji, F. Rupp, Phosphorylation of transcription factor CREB in rat spinal cord after formalin-induced hyperalgesia: relationship to c-fos induction, J. Neurosci. 17 (1997) 1776 – 1785.

202

K.J. Hilton et al. / Brain Research Reviews 46 (2004) 191–203

[36] R.R. Ji, C.J. Woolf, Neuronal plasticity and signal transduction in nociceptive neurons: implications for the initiation and maintenance of pathological pain, Neurobiol. Dis. 8 (2001) 1 – 10. [37] B.J. Kerr, E.J. Bradbury, D.L.H. Bennett, P.M. Trivedi, P. Dassan, J. French, D.B. Shelton, S.B. McMahon, S.W.N. Thompson, Brainderived neurotrophic factor modulates nociceptive sensory inputs and NMDA-evoked responses in the rat spinal cord, J. Neurosci. 19 (1999) 5138 – 5148. [38] S.G. Khasabov, S.D. Rogers, J.R. Ghilardi, C.M. Peters, P.W. Mantyh, D.A. Simone, Spinal neurons that possess the substance P receptor are required for the development of central sensitization, J. Neurosci. 22 (2002) 9086 – 9098. [39] A.E. King, M.A. Ackley, J.R. Slack, Profile of neuronal excitation following selective activation of the neurokinin-1 receptor in rat deep dorsal horn in vitro, Brain Res. 767 (1997) 55 – 63. [40] T.M. Klein, S. Fitzpatrick-McElligott, Particle bombardment: a universal approach for gene transfer to cells and tissues, Curr. Opin. Biotechnol. 4 (1993) 583 – 590. [41] J.E. Krause, J.M. Chirgwin, M.S. Carter, Z.S. Xu, A.D. Hershey, Three rat preprotachykinin mRNAs encode the neuropeptides substance P and neurokinin A, Proc. Natl. Acad. Sci. U. S. A. 84 (1987) 881 – 885. [42] D.S. Latchman, How can we use our growing understanding of gene transcription to discover effective new medicines? Curr. Opin. Biotechnol. 8 (1997) 713 – 717. [43] J. Leah, D. Menetrey, J. de Pommery, Neuropeptides in long ascending spinal tract cells in the rat: evidence for parallel processing of ascending information, Neuroscience 24 (1988) 195 – 207. [44] T.A. Leslie, P.C. Emson, P.M. Dowd, C.J. Woolf, Nerve growth factor contributes to the up-regulation of growth-associated protein 43 and preprotachykinin A messenger RNAs in primary sensory neurons following peripheral inflammation, Neuroscience 67 (1995) 753 – 761. [45] D. Lima, A. Avelino, A. Coimbra, Morphological characterization of marginal (lamina I) neurons immunoreactive for substance P, enkephalin, dynorphin and gamma-aminobutyric acid in the rat spinal cord, J. Chem. Neuroanat. 6 (1993) 43 – 52. [46] R.C. Lin, Reactive astrocytes express substance-P immunoreactivity in the adult forebrain after injury, NeuroReport 7 (1995) 310 – 312. [47] R.M. Lindsay, A.J. Harmar, Nerve growth factor regulates expression of neuropeptide genes in adult sensory neurons, Nature 337 (1989) 362 – 364. [48] N.K. Littlewood, A.J. Todd, R.C. Spike, C. Watt, S.A.S. Shehab, The types of neuron in spinal dorsal horn which possess neurokinin-1 receptors, Neuroscience 66 (1995) 597 – 608. [49] D.C. Lo, A.K. McAllister, L.C. Katz, Neuronal transfection in brain slices using particle-mediated gene transfer, Neuron 13 (1994) 1263 – 1268. [50] D.R. Marsh, K.D. Holmes, G.A. Dekaban, L.C. Weaver, Distribution of an NMDA receptor:GFP fusion protein in sensory neurons is altered by a C-terminal construct, J. Neurochem. 77 (2001) 23 – 33. [51] G.E. Marshall, S.A.S. Shehab, R.C. Spike, A.J. Todd, Neurokinin-1 receptors on lumbar spinothalamic neurons in the rat, Neuroscience 72 (1996) 255 – 263. [52] V.J. Massari, Y. Tizabi, C.H. Park, T.W. Moody, C.J. Helke, T.L. O’Donohue, Distribution and origin of bombesin, substance P and somatostatin in cat spinal cord, Peptides 4 (1983) 673 – 681. [53] K.E. McCarson, Central and peripheral expression of neurokinin-1 and neurokinin-3 receptor and substance P-encoding messenger RNAs: peripheral regulation during formalin-induced inflammation and lack of neurokinin receptor expression in primary afferent sensory neurons, Neuroscience 93 (1999) 361 – 370. [54] K.E. McCarson, J.E. Krause, The formalin-induced expression of tachykinin peptide and neurokinin receptor messenger RNAs in rat sensory ganglia and spinal cord is modulated by opiate preadministration, Neuroscience 64 (1995) 729 – 739.

