Shedding new light on Pain pathways using Jelly-fish genes.

Richard Morris and Richard Barrett-Jolley

 

Veterinary Neurobiology, University of Liverpool
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Studies to further the development of pain relief in humans and animals

Description of research

Aims

Recent developments in molecular biology methods have permitted the incorporation of marker fluorescent proteins into transgenic mice. One of the most commonly used marker proteins fluoresces green (GFP) and was originally identified in a jellyfish. This fluorescent protein permits labeling of cells on the basis of the genes they express. The huge advantage of this approach is that these proteins can be seen in living cells and do not alter the normal function of these cells. Thus for example, in principle, it would be possible to produce a transgenic mouse in which all the neurons that express m-opioid receptors are bright green. This can then be used to target these cells for electrophysiological recording, track cells in development and in cell culture and to sort these cells for gene analysis. This technology is revolutionizing neurobiology and as I will show below is already generating startling new results.

     Expressed simply our overall aim is to start to exploit this new technology fully to investigate nociceptive systems. This technology is quite new and developing rapidly. As discussed below several animals have already been produced in which genes of interest in nociceptive pathways have been coupled to GFP. Through collaborations we have been able to obtain some of these mouse strains and are in the process of acquiring others. Currently we are breeding a very interesting mouse in which most of the primary afferents innervating the epidermis express GFP. Most of this application concerns this mouse. In addition we will be establishing a colony of mice in which GABAergic inhibitory neurons are labeled with GFP and some of the studies this will be used for are also discussed. Several other developments are also discussed to indicate the overall direction in which this research is developing.

 

b. Background.

 

i. Promoters driving production of marker proteins.

          With the sequencing of the human genome and subsequently that of the mouse it has been possible to identify the regulatory regions controlling the expression of many genes. Typically this is a sequence immediately preceding the first translated region of the gene (exon1). These promoter regions can be used to drive other genes including those for the fluorescent protein, green fluorescent protein (GFP). If a DNA sequence consisting of a promoter and GFP is inserted into the genome it will have in addition to its own copy of this promoter driving its normal gene, the additional transgene. Now when the normal promoter is activated to produce its gene product, the transgene will also be activated to produce GFP. For example, we have recently started investigating a mouse in which a promoter called Thy 1.2 drives the production of GFP. In this mouse a group of primary afferents innervating the epidermis is bright green (Figure 1). These fibres terminate in LII of the spinal cord. Findings on a similar mouse in which a G-protein couple receptor drives the production of GFP, which also has labeled epidermal fibres, were reported in January (Zylka et al 2005).

 

ii. Viral delivery of promoters driving production of marker proteins.

 

An alternative route to introducing the transgene is to use a virus vector such as the adeno-associated virus (AAV). The viral gene is altered so that it now contains a promoter driving production of GFP. Now cells taking up the virus and activating this promoter will turn green. This has been very successfully applied to neurons expressing melanin concentrating hormone (MCH) in the hypothalamus (van den Pol et al 2004). The MCH neurons taking up the virus turned green permitting the researchers to selectively target these neurons with patch recording electrodes. As only the MCH neurons turn green this greatly facilitates recording from specific neurone types that occur in low density.

 

Figure 1. Section through the skin of transgenic mouse expressing GFP in epidermal fibres (Fresh frozen section no staining)

 

 

iii. Reporter mice and cre-loxP technology.

          Another development in these methods is to use a system known as the cre-loxP system. LoxP sequences are short DNA sequences. Typically two or more of these are introduced into a gene and the gene is said to be “floxed”. Cre-recombinase is an enzyme from bacteriophages that cross links two loxP sequence and excises one loxP sequence and the intervening DNA. This has been used to inactivate or “knockout” genes. However, another interesting use is to label cells. Mice have been produced that are called reporter mice. These have the sequence promoter-loxP-stop-loxP-reporter gene. The reporter gene is commonly a gene for a fluorescent protein such as GFP. In this mouse the gene is not expressed due to the stop sequence. However, when cre-recombinase is introduced cells in which this is produced turn green. As discussed above, for promoters driving GFP, the cre-recombinase can either be produced genetically or via a viral vector. For example, if a mouse is produced in which a homeobox (Hox) gene drives the production of cre-recombinase, and this is then crossed with a reporter mouse in which the neuronal specific promoter tau is linked to GFP, then all the neurons activating tau, which also activate the hox gene, will turn green. This technology is rapidly identifying neuronal lineages and has for example revealed how the ventral floor plate of the spinal cord differentiates to produce different groups of neurones. The hox genes regulating the dorsal plate differentiation into dorsal horn neurones are now being identified. This technology will permit different groups to dorsal horn neurones to tracked through development and targeted with whole-cell patch recording electrodes.

