General health and behavioural phenotyping

The observation of over 600 animals did not reveal any overt clinical impairment under standard diet (Supplementary Table 1) and normal housing conditions, nor increase in incidental pathologies in either heterozygous Tspo^+/− or homozygous Tspo^−/− animals, both having the same litter size, length of 1-day-old pups, sex ratio, and subsequent growth rate and weight increase from 3 to 50 weeks (weekly measurement) as the colony control wild-type Tspo^+/+ animals (Fig. 8a,b). Follow-up monthly measurements from 50 to 80 weeks failed to reveal any significant genotype-dependent weight differences in older animals.

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Figure 8

Functional characterization of global Tspo^−/− mice.

(a) No significant differences in body length between Tspo^+/+ (n=5) and Tspo^−/− (n=9) neonatal 1-day-old mice are apparent (Student’s t-test). (b) Likewise, there is no significant difference (ANOVA) in the weight gain trajectory measured from 4 to 83 weeks in Tspo^+/+, Tspo^+/− and Tspo^−/− mice within the same sex (n=10–80 animals per time point for weeks 4–43 per group; n=3–44 per time point for weeks 44–83). Independent from genotypes, females weigh significantly less than males, as is known to be the case for this mouse strain (b). (c–e) No significant differences between Tspo^+/+ and Tspo^−/− mice are found in blood pregnenolone concentrations (n=4 for all groups; Student’s t-test) (c); blood protoporphyrin IX (PPIX; left: three typical closely overlapping spectra from the different genotypes after addition of 5-aminolevulinic acid (ALA), right: no significant differences in levels of PPIX (PPIX per ml of blood in arbitrary units (A.U.); n=3–5 per genotype; Student’s t-test)) (d); and mRNA expression of steroidogenic acute regulatory (StAR) protein, P450scc and Tspo2 in nine organs (normalized to reference genes Actb and Gapdh, n=3 per genotype) (e). Error bars denote standard deviation.

Likewise, there was no indication of a decrease in fertility or lifespan, the oldest animals having so far exceeded 24 months without illness. Tests of sperm viability and function yielded no differences between sperm from Tspo^+/+ (mean motility: 82.7±9.33%; mean progress: 47.7±10.2%; mean velocities average path velocity (VAP): 98.6±13.0μms⁻¹, straight line velocity (VSL): 65.7±8.90μms⁻¹ and curvilinear velocity (VCL): 179±20.2μms⁻¹) and Tspo^−/− animals (mean motility: 86.1±5.74%; mean progress: 49.6±5.32%; mean velocities VAP: 105±6.54μms⁻¹, VSL: 67.5±5.08μms⁻¹ and VCL: 197±11.2μms⁻¹). Standard open field, emergence, light/dark preference and elevated plus maze tests revealed similar behaviour across all genotypes and both sexes (Supplementary Table 2).

Cholesterol and pregnenolone biosynthesis

In both, male and female Tspo^−/− mice, all serum pregnenolone concentrations, as measured by enzyme-linked immunosorbent assay (ELISA), were within the normal range seen in the colony control wild-type (Tspo^+/+: males 131±46.5ngml⁻¹, females 150±36.5ngml⁻¹; Tspo^−/−: males 145±44.8ngml⁻¹, females 141±26.8ngml⁻¹) (Fig. 8c).

The enzymatic conversion of ³H-cholesterol to pregnenolone by P450scc over a period of up to 2h as a read-out of mitochondrial cholesterol transport revealed no statistically significant differences in mean percentage conversion rates between all genotypes (Tspo^+/+: 14.35±4.6% (std); Tspo^+/−: 14.28±3.6% (std); Tspo^−/−: 15.6±4.0% (std)).

Haematological analysis and blood biochemistry

Analysis of blood, including cell sorting, demonstrated that the blood phenotype in both sexes remained largely unaffected by the heterozygous or homozygous loss of the Tspo gene, the only exception being a statistically significant trend increase in natural killer (NK) cells in female Tspo^−/− mice compared to female Tspo^+/+ (Supplementary Table 3). Thus, the Tspo^−/− mice did not reveal the changes in cellular blood composition seen in zebrafish tspo gene silencing experiments15.

