- Email: [email protected]
Mu opioid receptors are in somatodendritic and axonal compartments of GABAergic neurons in rat hippocampal formation
Brain Research 849 Ž1999. 203–215 www.elsevier.comrlocaterbres
Research report
Mu opioid receptors are in somatodendritic and axonal compartments of GABAergic neurons in rat hippocampal formation Carrie T. Drake ) , Teresa A. Milner Department of Neurology and Neuroscience, DiÕision of Neurobiology, Weill Medical College of Cornell UniÕersity, 411 East 69th Street, New York, NY 10021, USA Accepted 27 July 1999
Abstract Activation of mu opioid receptors ŽMORs. has a net excitatory effect in the hippocampal formation through inhibition of gamma-amino butyric acid ŽGABA.-containing interneurons. To determine the precise subcellular targets of MOR agonists, immunoreactivity against MOR1 and GABA was examined in single sections of the hippocampal formation prepared for dual-labeling electron microscopy. In both the CA1 region of hippocampus and the dentate gyrus, MOR-like immunoreactivity Ž-li. was present in neuronal somata, dendrites, axons, and axon terminals, as well as a very few glial processes. Axon terminals with MOR-li formed symmetric synapses with principal cell dendrites and somata. Many MOR-labeled profiles of all types also contained GABA-li, and the vast majority possessed the ultrastructural characteristics of interneurons. Additionally, in the dentate gyrus a very small proportion of granule cell dendrites contained MOR-li. MOR-li, identified using immunogold-silver particles, was often affiliated with the extrasynaptic regions of neuronal plasma membranes, consistent with responsiveness to diffusing endogenous neuropeptide ligands. Semiquantitative analysis of the distribution of MOR-li revealed significantly more ‘‘presynaptic’’ Žaxons and terminals. than ‘‘postsynaptic’’ Žsomata and dendrites. labeled profiles in most laminae. We conclude that in addition to previously described somatodendritic MOR-li, a substantial amount of MOR-li in hippocampal formation is presynaptic. Furthermore, MORs are almost exclusively in GABAergic interneurons. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Electron microscopy; Ultrastructure; Hippocampus; Dentate gyrus; Interneuron
1. Introduction In the rat hippocampal formation, mu opioid receptor ŽMOR. agonists have a net excitatory action which facilitates long-term potentiation ŽLTP. w7,8,31,68,69x and epileptogenesis w29,34,62x. Considerable evidence indicates that this excitation is produced through an inhibition of gamma-amino butyric acid ŽGABA.-mediated neurotransmission w35,44,45,48,53,69,70,72x, and recordings from local-circuit neurons Žinterneurons. have shown that MOR activation directly inhibits these GABAergic cells w36,59,60x. An additional, and opposing, action for MORs has been observed in the dentate gyrus, where a few of the glutamatergic dentate granule cells are hyperpolarized by selective MOR agonists w48x. In hippocampal formation, the inhibitory neurotransmitter GABA is present in interneurons Žapproximately ) Corresponding author. [email protected]
Fax:
q 1-212-988-3672;
e-mail:
10% of all hippocampal neurons. and afferents originating in the septum w24,25x. Recent light microscopic ŽLM. studies of MOR-like immunoreactivity Ž-li. in the rat brain have indicated that MOR-li is present in somatodendritic compartments of a subpopulation of neurons in the hippocampal formation w1,3,4,17,32,37x. In addition, some conventional and confocal LM evidence has suggested that MORs may be expressed in a subset of excitatory Žglutamatergic. principal neurons w1,17,32x. In the dentate gyrus, a few granule cell perikarya and dendrites appeared to contain MOR-li w1,32x and in hippocampus proper, an LM study has suggested that some pyramidal cells may contain MORs w17x. However, most MOR-labeled neurons possess the morphology and location of inhibitory interneurons, and overlapping immunoreactivity for MOR and GABA has been observed in some hippocampal neurons w2,32x. Within interneurons, physiological evidence suggests that MORs may have a widespread subcellular distribution. MOR agonists strongly hyperpolarize some GABAergic
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 9 1 0 - 1
204
C.T. Drake, T.A. Milnerr Brain Research 849 (1999) 203–215
neurons w36,59,60,67x, consistent with a somatodendritic location of MORs. MOR agonists also have been shown to inhibit spontaneous GABA release onto CA1 pyramidal cells w9,14,35x, indicating that MORs may be on axon terminals as well. Although previous confocal and conventional LM studies have attempted to determine whether MORs are present in axons and terminals in the hippocampal formation w1,4,32,37x, conflicting data have emerged. This question can be definitively answered using the high resolution provided by electron microscopy ŽEM., which allows both clarification of the identity of neurons based on their ultrastructural characteristics and detection of low levels of MORs not visible with LM. Thus, in the present study, we used EM to extend the characterization of neurons with MOR-li in the hippocampal formation. We determined Ž1. whether MOR-li is present in axon terminals as well as somatodendritic compartments, Ž2. whether profiles with MOR-li resemble interneurons and contain immunoreactivity for GABA, and Ž3. whether MOR-li is present in principal cells. These results have been previously presented in abstract form w18,19x.
2. Materials and methods 2.1. Antisera 2.1.1. Mu opioid receptor A polyclonal rabbit antiserum against MOR was purchased from Incstar ŽStillwater, MN.. This antiserum was raised against a 15 amino acid peptide sequence Žresidues 384–398. from the predicted amino terminus of MOR1, a cloned rat mu opioid receptor. Specificity of the MOR antiserum was demonstrated previously using epitope-expressing cell lines, Western blotting, and adsorption controls w1x. Controls in the present study included: Ž1. preadsorbing the MOR antiserum with the antigenic peptide Ž10 mgrml. to ascertain its specificity, and Ž2. omitting the MOR antiserum in some sections as a control for the specificity of secondary antibodies. The antiserum was diluted 1r5000–1r6500 for immunoperoxidase labeling or 1r1000–1r1500 for immunogold labeling.
2.1.2. GABA A rat polyclonal antiserum against GABA was used. Specificity of this antiserum has been previously demonstrated by dot blots and immunocytochemical adsorption controls w5x. The antiserum was diluted 1r4000 for immunoperoxidase labeling or 1r1000 for immunogold labeling. 2.2. Immunocytochemistry All procedures were approved by the Institutional Animal Care and Use Committee of Weill Medical College of Cornell University. Fifteen adult male Sprague–Dawley rats Ž250–350 g; Taconic, Germantown, NY. were deeply anesthetized with sodium pentobarbital Ž150 mgrkg, i.p.. and perfused sequentially with: Ž1. 10–20 ml normal saline containing 1000 unitsrml heparin, Ž2. 50–55 ml 3.75% acrolein and 2% paraformaldehyde in 0.1 M phosphate buffer ŽPB; pH 7.4., and Ž3. 200 ml 2% paraformaldehyde in PB. Before anesthetizing, rats used for duallabeling experiments were treated with DEDTC to diminish artifactual labeling of neuronal zinc stores w64x. The brains were removed, cut into 5–6 mm coronal blocks, and immersed in the last fixative for 30 min. Sections Ž40 mm. through the hippocampal formation were cut on a vibrating microtome ŽVibratome. and collected in PB. Free-floating sections were pretreated with 1% sodium borohydride to restore immunoreactivity, as described previously w22x. For dual-labeling, sections were processed with the combined immunoperoxidase–immunogold procedure described by Chan et al. w10x. Briefly, the sections were incubated in a cocktail of MOR and GABA antisera diluted in 0.1 M Tris–saline buffer ŽTS; pH 7.6. containing 0.1% bovine serum albumen ŽBSA. for 36–42 h at 48C. For LM, 0.25% Triton X-100 was included in the diluent. Tissue was incubated in biotinylated secondary antibody goat anti-rabbit IgG ŽVector, Burlingame, CA. for MOR or donkey anti-rat ŽJackson, West Grove, PA. for GABA4 diluted 1r400 in TS with 0.1% BSA, rinsed in TS, then incubated in avidin–biotin peroxidase complex ŽVectastain Elite kit; Vector. at twice the recommended dilution in TS. Immunoperoxidase labeling was visualized
Fig. 1. By light microscopy, specific MOR-labeling is present in the hippocampus ŽA–F. and the dentate gyrus ŽG–H.. ŽA. In the CA1 region of hippocampus, a pyramidal-shaped MOR-immunoperoxidase-labeled neuron Žopen arrow. in s. pyramidale extends a large apical dendrite into s. radiatum. Numerous labeled processes are also present in s. pyramidale. Two multipolar MOR-labeled neurons Žopen arrows. are present at the border of s. radiatum and s. lacunosum-moleculare. Fine processes Žarrow. are also present in s. lacunosum-moleculare. ŽB. In the CA1 region of a preadsorption control, no labeling is present. ŽC. A fusiform MOR-labeled neuron Žopen arrow. is present in the alveus of the CA1 region of hippocampus. ŽD. A pyramidal-shaped neuron in s. pyramidale of the CA1 region extends its apical dendrite into s. radiatum. ŽE. In the CA1 region of hippocampus, MOR-li is present in a neuron Žopen arrow. in s. radiatum and fine processes Žarrow. in s. lacunosum-moleculare. ŽF. The CA3 region of hippocampus contains many MOR-labeled processes Žarrow indicates example. in s. pyramidale, s. oriens, s. lucidum, and s. radiatum, and a pyramidal-shaped neuron with MOR-li Žopen arrow. in s. radiatum. ŽG. In the dentate gyrus, MOR-labeled neurons Žopen arrows. are present in s. moleculare, along the border of s. granulosum and the hilus, and within the hilus. All laminae also contain processes with MOR-li Žexample indicated by arrow.. ŽH. Neurons with MOR-li Žopen arrows. are present in the subgranular hilus and in s. granulosum. Fine processes are also present in both hilus Žarrow. and s. granulosum. h s Hilus, sg s s. granulosum, slm s s. lacunosum-moleculare, sl s s. lucidum, sm s s. moleculare, so s s. oriens, sp s s. pyramidale, and sr s s. radiatum. Scale bars s 50 mm.
C.T. Drake, T.A. Milnerr Brain Research 849 (1999) 203–215
205
206
C.T. Drake, T.A. Milnerr Brain Research 849 (1999) 203–215
C.T. Drake, T.A. Milnerr Brain Research 849 (1999) 203–215
207
Fig. 3. In the CA1 region of hippocampus, MOR-li is common in axon terminals, many of which form symmetric synapses and contain GABA-li. ŽA. A terminal in s. pyramidale with both MOR-immunogold Žarrows. and GABA-immunoperoxidase labeling contacts an unlabeled perikaryon Žu-p.. MOR-immunogold particles are distributed along the terminal’s plasma membrane. ŽB. In s. radiatum near s. pyramidale, an axon terminal contains GABA-immunogold particles Žarrows. over cytoplasmic organelles and diffuse MOR-immunoperoxidase labeling. The dual-labeled terminal forms a symmetric synapse Žwide arrow. with a large unlabeled dendritic shaft Žu-d.. Scale bar s 0.5 mm.
by incubating sections in PB containing 3,3X-diaminobenzidine ŽDAB, 0.22% . and hydrogen peroxide Ž0.00003%.. Sections were rinsed in 0.01 M phosphate buffered saline, ŽPBS, pH 7.4. followed by incubation buffer ŽPBS containing 0.1% gelatin and 0.8% BSA. for 30 min. Sections were incubated 2 h in secondary antibody conjugated to 1 nm gold particles goat anti-rabbit IgG ŽAmersham, Arlington Heights, IL. for MOR, goat anti-rat IgG ŽGoldmark Biologicals, Phillipsburg, NJ. for GABA4 , diluted 1r50 in the incubation buffer. The sections were rinsed in PBS, incubated 10 min in 2% glutaraldehyde, transferred to 0.2 M sodium citrate buffer ŽpH 7.4., and intensified in silver ŽAmersham IntenSE kit. for 6–8 min. In addition to the adsorption controls described above, specificity controls included omitting one of the primary antibodies and processing sections through both immunoperoxidase and immunogold procedures. Furthermore, the gold and peroxidase labels were reversed to confirm that the labeling pattern was not dependent on the visualization technique used. For LM, single- and dual-labeled sections were mounted onto gelatin-coated glass slides, air dried, dehydrated
through alcohols and xylenes, and coverslipped in DPX mounting medium ŽAldrich; Milwaukee, WI.. The hippocampal formation was examined and photographed on a Nikon Microphot microscope using brightfield and differential interference contrast ŽDIC. optics. For EM, labeled sections were fixed in 2% osmium tetroxide for 1 h and embedded in EMbed ŽEMS; Ft. Washington, PA. as previously described w22x. Immunolabeled regions of the hippocampus proper or dentate gyrus were glued onto blocks. Ultrathin Žapproximately 60 nm. sections were cut on an ultratome, collected on copper grids, and counterstained with uranyl acetate and lead citrate w49x. The sections were examined and photographed on Philips 201 and CM10 electron microscopes. To prepare figures, photographs were scanned, and final images were generated using PhotoShop ŽAdobe; Mountain View, CA. to adjust brightness and contrast and Xpress ŽQuark; Denver, CO. to assemble composite figures. 2.3. Data analysis For qualitative EM analysis, a total of 26 40-mm sections from six animals were thin sectioned and examined.
