Subcellular organization of camkii in rat hippocampal pyramidal neurons

Calcium/calmodulin‐dependent protein kinase II (CaMKII) plays a key role in N‐methyl‐D‐aspartate (NMDA) receptor‐dependent long‐term synaptic plasticity; its location is critical for signal transduction, and may provide clues that further elucidate its function. We therefore examined the subcellular localization of CaMKII in CA1 stratum radiatum of adult rat hippocampus, by using immuno‐electron microscopy after chemical fixation. When tissue was fixed quickly, the concentration of CaMKIIα (assessed by pre‐embedding immunogold) was significantly higher in dendritic shafts than in spine heads. However, when tissue was fixed 5 minutes after perfusion with normal saline, the density of labeling decreased in dendritic shaft while increasing in spine heads, implying rapid translocation into the spine during brief perimortem stress. Likewise, in quickly fixed tissue, CaMKII within spine heads was found at comparable concentrations in the “proximal” half (adjacent to the spine neck) and the “distal” half (containing the postsynaptic density [PSD]), whereas after delayed fixation, label density increased in the distal side of the spine head, suggesting that CaMKII within the spine head moves toward the PSD during this interval. To estimate its distribution at the synapse in vivo, we performed postembedding immunogold staining for CaMKII in quick‐fixed tissue, and found that the enzyme did not concentrate primarily within the central matrix of the PSD. Instead, labeling density peaked ～40 nm inside the postsynaptic membrane, at the cytoplasmic fringe of the PSD. Labeling within 25 nm of the postsynaptic membrane concentrated at the lateral edge of the synapse. This lateral “PSD core” pool of CaMKII may play a special role in synaptic plasticity. J. Comp. Neurol. 521:3570‐3583, 2013. © 2013 Wiley Periodicals, Inc.     

Estradiol increases the frequency of multiple synapse boutons in the hippocampal CA1 region of the adult female rat

The effect of estradiol to increase the density of dendritic spines and axospinous synapses on hippocampal CA1 pyramidal cells in the adult female rat has been well-documented. However, presynaptic involvement in this process of synapse elimination and formation in the adult is unknown. To address this issue, we have reconstructed 410 complete presynaptic boutons through coded serial electron micrographs of CA1 stratum radiatum to determine the: (1) frequency of multiple (MSB) vs. single (SSB) synapse boutons; (2) number of synaptic contacts per MSB; (3) bouton volume and surface area; and (4) types of spines in synaptic contact with MSBs and SSBs in ovariectomized, estradiol-treated animals (OVX + E) versus ovariectomized oil-treated controls (OVX + O). Quantitative analysis of this tissue revealed that, in OVX + E animals, 45.0% of presynaptic boutons form multiple synaptic contacts with dendritic spines compared to 27.3% in controls (P < 0.01); the average number of synapses per MSB was 2.7 in OVX + E animals compared to 2.3 in controls (P < 0.05). This represents a 25.5% increase in the number of synapses formed by a given number of presynaptic boutons in estradiol-treated animals (P < 0.01) which largely accounts for the previously observed estradiol-induced increase in axospinous synapse density. There was no treatment effect on bouton size; however, because MSBs are larger than SSBs, the increased frequency of MSBs in estradiol-treated tissue results in a trend toward an estradiol-induced increase in average bouton size. Additionally, MSBs were found to be more irregular in shape, i.e., significantly less spherical, than SSBs. Our results indicate that estradiol-induced dendritic spines form synapses primarily with preexisting boutons in stratum radiatum and that these boutons enlarge and change shape as they accommodate new synapses. Such findings suggest a relatively active role for dendrites in the process of adult synapse formation. © 1996 Wiley-Liss, Inc.     

