OBJECTIVE: Both orexin (ORX)- and melanin-concentrating hormone (MCH) are expressed in different neurons in the lateral hypothalamic area (LH), and are considered to have common effects on stimulating food intake. There are no reports to demonstrate neural interactions between them at the ultrastructural level. We observed these neurons in the LH to evaluate the relationships between them.
DESIGN: We used two different types of double immunostaining to reveal the ultrastructure of both the ORX- and MCH-containing neurons. A preembedding double immunostaining technique was used to study the synaptic relationships between the two kinds of neuron.
RESULTS: The main new findings are as follows: 1) Both ORX- and MCH-containing neurons received other synaptic input and made synaptic input to other neurons; 2) Reciprocal synaptic relationships were observed between the ORX- and MCH-containing neurons.
CONCLUSION: The ORX- and MCH-containing neurons in the lateral hypothalamic area may influence food intake through synapse with each other.
The lateral hypothalamus (LH) has been described as a ‘feeding center’ since 1951.1 Lesions of the LH have been shown to produce temporary aphagia, adipsia and loss in body weight.1,2,3 Electrical stimulation of the LH has the opposite effect inducing vigorous feeding with a resultant increase in body weight.4 Several neuromediators have been implicated in this behavior, including melanin-concentrating hormone (MCH)5,6,7 and orexins (ORX).8 Although it had been proposed that MCH played a role in stimulation of food intake5,6,7 or acted as an anorectic peptide,9 some of the functions were revealed by a study using MCH gene-depleted mice.10 Likewise, there is no doubt that ORX acts to increase food intake and thus body weight.8 Using an immunostaining technique, the distributions of MCH-11 and ORX-12,13 containing neurons have been revealed, as mostly concentrated in the LH.
Bittencourt et al11 produced electron micrographs of the MCH-containing neurons, however, they did not indicate whether these micrographs were actually taken from the LH, and the ultrastructure of MCH-containing neurons in the LH rely on the results of an earlier report.14 In the case of the ORX-containing neurons, their ultrastructure has recently been examined in the cat LH,15 but a comparable electron microscopic study has not been reported in the rat. Given that both MCH- and ORX-containing neurons are involved in the regulation of food-intake behavior, one may imagine the possibility of direct synaptic relationships between them. Furthermore, ORX16 and MCH17 receptors have been found in the LH. The findings increase the possibility of reciprocal synaptic relationships between the ORX- and MCH-containing neurons. For this reason, we investigated their possible synaptic relationships with the use of a pre-embedding double immunostaining technique along with studying the ultrastructure of both the MCH- and ORX-containing neurons in the LH.
Materials and methods
All animal protocols were approved by the Ethics Review Committee for Animal Experimentation of Showa University School of Medicine.
Goat polyclonal anti-orexin-A antibody (affinity-purified, Santa Cruz Biotechnology, Inc. Raised against a peptide mapping to the carboxy terminus of orexin-A of human origin. Passed Western blotting test, not cross reactive with orexin-B). The specificity was also checked in one of our previous studies.18
Rabbit anti-rat MCH antibody (Code #PBL 234; a gift from Drs Vaughan and Vale, The Salk Institute), produced using rat MCH coupled to human α-globulins via glutaraldehyde, absorbed with human α-globulins. Passed preabsorption test with 40 µM concentration of the antigen which deleted immunostaining.11
The other antisera including the biotinylated anti-goat and anti-rabbit antibodies, as well as the avidin–biotin–peroxidase–complex (ABC) solutions were purchased from Vector Laboratories (Burlingame, CA, USA).
