Neurogenesis in the visual system of the rat. An autoradiographic investigation.

Rats of the BD III strain were injected with a single dose of 3H-thymidine on either the twelfth, fourteenth, sixteenth, eighteenth or twentieth day of gestation (ED 12. . . . .ED 20) or on the postnatal day one, three, or seven. Animals were killed at age 22 to 24 days. DNA synthesis, as an indicator of cell division, was studied in matrix precursors of nerve and glial cells in the visula centers, including the lateral geniculate body (LGB), the superior colliculus (SC) and the visual cortex (VC). It was found that proliferation of matrix precursors of nerve cells destined for all the regions studied was in progress on ED 12. In the subcortical regions (LGB, SC) this process was substantially more advanced than in the VC. The first neuroblasts appeared in the SC (ED 12) and only later (ED 14) in the LGB and VC. In comparison with the LGB, VC neuroblasts were quite rare on ED 14 and were present only in layer VI. They appeared more frequently in this region only after injection of isotope on ED 16. Matrix cell proliferation and nerve cell formation ceased in the LGB between ED 16 and ED 18. The number of labeled cells arising after injection of the isotope on ED 16 indicates that neurogenesis ceased somewhat earlier in the dorsal nucleus of the LGB than in the ventral. In the SC the last neurons arose between ED 18 and ED 20, and in the VC, with the possible exception of a few granular neurons (which may continue division into the first few days postnatally), proliferation continued until the end of gestation. The origin of neuroblasts initially followed a caudo-rostral gradient. Later, the times of neurogenesis in the regions studied overlapped significantly. This is clear, for example. on ED 16, when neurogenesis in the mesencephalic SC continued for about two days longer than in the more postral LGB, and coincided with that in the VC, especially in the deep layers. The end of neurogenesis in the LGB, especially in the ventral nucleus, coincided with the time of neurogenesis in the deep cortical layers. In the VC, and partly also in the SC, an inside-out pattern of proliferation and neuron formation was confirmed. The times of proliferation of precursor cells, with the exception of the very end of neurogenesis, substantially overlapped within both these regions. The degree of this overlapping, described in terms of Labeling Index values, decreased towards the end of the neurogenetic period. Division of neuroglial cell precursors, started as early as on ED 14 in/for subcortical centers (LGB, SC), but not until ED 18 in/for the VC. A few labeled endothelial-like cells were observed in all regions studied after isotope injection on ED 12.

Functional regions within the map of a single digit in raccoon primary somatosensory cortex.

Electrophysiological recordings were made at a large number of sites in the primary somatosensory cortex of six anesthetized raccoons. A high density of penetrations (110-229 per animal), within or near the representation of the fourth digit, allowed identification of three cortical regions with different physiological properties: a glabrous zone, containing a highly detailed, somatotopically ordered representation of the glabrous surface of the digit; rostral to this a claw-dominant zone, in which the neurons at most penetrations respond to stimulation of the claw of the fourth digit, but may also receive input from the hairy skin or surrounding glabrous skin; and a more rostral multidigit zone, in which the neurons respond to stimulation of two to five digits, with the dominant digit usually being the one represented caudally (i.e., the fourth digit at most of the sites sampled here). Claw-dominant zones with receptive fields restricted to digit three or five are also found rostral to the representations of the glabrous skin of the corresponding digit. The glabrous and claw-dominant zones constitute a complete map of the fourth digit. The multidigit region presumably is a separate map, since its neurons have different spatial convergence, higher thresholds, and a lower incidence of slowly adapting inputs than those in the claw-dominant and glabrous zones. A comparison between animals with lesions of the basal forebrain and intact animals found no differences in the organization of these zones or in the responses to peripheral input, suggesting that cholinergic inputs to the cortex are not essential to these properties. The detailed description of these regions and the proposed terminology should resolve some inconsistencies in the use of the term "heterogeneous zone" in this species.

Regional and cellular differences in fucosylation of glycomacromolecules in the mouse brain. A biochemical and autoradiographic study of early postnatal and adolescent animals.

