All procedures complied with the United Kingdom Animals (Scientific Procedures) Act 1986. Experiments were performed on male Sprague–Dawley postnatal day 3 (P3), P10, P20 and adult rats (P48 onwards; 200–250 g) from the colony at University College London. Animals were kept in their home cages at 21°C and 55% relative humidity with a 12 hour light/dark cycle (lights on at 08:00 h) and had unlimited access to food and water. All efforts were made to minimize animal suffering and to reduce the number of animals used.
Antibodies and drugs
Anti-NK1 receptor antibody was a gift from S. Vigna; anti-c-fos was from Calbiochem (Darmstadt, Germany); anti-Fluorogold (anti-FG) from Fluorochrome (Englewood, NJ, USA); anti-NeuN, anti-5-HT and anti-CGRP from Chemicon (CA, US); anti-GFAP from DakoCytomation (Denmark); anti-PKC gamma from Santa Cruz (CA, USA); and anti-isolectin B4 from Vector Laboratories (CA, USA). Formalin was purchased from BDH; Fluorogold (FG) was from Fluorochrome (Englewood, NJ, USA). Substance P-Saporin (SP-SAP) and Blank- Saporin (blank-SAP) were from Advanced Targeting Systems (San Diego, California).
2.5% formalin diluted in 0.9% normal saline was used to induce expression of c-fos in the spinal cord. To take into account the postnatal increase in the size of hindpaw, the volumes of formalin solution injected in P3, P10, P20 and adult rats were 10 μl, 20 μl, 40 μl and 100 μl respectively. These injection volumes were optimized in pilot studies to cover a similar proportion of the animals’ hindpaw across all ages. Injection was delivered with a 0.5 ml insulin syringe with a 28 G needle. Prior to injection of formalin, animals were manually restrained, without anaesthesia, in plantar flexion. Subcutaneous injection of formalin was performed on the plantar surface of the left hindpaw. To control for restraint-induced stress in formalin-injected animals, control animals were also briefly restrained in plantar flexion. Following injection of formalin or brief restraint, P3 and P10 rats were separated from their mothers and kept in a box lined with a thermal pad for two hours. P20 rats and adult rats were returned to their home cages and kept for two hours. Maternal separation of young rats was performed to reduce variability in basal c-fos expression induced by maternal grooming and feeding . Expression of c-fos typically peaks at 2 hours following physiological stimulation (Hunt et al., 1987; Herdegen and Leah, 1998). Therefore, animals were perfused at 2 hours after injection of formalin
4% Fluorogold (FG) mixed in distilled water was prepared on the day of injection. A gas mixture of 4% halothane and 100% oxygen delivered at 2 L/min in a close chamber was used to induce anaesthesia in P3 rats and adult rats. The rats were placed in a Kopf stereotaxic frame and anaesthesia was maintained by the delivery of 1.5 – 2% halothane combined with 100% O2 (1 L/min) via a face mask. A small incision was made in the scalp to expose the skull and reveal bregma. Following craniotomy, animals received an injection of 4% FG into the lateral parabrachial nucleus (PB) on the right side (coordinates for P3: –5.2 mm anteroposterior, 1.3 mm mediolateral and −5.0 mm dorsoventral; coordinates for adult: -9.2 mm anteroposterior, 1.7 mm mediolateral and −6.4 mm dorsoventral) delivered by a 2.5 μl Hamilton syringe. These coordinates were obtained from pilot studies based on an atlas of the rat brain . The injection volumes of FG in P3 and adult rats obtained from pilot studies were 50 nl and 300 nl respectively. Rats were allowed to recover from anaesthesia in an incubation chamber and then transferred back to their home cages until perfusion.
On the day of perfusion (1 day after FG injection in P3 rats and 3 days after FG injection in adult rats), rats were perfused in their naïve state or received a subcutaneous injection of formalin in the left hindpaw, and perfused two hours later. Following fixation and cryoprotection, the brain and the spinal cord were then sectioned on a freezing microtome. Sections were then mounted onto glass slides under a fluorescence microscope equipped with a wide band UV filter.