[55] L.T. McPhail, C.B. McBride, J. McGraw, J.D. Steeves, W. Tetzlaff, Axotomy abolishes NeuN expression in facial but not rubrospinal neurons, Exp. Neurol. 185 (2004) 182 – 190. [56] A. Merighi, G. Carmignoto, S. Gobbo, L. Lossi, C. Salio, A.M. Vergnano, M. Zonta, Neurotrophins in spinal cord nociceptive pathways, Prog. Brain Res. 146 (2004) 291 – 321. [57] G.J. Michael, E. Kaya, S. Averill, M. Rattray, D.O. Clary, J.V. Priestley, TrkA immunoreactive neurones in the rat spinal cord, J. Comp. Neurol. 385 (1997) 441 – 455. [58] M. Minami, Y. Kuraishi, M. Kawamura, T. Yamaguchi, Y. Masu, S. Nakanishi, M. Satoh, Enhancement of preprotachykinin A gene expression by adjuvant-induced inflammation in the rat spinal cord: possible involvement of substance P-containing spinal neurons in nociception, Neurosci. Lett. 98 (1989) 105 – 110. [59] P.K. Mulderry, Neuropeptide expression by newborn and adult rat sensory neurons in culture: effects of nerve growth factor and other neurotrophic factors, Neuroscience 59 (1994) 673 – 688. [60] R.J. Mullen, C.R. Buck, A.M. Smith, NeuN, a neuronal specific nuclear protein in vertebrates, Development 116 (1992) 201 – 211. [61] R. Munglani, S.P. Hunt, Molecular biology of pain, Br. J. Anaesth. 75 (1995) 186 – 192. [62] R. Munglani, S.P. Hunt, Proto-oncogenes: basic concepts and stimulation induced changes in the spinal cord, Prog. Brain Res. 104 (1995) 283 – 298. [63] J.I. Nagy, S.R. Vincent, W.A. Staines, H.C. Fibiger, T.D. Reisine, H.I. Yamamura, Neurotoxic action of capsaicin on spinal substance P neurons, Brain Res. 186 (1980) 435 – 444. [64] R.L. Nahin, Immunocytochemical identification of long ascending peptidergic neurons contributing to the spinoreticular tract in the rat, Neuroscience 23 (1987) 859 – 869. [65] V. Neugebauer, H.-G. Schaible, F. Weiretter, U. Freudenberger, The involvement of substance P and neurokinin-1 receptors in the responses of rat dorsal horn neurons to noxious but not to innocuous mechanical stimuli applied to the knee joint, Brain Res. 666 (1994) 207 – 215. [66] V. Neugebauer, F. Weiretter, H.-G. Schaible, Involvement of substance P and neurokinin-1 receptors in the hyperexcitability of dorsal horn neurons during development of acute arthritis in rat’s knee joint, J. Neurophysiol. 73 (1995) 1574 – 1583. [67] S. Neumann, T. Doubell, T. Leslie, C.J. Woolf, Inflammatory pain hypersensitivity mediated by phenotypic switch in myelinated primary sensory neurons, Nature 384 (1996) 360 – 364. [68] M.M. Niblock, J.K. Brunso-Bechtold, D.R. Riddle, Laminar variation in neuronal viability and trophic dependence in neocortical slices, J. Neurosci. Res. 65 (2001) 455 – 462. [69] K. Noguchi, M.A. Ruda, Gene regulation in an ascending nociceptive pathway: inflammation-induced increase in preprotachykinin mRNA in rat lamina I spinal projection neurons, J. Neurosci. 12 (1992) 2563 – 2572. [70] J. Noraberg, B.W. Kristensen, J. Zimmer, Markers for neuronal degeneration in organotypic slice cultures, Brain Res. Brain Res. Protoc. 3 (1999) 278 – 290. [71] M. Otsuka, M. Yanagisawa, Pain and neurotransmitters, Cell. Mol. Neurobiol. 10 (1990) 293 – 302. [72] M. Otsuka, K. Yoshioka, Neurotransmitter functions of mammalian tachykinins, Physiol. Rev. 73 (1993) 229 – 308. [73] M. Otsuka, S. Konishi, M. Yanagisawa, A. Tsunoo, H. Akagi, Role of substance P as a sensory transmitter in spinal cord and sympathetic ganglia, Ciba Found. Symp. (1982) 13 – 34. [74] J.A. Palmer, C. De Felipe, J.A. O’Brien, S.P. Hunt, Disruption of the substance P receptor (neurokinin-1) gene does not prevent upregulation of preprotachykinin-A mRNA in the spinal cord of mice following peripheral inflammation, Eur. J. Neurosci. 11 (1999) 3531 – 3538. [75] J.M. Paterson, S.C. Mendelson, J. McAllister, C.F. Morrison, S. Dobson, C. Grace, J.P. Quinn, Three immediate early gene response elements in the proximal preprotachykinin-A promoter in two functionally distinct domains, Neuroscience 66 (1995) 921 – 932.

