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CCK mRNA expression in the hippocampus following deafferentiation. Horizontal sections of adult control and lesioned hippocampus were hybridized with a 35 S-labeled CCK probe. The left column represents the ipsilateral side (A, C, E, G) and the right column the contralateral side (B, D, F, H) of the hippocampus. A down-regulation of transcripts is visible in the ipsilateral and contralateral hippocampus one day after lesion (C, D) as well as in the contralateral cortex (D). An increased expression level occurs in both the ipsi-and contralateral regions of the hippocampus and cortex 5 dal onwards. (E, F) and reaches control levels at 21-28 dal (G, H). Note the decreased hybridisation signals in the thalamus 1 dal and their recovery at 28 dal (C, G). The scale bar in (H)200 m. Asterisks indicate the side of the lesion. Arrows mark the rhinal fissure.
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The cortical information flow via the perforant path represents a major excitatory projection to the hippocampus. Lesioning this projection leads to massive degeneration and subsequently to reorganization in its termination zones as well as in primary non-affected subfields of the hippocampus. The molecular mechanisms and factors which are involved...
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Context 1
... keeping with data from earlier studies (Hö kfelt et al., 1991;De Belleroche et al., 1990), we found high levels of CCK transcripts in CA3 and CA1 hippocampal regions and in temporal cortical layers I, III, V and VI. CCK mRNA was observed in smaller amounts in a dot-like fashion in the hilar region of the hippocampus (Fig. 4A, B). In our investigation, we focused on the CA3 and CA1 regions and on the cortex. One day after ECL, the hybridization signal for CCK transcripts decreased significantly in the ipsilateral and contralateral CA regions and recovered to control levels at 3 dal ( Fig. 4C-D; Fig. 5). However, at 5 dal the expression levels of CCK mRNA ...
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... smaller amounts in a dot-like fashion in the hilar region of the hippocampus (Fig. 4A, B). In our investigation, we focused on the CA3 and CA1 regions and on the cortex. One day after ECL, the hybridization signal for CCK transcripts decreased significantly in the ipsilateral and contralateral CA regions and recovered to control levels at 3 dal ( Fig. 4C-D; Fig. 5). However, at 5 dal the expression levels of CCK mRNA increased and peaked with maximal intensity in the ipsilateral and contralateral CA regions (41% and 70% increase, respectively). In longer survival stages (10 -15 dal), the hybridization levels again decreased in the CA regions of both hemispheres and reached control levels at 28 ...
Context 3
... Fig. 5). However, at 5 dal the expression levels of CCK mRNA increased and peaked with maximal intensity in the ipsilateral and contralateral CA regions (41% and 70% increase, respectively). In longer survival stages (10 -15 dal), the hybridization levels again decreased in the CA regions of both hemispheres and reached control levels at 28 dal ( Fig. 4G, H; Fig. ...
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... CCK ability to stimulate the synthesis of neurotransmitters, neuropeptides, and neurotrophic factors, besides being an inductor of good number of proteins and enzymes responsible of the circadian rhythms, strengthens the idea that systemic administered CCK produces its effects in the CNS through CCK-A vagal receptors activation (Hansen et al., 2008;Luckman et al., 1993;Morton et al., 2006;Wang et al., 1998) by sharply increasing c-Fos protein induction in the hindbrain (AP, NST), limbic (CeA), and hypothalamus (arcuate nucleus) nuclei providing afferent inputs to the PVN known to be involved in the regulation of food intake, blood pressure, anxiety and stress-response, reproduction, and lactation, Section 4 (Blevins et al., 2003(Blevins et al., , 2004Blouet et al., 2009;Cota et al., 2006;Luckman et al., 1993;Micevych and Sinchak, 2001;Wang et al., 1998), exerting both neuromodulatory and neuroprotective actions that lead to the recovery of damaged neuronal functionality (Brä uer et al., 2003;Gong et al., 2006;Horner and Gage, 2000;Raivich and Behrens, 2006;Sutton et al., 2004;Tirassa and Costa, 2007). Indeed, systemic administration of CCK induces the functional and structural recovery of brain damage in animal models of ischemia (Eigyo et al., 1992), lesions of the nucleus basalis magnocellularis, and fimbria-fornix transection (Takahashi et al., 1993;Tirassa et al., 1999). ...
