癫痫大鼠海马出芽苔藓纤维突触的超微结构特征

目的:探讨匹罗卡品颞叶癫痫大鼠海马出芽苔藓纤维突触的超微结构特征及其在颞叶癫痫发病机制中的作用。方法:采用Timm组化染色标记出芽苔藓纤维突触末端,在电镜下观察新生突触的类型、比例、定位、以及突触后靶成分。结果:颞叶癫痫大鼠齿状回内分子层可见到银标记的突触末端,出芽苔藓纤维突触主要是轴棘型非对称性突触,其次是轴树型非对称性突触,偶可看到出芽轴突和颗粒细胞体形成突触联系。结论:轴棘型非对称性突触是颞叶癫痫大鼠海马出芽苔藓纤维突触的主要类型,出芽苔藓纤维突触的超微结构特性支持重组突触形成重复的兴奋性环路,而且形成的新的兴奋性环路可能在颞叶癫痫的发生与发展中起重要作用。     

Ultrastructural features of sprouted mossy fiber synapses in kindled and kainic acid-treated rats

The mossy fiber pathway in the dentate gyrus undergoes sprouting and synaptic reorganization in response to seizures. The types of new synapses, their location and number, and the identity of their postsynaptic targets determine the functional properties of the reorganized circuitry. The goal of this study was to characterize the types and proportions of sprouted mossy fiber synapses in kindled and kainic acid-treated rats. In normal rats, synapses labeled by Timm histochemistry or dynorphin immunohistochemistry were rarely observed in the supragranular region of the inner molecular layer when examined by electron microscopy. In epileptic rats, sprouted mossy fiber synaptic terminals were frequently observed. The ultrastructural analysis of the types of sprouted synapses revealed that 1) in the supragranular region, labeled synaptic profiles were more frequently axospinous than axodendritic, and many axospinous synapses were perforated; 2) sprouted mossy fiber synaptic terminals formed exclusively asymmetric, putatively excitatory synapses with dendritic spines and shafts in the supragranular region and with the soma of granule cells in the granule cell layer; 3) in contrast to the large sprouted mossy fiber synapses in resected human epileptic hippocampus, the synapses formed by sprouted mossy fibers in rats were smaller; and 4) in several cases, the postsynaptic targets of sprouted synapses were identified as granule cells, but, in one case, a sprouted synaptic terminal formed a synapse with an inhibitory interneuron. The results demonstrate that axospinous asymmetric synapses are the most common type of synapse formed by sprouted mossy fiber terminals, supporting the viewpoint that most sprouted mossy fibers contribute to recurrent excitation in epilepsy.     

Ultrastructural localization of neurotransmitter immunoreactivity in mossy cell axons and their synaptic targets in the rat dentate gyrus

Electrophysiologically identified and intracellularly biocytin-labeled mossy cells in the dentate hilus of the rat were studied using electron microscopy and postembedding immunogold techniques. Ultrathin sections containing a labeled mossy cell or its axon collaterals were reacted with antisera against the excitatory neurotransmitter glutamate and against the inhibitory neurotransmitter γ-aminobutyric acid (GABA). From single- and double-immunolabeled preparations, we found that 1) mossy cell axon terminals made asymmetric contacts onto postsynaptic targets in the hilus and stratum moleculare of the dentate gyrus and showed immunoreactivity primarily for glutamate, but never for GABA; 2) in the hilus, glutamate-positive mossy cell axon terminals targeted GABA-positive dendritic shafts of hilar interneurons and GABA-negative dendritic spines; and 3) in the inner molecular layer, the mossy cell axon formed asymmetric synapses with dendritic spines associated with GABA-negative (presumably granule cell) dendrites. The results of this study support the view that excitatory (glutamatergic) mossy cell terminals contact GABAergic interneurons and non-GABAergic neurons in the hilar region and GABA-negative granule cells in the stratum moleculare. This pattern of connectivity is consistent with the hypothesis that mossy cells provide excitatory feedback to granule cells in a dentate gyrus associational network and also activate local hilar inhibitory elements. Hippocampus 1997;7:559–570. © 1997 Wiley-Liss, Inc.     