Calcium-binding protein Caldendrin and CaMKII are localized in spinules of the carp retina

Calcium-binding proteins translate the influx of Ca(2+) at excitatory synapses into spatiotemporal signals that regulate a variety of processes underlying synaptic plasticity. In the fish retina, the synaptic connectivity between photoreceptors and horizontal cells undergoes a remarkable plasticity, triggered by the ambient light conditions. With increasing light, the synaptic dendrites of horizontal cells form numerous spinules that are dissolved during dark adaptation. The dynamic regulation of this process is calcium-dependent and involves the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), but astonishingly its principal regulator Calmodulin (CaM) could not be localized to spinules. Here, we show that antibodies directed against Caldendrin (CaBP1), a member of the EF-hand calcium-binding protein family, strongly label the terminal dendrites of horizontal cells invaginating cone pedicles. Double-labeling experiments revealed that this label is closely associated with label for CaMKII. This association was confirmed at the ultrastructural level. Caldendrin immunoreactivity and CaMKII immunoreactivity are both present in horizontal cell dendrites flanking the synaptic ribbon within the cone pedicle and in particular in spinules formed by these terminals. Comparison of light- and dark-adapted retinas revealed a shift of the membrane-associated label for Caldendrin from the terminal dendrites into the spinules during light adaptation. These results suggest that Caldendrin is involved in the dynamic regulation of spinules and confirms the assumed potential of Caldendrin as a neural calcium sensor for synaptic plasticity.     

Role of Ca2+/calmodulin‐dependent protein kinase II in dendritic spine remodeling during epileptiform activity in vitro

Epileptiform activity (EA) in vivo and in vitro induces a loss of dendritic spines and synapses. Because CaMKII has been implicated in synaptogenesis and synaptic plasticity, we investigated the role of CaMKII in the effects of EA on spines, using rat hippocampal slice cultures. To visualize dendrites and postsynaptic densities (PSDs) in pyramidal neurons in the slices, we used biolistic transfection to express either free GFP or a PSD95‐YFP construct that specifically labels PSDs. This allowed us to distinguish two classes of dendritic protrusions: spines that contain PSDs, and filopodia that lack PSDs and that are, on average, longer than spines. By these criteria, 48 hr of EA caused a decrease specifically in the number of spines. Immunoblots showed that EA increased CaMKII activity in the slices. Inhibition of CaMKII by expression of AIP, a specific peptide inhibitor of CaMKII, reduced spine number under basal conditions and failed to prevent EA‐induced spine loss. However, under EA conditions, AIP increased the number of filopodia and the number of PSDs on the dendritic shaft. These data show at least two roles for CaMKII activity in maintenance and remodeling of dendritic spines under basal or EA conditions. First, CaMKII activity promotes the maintenance of spines and spine PSDs. Second, CaMKII activity suppresses EA‐induced formation of filopodia and suppresses an increase in shaft PSDs, apparently by promoting translocation of PSDs from dendritic shafts to spines and/or selectively stabilizing spine rather than shaft PSDs. © 2009 Wiley‐Liss, Inc.     

Chemically induced long-term potentiation increases the number of perforated and complex postsynaptic densities but does not alter dendritic spine volume in CA1 of adult mouse hippocampal slices

Examination of the morphological correlates of long-term potentiation (LTP) in the hippocampus requires the analysis of both the presynaptic and postsynaptic elements. However, ultrastructural measurements of synapses and dendritic spines following LTP induced via tetanic stimulation presents the difficulty that not all synapses examined are necessarily activated. To overcome this limitation, and to ensure that a very large proportion of the synapses and spines examined have been potentiated, we induced LTP in acute hippocampal slices of adult mice by addition of tetraethylammonium (TEA) to a modified CSF containing an elevated concentration of Ca(2+) and no Mg(+). Quantitative electron microscope morphometric analyses and three-dimensional (3-D) reconstructions of both dendritic spines and postsynaptic densities (PSDs) in CA1 stratum radiatum were made on serial ultrathin sections. One hour after chemical LTP induction the proportion of macular (unperforated) synapses decreased (50%) whilst the number of synapses with simple perforated and complex PSDs (nonmacular) increased significantly (17%), without significant changes in volume and surface area of the PSD. In addition, the surface area of mushroom spines increased significantly (13%) whilst there were no volume differences in either mushroom or thin spines, or in surface area of thin spines. CA1 stratum radiatum contained multiple-synapse en passant axons as well as multiple-synapse spines, which were unaffected by chemical LTP. Our results suggest that chemical LTP induces active dendritic spine remodelling and correlates with a change in the weight and strength of synaptic transmission as shown by the increase in the proportion of nonmacular synapses.     

