Expression of orphanin FQ and the opioid receptor-like (ORL1) receptor in the developing human and rat brain

Journal of Chemical Neuroanatomy 22 (2001) 219– 249 www.elsevier.com/locate/jchemneu

Expression of orphanin FQ and the opioid receptor-like (ORL1) receptor in the developing human and rat brain Charles R. Neal Jr. a,b,*, Huda Akil a, Stanley J. Watson Jr. a,c a

Mental Health Research Institute, Uni6ersity of Michigan, 205 Zina Pitcher Place, Ann Arbor, MI 48109 -0720, USA b Department of Pediatrics, Uni6ersity of Michigan, Ann Arbor, MI 48109 -0720, USA c Department of Psychiatry, Uni6ersity of Michigan, Ann Arbor, MI 48109 -0720, USA Received 1 February 2001; accepted 14 June 2001

Abstract The orphanin peptide system, although structurally similar to the endogenous opioid family of peptides and receptors, has been established as a distinct neurochemical entity. The distribution of the opioid receptor-like (ORL1) receptor and its endogenous ligand orphanin FQ (OFQ) in the central nervous system of the adult rat has been recently reported, and although diffusely disseminated throughout the brain, this neuropeptide system is particularly expressed within stress and pain circuitry. Little is known concerning the normal expression of the orphanin system during gestation, nor how opiate or stress exposure may influence its development. Using in situ hybridization techniques, the present study was undertaken to determine the normal pattern of expression of ORL1 mRNA in the human and rat brain at various developmental stages. Rat embryos, postnatal rat brains and postmortem human brains were collected, frozen and cut into 15 mm coronal sections. In situ hybridization was performed using riboprobes generated from cDNA containing representative human and rat ORL1 and OFQ sequences. Both ORL1 and OFQ mRNA is detected as early as E12 in the cortical plate, basal forebrain, brainstem and spinal cord. Expression for both ORL1 and OFQ is strongest during the early postnatal period, remaining strong in the spinal cord, brainstem, ventral forebrain, and neocortex into the adult. Human ORL1 and OFQ expression is observed at 16 weeks gestation, remaining relatively unchanged up to 36 weeks. The influence of early orphanin expression on maturation of stress and pain circuitry in the developing brain remains unknown. © 2001 Elsevier Science B.V. All rights reserved. Keywords: In situ hybridization; Nociceptin; Ontogeny; Opiate; Primate

1. Introduction The opioid receptor-like receptor (ORL1), a seven transmembrane member of the G-protein family of receptors, shares significant amino acid homology with the opioid receptors (Bunzow et al., 1994; Chen et al., 1994; Fukuda et al., 1994; Marchese et al., 1994; Mollereau et al., 1994; Wick et al., 1994; Wang et al., 1994; Lachowicz et al., 1995). In spite of these similarities, opioid peptides and opiate alkaloids have little affinity for the orphanin receptor (Bunzow et al., 1994; Chen et al., 1994; Fukuda et al., 1994; Lachowicz et al., 1995; Mollereau et al., 1994; Wick et al., 1994; Ma et * Corresponding author. Tel.: + 1-734-936-2036; fax: +1-734-6474130. E-mail address: [email protected] (C.R. Neal, Jr.).

al., 1997; Nicholson et al., 1998). Functional studies of ORL1 have shown it to exclusively bind an endogenous ligand, a heptadecapeptide referred to as nociceptin or orphanin FQ (OFQ). Although OFQ demonstrates high affinity binding to ORL1 and inhibits cAMP formation similar to endogenous opioid peptides (Meunier et al., 1995; Reinscheid et al., 1995; Dooley and Houghten, 1996; Reinscheid et al., 1996; Saito et al., 1995, 1996, 1997; Shimohigashi et al., 1996; Ardati et al., 1997; Butour et al. 1997; Civelli et al., 1997; Guerrini et al., 1997), it demonstrates no binding affinity for endogenous opioid receptors. Orphanin FQ has an amino acid sequence strikingly similar to the endogenous opioid peptide dynorphin A1 – 17 and is derived from a larger precursor molecule referred to as preproorphanin, which itself shares structural homology to the opioid precursor prodynorphin

0891-0618/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S0891-0618(01)00135-1

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(Houtani et al., 1996; Mollereau et al., 1996; Nothacker et al., 1996; Pan et al., 1996). Recent studies have demonstrated that, similar to endogenous opioid precursors, preproorphanin contains additional neuropeptides that may be biologically active (Okuda-Ashitaka et al., 1998; Rossi et al., 1998; Hiramatsu and Inoue, 1999; Xu et al., 1999; Yamamoto et al., 1999; Zhao et al., 1999; Nakano et al., 2000), suggesting a coordinated mechanism of evolution that has separated the orphanin FQ and opioid systems (Reinscheid et al., 1998; Danielson and Dores, 1999). Functionally, numerous specific effects have been demonstrated by the binding of OFQ to the ORL1 receptor. At the cellular level ORL1 activation leads to inhibition of cAMP formation, stimulation of protein kinase C, and neuronal Ca2 + and K+ conductance changes. Systemic and central nervous system (CNS) infusions of OFQ have been shown to modulate several complex physiologic functions and behaviors, including pituitary function, cardiovascular control, sodium balance, allodynia and nociception, feeding, learning, locomotion, stress response and sexual behavior (for reviews; Civelli et al., 1998; Darland et al., 1998; Harrison and Grandy, 2000; Mollereau and Mouledous, 2000). Several studies have confirmed an extensive distribution of the orphanin system throughout the central nervous system. General descriptions of [3H]orphanin receptor binding in the mouse (Florin et al., 1997), 125 I-labeled orphanin binding in the rat and human hypothalamus (Makman et al., 1997), and preproOFQ and ORL1 mRNA distribution in the developing mouse brain (Ikeda et al., 1998) have been reported. Detailed descriptions of the distribution of preproorphanin and ORL1 mRNA, OFQ peptide immunoreactivity, and 125I-OFQ binding to the ORL1 receptor in the adult rat CNS has also been reported (Neal et al., 1999a,b). In these studies, OFQ and ORL1 were shown to be widely distributed throughout the brain and spinal cord, with prominent representation in stress and pain circuitry. In spite of detailed anatomical investigations focusing on OFQ and its relation to ORL1, no anatomical report to date has analyzed the expression of the orphanin system during gestational or early postnatal development. Equally lacking is an analysis of OFQ and ORL1 distribution in the human central nervous system. A knowledge of the distribution of the orphanin system during development, in human as well as the rodent, is crucial in understanding what role, if any, orphanin may play in the evolution of stress and pain circuitry. Exposure to stress, both prenatal (Weinstock, 1997; Williams et al., 1999) and postnatal (Suchecki et al., 1995; Johnson et al., 1996; Meaney et al., 1996; Sutanto et al., 1996; Va´ zquez et al., 1996; Williams et al., 1999), has been shown to have long-term effects on

the development and function of the limbic– hypothalamic –pituitary adrenal axis in the human, primate and rat. Although exposure to morphine in the neonatal period does not seem to have any adverse effects on intelligence, motor function, or behaviour (MacGregor et al., 1998), exposure to pain and exogenous opiates does appear to have a long-term effect on not only tolerance to opioid peptides, but also to painful stimuli (Rots et al., 1996; Anand et al., 1999; Whitfield and Grunau, 2000). Moreover, not only is OFQ and ORL1 distinctly distributed throughout this pain and stress neurocircuitry, there is a significant amount of behavioral data implicating OFQ in the stress response (Jenck et al., 1997; Griebel et al., 1999; Koster et al., 1999; Jenck et al., 2000; Martin-Fardon et al., 2000) and pain perception (Grisel et al., 1996; Mogil et al., 1996a,b; Rossi et al., 1996, 1997; Stanfa et al., 1996; Xu et al., 1996; Dawson-Basoa and Gintzler, 1997; Hara et al., 1997; Heinricher et al., 1997; King et al., 1997; Liebel et al., 1997; Minami et al., 1997; Morgan et al., 1997; Nishi et al., 1997; Tian et al., 1997a,b; Yamamoto et al., 1997, 1999; Zhu et al., 1997; Kolesnikov and Pasternak, 1997; Mogil et al., 1999; Pan et al., 2000). Given the high likelihood for an important role of orphanin in stress and pain systems, and the profound influence that prenatal or early postnatal stress and/or pain exposure can have on the development of these systems, it is important to better understand the ontological expression of OFQ and its receptor in the brain. We present results of an ontological study examining the expression of preproorphanin and ORL1 mRNA in the rat brain at various gestational ages, as well as ORL1 and OFQ mRNA expression in select regions of the human brain tissue at various gestational ages during the second and third trimester.

2. Materials and methods

2.1. Animals Time-pregnant adult female Sprague–Dawley rats were obtained from Charles River at various gestational ages. Prior to sacrifice, handling and use of all animals strictly conformed to NIH guidelines. Additionally, protocols for animal use in this study were approved by the university unit for lab animal medicine (ULAM) at the University of Michigan Medical Center.

2.2. Rat tissue Age of gestation of pregnant females was provided by Charles River, and fetal age was assigned as embryonic (E) days 12–22 prior to sacrifice. Postnatal (P) pup ages were assigned based on day of birth designated as

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P1. Pregnant rats and postnatal pups were deeply anesthetized with 75 mg/kg of sodium pentobarbital i.p. and transcardially perfused with 0.9% NaCl containing 2.2% sodium nitrite. This was followed by tissue fixation using Zamboni’s fixative (Zamboni and DeMartino, 1967). After perfusion was completed, whole embryos (E12–E14) or fetal brains (E16– E22) were dissected from pregnant females. Whole brains were removed from postnatal pups (P1– P21) after perfusion. All embryos and embryonic brains were postfixed for 24 h in Zamboni fixative after perfusion. Postnatal brains (P1, P4, P7, P14, P21 and adult) were postfixed for 1 h in Zamboni fixative after perfusion. After postfixation, all rat brain tissue was handled alike. Tissue was transferred to a solution of 10% sucrose in 50 mM potassium phosphate buffered saline for 48 h. Brains were frozen in liquid isopentane at − 30 °C, then stored at −80 °C until sectioning. All material was sectioned in the coronal plane on a Leica CM1800 cryostat at 15 mm and thaw-mounted on polylysinetreated microscope slides, then stored at −80 °C until used.

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In all specimens used, external examination of brain, meninges and cerebral spinal fluid was unremarkable. All human brain specimens remain immersed in Zamboni’s fixative for a minimum of 80 days before use. Prior to processing, whole brains were placed in 10% sucrose for 72 h, with a fresh sucrose change every 24 h. Whole brains were then photographed and cut into 3–5 cm thick blocks. Blocks were quick-frozen at − 50 °C in isopentane and stored at − 80 °C until sectioned. Selected blocks of tissue were carefully mounted based on known anatomical landmarks then sectioned in the coronal plane at 15 mm. All human sections were thaw-mounted on polylysine-treated slides then stored at − 80 °C until used.