Chromogranins as markers of altered hippocampal circuitry in temporal lobe epilepsy

Chromogranins are polypeptides which are widely expressed in the central nervous system. They are stored in dense core vesicles of nerve terminals, from where they are released upon stimulation. Using immunocytochemistry, we investigated the distribution of chromogranin A, chromogranin B, secretoneurin, and, for comparison, dynorphin in hippocampal specimens removed at routine surgery from patients with drug-resistant mesial temporal lobe epilepsy and in autopsy tissues from nonneurologically deceased subjects. In post mortem controls (n = 21), immunoreactivity for all 4 peptides (most prominently for chromogranin B and dynorphin) was observed in the terminal field of mossy fibers. For chromogranins, staining was observed also in sectors CA1 to CA3 and in the subiculum. Chromogranin B immunoreactivity was found in the inner molecular layer of the dentate gyrus, the area of terminating associational-commissural fibers. Secretoneurin and dynorphin immunoreactivity labeled the outer molecular layer and the stratum lacunosum moleculare of sectors CA1 to CA3, where projections from the entorhinal cortex terminate. In specimens with Ammon's horn sclerosis (n = 25), staining for all 3 chromogranins and for dynorphin was reduced in the hilus of the dentate gyrus. Instead, intense staining was observed in the inner molecular layer, presumably delineating terminals of sprouted mossy fibers. Specimens obtained from temporal lobe epilepsy patients without Ammon's horn sclerosis (n = 4) lacked this pronounced rearrangement of mossy fibers. In the stratum lacunosum moleculare of sector CA1, secretoneurin and dynorphin immunoreactivity was reduced in sclerotic, but not in nonsclerotic, specimens, paralleling the partial loss of fibers arising from the entorhinal cortex. Instead, presumably sprouted secretoneurin-immunoreactive fibers were found in the outer dentate molecular layer in sclerotic specimens. These changes in staining patterns for chromogranins and dynorphin mark profound plastic and functional rearrangement of hippocampal circuitry in temporal lobe epilepsy.     

Status epilepticus-induced hilar basal dendrites on rodent granule cells contribute to recurrent excitatory circuitry

Mossy fiber sprouting into the inner molecular layer of the dentate gyrus is an important neuroplastic change found in animal models of temporal lobe epilepsy and in humans with this type of epilepsy. Recently, we reported in the perforant path stimulation model another neuroplastic change for dentate granule cells following seizures: hilar basal dendrites (HBDs). The present study determined whether status epilepticus-induced HBDs on dentate granule cells occur in the pilocarpine model of temporal lobe epilepsy and whether these dendrites are targeted by mossy fibers. Retrograde transport of biocytin following its ejection into stratum lucidum of CA3 was used to label granule cells for both light and electron microscopy. Granule cells with a heterogeneous morphology, including recurrent basal dendrites, and locations outside the granule cell layer were observed in control preparations. Preparations from both pilocarpine and kainate models of temporal lobe epilepsy also showed granule cells with HBDs. These dendrites branched and extended into the hilus of the dentate gyrus and were shown to be present on 5% of the granule cells in pilocarpine-treated rats with status epilepticus, whereas control rats had virtually none. Electron microscopy was used to determine whether HBDs were postsynaptic to axon terminals in the hilus, a site where mossy fiber collaterals are prevalent. Labeled granule cell axon terminals were found to form asymmetric synapses with labeled HBDs. Also, unlabeled, large mossy fiber boutons were presynaptic to HBDs of granule cells. These results indicate that HBDs are present in the pilocarpine model of temporal lobe epilepsy, confirm the presence of HBDs in the kainate model, and show that HBDs are postsynaptic to mossy fibers. These new mossy fiber synapses with HBDs may contribute to additional recurrent excitatory circuitry for granule cells.     