... In fact, oxidative stress is a main inductor of the metabolic syndromes diseases including renal injury, hippocampal damage, cardiovascular disease, diabetes, and neurodegenerative diseases (Bashan et al., 2009;Halliwell, 2006;Nath and Norby, 2000;McMillen and Robinson, 2005;Sapolsky, 2001;Vargas-Martìnez et al., 2012). Antioxidants produced in the brain and peripheral organs by OT, ANF, CCK, acetylcholine (muscarinic type receptor agonists, MuAchR), ANG (1-7), adiponectin, etc., play a diminished role in deregulated HPA axis animals and humans, given its downregulation by excess of GCs (Brä uer et al., 2003;Coupé et al., 2012;Deepa and Dong, 2009;Du et al., 2008;Espada et al., 2009;Jankowski et al., 2004;Kensler et al., 2007;Staab and Maser, 2010;Wolkowitz et al., 2009). Thus, under oxidative stress it is necessary to increase antioxidant hormones (vasorelaxant) activity that promote the synthesis of reductants that abate ROS accumulation. ...
... Once in the nuclei Nrf2 interacts with AREs activating the transcription of these reducing enzymes battery, known as the phase II antioxidant response (Kensler et al., 2007); as has been shown by Espada et al. (2009) for the MuAchR in hippocampal primary and cerebellar granule neuron cultures. CCK neuroprotective effects in lesioned brain pathways and brain ischemic preparations for CCK-A type receptor and for estradiol has been reported, see Sections 6.2.2 and 7.2.2 (Brä uer et al., 2003;Dufresne et al., 2006;Eigyo et al., 1992;Tirassa and Costa, 2007). Surely, the neuroprotective action of CCK in these aforementioned preparations includes the high potential of the CCK signaling to promote redox balance similarly to OT's. ...
... Among neuropeptides in CA1 and CA3 pyramidal cell layers, expression of Cck represents 66%. In situ hybridization studies showed robust expression of Cck transcript in CA1 and CA3 pyramidal cell layers and scattered expression in the other sublayers of the hippocampus (Lucas et al., 1998;Zachrisson et al., 2000;Brauer et al., 2003;Nakamura and McEwen, 2005). The distribution of Cck octapeptide sulfated, which is the active form of Cck peptides, was mostly found in scattered basket interneurons of rats and mice (Freund and Buzsaki, 1996;Freund, 2003;Matyas et al., 2004). ...
We have shown quantitative expression levels of genes coding for the "ligand-receptor system" for classical neurotransmitters and neuropeptides in hippocampal subregions CA1, CA3, and dentate gyrus (DG). Using a combination of DNA microarray and quantitative PCR methods, we found that the three subregions have relatively similar expression patterns of ionotropic receptors for classical neurotransmitters. Expression of ionotropic receptors for glutamate and GABA represents more than 90% of all ionotropic receptors for classical neurotransmitters, and the expression ratio between ionotropic receptors for glutamate and GABA is constant (1.2:1-1.6:1) in each subregion. Meanwhile, the three subregions have different expression patterns of neuropeptide receptors. Furthermore, there are asymmetric expression patterns between neuropeptides and their receptors. Expression of Cck, Npy, Sst, and Penk1 represents 90% of neuropeptides derived locally in the hippocampus, whereas expression of these four neuropeptide receptors accounts for 50% of G protein-coupled receptors for neuropeptides. We propose that CA1, CA3, and DG have different modalities based on the ligand-receptor system, particularly the "neuropeptidergic system." Our quantitative gene-expression analysis provides fundamental data to support functional differences between the three hippocampal subregions regarding ligand-receptor interactions.
... The abundant CCK transcript expression in CA1 and CA3 pyramidal cell layers of the hippocampus is also supported by previous in situ hybridization studies that showed robust expression of CCK mRNAs in CA1 and CA3 pyramidal cell layers including pyramidal cells and interneurons [3][4][5][6]. CCK peptides are known to be found in scattered interneurons, but not in pyramidal cells [7][8][9]. The distribution discrepancy between CCK transcripts and peptides in the hippocampus provides an interesting subject to be investigated. ...
We examined gene expression profiling in single neuron types and small regions of the nervous system.
