The abundance of nAChRs can be measured in vitro and ex vivo using selective radioligands in conjunction with quantitative autoradiography. However, molecular imaging with positron emission tomography (PET) or single photon emission computed tomo-graphy (SPECT) makes possible the detection of neuroreceptors in the living brain. Contrary to autoradiography in vitro, in vivo imaging procedures allow for longitudinal studies in individual animals, thereby minimising issues arising from intersubject variability and allowing intervention or challenge studies. Until recently, animal PET studies were carried out using clinical scanners, most of which having a spatial resolution of approximately 5 mm; this is hardly adequate for resolution of structures within rodent brain. Only in the past 10 years have dedicated small-animal PET scanners become widely available. The best current instruments can attain a spatial resolution of little more than 1 mm full width at half maximum (FWHM) in the centre of the field of view, which is adequate to resolve structures as small as the mouse striatum.

PET technology is based upon coincident detection of two gamma photons (511 keV) arising from positron-electron annihilation subsequent to positron emission by decay of a neutron-deficient radionuclide incorporated in the tracer molecule. As such, the positron range prior to annihilation ultimately limits the spatial resolution of small-animal PET. The positron range is somewhat greater for positrons arising from the decay of carbon-11 than for the less energetic positrons emitted by fluorine-18. Iterative image reconstruction algorithms can approach a final resolution for ¹⁸F sources of little more than 0.7 mm FWHM (Reader 2008).

Some of the dedicated small-animal scanners such as the Focus 120 (Siemens) are designed to be readily movable between research units (Tai et al. 2001). Other systems, e.g. the microPET P4, Focus 220 (Siemens) and ClearPET (Raytest), can accommodate rodents and small non-human primates, whereas the larger Hamamatsu SHR-7700 instrument is designed specifically for non-human primate studies. The aperture and field of view of PET imaging systems designed for animal studies allow simultaneous recordings from up to eight mice (Rominger et al. 2010a), which should present a considerable economy in the experimental design of studies with transgenic animals. The most recent generation of preclinical PET scanners, such as the Inveon® PET-CT (Siemens), affords a considerable advantage in terms of anatomic information provided by the CT modality, but at the expense of lesser mobility for the hybrid scanners.

Small-animal PET and SPECT images are frequently characterised by a lack of anatomic information. In order to interpret the emission images, it has generally been necessary to employ some manual procedure for registration of the emission images to digitised brain atlases, based on histology or magnetic resonance imaging (MRI) atlases for rodent brain (Jupp et al. 2007; Rominger et al. 2010b). Contemporary multimodal imaging systems combine small-animal PET with SPECT, X-ray computed tomography (CT) and/or MRI. As noted above, PET-CT presents a great advantage for brain studies in that a high-resolution structural brain image in perfect registration with the PET image is obtained for each individual animal, rather than resorting to some standard atlas. In addition, the CT scan can be used to correct the PET images for attenuation by tissue, thus providing absolute quantitation of radioactivity concentrations in the brain without requiring an additional time-consuming transmission scan. MRI offers better tissue contrast than CT, but the combining of PET and MRI instrumentation has presented a greater technical challenge, due to incompatibility of traditional electronic components with a strong magnetic field. Nonetheless, a number of prototype PET/MRI scanners have been constructed, including the split magnet design in an animal microPET^(R)MR system at the University of Cambridge (Lucas et al. 2007). A commercial PET/MRI system for rats and mice was developed by Mediso; its 1 T permanent magnet limits MRI applications but offers great flexibility for animal PET studies.

At the cutting edge of small-animal imaging is the head-mounted PET detector developed at Brookhaven (“ratCAP”), which is compatible with imaging in awake, behaving rats (Schulz et al. 2011). This technology promises to revolutionise preclinical molecular brain imaging, by avoiding the well-known pharmacological confounds presented by anaesthesia. The ratCAP has recently been tested within the bore of a very high-field microMRI scanner, with minimal loss of sensitivity arising from the requirement for gating of the PET acquisition (Maramraju et al. 2011). Likewise, a SPECT camera which can be placed within an MRI magnet has recently been developed for small-animal studies (Meier et al. 2011). Whereas attenuation correction is usually obtained by CT scanning, MR-based attenuation correction has been demonstrated for PET and SPECT imaging using clinical scanners (Marshall et al. 2011) but has yet to be developed for small-animal studies. Although this technology remains at an early stage of development, hybrid PET-SPECT should eventually be permissive to multiple tracer studies with simultaneous acquisitions.

Many autoradiographic studies of nAChRs particularly in human brain have focussed on comparing the agonist-sensitive sites using rather non-selective ligands such as [³H]acetylcholine, [³H]nicotine, [³H/¹²⁵I]epibatidine or [³H]cytisine with those of the α7-selective antagonist [³H/¹²⁵I]α-bungarotoxin (Paterson and Nordberg 2000). This approach suggested the existence of at least three native receptor subtypes with multiple affinity states, such that the α-bungarotoxin-sensitive receptors predominantly represent homomeric α7 nAChRs, whereas the acetylcholine/nicotine binding sites represent mainly heteromeric α4β2 nAChRs and a small population of heteromeric receptors containing the α3 subunit. Given the predominance α4β2 and the α7 subtypes in the brain as documented above, they have presented the main targets for molecular imaging of nAChRs. The available information on the distribution, density and functional role of other subtypes in the brain is relatively sparse (Gotti et al. 2006; Sharma and Vijayaraghavan 2008). Furthermore, the current emphasis on α4β2 and the α7 subtypes as targets for pharmaceutical development may account for the paucity of selective high-affinity drugs for the other subtypes (Gündisch and Eibl 2011).

