Experimental Modeling and Discussion of Syncytial Connections in the Nervous System

This article addresses the question of the possible existence of syncytial connections in the nervous system by presenting the first data on experimental syncytial fusion of neurons. Neurons from the ganglia of the mollusk Lymnaea stagnalis without their surrounding glial cells, isolated using pronase, were brought together by centrifugation and remained in the aggregated state for two days in culture medium. The neurons retained the ability to form normal processes. At the boundaries of adjacent cells, contacting mutual invaginations (podia) appeared, separated from each other by vacuole-like enlargements of intercellular clefts. Electron microscopy showed that the outer cell membranes were disrupted at contact sites between podia. Only residual fragments of degraded membrane were seen. The cytoplasm of one adjacent cells merged continuously with the cytoplasm of the other. Thus, these experiments provide further support for the cellular theory in relation to the common main properties of all cells and extend the view of the neuron theory that the nervous system, apart from chemical synapses and electrical membrane contacts, may also include syncytial interneuron contacts.     

Increased excitatory synaptic activity and local connectivity of hippocampal CA1 pyramidal cells in rats with kainate-induced epilepsy

Formation of local excitatory circuits may contribute to epileptogenesis. We tested the hypothesis that epileptogenesis is associated with increased recurrent excitation in the hippocampal CA1 area of rats with kainate-induced epilepsy. Whole cell recordings were obtained during focal flash photolysis of caged glutamate, which served as a focal excitant to activate local pyramidal cells and to study possible connections between neurons. Kainate-treated rats with spontaneous seizures were studied months after status epilepticus and were compared with saline-injected control rats. Experiments were done in isolated CA1 minislices and in bicuculline to block GABA(A) receptors. Spontaneous excitatory postsynaptic currents (sEPSCs) were present in 42% of the CA1 pyramidal cells from controls and 62% from kainate-treated rats. The frequency of sEPSCs in the kainate group was significantly higher than that in the control group, but mean amplitude was not different. Flash photolysis of caged glutamate on the somatodendritic area of CA1 pyramidal neurons caused a burst of action potentials. Local excitatory connections between CA1 pyramidal cells were found in 4 of 48 neurons (8%) in slices from control animals, but in significantly more neurons (12 of 37; 32%) from rats with kainate-induced epilepsy exhibited interconnections (P < 0.001). Photoactivation of glutamate on recorded CA1 pyramidal cells in the kainate group sometimes caused afterdischarges, but not in controls. The kainate-treated rats with pyramidal cells that responded to photostimulaltion with repetitive EPSCs appeared to have experienced more severe seizures. These data provide new electrophysiological evidence for the formation of recurrent excitatory circuits in the CA1 area of rats with kainate-induced epilepsy.     

Glial cell plasticity in sensory ganglia induced by nerve damage

Numerous studies have been done on the effect of nerve injury on neurons of sensory ganglia but little is known about the contribution of satellite glial cells (SCs) in these ganglia to post-injury events. We investigated cell-to-cell coupling and ultrastructure of SCs in mouse dorsal root ganglia after nerve injury (axotomy). Under control conditions SCs were mutually coupled, but mainly to other SCs around a given neuron. After axotomy SCs became extensively coupled to SCs that enveloped other neurons, apparently by gap junctions. Serial section electron microscopy showed that after axotomy SC sheaths enveloping neighboring neurons formed connections with each other. Such connections were absent in control ganglia. The number of gap junctions between SCs increased 6.5-fold after axotomy. We propose that axotomy induces growth of perineuronal SC sheaths, leading to contacts between SCs enveloping adjacent neurons and to formation of new gap junctions between SCs. These changes may be an important mode of glial plasticity and can contribute to neuropathic pain.     

Specificity of interneuronal connections.

