Characterization of input synapses on intracellularly stained neurons in hippocampal slices: an HRP/EM study

Summary This study describes the fine structure of input synapses on identified neurons in slices of the guinea pig hippocampus. For morphological identification, granule cells of the fascia dentata and pyramidal neurons of regio inferior of the hippocampus were impaled and intracellularly stained with horse-radish peroxidase (HRP). Input synapses on the HRP-stained neurons were identified in the electron microscope by the location of the synapses in inner or outer zones of the dentate molecular layer, as in the case of the synaptic contacts on injected granule cells, or by unique fine structural characteristics, as in the case of the giant mossy fiber boutons on CA3 pyramidal cells. As in tissue fixed in situ by transcardial perfusion, a large number of terminals arising from the different afferents in inner and outer zones of the dentate molecular layer were well preserved and formed synaptic contacts with small spines, large complex spines, and dendritic shafts of the HRP-filled granule cells. Mossy fiber synapses on the stained CA3 neurons were densely filled with clear vesicles, contained a few dense-core vesicles, and formed synaptic contacts with large spines or excrescences. Occasionally electrondense degenerating boutons were also found impinging on the stained dendrites and spines. The significance of the present findings for electrophysiological and pharmacological studies on brain slices is discussed.     

Large variability in synaptic N-methyl-D-aspartate receptor density on interneurons and a comparison with pyramidal-cell spines in the rat hippocampus

Pyramidal cells receive input from several types of GABA-releasing interneurons and innervate them reciprocally. Glutamatergic activation of interneurons involves both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) type glutamate receptors expressed in type I synapses, mostly on their dendritic shafts. On average, the synaptic AMPA receptor content is several times higher on interneurons than in the spines of pyramidal cells. To compare the NMDA receptor content of synapses, we used a quantitative postembedding immunogold technique on serial electron microscopic sections, and analysed the synapses on interneuron dendrites and pyramidal cell spines in the CA1 area. Because all NMDA receptors contain the obligatory NR1 subunit, receptor localisation was carried out using antibodies recognising all splice variants of the NR1 subunit. Four populations of synapse were examined: i). on spines of pyramidal cells in stratum (str.) radiatum and str. oriens; ii). on parvalbumin-positive interneuronal dendritic shafts in str. radiatum; iii). on randomly found dendritic shafts in str. oriens and iv). on somatostatin-positive interneuronal dendritic shafts and somata in str. oriens. On average, the size of the synapses on spines was about half of those on interneurons. The four populations of synapse significantly differed in labelling for the NR1 subunit. The median density of NR1 subunit labelling was highest on pyramidal cell spines. It was lowest in the synapses on parvalbumin-positive dendrites in str. radiatum, where more than half of these synapses were immunonegative. In str. oriens, synapses on interneurons had a high variability of receptor content; some dendrites were similar to those in str. radiatum, including the proximal synapses of somatostatin-positive cells, whereas others had immunoreactivity for the NR1 subunit similar to or higher than synapses on pyramidal cell spines.These results show that synaptic NMDA receptor density differs between pyramidal cells and interneurons. Some interneurons may have a high NMDA receptor content, whereas others, like some parvalbumin-expressing cells, a particularly low synaptic NMDA receptor content. Consequently, fast glutamatergic activation of interneurons is expected to show cell type-specific time course and state-dependent dynamics.     