Substance P-Saporin (SP-SAP) injections
P3 rats were briefly anaesthetized with a mixture of 4% halothane and 100% oxygen delivered at 2 L/min. A 5 μl Hamilton syringe (model 84851, Essex Scientific Laboratory Support) with a removable needle (gauge 32, 25 mm in length, point style 4) was used to deliver the drug intrathecally. Intrathecal injection was targeted at the level of the sixth lumbar vertebrae to minimize damage to the neonatal spinal cord. The needle was aimed at the midpoint of the vertebral column just above the pelvic girdle and carefully advanced until a slight decrease in resistance was felt in the path of the needle and a small flick of the tail or of the lower limb was observed. These signs indicated entry of the needle into the intrathecal space. 2 μl of SP-SAP or blank-SAP at 5 μM was then injected manually over one minute in P3 rats. At the end of the injection, the needle was slowly withdrawn. Rats were allowed to recover from anaesthesia in an incubation chamber and then transferred back to their home cages. Locomotive abilities, feeding behaviours and maternal-neonatal interaction were monitored every day in the first week and then once every 3 days until P48. Body weights of animals were also recorded postnatally to monitor growth. Mechanical threshold of the left hindpaw was measured prior to treatment at P3 and postnatally to assess cutaneous reflex of the lower limbs until P48. Animals were then terminally anaesthetized and perfused for histology.
Von Frey filaments (Stoelting, IL, USA) were used to test mechanical threshold of the hindpaw of postnatal rats treated with SP-SAP and control postnatal rats. All measurements were performed blind to treatment allocation. Testing was an adapted ‘up-down’ method . Prior to testing, animals were placed in clear perspex compartments situated above a metal wire mesh that allowed access to the plantar surfaces of the animals’ hindpaws. Animals were allowed to habituate to the testing environment for 15 min prior to testing. A series of up to 13 Von Frey filaments with logarithmically incremental stiffness was applied perpendicularly to the mid-plantar surface of the hindpaw until slightly bent. Each filament was applied 5 times with 2 to 3 seconds in between applications. A positive response was inferred from a reflex withdrawal of the stimulated hindpaw from a filament. Mechanical threshold was the lowest Von Frey filament that elicited more than 2 positive responses out of 5 applications.
Rats were deeply anaesthetized with intraperitoneal pentobarbital and perfused transcardially with saline containing 5 000 I.U./ml heparin followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (50 ml, 100 ml, 150 ml and 250 ml in P3, P10, P20 and adult rats respectively). Lumbar spinal cord was dissected out, post-fixed in the same PFA solution for 2 hours and transferred into a 30% sucrose solution in phosphate buffer containing 0.01% azide, for a minimum of 24 hours, at 4°C. Spinal cords were cut on a freezing microtome set at 40 μm. All antibody solutions contained 0.01% triton X-100 and 0.3% serum of the host species of a secondary antibody to block non-specific background staining. Sections were incubated with primary antibodies for 48 hours at 4°C. The primary antibodies used were anti-NK1 receptor (1:5000 for biotin protocol or 1:100,000 with tyramide signal amplification (TSA) protocol), anti-c-fos (1:5000 with biotin protocol and 1:100,000 with TSA protocol), anti-Fluorogold (1:50,000 for direct stain), anti-NeuN (1:1000 for direct stain), GFAP (1:1000 with biotin protocol), PKC gamma (1:2000 with biotin protocol), CGRP (1:2000 with biotin protocol), 5-HT (1:100, with biotin protocol) and isolectin B4 (IB4) (1:500, with biotin protocol). For the biotin protocol, appropriate secondary biotinylated antibodies were used (1:500, 2 h) followed by avidin-Cy3 (1:4000, 1 hour, Vector Labs) or avidin-FITC (1:2000, 2 hours, Vector Labs).