A fundamental problem of neurobiological research is how specific connections between individual neurons are established and maintained. In this report different levels of neuronal specificity are described. Some neuronal populations display region specificity, but within the target region they establish synapses with a variety of neurons. A characteristic feature of the afferent innervation of hippocampal neurons is that many fibers terminate in a laminated fashion. Such a layer specificity is known for the afferents from the entorhinal cortex and for the mossy fibers. The entorhinal afferents terminate in the outer molecular layer of the fascia dentata and in the stratum lacunosum-moleculare of the hippocampus proper. The mossy fibers display both region specificity and layer specificity: they form numerous synapses in hippocampal region CA3 but never invade CA1; in CA3 they are restricted to stratum lucidum. An extremely high degree of neuronal specificity is observed in the case of the axo-axonic or chandelier cells. The axons of these neurons specifically terminate on the axon initial segments of projection neurons in the neocortex, hippocampus and fascia dentata. Thus, these cells not only display a target cell specificity but a selectivity for a distinct portion of the target cell's membrane. Some of the factors that contribute to these different levels of neuronal specificity are briefly discussed. Positional cues as well as diffusible molecules from the target region may guide the outgrowing growth cone to its target. Molecular interactions between pre- and postsynaptic membranes, the functional load of the synaptic contact, and the selective death of a number of neurons and synapses further determine the specificity of interneuronal connections.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphological and ultrastructural changes in the placenta of the diabetic pregnant Egyptian women

Diabetes mellitus (DM) is a chronic metabolic disease in which the body fails to produce enough insulin or increased tissue resistance to insulin. The diabetes may have profound effects on placental development and function. This study was designed to detect the placental changes in pregnancy associated with DM comparing these changes with normal placenta. The study was carried out on sixty full-term placentae; divided into three equal groups; control group (group I): placentae of normal pregnancy, uncontrolled diabetes (group II): placentae from pregnant women whose blood glucose is poorly controlled during pregnancy. Controlled diabetes (group III): includes placentae from diabetic women whose blood glucose is controlled during pregnancy. The placentae from group II tend to be heavier and exhibited immaturity of villi, villous edema, fibrosis, excessive syncytial knots formation and infarctions. In addition to, fibrinoid necrosis, increased thickness of vasculosyncytial membrane, syncytial basement membrane, microvillous abnormalities and vascular endothelial changes were demonstrated. The syncytial multivesicular knots were present in placentae of group II. The nuclei within these syncytial knots display condensed chromatin, either dispersed throughout the nucleus or in the form of dense peripheral clumps with and numerous cytoplasmic vacuoles. The syncytial basement membrane showed focal areas of increase in its thickness and irregularity. Villous cytotrophoblasts showed increased number and activity in the form of numerous secretory granules, abundant dilated RER, larger distorted mitochondria. Villous vessels showed various degrees of abnormalities in the form of endothelial cell enlargement, folding, thickening and protrusion of their luminal surfaces into vascular lumen making it narrower in caliber. In placentae of group III, most of these abnormalities decreased. In most of placentae of group III, the VSM appeared nearly normal in thickness and showed nearly normal composition of one layer of syncytiotrophoblastic cells, one layer of smooth, regular capillary endothelium and the space between them. Mild microvillous abnormalities were noted in few placentae as they appeared short and blunted with mild decrease in their number per micron. The electron picture of syncytial knots appeared nearly normal containing aggregations of small, condensed hyperchromatic nuclei, minimal vacuoles could be seen in the cytoplasm of syncytial knots. Syncytial basement membrane appeared regular and nearly normal in its thickness and composition coming in direct contact with fetal blood capillaries but mild abnormalities were noted in the basement membrane in few placentae as increased its thickness and deposition of fibers or fibrinoid. Regarding cytotrophoblasts in the terminal villi of placentae with controlled diabetes, these cells appeared nearly normal. They were scattered beneath the syncytium and were active containing mitochondria, rough endoplasmic reticulum, free ribosomes and a large nucleus with fine dispersed chromatin. The vascular ultrastructural pattern in terminal villi of placentae of this group showed no significant abnormalities and was normally distributed in the villous tree. The luminal surface of the vascular endothelium appeared regular smooth in the majority of placentae of this group. The endothelial cells appeared connected to each other with tight junctions. It could be concluded that whether if long-term diabetes is controlled or not, placentae of diabetic mother showed a variety of significant histological structural changes seen more frequently than in the placentae of pregnant women without diabetes.     