 

iv. Conditional gene knockout using cre-loxP technology.

The implications of the cre-loxP system do not stop at simply labeling neurones. If a mouse is generated in which a specific promoter drives the production of cre-recombinase then when this is crossed with animals containing “floxed” genes the gene will be inactivated. Recently, for example a mouse has been produced in which the promoter for a sodium channel (NaV 1.8) that is unique to small dorsal root ganglion (DRG) neurons drives the production of cre-recombinase (Stirling et al 2005). Crossing this animal with one in which a gene has been “floxed” will now inactivate this gene in just the neurons activating NaV1.8. So we now have a method for inactivating genes in just one group of neurons. An alternative approach is to introduce the cre-recombinase via a viral vector. This has been used to examine the role of the NR1 subunit of the NMDA receptor in nociception. A mouse was produced in which the NR1 gene was “floxed” and adeno-associated virus expressing the cre-recombinase gene under the regulation of the human cyclomegalavirus promoter (HCMV) was injected in the cord. This inactivated NR1 subunit synthesis in the transfected neurons and led to changes in the development of the second phase response in the formalin test (South et al 2003). This approach is very valuable in testing new targets for analgesics when no specific antagonists have been developed.

 

v. Switching on neurotoxic genes.

          In the same way as discussed for reporter mice it is possible to selectively lesion neurons. If a mouse with the following sequence promoter-loxP-Stop-loxP-neurotoxin gene is crossed with an animal in which cre-recombinase is produced by a neuron specific promoter, such as the NaV1.8 promoter, just those neurons expressing this gene would die. This provides a method for selectively lesioning component neurons in pain pathways to see what changes occur. For example applying this to the neurokinin 1 (NK1) receptor expressing neurons would be a much cleaner method of lesioning these neurons than the existing method with saporin conjugated substance P.

 

vi. Summary.

Essentially neurobiology is entering a new era in which new genomic methods open huge possibilities. In theory we could have a colour coded nervous system, lesion any selected neuronal group, separate any neurone type for gene expression analysis and track any neurone type through development, in culture or as it responds to insults such as nerve injury.

 

vii. Evaluation and exploitation of these technologies.

          In view of this potential we have been rapidly reconfiguring our laboratories to exploit these methods. We have assembled several electrophysiology systems equipped for whole-cell patch electrode studies from fluorescently labeled neurons in tissue slices and dissociated cultures. Several strains of mice have been imported in which different populations of neurons fluoresce green. Several reporter mice lines have been established. One mouse line expressing cre-recombinase has been imported and contacts have been established with several other groups breeding these mice. In collaboration with Professor John Quinn several viral vectors are being constructed including ones in which galanine and the NK1 promoter regions drive production of GFP. We are in position immediately to investigate the functions of epidermal primary afferents and a number of specific experiments to be conducted with these animals are discussed. We also have on order a mouse in which inhibitory interneurones containing GABA are labelled.  Other work at an earlier stage of development is also discussed.

 

 

1.Thy1.2-gfp mice.

          We have been given a remarkable mouse in which all the small DRG neurons that bind IB4 lectin express GFP. There is no expression in calcitonin gene related peptide (CGRP) or substance P containing primary afferents. All the GFP labeled afferents innervate hairy skin and reveal fine details of this innervation (Figure 1). Most cross the dermis without branching and enter the epidermis where they break up into huge numbers of fine terminal branches. The central terminals of these primary afferents innervate LII, the substantia gelatinosa (SG). Hence, we now know that one of the primary functions of the SG is analysis of epidermal information. Many of these IB4 binding afferents express TRPV1 channels that are activated by noxious heat, acid and capsaicin. Activation of these afferents would produce the sensation of burning pain, which can be so debilitating in some neuropathic pain patients.  We propose that this system of primary afferents transducer signals that have to be detected in the epidermis. Thus they would be concerned with noxious and non-noxious thermal reception, itch and probably pleasurable touch. We would hypothesise that different groups of these afferents would express different TRP channels and different Mgr-family G-protein coupled receptors. This will be tested with by whole-cell patch recording from the dissociated DRG.  The expression of GFP will permit direct targeting of electrodes on these specific cells. Additionally this opens up a range of other possibilities including studies of : the effects of peripheral applications of substances such as capsaicin (they could also be used to evaluate any topically applied compound), the effects of peripheral nerve damage, dorsal root damage and dorsal root regeneration. These cells can also be separated in a fluorescent cell sorter for gene expression studies (not part of the current proposal due to expense).