2.4. Preproorphanin and ORL1 cRNA probes Hybridization of rat developmental brain tissue was performed using 35S-labeled cRNA riboprobes generated from the same cDNA utilized in previous adult studies (Neal et al., 1999a,b). The preproorphanin riboprobe was generated from a PCR fragment corresponding to the 5% portion of the preproOFQ sequence. This cDNA segment spans 580 base pairs (bases 106–686) and contains the entire open reading frame of the preproOFQ precursor molecule (Houtani et al., 1996; Mollereau et al., 1996; Nothacker et al., 1996). The orphanin receptor riboprobe was generated from a 700 base cDNA extending from the 5% untranslated region to 611 bases within the protein coding region of ORL1 (Bunzow et al., 1994; Chen et al., 1994; Fukuda et al., 1994; Mollereau et al., 1994; Wang et al., 1994; Wick et al., 1994; Lachowicz et al., 1995). In situ hybridization on human developmental tissue was performed using 35S-labeled cRNA riboprobes generated against cDNA generously provided by Hans-Peter Nothacker and Olivier Civelli (University of California, Irvine). The preproorphanin riboprobe was generated from a 900 base cDNA segment spanning

2.3. Human tissue Consent for use of donated materials was obtained from the University of Michigan Institutional Review Board for human subject use. Whole human brains (range 16–36 weeks gestation) were collected from teratology donations to the University of Michigan Pathology Department. Tissues are most commonly are donated to teratology following spontaneous fetal loss or neonatal death due to nonviable gestational age. These are primarily specimens that did not survive in the neonatal intensive care setting and, therefore, had not been exposed to prolonged therapy or stresses (Table 1). During autopsy, whole brain specimens were removed, weighed, and immersed in Zamboni’s fixative. Table 1 Fetal and neonatal brain specimens Specimen

Gestation

CNH1 CNH3 CNH4 CNH5 CNH7 CNH8 CNH9 CNH10 CNH12

175 175 116 245 207 180 138 138 138

days, days, days, days, days, days, days, days, days,

25 25 16 35 30 25 19 19 19

weeks weeks weeks weeks weeks weeks weeks weeks weeks

Fixative

History

108 days in fixative 100 days in fixative 88 days in fixative 157 days in fixative 210 days in fixative 168 days in fixative 110 days in fixative 110 days in fixative 110 days in fixative

25 25 16 35 28 25 19 21 21

week week week week week week week week week

twin A, born at 512 g. Nonviable. twin B, born at 502 g. Severe respiratory failure. singleton, born at 131 g. Nonviable. singleton, born at 3092 g. Pulmonary hypoplasia. singleton, born at 1323 g. Pulmonary hypoplasia. singleton, born at 461 g. Severe respiratory failure. singleton, born at 268 g. Nonviable. male twin B, born at 450 g. Nonviable. female twin A, born at 350 g. Nonviable.

Compilation of pertinent information regarding fetal and neonatal brain specimens used in the present study. Information provided for each specimen includes gestational age, total time in fixative until processed for in situ hybridization, birth weight and cause of death. Specific obstetrical histories are not provided. In all cases, obstetrical histories provide no significant information relevant to the integrity of the brain specimens received.

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open reading frame of the preproOFQ precursor molecule. The orphanin receptor riboprobe was generated from a 1200 base cDNA fragment encompassing the entire reading frame of the human ORL1 receptor.

2.5. In situ hybridization The in situ hybridization technique used for detection of preproOFQ and ORL1 mRNA in rat brain tissue has been described previously (Neal et al., 1999a,b). For the present study, this same technique was applied to both rat and human tissue, with minor alterations to accommodate human tissues. Those modifications are presented below. Adjacent sections of frozen brain were removed from − 80 °C storage and placed into 4% paraformaldehyde for 60 min at room temperature. Further fixation in paraformaldehyde was crucial in this protocol, as it minimized loss of fragile developmental tissue during the in situ procedure. Sections were next given three 5 min rinses in a solution of 300 mM sodium chloride and 30 mM sodium citrate, pH 7.2 (2× SSC). Sections were rinsed once in water then treated with 0.1 M triethanolamine containing acetic anhydride diluted to 400:1 vol/vol, pH 8.0, for 10 min at room temperature. Sections were rinsed again in water, dehydrated in graded alcohols, then air-dried. Prepared tissue was hybridized with a 35S-UTP and 35 S-CTP-labeled riboprobe generated to either the rat preproorphanin, rat ORL1, human preproorphanin or human ORL1 sequence as described above. The cRNA probe was diluted using a hybridization buffer composed of 75% formamide, 10% dextran sulfate, 3× SSC, 0.1 mg/ml yeast tRNA, 1 × Denhardt’s and 10 mM dithiothreitol in 50 mM Na2 PO4 (pH 7.4). The activity of 35S-labeled cRNA used for hybridization was in the range of 1.5– 2 × 106 cpm/35 ml for rat sections, and 1 –2×106 cpm/300 ml for human tissue sections. For hybridization, 35 ml of diluted probe was applied to rat tissue sections and 300 ml of diluted probe was applied to human brain sections. Coverslips (22×22 mm for rat slides and 48×65 mm for human slides) were applied to keep the hybridization buffer in contact with tissue. Tissue sections were placed in sealed humidifying chambers containing 50% formamide and hybridized overnight in a VRW 1535 incubator (Cornelius, OR) at 55 °C. On day 2, glass coverslips were removed and slides were rinsed two times in 2× SSC for 5 min, then treated with RNase A for 60 min at 37 °C (200 mg/ml RNase A and 0.5 M NaCl in 100 mM Tris, pH 8.0). Following RNase A treatment, sections were washed in 2× SSC for 5 min, followed by 1 × SSC for 5 min and 0.5 × SSC for 5 min, all at room temperature. The low salt wash was completed with incubation in 0.1× SSC for 60 min at 65 °C, followed by a water rinse at room

temperature. Rat brain sections were dehydrated through graded alcohols and air-dried. To minimize tissue loss, human brain sections were air-dried without dehydration upon completion of the hybridization procedure. Upon completion of hybridization, slide mounted rat and human sections were opposed to Kodak XAR-5 X-ray film. Film was exposed to rat ORL1 and OFQ sections for 5 days, and human OFQ and ORL1 sections for 2–3 weeks. Following X-ray film exposure, slides were dipped in NTB2 film emulsion. Rat OFQ sections were developed following a 14 day exposure to NTB2 emulsion and ORL1 sections were developed following a 16 day exposure. In contrast, human OFQ sections were developed following an 84 day exposure to NTB2 and ORL1 sections were developed following a 90 day exposure. Exposure time was chosen to maximize the detection of in situ hybridization grains, and was determined empirically via periodic development of test slides of tissue sections dipped in the NTB2 emulsion. Following development of the NTB2 film emulsion, all slides were rinsed in running water at room temperature for 30 min and Nissl counterstained with cresyl violet. Slides were dehydrated in graded alcohols followed by xylene, and coverslipped with Permount. Hybridized mRNA expression was analyzed on images captured from film using Scion Image (NIH image modified by Scion Corporation for the National Institutes of Health, Frederick, MD). For precise anatomical localization, mRNA expression was analyzed using a ‘Dark-Lite’ darkfield attachment on a Leitz DM RD microscope with camera attachment. Representative sections were captured using Scion Image and photographic plates prepared using Photoshop version 5.5 (Adobe Systems Inc., San Jose, CA).

2.6. In situ hybridization controls The specificity of the rat and human preproOFQ and ORL1 riboprobes was determined using standard control measures with two separate control conditions. After the 60 min incubation in 4% paraformaldehyde, sections from representative rat and human brain regions were incubated in RNase A for 60 min at 37 °C (200 mg/ml RNase A and 0.5 M NaCl in 100 mM Tris, pH 8.0). They were then run through the entire hybridization procedure with 35S-labeled cRNA, as described above. A separate set of adjacent, representative sections were run through the entire hybridization procedure, as described above, with the exception that a 35 S-labeled mRNA (sense strand) was used for the hybridization. Other than the alterations described above (RNase A and sense controls), all human and rat control tissue was treated identically and run alongside adjacent sections under normal conditions for comparison.

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Fig. 1. In situ hybridization controls. (A) autoradiogram showing OFQ mRNA expression obtained after hybridization with a 35S-labeled cRNA (antisense) generated against the 5% portion of the preproorphanin sequence at the level of the mid-forebrain. (B) mRNA expression is absent in an adjacent section hybridized with a 35S-labeled mRNA (sense) generated against the same region of the preproorphanin sequence. (C) autoradiogram demonstrating ORL1 mRNA expression obtained after hybridization with a 35S-labeled cRNA (antisense) generated against the 5’UT portion of the ORL1 sequence. (D) Labeling is absent in an adjacent section treated with RNase A prior to hybridization with the same 35 S-labeled cRNA. Scale bar = 2000 mm.

3. Results

3.1. Tissue fixation In our hands, the distribution of preproorphanin and ORL1 mRNA expression in adult rat tissue is identical to what has been observed using fresh frozen tissue in previous studies (Neal et al., 1999a,b). Initial studies with fresh frozen developmental rat tissue also demonstrated no difference in mRNA expression between Zamboni-fixed and fresh frozen tissue. However, integrity of embryonic and early postnatal rat tissue was superior in previously fixed tissue after exposure to in situ hybridization conditions. Additionally, given our experiences with Zamboni fixation and in situ hybridization, all human tissue was immersion fixed in Zamboni fixative. Half of one brain (Table 1) was immersion fixed in 4% paraformaldehyde. Although not presented, the general quality of in situ hybridization was superior in Zamboni-fixed human tissue. Given the several advantages of Zamboni fixation in both human and rat tissue, only Zamboni-fixed brains

were used for in situ hybridization and analysis of distribution of preproOFQ and ORL1 mRNA in the developing human and rat brain.

3.2. Controls No mRNA expression was detected in tissues pretreated with RNase A prior to in situ hybridization using 35S-labeled cRNA (antisense) directed to the rat preproOFQ cDNA sequence, the human preproOFQ cDNA sequence, the rat ORL1 cDNA sequence (Fig. 1C and D) or the human OLR1 mRNA sequence. Messenger RNA levels in these tissues were negligible in all levels of the forebrain, brainstem or spinal cord analyzed. In addition, no mRNA-expressing cells were detected in tissues hybridized with a 35S-labeled mRNA (sense strand) directed against any of the cDNA sequences above. As with the RNase A treatment, these tissues contained no mRNA-expressing cells at all levels studied (Fig. 1A and B, Fig. 12A and B, Fig. 13B and C, Fig. 16B and C).

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3.3. Nomenclature Descriptive analyses of ORL1 and OFQ expression in developing human and rat brain are provided below. No widely accepted nomenclature exists for structural identification of developing structures in the central nervous system of the rat and human brain. However, in order to maintain consistency, human material was labeled in accordance with accepted structural nomenclature for adult anatomical analysis when appropriate. For developing structures, when identified, nomenclature was used in accordance with that proposed by Feess-Higgins and Larroche (1987) for development of the human fetal brain from 10 to 40 weeks gestational age. For developing rat brain, nomenclature used for most structures in embryonic stages of development were in accordance with those proposed by Altman and Bayer (1995) and Paxinos et al. (1994). These sources were used principally for all anatomical analyses as well as identification of structures in figures. Nomenclature for structures differs markedly between the atlas of Altman and Bayer and Paxinos et al. However, for simplicity, structure abbreviations were used interchangeably throughout all rat figures. The reader is asked to refer to the table of abbreviations for definitive identification of labeled structures in figures.