Combining the Cell-Encapsulation Technique and Axon Guidance for Cell Transplantation Therapy

In cell transplantation therapy for the treatment of neurodegenerative disorders, encapsulation of implanted cells in a semipermeable membrane is a promising approach to protect the implanted cells from host immune rejection and inhibit the invasion of tumor into surrounding tissue if the implanted cells form a tumor after transplantation. However, implanted neurons isolated by capsules could not build connections with host neurons, preventing the implanted neurons from responding to stimuli from host neurons. In the present study, we focused on the passage of neurites and axons navigated by axon guidance molecules through membrane pores to enable encapsulated neurons and host neurons to form connections. The type of matrix coated on membranes and the pore size of the membranes greatly affected the successful passage of PC12 neurites through membrane pores. PC12 neurites preferably passed through collagen-coated membranes with pores greater than 0.8 μm in diameter, but the neurites did not pass through albumin- or fibronectin-coated membranes or membranes with pores less than 0.1 μm in diameter. We could navigate the direction of commissural neural axon extensions by utilizing the axon guidance molecules secreted from floor plate and make guided axons pass through the membrane pores. These results suggest the feasibility of building connections between encapsulated neurons and host neurons by encapsulating the implanted neurons and axon guidance molecules, which attract the axons of host neurons into the capsule, in the porous membranes with suitable pore size and matrix coating.     

A combined Golgi-electron microscopic study of non-pyramidal neurons in the CA 1 area of the hippocampus

Summary Non-pyramidal neurons of the CA 1 area of the rat hippocampus were identified with a combined Golgi-electron microscopic method. They were observed to have distinctive light and electron microscopic characteristics that are different from those of pyramidal cells. These features included smooth dendrites, locally arborizing axons, infolded cell nuclei with intranuclear rods or sheets, and a well-developed perikaryal cytoplasm with many organelles. In addition, the axon terminals that contact the somata and dendrites of local circuit neurons may form asymmetric as well as symmetric synapses. The axons of these cells form symmetric synapses with dendrites and somata of pyramidal cells. Some of these features were utilized to identify non-pyramidal neurons of the CA 1 area for studies of connectivity. Degenerating commissural terminals were found to form synapses with the dendrites and somata of non-pyramidal neurons. These results indicate that these neurons are a significant population of hippocampal neurons that may provide feed-forward inhibition of pyramidal neurons.     

Initiation and spread of epileptiform discharges in the methylazoxymethanol acetate rat model of cortical dysplasia: functional and structural connectivity between CA1 heterotopia and hippocampus/neocortex

Neuronal migration disorders (NMDs) are often associated with medically intractable epilepsy. In utero injection of methylazoxymethanol acetate into pregnant rats gives rise to dysplastic cell clusters ("heterotopia") in hippocampus (and nearby regions), providing an animal model of NMD. In the present study, we have examined the structural and functional integration of hippocampal heterotopic cells into circuits that link the heterotopia with surrounding "normal" brain. Bi-directional morphological connectivity between the heterotopia and hippocampus/neocortex was demonstrated using the neurotracer, biotinylated dextran amine. Single cell recordings in hippocampal slices showed that heterotopia neurons form functional connections with the surrounding hippocampus and neocortex. However, simultaneous field recordings from the CA1 heterotopia, normotopic hippocampus, and neocortex indicated that epileptiform discharges (spontaneous events seen in slices bathed with high [K+]o and bicuculline) were rarely initiated in the heterotopia (although the heterotopia was capable of generating epileptiform discharges independently of normal brain regions). Further, in most of the experiments, the aberrant connectivity provided by CA1 heterotopia failed to function as a "bridge" for epileptiform discharges to propagate directly from low-threshold hippocampus to neocortex. These data do not support the hypothesis that NMDs (heterotopic cell populations) serve as a focus and/or trigger for epileptiform activity, and/or facilitate propagation of epileptiform events.     

Postnatal development of nonpyramidal neurons in the rat hippocampus (areas CA1 and CA3): a combined Golgi/electron microscope study