Two-color diaminobenzidine technique for light microscopic study
Two adult male Wistar rats (190–210 g body weight) were used in this study. The animals were injected with colchicine treatment by injecting colchicine (200 µg olchicine/5 µl in sterile normal saline) into the lateral ventricle under nembutal anesthesia (40 mg/kg body weight, i.p.). Two days later, each animal was again deeply anesthetized with sodium pentobarbital (75 mg/kg body weight, i.p.), then perfused via a cardiac ventricle with 100 ml of 0.9% sodium chloride, followed by a fixative solution of 4% paraformaldehyde in 0.1 M phosphate buffer solution (PBS, pH 7.4). Tissue blocks containing the hypothalamus were dissected and immersed overnight in 20% sucrose (w/v) in 0.1 M PBS followed by a further day in 30% sucrose (w/v) in PBS at 4°C. After freezing in dry ice, the blocks were cut into 15 µm coronal sections with a Microm Heidelberg cryostat. All the sections were collected and rinsed overnight at 4°C in 0.1 M PBS containing 0.3% Triton X-100. The next day, the floating sections were double immunostained according to the following series of incubation steps: 1) 5% normal horse serum for 20 min; 2) 1:20 000 diluted goat polyclonal anti-orexin-A antibody (Santa Cruz Biotechnology Inc.) overnight; 3) biotinylated anti-goat IgG for 1 h at room temperature; 4) ABC complex for 45 min at room temperature; 5) 3,3′diaminobenzidine (DAB, 0.2 mg/ml) and hydrogen peroxide (0.005%) in 0.05 M Tris buffer containing NiCl according to the protocols described in the data sheet provided by the supplier (Vector Laboratories, Inc); 6) 5% normal goat serum 20 min; 7) 1:128 000 diluted rabbit anti-rat MCH antibody (Code #PBL 234; a gift from Drs Vaughan and Vale); 8) biotinylated anti-rabbit IgG for 1 h at room temperature; 9) ABC complex for 45 min at room temperature; and 10) DAB (0.2 mg/ml) and hydrogen peroxide (0.005%) in 0.05 M Tris buffer without NiCl. After dehydration, the sections were carefully rinsed and mounted on glass slides for light microscopy.
Ultrastructural study of melanin-concentrating hormone and orexin-containing neurons
Two adult male Wistar rats (190–210 g body weight) were used for studying the distribution and ultrastructure of the MCH- and the ORX-containing neurons in the LH. The perfusing and fixation techniques were similar to those described above except that 0.1% glutaraldehyde was added to the fixative. Tissue blocks containing the hypothalamus were dissected in coronal sections from each brain and rinsed with PBS. After 2 h of postfixation in 4% paraformaldehyde prepared with PBS, the blocks were cut into 30 or 40 µm thick frontal sections using a Vibratome. The sections were rinsed in PBS (pH 7.4) overnight to wash out the fixative and then used for immunostaining. An avidin–biotin–peroxidase complex (ABC) immunoreaction19 was used to detect ORX or MCH antigens. The steps of the protocol were as follows: 1) normal goat serum (for MCH detection) or normal horse serum (for ORX detection) diluted 1:20 at room temperature for 30 min; 2) primary antisera (diluted 1:30 000 for ORX and 1:128 000 for MCH) for 48 h at 4°C; 3) biotinylated anti-rabbit IgG for 1 h at room temperature; 4) ABC complex for 1 h at room temperature; 5) DAB (0.2 mg/ml) and hydrogen peroxide (0.005%) in 0.05 M Tris buffer. The reaction time was controlled by direct observation with a light microscope during the reaction process. The sections were rinsed for 5 min with 0.1 M phosphate buffer three times after each step, except the first. After the immunostaining, the tissue sections were postfixed with 1% OsO4 in PBS for 1 h at 4°C, dehydrated in a series of ethanol solutions and embedded in a mixture of Epon Araldite. Ultrathin sections were cut and stained with lead citrate and uranyl acetate and examined under a Hitachi H-7000 electron microscope.
Double immunostaining with silver–gold-intensification method for examination at the light and electron microscopic level
Six adult male Wistar rats (190–210 g) were used to double immunostain for both the ORX and MCH with the ABC–DAB–silver–gold intensification (SGI) method.20 The tissue preparation procedure was the same as that described above for single immunostaining prior to electron microscopic study. Orexin-like immunoreactivity was detected by incubation of sections using the following protocol. At first, tissue sections were incubated in 5% normal horse serum for 20 min at room temperature, after which they were incubated in 1:30 000 diluted anti-ORX-A antiserum (Santa Cruz Biotechnology, Inc.) overnight at 4°C. The next day, the sections were incubated with biotinylated anti-goat IgG for 45 min, and then with ABC complex for 1 h at room temperature. Following this, they were treated for 5–7 min in the dark with DAB and 0.005% H2O2 in Tris-buffer (pH 7.2). Between the above steps, each section was carefully rinsed three times with PBS, except between the steps using normal horse serum and the primary antibody. Sections that showed a DAB reaction were treated with silver–gold intensification20 to darken the brown-colored DAB reaction product to black. The sections were then treated to detect MCH. First, they were incubated in 5% normal goat serum for 20 min, followed by 1:128 000 diluted MCH antiserum (Code #PBL 234; a gift from Drs Vaughan and Vale) for two days at 4°C. After that, the sections were incubated with biotinylated anti-rabbit IgG for 45 min and then with ABC complex for 1 h at room temperature. This was followed by a DAB reaction similar to the procedure previously described for detecting ORX, except that SGI was not applied. Some of the double stained sections were used for light microscopy, while the others were used for ultrastructural study. Similar to the procedure described in the single immunostaining for electron microscopy, the sections were postfixed, dehydrated, embedded in Epon Araldite, then ultrathin-sectioned and stained with lead citrate and uranyl acetate for examination under a Hitachi H-7000 electron microscope.
