Effects of dark-rearing on the vascularization of the developmental rat visual cortex

Cerebral vascular density corresponds to metabolic demand, which increases in highly active areas. External inputs play an important role in the modeling and development of the visual cortex. Experience-mediated development is very active during the first postnatal month, when accurate simultaneous blood supply is needed to satisfy increased demand. We studied the development of visual cortex vascularization in relation to experience, comparing rats raised in darkness with rats raised in standard conditions. The parameters measured were cortical thickness, vascular density and number of perpendicular vessels, constituting the first stage of cortical vascular development. Vessels were stained using butyryl cholinesterase histochemistry, which labels some neurons and microvascularization (vessels from 5 to 50 μm). Animals from both groups were sampled at 0, 7, 14, 21 and 60 days postnatal. Vascularization of the brain starts with vertically oriented intracortical vascular trunks whose density decreases notably after birth in rats reared in standard laboratory conditions. The most striking finding of our work is the significantly lower decrease in the number of these vessels in dark-reared rats. Our results also show that cortex thickness and vessel density are significantly lower in dark-reared rats. These results suggest that the absence of visual stimuli retards the maturation of the visual cortex including its vascular bed.     

Lack of experience-mediated differences in the immunohistochemical expression of blood-brain barrier markers (EBA and GluT-1) during the postnatal development of the rat visual cortex

The development of the cortical vascular tree depends on functional development. External inputs are an essential requirement in the modeling of the visual cortex, mainly during the critical period, when congruous blood supply is needed. The blood brain barrier (BBB) function regulates the passage of substances between the blood and the brain parenchyma, which is one of the main differential features of central nervous system (CNS) microvessels. The endothelial barrier antigen (EBA) has been reported as a specific marker for the BBB physiological function in rats. We studied the postnatal development of EBA expression in the visual cortex of rats reared under opposite paradigms of visual experience, e.g., standard laboratory conditions, dark rearing, and enriched environment at 14, 21, 28, 35, 42, 49, 56, and 63 days postnatal (dpn). Parallel sections were immunohistochemically processed for endothelial barrier antigen (EBA) and glucose transporter-1 (GluT-1). Total vasculature was quantified by Lycopersicon esculentum (LEA) lectin histochemistry. No differences in EBA expression were found between groups, although quantitative differences were recorded paralleling differences in vascular density. Paradoxically, there was no expression in certain cortical vessels which were GluT-1 immunopositive and positivity was consistent in non-barrier areas such as the pineal gland. These findings were completely independent of age or experimental conditions. Therefore, the role of the EBA antigen in the BBB remains unclear: it has been undeniably linked to vascular permeability, but its presence in non-barrier vessels suggests another vascular function. Although visual experience modifies vascular density in the visual cortex, it has not been shown to have an influence on the maturation of the BBB function.     

Visual deprivation effects on the s100beta positive astrocytic population in the developing rat visual cortex: a quantitative study

After birth, exposure to visual inputs modulates cortical development, inducing numerous changes of all components of the visual cortex. Most of the cortical changes thus induced occur during what is called the critical period. Astrocytes play an important role in the development, maintenance and plasticity of the cortex, as well as in the structure and function of the vascular network. Dark-reared Sprague-Dawley rats and age-matched controls sampled at 14, 21, 28, 35, 42, 49, 56 and 63 days postnatal (dpn) were studied in order to elucidate quantitative differences in the number of positive cells in the striate cortex. The astrocytic population was estimated by immunohistochemistry for S-100beta protein. The same quantification was also performed in a nonsensory area, the retrosplenial granular cortex. S-100beta positive cells had adult morphology in the visual cortex at 14 dpn and their numbers were not significantly different in light-exposed and nonexposed rats up to 35 dpn, and were even higher in dark-reared rats at 21 dpn. However, significant quantitative changes were recorded after the beginning of the critical period. The main finding of the present study was the significantly lower astroglial density estimated in the visual cortex of dark-reared rats over 35 dpn as well as the lack of difference at previous ages. Our results also showed that there were no differences when comparing the measurements from a nonsensory area between both groups. This led us to postulate that the astrocytic population in the visual cortex is downregulated by the lack of visual experience.     