Summary This study describes the morphological differentiation of nonpyramidal neurons in areas CA1 and CA3 of the rat hippocampus as seen after Golgi-impregnation. Representative neurons were gold-toned and processed for an electron microscopic study of identified cells. We analyzed the postnatal stages P0 (day of birth), P5, P10 and P20. The results can be summarized as follows: 1. On the day of birth nonpyramidal neurons display relatively large cell bodies with short, clumsy dendrites. Great variability of the shape of the cell body and of the orientation of dendrites was observed when compared with the more stereotyped pyramidal neurons. Electron microscopy of identified nonpyramidal neurons revealed small infoldings of the nuclear membrane and immature synapses on the short dendritic shafts of these cells. 2. Developing nonpyramidal neurons from P0 and P5 display growth cones, filopodia, preterminal growth buds, and irregular varicose swellings along the dendrites. 3. Further postnatal development of nonpyramidal neurons is mainly characterized by an increase in dendritic length, paralled by a decrease in growth cones and preterminal growth buds. By means of the electron microscope an increase in the number of mature input synapses on the gold-toned dendritic shafts of identified nonpyramidal neurons was observed. 4. There is a significant developmental difference between nonpyramidal neurons in CA1 and CA3 that was most obvious on P5. Nonpyramidal neurons in CA3 appear more mature, displaying longer dendrites that sometimes traverse through several hippocampal layers. In contrast, the dendrites of nonpyramidal neurons in CA1 are still restricted to the layer of the parent cell body. The earlier differentiation of nonpyramidal neurons in CA3 may result from the earlier formation of neurons in CA3 than in CA1. Longer dendrites of nonpyramidal neurons in CA3, together with an earlier arrival of afferent fibers in this region, suggest that nonpyramidal neurons in CA3 are integrated into inhibitory hippocampal circuits earlier than their counterparts in CA1. 5. On P20, hippocampal nonpyramidal neurons showed all structural characteristics as observed in adult animals both at light and electron microscopic levels. It is concluded that the structural maturation of hippocampal nonpyramidal cells is completed by that postnatal age.In partial fulfilment of the requirements for the degree of Dr. med. at the University of Frankfurt/Main     

Synaptic connections from multiple subfields contribute to granule cell hyperexcitability in hippocampal slice cultures

Limbic status epilepticus and preparation of hippocampal slice cultures both produce cell loss and denervation. This commonality led us to hypothesize that morphological and physiological alterations in hippocampal slice cultures may be similar to those observed in human limbic epilepsy and animal models. To test this hypothesis, we performed electrophysiological and morphological analyses in long-term (postnatal day 11; 40-60 days in vitro) organotypic hippocampal slice cultures. Electrophysiological analyses of dentate granule cell excitability revealed that granule cells in slice cultures were hyperexcitable compared with acute slices from normal rats. In physiological buffer, spontaneous electrographic granule cell seizures were seen in 22% of cultures; in the presence of a GABA(A) receptor antagonist, seizures were documented in 75% of cultures. Hilar stimulation evoked postsynaptic potentials (PSPs) and multiple population spikes in the granule cell layer, which were eliminated by glutamate receptor antagonists, demonstrating the requirement for excitatory synaptic transmission. By contrast, under identical recording conditions, acute hippocampal slices isolated from normal rats exhibited a lack of seizures, and hilar stimulation evoked an isolated population spike without PSPs. To examine the possibility that newly formed excitatory synaptic connections to the dentate gyrus contribute to granule cell hyperexcitability in slice cultures, anatomical labeling and electrophysiological recordings following knife cuts were performed. Anatomical labeling of individual dentate granule, CA3 and CA1 pyramidal cells with neurobiotin illustrated the presence of axonal projections that may provide reciprocal excitatory synaptic connections among these regions and contribute to granule cell hyperexcitability. Knife cuts severing connections between CA1 and the dentate gyrus/CA3c region reduced but did not abolish hilar-evoked excitatory PSPs, suggesting the presence of newly formed, functional synaptic connections to the granule cells from CA1 and CA3 as well as from neurons intrinsic to the dentate gyrus. Many of the electrophysiological and morphological abnormalities reported here for long-term hippocampal slice cultures bear striking similarities to both human and in vivo models, making this in vitro model a simple, powerful system to begin to elucidate the molecular and cellular mechanisms underlying synaptic rearrangements and epileptogenesis.     

Atrophy of pyramidal neurons and increased stress-induced glutamate levels in CA3 following chronic suppression of adult neurogenesis

Following their birth in the adult hippocampal dentate gyrus, newborn progenitor cells migrate into the granule cell layer where they differentiate, mature, and functionally integrate into existing circuitry. The hypothesis that adult hippocampal neurogenesis is physiologically important has gained traction, but the precise role of newborn neurons in hippocampal function remains unclear. We investigated whether loss of new neurons impacts dendrite morphology and glutamate levels in area CA3 of the hippocampus by utilizing a human GFAP promoter-driven thymidine kinase genetic mouse model to conditionally suppress adult neurogenesis. We found that chronic ablation of new neurons induces remodeling in CA3 pyramidal cells and increases stress-induced release of the neurotransmitter glutamate. The ability of persistent impairment of adult neurogenesis to influence hippocampal dendrite morphology and excitatory amino acid neurotransmission has important implications for elucidating newborn neuron function, and in particular, understanding the role of these cells in stress-related excitoxicity.     