Neural grafting to ischemic lesions of the adult rat hippocampus

Summary The purpose of this study was to examine the structural and connective integration of developing hippocampal neurons grafted to ischemic lesions of the adult rat hippocampus. The 4-vessel occlusion model was used to cause transient cerebral ischemia which damages CA1 pyramidal cells in the dorsal hippocampus, but spares nonpyramidal neurons and afferents in the area. One week later, cell suspensions were made from the CA1 region of fetal (E18-20) rats and injected stereotaxically into the lesion. The recipient brains were examined 6 weeks to 6 months later for survival, morphology, and intrinsic and extrinsic connections of the grafts. The methods used included cell stains, histochemical staining for acetylcholinesterease (AChE), immunocytochemical staining for neuropeptides (cholelecystokinin (CCK), somatostatin (SS), enkephalin (Enk) and an astrocytic marker, glial fibrillary acidic protein (GFAP), as well as tracing by retrograde axonal transport of fluorochromes and light and electron microscopy of anterograde axonal degeneration. The grafts survived well (80%) and were often quite large. They were well integrated in the lesioned host brain area, contained both pyramidal cells and neuropeptidergic neurons and displayed a near normal GFAP immunoreactivity for astrocytes. The latter contrasted the dense gliosis of the host ischemic lesion. Judged by the AChE staining the grafts were innervated by cholinergic host septohippocampal fibers. Ingrowth of host hippocampal commissural fibers was demonstrated by Fink-Heimer staining for degenerating nerve terminals following acute lesions of the hippocampal commissures. At the ultrastructural level degenerating, electron dense terminals of host commissural origin were found even deep inside the graft neuropil in synaptic contact with mainly dendritic spines. A transplant efferent connection to the host brain was demonstrated by retrograde fluorochrome tracing and consisted of a homotypic projection to more posterior levels of the ipsilateral host CA1 and subiculum. Minor abnormal, efferent projections to the host dentate molecular layer were shown in Timm staining. We conclude that fetal CA1 neurons grafted to one week old ischemic lesions of the dorsal CA1 in adult rats become structurally well incorporated and can establish nerve connections with the host brain.     

Total number and distribution of inhibitory and excitatory synapses on hippocampal CA1 pyramidal cells

The integrative properties of neurons depend strongly on the number, proportions and distribution of excitatory and inhibitory synaptic inputs they receive. In this study the three-dimensional geometry of dendritic trees and the density of symmetrical and asymmetrical synapses on different cellular compartments of rat hippocampal CA1 area pyramidal cells was measured to calculate the total number and distribution of excitatory and inhibitory inputs on a single cell.A single pyramidal cell has approximately 12,000 microm dendrites and receives around 30,000 excitatory and 1700 inhibitory inputs, of which 40 % are concentrated in the perisomatic region and 20 % on dendrites in the stratum lacunosum-moleculare. The pre- and post-synaptic features suggest that CA1 pyramidal cell dendrites are heterogeneous. Strata radiatum and oriens dendrites are similar and differ from stratum lacunosum-moleculare dendrites. Proximal apical and basal strata radiatum and oriens dendrites are spine-free or sparsely spiny. Distal strata radiatum and oriens dendrites (forming 68.5 % of the pyramidal cells' dendritic tree) are densely spiny; their excitatory inputs terminate exclusively on dendritic spines, while inhibitory inputs target only dendritic shafts. The proportion of inhibitory inputs on distal spiny strata radiatum and oriens dendrites is low ( approximately 3 %). In contrast, proximal dendritic segments receive mostly (70-100 %) inhibitory inputs. Only inhibitory inputs innervate the somata (77-103 per cell) and axon initial segments. Dendrites in the stratum lacunosum-moleculare possess moderate to small amounts of spines. Excitatory synapses on stratum lacunosum-moleculare dendrites are larger than the synapses in other layers, are frequently perforated ( approximately 40 %) and can be located on dendritic shafts. Inhibitory inputs, whose percentage is relatively high ( approximately 14-17 %), also terminate on dendritic spines.Our results indicate that: (i) the highly convergent excitation arriving onto the distal dendrites of pyramidal cells is primarily controlled by proximally located inhibition; (ii) the organization of excitatory and inhibitory inputs in layers receiving Schaffer collateral input (radiatum/oriens) versus perforant path input (lacunosum-moleculare) is significantly different.     

The lateral cervical nucleus in the cat III. An electron microscopical study after transection of spinal afferents

Summary The ultrastructure of terminal degeneration within the lateral cervical nucleus (LCN) after transection of its spinal afferent fibers 2 days–2 years earlier is described. The degeneration after 2 days was of both the neurofilamentous and dense type. The highest number of degenerating terminals, about 15%, was found after 4–5 days. Then most of the degenerating boutons were of the dense type. The degenerating terminals had synaptic contact with cell bodies and dendrites of LCN-neurons. Removal of the degenerating boutons seemed to be effected by a phagocytic cell present in increased number compared to the normal LCN. In cases with long survival times an increase in the number of astroglial filaments was observed. In an animal where the spinal afferents to the LCN had been cut 2 years earlier a decrease in medium size of the neurons was observed. The amount of dendritic spines was also considerably smaller than normally.     