The concentrations of protoporphyrin IX (PPIX), thought to be an endogenous PBR/TSPO ligand, in the blood of Tspo^+/+, Tspo^+/− and Tspo^−/− mice were indistinguishable and increased similarly after treatment with 5-aminolevulinic acid (ALA), the first intermediate in haem biosynthesis and a precursor of PPIX (Fig. 8d), thus indicating normal haem metabolism.

Absence of compensatory transcriptomic regulation

Likewise, quantitative RT-PCR showed that the gene expression profiles for StAR, P450scc and Tspo2 across the major organs known to express the Tspo were similar across genotypes, indicating that the global loss of Tspo had not led to any compensatory transcriptomic regulation of those genes thought to be most closely linked to PBR/TSPO as a regulator of steroidogenesis (Fig. 8e).

Immunoblotting

Lysates (20μg protein) were mixed with 2 × Laemmli sample buffer (Bio-Rad, Hercules, CA, USA), heated to 70°C for 10min and resolved on a 4–20% Mini-PROTEAN TGX gel (Bio-Rad). MagicMark XP (Life Technologies, Carlsbad, CA, USA) was used as the molecular weight marker. Proteins were transferred to a nitrocellulose membrane using the Trans-Blot Turbo transfer system (Bio-Rad). The membrane was incubated with anti-TSPO antibody (#109497, Abcam, Cambridge, UK) diluted 1:10,000 or anti-GAPDH (#37168, Abcam) diluted 1:1,000 and peroxidase-conjugated anti-rabbit antibody (#A0545, Sigma-Aldrich, St Louis, MO, USA) diluted 1:10,000. Pierce ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL, USA) was used and the membrane visualized using an ImageQuant LAS 4000 (GE Healthcare, Little Chalfont, Buckinghamshire, UK).

RT-PCR and quantitative real-time PCR

Tissue samples were homogenised in TRIzol (Life Technologies) and total RNA isolated using the PureLink RNA mini kit (Life Technologies) with on-column DNase treatment following the manufacturer’s instructions. Purified RNA (1μg) was reverse-transcribed to cDNA using the SuperScript III First-Strand Synthesis kit (Life Technologies). For RT-PCR, cDNA from the kidneys of Tspo^+/+ and Tspo^−/− mice was amplified using primers (Supplementary Table 4) located in exons 1 and 4. The PCR consisted of an initial incubation at 95°C for 2min, followed by 34 cycles at 95°C for 30s, 55°C for 30s and 72°C for 1min, and a final step at 72°C for 5min. The PCR products were confirmed by sequencing. For quantitative real-time PCR, cDNA was added to 2.5μl of SsoFast EvaGreen Supermix (Bio-Rad) and 0.5μM of each primer (Supplementary Table 4) in a final reaction volume of 5μl. Reactions were performed on a CFX 384 Real-Time PCR detection System (Bio-Rad) by cycling at 98°C for 30s, followed by 45 cycles at 98°C for 5s and 63°C (61°C for the Tspo2 assay) for 10s. A melt curve analysis was performed to confirm the specificity of each reaction. Each sample was run in duplicate. The Tspo2 primers were designed using Primer-BLAST and the specificity confirmed by DNA sequencing.

Protoporphyrin IX

Blood from euthanized mice was placed into heparin tubes on ice, plasma removed, cells washed twice in ice-cold phosphate-buffered saline (PBS) and then resuspended in PBS to the original volume. Samples were evenly split (300μl each), one receiving 1mM 5-ALA and the other sterile H₂O, and incubated in a shaking incubator at 37°C for 4h. Ethyl acetate/acetic acid (4:1) was added to each sample, then mixed and centrifuged before transferring the supernatant (1ml) to a new tube with 1ml of 1.5M HCl. An aliquot of the lower HCl phase, after mixing and centrifuging the sample, was measured in a spectrofluorimeter (excitation wavelength 407nm, slit width 10nm, emission spectrum 450–800 in 1nm increments). Glass tubes and containers were used throughout and samples were protected from light.

Facial nerve axotomy

Facial nerve axotomy29,30 was performed on Tspo^+/+ and Tspo^−/− mice (n>4 per genotype) anaesthetized with 5% (v/v) isoflurane. A small incision (0.5–0.7cm) in the skin 0.5cm dorsal and lateral to the ear was made and the facial nerve transected under an operating microscope. The incision was closed with Vetbond Tissue Adhesive (3M, St Paul, MN, USA). The success of the operation was confirmed by the loss of ipsilateral whisker movement. Mice were monitored carefully and euthanized with CO₂ 3 days after the operation. Brains were dissected, placed in OCT, transferred to liquid nitrogen and stored at −80°C until cryo-sectioning.