Fig. 2. In the CA1 region of hippocampus, MOR-li often colocalized with GABA-li in somata and dendrites. ŽA. An interneuron soma at the border of s. pyramidale and s. radiatum contains MOR-immunoperoxidase and GABA-immunogold labeling. MOR-li is present over the Golgi apparatus ŽG., along the plasma membrane Žopen arrows., and rimming cytosolic vesicular structures. The soma size and the large apical dendrite are consistent with a pyramidal morphology, but the neuron can be identified as an interneuron both by the infolded nucleus Žn. and the presence of GABA-li Žarrow indicates a GABA-immunogold particle.. ŽB. A dendrite in s. lacunosum-moleculare contains MOR-immunogold Žarrows. and light GABA-immunoperoxidase labeling. A nearby unlabeled dendrite Žu-d. is indicated for comparison. In the labeled dendrite, MOR-immunogold particles are at extrasynaptic sites along the plasma membrane. ŽC. A dendrite in s. radiatum contains diffuse MOR-immunoperoxidase and GABA-immunogold Žarrow. labeling. This labeled dendrite is aspiny, and receives numerous asymmetric synapses Žexamples indicated by curved arrows. from unlabeled terminals. A neighboring spiny dendrite Žu-d. is unlabeled. Scale bars s 1 mm ŽA, C., 0.5 mm ŽB..
C.T. Drake, T.A. Milnerr Brain Research 849 (1999) 203–215
208
Table 1 Distribution of MOR-labeling in the hippocampal formation MOR-labeled profiles were counted in 55 = 55 mm fields Žone from each of three animals. through hippocampal and dentate gyrus laminae. The mean percentage" S.D. of each profile type is indicated. Total number of profiles
% Terminals
% Axons
% Dendrites
% Somataa
CA1 of hippocampus S. oriens S. pyramidale S. radiatum S. lacunosum-moleculare
93 147 54 78
41 " 19 42 " 17 32 " 12 17 " 7
32 " 10 46 " 19 40 " 11 52 " 14
21 " 9 4"2 16 " 5 24 " 5
0 0 0 0
Dentate gyrus S. moleculare Žouter. S. granulosum Hilus
89 77 150
18 " 9 40 " 18 31 " 12
23 " 11 34 " 13 42 " 17
31 " 15 13 " 9 14 " 6
0 1"1 1"1
Region
% Glia
% Unknown
2"1 0 1"1 1"1
4"5 8"3 11 " 3 6"5
11 " 6 1"1 3"3
17 " 8 11 " 6 10 " 4
a
Although not represented in these randomly chosen fields, MOR-labeled somata were observed in all laminae of the hippocampal formation.
These included 11 blocks single-labeled for MOR Žeight CA1, three dentate gyrus., and 15 blocks Ž11 CA1, four dentate gyrus. dual-labeled for MOR and GABA. Quantification of the EM distribution of MOR-immunoperoxidase-labeled profiles was performed in the CA1 region of the hippocampus and the dentate gyrus. Three 40-mm thick tissue sections, each from a different animal, were examined from each region. From each 40-mm section, ultrathin sections were prepared for EM examination, and one 55 mm = 55 mm field was selected from each lamina of the CA1 region Žstratum Žs.. oriens, s. pyramidale, s. radiatum, and s. lacunosum-moleculare., or dentate gyrus Žs. moleculare, s. granulosum, and hilus.. Criteria for field selection included good morphological preservation, the presence of immunolabeling in the field, and proximity to the plastic–tissue interface Žto minimize undercounting caused by limited reagent penetration w47x.. All immunolabeled profiles within each selected field were photographed. Labeled profiles were counted and scored by type, and synapses were classified as symmetric or asymmetric w46x. Profiles that lacked identifying characteristics were classified as ‘‘unknown’’. All profiles were counted before sorting by laminar location and by animal. Because the total number of labeled profiles varied between animals, values were converted to percentage of total labeled profiles per field, so that relative distributions could be compared between animals. The mean percentages of each profile type were calculated for each CA1 region and dentate gyrus lamina, and differences between mean presy-
naptic and postsynaptic profiles were evaluated using a Mann–Whitney rank–sum test ŽStatview; Abacus Concepts, Berkeley, CA., and setting the significance level at - 0.050.
3. Results 3.1. Light microscopy and antisera specificity By LM, MOR-li was present in neuronal somata throughout the hippocampal formation. In the CA1 and CA3 regions of the hippocampus proper, MOR-labeled somata were most common in s. pyramidale and the adjacent regions of s. oriens and s. radiatum, although the other laminae also contained some labeled cells. Morphologically, most MOR-labeled neurons were fusiform, multipolar, or ovoid ŽFig. 1A, C–F.. However, many of the MOR-labeled neurons in s. pyramidale were morphologically similar to pyramidal cells ŽFig. 1A, D., as previously reported by Arvidsson et al. w1x, Ding et al. w17x and Mansour et al. w37x. Additionally, fine punctate processes with MOR-li were abundant in s. pyramidale and in s. lacunosum-moleculare ŽFig. 1A, D–F.. These fine MORlabeled processes were denser in region CA3 than CA1. In the dentate gyrus, MOR-labeled neuronal somata and dendrites were observed in all laminae. Neurons with MOR-li were most abundant in s. granulosum and the subgranular zone of the hilus ŽFig. 1G, H., where they
Fig. 4. In dentate gyrus, MOR-li is commonly detected in GABA-immunoreactive somata and dendrites. ŽA. A soma in the subgranular hilus contains both MOR-immunogold Žarrows indicate examples. and diffuse GABA-immunoperoxidase labeling. MOR-immunogold particles are located along the plasma membrane and along cytoplasmic organelles. The infolded nucleus Žn. is characteristic of GABAergic neurons. ŽB. An aspiny dendrite in the subgranular hilus contains MOR-immunoperoxidase and GABA-immunogold particles Žarrows indicate examples.. This dendrite receives several asymmetric synapses Žcurved arrows. from unlabeled terminals. ŽC. A dendrite in the subgranular hilus contains MOR-immunogold Žarrows indicate examples. and GABA-immunoperoxidase labeling. The MOR-immunogold particles are distributed along extrasynaptic regions of the plasma membrane, while the GABA-li is along cytoplasmic membranes Žcompare to unlabeled dendrites Žu-d... This labeled dendrite extends a fine spine-like process Žopen arrow.. Scale bars s 1 mm ŽA., 0.5 mm ŽB, C..
C.T. Drake, T.A. Milnerr Brain Research 849 (1999) 203–215
often had a pyramidal or fusiform morphology. The central portion of the hilus contained fewer labeled neurons, which
209
were commonly multipolar or fusiform in shape Žnot shown.. MOR-labeled neurons were infrequently observed
210
C.T. Drake, T.A. Milnerr Brain Research 849 (1999) 203–215
in s. moleculare ŽFig. 1G.. Fine processes were observed primarily in the hilus and to a lesser extent in s. granulosum ŽFig. 1G, H.. In all regions of the hippocampal formation, labeling was absent in tissue that was incubated in preadsorbed MOR antiserum ŽFig. 1B., indicating that MOR antibody was specifically recognizing the antigenic peptide sequence taken from the mu opioid receptor. The CA1 region of hippocampus and the dentate gyrus were examined further by EM. By EM, omission of MOR or GABA antisera abolished labeling for that antibody. Reversing the immunoperoxidase and immunogold markers did not alter the labeling pattern for MOR or GABA, except that fewer labeled profiles overall were observed with immunogold due to its lower sensitivity. 3.2. Region CA1 of hippocampus By EM, many MOR-labeled profiles contained GABA-li ŽFigs. 2 and 3.. Interestingly, GABA-li in MOR-labeled profiles was often less dense than GABA-li in nearby profiles lacking MOR-labeling. Although GABA-immunoreactivity is not detectable in all interneurons w40,42,55x, the location and morphology of all types of profiles with MOR-li consistently resembled interneurons. In particular, all MOR-labeled somata observed possessed an indented nucleus ŽFig. 2A., which is characteristic of hippocampal inhibitory interneurons w50x. Somatic MOR-li was most often detected along the Golgi apparatus and was occasionally observed along the plasma membrane, using both immunoperoxidase ŽFig. 2A. and immunogold labels Žnot shown.. Most MOR-labeled dendrites likewise possessed the ultrastructural features of interneurons Žfor review, see Ref. w24x., specifically many asymmetric synapses along their shafts and a lack of dendritic spines ŽFig. 2B, C.. Using immunogold to precisely localize MOR-labeled sites, MOR-labeling in dendrites was often observed at the extrasynaptic portion of the plasma membrane ŽFig. 2B.. MOR-li was never detected in pyramidal cell somata, although a tiny fraction of labeled profiles Žsee below. resembled the spiny dendrites of pyramidal cells.
Fig. 5. In the dentate gyrus, MOR-li is present in axons and axon terminals containing GABA-li. ŽA. A terminal in s. granulosum contains MOR-immunogold Žarrow. and GABA-immunoperoxidase labeling. This dual-labeled terminal contacts an unlabeled granule cell perikaryon Žu-p. but does not form a clear synapse in this plane of section. ŽB. A terminal in the hilus contains MOR-immunogold Žarrow indicates example. and GABA-immunoperoxidase, and forms a symmetric synapse Žwide arrow. with the shaft of a small unlabeled spiny dendrite Žu-d.. Two of the MOR-immunogold particles are immediately adjacent to the synapse. The spine of this dendritic target receives an asymmetric synapse Žcurved arrow. from an unlabeled terminal Žu-t.. ŽC. A myelinated axon in the hilus contains MOR-immunogold Žexample indicated with arrow. and GABA-immunoperoxidase labeling. These MOR-immunogold particles are within the cytoplasm rather than along the plasma membrane. Scale bars s 0.5 mm.
C.T. Drake, T.A. Milnerr Brain Research 849 (1999) 203–215
MOR-li also was commonly observed in axons and axon terminals ŽFig. 3.. In the CA1 region, MOR-labeled ‘‘presynaptic profiles’’ Žaxons and terminals. were significantly more abundant than labeled ‘‘postsynaptic profiles’’ Žsomata and dendrites. ŽMann–Whitney rank–sum test, p s 0.049.. Axons with MOR-li were small-diameter Žapproximately 1 mm. and unmyelinated. MOR-labeled terminals either formed symmetric synapses ŽFig. 3B. or did not form synapses in the plane of section examined ŽFig. 3A.. In tissue labeled using the immunogold technique, MOR-li was often along extrasynaptic plasma membranes of axon terminals ŽFig. 3A., as well as within the cytoplasm. In axons, MOR-labeling was along the plasma membrane and within the cytoplasm, although the precise location of immunogold particles was often hard to resolve due to the small diameter of labeled axons. Finally, glia very rarely contained MOR-li ŽTable 1.. The abundance and type of MOR-labeled profiles varied between laminae ŽTable 1.. The largest number of labeled profiles was seen in s. pyramidale, followed by s. oriens, s. lacunosum-moleculare, and s. radiatum, respectively ŽTable 1.. S. pyramidale contained MOR-labeling in somata with indented nuclei ŽFig. 2A., in large dendrites ŽFig. 2B., and frequently in axons and terminals ŽFig. 3A.. One labeled dendritic profile in this lamina resembled a spiny dendrite. Some MOR-labeled terminals formed symmetric synapses, none formed asymmetric synapses, and many formed no distinguishable synapses in the plane of section analyzed. Most synaptic targets of MOR-labeled terminals resembled pyramidal cells Ži.e., dendritic targets extended spines and somatic targets contained smooth, round nuclei.. Synapses from MOR-labeled terminals onto axon initial segments were not observed. In s. oriens, many dendritic MOR-labeled profiles were observed near the border with s. pyramidale, and both somatic and dendritic MOR-labeled profiles were found near the alveus Ždefined by the increased abundance of myelin.. Very rarely Ž2r93 profiles., MOR-li was in dendrites that extended spine-like appendages or were themselves spines. Synapses between MOR-labeled terminals and the axon initial segments of pyramidal cells were not observed. S. lacunosum-moleculare contained a relatively large proportion of MOR-labeled dendrites ŽFig. 2C, Table 1., in addition to frequent labeled axons and terminals ŽTable 1.. Many of the MOR-labeled dendrites were small-to-medium diameter Ž1.5–2.5 mm., contained GABA-li, and received asymmetric synapses from unlabeled axon terminals. One MOR-labeled profile resembled a dendritic spine similar to those of pyramidal neurons. S. radiatum contained MOR-labeled somata that were most commonly located very near the border with s. pyramidale ŽFig. 2A.. Similarly, MOR-labeled axons and axon terminals in s. radiatum were most frequent near the border with s. pyramidale ŽFig. 3B.. Some labeled terminals formed symmetric synapses with the shafts or spine necks of pyramidal cell dendrites. In deeper portions of s. radiatum, MOR-labeled somata and dendrites were en-
211
countered rarely. Most of these labeled dendrites were large, aspiny, oriented perpendicular to s. pyramidale, and tapered toward s. lacunosum-moleculare ŽFig. 2C.. Labeled dendrites usually contained GABA-li and were contacted by several terminals that formed asymmetric synapses. However, a few Žthree of 54 profiles in the quantitative analysis. MOR-labeled profiles were spiny dendrites. 3.3. Dentate gyrus By EM, MOR-labeled profiles included somata, dendrites, axons, axon terminals, and a few glia. As in the hippocampus proper, many but not all neuronal MORlabeled profiles also contained GABA-li ŽFigs. 4 and 5., and the vast majority had the ultrastructural characteristics of interneurons w24,50x. Specifically, somata with MOR-li exhibited infolded nuclei and abundant organelles ŽFig. 4A.. Most dendrites with MOR-li lacked spines and received numerous asymmetric synapses ŽFig. 4B., and MOR-labeled terminals formed exclusively symmetric synapses ŽFig. 5A, B.. Most axons with MOR-li were small and unmyelinated, although a few examples of larger labeled myelinated axons were observed ŽFig. 5C.. As in the CA1 region, statistical analysis revealed that MORlabeled ‘‘presynaptic’’ profiles were significantly more abundant than ‘‘postsynaptic’’ profiles ŽMann–Whitney rank–sum test, p s 0.049.. MOR-labeled profiles were most abundant in the hilus, followed by s. moleculare and s. granulosum, respectively. The hilus contained MOR-li in somata ŽFig. 4A., dendrites ŽFig. 4B, C., unmyelinated axons, axon terminals ŽFig. 5B., and a few glial profiles Žnot shown.. Rarely, MORlabeled myelinated axons were observed ŽFig. 5C., suggesting a possible extrinsic origin w46x. Synaptic contacts between MOR-labeled terminals and the granule cell axon initial segments extending into the hilus were not observed. A few MOR-labeled hilar dendrites had spine-like extensions ŽFig. 4C., as has been described for dendrites of certain GABAergic hilar interneurons as well as the glutamatergic mossy cells w24x. The presence of GABA-li in most MOR-labeled spiny dendrites ŽFig. 4C. suggests they belong to the former population. S. moleculare contained MOR-labeled axons, terminals, dendrites, glia, and a few interneuron-like somata. In the outer portion of s. moleculare, which is innervated by enkephalin-containing afferents from entorhinal cortex w27x, MOR-li was frequently observed in all profile types except somata ŽTable 1.. Most of the labeled dendrites were aspiny and received many asymmetric synapses, although several examples of MORlabeled spiny dendrites Žeight of 89 labeled profiles. were also observed. In s. granulosum, MOR-labeled profiles included somata, dendrites, unmyelinated axons, and terminals. Somata with MOR-li contained infolded nuclei and were often ) 10 nm in diameter; thus they did not resemble the somata of granule cells. Labeled axons and termi-
212
C.T. Drake, T.A. Milnerr Brain Research 849 (1999) 203–215
nals were frequently observed ŽTable 1. within s. granulosum or at the border with the hilus ŽFig. 5A.. MOR-labeled terminals often contacted granule cell somata and shafts of large dendrites, and sometimes formed symmetric synapses with these structures.