Postnatal development of neurons and synapses in the visual and motor cortex of rabbits: a quantitative light and electron microscopic study

Introductory to a morphological investigation on the effects of early visual deprivation and on the critical periods in early postnatal life we have studied quantitatively the normal postnatal growth of neurons and synapses in the visual and motor cortex of rabbits. The major results of this analytical study are: (1) rapid decrease in neuron density and a rapid increase in neuronal volume are observed. They are almost completed at postnatal Day 10, i.e., before natural eye opening. The drop in neuron density is caused to a very large extent by an increase in cortical volume and not by a considerable disappearance of neurons; (2) the formation of synaptic contact zones starts at Day 6 to 7 and is most pronounced between Day 10 and Day 21, i.e., after natural eye opening. At Day 27 synaptic density has reached adult levels in the visual cortex and is in excess of the adult level in the motor cortex. In visual area I and in the motor cortex a significant difference in synaptic increase is observed between the left and right hemisphere, resulting in a lower synaptic density in the left counterparts at Day 27 and in adult animals [56,57]. In the visual cortex a small but highly correlated increase in synaptic vesicle density is observed. In the motor cortex no correlated relation between age and vesicle density is observed. In both cortical areas synaptic vesicle density has reached about 70 percent of the adult level at Day 27; and (3) in newborn and young rabbits the motor cortex seems to be more mature than the visual cortex.     

The effect of neonatal 6-hydroxydopamine treatment on synaptogenesis in the visual cortex of the rat

It has been proposed repeatedly that the noradrenergic (NE) system may exert an influence on cortical development. We have tested this proposition by examining synaptogenesis in the visual cortex of rats whose NE afferents were selectively lesioned by injections of the neurotoxin 6-hy-droxydopamine (6-OHDA). Control littermates were injected with equal volumes of vehicle. Montages of electron micrographs covering approximately 50 μm-wide strips of cortex were assembed from both groups of animals at 2,4,6,8,14, and 90 days of age. Symapses counts revealed a significantly higher density of synapses in the cortex of 6-OHDA-treated rats during the first week of postnatal life. The difference between the experimental and control rats was less apparent during the second postnatal week, and at day 90 the densities of synapses were similar for the two groups of animals. The enhanced density, which was the result of the increased number of Gray's type I synapses, was confined to the subplate region at day 2 but became more widespread in the cortex at subsequent stages of development. From these observations it would appear that the NE system exerts an inhibitory influence on synapse formation in the visual cortex in early postnatal life.     

A critical period of the development of beta-adrenergic receptor binding in the visual system of rat during visual deprivation

The density of [3H]dihydroalprenolol binding to beta-adrenergic receptors in the visual structures (visual cortex, superior colliculus and lateral geniculate nucleus) of rats raised under a normal 12 hr light-dark cycle was compared to those of rats visually deprived at different postnatal ages. Unilateral eyelid suture from postnatal days 10 or 16 to 3 months resulted in an increased [3H]dihydroalprenolol bilateral binding in the lateral geniculate nucleus compared to control animals. Monocular deprivation from postnatal days 25, 40, 60 and 90 had no effect on the density of [3H]dihydroalprenolol binding. After re-opening of the eyelid, which was sutured on postnatal day 10, at postnatal day 25 no changes in beta-adrenergic receptor binding in the lateral geniculate nucleus of the adult animal could be detected. After re-opening of the sutured eyelid on day 90, followed by examination of the adrenoceptor density 4 weeks later, the [3H]dihydroalprenolol binding in both lateral geniculate nuclei remained elevated as was also found in corresponding regions of monocular deprived animals. Binocular visual deprivation from postnatal day 10 until the age of 3 months had no effect on [3H]dihydroalprenolol binding in the visual centres in comparison to corresponding control animals. The data suggest that there exists a critical period for the ontogenetic development of beta-adrenergic receptors binding in the visual system of rats during which permanent alterations of receptor binding can be induced by monocular but not binocular visual deprivation.     