Postnatal development and synaptic connections of hilar mossy cells in the hippocampal dentate gyrus of rhesus monkeys

Mossy cells of the hippocampal dentate gyrus were analyzed through postnatal development. At birth, a few thorny excrescences were found on the proximal dendrites of mossy cells, whereas distal dendrites displayed pedunculate spines. Thorny excrescences increased in number and complexity until the third month. After that age, the complexity of thorny excrescences is so great that an increase in spine density can be seen only in electron microscopic preparations. An increase in the number of pedunculate spines per unit length of distal dendrite was detected via light microscopy during the first 9 postnatal months. The somata and dendrites of mossy cells displayed adult-like characteristics after the ninth postnatal month. Mossy fiber terminals at birth frequently displayed immature ultrastructural characteristies and formed synapses with dendritic shafts and spines. At later postnatal ages and in adults, axospinous synapses were found almost exclusively. This is consistent with the postnatal development of the complex spines of the mossy cells. Axons of mossy cells were generally confined to the hilus in our 150 -μm-thick sections, where they gave rise to several collaterals. The axon terminals from these collaterals formed asymmetric synapses with dendrites and dendritic spines in the hilar region of the dentate gyrus. These data provide the first anatomical evidence that hilar mossy cells of the primate dentate gyrus have excitatory projections similar to their equivalent cell type in subprimates. The present study indicates that mossy cells of the dentate gyrus are in a more advanced stage of development at birth and mature faster than similar neurons of the human hippocampus. This may represent a faster maturation of hippocampal circuitry in nonhuman primates compared to that in the human.     

Localization of EphA4 in axon terminals and dendritic spines of adult rat hippocampus

Eph receptors and their ephrin ligands assume various roles during central nervous system development. Several of these proteins are also expressed in the mature brain, and notably in the hippocampus, where EphA4 and ephrins have been shown to influence dendritic spine morphology and long-term potentiation (LTP). To examine the cellular and subcellular localization of EphA4 in adult rat ventral hippocampus, we used light and electron microscopic immunocytochemistry with a specific polyclonal antibody against EphA4. After immunoperoxidase labeling, EphA4 immunoreactivity was found to be enriched in the neuropil layers of CA1, CA3, and dentate gyrus. In all examined layers of these regions, myelinated axons, small astrocytic leaflets, unmyelinated axons, dendritic spines, and axon terminals were immunolabeled in increasing order of frequency. Neuronal cell bodies and dendritic branches were immunonegative. EphA4-labeled dendritic spines and axon terminals corresponded to 9-19% and 25-40% of the total number of spines and axon terminals, respectively. Most labeled spines were innervated by unlabeled terminals, but synaptic contacts between two labeled elements were seen. The vast majority of synaptic junctions made by labeled elements was asymmetrical and displayed features of excitatory synapses. Immunogold labeling of EphA4 was located mostly on the plasma membrane of axons, dendritic spines, and axon terminals, supporting its availability for surface interactions with ephrins. The dual preferential labeling of EphA4 on pre- or postsynaptic specializations of excitatory synapses in adult rat hippocampus is consistent with roles for this receptor in synaptic plasticity and LTP.     

Plasticity, Synaptic Strength, and Epilepsy: What Can We Learn from Ultrastructural Data?

Summary:  Central nervous system synapses have an intrinsic plastic capacity to adapt to new conditions with rapid changes in their structure. Such activity-dependent refinement occurs during development and learning, and shares features with diseases such as epilepsy. Quantitative ultrastructural studies based on serial sectioning and reconstructions have shown various structural changes associated with synaptic strength involving both dendritic spines and postsynaptic densities (PSDs) during long-term potentiation (LTP). In this review, we focus on experimental studies that have analyzed at the ultrastructural level the consequences of LTP in rodents, and plastic changes in the hippocampus of experimental models of epilepsy and human tissue obtained during surgeries for intractable temporal lobe epilepsy (TLE). Modifications in spine morphology, increases in the proportion of synapses with perforated PSDs, and formation of multiple spine boutons arising from the same dendrite are the possible sequence of events that accompany hippocampal LTP. Structural remodeling of mossy fiber synapses and formation of aberrant synaptic contacts in the dentate gyrus are common features in experimental models of epilepsy and in human TLE. Combined electrophysiological and ultrastructural studies in kindled rats and chronic epileptic animals have indicated the occurrence of seizure- and neuron loss-induced changes in the hippocampal network. In these experiments, the synaptic contacts on granule cells are similar to those described for LTP. Such changes could be associated with enhancement of synaptic efficiency and may be important in epileptogenesis.     