4. Discussion This study provides the first ultrastructural evidence that MOR-li is not only in somata and dendrites of GABAergic neurons, but is also in the terminals of these cells. Moreover, our results demonstrate that MORs are predominantly located on interneurons in both hippocampus proper and dentate gyrus. 4.1. Methodological considerations In this study, we used a well-characterized antiserum to GABA as a marker for inhibitory neurons. Although all interneurons are believed to use GABA as a neurotransmitter w30x, several studies have indicated that GABA-immunoreactivity is less abundant in specific subpopulations of interneurons w40,42,55x. We similarly observed that MOR-labeled profiles generally contained less GABA-li than many neighboring GABA-labeled profiles, suggesting that at least some MOR-containing neurons belong to a subpopulation of neurons with low levels of GABA. This observation also underscores the importance of using ultrastructural characteristics in addition to immunocytochemical markers to determine whether MOR-labeled profiles belong to interneurons or principal neurons. 4.2. MORs are primarily, but not exclusiÕely, on GABAergic interneurons The ultrastructural characteristics of MOR-labeled dendrites, somata, and terminals, and the presence of GABA-li in many MOR-containing profiles, suggest that the vast majority of MOR-labeled neuronal profiles in the hippocampal formation belong to GABAergic interneurons. In hippocampus proper, our data indicate that ambiguouslyshaped MOR-labeled somata belong to pyramidal-shaped interneurons, rather than to glutamatergic pyramidal cells. In contrast, in the dentate gyrus, MOR-li was observed in some granule cell dendrites, although MOR-li was never seen in granule cell somata. This is consistent with evidence suggesting that a small number of granule cells contain MOR mRNA w38x and are hyperpolarized by MOR-specific agonists w48x. Our data do not rule out the additional possibility that some of the GABA-lacking MOR-labeled axon terminals in the hilus may originate from extrahippocampal sources such as the noradrenergic neurons of the locus coeruleus or the cholinergic neurons of the medial septumrdiagonal band complex, both of which send sparse projections to the
hippocampal formation w13,26,41x. Consistent with this possibility, specific MOR agonists have been previously shown to decrease norepinephrine and acetylcholine release in the hippocampal formation w33,39,54,66x and MOR-li is present in the noradrenegic neurons of the locus coeruleus w43,63x. Further studies are required to determine directly whether subcortical afferents contain MORs. The presence of MOR-li in a few glial profiles Ž1% in CA1, 4% in dentate gyrus. suggests an additional, perhaps minor, site of action of MOR agonists. MOR-li has previously been observed in glial profiles in the nucleus accumbens w57x, locus coeruleus w43,63x, and MORs have been demonstrated in primary glial cultures w51,56x. The function of glial MORs in brain has not yet been established, although possibilities include modulation of glial-mediated Ca2q mobilization, cellular signaling, developmental influences, andror immune responses w28,51x. 4.3. MORs are present in both somatodendritic and axonal compartments of GABAergic neurons Our localization of MORs to GABAergic terminals provides the first anatomical support for the suggestion that MORs modulate hippocampal GABA release directly at the axon terminal w9,14,35x. Furthermore, our data suggest that MOR-labeled terminals originate from hippocampal interneurons, rather than from the GABAergic neurons in the septum. This conclusion is based on the observation that synaptic targets of MOR-labeled terminals included principal neurons but not GABAergic somata and dendrites. In contrast, septal GABAergic afferents synapse extensively on GABAergic interneurons w25,61x. The present colocalization of MOR-li and GABA-li in somatodendritic profiles confirms previous observations of overlap or colocalization of these molecules in several CNS regions w2,32,58x, and supports electrophysiological evidence that MOR agonists directly hyperpolarize or generate outward currents in some hippocampal nonpyramidal neurons w36,59,60,67x Our finding that MOR-li is in both somatodendritic and axonrterminal compartments is similar to the results of ultrastructural investigations in other brain regions. MOR-li has been observed in both dendrites and terminals in the nucleus accumbens w57,58x, caudate–putamen w65x, nucleus of the solitary tract w12x, and locus coeruleus w63x. As we observed in hippocampal formation, plasmalemmal MOR-li in other brain regions also was observed primarily at extrasynaptic sites w57,63,65x, suggesting a general situating of neuronal MORs to respond to diffusing extracellular ligands. 4.4. Functional implications MORs are positioned to modulate both somatodendritic excitability of neurons and evoked and spontaneous GABA release from axon terminals. This wide subcellular distri-
C.T. Drake, T.A. Milnerr Brain Research 849 (1999) 203–215
bution may facilitate maximal cellular sensitivity to diffusing endogenous MOR ligands. However, a clear understanding of how MOR-bearing neurons function in vivo will require determining which neurons are likely to be exposed to relevant concentrations of the endogenous ligands for these receptors. The enkephalins Žleu- and met-enkephalin. are present in several hippocampal formation locations, including scattered interneurons, granule cells, and lateral perforant path inputs to s. lacunosum-moleculare and outer s. moleculare w6,11,15,23,27x. Ultrastructural work in the rat has shown that profiles with enkephalin-li are often near GABA-containing profiles, suggesting potentially abundant paracrine interactions of these populations w16x. Moreover, in both the rat and the guinea pig, enkephalin-containing profiles have been shown to synapse directly onto GABAergic neurons w6,16x. In addition to enkephalins, the recently discovered endomorphin peptides have a very high affinity for MORs w71x. However, the role for these peptides in hippocampal formation appears to be small, since it has been reported that very few endomorphin-immunoreactive fibers are present there w52x. Many morphological, physiological, and neurochemical categories of interneurons have been described Žfor review, see Ref. w24x.. The present findings in hippocampus and dentate gyrus, along with several studies of MOR-responsive neurons in CA1 of hippocampus w36,59,60x, strongly indicate that MORs are present in a subset of hippocampal interneurons. The distribution and cytoarchitectural diversity of MOR-containing neurons, along with our preliminary evidence for a distinct neurochemical content w20,21x, suggests that MOR-labeled neurons may not fit into a single previously-described morphological and neurochemical category. Rather, several lines of research are more consistent with MOR-containing interneurons forming a functional category; namely, the selective inhibition of principal neurons. In the CA1 region of hippocampus, this concept is well supported by recent physiological data w9,35,59,60x, and we consistently observed labeled terminals synapsing with somata and spiny dendrites of pyramidal neurons but not interneurons. In the dentate gyrus, there is physiological evidence for MOR-mediated disinhibition of granule cells w44,48,70x, although the influence of somatodendritic vs. terminal actions has not been as extensively studied. Our demonstrations that MOR-li is in somata, dendrites, and terminals, and that MOR-labeled terminals form synaptic contacts with granule cell somata and dendrites, suggests that MOR-containing interneurons are also specialized to control principal neurons in the dentate gyrus.
Acknowledgements The authors would like to thank Ms. Sabrina Prince for technical assistance, and Ms. Joy Hornung for photographic assistance. This work was supported by DA08259
213
and MH48234 ŽT.A.M.., and an Aaron Diamond Postdoctoral Fellowship and DE12738 ŽC.T.D...