3.4. Expression of preproorphanin mRNA in the de6eloping rat brain 3.4.1. E12 –E14 (Figs. 2 and 3) Pronounced preproOFQ mRNA expression is observed in the developing rat brain as early as day 12 of embryonic development (Fig. 2). At this developmental age mRNA expression is moderate in early hypothalamic neuroepithelium. Moderate expression is also observed in the dorsal cervical spinal neuroepithelium. Light to moderate mRNA expression is noted in the medullary and pontine neuroepithelium, and light mRNA expression is observed in the optic vesicle. Orphanin mRNA expression increases dramatically in the day 14 embryo (Fig. 3). Very strong expression is observed rostrally in the olfactory bulb and rhinencephalic neuroepithelia, but there is no expression in the rhinoencephalon. No mRNA expression is seen in the adjacent septal region. Strong expression is present in the cingulate cortex neuroepithelium and moderate expression in hippocampal neuroepithelium (Fig. 3A). Expression is very strong within the basal telencephalon, in both the pallidum and striatal neuroepithelia. Expression in the pallidal subventricular zone is moderate. This pattern maintains caudally, with intense expression in ventral, intermediate and anterior hypothalamic neuroepithelium (Fig. 3A). Thalamic mRNA expression is strong and hippocampal expression is light. Moderate to strong expression is also observed in

the amygdala. In the caudal diencephalon expression is very strong in the posterior hypothalamus, zona incerta, medial and lateral geniculate bodies and pretectum. Robust mRNA expression is also observed in many brainstem structures at this developmental stage (Fig. 3B), including the superior colliculus neuroepithelium, tegmentum, isthmus region, pontine neuroepithelium, spinal trigeminal nucleus, the hypoglossal nucleus, medulla and intermediate reticular zone. No mRNA expression is observed in the cerebellar neuroepithelia. PreproOFQ is expressed strongly in the spinal cord and dorsal root by day E13, strong in the ventral horn and moderate in the dorsal horn (Fig. 3B).

3.4.2. E16 (Fig. 4) By embryonic day 16 preproorphanin mRNA is expressed broadly in many CNS regions. Rostrally, intense expression fills the septal region and diagonal band, extending to the olfactory tubercle and piriform cortex (Fig. 4A). Moderate expression is observed in the neocortex, the cingulate cortex, cingulate neuroepithelium, and the insular cortical region. Expression in the hippocampus at this stage is moderate to strong, primarily in the differentiating field. The striatum is devoid of mRNA expression, while the differentiating field of the pallidum contains strong mRNA expression. The claustrum contains strong mRNA expression. The bed nucleus of the stria terminalis is also noted to have strong expression.

Fig. 2. In situ autoradiogram demonstrating OFQ mRNA expression in the brain of a 12 day rat embryo. Hybridization was performed using a 35S-labeled cRNA generated against the rat preproorphanin sequence. Scale bar =1000 mm.

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by E16, is noted to have intense OFQ mRNA expression (Fig. 4B–D). In the hypothalamus, mRNA expression is strong in the caudal arcuate nucleus and ventral and lateral regions, moderate in the dorsomedial and paraventricular nuclei and light in the fields of Forel and intermediate hypothalamic neuroepithelium (Fig. 4B–D). In the amygdaloid region, OFQ expression is moderate in the lateral and anterior regions, and strong in the central and medial nuclei (Fig. 4B and C). In the rostral E16 midbrain, strong OFQ mRNA expression is observed in the differentiating tegmental field and medial geniculate body. Strong expression persists in the premammillary and early differentiating mammillary regions. Orphanin expression is strong in the subthalamic nucleus and substantia nigra, as well as in the pretectum and dorsal central gray.

Fig. 3. In situ autoradiogram demonstrating OFQ mRNA expression in the brain of a 14 day rat embryo. Hybridization was performed using a 35S-labeled cRNA generated against the rat preproorphanin sequence. Scale bar = 900 mm.

In the mid-caudal forebrain, mRNA expression is very intense in the hypothalamic, preoptic and thalamic neuroepithelia. The reticular thalamic nucleus, evident

3.4.3. E19 (Fig. 5) By embryonic day 19, preproorphanin mRNA expression is intense, with more distinct regional localization. Rostrally, robust expression fills the septum, piriform cortex and cingulate cortex (Fig. 5A). This expression is equally notable in the olfactory tubercle, insular cortex, and the frontal and parietal neocortex. Although expression remains light in the accumbens nucleus, moderate expression is noted in the region of the ventral pallidum and hindlimb of the diagonal band of Broca. Intense and localized expression is noted in the bed nucleus of the stria terminalis and medial preoptic area at this stage of development (Fig. 5B). Expression in the bed nucleus remains intense to its caudal pole and persists strongly in the thalamus. In more caudal forebrain regions, mRNA expression is strong in the amygdala, particularly in the central and medial nuclei as well as the anterior and posterolateral cortical amygdaloid nuclei (Fig. 5B and C). Moderate to strong expression is noted in the piriform cortex and globus pallidus, and cortical mRNA expression remains strong, particularly in the cortical plate and subventricular cortical layer. Hippocampal expression is evident, and moderate. In the thalamus, intense signals are noted in the paraventricular and reticular nuclei. In the hypothalamus, expression is strong in the anterior hypothalamic area, the arcuate and ventromedial nucleus, and the dorsomedial and posterior hypothalamus (Fig. 5D). Light to moderate expression is noted in the paraventricular nucleus. In the caudal forebrain and rostral midbrain, mRNA expression remains strong in the temporal and occipital neocortex, again predominant in the cortical plate and subventricular cortical layer. Strong expression is also noted in the retrosplenial cortex, subiculum and entorhinal cortex. Strong expression is also noted in the medial geniculate nucleus and zona incerta, the supramammillary and medial mammillary nuclei, the interpeduncular nucleus and the central gray. Moderate

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mRNA expression is noted in the ventral tegmental area. The substantia nigra, nucleus of Darkschewitsch and interstitial nucleus of the medial longitudinal fasciculus, each well defined by this stage of development, all contain intense OFQ mRNA expression.

3.4.4. E22 –P1 (Fig. 5) At parturition, preproorphanin mRNA is expressed in similar structures as is observed in the E19 brain, but with more intensity in some structures. Expression is well defined and significantly more intense than is observed in the adult brain. Moderate expression is still observed rostrally in the olfactory tubercle, tenia tecta and septal differentiating field (Fig. 5E). Moderate to strong expression is observed in the infragranular and

supragranular cortical plates of the neocortex, with mRNA expression more distinguishable in the subventricular zone at this developmental stage. The cingulate, orbital, insular and piriform cortices contain strong expression, as does the entire septal region, including the indusium griseum. Although OFQ expression is light in the medial septum of the adult rat, expression in this region is moderate to strong during development. As in earlier stages of development, orphanin expression is conspicuously absent in the striatum, and minimal in the accumbens nucleus (Fig. 5E). In the mid-caudal forebrain, preproOFQ expression is very strong in the bed nucleus of the stria terminalis, pallidum and preoptic region. Structures with light to moderate expression include the ventral pallidum, sub-

Fig. 4. In situ autoradiogram demonstrating OFQ mRNA expression in the brain of a 16 day rat embryo. Hybridization was performed using a 35S-labeled cRNA generated against the rat preproorphanin sequence. Scale bar = 900 mm.

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Fig. 5. In situ autoradiogram demonstrating OFQ mRNA expression in the developing rat brain on embryonic day 19 (A – D) and postnatal day 1 (E–H). Hybridization was performed using a 35S-labeled cRNA generated against the rat preproorphanin sequence. Scale bar = 900 mm.

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stantia innominata, endopiriform and piriform cortices and claustrum. Expression remains strong in the neocortical plate and subventricular zone, and the caudal cingulate cortex (Fig. 5F). In the thalamus, strong expression is observed in the reticular nucleus, the habenula, and the anterodorsal, centrolateral, anteromedial and periventricular nuclei (Fig. 5F). Expression observed in the hippocampal formation at parturition, fills the dentate gyrus and areas CA1– CA3 at rostral levels, with more moderate expression at caudal levels. Expression in the amygdala and hypothalamus is similar to that observed in the E19 embryo. Intense mRNA expression is observed in the central and medial amygdaloid nuclei, the basomedial nucleus and the posteromedial cortical amygdaloid nucleus (Fig. 5F and G). In the hypothalamus, strong expression is observed in the anterior hypothalamic area, the arcuate and ventromedial nucleus, and the dorsomedial and posterior hypothalamus (Fig. 5G). The paraventricular hypothalamic nucleus contains light expression. In the caudal forebrain and rostral midbrain, expression remains strong in the medial amygdala, with strong expression in the basomedial nucleus and the anterior and posterior cortical nuclei. Expression is moderate to strong in the temporal and occipital neocortex, with strong expression in the retrosplenial cortex, subiculum and entorhinal cortex. In the thalamus, expression is strong in the posterior paraventricular and medial geniculate nuclei, the reticular nucleus and the zona incerta (Fig. 5G). Dorsally, expression remains strong in the mesencephalon, in the pretectum, tegmental field and the superficial and deep layers of the superior colliculus (Fig. 5H). Ventrally, intense expression is noted in the mammillary region, particularly the lateral and medial mammillary nuclei. The ventral tegmental area contains diffuse, strong mRNA expression, with increased label noted in the paranigral nucleus (Fig. 5H). Expression is moderate in the caudal arcuate nucleus, and light in the central gray. As observed in the E19 brain, expression is intense in the developing substantia nigra, interpeduncular nucleus, nucleus of Darkschewitsch and interstitial nucleus of the medial longitudinal fasciculus, with diffuse and strong expression observed throughout the midbrain reticular formation.

3.4.5. P4 –P14 (Fig. 6) During the first 2 weeks of postnatal life, preproorphanin develops a distribution of mRNA expression similar to that observed in the adult brain. Expression is strong in the olfactory tubercle, cingulate and orbital cortex, lateral septum, tenia tecta, indusium griseum, diagonal band, bed nucleus of the stria terminalis, medial preoptic region, globus pallidus, granule cell layer of the dentate gyrus and areas CA1– CA3 of Ammon’s horn (Fig. 6A and D). Very strong expres-

sion is noted in the subiculum (Fig. 6C). In the thalamus, expression is strong in the reticular nucleus, the anterodorsal and anteroventral nuclei, the paratenial nucleus, the paraventricular nucleus and nucleus reunions (Fig. 6B and E). In the hypothalamus, expression is light in the paraventricular nucleus, stronger in the anterior, ventromedial and dorsomedial areas, and intense in the mammillary region. In the amygdala, strongest expression is again observed in the medial and central nuclei, with robust expression also observed in the anterior nucleus, posterior cortical nucleus and the lateral nucleus (Fig. 6B and E). In the caudal forebrain and rostral midbrain, OFQ distribution is well localized by postnatal day P7 (Fig. 6C). In the pretectum and tectum, expression is moderate, and expression is light to moderate in the medial and lateral geniculate bodies. In the interpeduncular nucleus, substantia nigra, paranigral nucleus, nucleus of Darkschewitsch and interstitial nucleus of the medial longitudinal fasciculus, mRNA expression remains intense. Strong expression is also observed in the ventral tegmental area and midbrain reticular formation. Moderate expression is evident at this stage of development in many midbrain structures, including the central gray and dorsal raphe (Fig. 6C and F).