Plasticity of the parallel Fiber-Purkinje cell synapse by spine takeover and new synapse formation in the adult rat

Alteration in synaptic connectivity between Purkinje cell spines and parallel fibers of the cerebellum were studied following partial deafferentation of Purkinje cells in the adult rat. Transection of parallel fibers by two lesions placed at a 1 mm interval on the folial crest were used to produce degeneration of these afferents. Ultrastructural analysis of synapses on Purkinje cell spines revealed degeneration with vacating of postsynaptic sites within 6 h. Reactive synaptogenesis as takeover of Purkinje cell spines by formation of new synapses from remaining parallel fibers occurred even before degenerating parallel fibers had vacated postsynaptic sites. This was accompanied by a marked increase in the number of dual innervations by reactive parallel fibers within one day. Some vacated postsynaptic sites were lost as indicated by a reduction in the number of synapses and others may have been taken over by newly formed synapses on spines. In addition, new synapses formed between the shafts of Purkinje cell branchlets and parallel fibers. Sprouting of parallel fibers occurred as small extensions without tubules while Purkinje cell spines reacted by forming elongated and multiple heads which contacted different parallel fibers. After 5 days degenerating boutons were rarely found. Enlarged spine heads were each capped by a proportionally enlarged parallel fiber bouton and joined by an elongated synaptic junction to parallel fibers. Some parallel fiber boutons were greatly enlarged and capped numerous profiles of spines.

This study shows that formation of new pre- and postsynaptic sites takes precedence over reoccupation of original contacts and that multiple synapses on individual spines are being eliminated to give rise to single contacts with boutons. This elimination resulted in enlargement of synaptic contact areas between Purkinje cell spines and parallel fibers by taking over postsynaptic sites from some vacated and eliminated boutons.     

Activity-dependent formation of perforated synapses in cultured hippocampal neurons

The study investigated the formation of perforated synapses in rat hippocampal cell cultures. Perforated synapses are defined by their discontinuous postsynaptic densities (PSDs) and are believed to occur in parallel with changes in synaptic activity and possibly also synaptic efficacy. Several in vivo studies have demonstrated an increase in the frequency of perforated synapses induced by development and environmental stimulation as well as long-term potentiation (LTP). Also in in vitro brain slices, LTP was associated with an elevated number of perforated spine synapses. Our study demonstrated for the first time that the formation of perforated synapses can be induced by a short-term increase in spontaneous neural activity in a hippocampal cell culture model. Stimulation with the GABAA-antagonist picrotoxin (PTX) induced a significant increase in the percentage of perforated synapses. This strong increase was blocked when APV was added together with PTX, indicating that the formation of perforated synapses depended on the activation of NMDA receptors. We also showed that inhibition of the tissue type plasminogen activator (tPA-stop/PAI-1) significantly interfered with the activity-induced increase in perforated synapses. This implies that the proteolytic activities of tPA might be involved in steps which are downstream from the NMDA receptor-mediated synaptic plasticity leading to structural changes at synaptic contacts. In contrast, even long-term inhibition of electrical network activity by tetrodotoxin had no effect on the number of perforated synapses, but almost completely abolished the formation of spine synapses. These results indicate that a short-term increase in neural activity via NMDA receptors and a proteolytic cascade involving tPA lead to the formation of perforated synapses.     