Mice of CBA strain of both sex were injected with [3H]fucose at age 2, 6, 12 or 30 days and the incorporation was determined biochemically 45, 90 and 180 min later. Biochemical measurements of the whole brain (at 90 min postinjection interval) revealed a stepwise age decrease in the amount of incorporated isotope (per mg protein). The amount of [3H]fucose available in the brain has however been found to decrease with age while the degree of its utilization increases. Thus, after correction of the data on [3H]fucose incorporation for the pool of the free isotope, a transient increase in fucosylation rate appeared at postnatal day 12 while the differences among 2-, 6- and 30-day-old animals became negligible. Further, the synthesis of fucosylated soluble glycomacromolecules appeared relatively higher at postnatal day 2 than in older age groups. Examination of different brain regions revealed that the rate of fucosylation is relatively highest in the olfactory bulbs; this prevalence starts appearing with age and becomes most evident in 30-day-old mice. Autoradiography carried out with 6- and 12-day-old animals revealed that the incorporation of [3H]fucose into meningovascular apparatus and the choroid plexus is a few times higher than into brain parenchyma. The regional differences appeared to be similar to those indicated by the biochemical data. Cellular analysis has shown that the incorporation is relatively higher in the cytoplasm of large projecting neurons of the cerebellum, hippocampus and the olfactory bulbs; in the latter region high amounts of macromolecule-bound [3H]fucose also appeared in the synaptic glomeruli.(ABSTRACT TRUNCATED AT 250 WORDS)

The basal forebrain cholinergic system in the raccoon.

The distribution of neurons displaying choline acetyltransferase (ChAT) immunoreactivity was examined in the raccoon basal forebrain using a rabbit antiserum and a monoclonal antibody. Alternating sections were used for Nissl staining. ChAT-positive neurons were arranged in a continuous mass extending from the medial septum to the caudal pole of the pallidum. Based upon spatial relations to fibre tracts, the clustering of neuronal groups, and cytological criteria, the basal forebrain magnocellular complex can be subdivided into several distinct regions. Although clear nuclear boundaries were often absent, the ChAT-positive neurons were divided into: the nucleus tractus diagonalis (comprising pars septi medialis, pars verticalis and pars horizontalis); nucleus praeopticus magnocellularis; substantia innominata; and the nucleus basalis of Meynert. Comparison with Nissl-stained sections indicated the presence of varying proportions of non-cholinergic neurons clustered or arranged loosely within these basal forebrain subdivisions. These data provide a structural basis for studies concerned with the topographical and physiological aspects of the raccoon basal forebrain cholinergic projections and its comparison with the basal forebrains of other species.

The effect of d-amphetamine and amitriptyline administered to pregnant rats on the locomotor activity and neurotransmitters of the offspring.

d-Amphetamine and amitriptyline (AT) were administered daily to female rats from day 7 of pregnancy until birth of the litters. Changes in the concentration of the biogenic amines, some of their metabolites, GABA, and the activities of glutamate decarboxylase, acetylcholinesterase (AChE), and choline acetyltransferase were determined in the whole brain of the offspring. The offspring of the amphetamine-treated rats showed a marked increase in serotonin concentration and that of its metabolite on postnatal day 1. Changes in the concentration of GABA were apparent on days 15 and 21 and were inversely correlated with changes in the activity of the synthesizing enzyme: Choline acetyltransferase and AChE activities were also increased at this time. Changes in neurotransmitter metabolism were not so evident in the offspring of rats treated with AT. The locomotor activity of the 8-, 15-, and 21-day offspring was also assessed. The offspring of the amphetamine-treated rats showed enhanced locomotor activity initially, but the activity decreased relative to the age-matched controls in the 21-day group. Offspring from the AT-treated group showed reduced locomotor activity.

A method for the dissection of the embryonic cerebral cortex into individual layers. An application to biochemical studies of glycan metabolism.