3.4.6. P21 –Adult (Fig. 6) By the 3rd week of life, preproorphanin mRNA expression is distributed in a manner essentially identical to that of the adult animal. However, intensity of expression remains slightly increased in some areas of the P21 brain in comparison to that of the adult. Strong expression in the cingulate cortex and tenia tecta is similar in P21 and adult brains (Fig. 6G). In the claustrum, expression is quite strong in the P21 brain, decreasing slightly by adulthood. Orphanin mRNA expression in the diagonal band of Broca, lateral septal nucleus, bed nucleus of the stria terminalis, medial preoptic area and hypothalamus remain generally strong, very similar to that observed in the adult. By postnatal day P21, mRNA expression in the medial septal nucleus becomes negligible, similar to what is observed in the adult. In the thalamus expression remains strong in the reticular nucleus and zona incerta (Fig. 6H), the paraventricular and paratenial nuclei, and the anteroventral nucleus. Some mRNA expression is also evident in the lateral habenula. However, strong expression is no longer noted in the anterodorsal or reunions nuclei. This pattern persists into adulthood. In the P21 amygdala, OFQ distribution is similar to that of the adult, with strong expression noted in the medial nucleus and the medial part of the central nucleus (Fig. 6H). Anterior and posterior cortical expression are light by postnatal day P21. PreproOFQ mRNA expression in the neocortex remains stronger in the P21 brain compared to the adult.

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Fig. 6. In situ autoradiogram demonstrating OFQ mRNA expression in the developing rat brain on postnatal days P7 (A – C), P14 (D–F) and P21 (G– I). Hybridization was performed using a 35S-labeled cRNA generated against the rat preproorphanin sequence. Scale bar = 900 mm.

In the cortical plate, distribution is light to moderate, as is observed in the fully layered cortex. In the subiculum and hippocampus, mRNA expression is also stronger in the P21 brain versus the adult (Fig. 6H and I). Rostrally, strongest expression is observed in areas CA1 and CA3, and the granule cell layer of the dentate gyrus. In caudal hippocampus, OFQ expression is similar to that observed in the adult dentate and Ammon’s horn. Expression in the subiculum is more intense than that observed in the adult at all rostral to caudal levels. In the rostral midbrain, expression is light in the pretectal region, the tectum and in the medial and lateral geniculate bodies. In the interpeduncular nucleus, substantia nigra, paranigral nucleus, nucleus of Darkschewitsch and interstitial nucleus of the medial longitudinal fasciculus, mRNA expression is intense (Fig. 6I). Strong expression is also observed diffusely throughout the ventral tegmental area (Fig. 6I). Moderate expression is observed in ventral part of the central gray and the dorsal raphe. This pattern is similar to that observed in the adult.

3.5. Expression of ORL1 receptor mRNA in the de6eloping rat brain 3.5.1. E12 –E14 (Fig. 7) Robust ORL1 receptor mRNA expression is observed in the day 12–13 embryo. At this age mRNA expression is moderate in neocortical neuroepithelium, much stronger in cingulate neuroepithelium. In the rostral differentiating hypothalamic field and hypothalamic neuroepithelium (Fig. 7A), ORL1 mRNA expression is moderate, becoming intense caudally in these regions. Expression in the thalamic neuroepithelium is moderate (Fig. 7A and B). In the pontine neuroepithelium mRNA expression is quite strong in the tegmental region, and light in the pretectum and isthmus (Fig. 7B). In medullary neuroepithelium, mRNA expression is moderate. Strong expression is observed in spinal cord neuroepithelium (Fig. 7A and B). In the day 14 embryo moderate ORL1 mRNA expression is observed rostrally in the olfactory bulb neuroepithelium, with light expression in the rhinen-

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cephalic neuroepithelium. Expression in the cingulate region remains strong, with moderate mRNA expression observed in the neocortical neuroepithelium. Light expression is present in hippocampal neuroepithelium, with moderate expression in basal telencephalon. Thalamic and amygdaloid ORL1 mRNA expression is strong at this stage, and very strong mRNA expression is observed in hypothalamic neuroepithelium, especially in the ventral region. In brainstem structures, light to moderate expression is observed in the superior colliculus neuroepithelium, strong expression in the isthmus region and light expression in the inferior colliculus. In the tegmentum and region of the central gray, mRNA expression is intense. Early moderate mRNA expression is observed in the cerebellar neuroepithelia at this stage, with intense expression in the region of the differentiating medulla and spinal trigeminal nucleus. Similar to preproOFQ, ORL1 mRNA is expressed strongly in the spinal cord by days E13–14, very strong in the ventral horn and moderate in the dorsal horn (Fig. 7A and B).

3.5.2. E16 (Fig. 8) On embryonic day 16 in rostral regions ORL1 mRNA is moderate to strong in the neocortex, moderate in the developing septum and strong in the developing piriform and cingulate cortices. Orbital and insular areas contain moderate expression. Strong expression in the septum persists caudally, with the medial septum and diagonal band also containing moderate to strong mRNA expression at this age. The pallidum contains moderate expression and the striatum is devoid of signal (Fig. 8B). The subicular area and developing Ammon’s horn contain moderate mRNA expression (Fig. 8B and C). In the caudal forebrain, intense mRNA expression fills the preoptic region (Fig. 8C). Strong expression is also observed at this level in the anterior hypothalamus. In the thalamus, strong expression is observed in the habenula, with moderate to strong expression diffusely filling the reticular nucleus and developing thalamic neuroepithelium (Fig. 8D). Strong mRNA expression is

Fig. 7. In situ autoradiogram demonstrating orphanin receptor mRNA expression in the brain of a 13 day rat embryo. Hybridization was performed using a 35S-labeled cRNA generated against the rat ORL1 sequence. Scale bar =900 mm.

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Fig. 8. In situ autoradiogram demonstrating orphanin receptor mRNA expression in the brain of a 16 day rat embryo. Hybridization was performed using a 35S-labeled cRNA generated against the rat ORL1 sequence. Scale bar =900 mm.

also observed in the amygdala, most notably in the medial, basomedial and basomedial nuclei (Fig. 8D). Expression in the cortical amygdaloid nucleus is moderate and in the lateral amygdala expression is light. Orphanin receptor mRNA expression is moderate in the hippocampus and subiculum in the E16 brain. Caudally, orphanin expression is intense in the ventral hypothalamus, particularly in the developing arcuate and ventromedial nuclei. In the thalamus, expression is

strong in the habenula and intermediate thalamus, moderate in the reticular thalamus, the medial geniculate and lateral geniculate nuclei.

3.5.3. E19 In the day 19 embryo, ORL1 mRNA expression in the cortical plate is strongest in the cingulate and insular cortex and moderate in the frontal neocortex and piriform cortex. Expression in the anterior olfac-

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tory nucleus, the septal region and tenia tecta is also strong. The nucleus accumbens has minimal signal. In the caudal ventral forebrain intense mRNA expression fills the region of the developing diagonal band and preoptic region. The bed nucleus of the stria terminalis contains intense ORL1 mRNA expression, with strong expression in the medial and lateral parts. Although a light signal is observed in the striatum, it is confined to the differentiating field, while the striatal subventricular zone is devoid of mRNA expression. The pallidum contains moderate to strong expression at rostral and caudal levels. In the hippocampal formation and subiculum, mRNA expression is moderate. In the caudal forebrain, the hypothalamus and developing hypothalamic neuroepithelium contain diffuse strong mRNA expression, particularly in the ventral zone, where intense expression is found in the ventromedial and arcuate nuclei. Moderate mRNA expression at this stage is observed in the paraventricular nucleus, anterior and lateral hypothalamus, and the dorsomedial region. The retrochiasmatic supraoptic nucleus also contains strong expression. In the thalamus, strong expression is present in the habenula, with a specific signal also observed in the paraventricular, mediodorsal and centromedial nuclei. In the amygdala, expression is strong in the medial nucleus, with moderate expression in the medial and lateral cortical amygdaloid nuclei. The central nucleus has no discernible mRNA expression and the basolateral and basomedial nuclei contain light expression. The bed nucleus of the lateral olfactory tract contains moderate to strong expression. In the hippocampal formation, ORL1 mRNA expression is light to moderate in Ammon’s horn and the dentate gyrus at caudal levels. In the E19 rostral midbrain, mRNA expression is moderate in the subthalamic nucleus and lateral geniculate nucleus. In the caudal thalamus, the signal in the habenula and paraventricular nucleus is moderate to strong. In the cortical region at this level, expression is strong in the cortical plate in the temporal and occipital cortices, with a lighter signal in the subplate region. Expression in the retrosplenial cortex and subiculum is strong. Light expression is observed in the midbrain pretectum, and moderate expression in the interpeduncular nucleus. The ventral tegmentum contains moderate ORL1 mRNA expression, with strong expression in the substantia nigra. Moderate expression is observed in the anterior pons, strongest in the region of the vestibular nuclei. Moderate to strong expression is observed in the medulla, particularly in the region of the developing medullary raphe and inferior olive.

3.5.4. E22 –P1 (Fig. 9) At parturition, ORL1 expression is well defined in most brain regions, with a distribution similar to that

observed in the adult. Rostrally, mRNA expression in the anterior olfactory nucleus is moderate in the lateral and dorsal parts, with light expression medially. Light to moderate expression is also noted in the rostral orbital and insular cortices. Cortical mRNA expression in the rostral forebrain is strongest in the insular and piriform cortices, with moderate expression in the cingulate cortex and tenia tecta. Neocortical mRNA expression is light to moderate, mostly observed in the granular part of the cortical plate. At the level of the nucleus accumbens, mRNA expression is strong throughout the cortical plate of the neocortex, with minimal subplate expression (Fig. 9A). The cingulate, insular and piriform cortices contain strong expression. Expression in the claustrum is moderate. The olfactory tubercle contains negligible signal. Interestingly, the rostral accumbens and differentiating striatum contain moderate mRNA expression at this developmental stage, although this signal becomes very light at more caudal levels (Fig. 9A and B). Orphanin receptor mRNA expression is very strong in the lateral septum and septal neuroepithelium at this age (Fig. 9B and C). Expression is light in the medial septum and diagonal band. The endopiriform cortex contains moderate to strong expression. In the ventral forebrain, the bed nucleus of the stria terminalis contains strong mRNA expression, in the ventral division rostrally, and in the medial and lateral divisions caudal. The preoptic region is filled with strong mRNA expression (Fig. 9D). No signal is observed in the diagonal band and substantia innominata. At this level, ORL1 mRNA expression remains moderate to strong in the insular and piriform cortices and moderate in the neocortex (Fig. 9C and D). At this level dorsally, the lateral septum and triangular septal nucleus contain strong expression. Expression becomes intense in the cingulate cortex at this level, with strong expression persisting into the developing retrosplenial cortical regions. Although some light mRNA expression is observed just lateral to the striatal subventricular zone, striatal mRNA expression is essentially negligible at mid to mid-caudal levels. At the emergence of the hippocampal formation, strong mRNA expression is observed in CA1 through CA3 of Ammon’s horn and the dentate gyrus (Fig. 9E–H). The subfornical organ also contains moderate expression. Intense expression is observed in the caudal bed nucleus of the stria terminalis, adjacent to the emergence of robust thalamic signal in the paraventricular, anteromedial and anteroventral nuclei (Fig. 9F– H). In the caudal forebrain, mRNA expression is moderate in the globus pallidus, with negligible expression observed in the entopeduncular nucleus. Ventrally, the nucleus of the lateral olfactory tract contains moderate to strong mRNA expression with moderate expression

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Fig. 9. In situ autoradiogram demonstrating orphanin receptor mRNA expression in the rat brain at term gestation (E22). Hybridization was performed using a 35S-labeled cRNA generated against the rat ORL1 sequence. Scale bar =900 mm.