Differential rearing effects on rat visual cortex synapses.: III. Neuronal and glial nuclei, boutons, dendrites, and capillaries

Morphological measures of neurons, astrocytes, oligodendrocytes, presynaptic boutons, dendrites and capillaries were examined in the upper 4 layers of occipital cortex in rats reared for 30 days postweaning in complex (EC), social (SC) or individual cage (IC) environments. EC rats had a lower numerical density of neuronal nuclei with a comparable volume fraction to SC and IC rats. The volume fraction of astrocyte and oligodendrocyte nuclei was significantly greater for EC rats than IC littermates, and IC rats also had more synapses and neurons/micron3 of glial nuclei. Environmental groups did not differ in the numerical density of presynaptic boutons but the number of boutons per neuron was greater in EC than in IC or SC rats. This result parallels the findings that EC rats have more synapses per neuron than IC rats. Electron microscopic estimates of dendritic volume fraction confirmed estimates from Golgi-impregnated neurons that there is more dendrite per neuron in the occipital cortex of EC rats than IC or SC rats. EC rats also had a larger capillary volume than SC or IC and these capillaries were closer together and had fewer synapses/micron3 of capillary in ECs. Another indicator of metabolic activity, mitochondria volume per neuron, gave similar results with ECs having a greater volume than ICs and SCs intermediate. These results indicate that not only are there more synapses per neuron in the visual cortex of rats from more complex environments but also that the brain appears to adjust to the metabolic requirements of its synapses or neurons, in terms of vascular, mitochondrial and glial support.     

Development of chloride homeostasis in albino and pigmented rat visual cortex neurons

Albinism has a profound effect on visual development and visual function. Pharmacologically significant alterations of the two most important chloride-transporters--KCC2 (outward transporter) and NKCC1 (inward transporter)--functions were found in albino visual cortex neurons, comprising a higher NKCC1 and a lower KCC2 action. In this study, we compare the early postnatal development of the reversal potential of gamma-aminobutyric acidAR-mediated currents in visual cortex neurons of albino and pigmented rats. At birth we found no differences. At the time of eye opening (second week postnatally) the reversal potential of gamma-aminobutyric acidAR-mediated currents is 15 mV more positive and intracellular Cl- concentration is higher in visual cortex neurons of albinos than of pigmented rats.     

Cell death in the development of the posterior cortex in male and female rats.

Work from our laboratory has shown that adult male rats have 19% more neurons than female rats in the binocular region and 18% more in the monocular region of the primary visual cortex (Reid and Juraska [1992] J Comp Neurol 321:448-455; Nu?ez et al., [1999] Soc Neurosci Abstr 25:229). In the current experiment, we investigated whether cell death in male and female rats (postnatal days 2-35) contributes to the formation of these differences. Using stereological techniques, we investigated neuron density along with pyknotic and apoptotic (TdT-mediated deoxyuridine triphosphate nick end-labeled) cell density in the developing posterior cortex (future primary visual cortex). Although no sex differences in neuronal density were found in early development, we observed a differential time course of cell death between the sexes. Consistent with earlier reports, males displayed a rapid rise in cell death, with a peak on day 7 followed by a sharp decline to negligible levels by day 15. Females, however, displayed moderate peaks of cell death on days 7 and 11, with the persistence of low-to-modest levels until day 25. Similar patterns were obtained from both pyknotic and apoptotic cell quantification. Also, a formula was developed to estimate the percentage of cells that die during development and the amount of time a dying cell is visible. This study demonstrates that there is a prolonged period of cell death in the posterior cortex of developing female rats that appears to result in more cell death in females than males. This may be an important mechanism by which the sex difference in adult neuron number is created.     