Ultrastructural quantitative analysis of glutamatergic and GABAergic synaptic terminals in the phrenic nucleus after spinal cord injury

Quantitative analysis of electron microscopic postembedding immunochemically stained material indicates that 48% of all terminals in the rat phrenic nucleus are glutamatergic and 33% are γ-aminobutyric acid (GABA) ergic. Three distinct types of glutamatergic terminals were observed in the rat phrenic nucleus: terminals characterized by large, loosely arranged spherical synaptic vesicles (SI) or small, compact spherical synaptic vesicles (Ss) and elongated terminals containing spherical synaptic vesicles with neurofilaments (NFs). All three types of glutamatergic terminals display asymmetrical synaptic membrane densities with postsynaptic dense bodies being present in some of the S-type terminals. The GABAergic immunoreactive terminals in the phrenic nucleus most closely resemble F-type terminals. They are characterized by flattened or pleomorphic synaptic vesicles and symmetric synaptic membrane densities. Among the 48% glutamatergic terminals, 27% are SI, 65% are Ss, and 8% are NFs, respectively. Significantly fewer glutamate, GABA, and unlabeled terminals per unit area are present in the phrenic nucleus 30 days after a C2 spinal cord hemisection as compared to nonhemisected controls. The average number of active zones per terminal, however, is greater in the hemisection group (1.45 ± 0.03) than in the control group (1.34 ± 0.03), with the active zones in the glutamate terminals mainly accounting for this difference. Moreover, the length of the active zones in the glutamate terminals was significantly longer in the hemisection group (0.37 ± 0.013 μm) as compared to the controls (0.24 ± 0.008 μm). In addition, the mean length of synaptic active zones in GABAergic terminals was also found to be longer in the hemisection group (0.36 ± 0.022 μm) as compared to controls (0.28 ± 0.014 μm). Finally, there is also a significantly higher ratio of synaptic active zones to the total number of glutamate-labeled terminals after injury (1.73 ± 0.08) as compared to controls (1.41 ± 0.04). The number of double/multiple synapses, the percentages of Sl, Ss, and NFs-type terminals, and the percentages of synaptic active zones contacting either distal dendrites or proximal dendrites/somata do not change significantly 30 days after injury. These results are important for a more complete understanding of the synaptic plasticity that occurs in the phrenic nucleus after spinal cord injury and to show how the plasticity may relate to the unmasking of latent bulbospinal respiratory connections which restore function to the hemidiaphragm paralyzed by an ipsilateral spinal cord hemisection. © 1996 Wiley-Liss, Inc.     

Changes in the hippocampal slices energy metabolism following stimulation and long-term potentiation of Schaffer collaterals-pyramidal cell synapses tested with the 2-deoxyglucose technique

Changes in the neuron metabolism in the hippocampal slices following stimulation and potentiation (induction of increase of the evoked extracellular potential) of Schaffer collaterals-pyramidal cell synapses were tested with 2-deoxyglucose (2DG) technique. 2DG uptake was used as an index of glucose utilization. Stimulation evoked calcium- and frequency-dependent increase in [³H]2DG uptake. Potentiation of the synaptic response increased [³H]2DG accumulation but only when potentiated synapses were pressed to be active. It is suggested that stimulation-dependent increase in [³H]2DG uptake is mainly related to neurotransmission. Potentiation-dependent increase of [³H]2DG uptake is probably due to phosphorylation (inhibition) of pyruvate dehydrogenase (PHD). Inactivation of PDH may partially change the nerve endings metabolism from the aerobic pathway into anaerobic. To get the same amount of energy after potentiation as before, the nerve endings have to increase the rate of metabolism.     