The projection of the visual cortex on the Clare-Bishop area in the cat

Summary Following large lesions of the cat visual cortex, the distribution of degenerating terminal boutons in the Clare-Bishop area was studied electron microscopically. Degenerating boutons were found throughout the cortical layers but mostly in layer III (51% of the total number of degenerating boutons) and layer V (24%). A smaller number of boutons were found in layers II (12%) and IV (9%), and very few in layers VI (3%) and I (1%). No degenerating terminals were observed in the upper two-thirds of layer I. Seventy-six per cent of the total degenerating boutons terminated on dendritic spines, 22% on dendritic shafts, and 2% on somata. Some degenerating boutons made synaptic contacts with somata and dendrites of nonpyramidal neurons. For example, one degenerating bouton was observed in contact with an apical dendrite of a fusiform cell. Three examples of dendritic spines, with which degenerating boutons made synaptic contacts, were found to belong to spinous stellate cells. No degenerating boutons were observed making synaptic contacts with profiles that could conclusively be traced to pyramidal cell somata.     

Substance P-containing hypothalamic afferents to the monkey hippocampus: an immunocytochemical,tracing, and coexistence study

In order to identify the synaptic connections of substance P-containing afferents within the hypothalamo-hippocampal projection of the monkey, we performed a combined light and electron microscopic, immunocytochemical study, made lesions of the fimbriafornix, and employed retrograde tracing using WGA-HRP. Furthermore, coexistence studies for substance P and GAD were performed to identify the putative transmitters of these hypothalamic projection neurons. A plexus of large substance P-immunoreactive terminals was identified in both the innermost portion of the molecular layer and in CA2. Axon terminals in both plexuses established exclusively asymmetric synapses with spines and dendritic shafts. Substance P-immuno-reactive boutons were degenerating 5 days after lesioning, and had disappeared 10 days after ipsilateral fimbria-fornix transection. Thus, these terminals were of extrinsic origin. In contrast, immunoreactive fibers in the outer third of the dentate molecular layer remained unaffected by the lesion. Retrograde tracing combined with immunostaining for substance P revealed the parent cell bodies of the extrinsic substance P-containing afferents in the supramammillary nucleus. Colocalization studies employing a consecutive semi-thin sections technique indicate that these large substance P-containing projection neurons lack GABA as an inhibitory transmitter. These results suggest that hypothalamic afferents of the monkey hippocampus contain substance P. Because these afferents lack GABA as an inhibitory transmitter and establish exclusively asymmetric synapses, this projection may excite hippocampal target neurons.     

Long-term potentiation induced by patterned stimulation of the commissural pathway to hippocampal CA1 region in freely moving rats.

In urethane-anesthetized rats, stimulation of the contralateral hippocampal CA1 region resulted in activation of the homotopic CA1 region. Current-source-density analysis revealed that both basal and apical dendrites were activated. However, alveolar and stratum oriens stimulation in CA1 gave about equal peak excitation of the basal and apical dendrites while CA1 stratum radiatum/moleculare and CA3c stimulation gave stronger apical than basal dendritic excitation. In chronically implanted and freely moving rats, tetanic patterned stimulation of the contralateral CA1, irrespective of depth, resulted in a robust long-term potentiation of the ipsilateral CA1 basal dendritic synapse. The population basal dendritic excitatory postsynaptic potential was initially potentiated to greater than 200% of the baseline and decayed with a 3 h time constant; it lasted at least two days. Patterned stimulation of the commissural inputs at 2 x threshold stimulus intensity seldom potentiated the apical dendritic synapse in CA1; rather, long-term depression was sometimes observed. After tetanic stimulations at 3 x threshold, a small potentiation of the apical dendritic excitation was seen in about half of the experiments. The average apical dendritic potentiation peaked at about 25% and persisted to at least one day. This study provides original evidence that the properties of long-term potentiation are different at the commissural basal dendritic and apical dendritic synapses in CA1 of the behaving rat. Basal dendritic potentiation is low-threshold, high-amplitude and decayed rapidly in the first 3 h. Apical dendritic potentiation is high-threshold, low-amplitude and not rapidly decaying. A long-lasting enhancement of synaptic transmission has been postulated as a physiological correlate of memory. This paper reports properties of this synaptic enhancement for two different types of synapses on the same cells in the behaving animal. The basal dendritic synapse on hippocampal pyramidal cells readily increased their efficacy, up to at least two days, after a brief, patterned stimulation. In the same preparation, it was difficult to obtain a long-lasting increase in the apical dendritic excitation, in contrast to studies on isolated hippocampal slices in vitro.     