The RNAs were extracted from mouse cerebellar Purkinje cells, granule cell layer, hippocampal CA1 and CA3 pyramidal cell layers, and three layers of the retina (outer nuclear layer, inner nuclear layer, and ganglion cell layer) were dissected by laser capture microdissection. The gene expression profiling of each sample was examined by Affymetrix GeneChip and real-time reverse transcription polymerase chain reaction. We studied the gene expression of 62 neuropeptide and hormone genes and 387 G-protein-coupled receptor (GPCR) genes.
Among them, cholecystokinin and neuropeptide Y genes were the most widely expressed. The gene expression of cholecystokinin was very high in the hippocampus, suggesting that cholecystokinin transcripts might have unknown roles in the hippocampus. More than 10 neuropeptide genes were expressed in the ganglion cell layer of the retina, whereas the outer nuclear layer of the retina did not express a considerable amount of neuropeptide mRNAs. In total 12 GPCR genes were found in all tissues examined, and half were orphans (6 of 12).
The high ratio of orphan GPCR genes suggests our limited knowledge of the ligand-receptor system in the nervous system. These results provide basic information for studying the function of neuropeptides.
... These controls showed only background hybridization signals. After exposure, the slides were counterstained with toluidine blue [43]. ...
... Immunocytochemistry. Animals were deeply anesthetized with a Ketamin mixture as described previously and perfused by vascular injection with 0.9% NaCl solution, followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4 [43]. Brains were removed and cut on a vibratome. ...
... Brains were removed and cut on a vibratome. The immunocytochemistry procedure and buffers have been described in detail previously [43]. Briefly, in horizontal brain sections (40 μm), the endogenous peroxidase was quenched in 0.5% hydrogen peroxide diluted in PBS. ...
Autotaxin is a secreted cell motility-stimulating exo-phosphodiesterase with lysophospholipase D activity that generates bioactive lysophosphatidic acid. Lysophosphatidic acid has been implicated in various neural cell functions such as neurite remodeling, demyelination, survival and inhibition of axon growth. Here, we report on the in vivo expression of autotaxin in the brain during development and following neurotrauma. We found that autotaxin is expressed in the proliferating subventricular and choroid plexus epithelium during embryonic development. After birth, autotaxin is mainly found in white matter areas in the central nervous system. In the adult brain, autotaxin is solely expressed in leptomeningeal cells and oligodendrocyte precursor cells. Following neurotrauma, autotaxin is strongly up-regulated in reactive astrocytes adjacent to the lesion. The present study revealed the cellular distribution of autotaxin in the developing and lesioned brain and implies a function of autotaxin in oligodendrocyte precursor cells and brain injuries.
... Riboprobes were generated by RT-PCR (Nakamura et al., 2004). DNA templates were determined to avoid homologous domains, as modeled after previously reported probe templates for PV Santhakumar et al., 2000), CB (Abe et al., 1992), CCK (Zachrisson et al., 2000;Brauer et al., 2003) and NPY (Smith, 1993). Two types of CR cDNA templates were designed from sequences in the 3=-UTR and 5=-UTR regions. ...
... In situ hybridization histochemistry detected the distribution of mRNA expression for different interneuronal phenotypes. As expected, mRNA expression differed between the hippocampus, dentate gyrus, reticular thalamic nucleus, cortex, and hypothalamus (Fig. 3B, E, H, K) in agreement to previous reports for PV (Seto-Ohshima et al., 1989;Bender et al., 2000;Santhakumar et al., 2000), CB (Abe et al., 1992;Frantz and Tobin, 1994), CR (Winsky et al., 1992;Kishimoto et al., 1998), CCK (Lucas et al., 1998b;Zachrisson et al., 2000;Brauer et al., 2003), and NPY (Smith, 1993;Li et al., 1998;Caberlotto et al., 1998). ...