Stereotactic implantation of GL261 mouse glioblastoma cells

The Tspo^+/+ (n=6) and Tspo^−/− mice (n=4) were operated under isofluorane anaesthesia and aseptic conditions using a Kopf stereotactic apparatus and precision dental drill (Kopf Instruments, Tujunga, CA, USA). A small burr hole was made 3mm anterior of the bregma and 2mm lateral over the right frontal lobe (3mm ventral). GL261 glioblastoma cells (NCI, Bethesda, MD, USA) at 3 × 10³ per μl in a volume of 10μl were slowly injected over 2min using a 10-μl Hamilton syringe connected to a pump by means of a 32-gauge needle. The animals were closely monitored daily for good recovery, including body weight and absence of neurological signs. Three and four weeks after implantation of the GL261 cells, the animals were imaged by means of microPET/CT and brains were dissected for histological studies and autoradiography.

PET and CT imaging

Mice, anaesthetized (5% (v/v) with isoflurane and maintained at 1–2%, were scanned using a small-animal Inveon PET/CT scanner (Siemens, Knoxville, TN, USA) according to the methods described previously31,32. Body temperature was maintained with a feedback regulated heating pad and respiration monitored (BioVet; m2m Imaging Corp, Cleveland, OH, USA). Scans started with the tail vein injection of [¹⁸F]PBR111 (8–18MBq per 100μl, 0.2nM). After 40min of imaging, PBR111 (1mg per kg in 2% acetic acid–saline) was injected to determine non-specific accumulation in organs and imaged for 10min. Then, mice underwent a 10-min CT scan for anatomical information. All PET data were corrected, normalized and reconstructed with an OSEM3D–MAP algorithm31 to produce PET volumes of activity concentration (kBq per ml).

Autoradiography

Tissue sections from Tspo^+/+, Tspo^+/− and Tspo^−/− mice were incubated at room temperature for 20min in 170mM Tris-HCl pH 7.4 containing 1nM [³H]PK11195 (specific activity 84 Ci per mmol; Perkin Elmer, Waltham, MA, USA), washed twice for 5min in 170mM Tris-HCl, rinsed with 3 dips in ice-cold MilliQ H₂O, and dried. Additional sections were incubated on ice for 1h in 50mM Tris-HCl pH 7.4 containing 3nM [¹²⁵I]CLINDE (specific activity 100 Ci per mmol; synthesized by ANSTO Life Sciences), washed twice for 2min in ice-cold 50mM Tris-HCl, for 1min in ice-cold MilliQ H₂O, and dried. Displacement binding was carried out in the presence of PK11195 (10μM), CLINDE (10μM) and PBR111 (10μM). Single-emulsion films were exposed to sections and standards (¹⁴C or ³H) for 3h ([¹²⁵I]CLINDE) or 10 weeks ([³H]PK11195).

Immunohisto- and cytochemistry

Immunohistochemistry was performed as previously described13,33,34. Tissue sections (used for autoradiography) were fixed with 3.7% formaldehyde in PBS for 5min, then permeabilized with ice-cold acetone. Non-specific antibody binding was blocked with 10% horse serum and 2% bovine serum albumin (BSA) in PBS. Sections were incubated with TSPO monoclonal antibody (#109497, Abcam) at 4°C overnight and secondary HRP-conjugated goat anti-rabbit antibody at RT for 1h (#A0545, Sigma-Aldrich). The activity of HRP was detected with the peroxidase 3,3′-diaminobenzidine tetrahydrochloride liquid substrate system (Sigma-Aldrich). Sections were dehydrated with ethanol, incubated with xylene, and slides were mounted in DPX mounting media with a coverslip. Sections were visualized under an inverted Olympus BX51 microscope (Olympus, Tokyo, Japan), and captured with a Q-imaging camera and ImagePro 5.1 program.

A Zeiss LSM 710 confocal microscope with a 5 × EC Plan-Neofluar NA 0.16 objective was used for the fluorescence microscopy of the facial nucleus. Alexa Fluor 568 was excited with a 561-nm laser, using a 488/561/633 dichroic mirror, and emission captured from 565 to 640nm.