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noted in the basolateral region. No signal is observed in the central amygdaloid nucleus. Orphanin receptor mRNA expression in the reminder of the amygdala is strong, with an intense signal in the medial nucleus, the basomedial nucleus and the posteromedial cortical nucleus. Light mRNA expression is observed in the lateral and basolateral nuclei. In the caudal amygdala, mRNA expression remains strong in the medial and basomedial nuclei, and moderate to strong in the posterior cortical nucleus and amygdalohippocampal area (Fig. 9H). The hippocampus contains strong mRNA expression. The strongest signal is noted in the subiculum, area CA3 and area CA1. Moderate ORL1 mRNA expression is observed in the dentate gyrus. In the hypothalamus, mRNA expression is strong rostrally in the anterior hypothalamic area, with minimal signal noted in the lateral hypothalamus. Moderate expression is observed in the suprachiasmatic nucleus (Fig. 9E and F). The paraventricular nucleus is filled with strong mRNA expression at all levels (Fig. 9F). At this developmental age, intense mRNA expression fills the ventromedial nucleus rostral to caudal, with the adjacent arcuate nucleus containing moderate expression (Fig. 9G and H). Strong expression persists in the ventral hypothalamus into the midbrain, where the mammillary nuclei contain a robust mRNA signal. In the caudal thalamus, ORL1 mRNA expression is strong in the medial habenula, the posterior paraventricular nucleus, and the laterodorsal, ventrolateral and reunions nuclei (Fig. 9G and H). Moderate expression is noted in the centromedian nucleus and the lateral geniculate nucleus. The lateral habenula, the reticular nucleus and the mediodorsal nucleus contain light mRNA expression. In the midbrain at parturition, moderate mRNA expression is observed in the subthalamic nucleus and lateral geniculate nucleus, and light expression is noted in the pretectum. At this level, strong expression is noted in the cortical plate of the temporal neocortex, with more moderate expression in the occipital cortex. The entorhinal cortex contains intense mRNA expression. Moderate to strong expression is observed in the parafascicular nucleus, medial geniculate nucleus and rostral central gray. The superior colliculus contains moderate mRNA expression. Moderate ORL1 expression is also noted ventrally in the substantia nigra and ventral tegmental area. At a mid to mid-caudal mesencephalic level, intense ORL1 mRNA expression fills the pars reticulata of the substantia nigra, with strong expression in the pars compacta. Other regions with strong mRNA expression at this level include the interpeduncular nucleus, red nucleus, medial geniculate nucleus and central gray. Moderate expression is noted in the dorsal raphe, superior colliculus and midbrain reticular formation of the caudal midbrain.

3.5.5. P4 –P14 (Fig. 10) After the first 2 weeks of postnatal life, ORL1 mRNA expression is similar to that observed in the adult brain with the exception of some increased intensity in the neocortex, hippocampus and thalamus. Rostrally, strong mRNA expression is observed in the anterior olfactory nucleus, in the lateral, dorsal and ventral divisions. Strong cortical expression is noted in the orbital cortex, cingulate, piriform and neocortical regions (Fig. 10A). Unlike earlier developmental ages, P4, P7 and P14 brains contain no mRNA expression in the striatum. Very light expression is noted in the nucleus accumbens core, with light to moderate expres-

Fig. 10. In situ autoradiogram demonstrating orphanin receptor mRNA expression in the brain of a 4-day old postnatal rat. Hybridization was performed using a 35S-labeled cRNA generated against the rat ORL1 sequence. Scale bar = 900 mm.

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sion in the shell region (Fig. 7A). Expression remains strong at this developmental stage in the lateral septum, the medial and lateral divisions of the bed nucleus of the stria terminalis, the medial preoptic region and the medial preoptic nucleus. Consistent with hippocampal ORL1 mRNA expression in the P1 brain, expression from P4 to P14 is strong in Ammon’s horn, and moderate to strong in the dentate gyrus (Fig. 10B and C). The subiculum at these ages contains diffuse, strong mRNA expression, mostly in the ventral part. This pattern persists throughout the extent of these regions. Within the amygdala, expression remains diffuse, with strong expression in the medial nucleus, basolateral nucleus and anterior cortical nucleus (Fig. 10B). Moderate to strong expression is observed in the posterior cortical nucleus. Hypothalamic mRNA expression remains generally robust at this developmental stage, with strong expression in the anterior hypothalamus, the paraventricular, ventromedial and the arcuate nuclei. The mammillary nuclei contain robust mRNA expression (Fig. 10C). In the thalamus, signal intensity is robust in the paraventricular nucleus and the anteromedial and anteroventral nuclei rostrally, with caudal thalamic mRNA expression strong in the medial habenula, the posterior paraventricular nucleus, the laterodorsal, ventrolateral and reunions nuclei (Fig. 10B and C). Orphanin receptor mRNA expression is also strong in the medial geniculate nucleus. Orphanin receptor mRNA expression in the midbrain is similar to that observed in the P1 brain. In the caudal cortical regions, expression is strong in the retrosplenial, entorhinal and temporal cortices. Messenger RNA expression is most robust in the midbrain in the parafascicular nucleus, medial geniculate nucleus, rostral central gray, superior colliculus, substantia nigra, ventral tegmental area, interpeduncular nucleus and red nucleus.

3.5.6. P21 –Adult (Fig. 11) In the P21 animal, ORL1 mRNA distribution is essentially identical to that of the fully developed animal. Similar to what is observed with preproorphanin at this developmental age, the intensity of ORL1 mRNA expression is slightly greater in some areas in comparison to that of the adult. Expression in the cingulate, retrosplenial, insular and piriform cortices and the neocortex remains generally similar in intensity than that observed in the adult. Neocortical expression is similar in intensity to that of the adult, but tends to be more diffusely distributed over layers II through VI. In cingulate and retrosplenial cortices, mRNA expression is significantly more intense at this age. Expression in the anterior olfactory nucleus and orbital cortex remains strong in the rostral P21 brain, but is similar to that observed in the adult. In the nucleus

Fig. 11. In situ autoradiogram demonstrating orphanin receptor mRNA expression in the brain of a 21-day old postnatal rat. Hybridization was performed using a 35S-labeled cRNA generated against the rat ORL1 sequence. Regions of artifact are denoted by asterisks (*). Scale bar =900 mm.

accumbens, mRNA expression is similar to adult distribution and is very light. The striatum has no observed signal (Fig. 11A and B). Expression in the lateral septum remains strong and is similar to the adult. The horizontal limb of the diagonal band of Broca is strongly labeled at this age, with more intensity than is observed in the adult. Claustrum and endopiriform nucleus expression are strong in both P21 and adult

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brains. The bed nucleus of the stria terminalis contains strong ORL1 mRNA expression in its lateral and medial divisions (Fig. 11A). The strongest signal in the preoptic region is in the medial preoptic area and medial preoptic nucleus. The intensity of signal in these structures is similar to that observed in the adult rat brain. Orphanin receptor mRNA expression in the hippocampal formation is robust in the P21 brain, and intensity of expression is stronger than that observed in the adult brain in Ammon’s horn, the dentate gyrus and the subiculum (Fig. 11B and C). In the amygdala, mRNA expression is similar to that observed in the adult with the strongest expression found in the medial nucleus, basolateral nucleus and anterior cortical nucleus (Fig. 11B). In the thalamus, mRNA expression in the paratenial nucleus and paraventricular nucleus is very strong (Fig. 11A–C). Paratenial signal intensity is stronger in the P21 brain than that observed in the adult. Expression in the remainder of the P21 thalamus is similar to that of the adult, with a strong signal observed in the anteromedial and anteroventral nuclei, the medial habenula, the posterior paraventricular nucleus, the laterodorsal, ventrolateral and reunions nuclei and the medial geniculate nucleus. Hypothalamic mRNA expression is generally robust in the P21 brain, with increased intensity relative to the adult in some regions, including the suprachiasmatic and supraoptic nuclei (Fig. 11A). Strong expression in the anterior hypothalamus, the paraventricular nucleus, ventromedial nucleus and mammillary region is similar to that observed in the adult. Orphanin receptor mRNA expression in the P21 arcuate nucleus is significantly stronger than is observed in the adult (Fig. 11B and C). In the midbrain, ORL1 mRNA expression in the caudal cortical regions remains strong in the retrosplenial, entorhinal and temporal cortices. Messenger RNA expression is also strong in the parafascicular nucleus, medial geniculate nucleus, rostral central gray, superior colliculus, substantia nigra, ventral tegmental area, interpeduncular nucleus and red nucleus. This pattern and intensity is identical to that observed in the adult.

sion. Adjacent to the internal capsule, the developing internal pallidum contains intense mRNA expression. Strong mRNA expression is also noted in the amygdala, particularly in the differentiating medial and central nuclei. Light to moderate expression is observed in the region of the developing posterior cortical nucleus, parahippocampal amygdaloid transition area and entorhinal cortex. At the midline, mRNA expression in the hypothalamus is most notable in the region of the developing paraventricular nucleus and dorsal hypothalamic area. By 19 weeks gestation, specific preproOFQ mRNA expression is detected in the cortical plate, with minimal subplate expression noted. No expression is noted in the claustrum, caudate nucleus or putamen, but strong expression is observed in the globus pallidus. In the thalamus, intense expression is still observed in the reticular nucleus and zona incerta, with light expression in the ventral thalamic neuroepithelium. The dorsal thalamus and subthalamic nucleus remain devoid of mRNA expression. Strong mRNA expression persists in the amygdala and hypothalamus at this gestational age (Fig. 13B).

3.6. Expression of preproorphanin mRNA in the de6eloping human brain Preproorphanin mRNA expression is observed robustly in the human brain by 16 weeks gestation (Fig. 12A). A specific signal is not detected in the cortical plate at this gestational age. Clear, strong expression is observed in the thalamus, particularly the reticular thalamic nucleus and zona incerta. Light to moderate expression is observed in the ventral and medial developing thalamic neuroepithelium. The dorsal thalamus and subthalamic nucleus are devoid of mRNA expres-

Fig. 12. In situ autoradiogram demonstrating OFQ mRNA expression in the human brain at 16-weeks gestation. (A) autoradiogram showing OFQ mRNA expression obtained after hybridization was performed using a 35S-labeled cRNA generated against the human preproorphanin sequence. (B) mRNA expression is absent in adjacent tissue hybridized with a 35S-labeled mRNA (sense) generated against the same region of the human preproorphanin sequence. Scale bar = 5250 mm.