Network interactions mediated by new excitatory connections between CA1 pyramidal cells in rats with kainate-induced epilepsy

Axon sprouting and synaptic reorganization in the hippocampus are associated with the development of seizures in temporal lobe epilepsy. Synaptic interactions among CA1 pyramidal cells were examined in fragments of hippocampal slices containing only the CA1 area from saline- and kainate-treated rats. Glutamate microapplication to the pyramidal cell layer increased excitatory postsynaptic current (EPSC) frequency, but only in rats with kainate-induced epilepsy. In bicuculline, action potentials evoked in single pyramidal cells increased the frequency of network bursts only in slices from rats with kainate-induced epilepsy. These data further support the hypothesis that excitatory connections between CA1 pyramidal cells increase after kainate-induced status epilepticus.     

The onset of synaptogenesis in rat temporal cortex.

The onset of synaptogenesis was studied in the temporal cortex of rat fetuses whose age ranged between 15 and 19 days of gestation. First synapses were found at a surprisingly early stage of cortical development: on day 16. These contacts showed relatively few vesicles and very inconspicuous membrane-thickenings. They were located in the marginal layer, above as well as below the narrow band formed by the newly arrived first neuroblasts of the prospective corticle plate. The postsynaptic structures were probably dendrites of the horizontally or obliquely orientated neurons scattered throughout the marginal layer (such neurons were seen even within the cell-dense band). On day 17, the cortical plate separated the differentiated cells definitely into a superficial and a deep population. As on the following days, synapses were found above and below the cortical plate but not within it. In addition to contacts showing the same features as those described on day 16, there were already synapses with numerous vesicles and clearly asymmetric membrane thickenings. On days 18 and 19 the borders of the cortical plate became more clear-cut. The well-differentiated neurons situated above and below this plate could now be identified as Retzius-Cajal cells of the prospective molecular layer and as polymorphous cells of the layer VI b respectively. The presence of axo-somatic contacts on these neurons provided direct evidence that both cell types are targets for synapses. Desmosome-like junctions were found even in the youngest fetuses studied. Their roughly symmetric membrane thickenings were clearly more conspicious than those of earliest synapses. Desmosome-like junctions occurred very frequently between structures which subsequently were never seen to become synaptically linked. During the entire period studied, numerous coated vesicles fused with cell membranes were noted. Such "open" vesicles were seen on neurons (sometimes in the immediate vicinity of synapses) but also on non-nervous, extracortical as well as intracortical structures. Thus there does not seem to be a specific relationship between desmosome-like junctions and coated vesicles on the one hand and synapse formation on the other.     

Electrophysiological evidence using focal flash photolysis of caged glutamate that CA1 pyramidal cells receive excitatory synaptic input from the subiculum

The hippocampus sends efferent fibers to the subiculum, which projects to the entorinal cortex. Previous studies suggest that the hippocampal CA1 area may receive a projection back from the subiculum. This hypothesis was tested using whole cell recording from CA1 pyramidal cells while subicular neurons were selectively stimulated with focal flash photolysis of caged glutamate, which avoids stimulation of fibers of passage. Control experiments showed that focal flash stimulations caused direct glutamate-mediated depolarizations and bursts of action potentials in the recorded CA1 pyramidal cells, but only when the stimulation targeted the somatodendritic regions of a neuron, not the axons. To block GABA(A)-mediated inhibition and isolate local excitatory circuits, bicuculline was applied to minislices containing only the isolated CA1 area and the subiculum. Of 24 CA1 pyramidal cells, 25% (6 of 24) consistently generated repetitive excitatory postsynaptic currents (EPSCs) in response to flash stimulation in the subiculum. The responsive neurons were located 200-500 microm from the distal end of CA1 and 400-1,100 microm from the stimulation sites in subiculum, suggesting excitatory synaptic projections from the subicular neurons to CA1 pyramidal cells. This study provides new electrophysiological evidence that CA1 pyramidal cells receive excitatory synaptic input from the subiculum. Thus a reciprocal excitatory synaptic circuit connects the subiculum and the CA1 area in the normal adult rat.     