Ovarian hormones regulate pyramidal cell synapse formation and excitability and interneuronal GABAergic tone in the CA1 region of the adult female rat hippocampus. The role of 17beta-estradiol in these effects is complex and appears to involve a subset of hippocampal interneurons, which express different calcium-binding protein and neuropeptide phenotypes and nuclear estrogen receptor alpha. We found that, in the hippocampus, nuclear estrogen receptor alpha-immunoreactive interneurons co-express neuropeptide Y, calbindin-D28k and calretinin but do not parvalbumin or cholecystokinin. Moreover, a proportion of neuropeptide Y-immunoreactive interneurons co-expresses calbindin-D28k and calretinin. This pattern is similar in the presence or absence of 17beta-estradiol treatment in ovariectomized rats. We then used immunohistochemistry and in situ hybridization to determine whether 17beta-estradiol treatment regulates expression of CA1 interneuronal phenotypic markers via nuclear estrogen receptor alpha activation. We found that 17beta-estradiol treatment of ovariectomized rats increased neuropeptide Y mRNA levels (25%) and the neuropeptide Y mRNA-associated grain density per cell (11%), as well as the number of neuropeptide Y-immunoreactive cells (11%), predominantly in the pyramidal cell layer (stratum pyramidale). Treatment with CI628, a selective estrogen response modulator that acts as an antagonist for nuclear estrogen receptor, blocked 17beta-estradiol-induced increase of neuropeptide Y mRNA levels. 17beta-Estradiol treatment did not alter the number of parvalbumin, calretinin, and cholecystokinin immunoreactive cells, nor mRNA levels for parvalbumin and cholecystokinin. Therefore, the present study has identified neuropeptide Y expression as the main interneuronal phenotype that co-expresses nuclear estrogen receptor alpha and shown that neuropeptide Y is responsive to 17beta-estradiol in CA1 pyramidal cell layer. We suggest that 17beta-estradiol may regulate neuropeptide Y expression mediated by nuclear estrogen receptor alpha-dependent activation in a subset of hippocampal interneurons, and we speculate that subsequent neuropeptide Y release may indirectly contribute to regulate glutamate-dependent neuronal activity in the adult rat hippocampus.
... A standard electrocoagulator was used to make unilateral incisions [with four single pulses (2.5 mA), 3 s each] in the frontal and sagittal planes between the entorhinal cortex and hippocampus. We used the following coordinates measured from lambda: frontal incision: AP + 1.2; L + 3.1 to + 6.1; V down to the interior of the cranium; sagittal incision: AP + 1.2 to + 4.2; L 6.1; V down to the interior of the cranium [38]. Rats were allowed to survive for 2, 5, 10, 28 and 60 days (± 0.5 days; three lesioned animals at each time). ...
... Brains were removed and immerse-fixed overnight in the same fixative. Both for immunocytochemistry and Fluoro-Jade staining, horizontal serial sections of the entorhino-hippocampal region were cut on a vibratome at 50 mm for immunocytochemistry and 30 mm for Fluoro- Jade staining [38, 39]. Cryocut sections (30 – 50 mm) were used for Black Gold labeling. ...
Myelin is crucial for the stabilization of axonal projections in the developing and adult mammalian brain. However, myelin components also act as a non-permissive and repellent substrate for outgrowing axons. Therefore, one major factor which accounts for the lack of axonal regeneration in the mature brain is myelin. Here we report on the appearance of mature, fully myelinated axons during hippocampal development and following entorhinal lesion with the myelin-specific marker Black Gold. Although entorhinal axons enter the hippocampal formation at embryonic day 17, light and ultrastructural analysis revealed that mature myelinated fibers in the hippocampus occur in the second postnatal week. During postnatal development, increasing numbers of myelinated fibers appear and the distribution of myelinated fibers at postnatal day 25 was similar to that found in the adult. After entorhinal cortex lesion, a specific anterograde denervation in the hippocampus takes place, accompanied by a long-lasting loss of myelin. Quantitative analysis of myelin and myelin breakdown products at different time points after lesion revealed a temporally close correlation to the degeneration and reorganization pha-ses in the hippocampus. In contrast, electroconvulsive seizures resulted in brief demyelination and a faster recovery time course. In conclusion, we could show that the appearance of mature axons in the hippocampus is temporally regulated during development. In the adult hippocampus, demyelination was found after anterograde degeneration and also following seizures, suggesting that independent types of insult lead to demyelination. Reappearing mature axons were found in the hippocampus following axonal sprouting. Therefore, our quantitative analysis of mature axons and myelination effectively reflects the readjusted axonal density and possible electrophysiological balance following lesion.