Reductions in cortical nAChR binding have been found in patients with diverse forms of dementia, including Alzheimer’s disease, Parkinson’s disease, Lewy body disease, progressive supranuclear palsy and Down’s syndrome (Perry et al. 1986; Picciotto and Zoli 2002). However, there have been few preclinical PET and SPECT studies of nAChRs in animal models of neurodegenerative disease. This is attributable to two considerations: First, models in transgenic mice have only recently become available for some of these diseases while remaining lacking for others, and second, the spatial resolution of small-animal PET and SPECT instruments has until recently been inadequate for regional analysis of neuroreceptors in rodent brain, such that the majority of such investigations have been conducted using autoradio-graphy in vitro. Furthermore, interpretation of preclinical imaging studies are hindered by the occurrence of species differences (Pauly et al. 1989; Quik et al. 2000; Han et al. 2003). Despite these limitations, molecular imaging is emerging as a powerful tool for investigating pathophysiological changes in animal models of neurodegenerative diseases, especially when conducted in conjunction with techniques such as in vivo microdialysis, electrophysiology and histopathology (Higuchi et al. 2012).

Stroke is the leading cause of adult disability in the United States and Europe and the second leading cause of death worldwide. Stroke is characterised by a loss of brain functions due to rapid disturbances in cerebral blood supply, either as reduced blood flow by thrombosis or embolism (ischemic stroke) or blood leakage (haemorrhagic stroke). Hyperacute mechanisms of stroke-related brain tissue damage, such as excitotoxicity, can be discriminated from delayed factors such as inflammation and apoptosis. Cellular components of the so-called neurovascular unit, which includes neurons, astrocytes and endothelial cells, all express nAChRs (Paulson et al. 2010). Insofar as long-term tobacco smoking is a risk factor for ischemic stroke (Hawkins et al. 2002), it may be relevant that nAChRs were altered in a post-mortem study of smokers; α4 expression was increased in neurons and dendritic processes and α7 expression was decreased in hippocampal neurons and astrocytes (Teaktong et al. 2004). In hypertensive stroke-prone rats, cortical α7 nAChRs are reduced, without concomitant changes in the α4β2 nAChRs (Ferrari et al. 1999). Activation of nAChRs by nicotine promotes endothelial cell proliferation (Villablanca 1998) and leucocyte migration (Yong et al. 1997), which together may increase thrombotic risk. However, nAChR agonism was neuroprotective against excitotoxicity in vitro, an effect which was mediated by growth factors (Belluardo et al. 1998) and also by inactivation of the toxins (O’Neill et al. 2002). These effects were blocked by the α7 nAChR antagonist α-bungarotoxin (Donnelly-Roberts et al. 1996). Interestingly, nicotine increased oedema/infarct size in a rodent stroke model (Paulson et al. 2010). A particular contribution of α7 nAChRs to stroke-related excitotoxicity might reflect their high Ca²⁺ permeability, especially under depolarising conditions. Treatment with the acetylcholinesterase inhibitor methanesulfonyl fluoride attenuated stroke-induced learning and memory deficits in rats (Borlongan et al. 2005).

These rather discordant findings of effects of nAChR agonism in stroke models may reflect the different contributions of excitotoxicity and also neuroinflammatory processes. The α7 nAChRs expressed on microglia (Shytle et al. 2004) seem particularly posed to mediate inflammatory responses. Nicotinic agonism at these sites suppressed inflammation by decreasing TNF-α production, while nicotine antagonists had the opposite effect (Shytle et al. 2004). In addition to central mechanisms, cholinergic signalling in the “cholinergic anti-inflammatory pathway” involving the vagus nerve may suppress the release of pro-inflammatory cytokines and influence migration T cells from the periphery to brain areas affected by stroke or multiple sclerosis (Borovikova et al. 2000) by a mechanism sensitive to α-bungarotoxin (Pavlov et al. 2003).

Despite this extensive background, there have been very few molecular imaging studies of nAChRs in relevant neuroinflammatory models. We have undertaken PET studies to assess α7 nAChR alterations in the sheep stroke model (Boltze et al. 2008). Recently, we measured [¹⁸F]NS10743 binding at 6 months after permanent medial cerebral artery occlusion (pMCAO) by dynamic PET imaging using a clinical tomograph (unpublished data). We found reduced tracer uptake in cortex ipsilateral to the lesion, but increased uptake in the brainstem. The declining α7 nAChR availability doubtless indicates degeneration of neurons in the infarct area, but the distal increases are of uncertain significance. Ongoing studies aim to establish better the time course and histological correlates of the α7 nAChR changes in our stroke model.

Traumatic brain injury is a permanent or temporary impairment of brain functions with an associated diminished or altered state of consciousness caused by external mechanical force. In the western world, it is the most important cause of death in young adults (Olesen and Leonardi 2003). In a human post-mortem study, performed after traumatic brain injury, reduced choline acetyl transferase activity and synaptophysin immunoreactivity in cerebral cortex were found, indicating damage of the cholinergic innervation, but nAChR binding determined by either [³H]nicotine or α-[¹²⁵I]bungarotoxin was unchanged (Murdoch et al. 1998), suggesting mainly presynaptic mechanisms.

Among various animal models which have been developed to experimentally investigate traumatic brain injury, the fluid-percussion (FPI), weight drop and controlled cortical impact (CCI) models are the most common. The FPI model, which entails transduction of mechanical force to the brain via a fluidic wave (Thompson et al. 2005), causes injuries resembling those observed in the clinic. The CCI model, which was first developed in small animals (Lighthall 1988) and later used in pigs (Duhaime et al. 2000; Alessandri et al. 2003; Manley et al. 2006), entails a piston strike, which results in more focal brain injury than is afforded by the FPI model.