A freeze-fracture study of synaptogenesis in the distal retina of larvalXenopus

Summary Synapse formation between photoreceptor, bipolar and horizontal cells of the larvalXenopus retina was studied by the freeze-fracture technique. Photoreceptors and horizontal cells were joined by ribbon synapses; photoreceptor and bipolar cells by basal junctions. Gap junctions were found between photoreceptors and between horizontal cells. Horizontal cell dendrites invaginated receptor bases before the plasma membrane of either cell showed zones of intramembrane (IMP) particle accumulation. Subsequently the receptor cell began to form a synaptic ridge where P-face IMPs aggregated at a protrusion of the surface membrane. The length of the ridge and the density of its IMPs increased between larval stages 40 and 56. Cross-fractured views of receptor cytoplasm at different larval stages showed that synaptic ribbons and synaptic vesicles developed in conjunction with the ridge. Plasmalemmal deformations suggesting sites of vesicle fusion or uptake were noted adjacent to the apex of the ridge. Horizontal cell dendritic membrane first accumulated P-face IMPs at several small regions; subsequently the IMPs became aligned over a broad membrane area. Both rod- and cone-related horizontal cell dendrites also manifested a loose patch of E-face IMPs which subsequently was transformed into a linear array. Basal junctions were characterized by a P-face IMP aggregate in the photoreceptor membrane and an E-face IMP aggregate in the bipolar cell membrane. Basal junctions appeared suddenly in a mature configuration at larval stage 42.     

Regulatory role of the cell adhesion molecule nectin‐1 in GABAergic inhibitory synaptic transmission in the CA3 region of mouse hippocampus

Proper operation of a neural circuit relies on both excitatory and inhibitory synapses. We previously showed that cell adhesion molecules nectin‐1 and nectin‐3 are localized at puncta adherentia junctions of the hippocampal mossy fiber glutamatergic excitatory synapses and that they do not regulate the excitatory synaptic transmission onto the CA3 pyramidal cells. We studied here the roles of these nectins in the GABAergic inhibitory synaptic transmission onto the CA3 pyramidal cells using nectin‐1‐deficient and nectin‐3‐deficient cultured mouse hippocampal slices. In these mutant slices, the amplitudes and frequencies of miniature excitatory postsynaptic currents were indistinguishable from those in the control slices. In the nectin‐1‐deficient slices, but not in the nectin‐3‐deficient slices, however, the amplitude of miniature inhibitory postsynaptic currents (mIPSCs) was larger than that in the control slices, although the frequency of the mIPSCs was not different between these two groups of slices. In the dissociated culture of hippocampal neurons from the nectin‐1‐deficient mice, the amplitude and frequency of mIPSCs were indistinguishable from those in the control neurons. Nectin‐1 was not localized at or near the GABAergic inhibitory synapses. These results indicate that nectin‐1 regulates the neuronal activities in the CA3 region of the hippocampus by suppressing the GABAergic inhibitory synaptic transmission.     

Cell junctions and membrane specializations in the ventricular zone (germinal matrix) of the developing sheep brain: A CSF-brain barrier

Summary Cell junctions in the ventricular zone (germinal matrix) of the embryonic and foetal sheep brain were examined with thin-section and freeze-fracture electron microscopy. Neuroependymal cells in the early ventricular zone (days 19–40 of embryonic development, gestation period 147 days) exhibit a novel arrangement of cell junctions that connect adjacent neuroependymal cells at their lateral cell membranes next to the ventricular system. Small but typical gap junctions were also identified from the earliest stages examined. In serial thin sections and using a goniometer with a tilting device, the cell contacts showed a tight junction-like appearance of close and continuous fusion between neighbouring cell membranes. However, they were not arranged in a belt-like fashion close to the ventricular surface, but spiralled from the ventricular pole of the cells along the lateral cell membrane towards the deeper parts of the ventricular zone. Their freeze fracture appearance was different from that of single-stranded tight junctions in that the dimensions of their ridges and grooves were generally greater and the E-face grooves contained many particles. The junctions were especially prominent where more than two cells made contact. At mid-gestation they were less prominent than earlier and at 125 days gestation the neuroependymal layer was replaced by a mature-looking normal ependymal layer in which individual ependymal cells were connected by zonulae adherentes and large gap junctions; orthogonal arrays were also prominent. The close contact between gap junctions and single-stranded junctions found early in gestation suggests that there may be some developmental relation between these two membrane specializations.The transient single-strand junctions presumably form the morphological basis for a recently described CSF-brain barrier in the early foetal sheep brain. They may also have some mechanical function in anchoring neighbouring cells together in the region of the developing brain where cells are continuously dividing and migrating.     