... In an attempt to identify which molecules are associated with the activation of microglial cells and secondary damage to neurons, we performed a di¡erential display screening with cDNAs obtained from lesioned hippocampi [15]. Here we report on the identi¢cation of a new macrophage/microglia activation factor (MAF). ...
... All surgical procedures were performed in agreement with the German law on the use of laboratory animals. For stereotactic surgery, rats were anesthetized with a mixture of 25 mg/ml ketamine (CuraMed Pharma GmbH, Karlsruhe, Germany), 1.2 mg/ml xylazine (Bayer, Leverkusen, Germany) and 0.35 mg/ml acepromazine (Sano¢ GmbH, Du « sseldorf, Germany) in 0.9% sterile NaCl (2.5 ml/kg body weight intraperitoneally (i.p.)) and received a unilateral entorhinal cortex lesion (ECL) through the use of a stereotaxic headholder (Stoelting, Germany) [15]. In brief, a standard electrocoagulator was used to make bilateral incisions (with four single pulses (2.5 WA) for 3 s each) in the frontal and sagittal planes between the entorhinal cortex and hippocampus. ...
Damage to the central nervous system triggers rapid activation and specific migration of glial cells towards the lesion site. There, glial cells contribute heavily to secondary neuronal changes that take place after lesion. In an attempt to identify the molecular cues of glial activation following brain trauma we performed differential display reverse transcription-polymerase chain reaction screenings from lesioned and control hippocampus. Here we report on the identification of the macrophage/microglia activation factor (MAF), a new membrane protein with seven putative transmembrane domains. Expression analysis revealed that MAF is predominantly expressed in microglial cells in the brain, and is upregulated following brain lesion. Overexpression of MAF in non-glial cells shows an intracellular codistribution with the lysosomal marker endosome/lysosome-associated membrane protein-1 (lamp-1). Furthermore, MAF-transfected cells show that MAF is primarily associated with late endosomes/lysosomes, and that this association can be disrupted by activation of protein kinase C-dependent pathways. In conclusion, these results imply that MAF is involved in the dynamics of lysosomal membranes associated with microglial activation following brain lesion.
... To quantify the intensity at a pixel level, we used the computerized videodensiometry system (Metamorph; Universal Imaging, Downingtown, PA, USA). A visually established pixel intensity threshold was set to remove the unlabelled portion of the image as described previously (Brauer et al., 2003b). The standard rectangle (1.5 mm 2 ) was de®ned and placed in six different positions over the pyramidal cell layers of the CA1 and CA2/CA3 and the granule cell layer of the dentate gyrus. ...
Phospholipid-mediated signalling on neurons provokes diverse responses such as neurogenesis, pattern formation and neurite remodelling. We have recently uncovered a novel set of molecules in the mammalian brain, named plasticity-related genes (PRGs), which mediate lipid phosphate phosphatase activity and provide evidence for their involvement in mechanisms of neuronal plasticity. Here, we report on a new member of the vertebrate-specific PRG family, which we have named plasticity-related gene-3 (PRG-3). PRG-3 is heavily expressed in the brain and shows a specific expression pattern during brain development where PRG-3 expression is found predominantly in neuronal cell layers and is already expressed at embryonic day 16. In the mature brain, strongest PRG-3 expression occurs in the hippocampus and cerebellum. Overexcitation of neurons induced by kainic acid leads to a transient down-regulation of PRG-3. Furthermore, PRG-3 is expressed on neurite extensions and promotes neurite growth and a spreading-like cell body in neuronal cells and COS-7 cells. In contrast to previously described members of the PRG family, PRG-3 does not perform its function through enzymatic phospholipid degradation. In summary, our findings feature a new member of the PRG family which shows dynamic expression regulation during brain development and neuronal excitation.
