Experiments were performed on mice (Swiss CD-1) about 3 weeks old. All experimental procedures involving animals were approved by the Ministero Italiano della Sanità, and comply with the European international laws on animal research. Prior to any surgery, deep and irreversible anesthesia was obtained by means of alothane.
For RT-PCR experiments, brain and vestibular ganglia were removed immediately after decapitation of the animal. The superior and inferior vestibular ganglia were removed together. Since both ganglia similarly stained for the H3 receptor (H3R) antibody, no differential analysis was performed.
Total RNA was extracted from mouse brain (1 mouse/experiment for 3 experiments) and Scarpa's vestibular ganglia (50–100 mice/experiment for 3 experiments) using QIAzol Lysis Reagent (QUIAGEN, Italy). Single-strand cDNA was synthesized from RNA (1 μg) using random hexamers and M-MLV Reverse Transcriptase (Invitrogen, USA). Subsequently, PCR (30 s at 96°C, then 30 s at 62°C or 69°C for β-actin and H3 respectively, followed by 30 s at 72°C for 33 cycles) was performed on 2.5 μl cDNA using specific primers for H3 receptors designed in accordance with the published sequence. H3 receptors display alternative splicing mainly in the region of the third intracellular loop . Primers for H3 receptors were designed around a nonspliced region, so that all variants would be detected, although not differentiated. The forward and reverse sequences were: 5'-TTCAACATCGTGCTGATCAG-3' 5'-TGTTCAGGTAGATGCTGAGG-3. As an internal control for cDNA yield, parallel RT-PCR was performed using specific primers for mouse β-actin. The primer sequences were: 5'-CAGATCATGTTTGAGACCTT-3' 5'-CGGATGTCMACGTCACACTT-3'. Negative control experiments were always performed by omitting the reverse transcriptase. The molecular weight (MW) of PCR products was estimated using the DNA MW marker VIII (Roche Molecular Biochemicals, Italy).
For immunofluorescence experiments ganglia were dissected from mice (n = 8), embedded in Jung Tissue Freezing Medium (Leica Microsystems, Italy) and immediately frozen in liquid nitrogen. Multiple 10 μm cryostat sections were obtained from the frozen samples, washed with phosphate-buffered saline (PBS) solution, blocked for 60 min with bovine serum albumin (BSA) 3%, rinsed with PBS and then incubated for 2 hours with rabbit anti-rat H3R IgG (Alpha Diagnostic International, USA), diluted 1:50 in PBS. After a rinse in PBS (15 min), the sections treated with anti-H3 were incubated (30 min) with Alexa-fluor 488-conjugated anti-rabbit IgG (1:1000) (Invitrogen, Italy).
No immunostaining was observed in control sections incubated with H3R antibodies preadsorbed with 20 M excess of the relative control peptide (Alpha Diagnostic International, USA), thus confirming anti-H3R antibody specificity.
For the colocalization study of histamine receptors and calretinin or calbindin, slices were incubated with rabbit anti-rat H3R IgG (Alpha Diagnostic International, USA) plus goat calretinin antiserum (Alpha Diagnostic International, USA), or plus goat calbindin antiserum (D28k-c20; Santa Cruz Biotechnology Inc., Germany), all diluted 1:50 in PBS except for calretinin which was diluted 1:250. After a rinse in PBS (15 min) the sections were incubated with Alexa Fluor 488-conjugated anti-rabbit IgG (1:1000) and Alexa Fluor 546-conjugated anti-goat IgG (1:1000). The immunostained slices were examined using the AFTER® fluorescence LED module (Fraen Corporation, Italy). Nuclei were counterstained with DAPI (ProLong® Gold antifade reagent with DAPI, Invitrogen, Italy). Slides were mounted on an Olympus BX41 light microscope, and digital images were captured with an Olympus Camedia C-5050 zoom digital camera (Olympus Italia, Italy). Several images were also acquired by mean of a TCS SP2 LEICA confocal microscopy system equipped with a LEICA DM IRBE inverted microscope.
In two additional experiments ganglia were not dissected directly from the mouse, but the petrous bone containing the whole inner ear was dissected out surgically and post-fixed in 4% paraformaldehyde in 0.1 M PB for 2–3 hr. Bone was decalcified for at least 1 week in a 120 mM EDTA solution at 4°C before further processing into paraffin. Serial paraffin sections (10 μm) were brought to water, washed with phosphate-buffered saline (PBS) solution, blocked for 60 min with BSA 3%, rinsed with PBS and then incubated for 2 hr with anti-H3R antibody (Alpha Diagnostic International, USA) diluted 1:50 in PBS. After a rinse in PBS (15'), the slices were treated with anti-H3R and incubated (30') with Alexa-fluor 488 anti-rabbit IgG (Invitrogen, Milan, Italy). Slices were then washed 3 × 5 min with PBS, mounted in ProLong® Gold antifade reagent with DAPI (Molecular Probes) and examined with a TCS SP2 LEICA confocal microscopy system equipped with a LEICA DM IRBE inverted microscope.
Neuron count and size
To estimate the percentage of ganglia neurons expressing H3 receptors, we calculated the ratio of cells staining for both H3 antibody and DAPI vs. cells staining for DAPI only in ganglia slices. All nuclei that were clearly recognizable were counted in slices. Since slices were 10 μm thick, and neuron soma ranged between 10 and 30 μm, in serial sections a same neuron could be counted twice. This would have yielded to an overestimation of H3 expressing neurons as they showed a larger soma (see Result sections). Therefore, we skipped one slice every count. The same was done for calculating the percentage of calretinin-positive neurons.
Finally, in some experiments ganglia tissue was stained with toluidine blue or hematoxylin/eosin, which stain neuron nuclei and cytoplasm. The diameter of each nucleus and soma was obtained by averaging the largest and the shortest diameters measured.
Analysis was performed with Microcal Origin (Version 6.0. Microcal Software, Northampton, MA, USA) and Microsoft Excel V. 5 (Microsoft, Redmond, WA, USA). Data are presented as means ± standard deviation; n = number of cases.