Fluorescent imaging of nitric oxide production in neuronal varicosities associated with intraparenchymal arterioles in rat hippocampal slices L.A. Brown a , B.J. Key b , T.A. Lovick a, * a Department of Physiology, The Medical School, University of Birmingham, Birmingham, B152TT, UK b Department of Pharmacology, University of Birmingham, Birmingham, B152TT, UK Received 16 August 2000; received in revised form 13 September 2000; accepted 13 September 2000 Abstract The ¯uorescent indicator 4,5-diamino¯uorescein (DAF-2) has been used to investigate the production of nitric oxide in the vicinity of intraparenchymal cerebral blood vessels. Slices of rat hippocampus 300±350 mm thick, were loaded with 5 mM DAF-2 diacetate. On exposure to light of 450±490 nm wavelength, point sources of ¯uorescence, 1.8 ^ 0.2 mm in diameter (mean ^ SEM), were observed in close apposition to the outer surface of the vascular smooth muscle wall of 10/15 arterioles. In ®xed slices, resectioned and processed for nicotinamide adenine dinucleotide phosphate-dependent diaphorase, stained varicose ®bres were also seen in close association with the smooth muscle wall of small arterioles. These ®ndings suggest that tonic activity in perivascular nitrergic nerve ®bres lying in close proximity to intraparench- ymal microvessels may be a source of dilator tone within the parenchyma. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Hippocampus; Cerebral microvessels; Nitric oxide; 4,5-Diamino¯uorescein; Imaging; Perivascular nerves The cerebral circulation appears to be subject to a tonic, neurally-mediated nitrergic vasodilator in¯uence since cortical blood ¯ow is reduced following systemic adminis- tration of selective inhibitors of the neuronal isoform of nitric oxide synthase (NOS) [7,14,18]. Similarly, intrapar- enchymal microvessels observed in situ in hippocampal slices were found to constrict when extravascular levels of NO were reduced by the addition of a NO scavenger [10]. The origin of this dilator tone is unclear. It is known that the larger vessels of the pia mater are innervated by nitrergic dilator nerves and that these nerves originate principally from NOS-containing nerve cell bodies located in the sphe- nopalatine ganglion [1,6,11,13,15]. Stimulation of this ganglion produces an increase in pial arterial diameter and cortical blood ¯ow [12]. However, inhibition of ganglionic activity by cooling the ganglion was not associated with a signi®cant change in cerebral blood ¯ow [2]. Moreover, since the extrinsic innervation does not extend distal to the larger pial arteries [1,6,11], it is unlikely that the post- ganglionic nitrergic dilator nerves make a signi®cant contri- bution to resting dilator tone of blood vessels within the brain parenchyma. An alternative explanation is that the nitrergic dilator tone originates from intracranial sources. NOS-containing nerve ®bres have been described in association with cerebral capillaries [5,6], but there is less information regarding the relationship between nitrergic nerves and arterioles which constitute the major source of vascular resistance within the parenchyma. Moreover, the presence of its synthesizing enzyme can at best, indicate only the potential of the tissue to generate NO. It does not provide an indica- tion of ongoing functional activity. The recent development of NO-sensitive dyes [8] now offers the possibility of over- coming some of these problems by visualizing the produc- tion of NO in living tissue. One such compound, 4,5- diamino¯uorescein (DAF-2) has recently been character- ized and validated as a selective marker for the generation of NO in neural tissue [3,9]. We have therefore used DAF-2 to investigate the generation of NO in the vicinity of micro- vessels in hippocampal slices in an attempt to identify a morphological basis for nitrergic vasodilator tone in a deep brain structure. Neuroscience Letters 294 (2000) 9±12 0304-3940/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S0304-3940(00)01521-4 www.elsevier.com/locate/neulet * Corresponding author. Tel.: 144-121-414-6929; fax: 144-121- 414-6919. E-mail address: t.a.lovick@bham.ac.uk (T.A. Lovick).