References w1x U. Arvidsson, M. Riedl, S. Chakrabarti, J.H. Lee, A.H. Nakano, R.J. Dado, H.H. Loh, P.Y. Law, M.W. Wessendorf, R. Elde, Distribution and targeting of a mu opioid receptor ŽMOR1. in brain and spinal cord, J. Neurosci. Ž1995.. w2x S.B. Bausch, C. Chavkin, Colocalization of mu and delta opioid receptors with GABA, parvalbumin, and a G-protein-coupled inwardly rectifying potassium channel ŽGIRK1. in the rodent brain, Analgesia 1 Ž1995. 282–285. w3x S.B. Bausch, C. Chavkin, Changes in hippocampal circuitry after pilocarpine-induced seizures as revealed by opioid receptor distribution and activation, J. Neurosci. 17 Ž1997. 477–492. w4x S.B. Bausch, T.A. Patterson, M.U. Ehrengruber, H.A. Lester, N. Davidson, C. Chavkin, Colocalization of mu opioid receptors with GIRK1 potassium channels in the rat brain: an immunocytochemical study, Receptors and Channels 3 Ž1995. 221–241. w5x V.E. Bayer, V.M. Pickel, GABA-labeled terminals form proportionally more synapses with dopaminergic neurons containing low densities of tyrosine hydroxylase-immunoreactivity in rat ventral tegmental area, Brain Res. 559 Ž1991. 44–55. w6 x J.M. Blasco-Ibanez, F.J. Martinez-Guijarro, T.F. Freund, Enkephalin-containing interneurons are specialized to innervate other interneurons in the hippocampal CA1 region of the rat and guinea pig, Eur. J. Neurosci. 10 Ž1998. 1784–1795. w7x C.R. Bramham, Opioid receptor dependent long-term potentiation: peptidergic regulation of synaptic plasticity in the hippocampus, Neurochem. Int. 20 Ž1992. 441–455. w8x C.R. Bramham, J.M. Sarvey, Endogenous activation of mu and delta-1 opioid receptors is required for long-term potentiation induction in the lateral perforant path: dependence on GABAergic inhibition, J. Neurosci. 16 Ž1996. 8123–8131. w9x M. Capogna, B.H. Gahwiler, S.M. Thompson, Mechanism of mu opioid receptor-mediated presynaptic inhibition in the rat hippocampus in vitro, J. Physiol. 470 Ž1993. 539–558. w10x J. Chan, C. Aoki, V.M. Pickel, Optimization of differential immunogold-silver and peroxidase labeling with maintenance of ultrastructure in brain sections before plastic embedding, Journal of Neuroscience Methods 33 Ž1990. 113–127. w11x C. Chavkin, W.J. Shoemaker, J.F. McGinty, A. Bayon, F.E. Bloom, Characterization of the prodynorphin and proenkephalin neuropeptide systems in rat hippocampus, J. Neurosci. 5 Ž1985. 808–816. w12x P.Y. Cheng, A. Moriwaki, J.B. Wang, G.R. Uhl, V.M. Pickel, Ultrastructural localization of mu opioid receptors in the superficial layers of the rat cervical spinal cord: extrasynaptic localization and proximity to leu5-enkephalin, Brain Res. 731 Ž1996. 141–154. w13x D.J. Clarke, Cholinergic innervation of the rat dentate gyrus: an immunocytochemical and electron microscopical study, Brain Res. 360 Ž1985. 349–354. w14x G.A. Cohen, V.A. Doze, D.V. Madison, Opioid inhibition of GABA release from presynaptic terminals of rat hippocampal interneurons, Neuron 9 Ž1992. 325–335. w15x K.G. Commons, T.A. Milner, Ultrastructural heterogeneity of enkephalin-containing terminals in the rat hippocampal formation, J. Comp. Neurol. 358 Ž1995. 324–342. w16x K.G. Commons, T.A. Milner, Ultrastructural relationships between leu-enkephalin- and GABA-containing neurons differ within the hippocampal formation, Brain Res. 724 Ž1996. 1–15. w17x Y.Q. Ding, T. Kaneko, S. Nomura, N. Mizuno, Immunohistochemical localization of mu opioid receptors in the central nervous system of the rat, J. Comp. Neurol. 367 Ž1996. 375–402. w18x C.T. Drake, T.A. Milner, Ultrastructural localization of mu opioid
214
w19x
w20x
w21x
w22x
w23x
w24x w25x
w26x
w27x
w28x
w29x
w30x
w31x
w32x
w33x
w34x
w35x
w36x w37x
w38x
C.T. Drake, T.A. Milnerr Brain Research 849 (1999) 203–215 receptor immunoreactivity to GABAergic neurons in rat dentate gyrus, Soc. Neurosci. Abstr. 22 Ž1996. 72. C.T. Drake, T.A. Milner, Ultrastructural localization of mu opioid receptor immunoreactivity to GABAergic neurons in rat hippocampus, Int. Narc. Res. Council Abstr., Long Beach, CA, 1996. C.T. Drake, T.A. Milner, Discrete subpopulations of nonprincipal neurons contain mu opioid receptor immunoreactivity in rat hippocampus, Soc. Neurosci. Abstr. 24 Ž1998. 853. C.T. Drake, T.A. Milner, Mu opioid receptors are present in distinct hippocampal interneuron subpopulations, Int. Narc. Res. Council Abstr., 1998. C.T. Drake, T.A. Patterson, M.L. Simmons, C. Chavkin, T.A. Milner, Kappa opioid receptor-like immunoreactivity in guinea pig brain: ultrastructural localization in presynaptic terminals in hippocampal formation, J. Comp. Neurol. 370 Ž1996. 377–395. K. Fredens, K. Stengaard-Pedersen, L.I. Larsson, Localization of enkephalin and cholecystokinin immunoreactivities in the perforant path terminal fields of the rat hippocampal formation, Brain Res. 304 Ž1984. 255–263. T. Freund, G. Buzsaki, Interneurons of the hippocampus, Hippocampus 6 Ž1996. 347–470. T.F. Freund, M. Antal, GABA-containing neurons in the septum control inhibitory interneurons in the hippocampus, Nature 336 Ž1988. 170–173. M. Frotscher, C. Leranth, The cholinergic innervation of the rat fascia dentata: identification of target structures on granule cells by combining choline acetyltransferase immunocytochemistry and Golgi impregnation, J. Comp. Neurol. 243 Ž1986. 58–70. C. Gall, N. Brecha, H.J. Karten, K.J. Chang, Localization of enkephalin-like immunoreactivity to identified axonal and neuronal populations of the rat hippocampus, J. Comp. Neurol. 198 Ž1981. 335–350. K.F. Hauser, A. Stiene-Martin, M.P. Mattson, R.P. Elde, S.E. Ryan, C.C. Godleske, Mu-opioid receptor-induced Ca2q mobilization and astroglial development: morphine inhibits DNA synthesis and stimulates cellular hypertrophy through a CaŽ 2q .-dependent mechanism, Brain Res. 720 Ž1996. 191–203. J.S. Hong, J.F. McGinty, P. Lee, C.W. Xie, C.L. Mitchell, Relationship between hippocampal opioid peptides and seizures, Prog. Neurobiol. 40 Ž1993. 507–528. C.R. Houser, M. Esclapez, Localization of mRNAs encoding two forms of glutamic acid decarboxylase in the rat hippocampal formation, Hippocampus 4 Ž1994. 530–545. W. Jin, C. Chavkin, Mu opioids enhance mossy fiber synaptic transmission indirectly by reducing GABA B receptor activation, Brain Res. 821 Ž1999. 286–293. A.E. Kalyuzhny, M.W. Wessendorf, CNS GABA neurons express the mu opioid receptor: immunocytochemical studies, NeuroReport 8 Ž1997. 3367–3372. P.A. Lapchak, D.M. Araujo, B. Collier, Regulation of endogenous acetylcholine release from mammalian brain slices by opiate receptors: hippocampus, striatum and cerebral cortex of guinea pig and rat, Neuroscience 31 Ž1989. 313–325. P.H. Lee, J. Obie, J.S. Hong, Opioids induce convulsions and wet dog shakes in rats: mediation by hippocampal mu, but not delta or kappa opioid receptors, J. Neurosci. 9 Ž1989. 692–697. C.R. Lupica, Delta and mu enkephalins inhibit spontaneous GABAmediated IPSCs via a cyclic AMP-independent mechanism in the rat hippocampus, J. Neurosci. 15 Ž1995. 737–749. D. Madison, R. Nicoll, Enkephalin hyperpolarizes interneurones in rat hippocampal formation, J. Physiol. 398 Ž1988. 123–130. A. Mansour, C.A. Fox, S. Burke, H. Akil, S.J. Watson, Immunohistochemical localization of the cloned mu opioid receptor in the rat CNS, J. Chem. Neuroanat. 8 Ž1995. 283–305. A. Mansour, C.A. Fox, R.C. Thompson, H. Akil, S.J. Watson, Mu opioid receptor mRNA expression in the rat CNS: comparison to mu-receptor binding, Brain Res. 643 Ž1994. 245–265.
w39x M. Matsumoto, M. Yoshioka, H. Togashi, M. Hirokami, M. Tochihara, T. Ikeda, C.B. Smith, H. Saito, Mu opioid receptors modulate noradrenaline release from the rat hippocampus as measured by brain microdialysis, Brain Res. 636 Ž1994. 1–8. w40x T.A. Milner, C.E. Bacon, GABAergic neurons in the rat hippocampal formation: ultrastructure and synaptic relationships with catecholaminergic terminals, J. Neurosci. 9 Ž1989. 3410–3427. w41x T.A. Milner, C.E. Bacon, Ultrastructural localization of tyrosine hydroxylase-like immunoreactivity in the rat hippocampal formation, J. Comp. Neurol. 281 Ž1989. 479–495. w42x T.A. Milner, R.G. Wiley, O.S. Kurucz, S.R. Prince, J.P. Pierce, Selective changes in hippocampal neuropeptide Y neurons following removal of the cholinergic septal inputs, J. Comp. Neurol. 386 Ž1997. 46–59. w43x E. Moyse, D. Marcel, K. Leonard, A. Beaudet, Electron microscopic distribution of mu opioid receptors on noradrenergic neurons of the locus coeruleus, Eur. J. Neurosci. 9 Ž1997. 128–139. w44x J.F. Neumaier, S. Mailheau, C. Chavkin, Opioid receptor-mediated responses in the dentate gyrus and CA1 region of the rat hippocampus, J. Pharmacol. Exp. Ther. 244 Ž1988. 564–570. w45x K. Pang, G.M. Rose, Differential effects of methionine 5-enkephalin on hippocampal pyramidal cells and interneurons, Neuropharmacology 28 Ž1989. 1175–1181. w46x A. Peters, S.L. Palay, H.D. Webster, The Fine Structure of the Nervous System, Oxford Univ. Press, New York, 1991. w47x V.M. Pickel, J. Chan, C. Aoki, Electron microscopic immunocytochemical labeling of endogenous andror transported antigens in rat brain using silver-intensified one-nanometre colloidal gold, in: A. Cuello ŽEd.., Immunohistochemistry II, Wiley, New York, NY, 1993, pp. 265–280. w48x P. Piguet, R.A. North, Opioid actions at mu and delta receptors in the rat dentate gyrus in vitro, J. Pharmacol. Exp. Ther. 266 Ž1993. 1139–1149. w49x E.S. Reynolds, The use of lead citrate at high pH as an electronopaque stain in electron microscopy, Journal of Cell Biology 17 Ž1963. 208–212. w50x C.E. Ribak, L. Seress, Five types of basket cell in the hippocampal dentate gyrus: a combined Golgi and electron microscopic study, J. Neurocytol. 12 Ž1983. 577–597. w51x B.B. Ruzicka, C.A. Fox, R.C. Thompson, F. Meng, S.J. Watson, H. Akil, Primary astroglial cultures derived from several rat brain regions differentially express mu, delta and kappa opioid receptor mRNA, Brain Res. Mol. Brain Res. 34 Ž1995. 209–220. w52x M. Schreff, S. Schulz, D. Wiborny, V. Hollt, Immunofluorescent identification of endomorphin-2-containing nerve fibers and terminals in the rat brain and spinal cord, NeuroReport 9 Ž1998. 1031– 1034. w53x G.R. Siggins, W. Zeiglgansberger, Morphine and opioid peptides reduce inhibitory synaptic potentials in hippcampal pyramidal cells in vitro without alteration of membrane potential, Proc. Natl. Acad. Sci. 78 Ž1981. 5235–5239. w54x M.L. Simmons, J.J. Wagner, R.M. Caudle, C. Chavkin, Endogenous opioid regulation of norepinephrine release in guinea pig hippocampus, Neurosci. Lett. Ž1992.. w55x R.S. Sloviter, G. Nilaver, Immunocytochemical localization of GABA-, cholecystokinin-, vasoactive intestinal polypeptide-, and somatostatin-like immunoreactivity in the area dentata and hippocampus of the rat, J. Comp. Neurol. 256 Ž1987. 42–60. w56x A. Stiene-Martin, R. Zhou, K.F. Hauser, Regional, developmental, and cell cycle-dependent differences in mu, delta, and kappa opioid receptor expression among cultured mouse astrocytes, Glia 22 Ž1998. 249–259. w57x A.L. Svingos, A. Moriwaki, J.B. Wang, G.R. Uhl, V.M. Pickel, Ultrastructural immunocytochemical localization of m opioid receptors in rat nucleus accumbens: extrasynaptic plasmalemmal distribution and association with leu5-enkephalin, J. Neurosci. 16 Ž1996. 4162–4173.
C.T. Drake, T.A. Milnerr Brain Research 849 (1999) 203–215 w58x A.L. Svingos, A. Moriwaki, J.B. Wang, G.R. Uhl, V.M. Pickel, m opioid receptors are localized to extrasynaptic plasma membranes of GABAergic neurons and their targets in the rat nucleus accumbens, J. Neurosci. 17 Ž1997. 2585–2594. w59x K.R. Svoboda, C.E. Adams, C.R. Lupica, Opioid receptor subtype expression defines morphologically distinct classes of hippocampal interneurons, J. Neurosci. 19 Ž1999. 85–95. w60x K.R. Svoboda, C.R. Lupica, Opioid inhibition of hippocampal interneurons via modulation of potassium and hyperpolarizationactivated cation ŽIh. currents, J. Neurosci. 18 Ž1998. 7084–7098. w61x K. Toth, T.F. Freund, R. Miles, Disinhibition of rat hippocampal pyramidal cells by GABAergic afferents from the septum, J. Physiol. ŽLondon. 500 Ž1997. 463–474. w62x G. Urca, H. Frenk, J.C. Liebeskind, A.N. Taylor, Morphine and enkephalin: analgesic and epileptic properties, Science 197 Ž1977. 83–86. w63x E.J. Van Bockstaele, E.E. Colago, P. Cheng, A. Moriwaki, G.R. Uhl, V.M. Pickel, Ultrastructural evidence for prominent distribution of the mu opioid receptor at extrasynaptic sites on noradrenergic dendrites in the rat nucleus locus coeruleus, J. Neurosci. 16 Ž1996. 5037–5048. w64x E. Veznedaroglu, T. Milner, Elimination of artifactual labeling of hippocampal mossy fibers seen following preembedding immunogold-silver technique by pretreatment with zinc chelator, J. Microsc. Res. 23 Ž1992. 100–101.
215
w65x H. Wang, A. Moriwaki, J.B. Wang, G.R. Uhl, V.M. Pickel, Ultrastructural immunocytochemical localization of mu opioid receptors and leu5-enkephalin in the patch compartment of the rat caudate– putamen nucleus, J. Comp. Neurol. 375 Ž1996. 659–674. w66x L.L. Werling, S.R. Brown, B.M. Cox, Opioid receptor regulation of the release of norepinephrine in brain, Neuropharmacology 26 Ž1987. 987–996. w67x T.L. Wimpey, C. Chavkin, Opioids activate both an inward rectifier and a novel voltage-gated potassium conductance in the hippocampal formation, Neuron 6 Ž1991. 281–289. w68x C.-W. Xie, C.V. Lewis, Opioid-mediated facilitation of long-term potentiation at the lateral perforant path-dentate granule cell synapse, J. Pharmacol. Exp. Ther. 256 Ž1991. 289–296. w69x C.W. Xie, D.V. Lewis, Endogenous opioids regulate long-term potentiation of synaptic inhibition in the dentate gyrus of rat hippocampus, J. Neurosci. 15 Ž1995. 3788–3795. w70x C.W. Xie, R.A. Morrisett, D.V. Lewis, Mu opioid receptor-mediated modulation of synaptic currents in dentate granule cells of rat hippocampus, J. Neurophysiol. 68 Ž1992. 1113–1120. w71x J.E. Zadina, L. Hackler, L.J. Ge, A.J. Kastin, A potent and selective endogenous agonist for the mu opiate receptor, Nature 386 Ž1997. 499–502. w72x W. Zieglgansberger, E.D. French, G.R. Siggins, F.E. Bloom, Opioid peptides may excite hippocampal pyramidal neurons by inhibiting adjacent inhibitory interneurons, Science 205 Ž1979. 415–417.