Ultrastructure of degenerating cerebellothalamic terminals in the ventral medial nucleus of the cat

Summary Terminal degeneration of cerebellar afferents in the ventral medial thalamic nucleus (VM) was studied in cats at the ultrastructural level after uni- or bilateral lesions in the brachium conjunctivum (BC). To achieve discrete lesions within the BC, a new very accurate stereotaxic technique was used. Numerous large terminals belonging to a population of so-called LR boutons were observed degenerating in the VM. The boutons displayed a wide variety of degenerative changes. Some revealed the features of the classical neurofilamentous type of degeneration. Others, although containing a slightly increased number of neurofilaments, featured much more prominently large numbers of coated vesicle shells and heavy accumulations of a flocculent electrondense material. Degeneration in a third group of boutons similar to some extent to the light type of degeneration was characterized by tight clumping of enormously swollen or distorted synaptic vesicles within a light matrix. At later stages, however, all these boutons were believed to become shrunken and electron-dense since intermediate stages between the light- and dark-appearing boutons were observed. The degenerating cerebellar boutons formed asymmetrical synaptic contacts. Groups of 3 or 4 boutons terminated upon dendrites of projection neurons synapsing more frequently on spines than on dendritic stems. The synaptic contacts between cerebellar boutons and the vesicle-containing dendrites of local circuit neurons were encountered as often if not more than the contacts on projection neuron dendrites. Triads consisting of cerebellar boutons and dendrites of both types of neurons were observed very regularly. This synaptic arrangement provides the anatomical basis for the modification of cerebellar input in the VM by interneurons.     

Membrane structure at synaptic junctions in area CA1 of the rat hippocampus

In tissue from area CA1 of the rat hippocampus prepared for electron microscopic study by thin-sectioning, asymmetric synaptic junctions were found on dendritic spines, spiny dendritic shafts, and non-spiny dendritic shafts. In freeze-fractured preparations, aggregates of large particles were found on the extracellular half of the postsynaptic membrane at each of these synaptic junctions. Particle aggregate areas were measured and particle packing densities were computed at dendritic spine synapses and dendritic shaft synapses in area CA1, and compared to similar measures of particle aggregates on dendritic spines of cerebellar Purkinje cells. All of these CA1 and cerebellar synapses are excitatory and are thought to use glutamate as a neurotransmitter. There was a tendency for the dispersion of particles within individual aggregates to be less uniform in larger aggregates in both area CA1 and cerebellar cortex. Distinct particle-free zones could be distinguished in the center of particle aggregates on large "mushroom-shaped" spines in area CA1. There was no statistically significant difference between the particle densities at CA1 dendritic spines (2848 +/- 863 particles/micron2) and CA1 dendritic shafts (2707 +/- 718 particles/micron2). However, the average density of particles at cerebellar dendritic spine synapses (3614 +/- 1081 particles/micron2) was significantly greater than at dendritic spine or shaft synapses found in area CA1. Symmetric synaptic junctions were observed on the CA1 pyramidal cell somas and dendritic shafts in thin-sectioned preparations. These synapses typically exert an inhibitory action mediated by gamma-aminobutyric acid. In freeze-fracture preparations, large varicosities were found apposed to the pyramidal somal and dendritic membranes, but there were no specializations of particle distribution on either the extracellular or the cytoplasmic half of the fractured postsynaptic membranes. This finding parallels observations from freeze-fracture preparations of other GABAergic synapses in the central nervous system.     

Synaptic organization of intracellularly stained CA3 pyramidal neurons in slice cultures of rat hippocampus

