Expression of certain proteins in the subfornical organ and cerebrospinal fluid of spontaneously hypertensive rats.

The objective of this study was to analyze the proteins in the cerebrospinal fluid of spontaneously hypertensive rats and to study their possible role in the relationship between hydrocephalus, arterial hypertension and variations in the subfornical organ. Brains and cerebrospinal fluid from control Wistar-Kyoto rats and spontaneously hypertensive rats sacrificed with chloral hydrate were used. Cerebrospinal fluid and extract of subfornical organ were processed by protein electrophoresis. Antisera against protein bands of 141, 117 and 48 kDa and Concanavalin A were used for immunohistochemical and western blot study of the subfornical organ, adjacent circumventricular structures and cerebrospinal fluid. Ventricular dilation in the spontaneously hypertensive rats and the presence of quite a lot of protein bands in the cerebrospinal fluid of the hypertensive rats, which were either not observed or scarcely present in the cerebrospinal fluid of the Wistar-Kyoto rats, were confirmed. The subfornical organ, third ventricle ependyma and choroideus plexus showed immunoreactive material for antibodies against 141kDa, 117 and 48 kDa proteins band (anti-B1, anti-B2 and anti-B3). The larger amount of the immunoreactive material was found in the subfornical organ of the spontaneously hypertensive rat. Our results and the alterations observed by other authors in the subfornical organ in hydrocephalic and hypertensive rats support the possibility that this circumventricular organ, some proteins of the cerebrospinal fluid and ventricular dilation could be connected with the physiopathology of this type of hypertension.

Alterations of the cerebrospinal fluid proteins and subcommissural organ secretion in the arterial hypertension and ventricular dilatation. A study in SHR rats.

The aim of this work was to analyze the proteins in the cerebrospinal fluid (CSF) of spontaneously hypertensive rats, to study their possible role in the relationship between hydrocephalus, arterial hypertension and alterations in the subcommissural organ. Brains from control Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) sacrificed with chloral hydrate were used. Antiserums against some cerebrospinal fluid protein bands and Reissner's fiber (RF) were used for immunohistochemical study of the SCO. Ventricular dilation was observed in the lateral and third ventricle of the SHR. Third ventricle ependyma showed immunoreactive material (IRM) for antibody against 141 kDa protein band anti-B1 and 117 protein band anti-B2 and the SCO of the SHR showed a decrease of the IRM when compared with WKY rats. An alteration in the expression of anti-RF was found to compare the SCO of the WKY and SHR groups. Our results demonstrate that hydrocephalus and hypertension are interconnected in this kind of rat which produce alterations in SCO secretions and some proteins of the CSF.

Effect of the arterial hypertension and captopril treatment on the angiotensin II content in the subfornical organ. A study in SHR rats.

We studied the effects of spontaneous high blood pressure and the captopril treatment on the subfornical organ (SFO) of rats. The brains of control Wistar-Kyoto rats (WKY), WKY rats treated with captopril (WKY-T), spontaneously hypertensive rats (SHR) and SHR rats treated with captopril (SHR-T) were processed immunohistochemically using anti-angiotensin II as primary antibody. Immunorective material (IRM) for angiotensin II was observed in a group of neurons and some cells of the ependymal layer of the SFO in WKY rats. The angiotensin II immunoreactive (AGII-ir) in the SHR rats was decreased, showing positive reaction only in a few neurons, while captopril treatment induced an increase in immunoreactive material in hypertensive rats, but contrarily, the expression of AGII-ir in the WKY-T group was scarce. The variations of the angiotensin II observed in the SFO could be owing to an interaction between the hypertension and its captopril treatment.

Reelin-immunoreactive neurons in the adult vertebrate pallium.

Reelin, an extracellular matrix protein, plays a crucial role in cortical development. By using Reelin-immunohistochemistry in different vertebrates (fish, amphibians, reptiles, and mammals : insectivores, odontocetes, rodents, carnivores and man) we show here that Reelin is also expressed by a variety of neurons in the adult pallium. In the everted telencephalon of the zebrafish, Reelin-positive neurons are widely distributed over the dorsal pallium. In land vertebrates, the most consistent and evolutionary conserved location of Reelin-expressing neurons is in the cell-sparse molecular layer associated with laminated cortical organization. We describe an additional heterogeneous population of Reelin-positive neurons outside the molecular layer, the location and distribution of which are more variable, and which may reflect major evolutionary changes in cortical architecture. In squamate reptiles, the Reelin-negative main cell layer is flanked by a superficial and a deep plexiform layer which both contain Reelin-expressing neurons. In mammals, Reelin-positive interneurons are dispersed throughout layers II--VI; the human neocortex is particularly poor in Reelin-positive interneurons. Reelin is also expressed by large stellate and modified pyramidal neurons in layer II of the mammalian entorhinal cortex, and in the superficial lateral cortex of lizards. Examination of this cell population (layer II Pre-alpha) in human brains of different age groups points to a decrease in Reelin-expression in the course of adult life.