A method is presented for the isolation of defined cerebral cortical layers from the prenatal rat brain. By this procedure cells at different stages of proliferation and/or differentiation can be obtained as relatively homogeneous populations. The principle of the proposed isolation procedure consists of freezing the isolated embryonic brain in a mould which flattens the two hemispheres. The different cortical layers are then isolated by carefully cutting serial cryostat sections. On embryonic day 16 (ED 16), 3 individual layers could be sampled from the cortex: the marginal zone, the zone of DNA synthesis and the mitotic region of the ventricular germinative zone. On ED 18 and ED 20, a further 3 layers could be isolated: the cortical plate, the sub-plate region, and the intermediate zone. As an example of an application of the isolation procedure for biochemical studies, maturation-dependent changes in the protein and Concanavalin A-binding glycoprotein patterns together with the activity of beta-N-acetylglucosaminidase in the different cortical layers of the embryonic brain are demonstrated at different stages of brain development.

Stereotaxic preparation of circumscribed cortical areas from rat brain for biochemical studies.

A method for the rapid dissection of circumscribed areas of rat cortex is described. The technique does not depend on skull-derived landmarks but uses for stereotaxic orientation the cross-point of the interhemispheric gap with the caudal margin of the cortex. An application of this dissection method to the biochemical analysis of cholinergic markers within the hindlimb representation of the primary somatosensory cortex revealed that both the activity of the enzyme choline acetyltransferase as well as the binding of [3H] quinuclidinyl benzilate to muscarinic cholinergic receptors do not seem to be affected drastically three days after unilateral transection of the sciatic nerve. The only significant effect detected was a slight decrease in the activity of the choline acetyltransferase within the hindlimb representation of the primary somatosensory cortex contralateral to the transected sciatic nerve. In the primary visual cortex, the cholinergic markers investigated did not show significant alterations after sciatic nerve injury.

An autoradiographic study of gliogenesis in the rat lateral geniculate nucleus (LGN).

Rats pregnant for 14, 16, 18 and 20 days, as well as 1-, 3-, 7-, 12-, 16- and 21-day-old offspring, received a single injection of [3H]thymidine and were killed at postnatal ages of 22-27 days. An autoradiographic study of DNA synthesis revealed that proliferation of non-neuronal matrix cell precursors destined for the dorsal part of the lateral geniculate nucleus had already started by gestational day 14. Quantiative evaluation of the incidence of labelled cells revealed 2 peaks occurring in both groups of non-neuronal cells at postnatal days (PD) 7 and 16. It has been assumed that the accelerated production of ectodermally derived glial cells, observed towards PD 7, relfects the enhanced growth of the neuronal surface covered with glial cell processes, and that at PD 16 formation of myelin sheaths. Changes in the number of labelled cells of mesodermal origin in the course of development can probably be attributed to the rate of vasculogenesis of the studied region.

Stimulation of the nucleus basalis of Meynert and substantia innominata produces widespread increases in cerebral blood flow in the frontal, parietal and occipital cortices.

The effect of a focal stimulation of the magnocellular nucleus of the basal forebrain at two different areas, the nucleus basalis of Meynert (NBM) and the substantia innominata (SI), on local cerebral blood flow (CBF) in the frontal, parietal and occipital cortices was examined in urethane-anesthetized rats. The stimulation, either electrically or chemically, of both the NBM and SI produced significant CBF increase in all these 3 cortices ipsilateral to the stimulation site. This fact suggests that activation of neurons originating in the NBM and SI produces widespread increases in local CBF in the ipsilateral cerebral cortex.

Long-term enhancement of evoked potentials in raccoon somatosensory cortex following co-activation of the nucleus basalis of Meynert complex and cutaneous receptors.

Long-term enhancement of the evoked potential was induced in the primary somatosensory cortex of anaesthetized raccoons after mechanical stimulation of the skin was paired with electrical stimulation of the nucleus basalis of Meynert (NBM). Sets of 4 pulses, 0.5 ms duration at 300 Hz were delivered at 2-s intervals to the basal forebrain 80 ms before the glabrous skin on the 4th digit of the contralateral forepaw was stimulated mechanically. The average waveform of 30 evoked potentials was separated into an initial positive, a negative and a second positive component. During pairing of the skin and NBM stimuli, the area under the initial positive component was smaller than before or after pairing. The negative and second positive waves were unchanged. One minute after pairing, the initial positive wave returned to control values and continued to increase until the end of the experiment 50 min later, at which time it was 300% above control. The negative and second positive waves increased after the pairing to between 130 and 200% and remained at that level for the duration of the experiment. The effective NBM site for stimulation was the area rich in cholinergic neurons corresponding to the NBM. In control animals, repeated stimulation of the skin or NBM alone, or their random, unpaired stimulation together, did not enhance the somatosensory evoked potential. The results suggest that the NBM input enhances the efficacy of cortical responses to cutaneous input and thus may play a role in cortical neuronal plasticity.