Pyramidal cells of regio inferior in slice cultures of the rat hippocampus were impaled and intracellularly stained with horseradish peroxidase. A correlated light- and electron-microscopic analysis was then performed to study the properties of these neurons under culture conditions with particular emphasis on input synapses onto these cells. Like pyramidal cells in situ, CA3 pyramidal neurons in slice cultures had a triangular cell body with an apical stem dendrite emerging from it. Several basal dendrites and the axon arose from the basal pole of the cell body. The peripheral thin branches of both apical and basal dendrites were covered with small spines, whereas proximal thick dendritic segments and portions of the cell body exhibited large spines or excrescences. The axon gave off numerous fine varicose collaterals which projected to stratum radiatum of CA1 (Schaffer collaterals), to the alveus and to the hilar region. In one case a collateral could be followed to stratum moleculare of the fascia dentata. Electron-microscopic analysis of the injected pyramidal neurons revealed that their cell bodies, dendritic shafts and spines formed synaptic contacts with presynaptic terminals. Mossy fiber endings were identified by their large size and their numerous clear synaptic vesicles with some dense-core vesicles intermingled, and were observed to form synaptic contacts on the large spines or excrescences. Since extrinsic afferents degenerate in slice cultures, the numerous synaptic boutons on the identified pyramidal neurons probably arise from axons of intrinsic neurons that have sprouted in response to deafferentation. This assumption is supported by the finding that collaterals of the injected neurons formed abundant synaptic contacts on dendritic shafts and spines of other cells. These results suggest that, although pyramidal cells under culture conditions retain a remarkable number of their normal characteristics, considerable synaptic reorganization does take place.     

The study of Golgi stained cells and of experimental degeneration under the electron microscope: a direct method for the identification in the visual cortex of three successive links in a neuron chain.

A direct method is presented which makes it possible to identify, from synapse to synapse, three successive links of a neuron chain. The potentialities of the method are shown by examples of the termination of specific afferents in the visual cortex. Following unilateral lesion of the lateral geniculate nucleus in the rat, the distribution of degenerating geniculocortical boutons was studied on two Golgi-stained cells in layer IV of the primary visual cortex. One of the cells was definitely a small pyramidal cell; the other was identified as a spiny stellate (although the possibility that it too was a small pyramidal cell was not rigorously excluded). Both cells received monosynaptic input from the specific afferents as proved by the existence of degenerating boutons synapsing on their dendritic spines. The axonal arborizations of both Golgi-stained cells were traced at the electron microscopic level in thin section series in order to identify the postsynaptic structures contacted by their boutons. All boutons studied established asymmetrical contacts and about 50% of the synapses given by the impregnated boutons were found on smooth dendritic shafts of stellate cells, the rest on spines. The results obtained suggest a neuron circuit involving, successively, the visual afferents, spiny interneurons or monosynaptic visual target pyramidal cells and nonspiny stellate cells.It is suggested that a similar approach might provide direct information about the connectivity in neuron networks in many other parts of the central nervous system hitherto defying elucidation with conventional methods.     

The termination of callosal fibres in the auditory cortex of the rat. A combined Golgi--electron microscope and degeneration study

When the corpus callosum of the rat is sectioned, the callosal fibres in the cerebral cortex undergo degeneration. In the auditory cortex (area 41) the degenerating axon terminals form asymmetric synapses, and the vast majority of them synapse with dendritic spines. Some other synapse with the shafts of both spiny and smooth dendrites, and a few with the perikarya of non-pyramidal cells. The degenerating axon terminals are contained principally within layer II/III, in which they aggregate in patches. Using a technique in which neurons within the cortex are Golgi-impregnated, then gold-toned and examined in the electron microscope, it has been shown that the dendritic spines of pyramidal neurons with cell bodies in different layers receive the degenerating callosal afferents. The spines arise from the main apical dendritic shafts and their branches, from the dendrites of the apical tufts, and in some cases from the basal dendrites of the pyramidal neurons. The shafts of some pyramidal cell apical dendrites also form asymmetric synapses with callosal afferents. Since we have encountered no spiny non-pyramidal neurons in Golgi preparations of rat auditory cortex, and because other types of non-pyramidal cells have few dendritic spines, it is concluded that practically all of the dendritic spines synapsing with callosal afferents originate from pyramidal neurons.     

Ultrastructure of vestibular commissural neurons related to velocity storage in the monkey.