Axospinous synaptic subtype‐specific differences in structure,size, ionotropic receptor expression,and connectivity in apical dendritic regions of rat hippocampal CA1 pyramidal neurons

The morphology of axospinous synapses and their parent spines varies widely. Additionally, many of these synapses are contacted by multiple synapse boutons (MSBs) and show substantial variability in receptor expression. The two major axospinous synaptic subtypes are perforated and nonperforated, but there are several subcategories within these two classes. The present study used serial section electron microscopy to determine whether perforated and nonperforated synaptic subtypes differed with regard to their distribution, size, receptor expression, and connectivity to MSBs in three apical dendritic regions of rat hippocampal area CA1: the proximal and distal thirds of stratum radiatum, and the stratum lacunosum‐moleculare. All synaptic subtypes were present throughout the apical dendritic regions, but there were several subclass‐specific differences. First, segmented, completely partitioned synapses changed in number, proportion, and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) receptor expression with distance from the soma beyond that found within other perforated synaptic subtypes. Second, atypically large, nonperforated synapses showed N‐methyl‐D ‐aspartate (NMDA) receptor immunoreactivity identical to that of perforated synapses, levels of AMPA receptor expression intermediate to that of nonperforated and perforated synapses, and perforated synapse‐like changes in structure with distance from the soma. Finally, MSB connectivity was highest in the proximal stratum radiatum, but only for those MSBs composed of nonperforated synapses. The immunogold data suggest that most MSBs would not generate simultaneous depolarizations in multiple neurons or spines, however, because the vast majority of MSBs are comprised of two synapses with abnormally low levels of receptor expression, or involve one synapse with a high level of receptor expression and another with only a low level. J. Comp. Neurol. 512:399–418, 2009. © 2008 Wiley‐Liss, Inc.     

Synaptic plasticity of 5-hydroxytryptamine–immunoreactive terminals in the phrenic nucleus following spinal cord injury: A quantitative electron microscopic analysis

The present study was conducted to examine the plasticity of 5-hydroxytryptamine (5-HT)–immunoreactive terminals in the rat phrenic nucleus following an ipsilateral C2 spinal cord hemisection and 30-day survival period. A retrograde horseradish peroxidase (HRP) labeling technique was used to identify the phrenic motoneurons at the electron microscopic (EM) level. After employing a pre-embedding immunocytochemical technique, the ultrastructural characteristics of 5-HT–immunoreactive terminals were qualitatively and then quantitatively analyzed with a computerized morphometric system before and after injury in separate groups of rats. The results indicated that the majority of the 5-HT–labeled terminals formed axodendritic contacts, but some 5-HT–labeled terminals made axosomatic contacts. 5-HT terminals were associated with either asymmetrical or symmetrical synapses, and some displayed postsynaptic dense bodies. Approximately 2% of the 5-HT terminals had dense-core vesicles. Although the total number of labeled and unlabeled terminals in the phrenic nucleus was reduced after hemisection, the number of 5-HT terminals in the hemisected group was greater than that of the control group. Moreover, the total number and length of asymmetrical and symmetrical synaptic active zones per 5-HT terminal were significantly greater after injury. Finally, the total number of 5-HT terminals with multiple synapses was significantly greater in the hemisected group as compared to controls. It is possible that 5-HT synaptic plasticity may be part of the morphological substrate for the unmasking of the latent crossed phrenic pathway which mediates recovery of the ipsilateral hemidiaphragm paralyzed by C2 spinal cord hemisection. J. Comp. Neurol. 386:613–624, 1997. © 1997 Wiley-Liss, Inc.     

Perforated axospinous synapses with multiple,completely partitioned transmission zones: Probable structural intermediates in synaptic plasticity

