0270.6474/85/0502-0551$02.00/O The Journal of Neuroscience Copyright 0 Society for Neuroscience Vol. 5, No. 2, pp. 551-561 Printed in U.S.A. February 1985 THYROTROPIN-RELEASING HORMONE (TRH) RECEPTORS Localization by Light Microscopic Autoradiography in Rat Brain Using [3H][3-Me-His2]TRH as the Radioligand’ PATRICK W. MANTYH* AND STEPHEN P. HUNT Medical Research Council Neurochemical Pharmacology Unit, Medical Research Council Centre, Medical School, Cambridge, CB2-2QH United Kingdom Abstract Thyrotropin releasing hormone (TRH) is a putative neurotransmitter in both the central and peripheral nervous system. In the present report, we have used autoradiography coupled with densitometric analysis of tritium-sensitive film to investigate the distribution of [3H][3-Me-His2]TRH ([3H]MeTRH)-binding sizes in the rat brain. Previous pharmacological reports have established that many of these [3H]MeTRH-binding sites have a structure-activity profile consistent with being a physiological TRH receptor. A high level of TRH receptors were observed in the accessory olfactory bulb, lateral nucleus of the amygdala, dentate gyrus, and entorhinal cortex. Moderate levels of TRH receptors were observed in the rhinal cortex, hypothalamus, superior colliculus, several brainstem motor nuclei, and lamina I of the spinal trigeminal nucleus pars candalis, while low concentrations of receptors are present in the cerebral cortex, striatum and ventral horn-of the spinal cord. Very low levels of receptors were observed in the globus pallidus and in most nuclei of the dorsal thalamus. Comparisons of the distribution of TRH receptors to TRH-immunoreactive content indicates that, while in some areas of the brain there is a rough correlation between levels of TRH peptide and its receptor, in most brain areas there is little obvious correlation between the two. While such a discrepancy has been observed for other peptides and their receptors, the extensive distribution of TRH receptors in the central nervous system does provide an explanation for the variety of behavioral effects observed when TRH is infused into the central nervous system. The tripeptide thyrotropin-releasing hormone (TRH, pGlu- (Sharif and Burt, 1983; Sharif et al., 1983), and some peripheral His-Pro-NH2) was originally isolated from the hypothalamus tissues (Burt and Snyder, 1975; Taylor and Burt, 1982). That of pigs and sheep and shown to act upon the anterior pituitary these TRH receptors are functional is suggested by electro- to evoke release of thyrotropin-stimulating hormone (Guille- physiological studies which have demonstrated that neurons min, 1978; Schally, 1978). Since this original isolation, it has responsive to TRH are found in cerebral cortex (Renaud and become apparent that thyrotropin release is only one of the Martin, 1975; Braitman et al., 1980), septum (Winokur and many actions of TRH. Using radioimmunoassay (Brownstein Beckman, 1978), hypothalamus (Renaud and Martin, 1975; et al., 1974; Jackson and Reichlin, 1974; Winokur and Utiger, Winokur and Beckman, 1978), cerebellum (Renaud and Mar- 1974; Kardon et al., 1977) and immunohistochemistry (Hokfelt tin, 1975), and the spinal cord (Nicoll, 1977). Behavioral data et al., 1975a, 1975b), TRH (Jackson, 1980) has been shown to also supports the hypothesis that TRH interacts with its recep- be present in both the CNS and in a variety of peripheral tors outside the hypothalamo-pituitary axis. Thus, TRH infu- tissues including the pancreas and digestive system (Morley, sion in the CNS has been shown to produce a pressor response 1979). Interestingly, the distribution of TRH receptors has (Beale et al., 1977), tachycardia (Hine et al., 1973), hyperther- been shown to be even more ubiquitous than the peptide TRH. mia (Metcalf, 1974), satiety (Vijayan and McCann, 1979; Mor- Using homogenate tissue binding, it was shown that TRH ley and Levine, 1980), locomotor activity (Vogel et al., 1979), arousal (Stanton et al., 1980), and to antagonize the effects of receptors are also present in the forebrain, brainstem (Burt and barbiturates and alcohol in the CNS (Kalivas and Horita, 1980). Snyder, 1975; Ogawa et al. 1981; Taylor and Burt, 1982; Si- Other more controversial reports have suggested that TRH masko and Horita, 1982), retina (Burt, 1979), spinal cord administration may be of therapeutic benefit for patients af- flicted with depression (Kastin et al., 1972; Prange et al., 1972; ’ We would like to express our appreciation to Drs. I. Abols and N. however, see Coppen et al., 1974, and Montjoy et al., 1974), Brecha for helpful criticism of the manuscript, A. Bond for excellent schizophrenia (Inanaga et al., 1978), amyotrophic lateral scle- technical assistance, and A. Boesman for typing the manuscript. P. W. rosis (Engel et al., 1983), or spinal trauma (Faden et al., 1981). M. is a National Institute of