The angular vestibulo-ocular reflex maintains gaze during head movements. It is thought to be mediated by two components: direct and velocity storage pathways. The direct angular vestibulo-ocular reflex is conveyed by a three neuron chain from the labyrinth to the ocular motoneurons. The indirect pathway involves a more complex neural network that utilizes a portion of the vestibular commissure. The purpose of the present study was to identify the ultrastructural characteristics of commissural neurons in the medial vestibular nucleus that are related to the velocity storage component of the angular vestibulo-ocular reflex. Ultrastructural studies of degenerating medial vestibular nucleus neurons were conducted in monkeys following midline section of rostral medullary commissural fibers with subsequent behavioral testing. After this lesion, oculomotor and vestibular functions attributable to velocity storage were abolished, whereas the direct angular vestibulo-ocular reflex pathway remained intact. Since this damage was functionally discrete, degenerating neurons were interpreted as potential participants in the velocity storage network. Ultrastructural observations indicate that commissural neurons related to velocity storage are small and medium sized cells having large nuclei with deep indentations and relatively little cytoplasm, which are located in the lateral crescents of rostral medial vestibular nucleus. The morphology of degenerating dendritic profiles varied. Some contained numerous round or tubular mitochondria in a pale cytoplasmic matrix with few other organelles, while others had few mitochondria but many cisterns and vacuoles in dense granular cytoplasm. The commissural nature of these cells was further suggested by the presence of two different types of degenerating axon terminals in the rostral medial vestibular nucleus: those with a moderate density of large spherical synaptic vesicles, and those with pleomorphic, primarily ellipsoid synaptic vesicles. The recognition of two types of degenerating terminals further supports our interpretation that at least two morphological types of commissural neurons participate in the velocity storage network. The degenerating boutons formed contacts with a variety of postsynaptic partners. In particular, synapses were observed between degenerating boutons and non-degenerating dendrites, and between intact terminals and degenerating dendrites. However, degenerating pre- and postsynaptic elements were rarely observed in direct contact, suggesting that additional neurons are interposed in the indirect pathway commissural system. On the basis of these ultrastructural observations, it is concluded that vestibular commissural neurons involved in the mediation of velocity storage have distinguishing ultrastructural features and synaptology, that are different from those of direct pathway neurons.     

Convergent vasopressinergic and hippocampal input onto somatospiny neurons of the rat lateral septal area

Electron microscopic immunocytochemistry, was combined with acute anterograde axon degeneration, following transection of the fimbria-fornix, to describe the innervation of somatospiny neurons by vasopressin-immunoreactive and degenerated hippocamposeptal axon terminals in the rat lateral septal area. Vasopressin-immunopositive boutons characterized by symmetric synaptic membrane specializations, and the degenerated hippocamposeptal axon terminals which form asymmetric synaptic contacts, frequently terminate on the same dendritic and somatic profiles, and particularly on the somata of somatospiny neurons. Although hippocamposeptal fibers predominantly form axospinous synapses in the lateral septal area, they terminate mainly on the dendritic shafts and soma of the vasopressin-receptive neurons. Of 720 vasopressin-immunoreactive terminals in the mediolateral part of the lateral septal area, 80% form synaptic contacts with dendritic shafts; 50% on small (distal) dendritic profiles and 30% on large (proximal) dendrites. Synaptic contacts between vasopressin-immunoreactive terminals and dendritic spines were not observed. The remaining 20% of immunoreactive boutons formed axosomatic synaptic contacts with a total of 58 neurons; 31% of these neurons exhibited somatic spines in the plane of the section analysed. Previous studies have demonstrated that in the lateral septal area vasopressin modulates the action of the excitatory amino acid-containing hypocamposeptal fibers, and also plays a role in the maintenance of long term potentiation evoked by fimbria-fornix stimulation. The convergent vasopressinergic and hippocampal input onto the same somatospiny neurons of the lateral septal area suggests that these neurons are targets of these physiological actions.     

Proliferation of thalamic afferents in cerebral cortex altered by callosal deafferentation

Summary In area 41, the auditory region of rat neocortex, callosal afferents project to layers I through III and thalamic afferents project to deep layer III through IV. Thus, these two extrinsic systems of afferents project simultaneously to only a narrow lamina in mid to low layer III. For this study, this narrow region of overlap is quantitatively examined to determine the distribution of callosal and thalamic afferents by observing degenerating terminals produced by separate callosal and thalamic lesions. The results show that of all asymmetric synapses observed in the neuropil of this narrow zone, 84% are dendritic spines and the balance are dendritic shafts. Although both callosal and thalamic afferents prefer to synapse with dendritic spines in the neuropil, 78% of the thalamic afferents synapse with dendritic spines while 93% of the callosal afferents synapse with dendritic spines.Vaughan & Foundas (1982) have shown that 3 months after callosal lesions in 1-month-old animals, additional thalamic axons have grown into, and proliferated in, this part of mid to low layer III. Quantitative analysis of the distribution of the degenerating thalamic axon terminals in these long-term callosally lesioned animals has been used to determine whether the proliferating thalamic afferents demonstrate any specificity in the pattern of synapses they make or whether the callosally deafferented neurons determine the pattern of synapses. The results indicate that thalamic axons do exhibit axon specificity, for after they have proliferated into the callosal domain, 80% of the degenerating terminals synapse with dendritic spines and 20% synapse with shafts. This distribution is most comparable to the normal distribution of thalamic axons in this region.     