Forward into our next century—A letter from the editor-in-chief to the readers of the Journal of Comparative Neurology

Dynorphin facilitates conditioned place aversion and reduces locomotor activity through mechanisms potentially involving direct activation of target neurons or release of catecholamines from afferents in the nucleus accumbens. We examined the ultrastructural substrates underlying these actions by combining immunoperoxidase labeling for dynorphin 1–8 and immunogold silver labeling for the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH). The two markers were simultaneously visualized in single coronal sections through the rat nucleus accumbens. By light microscopy, dynorphin immunoreactivity was seen as patches of immunoreactive varicosities throughout all rostrocaudal levels of the nucleus accumbens. The dynorphin-immunoreactive terminals identified by electron microscopy ranged from 0.2 to 1.5 μm in cross-sectional diameter, contained numerous small (30–40 nm) clear vesicles, as well as one or more large (80–100 nm) dense core vesicles. From the dynorphin-immunoreactive terminals quantitatively examined in single sections, 74% (173/370) showed symmetric synaptic junctions mainly with large unlabeled dendrites. Of the dynorphin-immunoreactive terminals forming identifiable synapses, approximately 30% contacted more than one dendritic target. In addition, single dendrites frequently received convergent input from more than one dynorphin-labeled terminal. Irrespective of their dendritic associations, dynorphin-immunoreactive terminals also frequently showed close appositions with other axons and terminals; these included unlabeled (41%), TH-labeled (10%) or dynorphin-labeled axons (14%). In contrast to dynorphin-immunoreactive terminals, TH-labeled terminals formed primarily symmetric synapses with small dendrites and spines or lacked recognizable specializations in the plane of section analyzed. In some cases, single dendrites were postsynaptic to both dynorphin and TH-immunoreactive terminals. We conclude that dynorphin-immunoreactive terminals potently modulate, and most likely inhibit, target neurons in both subregions of the rat nucleus accumbens. This modulatory action could attenuate or potentiate incoming catecholamine signals on more distal dendrites of the accumbens neurons. The findings also suggest potential sites for presynaptic modulatory interactions involving dynorphin and catecholamine or other transmitters in apposed terminals.     

Lesion-induced mossy fibers to the molecular layer of the rat fascia dentata: identification of postsynaptic granule cells by the Golgi-EM technique

The axons of the dentate granule cells, the hippocampal mossy fibers, sprout "backward" into the dentate molecular layer when this is heavily denervated. Using the combined Golgi-electron microscopy (EM) technique we now demonstrate that these aberrant supragranular mossy fibers at least in part terminate on granule cell dendrites. Sprouting of mossy fibers into the dentate molecular layer was induced in adult rats by simultaneous surgical removal of the commissural and entorhinal afferents to the fascia dentata. After at least 7 weeks survival, the presence of mossy fiber terminals in the inner part of the dentate molecular layer was demonstrated by light microscopy. In the electron microscope the mossy fiber terminals were identified by their unique structural characteristics, namely, the unusually large size of the terminals, the dense packing of clear synaptic vesicles with a few dense core vesicles intermingled, the presence of asymmetric synaptic contacts with spines and desmosome-like contacts with dendritic shafts, and the continuity with a thin unmyelinated preterminal axon. Golgi-stained granule cells were first identified in the light microscope, and then, after deimpregnation, the same cells were examined in the electron microscope. In ultrathin, serial sections lesion-induced mossy fiber terminals were found in synaptic contact with spines on proximal dendritic segments of such identified Golgi-impregnated granule cells. From this we conclude that the aberrant, supragranular mossy fibers can innervate dendrites of the parent cell group, the dentate granule cells. The results, moreover, provide an example of reactive synaptogenesis where both the sprouted afferents and its postsynaptic element have been identified.     

Kainic acid-induced mossy fiber sprouting and synapse formation in the dentate gyrus of rats

In the kainic acid (KA) model of temporal lobe epilepsy, mossy fibers (MFs) are thought to establish recurrent excitatory synaptic contacts onto granule cells. This hypothesis was tested by intracellular labeling of granule cells with biocytin and identifying their synaptic contacts in the dentate molecular layer with electron microscopic (EM) techniques. Twenty-three granule cells from KA-treated animals and 14 granule cells from control rats were examined 2 to 4 months following KA at the light microscopic (LM) level; four cells showing MF sprouting were further characterized at the EM level. Timm staining revealed a time-dependent growth of aberrant MFs into the dentate inner molecular layer. The degree of sprouting was generally (but not invariably) correlated with the severity and frequency of seizures. LM examination of individual biocytin-labeled MF axon collaterals revealed enhanced collateralization and significantly increased numbers of synaptic MF boutons in the hilus compared to controls, as well as aberrant MF growth into the granule cell and molecular layers. EM examination of serially reconstructed, biocytin-labeled MF collaterals in the molecular layer revealed MF boutons that form asymmetrical synapses with dendritic shafts and spines of granule cells, including likely autaptic contacts on parent dendrites of the biocytin-labeled granule cell. These results constitute ultrastructural evidence for newly formed excitatory recurrent circuits, which might provide a structural basis for enhanced excitation and epileptogenesis in the hippocampus of KA-treated rats.     