Effect of hypertension and captopril treatment on the vasopressin in the rat median eminence and posterior lobe of the hypophysis. An immunohistochemical study.

The present study analyses the effects of hypertension and/or its oral treatment with captopril (angiotensine-converting enzyme inhibitor) on the rat median eminence (ME) and the posterior lobe of the hypophysis (PL). After an immunohistochemical reaction using an antibody against arginine-vasopressin, we compared by densitometry the amount of vasopressin immunoreactive material (vasopressin-ir) of these centers in 4 groups of animals: control Wistar Kyoto rats (WKY), spontaneously hypertensive rats (SHR), WKY rats treated with captopril (WKY-T) and SHR rats also treated with the same drug (SHR-T). Captopril was administrated at a dosage of 0.1 mg/ml in the drinking water from the 8th to the 15th weeks. We have found that the rats showing the lowest level of vasopressin-ir, in both ME and PL, were those from the SHR group, the concentration increasing after oral captopril treatment (SHR-T), although without reaching the values of WKY rats. Then, ACE inhibition by captopril influences vasopressin content in brain areas where the hormone is concentrated before being released, which supports the hypothesis that suggests a central modulatory effect of ACE inhibitors, contributing to their therapeutic action on hypertension.

Effects of alcohol and aging on the cingular (area 24) and frontal (area 6) cortical areas of the mouse.

We have studied the morphometric changes of the neurons of the cingular area 24 and frontal area 6 of the mouse, produced by age and/or chronic alcohol intake. The parameters analyzed were nuclear area of these cortical neurons and cellular density (cell/neuropil coefficient). We detected a decrease in the number of neurons with age in practically all layers of the control animals. In the animals that chronically ingested the alcoholic solution, we also detected a decrease in the number of neurons with age, but only in layer V of the frontal cortex and in layer VI of the cingular area 24. The comparison between the control and the alcoholic group showed that alcohol intake caused an increase in the nuclear area of the neurons in layer II-III of the frontal cortex at 180 days, while in the cingular cortex the increase in nuclear area of its neurons was significative at 180 days in layer II-III and at 35 and 180 days in layers V and VI. We think that these changes are the expression of the neuronal plasticity in both cortical areas in response to the alcohol exposure.

The effects of chronic administration of captopril on the mouse median eminence.

The effects of Captopril (an angiotensin-converting enzyme inhibitor) on the median eminence (ME) of the male albino mouse have been examined using morphometric and immunohistochemical procedures. We measured the nuclear area of the ependymocytes of the ME and of the glial cells of the reticular external zone of the ME. We also determined the cell/neuropil coefficient (CNC), which expresses the relation between cellular area and neuropil of the ME, and the global volume of the ME in each animal. For the immunohistochemical study we used rabbit antiarginine-vasopressin, and compared the results in the different groups of mice. We detected an increased in the immunoreactive material (arginine-vasopressin, A-V) and an increase in the global volume of the organ and also an increase of the neuropil of the ME after the longest exposure to the drug. These alterations could be related to the inhibition of the brain angiotensin II by captopril and the accumulation of vasopressin in the fibrous tract that runs from the paraventricular nucleus (PVN) to the neurohypophysis.

Effects of chronic alcohol intake on the vasopressin content in the hypothalamic paraventricular and supraoptic nuclei of the mouse. An immunohistochemical and morphometric study.

The present study analyses the response of the paraventricular (PVN) and supraoptic (SPO) nuclei of the hypothalamus of the male mouse to chronic alcohol intake by immunohistochemical and morphometric methods. We relate the intensity of the reaction to A-V with the vasopressin content of the nucleus, as all the slides, from the control and experimental groups, were processed at the same time and with the same solutions of the antibodies. We suggest that the accumulation of vasopressin, observed in the alcohol-treated animals, of both hypothalamic nuclei could be related to an inhibition of vasopressin release and/or transport from the SPO and PVN to the neurohypophysis and to an increase in vasopressin synthesis in the SPO as this nucleus shows an increase in its nuclear sizes, an index of the function of the neurons.

Effects of chronic alcohol exposure on the morphometric development of medial preoptic area neurons of the male mouse.