For immunocytochemistry, peritoneal macrophages were isolated according to Zhang et al.35. After 2 days in complete cultural medium the purity of macrophages was >97% as confirmed with the macrophage-specific marker IB4 conjugated with FITC (Sigma-Aldrich). Cells on coverslips were fixed and incubated using the same method as for tissue sections, with the addition of a mouse anti-human/mouse mitochondrial electron transport chain complex IV antibody (#AB14705, Abcam), then incubated with the secondary antibodies Alexa Fluor (AF) 594-conjugated goat anti-rabbit antibody (Life Technologies) and AF488-conjugated goat anti-mouse antibody (Life Technologies). Cells on coverslips were mounted on microscope slides with ProLong Gold antifade reagent containing DAPI (Life Technologies) and viewed under a BX61WI Olympus microscope. Images were acquired with a digital camera (CoolSNAP, Photometrics, Tucson, AZ, USA) and the Image InVivo program (Photometrics). Deconvolution of images was performed with the AutoDeblur program (Photometrics) and further processed with ImageJ (NIH, Baltimore, MD, USA).

Primary microglial cell culture

Microglia were cultured from 0–2-day-old Tspo^+/+ and Tspo^−/− mice according to methods described previously33. Briefly, whole brains were dissected, cut into fine pieces and treated with 0.025% trypsin (Sigma-Aldrich). They were cultured in Dulbecco’s modified Eagle’s medium (DMEM/F12; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Life Technologies), 1% penicillin-streptomycin-glutamine (Sigma-Aldrich) and 0.5ng per ml GM-CSF (Abcam).

Microglia were purified by shaking at 350 revolutions per minute (r.p.m.) for 50min at 37°C, pelleted by centrifugation, resuspended in supplemented DMEM/F12 and placed (4 × 10⁴ cells per well) into a 96 well Seahorse XF cell culture plate (Seahorse Bioscience, North Billerica, MA, USA) pre-coated with FBS for 15min at room temperature. After 15min the wells were washed twice to remove unattached cells. The purity of microglial cultures was >98% as confirmed by staining with the microglial marker Alexa Fluor-conjugated isolectin GS-IB4 (Life Technologies). The 5 × 10⁴ microglia from wild-type and knockout mice were plated into each well of the XF96 plate and were grown for 2–3 days before evaluating mitochondrial functions with the Seahorse XF 96 Cell Analyzer (Seahorse Bioscience). To rule out systematic differences in cell density and viability between Tspo^+/+ and Tspo^−/− microglia, cell density was monitored and assured throughout the experimental measurements, while viability, including apoptosis, was confirmed using caspase 3/7 (ref. 36).

Measurements of mitochondrial respiration

The oxygen consumption rate (OCR) and extracellular acidification rate in primary microglial cultures were determined using the Seahorse XF 96 Cell Analyzer. OCR is an indicator of mitochondrial respiration37,38. The day before the experiment 200μl of the Seahorse XF Calibrant (pH 7.4) was added to each well of the XF 96-well utility plates. The sensor cartridge was placed on top of the plate and hydrated for 16h at 37°C in a non-CO₂ incubator. The protocol for basal measurements of the cells at 37°C (start, calibrate probes, equilibrate, mix 2min, 1min delay, measure 3min) was repeated twice. Mitochondrial stress compounds (oligomycin, rotenone combined with antimycin A) were then injected, followed by 2min mixing, 1min delay, measure 3min (repeated 3 times).

On the day of the experiment, microglia (plating density 5 × 10⁵ per well, purity >98% and viability >92% for both wild-type and knockout microglia) were washed 3 times with un-buffered DMEM plus 20mM glucose (no buffers) and incubated at 37°C (no CO₂) for 1h. Oligomycin and rotenone combined with antimycin A were used to determine mitochondrial ATP production and basal mitochondrial OCR37,38. Oligomycin and the combination of rotenone and antimycin A were injected at concentrations of 0.3, 1 and 3μM (optimal for oligomycin) and 10μM (optimal for rotenone and antimycin A).

The data for each reported parameter reflect 11–14 independent Seahorse XF measurement series from a total of seven independently repeated microglial cell culture preparations.