Postnatal development of CA3 pyramidal neurons and their afferents in the Ammon's horn of rhesus monkeys

Previous studies described the postnatal development of CA3 pyramidal neurons and their afferents in the rat. However, the postnatal development of the primate hippocampus was not previously studied. Thus, pyramidal neurons of the CA3 area of the monkey hippocampus were analyzed postnatally in the present study. At birth, a few thorny excrescences, the complex spines postsynaptic to mossy fibers, were found on the proximal segments of both apical and basal dendrites, whereas distal dendrites displayed pedunculate spines. Thorny excrescences increased in number until the third month. A continuous increase in the number of spines per unit length along the distal dendrites was observed during the first 12 months. The ultrastructural features of somata and dendrites of pyramidal cells in newborn monkeys were similar to those of adults. The analysis of the afferents to the CA3 pyramidal neurons was limited to the development of mossy fibers, the axons of granule cells, and myelinated axons in the alveus, stratum oriens, and stratum lacunosum-moleculare. At birth, most mossy fiber terminals were densely packed with synaptic vesicles and formed mainly axospinous synapses with CA3 pyramidal cells. By 1 month of age, the number of mitochondria and embedded spines increased to mature amounts. In the first postnatal month, degenerating axons and axon terminals were frequently observed in the mossy fiber bundles in stratum lucidum. The proportion of myelinated axons increased simultaneously in all three examined layers. At birth most axons were unmyelinated, whereas at 7 months of age the proportion of myelinated axons was similar to that found in adults. The present study indicates that most pyramidal neurons of the CA3 region in monkeys are in an advanced stage of development at the time of birth. Thus, mossy fibers from granule cells in the dentate gyrus have established mature-looking synapses, and the thorny excrescences of pyramidal cells that are postsynaptic to mossy fibers are also adult-like. Nevertheless, several of the adult features, such as the spine density of distal dendrites of pyramidal neurons and the myelination of afferent axons, develop during an extended period of time in the first year. The significance of this early anatomical maturation in a brain region involved in memory function is consistent with recent behavioral data that show a rapid postnatal maturation of limbic-dependent recognition memory in rhesus monkeys. © 1995 Wiley-Liss, Inc.     

Axon arbors and synaptic connections of a vulnerable population of interneurons in the dentate gyrus in vivo

The predominant gamma-aminobutyric acid (GABA)ergic neuron class in the hilus of the dentate gyrus consists of spiny somatostatinergic interneurons. We examined the axon projections and synaptic connections made by spiny hilar interneurons labeled with biocytin in gerbils in vivo. Axon length was 152-497 mm/neuron. Sixty to 85% of the axon concentrated in the outer two thirds of the molecular layer of the dentate gyrus. The septotemporal span of the axon arbor extended over 48-82% of the total hippocampal length, which far exceeds the septotemporal span of axons of granule cells whose complete axon arbors extended over 15-29%. A three-dimensionally reconstructed 216-microm-long spiny hilar interneuron axon segment in the outer third of the molecular layer formed an average of 1 synapse every 5.1 microm. Of the 42 symmetric (inhibitory) synapses formed by the reconstructed segment, 88% were with spiny dendrites of presumed granule cells, and 67% were with dendritic spines that also receive an asymmetric (excitatory) contact from an unlabeled axon terminal. Postembedding GABA-immunocytochemistry revealed that 55% of the GABAergic synapses in the outer third of the molecular layer were with spines. Therefore, in the outer molecular layer, spiny hilar interneurons form synaptic contacts that appear to be positioned to exert inhibitory control near sites of excitatory synaptic input from the entorhinal cortex to granule cell dendritic spines. These findings demonstrate far-reaching, yet highly specific, connectivity of individual interneurons and suggest that the loss of spiny hilar interneurons, as occurs in temporal lobe epilepsy, may contribute to hyperexcitability in the hippocampus.     