We have performed a karyometric study of the medial preoptic area of male mice from mothers that ingested chronically a solution of 20% of ethanol added to the drinking water. Pups then were exposed prenatally to alcohol. After parturition, pups were also exposed to alcohol, first through their mother's milk and after weaning by direct ingestion of the same solution of 20% of alcohol until the day of sacrifice. Animals were sacrificed at the 25th, 35th, 45th, 55th and 100th day and the results compared with those obtained in another group of control animals, sacrificed at the same ages. Chronic alcohol exposure reduces the studied nuclear sizes (perimeter, area and maximum diameter) in adult animals of 100 days of life, but does not produce significative changes in nuclear sizes of younger animals. However, nuclear shape, another of the nuclear parameters analysed, did show significative alterations in relation with the puberal age. These morphometric effects could be due to the reduction of plasmatic testosterone levels produced by alcohol and/or to a direct toxic effect of the alcohol on central nervous system neurons.

Alcohol effects on paraventricular hypothalamic nucleus morphometry.

The morphometric effects of postnatal exposure to alcohol on the neurons of the paraventricular hypothalamic nucleus (PVN) have been studied in four different topographic subdivisions of the nucleus: rostral, intermediate medial, intermediate lateral and caudal. Male mice were exposed to alcohol during lactation and after weaning by addition of 20% of ethanol to the drinking water that was first ingested by the mother and thereafter by the experimental animals themselves. Animals were sacrificed at the 25th, 35th, 45th, 55th and 100th postnatal day. Nuclear sizes of the PVN neurons (Perimeter, area and maximum diameter) were determined in both control and experimental alcohol-treated groups. The shape of these neuronal nuclei was also determined and compared. PVN responds globally to alcohol exposure, showing a decrease in the neuronal nuclear sizes in the four studied PVN subdivisions of the alcohol-treated mice at the 35th and 45th and 100th day. We suggest that these decreases could be related to changes in gonadal hormone levels induced by alcohol exposure and/or disturbances of brain neurotransmitter and neuropeptide metabolism caused by ethanol.

Postnatal development of the Ammon's horn (CA1 and CA3 fields). A karyometric and topographic study.

We have performed a karyometric study of the pyramidal neurons of CA1 and CA3 fields of the Ammon's horn, in male mice aged from the 5th to the 190th postnatal day. Nuclear sizes were measured with the aid of a Magiscan Analysis System, used in an interactive form, in both superficial and deep layers of the stratum pyramidal in those fields. The measurements were made at three different topographic levels: rostral; intermediate; and caudal, to detect any possible difference related to the topography of the neuron in the same field. We have found that both CA1 and CA3 fields are correlated in the postnatal development of their nuclear pyramidal sizes and that all topographic levels of the hippocampus reach their highest karyometric sizes at the 10th-15th postnatal day. Caudal levels show higher karyometric values than the other levels and some differences between neurons of the superficial and deep layers of both fields are also described here and analysed in relation to the different ontogenetic gradients of these cells.

Alterations of the subcommissural organ in the hydrocephalic human fetal brain.

We have studied the subcommissural organ of two hydrocephalic brains, of 20 and 21 gestational weeks and of two normal brains, aged 19 and 23 gestational weeks. Both hydrocephalic cases presented a size reduction of the subcommissural organ compared to the normal cases; only in one case, there were also alterations of the morphological components of the subcommissural organ, suggesting different pathogenic relationships between hydrocephalus and dysplasia of the subcommissural organ.

NADPH-d activity in the islands of Calleja: a regulatory system of blood flow to the ventral striatum/pallidum?

We have used dihydronicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry to study the anatomical relationships between the islands of Calleja (ICs), ventral striatum (VS) and ventral pallidum (VP), and the perforating branches of the anterior communicating and anterior cerebral arteries traversing the olfactory tubercle. The granule cells of the ICs are intensely positive for NADPH-d, a marker for neuronal nitric oxide synthetase (NOS), and closely surround all arterioles perfusing the VP and most of the arterioles supplying the VS. In contrast, they are not related to the arteries destined for the dorsal striatum. On the ground of the vasodilatory properties of the nitric oxide, we propose that the ICs may play a role in the regulation of blood flow to specific centres of the limbic forebrain.

Transient NADPH-diaphorase activity in motor nuclei of the foetal human brain stem.

NADPH-d activity is a marker for neurones which synthesize nitric oxide (NO). In the present paper we study the NADPH-d activity in the human brain stem between 16 and 24 gestational weeks (GW). The adult distribution of NADPH-d containing neurones is established at 24 GW. Between 19 and 21 GW, neurones of the facial, hypoglossal and ambiguus nuclei show transient NADPH-d positivity. Therefore, in the human brain stem there are two chronological patterns of neuronal nitric oxide synthase (NOS) expression, one that appears during prenatal development and persists during the whole life, and another one, present only at a precise foetal moment. The transient NADPH-d activity in motor nuclei suggests that NO is involved in early regulatory processes of the human brain development.