Neurological and Communicative Disor- In the present study, we have examined the distribution of ders and Stroke postdoctoral fellow. TRH receptors in the rat CNS using a modification of the * To whom correspondence should be addressed at current address: Young and Kuhar (1979) autoradiographic binding technique. Center for Ulcer Research and Education, Veterans Administration Previous in vitro binding studies using rat brain homogenates Center, Building 115, Room 217, Los Angeles, CA 90073. have established that the ligand we have used [3H][3-methyl- 551 552 Mantyh and Hunt Vol. 5, No. 2, Feb. 1985 Figure 1. a and b The Journal of Neuroscience Distribution of TRH Receptors in Rat Brain Figure 1. c and d 554 Mantyh and Hunt Vol. 5, No. 2, Feb. 1985 Figure 1. e and f The Journal of Neuroscience Distribution of TRH Receptors in Rat Brain Figure 1. g and h Mantyh and Hunt Vol. 5, No. 2, Feb. 1985 The Journal of Neuroscience Distribution of TRH Receptors in Rat Brain 557 His’lthyrotropin-releasing hormone ( [3H]MeTRH) displays a formed as previously described (Palacios et al., 1981; Hunt and Mantyh, similar pharmacological structure-activity profile as TRH itself 1984). The exposed film was placed onto the stage of a Leitz Orthoplan (Taylor and Burt, 1981b; Simasko and Horita, 1982). Thus, we microscope equipped with a Vario-Orthomat camera system which have used this radiolabeled analogue to visualize the location incorporates a back-projected exposure guide. A small and variable sized spot of light indicates the area of tissue or negative from which of TRH receptors in the CNS. exposure is being read. Direct reading of the voltage content from the photosensitive cell served as a density reading, being directly propor- Materials and Methods tional to the optical density of the film. Previous calibration experi- ments determined at which point given density was increasing in a Male Sprague-Dawley rats (150-250 gm) were killed by decapitation; linear fashion with increasing concentrations of isotope. Nonlinearity the brains rapidly were removed, blocked in the horizontal plane, placed occurs close to saturation of the film and this region was avoided. on brass microtome chucks, and frozen in dry ice. The brains were then serially sectioned (20 pm) and thaw-mounted on gelatin-coated micro- Background labeling was defined as the 5 nM [3H]MeTRH binding that was not displaced in the presence of 10 pM TRH. scope slides and stored at -20°C in boxes of desiccant for 48 hr. Previous brain homogenate studies have extensively defined the optimal incubation conditions necessary to achieve the highest specific/ Results nonspecific binding ratios using [3H]MeTRH as the radioligand (Taylor The results of the autoradiographic studies are shown in Fig. and Burt, 1981b; Simasko and Horita, 1982). In the present study, we 1, a to j and quantified in Table 1. In general, nearly all (95%) have used a nearly identical set of incubation conditions as previously described except that we have performed the incubations on slide- of the TRH receptors were located in the gray matter with very mounted tissue sections rather than brain homogenates. low levels being observed in most white matter tracts (i.e., The slide mounted 20-pm tissue sections were allowed to come to corpus callosum, internal and external capsule). In experiments room temperature and then placed in the incubation medium (0°C on testing for chemography artifacts, no positive or negative chem- ice for 2.5 hr in a cold room 4°C) of 0.1 M sodium phosphate buffer ography was observed in any area of the brain. Experiments to (pH 7.4) containing 5 nM [3H]MeTRH [L-histidyl-4-3H], L-prolyl-3,4- determine the ratio of specific to nonspecific binding showed 3H] (New England Nuclear; specific activity, 76.7 Ci/mmol) in the that the specific binding represented approximately 75% of absence (total binding) or presence (nonspecific binding) of 10 I.IM [3H]MeTRH binding to the slide. TRH. After this incubation period, the slide-mounted tissue sections In presenting the autoradiographic data, we used horizontal were rinsed with three washes of ice-cold 0.1 M sodium phosphate buffer (pH 7.4, 0°C on ice, 5 min each in the cold room at 4°C) and brain sections spaced at approximately 0.5-mm intervals in the three washes of dH,O (0°C on ice, 5 set each in the cold room at 4°C) dorsal-ventral plane so as to provide an atlas of TRH receptors and then quickly dried in the cold room using a stream of cold air. in the rat brain. In describing the distribution of TRH receptors Sections were left a further 3 hr to dry in the cold room and then stored a very low density of receptors will correspond to those areas in desiccant-filled boxes overnight at -20°C. The slides were then having an optical density (OD) of 0.1: low, an OD of 0.2 to 1.0; placed in apposition to LKB tritium-sensitive Ultrofilm.