Radioligand binding

Organs were homogenized using a T25 digital Ultra-Turrax homogenizer (Ika, Wilmington, NC, USA) at speed setting 5 and 20,000 r.p.m. in ~45ml of ice-cold TRIS buffer (pH 7.4) and washed twice in ice-cold 50mM Tris-HCl (pH 7.4) by centrifugation at 48,000g, resuspended in 50 volumes of ice-cold 50mM Tris-HCl, and protein concentration was determined using a BCA protein assay kit (Thermo Scientific). Bmax and Kd were determined with saturation binding by incubating membrane protein (60μl) with [³H]PK11195 (0.56–20nM) on ice for 90min before harvesting by rapid filtration through Whatman GF/C filters (GE Healthcare) presoaked in a 0.5% polyethylenimine solution. Non-specific binding was determined with 5μM PK11195. Filters were placed in scintillation cocktail (Perkin Elmer) for 12h and radioactivity was determined using a Tri-Carb 2100 TR Liquid Scintillation Counter (Perkin Elmer). Bmax and Kd values were obtained by non-linear regression using GraphPad Prism 5.04 (GraphPad Software, La Jolla, CA, USA) by fitting total and non-specific binding.

Cholesterol transport and biosynthesis

P450scc metabolism was measured in mouse testis according to Tuckey et al.39 and Slominski et al.40. Briefly, an enriched mitochondrial fraction was prepared by centrifugation in 250mM sucrose, 50mM Tris (pH 7.4), and the enzymatic conversion of cholesterol to pregnenolone was conducted under the following conditions out to a maximum 2-h time-point: 50mM HEPES pH 7.4, 250mM sucrose, 20mM KCl, 5mM MgSO₄, 0.2mM EDTA, 1mg per ml BSA, 8μM Trilostane, 0.05μCi ³H-cholesterol, 500mM isocitrate and 5mM NaNADP. Reactions were terminated with the addition of 4°C dichloromethane. The organic phase was retained and the aqueous phase was re-extracted twice more into dichloromethane. Extracts were combined, dried under nitrogen and dissolved in 100μl of ethyl acetate. Pregnenolone and cholesterol were separated by thin-layer chromatography, with hexane:diethylether:acetic acid (15:15:1) mobile phase, and the amount of radioactivity in each spot quantified by liquid scintillation counting.

Detection of serum pregnenolone

Serum pregnenolone of male and female Tspo^+/+ and Tspo^+/+mice was determined by ELISA performed according to the manufacturer’s instructions (Abnova Cooperation, Taiwan). Briefly, blood was collected, allowed to clot, and the serum collected. Standards, controls and samples were incubated in duplicate in a sealed plate with or without conjugate and antibody for 1h at room temperature on a plate shaker (KS4000ic, IKA, Selangor, Malaysia) at 200r.p.m. After the incubation, wells were emptied, washed five times, and the plate tapped on lint free paper towel to remove any remaining buffer. Wells were incubated with HRP conjugate on the plate shaker at 200r.p.m. for 30min at room temperature, washed five times, and incubated with TMB substrate for 10min at room temperature. Stop solution was added to each well and after 20min the optical density at 450nm was measured. Data were normalized against blood volume and analysed using a four-parameter logistic curve fitting program MasterPlex ReaderFit: Curve-Fitting Software for ELISA Analysis (Hitachi Solution America, San Francisco, CA, USA).

Blood phenotyping

Thirty mice (10 Tspo^+/+, 10 Tspo^+/−, 10 Tspo^−/−, 5 months old, males/females evenly distributed) used in behavioural experiments were euthanized with CO₂. Blood was collected from the pulmonary cavity by cutting the inferior vena cava and placed in microtubes with heparin (haematology and lymphocyte analysis) or gel clot activator (biochemical analysis). Spleens were collected in 10ml of ice-cold sterile PBS. Blood (room temperature) and spleens (on ice) were delivered (within 3–7h of collection) to the Australian Phenomics Facility for flow cytometric and biochemical analysis.

