In vitro development of vertebrate central synapses.

This article deals with basic determinants of synaptic efficacy during development of glutamatergic and GABAergic synaptic transmission: location and number of release sites, release probability and single cell-activated (unitary) conductances. We hypothesize that both types of neuronal connections differ in major aspects of synaptogenesis. Disregarding the fact that various test models and cell types could render diverging results, it can be observed that glutamatergic terminals display a preference for dendrites, whereas GABAergic terminals select soma locations at initial stages of development. Glutamatergic synapses are characterised by receptor accumulation in the region of terminal apposition, whereas in GABAergic synapses receptor concentration is weak, if present at all. The expression of glutamate receptors (GluRs), but not GABAA receptors is under control of interneurons. Developmental changes in glutamatergic synaptic transmission have not yet been assessed by quantal analysis. For GABAergic synapses, first results are now available from a culture preparation of the rat superior colliculus. In general terms, functional maturation seemed to lag behind the formation of structurally differentiated release sites. Compound binomial analysis revealed that during in vitro development a considerable fraction of GABAergic terminals remained in a low efficacy release state (p < 0.2). A developmental increase in synaptic strength was reached by the appearance of singular highly effective release sites. Presynaptic maturation could be manipulated by long-term drug treatment. Addition of GluR antagonists significantly increased amplitudes and decreased the coefficients of variations of evoked inhibitory postsynaptic currents. Thus, the strength of inhibitory synaptic transmission could be influenced by the status of heteronymous synaptic input.

Volumetric and histological changes in the cochlear nuclei of visually deprived rats: a possible morphological basis for intermodal sensory compensation.

The cochlear nucleus of 90 day-old rats was studied to determine whether bilateral visual deafferentation at birth induced compensatory structural changes in the auditory system. Computer-assisted reconstructions of the cochlear nucleus were used to demonstrate the three-dimensional organization of the nucleus and for calculating volumes of the subdivisions. In enucleated rats the volume of the granular region of the nucleus (GCD) increased 22.6% relative to controls, and the volume of the anterior ventral nucleus (AVCN) also increased by 10.0%. The posterior ventral nucleus (PVCN) and dorsal nucleus (DCN) were not affected. Since the volume change in GCD was substantial, this region was subjected to further analysis. Two types of granular cells were identified in the GCD of both control and enucleated rats with Kluver-Barrera staining. Type 1 cells contained an ovoid dark nucleus with clumpy chromatin and possessed only a very thin rim of cytoplasm. Type 2 cells were larger with pale spheroidal nuclei and with a more prominent cytoplasmic rim. There was an obvious stratification of these two cell types in the superficial layers over the lateral aspect of the AVCN in both groups. In contrast to this, the subpeduncular corner was not distinctively laminated and contained predominantly Type 2 cells. In enucleated animals the nuclear size of Type 1 cells increased by 3.8% while the size of Type 2 nuclei was not affected. Granule cell numbers increased by 28% in the visually deafferented rats. The GCD seems to be more highly developed in species which are dependent on non-visual modalities for spatial location. The regional hypertrophy of the GCD and AVCN seen in the present study may indicate that intermodal sensory compensation is taking place in the auditory system of visually deafferented rats.

Development of GABAergic synaptic connections in vivo and in cultures from the rat superior colliculus.

Synaptic activity in the superficial (i.e. visual) layer of the superior colliculus was investigated with intracellular microelectrodes using a preparation of the isolated superfused tectum from neonatal rat. It was found that by postnatal day 9 (i.e. before eye opening) the majority of neurons in the superficial gray layer (SGS, stratum griseum superficiale) were already capable of generating Cl(-)-dependent inhibitory postsynaptic potentials (IPSPs) in response to intracollicular stimulation. Properties and development of GABAergic synaptic connections were further characterized in a dissociated cell culture from the SGS. The cultures were prepared from E21 rat embryos and studied between 1 and 38 days in vitro (DIV). gamma-[3H]aminobutyric acid ([3H]GABA) uptake served to identify GABAergic neurons and to estimate their relative density. Axon terminals were labeled by indirect immunostaining for glutamic acid decarboxylase (GAD) and examined with light (LM) and electron microscopy (EM). Responsiveness to exogenous and endogenous GABA was investigated by recording ionic currents with patch clamp techniques. [3H]GABA uptake-positive neurons constituted about 40% of the whole cellular population dissociated from the SGS of E21 rats. After 2 weeks in culture, [3H]GABA uptake was observed in 45-60% of the cells with neuronal features. The relative number of GAD-immunoreactive neuronal perikarya ranged from 28 to 39%, after 2 weeks in vitro. Responsiveness to exogenous GABA was found in all freshly plated neurons. Release of GABA could be demonstrated after 2 DIV by recording spontaneous bicuculline-sensitive Cl- currents. These currents had the characteristics of GABAA receptor-mediated synaptic currents. However, even as late as DIV 6, very few vesicle-containing axonal terminals apposing postsynaptic specializations were revealed with EM. GAD-labeled puncta became clearly visible only after DIV 10-12. Between DIV 14 and 21, the intensity of immunostaining and the density of GAD-labeled synaptic contacts increased, reaching a maximum around DIV 28. GAD-positive puncta covered both neurons and non-neuronal cells. At the level of EM, GAD-positive terminals were shown to establish synaptic contacts with neuronal somata and processes, forming in the majority of cases (22 out of 32 stained terminals) symmetrical contacts. It is concluded that in the SGS of the rat superior colliculus GABAergic neurons and GABAA receptors are present before birth. In dissociated cell cultures ionic currents can be generated in response to endogenous GABA before axonal terminals of GABAergic neurons fully mature. Finally, our experiments show that visual activity is not a prerequisite for the formation of GABAergic synapses between neurons of the SGS.