In each of the experiments described above, some sections were incubated without the primary antiserum as controls. No immunostaining could be observed.
Observations at the light microscopic level
At the light microscopic level, with or without colchicine treatment, the MCH- and ORX-like immunoreactive neurons were found to be mostly distributed in the LH. The ORX-like immunoreactive (ORX-LI) and the MCH-like immunoreactive (MCH-LI) neurons could be clearly identified on the basis of color using either the two-color ABC–DAB technique or the ABC–DAB–SGI technique. In the two-color ABC–DAB immunostained sections, ORX-LI neurons were dark blue in color while the MCH-LI neurons appeared brown. In the ABC–DAB–SGI immunostained sections, the ORX-LI neurons were black while the MCH-LI neurons were brown. No significant difference could be found with the two different techniques. Both the ORX- and MCH-LI neurons were medium-sized and multipolar to fusiform in shape, with some projections that often showed varicosity-like enlargement/distension (Figure 1A and Figure 2A). In the ABC–DAB–SGI immunostained sections, longer projections were found from the MCH-LI neurons (Figure 2B). In this situation, some of the ORX-LI perikarya were found to have thin axons which projected over a long distance to contact with MCH-LI neurons (Figure 2B). No such phenomenon could be clearly identified between MCH-LI axons and ORX-LI perikarya. No immunostaining was visible in the sections incubated without the primary antisera.
Ultrastructure of the melanin-concentrating hormone and orexin-containing neurons
At the electron microscopic level of the single immunostaining sections, both the ORX- (Figure 3) and MCH-LI (Figure 4) neurons were immunostained with most of the organelles, except for the nuclei, mitochondria and multiple vesicular bodies. Sometimes the Golgi apparatus were only faintly stained. Similar invaginated nuclei and large dense-cored vesicles were found in both the ORX- and MCH-LI neurons. However, one striking difference was that they were not so developed in the MCH-LI neurons, while the mitochondria and rough endoplasmic reticulum could easily be found in ORX-LI neurons (Figure 3A). The immunoreactive large dense-cored vesicles were from 70–120 nm in size in the ORX-LI neurons and 90–150 nm in size in the MCH-LI neurons.
The ORX-LI neurons often received synapses from immunonegative axon terminals (Figures 3A and B). While most of the ORX-LI perikarya received symmetric synapses, the synapses received by ORX-LI dendrites were mostly asymmetrical because of the thicker postsynaptic membranes and wider synaptic clefts. Conversely, the ORX-LI axon terminals often made synapses on immunonegative perikarya (Figure 3C) and dendrites (not shown). Occasionally, some synapses were found between ORX-LI axon terminals and ORX-LI dendrites (Figure 3D). Apart from the large number of small, spherical synaptic vesicles, ORX-LI axon terminals often contained one or two large dense-cored vesicles (Figure 3D). Most of the synapses made by ORX-LI axon terminals were asymmetrical (Figure 3C), while the symmetrical synapses made by ORX-LI axon terminals were also found on ORX-LI dendrites (Figure 3D).
The MCH-LI neurons also received many synapses from immunonegative axon terminals. Similar to the ORX-LI neurons, the MCH-LI perikarya often received symmetrical synapses (Figure 4A). However, the synapses received by MCH-LI dendrites were mostly asymmetrical (Figures 4C and D). The presynaptic axon terminals also sometimes contained dense-cored vesicles (Figure 4D), but this was not always the case (Figures 4C and D). Occasionally, MCH-LI dendrites received multiple synapses from plural axon terminals including both MCH-LI and immunonegative axon terminals which also sometimes contained dense-cored vesicles (Figure 4D). Although not so frequently, the MCH-LI axon terminals were also found making synapses (Figure 4B). Sometimes, such axon terminals contained both immunopositive and immunonegative large dense-cored vesicles (Figure 4B).
