The MantelCHaenszel test was also used to determine whether the proportions of GRP+ and GRP-negative cells that showed pERK in response to noxious or pruritic stimuli differed significantly. that GRP cells are innervated by nociceptors and are strongly activated AC-42 by noxious stimuli, we found that very few GRP cells receive direct synaptic input from TRPV1-expressing afferents, and that they seldom phosphorylate extracellular signalCregulated kinases in response to noxious stimuli. These findings show that this SP and GRP cells differentially process somatosensory information. locus (Tac1Cre). Using this approach, we found that SP-expressing cells are located mainly in lamina IIo, somewhat dorsal to GRP cells, even though populations show some spatial overlap.23 Although SP-expressing neurons include some projection neurons and inhibitory cells, the great majority are excitatory interneurons.23,25 Despite the nonoverlapping expression pattern of these neuropeptides, we cannot be certain that GRP and SP cells symbolize distinct functional populations. Here, we used a variety of techniques to characterise and compare GRP- and SP-expressing neurons in the mouse superficial dorsal horn. We find that these 2 populations differ widely in anatomical, electrophysiological, and pharmacological properties, suggesting that they represent unique populations that are likely to contribute differentially to somatosensory processing. 2. Methods Experiments were approved by the Ethical Review Process Applications Panel of the University or college of Glasgow and were performed in accordance with the UK Animals (Scientific Procedures) Take action 1986 and the University or college of California, San Francisco’s Institutional Animal Care and Use Committee guidelines. 2.1. Animals We used 2 genetically altered mouse strains: a BAC transgenic Tg(GRP-EGFP) from GENSAT in which GFP is expressed under control of the GRP promoter,19,26,46,57 and a collection in which Cre-recombinase is inserted into the locus (Tac1-IRES2-Cre-D; Jackson Laboratory, Bar Harbor, ME; Stock number 021877).28 These lines are referred to as GRP::eGFP and Tac1Cre, respectively. GRP::eGFP mice were managed as heterozygotes, whereas most of the Tac1Cre mice were homozygous. Both strains were maintained on a C57BL/6 background. For some Mouse monoclonal to FGB experiments, the 2 2 lines were crossed to produce double transgenic mice (Tac1Cre;GRP::eGFP). Unless otherwise stated, mice of either sex weighing between 14 and 28 g were used in all parts of the study. Most of the mice that were utilized for anatomical studies underwent perfusion fixation. They were deeply anaesthetised with pentobarbitone (20 mg, intraperitoneally [i.p.]) and perfused through the heart with a fixative that contained 4% freshly depolymerised formaldehyde in phosphate buffer. Spinal cord tissue was rapidly dissected out and postfixed at 4C for 2 hours (unless stated normally). 2.2. Intraspinal injection Intraspinal injections were performed to deliver viral vectors coding for Cre-dependent constructs into Tac1Cre or Tac1Cre;GRP::eGFP mice, and to deliver the retrograde tracer cholera toxin B (CTb) subunit into GRP::eGFP mice. Table ?Table11 lists the viral vectors used. To identify SP cells, we used AAVs coding for Cre-dependent eGFP or tdTomato, and to investigate the somatodendritic morphology of these cells, we used AAV-Brainbow vectors.9 The injections used a modification of the method of Foster et al.17 as AC-42 described previously.23 The mice were anaesthetised with 1% to 2% isoflurane and placed in a stereotaxic frame. For the experiments including AAVs, 2 injections were made in each animal, either into the L3 and L5 segments on one side, or else bilaterally into either the L3 or L5 segments. The vertebral column was uncovered, and vertebral clamps were attached to the T12 and L1 vertebrae. The space between the laminae of T12 and T13 was utilized for L3 injections and that between laminae of T13 and L1 for L5 injections. In each case, a small incision was made in the dura to the side of the midline, and injections were made through glass micropipettes (inner diameter of tip 40 m) into the spinal dorsal horn. Injections were made 300 to 500 m lateral to the midline at a depth of 300 m below the pial surface and were administered at a rate of 30 nL/minute. The wound was then closed, and AC-42 animals were allowed to recover with appropriate analgesia (buprenorphine 0.3 mg/kg and carprofen 5 mg/kg). Injections of AC-42 CTb into the GRP::eGFP mice were targeted around the T13 spinal segment. These injections were performed as explained above, except that they were made through the space between the laminae of T11 and T12 vertebrae. 25 In each case, a single injection (300 nL of 1% CTb) was.