 

 

 

 

 

 

 

Figure 2. Section of DRG from Thy 1.2-gfp mouse. Stained with biotinylated IB4-lectin and strepatavidin Cy3 (red) showing colocalisation of IB4 and GFP (Note the GFP can be seen in intact ganglia with no staining).

 

         

 

2. Gad-GFP and VACh-cre mice.

One of the key problems in working out dorsal horn circuity is identification of neurone types. Recently the first studies exploiting GFP to identify spinal interneurones were published (Hantman et al 2004, Heinke et al 2004). Heinke et al (2004) used a commercially available (Jaxmice) mouse in which the promoter for enzyme glutamic acid decarboxylase (GAD) drives GFP production and hence, marks inhibitory neurons producing GABA. We are in the process of importing breeding pairs of these mice from Jackson Labs to establish a colony. Apart from their use to record from inhibitory interneurones in LI-LIII of the dorsal horn, they offer a simple way of evaluatating GABA neurone loss in neuropathic models. A collaborator (Dr Misawa) is also sending us mice in which the promoter for the vesicular transporter for acetylcholine (VACh) drives the production of cre-recombinase (Misawa H et al 2003). We will cross this animal with our reporter mice with the intention of producing green LIII cholinergic neurons. These are critically involved in inhibitory processes in the dorsal horn, but because of their low density it has not been possible to study them. If this works it will solve this problem.

 

3. AAV NK1-GFP, Galanine-GFP, NK1-cre.

          AAV are being made in Professor Quinn’s laboratory. These will be injected into the lumbar cords of neonatal rats (NK1-GFP, Galanine-GFP) or in adult reporter mice (NK1-cre). This is with the intention of producing green NK1 or galanine neurons in the lumbar spinal cord. If this is successful we will be able to target these for electrophysiological studies.

 

Methods.

 

Transgenic mouse breeding and screening. Virus production. Standard RT-PCR methods will be used to identify mice carrying the genes of interest in our breeding programs. Viruses expressing promoters linked to GFP or cre-recombinase have been produced or are being produced by Professor John Quinn as part of an ongoing collaboration.

 

Electrophsiology. Both the applicants have a strong background in electrophysiology. Dr Morris has developed and employed spinal cord recording methods for nearly twenty years (eg Cheunsuang et al 2002) and has extensively investigated cultured DRG (eg. Thippeswamy et al 2004). Dr Barrett-Jolley, has also an extensive background in patch-clamp work, particularly from the hypothalamus, but also brings to the project valuable experience in evaluating the biophysical properties of dissociated cells. Recordings will be made from neurones identified by their expression of GFP. In the thy1.2-gfp mouse recordings will be made from dissociated DRG neurons maintained in short term culture. Their biophysical properties and responses to a range of agonists will be examined. Although some studies have produced data for DRG neurons shown subsequently to bind IB4 lectin in the present studies we will have the advantage of being able to selectively target these neurons. Their expression of TRP channels, ASIC channels and H1 histamine receptors will be evaluated by local application of thermal stimuli, capsaicin, menthol, acid at different pH and histamine. The neurones will be tested under voltage clamp conditions and their current-voltage relations analysed. Some of the ligands for the recently identified Mrg family of G-protein coupled receptors will also be tested. In spinal cord slices preliminary recordings will also be made from GAD expressing neurons. Depending on the success of viral transfection and breeding similar studies will be undertaken of Ach, NK1 and Galanine neurons. These will act as proof of method pilot studies for further grant applications to the Wellcome trust and BBSRC.

 

Figure 3. Transverse section of the dorsal horn of the thy 1.2-gfp mouse to show distribution of GFP. Fresh frozen wet mounted section, no staining, the green is, as it would be observed in a living slice for recording.

 

 

 

 

Dorsal rhizotomy, sprouting, growth factors etc. It is immediately apparent that the Thy1.2-gfp mouse could also permit tracking of this fibre population following dorsal rhizotomy. Here we would get a definite loss of fibres in the spinal cord the territory denervated would be immediately apparent by the loss of fluorescence. It is possible that adjacent roots form sprouts into the denervated region and this could be examined. Some groups have also claimed that substantial regeneration of a sectioned dorsal root can be achieved by application of growth factors. However, this has been questioned by others. The Thy1.2-gfp mouse could solve this issue by having an inbuilt tracer which would permit detailed observations of fibres regeneration and immediately show any spared fibres.