NADPH-d (dihydronicotinamide adenine dinucleotide phosphate diaphorase) activity in geniculo-tectal neurons of the rat.

Retrograde transport of horseradish peroxidase (HRP) and dihydronicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry were used in order to determine whether NADPH-d-positive neurons of the ventral lateral geniculate nucleus (LGv) project to the superior colliculus (SC). Our results show that intensely stained NADPH-d neurons are restricted to the lateral half of the magnocellular division of LGv (LGv-MC), where they represent 50% of the total number of retrogradely labeled neurons. These findings indicate: (1) that LGv provides an important NADPH-d input to SC, and (2) that within the population of geniculo-tectal neurons, which constitute a morphologically well defined neuronal type, there are two different subclasses, one being NADPH-d positive and the other NADPH-d negative.

The effects of chronic administration of captopril on the mouse subfornical organ and area postrema.

We have studied by morphometric procedures the chronic effect of captopril on the subfornical organ (SFO) and area postrema (AP) of the adult mouse. Oral administration of captopril does not produce any change in the size of individual nuclei of the ependymocytes and neurons in both centers. However, there are other quantitative effects of captopril on the global volume of the SFO and on the neuropil and vascular elements of both the SFO and AP which present a significant increase. It is suggested that this increase is due to metabolic processes at the level of both circumventricular organs.

Postnatal development of the dentate gyrus: a karyometric and topographic study.

We have analysed the postnatal development of the nuclear sizes of the granular cells of the dentate gyrus in 5- to 190-day-old male mice. The study was performed in three topographic levels: rostral, intermediate and caudal. Three subdivisions were analysed in each level: suprapyramidal blade, infrapyramidal blade and the transition between them, the angular zone. Additionally, each of these subdivisions was measured in its external and internal layer, separately. Three gradients of postnatal karyometric development can be described: external-to-internal, suprapyramidal-to-infrapyramidal, and caudal-to-rostral, indicating that the external, suprapyramidal and caudal cells show higher karyometric sizes than the other subdivisions. These gradients are related to the ontogenetic gradients of these neurons.

Morphology of neurons in the white matter of the adult human neocortex.

Neurons in the human cerebral cortical white matter below motor, visual, auditory and prefrontal orbital areas have been studied with the Golgi method, immunohistochemistry and diaphorase histochemistry. The majority of white matter neurons are pyramidal cells displaying the typical polarized, spiny dendritic system. The morphological variety includes stellate forms as well as bipolar pyramidal cells, and the expression of a certain morphological phenotype seems to depend on the position of the neuron. Spineless nonpyramidal neurons with multipolar to bitufted dendritic fields constitute less than 10% of the neurons stained for microtubule associated protein (MAP-2). Only 3% of the MAP-2 immunoreactive neurons display nicotine adenine dinucleotide-diaphorase activity. The white matter pyramidal neurons are arranged in radial rows continuous with the columns of layer VI neurons. Neuron density is highest below layer VI, and decreases with increasing distance from the gray matter. White matter neurons are especially abundant below the primary motor cortex, and are least frequent below the visual cortex area 17. In contrast to other mammalian species, the white matter neurons in man are not only present during development, but persist throughout life.

Divergent projections of projecting neurons of the inferior colliculus to the medial geniculate body and the contralateral inferior colliculus in the rat.

The present paper shows, by means of the combination of two fluorescent tracers, that a significant number of neurons of the three divisions of the inferior colliculus in the rat project to the contralateral inferior colliculus and the ipsilateral medial geniculate body. The topographic distribution of double-labeled cells depends on the location of injections in each case. According to the soma size and the dendritic stem, different neuron types may be distinguished. Our results suggest that the inputs that converge in individual neurons of the inferior colliculus can diverge again.

The efferent projections of neurons in the white matter of different cortical areas of the adult rat.

Injection of Fast Blue into different cortical areas (frontal, parietal, anterior and posterior cingulate cortex) revealed that neurons in the white matter (interstitial neurons) give rise to association fibers which project mostly to the gray matter of the overlying cytoarchitectonic area, but which may extend also over different cytoarchitectonic areas. The rostrocaudal extent of the projecting axons was up to 1 mm in the frontal and parietal cortex, and up to 3.5 mm in the cingulate cortex. Concurrent processing for dihydronicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry showed that 70% of cortically projecting interstitial neurons were NADPH-d-positive. An analysis of neuronal morphology suggests that the FasT-Blue-labeled, NADPH-d-negative neurons may represent displaced pyramidal neurons of layer VIb; the Fast-Blue-labeled and NADPH-d-positive neurons have bipolar or multipolar dendritic trees, constituting a population of nonpyramidal interstitial neurons that project into the cortical gray matter.