Mismatch between BDNF mRNA and protein expression in the developing visual cortex: the role of visual experience

Brain-derived neurotrophic factor (BDNF) messenger RNA (mRNA) expression in the rat visual cortex of young and postnatal day 90 (P90) animals is developmentally regulated and influenced by visual experience. In the present paper we compared the expression of BDNF mRNA to the actual changes of BDNF protein occurring during postnatal development and verified whether BDNF protein distribution is controlled by visual activity. To achieve this aim we analysed BDNF mRNA and/or BDNF protein cellular distribution in the rat visual cortex at different postnatal ages by using immunohistochemistry and highly sensitive in situ hybridization. We found that before eye opening (P13), in all cortical layers a large number of visual cortical neurons contain BDNF mRNA with no detectable amount of BDNF protein. At later ages (P23 and P90), the number of BDNF-immunostained cells increases; most neurons are double labelled for BDNF mRNA and protein, and a small group of neurons is labelled only for BDNF protein. The cellular increase of BDNF immunolabelling is blocked in animals deprived of visual experience from birth (dark rearing), with a large population of neurons containing BDNF mRNA but not BDNF protein. This is similar to what is observed before eye opening. Exposure of dark-reared rats to a brief period (2 h) of light restores a good match between BDNF mRNA and BDNF protein cellular expression. We propose that visual experience controls the neuronal content of BDNF mRNA and BDNF protein in developing visual cortex.     

Sex differences in the number of synaptic junctions in the binocular area of the rat visual cortex

We had found that the binocular area of the visual cortex is larger in volume and has more neurons in male than in female rats. The present study examined whether the number of synaptic junctions in this area is sexually dimorphic. Ten littermate pairs of 90-day-old (socially housed) Long-Evans hooded rats were used. Synaptic junctions were counted and their lengths were measured on electron micrographs taken from layers II–III of the binocular visual cortex. There were no sex differences in the numerical density of synaptic junctions, the number of synaptic junctions per neuron, or the length of synaptic junctions within any synaptic category or of all synapses combined. Sex differences were found in the total number of synaptic junctions and in several categories (asymmetric synapses, spine synapses, asymmetric spine synapses): male rats had more synaptic junctions than female rats because of the larger volume of layers II--III in the binocular area of male rats. These data indicate that neurons in the binocular visual cortex of both male and female rats receive a characteristic number of synaptic junctions, but the greater number of neurons in the binocular area of male rats results in more synaptic junctions in the area. © 1995 Wiley-Liss, Inc.     

Differential rearing effects on rat visual cortex synapses. I. Synaptic and neuronal density and synapses per neuron

The bulk of the evidence indicating that different experiences can lead to differences in synapse numbers involves inference from measures of postsynaptic surface (spines and dendrites) in Golgi impregnated tissue. The capriciousness of Golgi impregnation and the absence of direct evidence regarding changes in afferents mandate confirmation of synapse changes by electron microscopy. We calculated the ratio of synapses per neuron in layers I-IV of occipital cortex of rats reared in complex (EC), social (SC), or isolated (IC) environments. Synaptic density estimates were derived from electron micrographs of osmium-uranyl-lead stained tissue and neuronal density estimates were derived from toluidine blue stained semithin sections using stereological methods which correct for group differences in the sizes of synapses and neuronal nuclei. The ratio of these densities, synapses per neuron, was highest in complex environment rats, intermediate in socially reared rats and lowest in isolates, in accordance with predictions from prior Golgi studies. The bulk of the differences were attributable to neuronal density, which was highest in IC rats and lowest in ECs. Synaptic density did not differ statistically across groups. These results indicate, at least within this area and paradigm, that differences in dendritic measures in Golgi impregnated tissue reflect differences in the number of synapses per neuron.     