 

Anticipated result

 

Firstly the experience we obtain in exploiting these new methods will constitute a major step forward. It is clear, that even if the specific examples given do not generate new data, the overall approach will. It is anticipated that the whole-cell patch studies of the Thy1.2-gfp DRG neurons will reveal a range of transduction properties. We already know that some express TRPV1 receptors activated by noxious heat, capsaicin and protons. Some will also express other thermal receptors. However, the majority are anticipated to express Mrg G-protein coupled receptors which we will test by applying FMRFamide peptides (Dong et al 2001). Activation of these receptors is thought to be noxious and these neurons would be an ideal test situation for antagonists.

 

The whole cell patch studies on GAD neurons will permit us to label these cells by intracellular injection and explore their axon trajectories and contacts. We anticipate that this will reveal several neurone populations. These studies will be used to apply for more extensive funding. Similarly the other studies targeted at labeling specific populations will be used as proof of principle studies.

 

The nerve injury studies are anticipated to result in a region of LII loosing its fluorescent labeling. As a result we will be able to target this region specifically. From earlier studies on complete sciatic nerve transection we anticipate that no evidence of Ab-fibre sprouting will be obtained. However, we do expect to see abnormal inhibitory processes with some neurons being strongly inhibited, and others loosing inhibition. The organization of these changes may contribute strongly to our understanding of neuropathic pain processes.

 

In the epidermis it is anticipated that application of capsaicin will cause many fine epidermal fibre terminals to shrink back from the epidermis as shown by others. However, we would anticipate that some fibres that do not express TRPV1 ion channels would not degenerate. Depending on the outcome of these experiments further studies of the functions of the remaining fibres would be possible. Conversely, if all the fibres do shrink back it presents an interesting question as to how capsaicin is acting.

 

The dorsal rhizotomy studies should reveal that GFP labeled afferents regenerate to the junction along the dorsal root where myelination by Schwann cells changes to myelination by oligodendrocytes. This junction is readily seen by staining for astrocytes. In the spinal cord a gap will be seen with no gfp in LII. We anticipate that no fibres will enter the dorsal root but that some branching will occur in the spinal cord form adjacent roots.

 

Relevant publications

 

Cheuansuang O and Morris R (2000) Spinal lamina I neurones which express neurokinin 1 receptors: 1. Morphological analysis, Neuroscience 97 :335-345

 

Nazli M., Hismiogullari E.S., Thippeswamy T and Morris R., (2001) How central is nitric oxide (NO) to the activation of c-fos in spinal neurones following noxious peripheral stimulation in the rat ? Brain Res 888 :172-157

 

Thippeswamy T. and Morris R., (2001) Evidence that nitric oxide induced sythesis of cGMP only occurs in a paracrine and not autocrine fashion and that the site of its release can be regulated: studies in dorsal root ganglia in vivo and in vitro. Nitric Oxide 5 : 105 – 115

 

Thippeswamy. T. and Morris R., (2001) Inhibition of nitric oxide synthesis in axotomised primary sensory neurones causes cell death : Evidence for a neuroprotective role for nitric oxide in injured neurones in vivo. Neuroscience Research  40 : 37 –44

 

Thippeswamy. T.  McKay J.S. and Morris R. (2001) Bax and caspases are inhibited by endogenous nitric oxide in dorsal root ganglion neurones in vitro. European  J. Neuroscience 14:1229-1236

       

Cheuansuang O., Maxwell D. and Morris R., (2002) Spinal lamina I neurones which express neurokinin 1 receptors: II. Electrophysiological characteristics,  responses to primary afferent stimulation and effects of a selective μ-opioid  receptor agonist. Neuroscience. 111 :423-434

 

Stewart A.L, Morris R, Bannatyne B.A., Gordon S.L.G., Singleton A.G. and Maxwell D.J. (2003). Preliminary evaluation of methods for the study of identified excitatory and inhibitory neurones in LI-LIII of the rat spinal dorsal horn. British Neurosci. Assoc. Abst 17: 34.03

 

Morris R, Cheunsuang O, Stewart A.L. and Maxwell.D., (2004) Spinal dorsal horn neurone targets for nociceptive primary afferents: do single neurone morphological characteristics suggest how nociceptive information is processed at the spinal level. Brain Research Review 46 :173 –190

 

Morris  R, Arber  S, Kramer I, Sigrist M, Belle  M and Cheunsuang O., (2004) Evaluation of the distribution of labeling in primary afferents in a Thy-1.2 promoter-enhanced green fluorescent protein (GFP) transgenic mouse: evidence that the substantia gelatinosa is primarily concerned with sensory information transduced in the epidermis.  Presented at the 2nd James Black Symposium, Cambridge.