Cytoarchitecture and ultrastructure of the avian ectostriatum: afferent terminals from the dorsal telencephalon and some nuclei in the thalamus

The cytoarchitecture, ultrastructure, and afferent terminals in the ectostriatal complex of the Japanese quail were examined. The complex consists of the central core (Ec) and peripheral belt (Ep). Terminals in the complex were categorized into three main groups according to the shape of synaptic vesicles: S (spherical), P (pleomorphic), and F (flat). S terminals are further classified into three types: Ss, small terminals which have densely packed vesicles and a long active zone and are presynaptic to large spines; Sm, medium-sized to large terminals which have a relatively short active zone and contact dendritic spines, trunks, and somata; Sl, large terminals which have many mitochondria and cored vesicles and form synapses only with somata. Some of the Sm terminals are derived from myelinated axons. The Sl terminals are frequently combined with gap junctions as so-called mixed synapses. The P terminal occasionally surrounds an axon hillock, making symmetric synaptic contacts. The F terminals often cover a wide area of the soma. A few gap junctions are also recognized between adjacent somata. Afferent sources of the ectostriatal complex were examined by means of horseradish peroxidase (HRP) retrograde transport. Many large HRP-labeled cells were recognized in the nucleus rotundus (Rt). HRP-labeled cells were seen in the nucleus triangularis (Tr), nucleus dorsolateralis posterior thalami (DLP), and a few labeled cells were scattered in the hyperstriatum ventrale (HV). Substantial numbers of Sm terminals in Ec degenerated after destruction of Rt; they made synaptic contacts with dendritic trunks (71.8%) and small spines (28.2%). Degenerating and intact Sm terminals were found to form synapses with the same dendritic trunk by the reconstruction of serial thin sections. Among the 167 Sm terminals, 20 terminals (12.0%) degenerated after lesions in Rt. The Sm terminals in Ec degenerated after destruction of Tr and the terminals formed synapses with somata as well as dendritic trunks and spines. After lesions of the dorsal telencephalon including HV, degenerating fibers sparsely entered the ectostriatal complex associated with the Ss terminal degeneration. DLP seemed to project mainly on the medial area of posterior Ep. The terminals from DLP made asymmetric synaptic contacts with dendritic spines, trunks, and somata. Some of the terminals from DLP were identified as the Sl type, but other S types could not be identified.     

Kainic acid-induced sprouting of dynorphin- and enkephalin- containing mossy fibers in the dentate gyrus of the rat hippocampus

This study utilized Timm histochemistry and immunocytochemistry to determine the prolonged effects of kainic acid on the distribution of dynorphin- and enkephalin-containing mossy fibers in the rat dentate gyrus at progressive time points following kainic acid injection. Beginning 1–2 weeks after kainic acid administration, a progressive increase in the distribution and intensity of staining for supragranular zinc, dynorphin and enkephalin was observed in the dentate gyrus. The kainic acid-induced sprouting of mossy fibers containing dynorphin and enkephalin strongly resembles the pattern observed in the dentate gyrus of humans with temporal lobe epilepsy.     

Ultrastructural heterogeneity of enkephalin-containing terminals in the rat hippocampal formation