Synaptogenesis in the stratum griseum superficiale of the rat superior colliculus.

The time course of synaptogenesis in the visual part of the superior colliculus (SC) of pigmented rats has been studied. The number of synaptic profiles per unit area and volume of neuropil in the stratum griseum superficiale (SGS) was estimated in seven groups of animals at ages 3, 9, 15, 21, 30, 49 and 85 days after birth. At 3 days only 1.5 +/- 0.06 synaptic contacts per unit area and 5.5 +/- 0.18 per unit volume were found. Most of them were immature contacts between growing processes. The density of synaptic contacts increased slowly during the first week. By day 9, 4.1 +/- 0.25 and 13.4 +/- 0.66 synaptic contacts were counted per unit of area and volume, respectively. A rapid synaptic proliferation occurred during the next 3 weeks and there were 7.7 +/- 0.27 and 25.5 +/- 1.04 synaptic contacts per unit area and volume at 15 days, 21.1 +/- 1.70 and 86.4 +/- 5.11 at 21 days, and 25.9 +/- 1.20 and 96.7 +/- 3.48 at 30 days. At the same time, the synaptic population gradually acquired more mature morphological characteristics: the pre- and postsynaptic structures became more specialized, the number of synaptic vesicles within presynaptic structures increased, and the synaptic junctional apposition became defined. After 30 days, a decrease in the density of synaptic profiles was recorded: 23.9 +/- 0.44 and 79.8 +/- 1.43 per unit area and volume of neuropil at 49 days and 13.4 +/- 0.53 and 49.7 +/- 2.40 at 85 days, respectively. Thus, after the phase of synaptic proliferation, a significant reduction of synaptic density occurred in the SGS neuropil until it was stabilized at the adult level by the third month of life. Considering the data available on the development of the retinal and visual cortical projections to the SC, the process of synaptic elimination, which takes place after the first postnatal month, does not appear to be directly connected with segregation of these particular projections.

Visual thalamocortical projections in normal and enucleated rats: HRP and fluorescent dye studies.

Visual thalamocortical projections of neonatally enucleated and control rats were studied after tracer injections into the striate and peristriate areas of adult pigmented rats. The distribution of retrogradely labeled neurons in the visual thalamic nuclei was mapped after (a) small localized injections of horseradish peroxidase into either area 17, 18, or 18a and (b) simultaneous injections of three different retrograde tracers (fast blue, HRP, and diamidino yellow) into the anterior, medial, and posterior regions of area 17. It was shown in both normal and neonatally enucleated rats, that the dorsal lateral geniculate nucleus projects to the striate cortex (area 17), whereas the laterodorsal thalamic nucleus of the lateral thalamus projects to the medial peristriate area 18, and the lateral posterior thalamic nucleus has a projection to the lateral peristriate area 18a. Additionally, both extrageniculate visual thalamic nuclei project to area 17. Neurons in the dorsoanterior region of the dorsal lateral geniculate nucleus project to the posterior part of area 17, while neurons in the ventroposterior region of the nucleus send their axons to the anterior part of area 17. A similarly inverted projection of anterior and posterior divisions of the lateral posterior thalamic nucleus to visual area 18a was detected. In enucleated rats, the general topography of the projections from the thalamic neurons to the striate and peristriate cortices was indistinguishable from that in the controls. Nonetheless, there was noticeable shrinkage of the dorsal lateral geniculate nucleus and lateral thalamus and a significant decrease in the size of the somata of projecting neurons. Mean somal area of the HRP-labeled neurons in the dorsal lateral geniculate nucleus of enucleated rats was reduced by 19.0% and the mean maximum cell diameter by 14.3% compared with controls.

The morphology of neurons in rat tectal transplants as revealed by Golgi-Cox impregnation.