Does bouton morphology optimize axon length?

The total length of cortical axons could be reduced if the parent axons maintained straight trajectories and simply connected to dendritic shafts via spine-like terminaux boutons and to dendritic spines via bead-like en passant boutons. Cortical axons from cat area 17 were reconstructed from serial electron micrographs and their bouton morphology was correlated with their synaptic targets. En passant or terminaux boutons did not differ in the proportion of synapses they formed with dendritic spines and shafts, and thus, the two morphological variants of synaptic bouton do not contribute directly to optimizing axon length.     

The development of the inferior olivary complex in preweanling opossums

Summary Pre- and postsynaptic elements within the developing inferior olive (IO) of both control and experimental opossums were examined via electron microscopy. Electron dense boutons identified di-/mesencephalic, cerebellar and spinal afferents within the IO of 8–71 day old animals, which survived 4–48 hours following either midbrain hemisections or spinal transections.During its initial stage of development (3–22 days) the neuropil of the IO is segregated into fields of small diameter neurites or flocculent profiles. Within the fields of flocculent profiles, synaptic interactions are established, which are both infrequent and immature. Although some flocculent profiles are presynaptic, most are postsynaptic and emanate from olivary somata and dendrites. Synaptic contacts also occur with olivary somata, dendritic shafts, spines and dendritic varicosities. Clear round vesicles (crv's; 40 m) predominate within all boutons, normal ones as well as those which degenerate after di-/mesencephalic, cerebellar and spinal lesions; however, larger (70 m) dense cored vesicles (dcv's) are occasionally observed within some boutons. Degenerating terminals from all three sources primarily contact flocculent profiles and dendritic shafts.As the opossum matures (42 days) dramatic increases occur in the number and complexity of both pre- and postsynaptic elements. Marked variations are observed in the matrix density of dendritic shafts. Although all terminal boutons predominantly contain crv's, the number of dcv's within the population of presynaptic elements increases markedly. Concurently, olivary neurons are profusely studded with spines. Simple dendritic spines and spiny appendages as well as dendritic shafts are the most frequent postsynaptic structures within the principal nucleus (PO). Olivary somata and their spines, however, are postsynaptic to degenerating de-/mesencephalic afferents within the PO. Flocculent profiles, which persist within the accessory nuclei, and dendritic shafts are postsynaptic to degenerating spinal boutons.By 70 days of age synaptic contacts appear more mature and more nearly approximate those seen in the adult (King 1980). Few somatic contacts, opaque dendrites, dendritic varicosities, and flocculent profiles are evident within the PO. Dendritic shafts and spines are the principal postsynaptic structures. Many di-/mesencephalic and cerebellar afferents synapse within maturing synaptic clusters on spines between which a rare gap junction is observed. Other di-/mesencephalic and cerebellar endings in the PO as well as spinal endings in the accessory nuclei are presynaptic to dendritic shafts and spines external to synaptic clusters. This predilection for contacting more specific loci on olivary neurons provides good evidence for synaptic remodeling.As the olivary nuclei develop further, the incidence of gap junctions increases and pleomorphic vesicles appear within boutons. The glial investment of neuronal elements, including synaptic clusters, also becomes more extensive.In conclusion, early di-/mesencephalic, cerebellar and spinal synaptic contacts appear qualitatively uniform in their synaptic features and postsynaptic interactions. As olivary development proceeds, however, the distinguishing synaptic features of the nuclear complex become more apparent. Synaptic remodeling occurs as some midbrain and cerebellar terminals are localized within synaptic clusters. The ultrastructural features characteristic of the adult IO are finally achieved by 80 days of age.This research was supported by N.I.H. Research Grant NS-08798     

Tyrosine hydroxylase-immunoreactive boutons in synaptic contact with identified striatonigral neurons, with particular reference to dendritic spines