Opioid peptides, including leu-enkephalin (LE), are important neuromodulators in the hippocampal formation where they may play a role in learning and memory as well as epileptogenesis. We examined the cellular substrates that underlie the function of LE in each lamina of the rat hippocampal formation by immunocytochemistry at the electron microscopic level in single section analysis. LE-like immunoreactivity (LE-LI) was primarily associated with large dense-core vesicles (80–100 nm), usually found in axons and axon terminals, but was also observed in perikarya and occasionally in dendrites. The morphology and synaptic associations of LE-LI-containing terminals were strikingly distinct in each region of the hippocampal formation. In the molecular layer of the dentate gyrus, terminals with LE-LI were typically small (0.6 μm) and formed primarily asymmetric (excitatory type) synapses on single dendritic spines, which is consistent with the presence of LE in the lateral perforant path. In the hilus of the dentate gyrus, twd types of LE-containing terminals were present: (1) small round terminals that were heterogeneous in size (0.4–1 μm) and in type of contact formed and (2) larger (3–5 μm) terminals exhibiting the characteristic morphology of mossy fiber boutons that formed asymmetric synapses on spines. This variation in morphology and the type of contact suggests LE may have a heterogeneous influence on diverse hilar interneurons. In the CA3 region of the hippocampus, LE-LI was localized to large mossy fiber boutons (3–7 μm) that formed multiple asymmetric synapses on complex spiny dendritic processes and often formed puncta adherentia with the shafts of large CA3 pyramidal cell dendrites, indicating that this peptide may be directly released onto pyramidal cells. At the border of stratum radiatum and lacunosum moleculare in the CA1 region of the hippocampus, LE-labeled terminals averaged 0.8 μm in diameter and often formed symmetric (inhibitory type) synapses on dendritic shafts, which is consistent with a role in disinhibition. In conclusion, these heterogeneous cellular interactions indicate that LE has diverse functional roles and mechanisms of action within each lamina of the hippocampal formation and may directly and indirectly modulate hippocampal cell activity. © 1995 Wiley-Liss, Inc.     

Plastische Veränderungen von Neuropeptiden bei Patienten mit Temporallappenepilepsie

Neuropeptides are stored together with the classical neurotransmitter in large dense core vesicles from where they are released upon stimulation. In animal models, neuropeptides have been shown to influence neuronal excitability. For example, an anticonvulsive action was found for neuropeptide Y (NPY), galanin, dynorphin and somatostatin. By contrast, substance P was found to have a proconvulsive action. We investigated the expression of NPY, dynorphin, secretoneurin and chromogranin B in hippocampi from patients with intractable temporal lobe epilepsy (TLE) (n=29; mean age=34.5 years; mean duration of epilepsy=21.4 years) and post mortem controls (n=21; mean age=57.7 years; mean post mortem delay=17.2 hours). In situ hybridization for dynorphin showed a marked increase of mRNA expression in granule cells of the dentate gyrus. For NPY, we found increased mRNA expression in hilar interneurons. Immunohistochemical staining showed a sprouting of neuropeptide-containing axons. Antibodies for dynorphin, secretoneurin and chromogranin B labeled mossy fiber terminals in the inner molecular layer of the denate gyrus of patients with, but not without, hippocampal sclerosis. NPY- and secretoneurin-containing interneurons revealed a dense plexus of fibers in the molecular layer and the hilus of the dentate gyrus as well as in stratum lucidum, the terminal field of mossy fibers. Radioligand binding to the NPY Y2 receptor, which mediates anticonvulsive actions of NPY, was increased in the hippocampus of TLE patients. In summary, our data show a marked reorganization of hippocampal circuits and an upregulation of the expression of dynorphin, NPY and the Y2 receptor in the epileptic hippocampus. These changes may contribute to endogenous anticonvulsive mechanisms in TLE patients.     

Cellular sites for dynorphin activation of kappa-opioid receptors in the rat nucleus accumbens shell.

The nucleus accumbens (Acb) is prominently involved in the aversive behavioral aspects of kappa-opioid receptor (KOR) agonists, including its endogenous ligand dynorphin (Dyn). We examined the ultrastructural immunoperoxidase localization of KOR and immunogold labeling of Dyn to determine the major cellular sites for KOR activation in this region. Of 851 KOR-labeled structures sampled from a total area of 10,457 microm2, 63% were small axons and morphologically heterogenous axon terminals, 31% of which apposed Dyn-labeled terminals or also contained Dyn. Sixty-eight percent of the KOR-containing axon terminals formed punctate-symmetric or appositional contacts with unlabeled dendrites and spines, many of which received convergent input from terminals that formed asymmetric synapses. Excitatory-type terminals that formed asymmetric synapses with dendritic spines comprised 21% of the KOR-immunoreactive profiles. Dendritic spines within the neuropil were the major nonaxonal structures that contained KOR immunoreactivity. These spines also received excitatory-type synapses from unlabeled terminals and were apposed by Dyn-containing terminals. These results provide ultrastructural evidence that in the Acb shell (AcbSh), KOR agonists play a primary role in regulating the presynaptic release of Dyn and other neuromodulators that influence the output of spiny neurons via changes in the presynaptic release of or the postsynaptic responses to excitatory amino acids. The cellular distribution of KOR complements those described previously for the reward-associated mu- and delta-opioid receptors in the Acb shell.     