The Golgi technique has been used to examine the morphology of neurons within tectal transplants. Embryonic tectal tissue was transplanted to the midbrain of newborn rats. Four to eight months later, host animals were decapitated under anaesthesia, the unfixed brains removed and processed by Golgi-Cox impregnation. In tectal grafts, different types of neuron were recognized on the basis of the size and shape of their somata and the morphology of their dendritic trees. Neuronal types found in transplants resembled cell classes found in normal rat superior colliculus (SC). Neurons characteristic of the superficial collicular layers such as marginal, ganglion type I, stellate and horizontal cells and multipolar cells typical of the deeper collicular layers were identified in the transplants. Compared with normal cells, grafted neurons often had smaller dendritic fields and fewer dendritic spines. No laminar organization was discernable in the grafts and there was commonly no preferential orientation of perikarya or dendrites. Small cells with similar dendritic morphology were sometimes found grouped together in patches within the graft neuropil. These patches resembled cytologically and histochemically distinct areas described in previous studies and may represent areas homologous to the superficial layers of normal SC.

Postnatal development of the superficial layers in the rat superior colliculus: a study with Golgi-Cox and Klüver-Barrera techniques.

The postnatal development of the superficial (optic) layers of the rat superior colliculus has been studied using Klüver-Barrera staining and Golgi impregnation in rats aged 3-45 days. The Klüver-Barrera staining reveals that the SC of 3 day old rats is morphologically immature with no obvious lamination. It contains densely packed cells of uniform size. The packing density of the cells gradually decreases between 9 and 15 days as the thickness of the layers increases. The first myelinated fibres in the SC appear at 15 days but the stratum opticum is still not recognizable. By 30 days, the SC has a distinctly laminated appearance, but the thickness of the superficial layers continues to increase until day 45 postnatal. Golgi-Cox impregnation displays the range of neuronal types in the superficial layers of the SC previously described by Langer and Lund (1974). Using the morphological criteria of these authors for classification of the neurons, the developmental changes of the marginal cells, horizontal cells, ganglion cells types I, II, III and stellate cells have been followed. The SC of 3 day old rats contains immature neurons; only a few larger cells have branched dendrites. In 9 days old SC the neuronal types present in the adult are recognizable, although their appearances are still immature. By 15 days neurons have adult-looking dendritic trees but dendritic growth continues beyond 30 days. The visual part of the SC has a protracted period of postnatal development, the sequence of developmental changes being similar for the different types of collicular neurons. Features common to development are the increasing size of neuronal somata, the increasing length of dendrites and the acquisition of a complex pattern of dendritic arborization. Larger cells appear to commence development earlier than small cells, although the rate of developmental changes is different for each of the various types of collicular neurons.

Postnatal development of cat superior colliculus. I. Presumptive neurons, immature neurons and degenerating cells in the intermediate and deep layers.

Presumptive neurons, immature neurons and degenerating structures were found in the intermediate and deep layers of the superior colliculus of normal cats over the age range 7-45 days postnatal. Electron microscopical analysis revealed these structures to be unexpectedly frequent in the early postnatal period. There were 34 immature neurons per 1000 neurons at 7-8 days and 78 per 1000 neurons at 14 days postnatal. Few were found in older cats. Two types of degenerative changes in the neurons were observed: nuclear and cytoplasmic. The number of degenerating structures to be unexpectedly frequent in the early postnatal period. There were 34 immature neurons per 1000 neurons at 7-8 days and 78 per 1000 neurons at 14 days postnatal. Few were found in older cats. Two types of degenerative changes in the neurons were observed: nuclear and cytoplasmic. The number of degenerating neurons was higher at 14 days (28 per 1000 neurons) than at 7-8 days (21 per 1000 neurons), but only a few were found at 30 and 45 days. Degenerative changes were noted in synaptic terminals at all ages studied. The greatest number of degenerating synaptic profiles were observed at 14 and 30 days. They were infrequent in younger or older animals. The number of presumptive neurons does not change significantly over 7-14 days. It is speculated that they persist as a reverse pool of neurons. The increasing number of immature neurons in the period between 7 and 14 days indicates that formation of new cells in the intermediate and deep layers of the superior colliculus continues into the early postnatal period. The degeneration of some neurons and synapses over the same developmental period suggest that the final adjustment of the neuronal composition of the superior colliculus occurs postnatally.

Ultrastructural organization of giant neurones of the mollusc Lymnaea stagnalis under different environmental temperatures.

The ultrastructure of identified giant neurones of the visceral ring of Lymnaea stagnalis ganglion alters with the seasonal change of the animal and, experimentally, from the inactive physiological state (winter time or at +4 degrees C) to an active one (spring-summer time or at +18 degrees C). The ultrastructural organization of the active animal's neurones is characterized by morphological alteration pointing to an increased metabolic activity, viz., an increased number of nucleoli, an enlarged surface of nuclear membrane and an increase in the nuclear membrane pores, appearance of a zone of free ribosomes near the nuclear membrane, changing the structure of cytosomes, abundant granular endoplasmic reticulum, increase in the number of mitochondria.