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Fig. 13. In situ autoradiogram demonstrating OFQ mRNA expression in the human brain at 19-weeks gestation using a generated against a cDNA encoding the human preproorphanin sequence. Scale bar =5250 mm.

By 21 –22 weeks gestation, OFQ expression increases dramatically in the developing cortex, with strong expression in the cortical plate, light mRNA expression throughout the subplate region and no expression in the marginal zone (Fig. 14A). No expression is noted in the head of the caudate, the putamen, the claustrum or the nucleus accumbens region (Fig. 14B). The septal region is filled with heavy OFQ mRNA expression. Robust expression persists in the reticular nucleus and zona incerta, in the globus pallidus and mammillary bodies and medial and central amygdaloid nuclei. At this gestational age, strong mRNA expression is also noted in the hippocampus, subthalamic nucleus and substantia nigra. The medial geniculate body contains intense mRNA expression and the lateral geniculate is without expression (Fig. 14A and B). There is little remarkable change in these expression patterns between 25 and 36 weeks gestation in the human forebrain regions analyzed. At 25 weeks gestation, cortical preproOFQ mRNA expression remains strong, but becomes more organized in a laminar distribution in the cortical plate. The strongest cortical expression is noted in the cingulate region. Thalamic expression remains strong in the reticular nucleus and zona incerta, and abundant preproOFQ is also observed in the anterior thalamic nucleus. At 30 weeks gestation, expression remains strong in the cortical plate (Fig. 15A). In the thalamus, expression in the anterior nucleus diminishes some, but remains strong in the reticular nucleus. Expression is also noted in the centromedian nucleus at this age, as is light expression in the ventrolateral thalamus. Globus pallidus expression remains strong, and light expression is observed in the claustrum at this gestational age (Fig. 15A). Expression in the amygdala remains strong. At 35 weeks gestation, OFQ mRNA expression is laminar in distri-

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S-labeled cRNA

bution and strong in the cortical plate (Fig. 15A and B). Specific OFQ mRNA expression is also observed early in the developing brainstem. In tissue analyzed at 16 weeks gestation, mRNA expression in the pons is noted near the vestibular nuclei, as well as the pontine neuroepithelium in the region of the developing locus coeruleus and midline raphe. In the medulla, mRNA expression is noted in the region of the developing reticular formation, nucleus ambiguous and nucleus of the solitary tract. In the 21-week brainstem, mRNA expression is observed in the lateral and ventral tegmental region and in the pontine neuroepithelium, particularly in the tegmentum, near the ventral aqueduct, and in the lateral developing reticular fields. In the medulla, expression is evident in the inferior olive and midline reticular region. Adjacent to the fourth ventricle OFQ mRNA expression is noted near the region of the developing vestibular and midline nuclei, and the locus coeruleus. In the 30-week brainstem, orphanin expression observed in the rostral pons is scattered, with most expression in the basal neuroepithelium and peri-aqueductal region. In the medulla, strong expression is observed in the spinal trigeminal nucleus, and in the caudal dorsal medulla, in the region of the developing dorsal horn of the spinal cord.

3.7. Expression of ORL1 mRNA in the de6eloping human brain Similar to preproorphanin mRNA, ORL1 mRNA expression is robust in the human brain by 16 weeks gestation (Fig. 16). At this gestational age no mRNA expression is found in the cortical plate, subplate region or germinal matrix. However, a light to moderate signal is detected in the head of the caudate and the putamen

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rostrally, with light expression in the claustrum and region of the developing olfactory tubercle. Further caudal, light expression is found in the putamen and head of the caudate, with strong expression in the thalamus, in the region of the anterior and ventral anterior thalamic nuclei. Light expression is also observed in the reticular thalamus. At the midline, ORL1 mRNA expression is observed in the developing rostral hypothalamus. A very similar pattern of mRNA expression is observed in the 19-week brain. However, at this gestational age, a moderate to strong signal is detected in the cortical plate, with light subplate expression. Very light expression is noted in the claustrum, and moderate expression is observed in the head of the caudate and putamen (Fig. 17). The globus pallidus contains light mRNA expression. In the thalamus, strong expression is still diffusely observed, particularly in the anterior and ventral anterior areas (Fig. 17). The rostral hypothalamus signal is light to moderate. In the 21–22-week brain, ORL1 expression remains strong in the cortical plate, and a negligible signal is observed in the subplate region. The cortical expression is much stronger and more laminar than that observed at 19 weeks (Fig. 18A). The olfactory region, caudate and putamen maintain moderate mRNA expression. At the level of the thalamus, strong expression is observed in the principal anterior nucleus, the ventral anterior

nucleus, the anteromedial, paratenial and paraventricular nuclei. Hypothalamic expression remains moderate, with a distinct signal in the region of the dorsal and dorsomedial areas, the ventromedial hypothalamus and the paraventricular nucleus. In the caudal forebrain, strong expression is observed in the subthalamic nucleus, the mediodorsal, centromedial, laterodorsal and parafascicular thalamic nuclei, and the red nucleus. A robust signal is observed by this gestational age in the dentate gyrus of the hippocampus, and Ammon’s horn (Fig. 18A). Strong ORL1 expression is observed in caudal cortical regions at 22-weeks gestation, including entorhinal, temporal and occipital lobes. At 25 weeks gestation, ORL1 mRNA expression remains strong in the caudate and putamen, and light in the claustrum. At this gestational age cortical expression is very strong in the cortical plate in the neocortex, cingulate cortex and insular region. Negligible expression is observed in subplate regions (Fig. 18B). In the 30-week brain, strong mRNA expression is observed in the developing hippocampus and subiculum. The dentate gyrus and areas CA2 and CA3 of Ammon’s horn contain a robust signal, with a strong expression extending into the subiculum (Fig. 18C). Lighter mRNA expression is observed in the parasubiculum. The lateral geniculate nucleus contains moderate to strong expression, and the medial geniculate has negligible expression (Fig. 18C). Cortical and

Fig. 14. In situ autoradiograms demonstrating OFQ mRNA expression in the human brain at 21-weeks gestation. The panels demonstrate signal generated using a 35S-labeled antisense riboprobe in the coronal (A) and sagittal (B) plane. Scale bar = 5250 mm.

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Fig. 15. In situ autoradiograms demonstrating OFQ mRNA expression in the human brain at gestational ages 30 weeks (A) and 35 weeks (B,C). In the 30-week fetal brain (A), cortical expression of OFQ mRNA reveals a more linear organization than that observed earlier in gestation. A higher power image in (B) demonstrates laminar cortical expression of preproOFQ mRNA in the parietal cortex at 35-weeks gestation. The autoradiogram in (C) demonstrates absence of mRNA expression in a section adjacent to (B) after hybridization with a 35S-labeled mRNA (sense) riboprobe. Scale bars = 5250 mm.

thalamic mRNA expression remain strong at this gestational age. In the 35-week brain, cortical ORL1 expression remains strong, particularly in deeper layers. A specific signal is still observed in the caudate and putamen, but decreased from that observed at earlier gestational ages. Robust expression is still observed in the dentate gyrus and Ammon’s horn, subiculum and entorhinal cortex. Orphanin receptor mRNA expression observed in the developing brainstem is less clearly distinguished than that observed with preproOFQ. In the 16-week brainstem, ORL1 expression is observed in the caudal midbrain and pontine region, primarily in the lateral reticular region and ventral tegmentum. Some moderate expression is observed in the ventral midline as well. In the 16-week medulla, ORL1 mRNA expression is observed in the region of the nucleus ambiguous, the hypoglossal nucleus and the caudal trigeminal nucleus.

A light signal is also noted in the region of the solitary nucleus and midline reticular formation. In the 22-week brainstem, light ORL1 expression is observed in the midbrain dorsal peri-aqueductal region, in the geniculate bodies, the peripeduncular and ventral tegmental region, and in the reticular formation. In the pons at this gestational age, a strong signal is observed primarily caudally, in the inferior olive, the vestibular and cochlear nuclei, the parabrachial nucleus, the reticular formation and the midline raphe region. In the 35-week brainstem tissue examined, light ORL1 mRNA expression in the midbrain is observed at the midline in the region of the dorsal raphe and in the ventral tegmentum. In the pontine region, most expression is light to moderate and observed in the ventral peri-aqueductal region and reticular formation. Moderate expression is observed in the region of the vestibular nuclei and trigeminal nucleus.

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4. Discussion Several studies have reported a widespread distribution of OFQ and the ORL1 receptor in the CNS of several species. Following the sequencing of the primary structure of the rat and human OFQ precursor, the general tissue distribution of preproorphanin mRNA was reported for the rat and mouse (Houtani et al., 1996; Mollereau et al., 1996; Nothacker et al., 1996; Pan et al., 1996), as well as the primate hypothalamus (Quigley et al., 1998). Orphanin FQ immunoreactivity has also been described in the spinal cord and other structures within pain-modulatory regions in the rat

(Riedl et al., 1996; Schulz et al., 1996; Lai et al., 1997; Schuligoi et al., 1997) and the mouse (Houtani et al., 2000). A recent extensive analysis of OFQ peptide and mRNA distribution in the rat brain and spinal cord has also been reported (Neal et al., 1999a). Following the cloning of the orphanin receptor, early descriptions of ORL1 mRNA distribution were also reported (Bunzow et al., 1994; Fukuda et al., 1994; Mollereau et al., 1994; Wick et al., 1994; Lachowicz et al., 1995). The first detailed description of anatomical expression of ORL1 was provided in rat, with a comprehensive presentation of ORL1 immunolabeling in brain and spinal cord (Anton et al., 1996). Although

Fig. 16. In situ autoradiograms demonstrating ORL1 mRNA expression in the rostral (A) and caudal (B,C) human forebrain at 16-weeks gestation. In (A) and (B), hybridization was performed using a 35S-labeled cRNA antisense probe generated against the human ORL1 sequence (AS). In (C), mRNA expression is absent in a section adjacent to (B), after hybridization using a 35S-labeled mRNA (sense) probe generated against the same ORL1 sequence (S). Regions of artifact are denoted by asterisks (*). Scale bar =5250 mm.