For general haematology, blood was diluted 1:2 and analysed with fluorescence-activated cell sorting using the Advia 2120 Haematology System (Siemens, Munich, Germany). Blood cell subsets, including white blood cells and platelets, were assessed. For lymphocyte analysis, blood was analysed for the presence or absence of abnormalities in T cells, B cells, NK cells and monocytes. The T-cell subsets included CD4+ and CD8+ subsets, while the B-cell subsets included immature and mature B cells and IgE+ monocytes. Spleens were analysed by flow cytometry and stained for NK cells, for B cells, including mature and immature subsets, and for T cells, including CD4+ and CD8+ cells and their activated subsets. See Supplementary Table 3 for a list of cell types examined.

For biochemistry, blood was centrifuged, the serum collected and run through the Olympus AU400 Chemistry Analyzer (Olympus), which detects levels of cholesterol, triglycerides, glucose, high-density lipoprotein (HDL), albumin, creatine kinase activity and alanine aminotransferase activity. See Supplementary Table 3 for a list of biochemical parameters examined.

Mouse sperm analysis

Sperm were released from the cauda epididymides of Tspo^+/+ and Tspo^−/− mice by cutting each cauda with a scalpel and incubating in 150μl of pre-warmed Biggers–Whitten–Whittingham (BWW) medium for 5min at 37°C. After the incubation, an additional 250μl of BWW medium was added, the tissue removed and the sperm assessed using a computer-assisted sperm analysis system (Hamilton Thorne, Beverly, MA, USA). Parameters assessed were total motile, progressive motile, VAP, VSL and VCL.

Assessment of anxiety-related behaviours

Behavioural tests were performed on mice aged between 3–4 months, handled regularly to reduce the impact of handling stress during testing. Anxiety-related behaviours were examined with the following: open-field test, emergence test, light/dark preference test and elevated plus maze. In each test the mice were allowed free exploration in the apparatus for 15min. The duration and frequency of entering each compartment, total distance travelled, time spent active and the number of stool droppings were assessed. In the elevated plus maze risk assessment was defined as when an animal’s head was facing towards an open arm with more than 15% of its body in an open arm while maintaining its centre of mass within the closed arm or central platform. The genotype of the animals was blind to the experimenter during testing. Tests were carried out at least 7 days apart to reduce the effects of training history41. All experiments were recorded with an overhead camera on to DVD and later analysed using Motman Tracker 4.5 software (Motion Mensura, Cooks Hill, NSW, Australia).

The authors would like to thank their colleagues at the Australian Nuclear Science and Technology Organisation (ANSTO): G. Dhand, E. Dodson, R. Shepherd and M. Harrison-Brown for technical laboratory support and assistance; F. Boisson, H. Hamze, A. Arthur, A. Reilhac-Laborde and A. Charil for the operation of the PET/CT; G. Rahardjo for PET image analysis; G. Perkins, R. Sheth and N. Wyatt for radiosynthesis and I. Greguric for advice on radiolabelling; A. Nguyen for assistance in the [¹²⁵I]CLINDE autoradiography; K. Belbine and M. Spoelder for assistance in collecting animal tissues; and G. Blackburn for proof-reading and editorial comments. We gratefully acknowledge the expert support from our colleagues at the University of Sydney, Australia: S. De Graaf for help in the measurement of sperm function, D. Lai for assistance in measurements of mitochondrial function with XF Seahorse Cell Analyser; and T. Burton (Bosch Institute Animal Behavioural Facility) for advice and assistance in the behavioural assessment of the animals. We thank R. Tuckey at the University of Western Australia for his valuable technical advice on the measurement of cholesterol transport and steroid biosynthesis, Ozgene Pty Ltd, Perth, Australia, for carrying out the generation of knockout mice according to the design of R.B.B., G.J.L. and R.J.M., and the Australian Phenomics Facility in Canberra, Australia, for blood phenotyping. The first confirmed Tspo^−/− mouse strain (13 Dec 2011) was named Guwiyang Wurra in the language of the local Dharawal people and we thank L. Bursill, Nowra, Australia, for his advice. RBB holds an ANSTO Distinguished Research Fellowship and gratefully acknowledges the support from Drs Ian Smith and George Collins, and the continued encouragement through the Strategic Research Initiative of Dr Adrian Paterson, ANSTO. Support from the Cure Brain Cancer Foundation, Australia, to MBG and RBB is gratefully acknowledged. The paper is in memoriam Dr Ralph Myers, pioneer in the field of PBR research at the former MRC Cyclotron Unit, London, and dedicated to G.W. Kreutzberg, Munich, Germany, and T. Jones, Manchester, UK.