Research report
Mu opioid receptors are in somatodendritic and axonal compartments of GABAergic neurons in rat hippocampal formation Carrie T. Drake ) , Teresa A. Milner Department of Neurology and Neuroscience, DiÕision of Neurobiology, Weill Medical College of Cornell UniÕersity, 411 East 69th Street, New York, NY 10021, USA Accepted 27 July 1999
Abstract Activation of mu opioid receptors ŽMORs. has a net excitatory effect in the hippocampal formation through inhibition of gamma-amino butyric acid ŽGABA.-containing interneurons. To determine the precise subcellular targets of MOR agonists, immunoreactivity against MOR1 and GABA was examined in single sections of the hippocampal formation prepared for dual-labeling electron microscopy. In both the CA1 region of hippocampus and the dentate gyrus, MOR-like immunoreactivity Ž-li. was present in neuronal somata, dendrites, axons, and axon terminals, as well as a very few glial processes. Axon terminals with MOR-li formed symmetric synapses with principal cell dendrites and somata. Many MOR-labeled profiles of all types also contained GABA-li, and the vast majority possessed the ultrastructural characteristics of interneurons. Additionally, in the dentate gyrus a very small proportion of granule cell dendrites contained MOR-li. MOR-li, identified using immunogold-silver particles, was often affiliated with the extrasynaptic regions of neuronal plasma membranes, consistent with responsiveness to diffusing endogenous neuropeptide ligands. Semiquantitative analysis of the distribution of MOR-li revealed significantly more ‘‘presynaptic’’ Žaxons and terminals. than ‘‘postsynaptic’’ Žsomata and dendrites. labeled profiles in most laminae. We conclude that in addition to previously described somatodendritic MOR-li, a substantial amount of MOR-li in hippocampal formation is presynaptic. Furthermore, MORs are almost exclusively in GABAergic interneurons. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Electron microscopy; Ultrastructure; Hippocampus; Dentate gyrus; Interneuron
1. Introduction In the rat hippocampal formation, mu opioid receptor ŽMOR. agonists have a net excitatory action which facilitates long-term potentiation ŽLTP. w7,8,31,68,69x and epileptogenesis w29,34,62x. Considerable evidence indicates that this excitation is produced through an inhibition of gamma-amino butyric acid ŽGABA.-mediated neurotransmission w35,44,45,48,53,69,70,72x, and recordings from local-circuit neurons Žinterneurons. have shown that MOR activation directly inhibits these GABAergic cells w36,59,60x. An additional, and opposing, action for MORs has been observed in the dentate gyrus, where a few of the glutamatergic dentate granule cells are hyperpolarized by selective MOR agonists w48x. In hippocampal formation, the inhibitory neurotransmitter GABA is present in interneurons Žapproximately ) Corresponding author. [email protected]
Fax:
q 1-212-988-3672;
e-mail:
10% of all hippocampal neurons. and afferents originating in the septum w24,25x. Recent light microscopic ŽLM. studies of MOR-like immunoreactivity Ž-li. in the rat brain have indicated that MOR-li is present in somatodendritic compartments of a subpopulation of neurons in the hippocampal formation w1,3,4,17,32,37x. In addition, some conventional and confocal LM evidence has suggested that MORs may be expressed in a subset of excitatory Žglutamatergic. principal neurons w1,17,32x. In the dentate gyrus, a few granule cell perikarya and dendrites appeared to contain MOR-li w1,32x and in hippocampus proper, an LM study has suggested that some pyramidal cells may contain MORs w17x. However, most MOR-labeled neurons possess the morphology and location of inhibitory interneurons, and overlapping immunoreactivity for MOR and GABA has been observed in some hippocampal neurons w2,32x. Within interneurons, physiological evidence suggests that MORs may have a widespread subcellular distribution. MOR agonists strongly hyperpolarize some GABAergic
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 9 1 0 - 1
204
C.T. Drake, T.A. Milnerr Brain Research 849 (1999) 203–215
neurons w36,59,60,67x, consistent with a somatodendritic location of MORs. MOR agonists also have been shown to inhibit spontaneous GABA release onto CA1 pyramidal cells w9,14,35x, indicating that MORs may be on axon terminals as well. Although previous confocal and conventional LM studies have attempted to determine whether MORs are present in axons and terminals in the hippocampal formation w1,4,32,37x, conflicting data have emerged. This question can be definitively answered using the high resolution provided by electron microscopy ŽEM., which allows both clarification of the identity of neurons based on their ultrastructural characteristics and detection of low levels of MORs not visible with LM. Thus, in the present study, we used EM to extend the characterization of neurons with MOR-li in the hippocampal formation. We determined Ž1. whether MOR-li is present in axon terminals as well as somatodendritic compartments, Ž2. whether profiles with MOR-li resemble interneurons and contain immunoreactivity for GABA, and Ž3. whether MOR-li is present in principal cells. These results have been previously presented in abstract form w18,19x.
2. Materials and methods 2.1. Antisera 2.1.1. Mu opioid receptor A polyclonal rabbit antiserum against MOR was purchased from Incstar ŽStillwater, MN.. This antiserum was raised against a 15 amino acid peptide sequence Žresidues 384–398. from the predicted amino terminus of MOR1, a cloned rat mu opioid receptor. Specificity of the MOR antiserum was demonstrated previously using epitope-expressing cell lines, Western blotting, and adsorption controls w1x. Controls in the present study included: Ž1. preadsorbing the MOR antiserum with the antigenic peptide Ž10 mgrml. to ascertain its specificity, and Ž2. omitting the MOR antiserum in some sections as a control for the specificity of secondary antibodies. The antiserum was diluted 1r5000–1r6500 for immunoperoxidase labeling or 1r1000–1r1500 for immunogold labeling.
2.1.2. GABA A rat polyclonal antiserum against GABA was used. Specificity of this antiserum has been previously demonstrated by dot blots and immunocytochemical adsorption controls w5x. The antiserum was diluted 1r4000 for immunoperoxidase labeling or 1r1000 for immunogold labeling. 2.2. Immunocytochemistry All procedures were approved by the Institutional Animal Care and Use Committee of Weill Medical College of Cornell University. Fifteen adult male Sprague–Dawley rats Ž250–350 g; Taconic, Germantown, NY. were deeply anesthetized with sodium pentobarbital Ž150 mgrkg, i.p.. and perfused sequentially with: Ž1. 10–20 ml normal saline containing 1000 unitsrml heparin, Ž2. 50–55 ml 3.75% acrolein and 2% paraformaldehyde in 0.1 M phosphate buffer ŽPB; pH 7.4., and Ž3. 200 ml 2% paraformaldehyde in PB. Before anesthetizing, rats used for duallabeling experiments were treated with DEDTC to diminish artifactual labeling of neuronal zinc stores w64x. The brains were removed, cut into 5–6 mm coronal blocks, and immersed in the last fixative for 30 min. Sections Ž40 mm. through the hippocampal formation were cut on a vibrating microtome ŽVibratome. and collected in PB. Free-floating sections were pretreated with 1% sodium borohydride to restore immunoreactivity, as described previously w22x. For dual-labeling, sections were processed with the combined immunoperoxidase–immunogold procedure described by Chan et al. w10x. Briefly, the sections were incubated in a cocktail of MOR and GABA antisera diluted in 0.1 M Tris–saline buffer ŽTS; pH 7.6. containing 0.1% bovine serum albumen ŽBSA. for 36–42 h at 48C. For LM, 0.25% Triton X-100 was included in the diluent. Tissue was incubated in biotinylated secondary antibody goat anti-rabbit IgG ŽVector, Burlingame, CA. for MOR or donkey anti-rat ŽJackson, West Grove, PA. for GABA4 diluted 1r400 in TS with 0.1% BSA, rinsed in TS, then incubated in avidin–biotin peroxidase complex ŽVectastain Elite kit; Vector. at twice the recommended dilution in TS. Immunoperoxidase labeling was visualized
Fig. 1. By light microscopy, specific MOR-labeling is present in the hippocampus ŽA–F. and the dentate gyrus ŽG–H.. ŽA. In the CA1 region of hippocampus, a pyramidal-shaped MOR-immunoperoxidase-labeled neuron Žopen arrow. in s. pyramidale extends a large apical dendrite into s. radiatum. Numerous labeled processes are also present in s. pyramidale. Two multipolar MOR-labeled neurons Žopen arrows. are present at the border of s. radiatum and s. lacunosum-moleculare. Fine processes Žarrow. are also present in s. lacunosum-moleculare. ŽB. In the CA1 region of a preadsorption control, no labeling is present. ŽC. A fusiform MOR-labeled neuron Žopen arrow. is present in the alveus of the CA1 region of hippocampus. ŽD. A pyramidal-shaped neuron in s. pyramidale of the CA1 region extends its apical dendrite into s. radiatum. ŽE. In the CA1 region of hippocampus, MOR-li is present in a neuron Žopen arrow. in s. radiatum and fine processes Žarrow. in s. lacunosum-moleculare. ŽF. The CA3 region of hippocampus contains many MOR-labeled processes Žarrow indicates example. in s. pyramidale, s. oriens, s. lucidum, and s. radiatum, and a pyramidal-shaped neuron with MOR-li Žopen arrow. in s. radiatum. ŽG. In the dentate gyrus, MOR-labeled neurons Žopen arrows. are present in s. moleculare, along the border of s. granulosum and the hilus, and within the hilus. All laminae also contain processes with MOR-li Žexample indicated by arrow.. ŽH. Neurons with MOR-li Žopen arrows. are present in the subgranular hilus and in s. granulosum. Fine processes are also present in both hilus Žarrow. and s. granulosum. h s Hilus, sg s s. granulosum, slm s s. lacunosum-moleculare, sl s s. lucidum, sm s s. moleculare, so s s. oriens, sp s s. pyramidale, and sr s s. radiatum. Scale bars s 50 mm.
C.T. Drake, T.A. Milnerr Brain Research 849 (1999) 203–215
205
206
C.T. Drake, T.A. Milnerr Brain Research 849 (1999) 203–215
C.T. Drake, T.A. Milnerr Brain Research 849 (1999) 203–215
207
Fig. 3. In the CA1 region of hippocampus, MOR-li is common in axon terminals, many of which form symmetric synapses and contain GABA-li. ŽA. A terminal in s. pyramidale with both MOR-immunogold Žarrows. and GABA-immunoperoxidase labeling contacts an unlabeled perikaryon Žu-p.. MOR-immunogold particles are distributed along the terminal’s plasma membrane. ŽB. In s. radiatum near s. pyramidale, an axon terminal contains GABA-immunogold particles Žarrows. over cytoplasmic organelles and diffuse MOR-immunoperoxidase labeling. The dual-labeled terminal forms a symmetric synapse Žwide arrow. with a large unlabeled dendritic shaft Žu-d.. Scale bar s 0.5 mm.
by incubating sections in PB containing 3,3X-diaminobenzidine ŽDAB, 0.22% . and hydrogen peroxide Ž0.00003%.. Sections were rinsed in 0.01 M phosphate buffered saline, ŽPBS, pH 7.4. followed by incubation buffer ŽPBS containing 0.1% gelatin and 0.8% BSA. for 30 min. Sections were incubated 2 h in secondary antibody conjugated to 1 nm gold particles goat anti-rabbit IgG ŽAmersham, Arlington Heights, IL. for MOR, goat anti-rat IgG ŽGoldmark Biologicals, Phillipsburg, NJ. for GABA4 , diluted 1r50 in the incubation buffer. The sections were rinsed in PBS, incubated 10 min in 2% glutaraldehyde, transferred to 0.2 M sodium citrate buffer ŽpH 7.4., and intensified in silver ŽAmersham IntenSE kit. for 6–8 min. In addition to the adsorption controls described above, specificity controls included omitting one of the primary antibodies and processing sections through both immunoperoxidase and immunogold procedures. Furthermore, the gold and peroxidase labels were reversed to confirm that the labeling pattern was not dependent on the visualization technique used. For LM, single- and dual-labeled sections were mounted onto gelatin-coated glass slides, air dried, dehydrated
through alcohols and xylenes, and coverslipped in DPX mounting medium ŽAldrich; Milwaukee, WI.. The hippocampal formation was examined and photographed on a Nikon Microphot microscope using brightfield and differential interference contrast ŽDIC. optics. For EM, labeled sections were fixed in 2% osmium tetroxide for 1 h and embedded in EMbed ŽEMS; Ft. Washington, PA. as previously described w22x. Immunolabeled regions of the hippocampus proper or dentate gyrus were glued onto blocks. Ultrathin Žapproximately 60 nm. sections were cut on an ultratome, collected on copper grids, and counterstained with uranyl acetate and lead citrate w49x. The sections were examined and photographed on Philips 201 and CM10 electron microscopes. To prepare figures, photographs were scanned, and final images were generated using PhotoShop ŽAdobe; Mountain View, CA. to adjust brightness and contrast and Xpress ŽQuark; Denver, CO. to assemble composite figures. 2.3. Data analysis For qualitative EM analysis, a total of 26 40-mm sections from six animals were thin sectioned and examined.
