Under physiological conditions, NO reacts with oxyhaemoglobin to form methaemoglobin, which levels can be used as indirect index of NO (Kelm et al. 1997). We have previously reported a method for in vivo spectroscopic recordings of methaemoglobin in anaesthetized rats (Gonzalez-Mora et al. 2002). The present work describes a method for using this technique to estimate NO levels in the brain of the freely moving rat.

Adult Sprague-Dawley rats were anaesthetized (ketamine- xylazine I.M.) for surgical procedure (approved by the local Animal Care and Use Committee). At the end of the experiment, animals were humanely killed by an overdose of anaesthetic. Two optical fibres and an attached polymicro tubing were placed together as previously described (Gonzalez-Mora et al. 2002). The stereotaxic surgical procedures consisted of the attachment to the skull of a carrier assembly that allows the fixation of the optical fibre tips into the striatum of a freely moving rat. Light from a halogen lamp was passed through an optical fibre and the scattered light was collected by another optical fibre and sent to a miniature spectrometer. The optical absorption spectra were obtained as previously described (Gonzalez-Mora et al. 2002).

In order to attribute in vivo changes in absorbance at 635 nm to NO, we studied the absorbance changes in response to manipulations of basal NO concentration in the tissue by the direct injection of NO solution close to the spectroscopic sensor.

The injection of NO solution drammatically increased the absorption band with a maximum at 635 nm, as reported in vitro (Kelm et al. 1997) and in anaesthetized rats (Gonzalez-Mora et al. 2002). To evaluate if this technique is sensitive enough to record basal levels of methaemoglobin and the spontaneous changes due to physiological activity, spectra were recorded every 10 min during 5 h. As illustrated, spontaneous changes in the absorption of the methaemoglobin band (maximum at 635 nm) were observed. This indicates that this technique is able to detect not only the basal levels but also their spontaneous modifications. Finally, as can be seen in Fig. 1, this technique allows one to record simultaneously changes at oxyhaemoglobin band (maximum at 577 nm). Thus, the method described here is a useful tool to gain insight in the interaction between NO and microcirculation and behaviour.

Felipe Martín was suported by Cajacanarias.