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Fig. 17. In situ autoradiogram demonstrating ORL1 mRNA expression in the human brain at 19-weeks gestation. Hybridization was performed using a 35S-labeled cRNA generated against a cDNA encoding the human ORL1 receptor. Regions of artifact are denoted by asterisks (*). Scale bar= 5250 mm.

the specificity of the antiserum used by Anton et al. to characterize the ORL1 receptor has been questioned (Evans, 1999), a recent comprehensive analysis of ORL1 distribution in the rat brain and spinal cord substantiated much of the pattern of distribution found using the ORL1 antiserum (Neal et al., 1999b). A recent analysis of ORL1 mRNA expression reported in mouse brain also confirms the diffuse pattern of distribution described in the rat (Houtani et al., 2000). Although the neuroanatomical distribution of orphanin FQ and its receptor is now well characterized, no data to date has emerged regarding the expression of this neurotransmitter system in human brain. Additionally, there has been minimal work reported on the expression of OFQ in developmental tissue. Ikeda et al. (1998) have reported on preproOFQ and ORL1 mRNA expression in the developing mouse brain. Although the authors include an analysis of developmental tissue in this study, they report primarily on the expression of OFQ and ORL1 in adult tissue. Minimal consideration was given to the ontological expression of this system. In the present study we have provided the first analysis of the localization of preproorphanin and ORL1 receptor mRNA during embryologic and postnatal development in the rat brain. We also have demonstrated a strong presence of preproorphanin and ORL1 mRNA

expression in the human brain as early as 16-weeks of gestation, at the start of the second trimester of fetal development.

4.1. Tissue fixation Due to concerns regarding preservation of tissue quality, particularly human developmental tissue, fresh frozen tissue was not used for this study. Our previous experiences using the in situ hybridization technique in this laboratory have demonstrated no difference in mRNA expression using fresh frozen tissue or Zamboni-fixed tissue. In situ hybridization in developmental human brain tissue is superior in fixed versus unfixed tissue, mostly for reasons of tissue integrity. Due to our early experiences with fetal brain tissue handling, we began collecting human brains at necroscopy, placing whole brains into Zamboni’s fixative. A minimum of 80 days in fixative was found to be necessary to maintain tissue quality, and for optimal neuroanatomical analysis. When possible, longer immersion times lead to better fixation and improved tissue quality upon sectioning. This treatment allows for in situ hybridization quality equivalent to that observed in fresh frozen or paraformaldehyde-fixed tissue. Additionally, Zamboni fixation allows for application of immunocytochemical

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techniques, permitting us to visualize immunolabeling of several proteins in the developing human brain, such as mu and kappa opioid receptor proteins (C.R. Neal and S.J. Watson, unpublished data). To maintain consistency in the present study, in situ hybridization was only performed on Zamboni-fixed rat tissue. Perfusion of pregnant females provided some added fixation, particularly in later gestational ages. Immersion fixation for 24 h postperfusion provided excellent fixation in fetal material. Comparisons demonstrated no difference in tissue quality or mRNA expression with in situ hybridization between fresh frozen versus Zamboni-fixed rat fetal or postnatal tissue. As with human tissue, immunohistochemical studies demonstrated excellent immunolabeling of several different proteins, at all gestational and postnatal ages examined (C.R. Neal and S.J. Watson, unpublished data). Recognizing the distinct advantage of this fixation method, all material used in the present study, rat and human, is Zamboni-fixed.

4.2. Extrapolation from rat to human neurode6elopment Much has been reported regarding development of the embryonic human brain, but much less is known regarding fetal and early postnatal development of specific neuroanatomical systems and regions (for reviews see Sidman and Rakic, 1982; O’Rahilly and Mu¨ ller, 1994). In contrast, much is known regarding neurodevelopmental stages and growth spurts in other species, such as the pig (Dickerson and Dobbing, 1967), guinea pig (Dobbing and Sands, 1970) and rat (Dobbing and

Sands, 1971, 1979; for reviews see Bayer and Altman, 1995a,b). Due to significant differences in nuclear and regional organization of the developing central nervous system across species, interspecies extrapolation had been very difficult to accomplish in this area of study (Dobbing, 1973). However, such an extrapolation between man and other species has been proposed by Dobbing (1981). Using a compilation of data from rather crude quantitative methods, Dobbing assumed that: (1) the general sequence of brain growth is equivalent in all mammalian species; and (2) the anatomical units composing the brain show no interspecies differences. In other words, a neuron in the rat is essentially the same as one in man, and myelin shows only minor compositional differences. Under the general assumptions listed above, it has been accepted that in evaluating the developing brain of one species with respect to that of another, it is stages of brain development that must be compared, not ages. Using growth spurt data with general brain growth velocity curves, he proposed that the newborn mouse, rat and rabbit have equivalent neuronal development to that of an 18-week human fetus, whereas a newborn guinea pig has development equivalent to that of a 2–3 year old human (Dobbing, 1981). To date, no other data comparing human and rat brain development has been reported. Modified only slightly in the past decade, Dobbing’s general extrapolation of brain growth from rat to human, remains generally accepted. Using these brain growth velocity and growth spurt extrapolations, one can predict that the newborn P1 rat brain (comparable to E21–E22) is equivalent to that of

Fig. 18. In situ autoradiograms of ORL1 mRNA expression in the human brain at different gestational ages. Orphanin receptor mRNA expression is demonstrated at gestational ages of 21 weeks (A), 25 weeks (B) and 30 weeks (C). Regions of artifact are denoted by asterisks (*). Scale bar=5250 mm.

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about 18 – 20 weeks in the human fetus, the P3 brain equivalent to a 27-week fetus, the P5 brain equivalent to approximately 36 weeks gestation with respect to neuronal development and the P7 brain equivalent to 40 – 41 weeks gestation (term newborn). Prenatal extrapolation allows one to approximate the E12 embryo equivalent to the human at about 6 weeks gestation, the E14 brain to about 9 weeks, the E16 brain to about 12 weeks gestation, and the E19 brain equivalent to about 16 – 17 weeks gestation. Postnatal extrapolation based on this data allows one to approximate that the P14 brain in equivalent to a 12-month human infant developmentally, and a P21 brain to that of a 24– 30-month human. Although crude estimates, they do provide for some reference point to make inference when comparing human and rat brain in comparative developmental studies, such as the present one.

4.3. OFQ and ORL1 mRNA expression during de6elopment As will be discussed below, orphanin FQ and its ORL1 receptor clearly demonstrate mRNA expression very early in development in the rat, particularly when compared with the endogenous opioid systems. This developmental trend is not specific just for the rat, as expression in the human brain is also quite robust when observed early in gestation. Earliest gestational ages we examined in human (16 weeks) correspond most closely with the E18 or E19 rat embryo, when OFQ and ORL1 expression is by now robust and similar to an adult pattern of distribution. This observation in the rat is supported in corresponding human tissue, with very strong mRNA expression noted at the earliest gestational ages examined.

4.3.1. Preproorphanin mRNA Orphanin expression, seen as early as E12, is robust in regions where OFQ is heavily expressed in adult brain, namely the cortex, hypothalamus and spinal cord. This robust expression continues in the E14 embryo, with heavy expression in the cortex, septum, pallidum, hypothalamus, thalamus brainstem and spinal cord. Developmentally, the E12 and E14 brains are most consistent with a human brain very early in gestation (6–9 weeks). No human tissue was obtained at this early gestational age for analysis. The earliest comparable age for analysis is the E19 rat embryo. At this age, OFQ expression is very strong and distinctly organized in an adult-like distribution, with robust expression in the septum, cingulate, insular and piriform cortex, neocortex, basal telencephalon, hypothalamus, amygdala, thalamus and numerous midbrain structures. Orphanin expression in both 16-week and 19-week human fetal brain was also robust. With very strong expression in the amygdala, hypothalamus,

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thalamic reticular nucleus and zona incerta, and the pallidum, a pattern of distribution and intensity is evident that corroborates very well with equivalent developmental tissue in the rat. The 21-week human brain and P1/E22 rat brain again demonstrate excellent matching of orphanin expression. In the P1 brain, cortical OFQ expression is quite robust, with prominent expression in the cortical plate and subventricular zone of the neocortex. This correlates well with the robust mRNA expression observed in the cortical plate region of the 21 week human cortex. This similarity is somewhat contrasted with strong subplate OFQ expression and the lack of signal found in the marginal zone. The remainder of OFQ expression by parturition is quite similar to adult distribution patterns, except for increased density of signal in many areas. Corresponding structures examined in the 21-week gestation human brain show the same robust mRNA expression, including the septal region, reticular nucleus and zona incerta of the thalamus, the globus pallidus, mammillary bodies, medial and central amygdaloid nuclei, hippocampus and substantia nigra. Orphanin mRNA expression in the rat is essentially adult-like in distribution and intensity by postnatal day 7. This gestational age correlates most closely with the term infant brain in neurodevelopment, a gestational age not examined in this study. In the gestational tissue examined, very little difference was noted in the regions examined between 25 and 36 weeks. There were no major changes noted in distribution patterns, even after 21-weeks gestation. However, a more localized distribution within structures was noted by 25 and 30-weeks gestation. No other changes were observed except in the neocortex. In the developing rat, orphanin expression in the neocortical region is very robust in the early postnatal brain, more localized and laminar by P14 to P21, as in the adult. A similar change in cortical distribution is observed in the human brain, with diffuse OFQ mRNA distribution throughout the cortical plate in the 21-week brain, which becomes more laminar by 25-weeks gestation. This change distribution pattern persisted up to 36-weeks gestation, most likely continuing into the postnatal period. Such a late presentation of neocortical modification in OFQ mRNA expression in the human is not surprising when compared to similar ongoing maturation observed in the rat cortex at later postnatal ages. In no other structure analyzed was such persistent development noted.

4.3.2. ORL1 receptor mRNA Orphanin receptor mRNA expression is not as robust in the E12 embryo as is OFQ. However, moderate expression present in the E12 rat embryo is diffuse, with signal observed in the neocortex, hypothalamus, thalamus, brainstem and spinal cord. This distribution becomes much more diffuse and robust by day 14

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gestation in the rat, with strong mRNA expression also observed in the developing hippocampus and amygdala. By E16, robust mRNA expression is observed in many regions similar to that observed in the adult, including the neocortex, cingulate and insular cortex, septal region, hippocampus, hypothalamus, thalamus, amygdala and brainstem. As previously stated, no correlating human tissue was obtained, at this early gestational age, for comparison. The E19 rat embryo, corresponding most closely with the 16-week human fetal brain, contains strong ORL1 expression in the cortical plate, septal region, ventral forebrain structures, hippocampus, hypothalamus and thalamus. Strong expression is also evident in the substantia nigra and lateral geniculate nucleus by this gestational age. Although ORL1 expression is robust by 16-weeks gestation in the human brain, unlike what is observed with OFQ expression, some differences are evident between rat and human at this correlating neurodevelopmental age. No ORL1 mRNA expression is observed in the cortical plate or subplate at this gestational age, and a significant signal is detected in the putamen and head of the caudate. In the E19 rat embryo, minimal signal is observed in the E19 embryo, with moderate striatal mRNA expression observed at parturition. This is in sharp contrast to the paucity of striatal ORL1 expression observed in the adult rat brain. This mRNA expression is transitory, as a negligible signal is observed at later postnatal ages in the rat. This temporary striatal mRNA expression in the developing rat and human is unique when compared to adult ORL1 expression in the rat. We identified no other CNS structure in which ORL1 or OFQ expression is negligible or minimal in the adult, yet clearly expressed during development. The significance of this striatal mRNA expression is unclear. By parturition, ORL1 expression in the rat is robust and well defined when compared to adult distribution. Strong mRNA expression is seen in piriform and insular cortices, the neocortex, septal region, bed nucleus of the stria terminalis and preoptic area, hippocampus, thalamus and hypothalamus. Several midbrain nuclei also contain adult-like ORL1 patterns by this gestational age. In corresponding human tissue at 21–22 weeks gestation, orphanin expression is quite robust in the cortical plate, but negligible in subplate regions. A laminar cortical pattern of expression is already beginning to emerge by this gestational age. Moderate ORL1 mRNA expression in the caudate and putamen persists in the human brain at this age. Distinct and robust expression is observed in hippocampal formation, thalamus and several hypothalamic nuclei, in a pattern similar to that observed in the P1 rat brain. Similar to OFQ mRNA expression, ORL1 expression in the P7 brain is similar to that observed in the adult brain, with the exception of some increased signal

intensity in the thalamus, neocortex and hippocampus. By this postnatal age, no mRNA expression is observed in the striatum. Human tissue examined at 25, 30 and 35 weeks gestation continues to demonstrate strong neocortical ORL1 expression, with increased laminar distribution. Strong expression persists in the developing hippocampus, thalamus and hypothalamus as well. Moderate ORL1 mRNA expression persists in the 25 and 30-week brain. By 35-weeks gestation, ORL1 expression in these structures is markedly less intense, but still present. Later gestational age tissue was not available to determine if this striatal signal persists to the term human. All other structures examined at these gestational ages were in a distribution pattern similar to that described for the adult rat.