Fig. 2. In the CA1 region of hippocampus, MOR-li often colocalized with GABA-li in somata and dendrites. ŽA. An interneuron soma at the border of s. pyramidale and s. radiatum contains MOR-immunoperoxidase and GABA-immunogold labeling. MOR-li is present over the Golgi apparatus ŽG., along the plasma membrane Žopen arrows., and rimming cytosolic vesicular structures. The soma size and the large apical dendrite are consistent with a pyramidal morphology, but the neuron can be identified as an interneuron both by the infolded nucleus Žn. and the presence of GABA-li Žarrow indicates a GABA-immunogold particle.. ŽB. A dendrite in s. lacunosum-moleculare contains MOR-immunogold Žarrows. and light GABA-immunoperoxidase labeling. A nearby unlabeled dendrite Žu-d. is indicated for comparison. In the labeled dendrite, MOR-immunogold particles are at extrasynaptic sites along the plasma membrane. ŽC. A dendrite in s. radiatum contains diffuse MOR-immunoperoxidase and GABA-immunogold Žarrow. labeling. This labeled dendrite is aspiny, and receives numerous asymmetric synapses Žexamples indicated by curved arrows. from unlabeled terminals. A neighboring spiny dendrite Žu-d. is unlabeled. Scale bars s 1 mm ŽA, C., 0.5 mm ŽB..
C.T. Drake, T.A. Milnerr Brain Research 849 (1999) 203–215
208
Table 1 Distribution of MOR-labeling in the hippocampal formation MOR-labeled profiles were counted in 55 = 55 mm fields Žone from each of three animals. through hippocampal and dentate gyrus laminae. The mean percentage" S.D. of each profile type is indicated. Total number of profiles
% Terminals
% Axons
% Dendrites
% Somataa
CA1 of hippocampus S. oriens S. pyramidale S. radiatum S. lacunosum-moleculare
93 147 54 78
41 " 19 42 " 17 32 " 12 17 " 7
32 " 10 46 " 19 40 " 11 52 " 14
21 " 9 4"2 16 " 5 24 " 5
0 0 0 0
Dentate gyrus S. moleculare Žouter. S. granulosum Hilus
89 77 150
18 " 9 40 " 18 31 " 12
23 " 11 34 " 13 42 " 17
31 " 15 13 " 9 14 " 6
0 1"1 1"1
Region
% Glia
% Unknown
2"1 0 1"1 1"1
4"5 8"3 11 " 3 6"5
11 " 6 1"1 3"3
17 " 8 11 " 6 10 " 4
a
Although not represented in these randomly chosen fields, MOR-labeled somata were observed in all laminae of the hippocampal formation.
These included 11 blocks single-labeled for MOR Žeight CA1, three dentate gyrus., and 15 blocks Ž11 CA1, four dentate gyrus. dual-labeled for MOR and GABA. Quantification of the EM distribution of MOR-immunoperoxidase-labeled profiles was performed in the CA1 region of the hippocampus and the dentate gyrus. Three 40-mm thick tissue sections, each from a different animal, were examined from each region. From each 40-mm section, ultrathin sections were prepared for EM examination, and one 55 mm = 55 mm field was selected from each lamina of the CA1 region Žstratum Žs.. oriens, s. pyramidale, s. radiatum, and s. lacunosum-moleculare., or dentate gyrus Žs. moleculare, s. granulosum, and hilus.. Criteria for field selection included good morphological preservation, the presence of immunolabeling in the field, and proximity to the plastic–tissue interface Žto minimize undercounting caused by limited reagent penetration w47x.. All immunolabeled profiles within each selected field were photographed. Labeled profiles were counted and scored by type, and synapses were classified as symmetric or asymmetric w46x. Profiles that lacked identifying characteristics were classified as ‘‘unknown’’. All profiles were counted before sorting by laminar location and by animal. Because the total number of labeled profiles varied between animals, values were converted to percentage of total labeled profiles per field, so that relative distributions could be compared between animals. The mean percentages of each profile type were calculated for each CA1 region and dentate gyrus lamina, and differences between mean presy-
naptic and postsynaptic profiles were evaluated using a Mann–Whitney rank–sum test ŽStatview; Abacus Concepts, Berkeley, CA., and setting the significance level at - 0.050.
3. Results 3.1. Light microscopy and antisera specificity By LM, MOR-li was present in neuronal somata throughout the hippocampal formation. In the CA1 and CA3 regions of the hippocampus proper, MOR-labeled somata were most common in s. pyramidale and the adjacent regions of s. oriens and s. radiatum, although the other laminae also contained some labeled cells. Morphologically, most MOR-labeled neurons were fusiform, multipolar, or ovoid ŽFig. 1A, C–F.. However, many of the MOR-labeled neurons in s. pyramidale were morphologically similar to pyramidal cells ŽFig. 1A, D., as previously reported by Arvidsson et al. w1x, Ding et al. w17x and Mansour et al. w37x. Additionally, fine punctate processes with MOR-li were abundant in s. pyramidale and in s. lacunosum-moleculare ŽFig. 1A, D–F.. These fine MORlabeled processes were denser in region CA3 than CA1. In the dentate gyrus, MOR-labeled neuronal somata and dendrites were observed in all laminae. Neurons with MOR-li were most abundant in s. granulosum and the subgranular zone of the hilus ŽFig. 1G, H., where they
Fig. 4. In dentate gyrus, MOR-li is commonly detected in GABA-immunoreactive somata and dendrites. ŽA. A soma in the subgranular hilus contains both MOR-immunogold Žarrows indicate examples. and diffuse GABA-immunoperoxidase labeling. MOR-immunogold particles are located along the plasma membrane and along cytoplasmic organelles. The infolded nucleus Žn. is characteristic of GABAergic neurons. ŽB. An aspiny dendrite in the subgranular hilus contains MOR-immunoperoxidase and GABA-immunogold particles Žarrows indicate examples.. This dendrite receives several asymmetric synapses Žcurved arrows. from unlabeled terminals. ŽC. A dendrite in the subgranular hilus contains MOR-immunogold Žarrows indicate examples. and GABA-immunoperoxidase labeling. The MOR-immunogold particles are distributed along extrasynaptic regions of the plasma membrane, while the GABA-li is along cytoplasmic membranes Žcompare to unlabeled dendrites Žu-d... This labeled dendrite extends a fine spine-like process Žopen arrow.. Scale bars s 1 mm ŽA., 0.5 mm ŽB, C..
C.T. Drake, T.A. Milnerr Brain Research 849 (1999) 203–215
often had a pyramidal or fusiform morphology. The central portion of the hilus contained fewer labeled neurons, which
209
were commonly multipolar or fusiform in shape Žnot shown.. MOR-labeled neurons were infrequently observed
210
C.T. Drake, T.A. Milnerr Brain Research 849 (1999) 203–215
in s. moleculare ŽFig. 1G.. Fine processes were observed primarily in the hilus and to a lesser extent in s. granulosum ŽFig. 1G, H.. In all regions of the hippocampal formation, labeling was absent in tissue that was incubated in preadsorbed MOR antiserum ŽFig. 1B., indicating that MOR antibody was specifically recognizing the antigenic peptide sequence taken from the mu opioid receptor. The CA1 region of hippocampus and the dentate gyrus were examined further by EM. By EM, omission of MOR or GABA antisera abolished labeling for that antibody. Reversing the immunoperoxidase and immunogold markers did not alter the labeling pattern for MOR or GABA, except that fewer labeled profiles overall were observed with immunogold due to its lower sensitivity. 3.2. Region CA1 of hippocampus By EM, many MOR-labeled profiles contained GABA-li ŽFigs. 2 and 3.. Interestingly, GABA-li in MOR-labeled profiles was often less dense than GABA-li in nearby profiles lacking MOR-labeling. Although GABA-immunoreactivity is not detectable in all interneurons w40,42,55x, the location and morphology of all types of profiles with MOR-li consistently resembled interneurons. In particular, all MOR-labeled somata observed possessed an indented nucleus ŽFig. 2A., which is characteristic of hippocampal inhibitory interneurons w50x. Somatic MOR-li was most often detected along the Golgi apparatus and was occasionally observed along the plasma membrane, using both immunoperoxidase ŽFig. 2A. and immunogold labels Žnot shown.. Most MOR-labeled dendrites likewise possessed the ultrastructural features of interneurons Žfor review, see Ref. w24x., specifically many asymmetric synapses along their shafts and a lack of dendritic spines ŽFig. 2B, C.. Using immunogold to precisely localize MOR-labeled sites, MOR-labeling in dendrites was often observed at the extrasynaptic portion of the plasma membrane ŽFig. 2B.. MOR-li was never detected in pyramidal cell somata, although a tiny fraction of labeled profiles Žsee below. resembled the spiny dendrites of pyramidal cells.
Fig. 5. In the dentate gyrus, MOR-li is present in axons and axon terminals containing GABA-li. ŽA. A terminal in s. granulosum contains MOR-immunogold Žarrow. and GABA-immunoperoxidase labeling. This dual-labeled terminal contacts an unlabeled granule cell perikaryon Žu-p. but does not form a clear synapse in this plane of section. ŽB. A terminal in the hilus contains MOR-immunogold Žarrow indicates example. and GABA-immunoperoxidase, and forms a symmetric synapse Žwide arrow. with the shaft of a small unlabeled spiny dendrite Žu-d.. Two of the MOR-immunogold particles are immediately adjacent to the synapse. The spine of this dendritic target receives an asymmetric synapse Žcurved arrow. from an unlabeled terminal Žu-t.. ŽC. A myelinated axon in the hilus contains MOR-immunogold Žexample indicated with arrow. and GABA-immunoperoxidase labeling. These MOR-immunogold particles are within the cytoplasm rather than along the plasma membrane. Scale bars s 0.5 mm.
C.T. Drake, T.A. Milnerr Brain Research 849 (1999) 203–215
MOR-li also was commonly observed in axons and axon terminals ŽFig. 3.. In the CA1 region, MOR-labeled ‘‘presynaptic profiles’’ Žaxons and terminals. were significantly more abundant than labeled ‘‘postsynaptic profiles’’ Žsomata and dendrites. ŽMann–Whitney rank–sum test, p s 0.049.. Axons with MOR-li were small-diameter Žapproximately 1 mm. and unmyelinated. MOR-labeled terminals either formed symmetric synapses ŽFig. 3B. or did not form synapses in the plane of section examined ŽFig. 3A.. In tissue labeled using the immunogold technique, MOR-li was often along extrasynaptic plasma membranes of axon terminals ŽFig. 3A., as well as within the cytoplasm. In axons, MOR-labeling was along the plasma membrane and within the cytoplasm, although the precise location of immunogold particles was often hard to resolve due to the small diameter of labeled axons. Finally, glia very rarely contained MOR-li ŽTable 1.. The abundance and type of MOR-labeled profiles varied between laminae ŽTable 1.. The largest number of labeled profiles was seen in s. pyramidale, followed by s. oriens, s. lacunosum-moleculare, and s. radiatum, respectively ŽTable 1.. S. pyramidale contained MOR-labeling in somata with indented nuclei ŽFig. 2A., in large dendrites ŽFig. 2B., and frequently in axons and terminals ŽFig. 3A.. One labeled dendritic profile in this lamina resembled a spiny dendrite. Some MOR-labeled terminals formed symmetric synapses, none formed asymmetric synapses, and many formed no distinguishable synapses in the plane of section analyzed. Most synaptic targets of MOR-labeled terminals resembled pyramidal cells Ži.e., dendritic targets extended spines and somatic targets contained smooth, round nuclei.. Synapses from MOR-labeled terminals onto axon initial segments were not observed. In s. oriens, many dendritic MOR-labeled profiles were observed near the border with s. pyramidale, and both somatic and dendritic MOR-labeled profiles were found near the alveus Ždefined by the increased abundance of myelin.. Very rarely Ž2r93 profiles., MOR-li was in dendrites that extended spine-like appendages or were themselves spines. Synapses between MOR-labeled terminals and the axon initial segments of pyramidal cells were not observed. S. lacunosum-moleculare contained a relatively large proportion of MOR-labeled dendrites ŽFig. 2C, Table 1., in addition to frequent labeled axons and terminals ŽTable 1.. Many of the MOR-labeled dendrites were small-to-medium diameter Ž1.5–2.5 mm., contained GABA-li, and received asymmetric synapses from unlabeled axon terminals. One MOR-labeled profile resembled a dendritic spine similar to those of pyramidal neurons. S. radiatum contained MOR-labeled somata that were most commonly located very near the border with s. pyramidale ŽFig. 2A.. Similarly, MOR-labeled axons and axon terminals in s. radiatum were most frequent near the border with s. pyramidale ŽFig. 3B.. Some labeled terminals formed symmetric synapses with the shafts or spine necks of pyramidal cell dendrites. In deeper portions of s. radiatum, MOR-labeled somata and dendrites were en-
211
countered rarely. Most of these labeled dendrites were large, aspiny, oriented perpendicular to s. pyramidale, and tapered toward s. lacunosum-moleculare ŽFig. 2C.. Labeled dendrites usually contained GABA-li and were contacted by several terminals that formed asymmetric synapses. However, a few Žthree of 54 profiles in the quantitative analysis. MOR-labeled profiles were spiny dendrites. 3.3. Dentate gyrus By EM, MOR-labeled profiles included somata, dendrites, axons, axon terminals, and a few glia. As in the hippocampus proper, many but not all neuronal MORlabeled profiles also contained GABA-li ŽFigs. 4 and 5., and the vast majority had the ultrastructural characteristics of interneurons w24,50x. Specifically, somata with MOR-li exhibited infolded nuclei and abundant organelles ŽFig. 4A.. Most dendrites with MOR-li lacked spines and received numerous asymmetric synapses ŽFig. 4B., and MOR-labeled terminals formed exclusively symmetric synapses ŽFig. 5A, B.. Most axons with MOR-li were small and unmyelinated, although a few examples of larger labeled myelinated axons were observed ŽFig. 5C.. As in the CA1 region, statistical analysis revealed that MORlabeled ‘‘presynaptic’’ profiles were significantly more abundant than ‘‘postsynaptic’’ profiles ŽMann–Whitney rank–sum test, p s 0.049.. MOR-labeled profiles were most abundant in the hilus, followed by s. moleculare and s. granulosum, respectively. The hilus contained MOR-li in somata ŽFig. 4A., dendrites ŽFig. 4B, C., unmyelinated axons, axon terminals ŽFig. 5B., and a few glial profiles Žnot shown.. Rarely, MORlabeled myelinated axons were observed ŽFig. 5C., suggesting a possible extrinsic origin w46x. Synaptic contacts between MOR-labeled terminals and the granule cell axon initial segments extending into the hilus were not observed. A few MOR-labeled hilar dendrites had spine-like extensions ŽFig. 4C., as has been described for dendrites of certain GABAergic hilar interneurons as well as the glutamatergic mossy cells w24x. The presence of GABA-li in most MOR-labeled spiny dendrites ŽFig. 4C. suggests they belong to the former population. S. moleculare contained MOR-labeled axons, terminals, dendrites, glia, and a few interneuron-like somata. In the outer portion of s. moleculare, which is innervated by enkephalin-containing afferents from entorhinal cortex w27x, MOR-li was frequently observed in all profile types except somata ŽTable 1.. Most of the labeled dendrites were aspiny and received many asymmetric synapses, although several examples of MORlabeled spiny dendrites Žeight of 89 labeled profiles. were also observed. In s. granulosum, MOR-labeled profiles included somata, dendrites, unmyelinated axons, and terminals. Somata with MOR-li contained infolded nuclei and were often ) 10 nm in diameter; thus they did not resemble the somata of granule cells. Labeled axons and termi-
212
C.T. Drake, T.A. Milnerr Brain Research 849 (1999) 203–215
nals were frequently observed ŽTable 1. within s. granulosum or at the border with the hilus ŽFig. 5A.. MOR-labeled terminals often contacted granule cell somata and shafts of large dendrites, and sometimes formed symmetric synapses with these structures.