Local circuit connections between hamster laminae III and IV dorsal horn neurons

To better understand the role of intrinsic spinal cord circuits in the integration of mechanosensory information, we studied synaptic transmission between neurons in Rexed's laminae III-IV, a major termination zone for cutaneous mechanoreceptor afferents, using dual, simultaneous whole cell electrophysiological recordings in young hamsters. Synaptic connections were detected between 32 of 106 cell pairs (linkage probability of 0.3) and were predominantly unidirectional (91%). Inhibitory connections outnumbered excitatory connections by 2:1. Amplitude of single-axon postsynaptic potentials (PSPs) was independent of postsynaptic cell input resistance. Intracellular labeling suggested that recordings were obtained from local axon interneurons. In connected cell pairs, the percentage of presynaptic action potentials that failed to evoke a postsynaptic response was 44 +/- 29%. Shape indices of PSPs suggested that synaptic contacts were widely distributed along the postsynaptic membrane. Linkage probability was unrelated to intrinsic firing properties, laminar position of the cells or the distance (<160 mum) separating them. However, PSPs in target cells following action potentials in neurons with phasic firing patterns had longer duration and lower failure rates than PSPs activated by neurons with tonic firing patterns. Thus transmission reliability at synapses between lamina III/IV interneurons overall is low, and efficacy of these connections is related to firing properties of the presynaptic cells. The observations also suggest that synaptic organization in LIII-IV is fundamentally different from the superficial dorsal horn (LI-II) where neural circuits may be composed of stereotyped units made from connections between a few functional types of neurons.     

Characterization of mitotic neurons derived from adult rat hypothalamus and brain stem

Embryonic or neonatal rat neurons retain plasticity and are readily grown in tissue culture, but neurons of the adult brain were thought to be terminally differentiated and therefore difficult to culture. Recent studies, however, suggest that it may be possible to culture differentiated neurons from the hippocampus of adult rats. We modified these procedures to grow differentiated neurons from adult rat hypothalamus and brain stem. At day 7 in tissue culture and beyond, the predominant cell types in hypothalamic and brain stem cultures had a stellate morphology and could be subdivided into two distinct groups, one of which stained with antibodies to the immature neuron marker alpha-internexin, while the other stained with the astrocyte marker GFAP. The alpha-internexin positive cells were mitotic and grew to form a characteristic two-dimensional cellular network. These alpha-internexin positive cells coimmunostained for the neuronal markers MAP2, type III beta-tubulin, and tau, and also bound tetanus toxin, but were negative for the oligodendrocyte marker GalC and also for the neurofilament triplet proteins NF-L, NF-M, and NF-H, markers of more mature neurons. Patch-clamp analysis of these alpha-internexin positive cells revealed small Ca(2+) currents with a peak current of -0.5 +/- 0.1 pA/pF at a membrane potential of -20 mV (n = 5) and half-maximal activation at -30 mV (n = 5). Na(+) currents with a peak current density of -154.5 +/- 49.8 pA/pF at a membrane potential of -15 mV (n = 5) were also present. We also show that these cells can be frozen and regrown in tissue culture and that they can be efficiently infected by viral vectors. These cells therefore have the immunological and electrophysiological properties of immature mitotic neurons and should be useful in a variety of future studies of neuronal differentiation and function.     

An electron microscope study of the development of the exocrine and endocrine pancreas with special reference to intercellular junctions

Intercellular junctions in pancreatic acinar, duct and endocrine cells were studied by thin section and freeze-fracture methods in developing rats and mice. Undifferentiated cells were joined by the zonula occludens and isolated fragments of tight junctional strands. Small gap junctions were either occasionally associated with tight junctional strands or appeared independent of them. During the morphological differentiation of acinar cells, strands of the zonula occludens developed to form a complicated meshwork while gap junctions rapidly increased in size. Duct cells were joined by the less-developed zonula occludens but gap junctions were rarely seen. In the neonate, intercellular junctions were similar to those in adult acini and intercalated ducts. Endocrine cells were joined by maculae occludentes and small gap junctions. During late prenatal days, the macula occludens increased in size and gap junctions in number. Sometimes tight junctional strands disappeared to leave membrane elevations, some of which were associated with small gap junctions. Maculae occludentes on endocrine cells were gradually fragmentized and diminished during postnatal development. They were completely lost in the rat. These results suggest that intercellular junctions play important roles in pancreatic development. In particular, the transient development of maculae occludentes is associated with endocrine cell development, and intercellular communication mediated by gap junctions may be important for the differentiation of acinar and endocrine cells.