For the TSA protocol, following incubation in primary antibody, sections were first incubated with appropriate secondary biotinylated antibodies (1:400, 1.5 hour) followed by avidin biotin complex (ABC Elite; 1:250 Vectastain A plus 1:250 Vectastain B; Vector Laboratories) for 30 minutes followed by a signal amplification step with biotinylated tyramide solution (1:75 for 7 minutes, Perkin Elmer). Sections were then incubated with FITC avidin for a further 2 hours (1:600). Finally, sections were incubated with the second primary antibody overnight at room temperature, followed by incubation in appropriate Alexa Fluor (1:500, 2 h).
NK1-c-fos, FG-NK1 and FG-c-fos double immunostaining were obtained with the TSA protocol. NeuN-c-fos double immunostaining and single immunostaining for NK1, NeuN, GFAP, PKC gamma, CGRP and 5-HT were obtained with the biotin protocol.
All sections were coverslipped with Gel Mount aqueous mounting medium (Sigma) to preserve fluorescence and stored in the dark at 4°C. Controls for immunohistochemistry were carried out omitting the first or second primary antibodies. In some cases, Fluorogold staining was directly visualized with UV detection.
Image acquisition and quantification of immunostaining
Imaging systems connected with Nikon Eclipse E800 microscope or Leica DMRBE confocal microscope equipped with SP2 confocal head were used to acquire images of spinal cord and brain sections immunostained with different antibodies. Images captured from the left dorsal horn of animals injected with formalin in the left hindpaw were used to quantify lamina I neurons immunostained for NK1, c-fos or FG in P3 and adult rats. Neurons with positive immunostaining for NK1, c-fos or FG along the whole length of lamina I were counted. Colocalization of NK1 with c-fos, NK1 with FG or FG with c-fos was assessed using single focal planes.
Images captured from the right lateral parabrachial nucleus (PB) and the left dorsal horn of animals injected with formalin in the left hindpaw were used to quantify parabrachial neurons and lamina I neurons immunostained for NeuN or c-fos in postnatal and adult rats. Colocalization of NeuN with c-fos was assessed using single focal planes. Using the drawing tool in Photoshop, a rectangular box (300 μm wide × 150 μm deep) was placed lateral to the superior cerebellar peduncle to quantify PB neurons immunostained for NeuN or c-fos. Likewise a rectangular box (250 μm wide × 50 μm deep) was placed on the medial part of lamina I to quantify lamina I neurons immunostained for NeuN or c-fos. Neuronal density was obtained by counting the number of neurons labelled with NeuN antibody within the rectangular boxes. A previous study showed that the superficial dorsal horn is approximately 200 μm, 250 μm and 300 μm deep from the dorsal white matter in P3, P10 and P21 rats respectively . To account for change in neuronal density with age, we calculated percentages of NeuN + ve lamina I and PB neurons that co-localized with c-Fos in order to compare levels of activation of lamina I projection pathway between different postnatal ages.
Independent t-test was applied to test for difference in mean number of FG-labelled lamina I projection neurons and mean percentage of FG-labelled lamina I projection neurons that expressed c-fos between P3 and adult rats. One-way ANOVA was used to test for effect of age on neuronal density in the PB and percentage of PB neurons that expressed c-fos. Multiple pairwise comparison with post-hoc Bonferroni test were carried out to test for difference between group means. Median test was used to test for effect of age on lamina I neuronal density as the data was non-parametric. Mann–Whitney test for non-parametric data was used to test for difference in lamina I neuronal density between age groups. One-way ANOVA was used to test for effect of age on the percentage of lamina I neurons that expressed c-fos.
Difference in mechanical threshold at different postnatal time points between postnatal rats treated with SP-SAP and control rats was assessed using repeated measure ANOVA and relevant post-hoc analysis. Since there was no significant difference in body weight or mechanical threshold between naïve rats and rats injected with blank-SAP across the whole study, data from these two groups were combined together into one control group. Statistical significance was set at a level of p < 0.05 in all statistical tests performed in this study.