4. Discussion This study provides the first ultrastructural evidence that MOR-li is not only in somata and dendrites of GABAergic neurons, but is also in the terminals of these cells. Moreover, our results demonstrate that MORs are predominantly located on interneurons in both hippocampus proper and dentate gyrus. 4.1. Methodological considerations In this study, we used a well-characterized antiserum to GABA as a marker for inhibitory neurons. Although all interneurons are believed to use GABA as a neurotransmitter w30x, several studies have indicated that GABA-immunoreactivity is less abundant in specific subpopulations of interneurons w40,42,55x. We similarly observed that MOR-labeled profiles generally contained less GABA-li than many neighboring GABA-labeled profiles, suggesting that at least some MOR-containing neurons belong to a subpopulation of neurons with low levels of GABA. This observation also underscores the importance of using ultrastructural characteristics in addition to immunocytochemical markers to determine whether MOR-labeled profiles belong to interneurons or principal neurons. 4.2. MORs are primarily, but not exclusiÕely, on GABAergic interneurons The ultrastructural characteristics of MOR-labeled dendrites, somata, and terminals, and the presence of GABA-li in many MOR-containing profiles, suggest that the vast majority of MOR-labeled neuronal profiles in the hippocampal formation belong to GABAergic interneurons. In hippocampus proper, our data indicate that ambiguouslyshaped MOR-labeled somata belong to pyramidal-shaped interneurons, rather than to glutamatergic pyramidal cells. In contrast, in the dentate gyrus, MOR-li was observed in some granule cell dendrites, although MOR-li was never seen in granule cell somata. This is consistent with evidence suggesting that a small number of granule cells contain MOR mRNA w38x and are hyperpolarized by MOR-specific agonists w48x. Our data do not rule out the additional possibility that some of the GABA-lacking MOR-labeled axon terminals in the hilus may originate from extrahippocampal sources such as the noradrenergic neurons of the locus coeruleus or the cholinergic neurons of the medial septumrdiagonal band complex, both of which send sparse projections to the
hippocampal formation w13,26,41x. Consistent with this possibility, specific MOR agonists have been previously shown to decrease norepinephrine and acetylcholine release in the hippocampal formation w33,39,54,66x and MOR-li is present in the noradrenegic neurons of the locus coeruleus w43,63x. Further studies are required to determine directly whether subcortical afferents contain MORs. The presence of MOR-li in a few glial profiles Ž1% in CA1, 4% in dentate gyrus. suggests an additional, perhaps minor, site of action of MOR agonists. MOR-li has previously been observed in glial profiles in the nucleus accumbens w57x, locus coeruleus w43,63x, and MORs have been demonstrated in primary glial cultures w51,56x. The function of glial MORs in brain has not yet been established, although possibilities include modulation of glial-mediated Ca2q mobilization, cellular signaling, developmental influences, andror immune responses w28,51x. 4.3. MORs are present in both somatodendritic and axonal compartments of GABAergic neurons Our localization of MORs to GABAergic terminals provides the first anatomical support for the suggestion that MORs modulate hippocampal GABA release directly at the axon terminal w9,14,35x. Furthermore, our data suggest that MOR-labeled terminals originate from hippocampal interneurons, rather than from the GABAergic neurons in the septum. This conclusion is based on the observation that synaptic targets of MOR-labeled terminals included principal neurons but not GABAergic somata and dendrites. In contrast, septal GABAergic afferents synapse extensively on GABAergic interneurons w25,61x. The present colocalization of MOR-li and GABA-li in somatodendritic profiles confirms previous observations of overlap or colocalization of these molecules in several CNS regions w2,32,58x, and supports electrophysiological evidence that MOR agonists directly hyperpolarize or generate outward currents in some hippocampal nonpyramidal neurons w36,59,60,67x Our finding that MOR-li is in both somatodendritic and axonrterminal compartments is similar to the results of ultrastructural investigations in other brain regions. MOR-li has been observed in both dendrites and terminals in the nucleus accumbens w57,58x, caudate–putamen w65x, nucleus of the solitary tract w12x, and locus coeruleus w63x. As we observed in hippocampal formation, plasmalemmal MOR-li in other brain regions also was observed primarily at extrasynaptic sites w57,63,65x, suggesting a general situating of neuronal MORs to respond to diffusing extracellular ligands. 4.4. Functional implications MORs are positioned to modulate both somatodendritic excitability of neurons and evoked and spontaneous GABA release from axon terminals. This wide subcellular distri-
C.T. Drake, T.A. Milnerr Brain Research 849 (1999) 203–215
bution may facilitate maximal cellular sensitivity to diffusing endogenous MOR ligands. However, a clear understanding of how MOR-bearing neurons function in vivo will require determining which neurons are likely to be exposed to relevant concentrations of the endogenous ligands for these receptors. The enkephalins Žleu- and met-enkephalin. are present in several hippocampal formation locations, including scattered interneurons, granule cells, and lateral perforant path inputs to s. lacunosum-moleculare and outer s. moleculare w6,11,15,23,27x. Ultrastructural work in the rat has shown that profiles with enkephalin-li are often near GABA-containing profiles, suggesting potentially abundant paracrine interactions of these populations w16x. Moreover, in both the rat and the guinea pig, enkephalin-containing profiles have been shown to synapse directly onto GABAergic neurons w6,16x. In addition to enkephalins, the recently discovered endomorphin peptides have a very high affinity for MORs w71x. However, the role for these peptides in hippocampal formation appears to be small, since it has been reported that very few endomorphin-immunoreactive fibers are present there w52x. Many morphological, physiological, and neurochemical categories of interneurons have been described Žfor review, see Ref. w24x.. The present findings in hippocampus and dentate gyrus, along with several studies of MOR-responsive neurons in CA1 of hippocampus w36,59,60x, strongly indicate that MORs are present in a subset of hippocampal interneurons. The distribution and cytoarchitectural diversity of MOR-containing neurons, along with our preliminary evidence for a distinct neurochemical content w20,21x, suggests that MOR-labeled neurons may not fit into a single previously-described morphological and neurochemical category. Rather, several lines of research are more consistent with MOR-containing interneurons forming a functional category; namely, the selective inhibition of principal neurons. In the CA1 region of hippocampus, this concept is well supported by recent physiological data w9,35,59,60x, and we consistently observed labeled terminals synapsing with somata and spiny dendrites of pyramidal neurons but not interneurons. In the dentate gyrus, there is physiological evidence for MOR-mediated disinhibition of granule cells w44,48,70x, although the influence of somatodendritic vs. terminal actions has not been as extensively studied. Our demonstrations that MOR-li is in somata, dendrites, and terminals, and that MOR-labeled terminals form synaptic contacts with granule cell somata and dendrites, suggests that MOR-containing interneurons are also specialized to control principal neurons in the dentate gyrus.
Acknowledgements The authors would like to thank Ms. Sabrina Prince for technical assistance, and Ms. Joy Hornung for photographic assistance. This work was supported by DA08259
213
and MH48234 ŽT.A.M.., and an Aaron Diamond Postdoctoral Fellowship and DE12738 ŽC.T.D...