Dynorphin-immunoreactive neurons in the rat nucleus accumbens: Ultrastructure and synaptic input from terminals containing substance P and/or dynorphin

The endogenous opioid peptide dynorphin is enriched in neurons in the nucleus accumbens, for which coexistence and synaptic interactions with substance P have been postulated. We examined the immunogold-silver localization of dynorphin and immunoperoxidase labeling for substance P in single coronal sections through the core subregion of the nucleus accumbens of acrolein-fixed rat brain tissue. Dynorphin-immunoreactive somata were more prevalent than substance P-containing neurons throughout the region sampled for ultrastructural analysis. Dynorphin-labeled cells were spherical, contained unindented nuclei, and were closely apposed to other somata and dendrites, some of which also contained dynorphin immunoreactivity. The appositions were characterized by the absence of glial processes and contiguous contacts between the plasma membranes. Smooth endoplasmic reticulum and coated vesicles could also be identified in the cytoplasms on either side of the somatic or dendritic appositions. The dynorphin somata and dendrites received synaptic input from numerous unlabeled as well as dynorphin-and/or substance P-labeled axon terminals. Both types of terminals were morphologically similar in their content of small and large dense core vesicles and their formation of mainly symmetric synaptic specializations. In addition to dynorphin-immunoreactive targets, numerous dynorphin-and substance P-labeled terminals also formed synapses with unlabeled somata and dendrites. In some cases, terminals separately labeled for dynorphin and substance P converged on common targets with or without detectable dynorphin immunoreactivity. Terminals colocalizing both peptides were also found to synapse on unlabeled or dynorphin-labeled somata and dendrites. Additionally, presynaptic interactions were suggested by close appositions between dynorphin-and/or substance P-labeled terminals and other terminals that were unlabeled, dynorphin labeled, or substance P labeled. These results provide morphological data suggesting nonsynaptic communication between dynorphin-immunoreactive neurons and other neurons possibly mediated through receptive sites or second messengers associated with smooth endoplasmic reticulum in the nucleus accumbens. They also indicate that, in this region, 1) the activity of dynorphin neurons may be dependent on activation of autoreceptors for dynorphin as well as substance P and 2) additional neurons lacking dynorphin immunoreactivity are most likely inhibited (symmetric junctions) by terminals containing either one or both peptides. The findings may have implications for motor and analgesic responses to aversive tonic pain transmitted through dynorphin and substance P pathways within the nucleus accumbens. © 1995 Willy-Liss, Inc.     

Novel expression of AMPA-receptor subunit GluR1 on mossy cells and CA3 pyramidal neurons in the human epileptogenic hippocampus

Previous immunocytochemical investigations performed in our laboratory on the human hippocampus surgically resected for the treatment of mesial temporal lobe epilepsy (MTLE) have demonstrated an increased expression of the AMPA-receptor subunit GluR1 on neurons in the hilus and area CA3. Light microscopically, many of these neurons exhibited peculiar filamentous extensions and grape-like excrescences that protruded from their somata and proximal dendrites, suggesting that these neurons may be mossy cells and CA3 pyramidal neurons, respectively. The present electron microscopic study was carried out to further characterize these cells. The filamentous extensions were identified as dendrites from which spines often protruded, and the grape-like excrescences represented clusters of closely associated dendrites and spines. A variety of synapses were formed by the GluR1-positive profiles. These arrangements ranged from simple contacts between a single unlabelled axon terminal and a single labelled postsynaptic element, to complex contacts involving multiple unlabelled axon terminals and labelled postsynaptic elements. Many of the axon terminals involved in these arrangements were mossy fibre boutons. Thus, a large proportion of the GluR1-positive neurons were identified as hilar mossy cells and CA3 pyramidal neurons, cells hitherto thought to be absent or greatly reduced in the MTLE hippocampus. Taken together, these data suggest the presence of a highly efficient excitatory circuit involving AMPA receptors, mossy cells and CA3 pyramidal neurons in the sclerotic hippocampus. Such a circuit could be critically involved in the genesis and maintenance of temporal lobe epilepsy.