Brain Research, 530 (1990) 196-204
Elsevier BRES 15928
Light-microscopic localization of somatostatin binding sites in the locus coeruleus of the rat* C. Gagne, E. Moyse, L. Kocher, H. Bour and J.E Pujol Laboratoire de Neuropharmacologie Mol~culaire (UMR 105), CNRS, Facult~ de M~decine A. Carrel, Lyon (France)
(Accepted 10 April 1990) Key words: Locus ceruleus; Somatostatin; Receptor; Autoradiography; Catecholamine; Rat
Somatostatin (SS14) binding sites within locus coeruleus (LC) were localized at the light microscope level by [125I][Tyr°,D-TrpS]SS14 radioautography combined with an immunohistochemical/neurotoxic lesioning approach. In intact rats, the dense accumulation of SS14 binding sites of LC conspicuously overlapped with the cluster of tyrosine hydroxylase (TH) immunoreactive neurons; SS14 specific binding was directly proportional to the number of TH immunostained (TH+) cell bodies per mg of tissue throughout LC. Complete lesion of catecholaminergic nerve cell bodies of LC by intracerebroventricular injection of 6-hydroxydopamine (6-OHDA) resulted in the total abolition of SS14 specific binding in the structure. In addition, specifically bound [125I][Tyr°,D-TrpS]SS14and TH+ cell density were quantified serially in a set of rats bearing various partial neurotoxic lesions; a highly significant correlation was found between the two parameters at each of the 16 coronal levels of LC examined. The coefficient of proportionality was identical at all levels. These results strongly suggest that somatostatin binding sites are uniformly localized on all noradrenergic neurons of LC. INTRODUCTION The locus coeruleus (LC) is a pair cluster of tightly packed noradrenergic neurons in the pontine tegmentum 5 which gives rise to massive ascending and descending projections in the central nervous system ~6. This nucleus is involved in numerous neural and neuroendocrine regulations such as sleep/waking cycle, arousal status, emotion and stress 24. However, the nature and mechanism of informative afference to LC are still poorly understood. Numerous neurotransmitters/neuromodulators have been visualized in nerve fibers and/or presynaptic axon terminals of LC by immunohistochemistry, such as substance p28, enkephalin28, neuropeptide y32, somatostatin12, neurotensinll, galanin3~, vasopressin 6 and A C T H 36. Although recent electrophysiological findings support the view of a single, glutamatergic type of synaptic input onto LC neurons 2, a variety of neuroactive compounds was found to modulate noradrenergic firing and/or norepinephrine synthesis rate in LC. It is tempting to relate such effects to the numerous neuroreceptors visualized within LC by radioligand binding and radioautography, a2-Adrenergic 38, kt-opioid 3, somatostatinergic 15"35, 5-HT1A (ref. 37) and 5-HT 2 (ref. 9), serotoninergic, M 2 muscarinic cholinergic TM, tachy-
kininergic 29, angiotensinergic 1° and D2-dopaminergic 4 receptors have been thus far identified in LC. However, the localization on the noradrenergic neurons in this structure was demonstrated only for #-opioid receptors 17' 22. A n o t h e r strong candidate for input signalling to LC neurons is the tetradecapeptide somatostatin (SS14). LC contains indeed a very high density of SS14-specific binding sites 15'23'35, and SS14 was recently shown to hyperpolarize individual noradrenergic neurons of LC in vitro19"25; further, somatostatinergic fibers arising from hypothalamic periventricular nucleus are known to project onto LC 8'26. In order to determine whether SS14specific binding sites of LC mediate the direct actions of the peptide on intrinsic noradrenergic neurons, we investigated the localization of SS14 binding sites within LC. This question was addressed in the present work by combining radioautographic detection of SS14 binding sites at the light-microscopic level with T H immunohistochemistry and selective lesioning of catecholaminergic neurons by 6-hydroxydopamine.
MATERIALS AND METHODS Animals
Twenty-five male Sprague-Dawley rats (Iffa-Credo, Lyon,
* Results presented in this paper were previously communicated at the 3rd International Symposium on Somatostatin held at Montr6al, Canada, in August 1989. Correspondence: E. Moyse, Laboratoire de Neuropharmacologie Mol6culaire (UMR 105), CNRS, Facult6 de M6decine A. Carrel, Rue G. Paradin, F-69372 Lyon Cedex 02, France. 0006-8993/90/$03.50 ~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)
197 SS14 binding
TH + ceils
Fig. 2. Comparative distribution of SS14 specific binding sites (left) and of TH+ cell bodies (right) over the caudo-rostral extent of LC. Camera lucida drawings from pairs of adjacent 10/zm thick sections, respectively processed for [125I][Tyr°,o-Trp8]SS14 'wet'. radioautography and for TH immnohistochemistry, b, Barrington's nucleus; LC, locus coeruleus; Me5, mesencephalic trigeminal nucleus; 4V, 4th ventricle.