 

Thippeswamy T, Jennifer S. McKay J.S, Morris R, Quinn J, Wong L-F and Murphy D., (2004) Glial-mediated neuroprotection: evidence for the protective role of the NO-cGMP pathway in the peripheral nervous system via neuron-glial communication. Glia 154 : 153-164

 

Thippeswamy T McKay J.S. Quinn J.P and Morris R., (2005) Either Nitric Oxide or Nerve Growth Factor is required for dorsal root ganglion neurons to survive during embryonic and neonatal development. Develop. Brain Res. 154 : 153-164

 

Abu Zaki & Barrett-Jolley, R., (2002). Rapid neuromodulation by cortisol in the rat paraventricular nucleus: An in vitro study. British Journal of Pharmacology 137:87-97.

Barrett-Jolley, R. (2001). Nipecotic acid directly activates GABA(A)-like ion channels.  British Journal of Pharmacology 133:673-678.

Barrett-Jolley, R, Pyner, S. & Coote, J.H. (2000). Measurement of Voltage-Gated Potassium Currents in Identified Spinally-Projecting Sympathetic Neurones of the Paraventricular Nucleus. Journal Neuroscience Methods. 102 :25-33.

 

Other relevant publications.

 

Dong X, Han S-K, Zylka M.J., Simon M.L. and Anderson D.J., (2001) A diverse family of GPCRs expressed in specific subset of nociceptive neurons. Cell 106 : 619-632.

 

Hantman AW, van den Pol AN, Perl ER. (2004)Morphological and physiological features of a set of spinal substantia gelatinosa neurons defined by green fluorescent protein expression. J Neurosci. 24 : 836-42.

 

Heinke B, Rischeweyh R., Fosthuber L., Wunderbaldinger G, and Sandkuhler J. (2004) Neurochemical and morphological properties of a subgroup of GABAergic spinal lamina II neurones identified by expression of green fluorescent protein in mice. J. Physiol 560 :249-266

 

Misawa H., Nakata K., Toda K., Matsuura J., Oda Y, Inoue H, Tatneno M. and Takahashi R., (2003) VAChT-Cre, fast and VAChT-cre slow: postanatal expression of cre recombinase in somatomotor neurones with different onset. Genesis 37 :44-50.

 

van den Pol AN, Acuna-Goycolea C, Clark KR, Ghosh PK. (2004) Physiological properties of hypothalamic MCH neurons identified with selective expression of reporter gene after recombinant virus infection. Neuron 42: 635-652

 

South S.M., et al (2003) A conditional deletion of the NR1 subunit of the NMDA receptor in adult spinal cord dorsal horn reduces NMDA currents and injury-induced pain. J Neuroscience 23 :5040-5031.

 

Stirling L.C., Forlani G., Baker M.D., Wood J.N., Matthews E.A., Dickenson A.H. and Nassar M.A., (2005) Nociceptive-specific gene deletion using heterozygous NaV 1.8-Cre recombinant mice. Pain 113 :27-36.

 

Zylka M.J., Rice F.L., and Anderson D.J. (2005) Topographically distinct epidermal nociceptive circuits revealed by axonal tracers targeted to Mrgprd. Neuron 46 :17-25.    

 

 

 

 

Justification of costs.

 

          The salary will be used to provide a stipend for a research student who would be registered for a Ph.D. The rate of the stipend is based on that provided by the BBSRC for research students. The student’s fees and further running expenses will be covered from funds in a Departmental account (see covering letter). The requested consumable costs will be spent on maintenance of mice, immunochemicals, drugs and other routine items. The travel money is the same amount as given to Research students by the BBSRC to cover travel to national and international meetings. The student will also be encouraged to apply for funds from other sources to attend International Pain meetings. Student stipends and their running costs are not subject to overhead costs by the University of Liverpool.

 

          There is a great shortage of trained research students entering Pain research and in particular into functional studies using electrophysiological methods. This is at a time when methods are emerging which will greatly advance this field. Apart from the importance of the work to be conducted this grant would have the added advantage of training a scientist at an early stage of their career who it is hoped would continue to contribute to this field after obtaining their doctorate.

 

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