Analysis of axospinous synapses in the rat dentate gyrus, using three-dimensional reconstructions from electron micrographs of serial sections, revealed a novel synaptic subtype. Synapses of this subtype exhibit partitions that emanate from the postsynaptic spine head and invaginate the presynaptic axon terminal, dividing its portion contacted by the spine into distinct protrusions. Such complete spine partitions provide barriers between two to four discrete transmission zones, each one being formed by a separate axon terminal protrusion and delineated by a separate segment of the postsynaptic density (PSD). Spine partitions that differ from the complete ones were found in two other synaptic subtypes. One of these is characterized by a sectional partition the base of which is placed between the arms of a horseshoeshaped PSD. Synapses of another subtype exhibit a focal partition the base of which is restricted to a perforation in a fenestrated PSD. Although both sectional and focal partitions invaginate a presynaptic axon terminal, they do not divide into separate protrusions and do not split a single transmission zone into disjointed entities. All three subtypes of partitioned synapses have nonpartitioned counterparts exhibiting segmented, horseshoe-shaped, or fenestrated PSDs. These observations suggest a model of structural modifications underlying synaptic plasticity. According to this model, synapses with multiple, completely partitioned transmission zones that appear to be designed as elements of an unusually high strength, represent pivotal structural intermediates in synaptic plasticity. The formation of such synapses from those that belong to other subtypes is postulated to result in a sustained increase in the efficacy of synaptic transmission. Conversely, a disassembly of complete partitions with the transformation of multiple transmission zones into a single one is proposed to lead to a persistent depression of synaptic respones.     

Three-dimensional organization of cell adhesion junctions at synapses and dendritic spines in area CA1 of the rat hippocampus

Recent work has emphasized the role of adhesion molecules in synaptic plasticity, including long-term potentiation in the hippocampus. Such adhesion molecules are concentrated in junctions that are characterized by dense thickenings on both sides of the junction and are called puncta adhaerentia (PA). Reconstruction from serial electron microscopy was used to determine the location and size of PA in the stratum radiatum of hippocampal area CA1, where many of the previous functional studies have been performed. PAs were found at the edges of synapses on 33% of dendritic spines. The areas occupied by PA were variable across different types of synapses, occupying 0.010 ± 0.005 μm² at macular synapses and 0.034 ± 0.031 μm² at perforated synapses. Another zone, called a vesicle-free transition zone (VFTZ), was identified. Like the PA, this zone also had no presynaptic vesicles and was located at the edges of synapses; however, unlike the PA, the presynaptic thickening was less than the postsynaptic thickening. Together, 45% of spine synapses had PA and/or VFTZ occupying 23 ± 11% of the total junctional area between axons and spines. PA also occurred at nonsynaptic sites involving neuronal as well as glial elements. Most (64%) of these PAs occurred between nonsynaptic portions of dendritic spines and neighboring astrocytic processes. Smooth endoplasmic reticulum was often apposed to one or both sides of the synaptic and the nonsynaptic PA. These findings provide further data as a structural basis for understanding the roles of cell adhesion junctions in hippocampal synaptic function and plasticity. J. Comp. Neurol. 393:58–68, 1998. © 1998 Wiley-Liss, Inc.     

Different phosphatase-dependent mechanisms mediate long-term depression and depotentiation of long-term potentiation in mouse hippocampal CA1 area

Two types of synaptic depression have been described in the hippocampus, long-term depression and depotentiation of long-term potentiation known to recruit the serine/threonine protein phosphatases PP1, PP2A and PP2B (calcineurin). The contribution of each of these protein phosphatases is controversial. To examine the role of the Ca2+/calmodulin-dependent protein phosphatase calcineurin in long-term depression and depotentiation, we analysed the effect of genetically inhibiting calcineurin reversibly in the hippocampus, using the doxycycline-dependent rtTA system in transgenic mice. We show that reducing calcineurin activity has no effect on long-term depression but reversibly affects depotentiation. Consistently, the calcineurin inhibitor FK-506 reproduces the depotentiation impairment observed in the mutant mice but does not affect long-term depression in control animals. In contrast, the PP1/PP2A inhibitor okadaic acid fully blocks both long-term depression and depotentiation. These data demonstrate that the nature of signalling cascades induced by synaptic activity depends on the initial synaptic state. While depression of potentiated synaptic responses requires activation of PP1/PP2A and/or calcineurin, depression of basal synaptic responses depends only on PP1/PP2A activation.     