The blood supply of the cat's visual cortex and its postnatal development

We examined the blood supply of the cat's visual cortex using alkaline phosphatase histochemistry to demonstrate the capillary endothelial cells. In the adult, layer 4 is marked by a band that is of obviously greater density, extends throughout areas 17 and 18, and ends abruptly at the 18/19 border. We quantified blood vessel density in area 17, observing a 23% greater density in layer 4 than in supragranular and infragranular layers. This difference reflects a laminar difference in metabolic rate. In three animals studied using the metabolic marker 2-deoxyglucose, layer 4 was 25% denser than the other layers. The band of greater density in layer 4 is not present in newborn kittens, but becomes apparent at about 5 weeks of age. Early in development, the endothelial cells form filopodia as the capillaries grow and branch. The density of blood vessels decreases slightly during the first week of postnatal life, but increases between 1 and 6 weeks of age, so that by 6 weeks, the blood supply of the visual cortex resembles that seen in the adult. This pattern resembles that of cortical metabolism seen with 2-deoxyglucose [J. Cereb. Blood Flow Metab. 11 (1991) 35], but the increase in vascular density precedes that in glucose metabolism.     

The development of somatostatin immunoreactive neurons in cat visual cortical areas

The development of somatostatin immunoreactive (SOM-ir) neurons in cat striate and extrastriate cortex was studied to determine whether temporal changes in the morphology, distribution and density of SOM-ir neurons during development would provide clues to the emergence of specific cortical areas. The visual cortical areas examined included areas 17–19 and 7, posteromedial lateral suprasylvian, posterolateral lateral suprasylvian cortex and splenial visual area. We observed that the pattern of SOM-ir neurons in the cortical plate reflects the maturation of the cortical plate. At 1 week of age, SOM-ir neurons were only found in layers V and VI of the developing cortex; by 2 weeks of age, SOM-ir neurons were found in layer IV; and by 3 weeks of age, SOM-ir neurons were located in all layers of the cortex except layer I. SOM-ir neurons in the subplate were much more numerous under lateral cortical areas than under medial areas. This difference decreased over the first 2 postnatal weeks and by the 14th day after birth (P14), the distribution and numbers of SOM-ir neurons in the subplate/white matter had reached the adult pattern. The timing of exuberant SOM expression in the subplate suggests a function in the formation of visual corticocortical connections which begin to develop during the first postnatal week in the kitten.     

Postnatal vascular growth in the neocortex of normal and protein-deprived rats

Summary The postnatal vascular growth in the neocortical area 18 of normal and pre- and postnatally protein-deprived rats was examined. For control rats the specific length, the specific surface and the volume fraction of vessels increased rapidly between 7 and 20 days of age. Thereafter, only a minor increase was seen. In protein-deprived rats there was no increase in the specific length of vessels between 7 and 10 days of age and this variable was still reduced at 30 days of age compared to controls. This reduction was due to a decrease in the specific length of thin vessels (Ø<8.25 ) whereas the specific length of wider vessels was not affected by the protein deprivation. There were no significant differences in the specific surface or volume fraction of vessels between control and protein-deprived rats. These findings indicate an adaptive increase in luminal diameter of vessels in the protein deprived rats during postnatal development. At 90 days of age no significant differences between vascular variables of control and protein-deprived rats were seen.     

Areal and laminar variations in the vascularity of the visual, auditory, and entorhinal cortices of the developing rat brain

Understanding of place-specific cortical cerebrovascular changes after insult and injury depends on the detailed knowledge of the areal and laminar variations in cortical vascularity. The present study examines comparatively the developmental changes of the total vascular density and the density of capillaries and medium- and large-sized vessels in the primary visual cortex (Oc1), the primary auditory cortex (Te1), and the lateral entorhinal cortex (EntL) of the developing rat brain. Vascular networks in the three cortical areas were marked after transcardial perfusion of India ink and quantified with an image analysis system. Parameters examined exhibited (i) peculiar developmental time course within individual cortical layers and (ii) area- and age-dependent variations. Angioarchitecture in Te1 layers was stabilized earlier than that in Oc1 layers and the period of postnatal development of the vascularity of neocortical sensory areas Oc1 and Te1 appeared to be more protracted compared to that of the phylogenetically older entorhinal cortex. By the end of the first postnatal month, vascular densities in the three cortical areas established a dorsoventral gradient (Oc1 > Te1 > EntL). Finally, in all areas, layer IV was the first layer to obtain adult values of capillary density.     

