Optical imaging spectroscopy in the unanaesthetised rat
Introduction
Neural activation causes a localised haemodynamic response that forms the basis of modern functional neuroimaging techniques such as PET and fMRI. A detailed investigation of the intrinsic signals upon which these techniques depend will enable the relationship between neural activity and the consequent changes in blood flow, volume and oxygenation to be better understood (Ogawa et al., 1993). Such understanding has wide implications for research on brain metabolism in addition to impacting upon the interpretation of data from increasingly abundant functional brain mapping studies. Optical imaging spectroscopy (OIS) is well placed to assist in such an investigation, as it provides concurrent measures of changes in the concentration of oxyhaemoglobin (HbO2), deoxyhaemoglobin (Hbr) and total haemoglobin (Hbt—which may be approximated to blood volume). Additionally, OIS has a spatial resolution of microns and a temporal resolution in milliseconds, which is beyond the current limits of both fMRI and PET. Briefly, changes in the reflection spectra of an imaged cortical tissue volume are recorded using a CCD camera. Given their known absorption spectra and assuming appropriate baseline values, one can calculate changes in the concentration of a variety of chromophores including HbO2 and Hbr (Mayhew et al., 2000). Our laboratory amongst others has used OIS to investigate the haemodynamic response to somatosensory activation in the rat following mechanical or electrical stimulation of the forepaw, hindpaw and whisker pad (Jones et al., 2001, Mayhew et al., 2000, Nemoto et al., 1997) or in cat visual cortex in response to visual stimuli (Malonek and Grinvald 1996, Malonek et al., 1997)
The major disadvantage of OIS in the visible spectrum range is that it is relatively invasive compared with fMRI and PET. Whilst some studies have made use of human patients undergoing neurosurgery (Cannestra et al., 2001), the research possibilities in humans are clearly limited for OIS in the visible wavelength range. Therefore, much of the OIS research has been carried out on anaesthetised animals and yet there are a number of important questions that can only be addressed in the context of an unanaesthetised preparation. Firstly, there is evidence to indicate that cerebral haemodynamics may be different under different anaesthetic regimes (Bonvento et al., 1994, Jones et al., 1995, Lindauer et al., 1993) and different again in the absence of anaesthesia. For example, fMRI studies in rat (Lahti et al., 1998, Lahti et al., 1999, Peeters et al., 2001) and OIS studies in monkey (Shtoyerman et al., 2000) indicate haemodynamic responses to stimulation are significantly larger in unanaesthetised animals. Secondly, development of an unanaesthetised OIS preparation would permit investigation of neural processes that are not accessible in an anaesthetised animal. Established paradigms used to study animal models of cognition and attention may be employed, and the effects of modulation by for example sensory or pharmacological stimuli, may be investigated. Thirdly, most human functional imaging studies are carried out using awake subjects whilst research using animal models to investigate homogolous systems usually involves anaesthesia. An anaesthetic-free animal preparation could either provide additional validation for the use of these models, or suggest caution is required in the interpretation of data from such comparative research. Intrinsic signal imaging has previously been performed in the alert monkey using implanted chambers (Grinvald et al., 1991). Establishment of a similar rodent preparation would complement this technique.
The aim of the present study was, therefore, to develop and evaluate a methodology enabling OIS studies of functional activation in the awake behaving rodent. A full investigation of the differences in the haemodynamic response to somatosensory stimulation between unanaesthetised and anaesthetised rats are reported in detail elsewhere (Berwick et al., 2002).
Section snippets
Experimental design
All aspects of this study were performed with Home Office approval under the Animals (Scientific Procedures) Act 1986. This study consisted of three experimental conditions. In condition 1, rats (n=4) were trained to accept restraint and then under recovery anaesthesia were surgically prepared for chronic optical imaging experiments. In condition 2, three of the four rats in condition 1 underwent additional surgery and were imaged under urethane anaesthesia as if an acute preparation. In
Haemodynamic responses
The main finding is a large, localised haemodynamic response to somatosensory stimulation in the unanaesthetised rat. Fig. 4a–c (left) show the mean OIS time series of the changes in HbO2, Hbr and Hbt concentration, respectively, across the three experimental conditions following stimulation of the whisker pad. This data is taken from the most responsive region, as located previously during single wavelength imaging experiments. It is clear that the responses are considerably larger in the
Discussion
The present report demonstrates the feasibility of performing OIS in the unanaesthetised, non-paralysed rat. The results lead to a number of conclusions. Firstly, a localised haemodynamic response in somatosensory cortex to whisker stimulation is observed in both anaesthetised and unanaesthetised rats. Secondly, the haemodynamic response observed in unanaesthetised rats was considerably larger than that found under anaesthesia. Thirdly, over the time period used, the chronic preparation did not
Acknowledgements
The authors gratefully acknowledge the support of the MRC (Co-operative Group Grant G9825307), EPSRC and Wellcome Trust. Chris Martin is supported by an MRC Research Studentship. Jason Berwick is supported by MRC Component Grant G9813068. David Johnston is supported by Wellcome Trust Project no. 050080/Z/97/Z. Thanks also to Michael Port for development and construction of equipment and also to the technical staff of the laboratory for their assistance.
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