Tyrosine hydroxylase-immunoreactive fibres in the rat neostriatum were studied in the electron microscope in order to determine the nature of the contacts they make with other neural elements. The larger varicose parts of such fibres contained relatively few vesicles and rarely displayed synaptic membrane specializations; however, thinner parts of axons (0.1-0.4 micron) contained many vesicles and had symmetrical membrane specializations, indicative of en passant type synapses. By far the most common postsynaptic targets of tyrosine hydroxylase-immunoreactive boutons were dendritic spines and shafts, although neuronal cell bodies and axon initial segments also received such input. Six striatonigral neurons in the ventral striatum were identified by retrograde labelling with horseradish peroxidase and their dendritic processes were revealed by Golgi impregnation using the section-Golgi procedure. The same sections were also developed to reveal tyrosine hydroxylase immunoreactivity and so we were able to study immunoreactive boutons in contact with the Golgi-impregnated striatonigral neurons. Each of the 280 immunoreactive boutons examined in the electron microscope displayed symmetrical synaptic membrane specializations: 59% of the boutons were in synaptic contact with the dendritic spines, 35% with the dendritic shafts and 6% with the cell bodies of striatonigral neurons. The dendritic spines of striatonigral neurons that received input from immunoreactive boutons invariably also received input, usually more distally, from unstained boutons that formed asymmetrical synaptic specializations. A study of 87 spines along the dendrites of an identified striatonigral neuron showed that the most common type of synaptic input was from an individual unstained bouton making asymmetrical synaptic contact (53%), while 39% of the spines received one asymmetrical synapse and one symmetrical immunoreactive synapse. It is proposed that the spatial distribution of presumed dopaminergic terminals in synaptic contact with different parts of striatonigral neurons has important functional implications. Those synapses on the cell body and proximal dendritic shafts might mediate a relatively non-selective inhibition. In contrast, the major dopaminergic input that occurs on the necks of dendritic spines is likely to be highly selective since it could prevent the excitatory input to the same spines from reaching the dendritic shaft. One of the main functions of dopamine released from nigrostriatal fibres might thus be to alter the pattern of firing of striatal output neurons by regulating their input.     

Synaptic relationships of a type of GABA-immunoreactive neuron (clutch cell), spiny stellate cells and lateral geniculate nucleus afferents in layer IVC of the monkey striate cortex

The precise stimulus specificity of striate cortical neurons is strongly influenced by processes involving gamma-aminobutyric acid (GABA). In the visual cortex of the monkey most afferents from the lateral geniculate nucleus terminate in layer IVC. We identified a type of smooth dendritic neuron (clutch cell) that was immunoreactive for GABA, and whose Golgi-impregnated dendrites and axon were largely restricted to layer IVC beta. The slightly ovoid somata were 8-12 micron by 12-15 micron and the dendritic field was often elongated, extending 80-200 micron in one dimension. The axonal field was 100-150 micron in diameter and densely packed with large bulbous boutons. Although mainly located in IVC beta both the dendritic and axonal processes entered IVC alpha. Fine structural features of GABA-immunoreactive and-impregnated clutch cells and impregnated spiny stellate cells were compared. Clutch cells had more cytoplasm, more densely packed mitochondria and endoplasmic reticulum, and made type II as opposed to type I synapses. A random sample of 159 elements postsynaptic to three clutch cells showed that they mainly terminated on dendritic shafts (43.8-58.5%) and spines (20.8-46.3%), rather than somata (10-17%). The majority of the postsynaptic targets were characteristic of spiny stellate cells. This was directly demonstrated by studying synaptic contacts between an identified GABA positive clutch cell and the dendrites and soma of an identified spiny stellate cell. The termination of clutch cells mainly on dendrites and spines of spiny stellate cells suggests that they interact with other inputs to the same cells. Following an electrolytic lesion in the ipsilateral lateral geniculate nucleus we examined the distribution of degenerating terminals on three identified spiny stellate neurons in layer IVC beta. Out of eight synapses from the lateral geniculate nucleus one was on a dendritic shaft, the rest on spines. Only a small fraction of all synapses on the cells were from degenerating boutons. A clutch cell within the area of dense terminal degeneration was not contacted by terminals from the lateral geniculate nucleus. The results show that layer IVC in the monkey has a specialized GABAergic neuron that terminates on spiny stellate cells monosynaptically innervated from the lateral geniculate nucleus. Possible functions of clutch cells may include inhibitory gating of geniculate input to cortex; maintenance of the antagonistic subregions in the receptive fields; and the creation from single opponent of double colour opponent receptive fields.