Variability in the terminations of GABAergic chandelier cell axons on initial segments of pyramidal cell axons in the monkey sensory-motor cortex

Chandelier cell axons were studied in the sensory-motor cortex of adult monkeys. The axonal fields of Golgi-impregnated chandelier cells in layer II in motor cortex are flattened sagittally. The vertical terminal portions of the axons varied both in length and in the numbers converging to form terminations of greater or lesser complexity. Golgi-impregnated plexuses were embedded in plastic and resectioned serially at 2.5-3.0 micrograms. A single axonal field could have as many as 400 terminal rows. All lie 3-13 micrograms beneath pyramidal cell somata. These terminations are not randomly distributed but instead, form clusters. Further resectioning the plastic sections for electron microscopy revealed that all the terminations are on the initial axon segments of pyramidal cells and all form symmetric synaptic contacts. In immunocytochemical material stained for glutamic acid decarboxylase (GAD), the enzyme involved in the synthesis of GABA, GAD-positive boutons were found to form symmetric synaptic contacts with a variety of postsynaptic elements including the axon hillocks and axon initial segments of pyramidal cells. Serial reconstructions from electron micrographs revealed GAD-positive terminals synapsing with the axon initial segment of pyramidal cells joined by cytoplasmic bridges and forming vertically oriented rows identical to those of chandelier cell terminals identified positively in the resectioned Golgi material. The GAD-positive terminals forming initial segment synapses were never continuous with GAD-positive terminals forming axo hillock synapses. The latter probably arise from basket cell axons. Initial segments of pyramidal cell axons in layers II and III were contacted by more GAD-positive terminals than the initial segments of pyramidal cell axons in layer V. The largest pyramidal cells in layer III received the most synapses. Many larger pyramidal cells, identified as callosally projecting cells by the retrograde transport of horseradish peroxidase (HRP), were shown in serial electron micrographs to possess large numbers of initial segment synapses, comparable to those seen in the immunocytochemical material. Serial reconstructions of pyramidal cell axons from axon hillock to the first myelin internode in resectioned Golgi, immunocytochemical and HRP material showed that the number of synapses varied from 2 to 52 for layers II and III and from 2 to 26 for layer V. The number of synapses on the axon hillocks varied from zero to 12. The variability in these terminations may be an important factor in the shaping of the functional properties of the pyramidal cells.     

Three-dimensional analysis of the structure and composition of CA3 branched dendritic spines and their synaptic relationships with mossy fiber boutons in the rat hippocampus.

This paper is the third in a series to quantify differences in the composition of subcellular organelles and three-dimensional structure of dendritic spines that could contribute to their specific biological properties. Proximal apical dendritic spines of the CA3 pyramidal cells receiving synaptic input from mossy fiber (MF) boutons in the adult rat hippocampus were evaluated in three sets of serial electron micrographs. These CA3 spines are unusual in that they have from 1 to 16 branches emerging from a single dendritic origin. The branched spines usually contain subcellular organelles that are rarely found in adult spines of other brain regions including ribosomes, multivesicular bodies (MVB), mitochondria, and microtubules. MVBs occur most often in the spine heads that also contain smooth endoplasmic reticulum, and ribosomes occur most often in spines that have spinules, which are small nonsynaptic protuberances emerging from the spine head. Most of the branched spines are surrounded by a single MF bouton, which establishes synapses with multiple spine heads. The postsynaptic densities (PSDs) occupy about 10-15% of the spine head membrane, a value that is consistent with spines from other brain regions, with spines of different geometries, and with immature spines. Individual MF boutons usually synapse with several different branched spines, all of which originate from the same parent dendrite. Larger branched spines and MF boutons are more likely to synapse with multiple MF boutons and spines, respectively, than smaller spines and boutons. Complete three-dimensional reconstructions of representative spines with 1, 6, or 12 heads were measured to obtain the volumes, total surface areas, and PSD surface areas. Overall, these dimensions were larger for the complete branched spines than for unbranched or branched spines in other brain regions. However, individual branches were of comparable size to the large mushroom spines in hippocampal area CA1 and in the visual cortex, though the CA3 branches were more irregular in shape. The diameters of each spine branch were measured along the cytoplasmic path from the PSD to the origin with the dendrite, and the lengths of branch segments over which the diameters remained approximately uniform were computed for subsequent use in biophysical models. No constrictions in the segments of the branched spines were thin enough to reduce charge transfer along their lengths.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fine structural organization of spinothalamic and trigeminothalamic lamina I terminations in the nucleus submedius of the cat