The synapses between melanin-concentrating hormone and orexin-containing neurons
In the sections double immunostained for electron microscopic study, the MCH- and ORX-LI neurons could also be easily found and identified (Figures 5 and 6). While the ORX-LI neurons could be distinguished by many silver–gold particles adhering to the DAB reaction product, the MCH-LI neurons had no such electron-dense coating on their DAB reaction product (Figures 5 and 6). The ultrastructure of both the MCH- and ORX-LI neurons appeared the same as that revealed with the single immunostaining technique.
A large number of ORX-LI dendrites were found in the present study, however, less than 20% of these dendrites received synapses from MCH-LI axon terminals by our estimation (Figure 5). The presynaptic MCH-LI axon terminals could contain dense-cored vesicles (Figure 5B) or not (Figure 5A). Sometimes, one ORX-LI dendrite was contacted by multiple axon terminals, including both MCH-LI and immunonegative axon terminals; however, only one of these could be identified as having a clear synaptic formation (Figure 5B).
Conversely, MCH-LI neurons received synapses from ORX-LI axon terminals (Figure 6A). These synapses were both axo-somatic and axo-dendritic. The axo-somatic synapses were usually symmetrical types as usual. In contrast to this, most of the ORX-LI axon terminals made asymmetrical synapses, although some axo-dendritic synapses between ORX-LI axon terminals and MCH-LI dendrites appeared symmetrical (Figures 6B, C and D). The MCH-LI dendrites were often observed to receive multiple synapses simultaneously from different types of axon terminals including the ORX-LI (Figures 6B, C and D), MCH-LI (Figures 6C and D) and immunonegative axon terminals (Figures 6B and D). The MCH-LI dendrite shown in Figure 6D received six synapses from three ORX-LI, one MCH-LI and two immunonegative axon terminals.
Orexin synapses in the lateral hypothalamus
The distribution of ORX-LI neurons in the brain has been reported in rat8,12,13 and cat.15 Our observations confirmed the results of these studies. Both the two-color ABC–DAB and the ABC–DAB–SGI techniques produced similar results. Although longer projections from the ORX-LI neurons were seen in the ABC–DAB–SGI reacted sections, no other significant difference could be identified. It seems that Triton X-100 treatment does not intensify the immunostaining as significantly as the SGI treatment.
Although the ultrastructure of ORX-LI axon terminals has been studied several times in the rat arcuate nucleus18,21 and locus coeruleus22 where the axon terminals have been demonstrated to synapse on neuropeptide Y,21 POMC18 and noradrenaline22 producing neurons, no work has dealt directly with ORX-LI neurons in the rat LH. For this reason, we examined the ORX-LI neuron in the LH at the electron microscopic level. The present study shows that the ORX-LI perikarya in the rat LH contains mitochondria, Golgi apparatus, lysosomes, rough endoplasmic reticulum and a moderate number of dense-cored vesicles, similar to those reported earlier in the cat.15 However, while the ORX-LI dendrites often receive multiple synapses simultaneously in the cat,15 we did not find this in the rat. Occasionally, an ORX-LI dendrite was contacted by two axon terminals but in each case only one of these axon terminals could be clearly identified as a presynaptic structure. Similar to the results reported in the cat,15 we also found synapses between ORX-LI axon terminals and ORX-LI dendrites. Although no dense-cored vesicles were reported in the ORX-LI axon terminals in previous studies,21,22 we did find that some of these axon terminals contained a few dense-cored vesicles. Our finding is in accordance with other reports.15,18,23 However, although the ORX-LI axon terminals have been reported as exclusively making asymmetrical synapses,21,22 we did find a low frequency of symmetrical synapses.
MCH synapses in the lateral hypothalamus
The distribution of MCH-LI neurons in the rat LH revealed by the present light microscopic study confirms previous results.11,24,25,26 Double staining of MCH-LI and ORX-LI has also been examined in the LH at the light microscopic level with fluorescence histochemistry,24in situ hybridization histochemistry25 and a combination of in situ hybridization and immunohistochemistry.25 These studies have indicated that, although the different neurotrasmitter-containing neurons having similar distributions, ORX does not coexist with MCH in the same neuron. In the present study, we used two different immunohistochemical methods and confirmed this finding.
The ultrastructure of MCH-LI neurons in the rat LH has been examined using antisera against rat11 or salmon14 MCH. However, these studies, particularly the former, did not emphasize on synaptic relationships. In the present study, we found that MCH-LI neurons in the LH have complex synaptic relationships. The MCH-LI dendrites often receive multiple synapses simultaneously. The complexity of these relationships indicates the diversity of their modulation functions in relation to other neurons. One striking finding was that we found some of the MCH-LI axon terminals contained both immunopositive and immunonegative dense-cored vesicles which means that some other neurotransmitter(s) could co-exist with the MCH in the same axon terminals.