Epilepsy in children is associated with a broad spectrum of cognitive deficits, which is associated with hippocampal mossy fiber sprouting. The underlying molecular mechanisms involved in mossy fiber sprouting in hippocampus following developmental seizures are not completely known. The timing of cognitive dysfunction and the relation of this cognitive impairment to calcium/calmodulin-dependent protein kinase II (CaMK- II ), plasticity related gene 3 (PRG-3), cholecystokinin (CCK), zinc transporter 1 and 3 (ZnT-1 and ZnT-3) in hippocampus were studied. A seizure was induced by penicillin quaque die alterna in Sprague-Dawley rats from postnatal day 29 (P29). Rats were assigned into the recurrent seizure group (RS, seizures were induced in eleven consecutive days, n=39) and the control group (NS, n =21). At 1.5 h, 3 h, 6 h and 12 h after the last seizure, apoptosis and autophagy were detected by transmission electronmicro-scopy (TEM) or in situ end labeling (TUNNEL). Electro-corticogram was observed after the last seizure. During P51 to P56, P81 to P84 and P92 to P95, the rats were tested for spatial learning and memory abilities with automatic Morris water maze task. On P95, mossy fiber sprouting and gene expressions in hippocampus were determined subsequently by Timm staining and real time RT-PCR methods. The results are as follows: a. TEM revealed the formation of autophagosomes and apoptosis in hippocampus after kindling. In the hippocampal neurons of the controls, chromatin and cytoplasmic organoids showed eumorphism with nuclear membrane integrity; 1.5 h after the last penicilin-induced recurrent seizures, "C" -morphous double membrane structure of dilated endocytoplasmic reticulum could be observed; 3 h after the last seizures, autophagosome appeared with manifest dilated endocytoplasmic reticulum; 6̃12 h after the last seizures, the hippocampal neurons showed apoptotic feature such as nuclear chromatinic pyknosis and verge-aggregation. TUNNEL staining also showed elevated number of apoptosis neurons in hippocampus of RS group(P< 0.05). b. Vertex sharp transient wave and sharp wave were soon detected just in a few minutes afer the last seizure in RS group by Electrocorticogram. c. Escape latency. The escape latencies from the water maze were significantly longer in rats of RS group than that of the control at d5 of the first test, at dl of the second test, and at d2 of the third test. d. Strategy analysis. RIDIT analysis showed that the scores were much worse in rats of RS group than that of the control at d4 and d5 of the first test. Moreover, the scores were much worse in rats of RS group than that of the control at the whole three days of both the second and third maze tests, e. Memory test. As for the value of distance in origin platform quadrant to total distance, the RS group seemed worse than the NS group in three maze tests(P< 0.05). f. Aberrant mossy fiber sprouting was seen in the inner molecular layer of the granule cells and the stratum pyramidale of CA3 subfield in RS group, g. Real time RT-PCR analysis showed decreased expression of CaMK II α and ZnT-3 in hippocampus of RS group than that in control group. Moreover, the four genes (CaMK II α, PRG-3, CCK and ZnT-3) in NS group and the two genes CaMK II α and ZnT-1 in RS group showed copolymerization characteristic by cluster analysis. In addition, correlation and regression analysis demonstrated positive linear correlation among CaMK II α, CCK, PRG-3 and ZnT-3 in NS group and between CaMK II α and ZnT-1 in RS group. It was concluded that recurrent developmental kindling induced by penicillin could cause not only autophagy and apoptosis in the earlier stage of brain damage, but also have long- term effects on cognitive function and hippocampal mossy fiber sprouting which may be associated with the down-regulated expression of CaMK II α and ZnT-1 in hippocampus.
Objective: We examined gene expression profiling in single neuron types and small regions of the nervous system. Methods: The RNAs were extracted from mouse cerebellar Purkinje cells, granule cell layer, hippocampal CA1 and CA3 pyramidal cell layers, and three layers of the retina (outer nuclear layer, inner nuclear layer, and ganglion cell layer) were dissected by laser capture microdissection. The gene expression profiling of each sample was examined by Affymetrix GeneChip and real-time reverse transcription polymerase chain reaction. We studied the gene expression of 62 neuropeptide and hormone genes and 387 G-protein– coupled receptor (GPCR) genes. Results: Among them, cholecystokinin and neuropeptide Y genes were the most widely expressed. The gene expression of cholecystokinin was very high in the hippocampus, suggesting that chole-cystokinin transcripts might have unknown roles in the hippocampus. More than 10 neuropeptide genes were expressed in the ganglion cell layer of the retina, whereas the outer nuclear layer of the retina did not express a considerable amount of neuropeptide mRNAs. In total 12 GPCR genes were found in all tissues examined, and half were orphans (6 of 12). Conclusion: The high ratio of orphan GPCR genes suggests our limited knowledge of the ligand-receptor system in the nervous system. These results provide basic information for studying the function of neuropeptides.