K.J. Hilton et al. / Brain Research Reviews 46 (2004) 191–203 [76] S. Pezet, B. Onte´niente, G. Grannec, B. Calvino, Chronic pain is associated with increased TrkA immunoreactivity in spinoreticular neurons, J. Neurosci. 19 (1999) 5482 – 5492. [77] J.P. Quinn, Neuronal-specific gene expression—the interaction of both positive and negative transcriptional regulators, Prog. Neurobiol. 50 (1996) 363 – 379. [78] J.P. Quinn, V.J. Bubb, Z.V. Marshall-Jones, J.M. Coulson, Neuron restrictive silencer factor as a modulator of neuropeptide gene expression, Regul. Pept. 108 (2002) 135 – 141. [79] V. Radhakrishnan, J.L. Henry, Antagonism of nociceptive responses of cat spinal dorsal horn neurons in vivo by the NK-1 receptor antagonists CP-96,345 and CP-99,994, but not by CP-96,344, Neuroscience 64 (1995) 943 – 958. [80] J.M. Rall, D.A. Matzilevich, P.K. Dash, Comparative analysis of mRNA levels in the frontal cortex and the hippocampus in the basal state and in response to experimental brain injury, Neuropathol. Appl. Neurobiol. 29 (2003) 118 – 131. [81] H.G. Schaible, B. Jarrott, P.J. Hope, A.W. Duggan, Release of immunoreactive substance P in the spinal cord during development of acute arthritis in the knee joint of the cat: a study with antibody microprobes, Brain Res. 529 (1990) 214 – 223. [82] E. Senba, C. Yanaihara, N. Yanaihara, M. Tohyama, Co-localization of substance P and Met–enkephalin–Arg6–Gly7–Leu8 in the intraspinal neurons of the rat, with special reference to the neurons in the substantia gelatinosa, Brain Res. 453 (1988) 110 – 116. [83] G.D. Smith, J.R. Seckl, W.J. Sheward, J.G. Bennie, S.M. Carroll, H. Dick, A.J. Harmar, Effect of adrenalectomy and dexamethasone on neuropeptide content of dorsal root ganglia in the rat, Brain Res. 564 (1991) 27 – 30. [84] L. Stoppini, P.A. Buchs, D. Muller, A simple method for organotypic cultures of nervous tissue, J. Neurosci. Methods 37 (1991) 173 – 182. [85] S. Sugiura, K. Kitagawa, E. Omura-Matsuoka, T. Sasaki, S. Tanaka, Y. Yagita, K. Matsushita, D.R. Storm, M. Hori, CRE-mediated gene transcription in the peri-infarct area after focal cerebral ischemia in mice, J. Neurosci. Res. 75 (2004) 401 – 407. [86] E. Theriault, M. Otsuka, T. Jessell, Capsaicin-evoked release of substance P from primary sensory neurons, Brain Res. 170 (1979) 209 – 213.

203

[87] A. Thomas, D.S. Kim, R.L. Fields, H. Chin, H. Gainer, Quantitative analysis of gene expression in organotypic slice-explant cultures by particle-mediated gene transfer, J. Neurosci. Methods 84 (1998) 181 – 191. [88] A.J. Todd, Anatomy of primary afferents and projection neurones in the rat spinal dorsal horn with particular emphasis on substance P and the neurokinin 1 receptor, Exp. Physiol. 87 (2002) 245 – 249. [89] A.J. Todd, M.M. McGill, S.A.S. Shehab, Neurokinin 1 receptor expression by neurons in laminae I, III and IV of the rat spinal dorsal horn that project to the brainstem, Eur. J. Neurosci. 12 (2000) 689 – 700. [90] H.P. Too, D.R. Marriott, G.P. Wilkin, Preprotachykinin-A and substance P receptor (NK1) gene expression in rat astrocytes in vitro, Neurosci. Lett. 182 (1994) 185 – 187. [91] F.D. Urnov, E.J. Rebar, Designed transcription factors as tools for therapeutics and functional genomics, Biochem. Pharmacol. 64 (2002) 919 – 923. [92] P.D. Walker, R. Andrade, J.P. Quinn, M.J. Bannon, Real-time analysis of preprotachykinin promoter activity in single cortical neurons, J. Neurochem. 75 (2000) 882 – 885. [93] M.K. Warden, W.S. Young III, Distribution of cells containing mRNAs encoding substance P and neurokinin B in the rat central nervous system, J. Comp. Neurol. 272 (1988) 90 – 113. [94] P. Washbourne, A.K. McAllister, Techniques for gene transfer into neurons, Curr. Opin. Neurobiol. 12 (2002) 566 – 573. [95] C.J. Woolf, M. Costigan, Transcriptional and posttranslational plasticity and the generation of inflammatory pain, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 7723 – 7730. [96] Z. Xia, H. Dudek, C.K. Miranti, M.E. Greenberg, Calcium influx via the NMDA receptor induces immediate early gene transcription by a MAP kinase/ERK-dependent mechanism, J. Neurosci. 16 (1996) 5425 – 5436. [97] X. Zhang, V.M. Verge, Z. Wiesenfeld-Hallin, F. Piehl, T. Hokfelt, Expression of neuropeptides and neuropeptide mRNAs in spinal cord after axotomy in the rat, with special reference to motoneurons and galanin, Exp. Brain Res. 93 (1993) 450 – 461.