References w1x U. Arvidsson, M. Riedl, S. Chakrabarti, J.H. Lee, A.H. Nakano, R.J. Dado, H.H. Loh, P.Y. Law, M.W. Wessendorf, R. Elde, Distribution and targeting of a mu opioid receptor ŽMOR1. in brain and spinal cord, J. Neurosci. Ž1995.. w2x S.B. Bausch, C. Chavkin, Colocalization of mu and delta opioid receptors with GABA, parvalbumin, and a G-protein-coupled inwardly rectifying potassium channel ŽGIRK1. in the rodent brain, Analgesia 1 Ž1995. 282–285. w3x S.B. Bausch, C. Chavkin, Changes in hippocampal circuitry after pilocarpine-induced seizures as revealed by opioid receptor distribution and activation, J. Neurosci. 17 Ž1997. 477–492. w4x S.B. Bausch, T.A. Patterson, M.U. Ehrengruber, H.A. Lester, N. Davidson, C. Chavkin, Colocalization of mu opioid receptors with GIRK1 potassium channels in the rat brain: an immunocytochemical study, Receptors and Channels 3 Ž1995. 221–241. w5x V.E. Bayer, V.M. Pickel, GABA-labeled terminals form proportionally more synapses with dopaminergic neurons containing low densities of tyrosine hydroxylase-immunoreactivity in rat ventral tegmental area, Brain Res. 559 Ž1991. 44–55. w6 x J.M. Blasco-Ibanez, F.J. Martinez-Guijarro, T.F. Freund, Enkephalin-containing interneurons are specialized to innervate other interneurons in the hippocampal CA1 region of the rat and guinea pig, Eur. J. Neurosci. 10 Ž1998. 1784–1795. w7x C.R. Bramham, Opioid receptor dependent long-term potentiation: peptidergic regulation of synaptic plasticity in the hippocampus, Neurochem. Int. 20 Ž1992. 441–455. w8x C.R. Bramham, J.M. Sarvey, Endogenous activation of mu and delta-1 opioid receptors is required for long-term potentiation induction in the lateral perforant path: dependence on GABAergic inhibition, J. Neurosci. 16 Ž1996. 8123–8131. w9x M. Capogna, B.H. Gahwiler, S.M. Thompson, Mechanism of mu opioid receptor-mediated presynaptic inhibition in the rat hippocampus in vitro, J. Physiol. 470 Ž1993. 539–558. w10x J. Chan, C. Aoki, V.M. Pickel, Optimization of differential immunogold-silver and peroxidase labeling with maintenance of ultrastructure in brain sections before plastic embedding, Journal of Neuroscience Methods 33 Ž1990. 113–127. w11x C. Chavkin, W.J. Shoemaker, J.F. McGinty, A. Bayon, F.E. Bloom, Characterization of the prodynorphin and proenkephalin neuropeptide systems in rat hippocampus, J. Neurosci. 5 Ž1985. 808–816. w12x P.Y. Cheng, A. Moriwaki, J.B. Wang, G.R. Uhl, V.M. Pickel, Ultrastructural localization of mu opioid receptors in the superficial layers of the rat cervical spinal cord: extrasynaptic localization and proximity to leu5-enkephalin, Brain Res. 731 Ž1996. 141–154. w13x D.J. Clarke, Cholinergic innervation of the rat dentate gyrus: an immunocytochemical and electron microscopical study, Brain Res. 360 Ž1985. 349–354. w14x G.A. Cohen, V.A. Doze, D.V. Madison, Opioid inhibition of GABA release from presynaptic terminals of rat hippocampal interneurons, Neuron 9 Ž1992. 325–335. w15x K.G. Commons, T.A. Milner, Ultrastructural heterogeneity of enkephalin-containing terminals in the rat hippocampal formation, J. Comp. Neurol. 358 Ž1995. 324–342. w16x K.G. Commons, T.A. Milner, Ultrastructural relationships between leu-enkephalin- and GABA-containing neurons differ within the hippocampal formation, Brain Res. 724 Ž1996. 1–15. w17x Y.Q. Ding, T. Kaneko, S. Nomura, N. Mizuno, Immunohistochemical localization of mu opioid receptors in the central nervous system of the rat, J. Comp. Neurol. 367 Ž1996. 375–402. w18x C.T. Drake, T.A. Milner, Ultrastructural localization of mu opioid
214
w19x
w20x
w21x
w22x
w23x
w24x w25x
w26x
w27x
w28x
w29x
w30x
w31x
w32x
w33x
w34x
w35x
w36x w37x
w38x
C.T. Drake, T.A. Milnerr Brain Research 849 (1999) 203–215 receptor immunoreactivity to GABAergic neurons in rat dentate gyrus, Soc. Neurosci. Abstr. 22 Ž1996. 72. C.T. Drake, T.A. Milner, Ultrastructural localization of mu opioid receptor immunoreactivity to GABAergic neurons in rat hippocampus, Int. Narc. Res. Council Abstr., Long Beach, CA, 1996. C.T. Drake, T.A. Milner, Discrete subpopulations of nonprincipal neurons contain mu opioid receptor immunoreactivity in rat hippocampus, Soc. Neurosci. Abstr. 24 Ž1998. 853. C.T. Drake, T.A. Milner, Mu opioid receptors are present in distinct hippocampal interneuron subpopulations, Int. Narc. Res. Council Abstr., 1998. C.T. Drake, T.A. Patterson, M.L. Simmons, C. Chavkin, T.A. Milner, Kappa opioid receptor-like immunoreactivity in guinea pig brain: ultrastructural localization in presynaptic terminals in hippocampal formation, J. Comp. Neurol. 370 Ž1996. 377–395. K. Fredens, K. Stengaard-Pedersen, L.I. Larsson, Localization of enkephalin and cholecystokinin immunoreactivities in the perforant path terminal fields of the rat hippocampal formation, Brain Res. 304 Ž1984. 255–263. T. Freund, G. Buzsaki, Interneurons of the hippocampus, Hippocampus 6 Ž1996. 347–470. T.F. Freund, M. Antal, GABA-containing neurons in the septum control inhibitory interneurons in the hippocampus, Nature 336 Ž1988. 170–173. M. Frotscher, C. Leranth, The cholinergic innervation of the rat fascia dentata: identification of target structures on granule cells by combining choline acetyltransferase immunocytochemistry and Golgi impregnation, J. Comp. Neurol. 243 Ž1986. 58–70. C. Gall, N. Brecha, H.J. Karten, K.J. Chang, Localization of enkephalin-like immunoreactivity to identified axonal and neuronal populations of the rat hippocampus, J. Comp. Neurol. 198 Ž1981. 335–350. K.F. Hauser, A. Stiene-Martin, M.P. Mattson, R.P. Elde, S.E. Ryan, C.C. Godleske, Mu-opioid receptor-induced Ca2q mobilization and astroglial development: morphine inhibits DNA synthesis and stimulates cellular hypertrophy through a CaŽ 2q .-dependent mechanism, Brain Res. 720 Ž1996. 191–203. J.S. Hong, J.F. McGinty, P. Lee, C.W. Xie, C.L. Mitchell, Relationship between hippocampal opioid peptides and seizures, Prog. Neurobiol. 40 Ž1993. 507–528. C.R. Houser, M. Esclapez, Localization of mRNAs encoding two forms of glutamic acid decarboxylase in the rat hippocampal formation, Hippocampus 4 Ž1994. 530–545. W. Jin, C. Chavkin, Mu opioids enhance mossy fiber synaptic transmission indirectly by reducing GABA B receptor activation, Brain Res. 821 Ž1999. 286–293. A.E. Kalyuzhny, M.W. Wessendorf, CNS GABA neurons express the mu opioid receptor: immunocytochemical studies, NeuroReport 8 Ž1997. 3367–3372. P.A. Lapchak, D.M. Araujo, B. Collier, Regulation of endogenous acetylcholine release from mammalian brain slices by opiate receptors: hippocampus, striatum and cerebral cortex of guinea pig and rat, Neuroscience 31 Ž1989. 313–325. P.H. Lee, J. Obie, J.S. Hong, Opioids induce convulsions and wet dog shakes in rats: mediation by hippocampal mu, but not delta or kappa opioid receptors, J. Neurosci. 9 Ž1989. 692–697. C.R. Lupica, Delta and mu enkephalins inhibit spontaneous GABAmediated IPSCs via a cyclic AMP-independent mechanism in the rat hippocampus, J. Neurosci. 15 Ž1995. 737–749. D. Madison, R. Nicoll, Enkephalin hyperpolarizes interneurones in rat hippocampal formation, J. Physiol. 398 Ž1988. 123–130. A. Mansour, C.A. Fox, S. Burke, H. Akil, S.J. Watson, Immunohistochemical localization of the cloned mu opioid receptor in the rat CNS, J. Chem. Neuroanat. 8 Ž1995. 283–305. A. Mansour, C.A. Fox, R.C. Thompson, H. Akil, S.J. Watson, Mu opioid receptor mRNA expression in the rat CNS: comparison to mu-receptor binding, Brain Res. 643 Ž1994. 245–265.
w39x M. Matsumoto, M. Yoshioka, H. Togashi, M. Hirokami, M. Tochihara, T. Ikeda, C.B. Smith, H. Saito, Mu opioid receptors modulate noradrenaline release from the rat hippocampus as measured by brain microdialysis, Brain Res. 636 Ž1994. 1–8. w40x T.A. Milner, C.E. Bacon, GABAergic neurons in the rat hippocampal formation: ultrastructure and synaptic relationships with catecholaminergic terminals, J. Neurosci. 9 Ž1989. 3410–3427. w41x T.A. Milner, C.E. Bacon, Ultrastructural localization of tyrosine hydroxylase-like immunoreactivity in the rat hippocampal formation, J. Comp. Neurol. 281 Ž1989. 479–495. w42x T.A. Milner, R.G. Wiley, O.S. Kurucz, S.R. Prince, J.P. Pierce, Selective changes in hippocampal neuropeptide Y neurons following removal of the cholinergic septal inputs, J. Comp. Neurol. 386 Ž1997. 46–59. w43x E. Moyse, D. Marcel, K. Leonard, A. Beaudet, Electron microscopic distribution of mu opioid receptors on noradrenergic neurons of the locus coeruleus, Eur. J. Neurosci. 9 Ž1997. 128–139. w44x J.F. Neumaier, S. Mailheau, C. Chavkin, Opioid receptor-mediated responses in the dentate gyrus and CA1 region of the rat hippocampus, J. Pharmacol. Exp. Ther. 244 Ž1988. 564–570. w45x K. Pang, G.M. Rose, Differential effects of methionine 5-enkephalin on hippocampal pyramidal cells and interneurons, Neuropharmacology 28 Ž1989. 1175–1181. w46x A. Peters, S.L. Palay, H.D. Webster, The Fine Structure of the Nervous System, Oxford Univ. Press, New York, 1991. w47x V.M. Pickel, J. Chan, C. Aoki, Electron microscopic immunocytochemical labeling of endogenous andror transported antigens in rat brain using silver-intensified one-nanometre colloidal gold, in: A. Cuello ŽEd.., Immunohistochemistry II, Wiley, New York, NY, 1993, pp. 265–280. w48x P. Piguet, R.A. North, Opioid actions at mu and delta receptors in the rat dentate gyrus in vitro, J. Pharmacol. Exp. Ther. 266 Ž1993. 1139–1149. w49x E.S. Reynolds, The use of lead citrate at high pH as an electronopaque stain in electron microscopy, Journal of Cell Biology 17 Ž1963. 208–212. w50x C.E. Ribak, L. Seress, Five types of basket cell in the hippocampal dentate gyrus: a combined Golgi and electron microscopic study, J. Neurocytol. 12 Ž1983. 577–597. w51x B.B. Ruzicka, C.A. Fox, R.C. Thompson, F. Meng, S.J. Watson, H. Akil, Primary astroglial cultures derived from several rat brain regions differentially express mu, delta and kappa opioid receptor mRNA, Brain Res. Mol. Brain Res. 34 Ž1995. 209–220. w52x M. Schreff, S. Schulz, D. Wiborny, V. Hollt, Immunofluorescent identification of endomorphin-2-containing nerve fibers and terminals in the rat brain and spinal cord, NeuroReport 9 Ž1998. 1031– 1034. w53x G.R. Siggins, W. Zeiglgansberger, Morphine and opioid peptides reduce inhibitory synaptic potentials in hippcampal pyramidal cells in vitro without alteration of membrane potential, Proc. Natl. Acad. Sci. 78 Ž1981. 5235–5239. w54x M.L. Simmons, J.J. Wagner, R.M. Caudle, C. Chavkin, Endogenous opioid regulation of norepinephrine release in guinea pig hippocampus, Neurosci. Lett. Ž1992.. w55x R.S. Sloviter, G. Nilaver, Immunocytochemical localization of GABA-, cholecystokinin-, vasoactive intestinal polypeptide-, and somatostatin-like immunoreactivity in the area dentata and hippocampus of the rat, J. Comp. Neurol. 256 Ž1987. 42–60. w56x A. Stiene-Martin, R. Zhou, K.F. Hauser, Regional, developmental, and cell cycle-dependent differences in mu, delta, and kappa opioid receptor expression among cultured mouse astrocytes, Glia 22 Ž1998. 249–259. w57x A.L. Svingos, A. Moriwaki, J.B. Wang, G.R. Uhl, V.M. Pickel, Ultrastructural immunocytochemical localization of m opioid receptors in rat nucleus accumbens: extrasynaptic plasmalemmal distribution and association with leu5-enkephalin, J. Neurosci. 16 Ž1996. 4162–4173.
C.T. Drake, T.A. Milnerr Brain Research 849 (1999) 203–215 w58x A.L. Svingos, A. Moriwaki, J.B. Wang, G.R. Uhl, V.M. Pickel, m opioid receptors are localized to extrasynaptic plasma membranes of GABAergic neurons and their targets in the rat nucleus accumbens, J. Neurosci. 17 Ž1997. 2585–2594. w59x K.R. Svoboda, C.E. Adams, C.R. Lupica, Opioid receptor subtype expression defines morphologically distinct classes of hippocampal interneurons, J. Neurosci. 19 Ž1999. 85–95. w60x K.R. Svoboda, C.R. Lupica, Opioid inhibition of hippocampal interneurons via modulation of potassium and hyperpolarizationactivated cation ŽIh. currents, J. Neurosci. 18 Ž1998. 7084–7098. w61x K. Toth, T.F. Freund, R. Miles, Disinhibition of rat hippocampal pyramidal cells by GABAergic afferents from the septum, J. Physiol. ŽLondon. 500 Ž1997. 463–474. w62x G. Urca, H. Frenk, J.C. Liebeskind, A.N. Taylor, Morphine and enkephalin: analgesic and epileptic properties, Science 197 Ž1977. 83–86. w63x E.J. Van Bockstaele, E.E. Colago, P. Cheng, A. Moriwaki, G.R. Uhl, V.M. Pickel, Ultrastructural evidence for prominent distribution of the mu opioid receptor at extrasynaptic sites on noradrenergic dendrites in the rat nucleus locus coeruleus, J. Neurosci. 16 Ž1996. 5037–5048. w64x E. Veznedaroglu, T. Milner, Elimination of artifactual labeling of hippocampal mossy fibers seen following preembedding immunogold-silver technique by pretreatment with zinc chelator, J. Microsc. Res. 23 Ž1992. 100–101.
215
w65x H. Wang, A. Moriwaki, J.B. Wang, G.R. Uhl, V.M. Pickel, Ultrastructural immunocytochemical localization of mu opioid receptors and leu5-enkephalin in the patch compartment of the rat caudate– putamen nucleus, J. Comp. Neurol. 375 Ž1996. 659–674. w66x L.L. Werling, S.R. Brown, B.M. Cox, Opioid receptor regulation of the release of norepinephrine in brain, Neuropharmacology 26 Ž1987. 987–996. w67x T.L. Wimpey, C. Chavkin, Opioids activate both an inward rectifier and a novel voltage-gated potassium conductance in the hippocampal formation, Neuron 6 Ž1991. 281–289. w68x C.-W. Xie, C.V. Lewis, Opioid-mediated facilitation of long-term potentiation at the lateral perforant path-dentate granule cell synapse, J. Pharmacol. Exp. Ther. 256 Ž1991. 289–296. w69x C.W. Xie, D.V. Lewis, Endogenous opioids regulate long-term potentiation of synaptic inhibition in the dentate gyrus of rat hippocampus, J. Neurosci. 15 Ž1995. 3788–3795. w70x C.W. Xie, R.A. Morrisett, D.V. Lewis, Mu opioid receptor-mediated modulation of synaptic currents in dentate granule cells of rat hippocampus, J. Neurophysiol. 68 Ž1992. 1113–1120. w71x J.E. Zadina, L. Hackler, L.J. Ge, A.J. Kastin, A potent and selective endogenous agonist for the mu opiate receptor, Nature 386 Ž1997. 499–502. w72x W. Zieglgansberger, E.D. French, G.R. Siggins, F.E. Bloom, Opioid peptides may excite hippocampal pyramidal neurons by inhibiting adjacent inhibitory interneurons, Science 205 Ž1979. 415–417.