Developmental changes in neuron size and density in the visual cortex and superior colliculus of the postnatal golden hamster

Postnatal changes in neuron cell body diameter and neuron density were observed in newborn, two-week old, and eight-week old golden hamsters. Quantitative data indicated that in both the visual cortex and superior colliculus, neuron cell body diameter increased nearly twofold during the first two-week period following birth and reached maximum size by approximately day 14, the stage at which eye opening usually occurs. Neuronal density was highest in the newborn and decreased with age in both visual centers. The decreased neuronal density and, thus, increased neuropil area may reflect a greater number of synaptic connections mediating visual function. Although the development in the visual cortex paralleled that in the superior colliculus, the superior colliculus seemed to mature earlier than the visual cortex, possibly related to its more primitive evolutionary role.     

Synaptic density of caudate-putamen and visual cortex following exposure to ethanol in utero

Pregnant Long-Evans rats were fed a liquid diet containing ethanol (30% of total calories) during days 3–19 of gestation. Controls were given ad libitum access to liquid diet lacking ethanol, or pair-fed isocaloric amounts based on consumption by the animals in the ethanol group. Brain development of female offspring was evaluated by analysis of electron micrographs of caudate-putamen and visual cortex. Numbers of presynaptic terminals and synaptic junctions (synaptic density) per unit area were compared for 14- and 28-day-old offspring of dams from the three treatment groups. Synaptic density of the caudate-putamen and visual cortex was not affected by ethanol at 14 or 28 days. Although exposure to ethanol during a period comparable to the first two trimesters of human development with minimal or no undernutrition did not affect numerical density of synapses in visual cortex or caudateputamen, synaptogenesis of caudate-putamen was altered in offspring of pair-fed animals.     

Long term depression is expressed during postnatal development in rat visual cortex: a role for visual experience

Long term forms of synaptic plasticity and in particular LTD/LTP are both present in the mammalian visual cortex. However, while LTP is not inducible in adulthood LTD can be elicited in the mature brain, but its developmental pattern is unknown. Aim of this work was to investigate whether LTD is expressed during postnatal development and if it is modulated by visual experience. To investigate these points we have used rat primary visual cortex slices taken at different stages of functional maturation process, i.e., postnatal day 17 (P17), P23 and P30-35. LTD was assessed by measuring the amplitude of extracellular field potentials recorded in cortical layers 2/3 and elicited by low frequency stimulation to the white matter. LTD was expressed at all ages investigated without significant differences between age groups. These data indicate that LTD developmental expression is not temporally related with the period of functional maturation of rat visual cortex. Dark rearing from birth to P23 resulted in a reduction of LTD amplitude while light deprivation from P17 to P30 did not affect LTD expression in comparison to age matched control values. We suggest that light imprinting is essential for a normal LTD expression during postnatal development.     

Androgens reduce cell death in the developing rat visual cortex

We have previously shown that males have more neurons than females in the primary visual cortex, and neonatal androgens play an important role in this difference. Also, we have found that females experience more cell death during development in this region than in males. Therefore, we hypothesized that the neonatal hormone environment directly influences the amount of cell death. In the present experiment, female rats were implanted with dihydrotestosterone (DHT) or estradiol at postnatal day 1. These animals, along with control males and females, were sacrificed on postnatal days 6, 11 and 25. Using unbiased stereology to quantify neuronal and pyknotic cell density, we observed that females implanted with DHT had a similar pattern and proportion of cells dying as control males. Additionally, developmental cell death in females implanted with estradiol was not significantly different than control females. Thus, neonatal androgens have an inhibitory effect on developmental cell death in the rat primary visual cortex.