Orexin–MCH synaptic interactions
At the light microscopic level using the ABC–DAB–SGI technique, we found many ORX-LI neuronal perikarya with projecting fiber and showed that these fibers sometimes directly contacted MCH-LI neurons. Although double staining of ORX- and MCH-containing neurons in the LH has been reported earlier,24,25 these studies could not reveal synaptic relationships. In the present study, at the electron microscopic level, at least some of the contacts revealed at the light microscopic level have been confirmed as synapses. The ORX-LI axon terminals not only make synapses on the MCH-LI perikarya, but also make synapses on the MCH-LI dendrites. One MCH-LI dendrite was found to receive multiple synapses from ORX-LI, MCH-LI and immunonegative axon terminals simultaneously, indicating that the MCH-LI neurons in the LH might have more complex relationships than ORX-LI neurons and diversity of their modulation functions in relation to other neurons.
In the present study, we could not clearly identify the contacts made by the axons projecting from MCH-LI neurons on ORX-LI neurons in the local LH at the light microscopic study. We presume this is due to the SGI treatment that inhibits the intense immunostaining of the second antigen.20 However, at the electron microscopic level, we did find that some ORX-LI neurons were synaptically innervated by MCH-LI axon terminals although such synapses were comparatively simpler than those between ORX-LI axon terminals and MCH-LI neurons although we did not find clear axo-somatic synapses between MCH-LI axon terminals and ORX-LI perikarya. This finding suggests that ORX-LI neurons could also influence MCH-neurons locally in the LH. The reciprocal synaptic innervation between the ORX- and MCH-containing neurons suggests that the two types of neurons can influence each other directly. Because many ORX receptors have been found in the LH,16 the ORXergic synaptic innervation of MCH-containing neurons may be through these ORX receptors. Conversely, a similar phenomenon could also be deduced for MCHergic synaptic innervation of ORX-containing neurons as many MCH receptors have been found in the LH.17
Orexin–MCH synaptic interactions in feeding regulation
To date, it has been demonstrated that ORX is involved in many physiological phenomena including food intake,27,28,29,30 sleep,31,32,33 endocrine function,34,35,36,37 narcolepsy38,39 and regulation of body temperature,40 muscle tone41 and even pain sensation.42 Likewise, MCH has been shown to play a role in food intake,5,6,7 endocrine function43,44,45,46 and pigmentation.47,48,49 From these reports, we know that both ORX and MCH play roles in the food intake and endocrine systems. Because the LH is known to act as a ‘feeding center’,1 we think it is most likely that the reciprocal synaptic relationships between the ORX- and MCH-LI neurons in the LH will be related to the regulation of food-intake, although the possibility of a role in the sleep–wake cycle still remains. As more synapses were made by ORX-LI axon terminals on MCH-LI neurons than vice versa, this may reflect the main synaptic direction with synapses from MCH-LI axon terminals to ORX-LI neurons representing a feedback system. Given that the synapses made by ORX-LI axon terminals have usually been identified as excitatory,23,50 similar to the synapses made by orexinergic axon terminals on neuropeptide Y21 and noradrenalin22 containing neurons, the synapses made by ORX-LI axon terminals on MCH-LI neurons found in the present study may also be excitatory. Unfortunately, as we do not know whether the synapses made by the MCH-LI axon terminals are excitatory or inhibitory, the physiological consequences of the feedback from MCH-LI axon terminals to ORX-LI neurons is still to be elucidated.
In the present study, we examined the detail structures of the ORX- and MCH-neurons in the LH with the use of double immunocytochemical methods. As a result, we found the reciprocal synaptic relationships between the ORX- and MCH-LI neurons in the LH. These results suggest that these neurons may interact with each other through the synapses. ORX and MCH may contribute to the feeding regulation in harmony with each other through neural interactions. Physiological evidence is needed to clarify the functional significance of these synapses.
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This study was supported in part by grants from the High-Technology Research Center project and the Ministry of Education, Science, Sports and Culture of Japan (to SS). We also express our thanks to Drs Vaughan and Vale (The Salk Institute, California).
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Guan, J., Uehara, K., Lu, S. et al. Reciprocal synaptic relationships between orexin- and melanin-concentrating hormone-containing neurons in the rat lateral hypothalamus: a novel circuit implicated in feeding regulation. Int J Obes 26, 1523–1532 (2002). https://doi.org/10.1038/sj.ijo.0802155
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