<-Fig. 1. Regional distribution of [12sI][Tyr°,D-TrpS]SS14 binding sites in the pontine tegmentum of the rat. A: film radioautograph from a 10 /~m thick coronal section. B: histological radioautograph obtained by liquid emulsion coating of a radiolabeled section; radioautographic silver grains are seen directly over the Cresyl violet-stained tissue. C: adjacent 10 pm thick section stained by TH immunohistochemistry, revealing the cluster of catecholaminergic nerve cell bodies of LC.
198 France), weighing 300 + 20 g, were used in the present study. They were kept at 25 °C under a 12h/12h dark-light cycle, and given food and water ad libitum. Prior to intracerebroventricular (i.c.v.) injections, rats were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally) and placed in a stereotaxic frame. Each rat received one unilateral, 50-~1 injection of 0.1% ascorbic acid containing saline without (sham-operated animals, n = 6) or with (n = 15) 300/tg 6-hydroxydopamine (6-OHDA, Sigma, 1 mg/kg, dissolved extemporaneously, on ice and light-sheltered), at the rate of 10/A/min, through a 22-gauge steel cannula implanted at bregma A -2 mm, L +1.4 mm, H -4.2 mm 27. The cannula was removed 2 min after the end of the injection, sterile sutures were applied onto the cutaneous incision and the animal allowed to recover. All animals were killed by decapitation 3 weeks after i.c.v, injections.
Preparation of tissue Brains were dissected out immediately after killing, snap-frozen in liquid isopentane at -40 °C and kept at -80 °C until use. For [~25I][Tyr°,o-TrpS]SS14 total and non-specific binding assays, pairs of 10 a m thick sections were cut on a cryostat (Reichert 2800) at 50-/,m intervals throughout the extent of the LC, mounted separately onto 2% gelatin-coated coverslips and kept at -80 °C. Adjacent 10/tm thick sections were systematically collected for TH immunocytochemistry, and immediately immersed in a fixative mixture of 4% paraformaldehyde and 0.1 M phosphate buffer at 4 °C for 18-24 h.
[Tyr°,o-TrpS]SS14 binding and radioautography [Tyr°,D-TrpS]SS14 (Peninsula) was iodinated by the chloramine-T method as previously described 7. Briefly, 5 /~g of peptide were incubated 40-45 s with 2 mCi [125I]Na (IMS-30, Amersham) and 4 /tg chloramine-T in 70/~1 solution buffered at pH 7.4; the reaction was stopped by addition of 12/zl Na2S205 and 100/~1 of 10% BSA solution. Monoiodinated peptide was purified on a carboxy methyl cellulose (CM-52, Whatmann) column pre-equilibrated in 2 mM ammonium acetate buffer at pH 4.6, by a stepwise elution with 2-200 mM ammonium acetate buffer at pH 4.6. Specific activity of the tracer was estimated from the elution profile at 700 Ci/mmol. Frozen brain sections were brought to room temperature, preincubated 15 min in 50 mM, pH 7.6 Tris-HCl buffer containing 0.25 M sucrose and 0.2% BSA. They were incubated 45 min at room temperature with 0.5 nM [125I][Tyr°,D-TrpS]SS14 in the same buffer supplemented with bacitracin (20 mg/1) and MgC! 2 (1 g/I), either in the absence (total binding) or in the presence (non-specific binding) of 1 a M non-radioactive SS14. All sections were rinsed in 2 consecutive baths of fresh buffer at 4 °C (5 min each) and a quick passage in distilled water. For film radioautography, sections were air-dried and apposed onto Hyperfilm sheets (Amersham) concomitantly with radioactive standards ([12SI]Microscales, Amersham). After 24-48 h exposure in the dark, films were developed routinely with Kodak D-19 at 20 °C. Radioautographic labeling was quantified by densitometry using the IMSTAR (France) image analysis system; optical density units were converted into radioactivities (nCi/mg tissue) by refer-
Fig. 3. Light-microscopic distribution of [t2sI][Tyr°,D-TrpS]SS14 binding sites within a 10/~m thick coronal section of LC, radioautographed by emulsion-coating and stained with Cresyl violet. Note, within LC, the even scattering of radioautographic silver grains both over nerve cell bodies (as those indicated by arrowheads) and in-between.
199 ence to the radioactive standards (Autorad Software). Radioautographic exposure time was set so as the labeling densities of LC fell within the linear range of the standard curve. For 'wet' radioautography, i.e. by liquid emulsion coating, sections were transferred immediately after rinsing to a fixative solution of 4% glutaraldehyde in 0.1 M pH 7.6 phosphate buffer, dried overnight at 37 °C, dehydrated-defatted through graded ethanols and methylcyelohexane, dried again and dipped in the dark into liquid Kodak NTB-2 emultions diluted 1:1. After 3-6 weeks exposure at 4 °C, sections were developed in Kodak Dektol 1:2 and fixed in Kodak Unifix 1:3. They were stained with Cresyi violet 0.5% and coverslipped for light microscopy.
Effect of 6-OHDA lesion on [Tyr°,D-TrpS]SS14specific labeling in the pontine tegmentum In the LC of 6 - O H D A - t r e a t e d rats (n = 15), both the staining intensity and the n u m b e r of T H + cells were lower than in the LC of sham animals (n = 6)....