We examined lamina I trigemino- and spinothalamic tract (TSTT) terminals labeled with Phaseolus vulgaris leucoagglutinin in the nucleus submedius (Sm), a nociceptive relay in the cat's thalamus. Volume-rendered (three-dimensional) reconstructions of ten lamina I TSTT terminals identified with light and electron microscopy were built from serial ultrathin sections by computer, which enabled the overall structures of the terminal complexes to be characterized in detail. Two fundamentally different terminations were observed: compact clusters of numerous boutons, which predominate in the dense focus of a lamina I terminal field in the Sm, and boutons-of-passage, which are present throughout the terminal field and predominate in its periphery. Reconstructions of cluster terminations reveal that all boutons of each cluster make synaptic contact with protrusions and branch points on a single dendrite and involve presynaptic dendrites (PSDs) in triadic arrangements, providing a basis for the secure relay of sensory information. In contrast, reconstructions show that boutons-of-passage are generally characterized by simple contacts with PSDs, indicating an ascending inhibitory lamina I influence. These different synaptic arrangements are consistent with physiological evidence indicating that the morphologically distinct nociceptive-specific and thermoreceptive-(cold)-specific lamina I TSTT neurons terminate differently within the Sm. Thus, a suitable structural substrate exists in the cat's Sm for the inhibitory effect of cold on nociception, a behavioral and physiological phenomenon of fundamental significance. We conclude that the Sm is more than a simple relay for nociception, and that it may be an integrative comparator of ascending modality-selective information that arrives from neurons in lamina I. © 1996 Wiley-Liss, Inc.     

Asynchronous changes in size, number, and shape of synapses in the developing superior colliculus

The ultra-structural development of synapses in retino-receptive layers of the opossum superior colliculus was studied by the ethanolic phosphotungstic acid (E-PTA) method. There was a tendency for a slight reduction in the diameter of synaptic disks, a rise and fall of numerical densities and, except for an ephemeral period, a general increase in the proportion of ‘‘frown’’ among curve synapses. The lack of strict synchrony and the occurrence of different patterns of changes suggest that multiple factors contribute to synaptic maturation.     

Drebrin a content correlates with spine head size in the adult mouse cerebral cortex

Synaptic activities alter synaptic strengths at the axospinous junctions, and such changes are often accompanied by changes in the size of the postsynaptic spines. We have been exploring the idea that drebrin A, a neuron-specific actin-binding protein localized on the postsynaptic side of excitatory synapses, may be a molecule that links synaptic activity to the shape and content of spines. Here, we performed electron microscopic immunocytochemistry with the nondiffusible gold label to explore the relationship among levels of drebrin A, the NR2A subunit of N-methyl-D-aspartate receptors, and the size of spines in the perirhinal cortex of adult mouse brains. In contrast to the membranous localization within neonatal spines, most immunogold particles for drebrin A were localized to the cytoplasmic core region of spines in mature spines. This distribution suggests that drebrin within adult spines may reorganize the F-actin network at the spine core, in addition to its known neonatal role in spine formation. Drebrin A-immunopositive (DIP) spines exhibited larger spine head areas and longer postsynaptic densities (PSDs) than drebrin A-immunonegative (DIN) spines (P < 0.001). Furthermore, spine head area and PSD lengths correlated positively with drebrin A levels (r = 0.47 and 0.40). The number of synaptic NR2A immunolabels was also higher in DIP spines than in DIN spines, whereas their densities per unit lengths of PSD were not significantly different. These differences between the DIP and the DIN spines indicate that spine sizes and synaptic protein composition of mature brains are regulated, at least in part, by drebrin A levels.