4.3.3. Comparison with opioid receptors In both rat and human tissues, expression of OFQ and the ORL1 receptor was observed at the earliest gestational ages studied (embryonic day 12 in rat and 16 weeks gestation in human). Due to limitations in tissue availability, earlier gestational ages were not examined. In the mouse, Ikeda et al. (1998) first observed preproorphanin and ORL1 mRNA expression in the E13 embryo. However, tissue from earlier developmental ages was not examined in their study and they were unable to comment on earlier expression. In order to characterize the establishment of the opioid system during prenatal mouse development, Zhu et al. (1998) examined the spatial and temporal expression patterns of m, k and d opioid receptor mRNA during development. In their study they found the k receptor to be the first opioid receptor expressed in peripheral gut epithelium (E9.5). However, in the brain, both m and k were first detected at E11.5, with m in the spinal cord and basal ganglia, and k in the midbrain. However, it is not until mid-gestation that the expression of both m and k receptors extended to other brain regions that exhibit high expression in the adult. In the mouse, by E17.5 most aspects of adult expression patterns of m and k receptors are established. In addition, unlike m and k receptors, the d receptor demonstrated quite late mRNA expression in mouse (Zhu et al., 1998), with mRNA expression observed in spinal cord by E15, but generally low as late as E19. Very little anatomical information has been provided regarding development of OFQ or ORL1 receptor expression in rodent brain. Although preproOFQ and ORL1 mRNA distribution in the developing mouse brain has been reported (Ikeda et al., 1998), more attention was focused on adult distribution, with little detail provided on the developmental expression of this system. Equally lacking is an analysis of OFQ and ORL1 distribution in the human central nervous system. As mentioned previously, a knowledge of the ontological expression of the orphanin system in the

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human and rodent brain is crucial in understanding any role orphanin may play in the evolution of stress and pain circuitry. The early, robust expression of OFQ and ORL1 in the developing rat presented here is similar to early signal noted in mouse by Ikeda et al. (1998). However, it differs significantly from the later onset of opioid receptors mRNA expression in mouse reported by Zhu et al. (1998). In the developing mouse, most aspects of endogenous opioid receptor expression seen in the adult rat (Mansour et al., 1994) are already present during late prenatal stages, with few exceptions. One example presented by Zhu et al. (1998), is the low expression of m receptor observed in the developing thalamus, an area of very robust m receptor expression in the adult rat (Mansour et al., 1994). Additionally, m receptor mRNA expression in the mouse striatum maintains a homogeneous distribution up to day E19.5. We have observed a similar distribution pattern in the rat, with a diffuse mRNA distribution in the striatum not developing an adult-like striatal patch pattern (Mansour et al., 1994) until after embryonic day E17 (C.R. Neal and S.J. Watson, unpublished data). Finally, mention should be made concerning the d opioid receptor which, in the mouse, was not detected until late prenatal stages (E17.5 and E19.5), and then restricted to few regions. In the rat, not only was robust orphanin expression observed very early on in gestation, an adult-like distribution pattern of OFQ and ORL1 mRNA expression was essentially in place by embryonic day 19. Although human tissue was not analyzed at earlier developmental ages, robust expression of OFQ as well as ORL1 as early as 16-weeks gestation supports the concept of very early expression of this neuropeptide system in both human and rat brain. Little anatomical data exists to compare our findings with opioid expression in human tissue at these early developmental ages. Enkephalin and dynorphin mRNA expression has been demonstrated in fetal human striatum, with expression observed by 13-weeks gestation (Brana et al., 1995). In the corresponding gestational age of the rat (E16), opioid receptor expression is strongly emerging, and OFQ/ORL1 mRNA expression is already robust. It is likely that in 13-week human tissue both preproorphanin and ORL1 mRNA expression would already be well established. The apparent early and robust expression of OFQ and ORL1 mRNA in the developing rat and human brain may imply an important role for orphanin during development, particularly in regions of high expression early in gestation, including the neocortex, limbic cortices, thalamus, hypothalamus, numerous brainstem structures and spinal cord. Moreover, it is possible that preproOFQ and ORL1 may emerge earlier in gestation than the endogenous opioid receptors, sug-

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gesting that early developmental events in neural tissues may be modulated not only by opioid receptors, but also by the orphanin system, signifying a role in developmental processes. 5. Conclusion In addition to the stress of critical illness, premature neonates are often exposed to multiple stressors during their stay in the neonatal intensive care unit, including handling, cold, pain, light and noise stress to name a few. As part of their management, most of these infants are also exposed to one or more neuroactive agents, such as benzodiazepines, glucocorticoids and opiates. These environmental exposures occur during a critical phase of development in the central nervous system. Little is known regarding the influence these compounds may have on the development of stress and pain circuitry. The ORL1 receptor and its endogenous ligand OFQ have been implicated in numerous behavioral and physiologic processes, including pain perception and stress. Our results demonstrate that mRNAs for preproorphanin and the ORL1 receptor are expressed early in rat and human gestation. Although no evidence for significant transient expression of OFQ or ORL1 has been observed, the presence of elements of the orphanin system on numerous populations of neurons soon after their differentiation, in the human and rat, suggests possible participation in early developmental events, particularly in the maturation of stress and pain neural circuitry. Acknowledgements Fetal human brain tissue used in this study was generously provided by Mason Barr, MD, Department of Pediatrics, Genetics Division. We are grateful for his generosity. Tissue was also obtained from the University of Miami Brain and Tissue Bank for Developmental Disorders through NICHD contract no. NO1-HD-8-3284. We also wish to thank Sharon Burke, Lisa Bain and James Stewart for their superb technical assistance. This work was supported by a Robert Wood Johnson Foundation Fellowship to CRNJ (RWJ 030811), a National Institute of Child Health and Development Junior Investigator Award to CRNJ (P30-HD28820) and a National Institute of Drug Abuse grant to HA and SJW (NIDA RO1 DA08920). Appendix A. Nomenclature 3V 4V

third ventricle fourth ventricle

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A6 A7 A9p A14 ac Acb ACe AH AHi AMe Amy Arc B1 B2 B3 B13 B23 BL BM C C11 CA1 CA3 cc Cg CG Cl cp CPu cx Cx CxP CxS DG DM DRG Ent g1 GP H Hl Hli Hlv H5 H6 H8 H9

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central nucleus of the amygdala (or differentiating field) basomedial nucleus of the amygdala (or differentiating field) posteromedial cortical nucleus of the amygdala (or differentiating field) basomedial nucleus of the amygdala (or differentiating field) anterior commissure nucleus accumbens central nucleus of the amygdala anterior hypothalamus amygdalohippocampal area medial nucleus of the amygdala amygdala arcuate nucleus striatal neuroepithelium striatal subventricular zone caudate–putamen (or differentiating field) pallidum (or differentiating field) bed nucleus of the stria terminalis (or differentiating field) basolateral nucleus of the amygdala basomedial nucleus of the amygdala head of the caudate nucleus cingulate cortex neuroepithelium area CA1 of Ammon’s horn area CA3 of Ammon’s horn corpus callosum cingulate cortex central gray claustrum cerebral peduncle caudate–putamen differentiating neocortex cortex cortical plate cortical subplate dentate gyrus dorsomedial thalamic nucleus dorsal root ganglion entorhinal cortex choroid plexus (lateral ventricle) globus pallidus hypothalamus hypothalamic neuroepithelium intermediate hypothalamic neuroepithelium ventral hypothalamic neuroepithelium suprachiasmatic nucleus of the hypothalamus (or differentiating field) paraventricular nucleus of the hypothalamus (or differentiating field) dorsomedial nucleus of the hypothalamus (or differentiating field) ventromedial nucleus of the hypothalamus (or differentiating field)

H20 Hb Hipp Hyp Ins Kl Kl1 K21 Ll LGN LH LM LOT LS LV MGN MM M11 M21 MS Mtx ne ot P4 P5 Pa Pir PMCo PT Pu PV R5 Re Rs Rt S S1 S6 S7 SC SN STh T Tl Tla Tli Tlp T5

arcuate nucleus of the hypothalamus (or differentiating field) habenula hippocampus hypothalamus insular cortex hippocampal neuroepithelium subicular neuroepithelium dentate gyrus germinal zone cerebellar neuroepithelium lateral geniculate nucleus lateral hypothalamus lateral mammillary nucleus nucleus of the lateral olfactory tract lateral septum lateral ventricle medial geniculate nucleus medial mammillary nucleus tegmental neuroepithelium pretectal neuroepithelium medial septum germinal matrix neuroepithelium optic tract medial preoptic region (or differentiating field) lateral preoptic region (or differentiating field) paraventricular nucleus of the hypothalamus piriform cortex posteromedial cortical nucleus of the amygdala pretectum putamen paraventricular nucleus of the thalamus endopiriform nucleus (or differentiating field) reuniens thalamic nucleus retrosplenial cortex reticular thalamic nucleus subiculum septal neuroepithelium medial septum (or differentiating field) diagonal band (or differentiating field) superior colliculus substantia nigra subthalamic nucleus thalamus thalamic neuroepithelium anterior thalamic neuroepithelium intermediate thalamic neuroepithelium posterior thalamic neuroepithelium lateral habenular nucleus (or differentiating field)

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T6 Thal Tu TT VDB VL VMH VTA Xl Xla Xlp Yl ZI Zld Zlv

paraventricular nucleus of the thalamus (or differentiating field) thalamus olfactory tubercle tenia tecta diagonal band of Broca, ventral limb ventrolateral thalamic nucleus ventromedial hypothalamic nucleus ventral tegmental area pontine neuroepithelium anterior pontine neuroepithelium posterior pontine neuroepithelium medullary neuroepithelium zona incerta dorsal spinal cord neuroepithelium ventral spinal cord neuroepithelium

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