A combined method of acetylcholinesterase histochemistry and [3H]thymidine autoradiography: application to neurogenesis of the rat basal forebrain cholinergic system.

Rats labelled with [3H]thymidine on embryonic day 15 were treated postnatally with diisopropylfluorophosphate and 2-3 h later perfused with 10% buffered formalin. Cryostat sections of the basal forebrain were autoradiographed and subsequently stained for acetylcholinesterase (AChE). It was shown that this procedure did not significantly influence either the AChE activity or the silver grains over [3H]thymidine-labelled nerve cell nuclei. The labelling index evaluated in cholinergic forebrain regions (medial septum, diagonal band of Broca, substantia innominata, nucleus basalis of Meynert) indicated a caudorostral gradient of the formation of AChE-containing neurons while formation of non-cholinergic neurons showed a more even pattern.

Stimulation of the nucleus basalis of Meynert increases cerebral cortical blood flow in rats.

Focal electrical stimulation of the magnocellular nucleus of the basal forebrain (nucleus basalis of Meynert; NBM) or a microinjection of L-glutamate (50 nmol) into the NBM increased cerebral cortical blood flow in the parietal lobe in urethane-anesthetized rats. The vasodilative responses were elicited only ipsilateral to the site of stimulation. Most of the vasodilative responses were abolished by intravenous administrations of muscarinic and nicotinic cholinergic blocking agents (atropine 0.5 mg/kg and mecamylamine 2 mg/kg). This suggests that the cholinergic projecting system sending fibers from the NBM to the parietal lobe contributes to the vasodilation of the cortex by activating muscarinic and nicotinic cholinergic receptors.

Age-dependent differences in cholinergic drug response in two strains of mice.

The behavioral effects of nicotine and oxotremorine were assessed in two strains of mice, C57BL/6 (C57) and DBA/2 (DBA), differing in the ontogeny of forebrain cholinergic structures. Both nicotine and oxotremorine depressed to a higher extent the behavior of DBA mice starting at day 45 of postnatal life.

Wheat germ agglutinin indicates the commencement of neuronal differentiation in the embryonic brain. A comparative carbohydrate histochemical study.

Paraffin sections prepared from prenatal rat brain (embryonic days 14, 16, 18 and 20) were incubated with buffered wheat germ agglutinin labelled with fluorescein isothiocyanate (FITC-WGA). In the embryonic brain, the proliferating cells of the ventricular zone showed a low binding of WGA. Differentiating neuroblasts and axonal bundles exhibited a significantly higher degree of labelling. This pattern was most evident in the cerebral cortex where the marginal zone and the sub-plate region were found to be most intensely strained. The structural basis of WGA-binding was specified by comparison with data obtained by the colloidal iron hydroxide staining procedure, the periodic acid Schiff's reagent method and Alcian Blue staining (critical electrolyte concentration method). The results show that at regions of intensive differentiation in the embryonic brain there is an increase in the concentrations of sialic acid and glycosaminoglycans.

Ultrastructure of symmetric interneuronal junctions after staining with ethanolic phosphotungstic acid.

1. The symmetric interneuronal junctions (attachment plaques) of large axon terminals in the dorsal lateral geniculate body of the rat were studied, applying besides the conventional staining the block staining method with ethanolic phosphotungstic acid (E-PTA). 2. After staining these contact zones with E-PTA 3 main components become visible: the interior presynaptic layer, the intermembranous material and the postsynaptic layer. 3. As opposed to the asymmetric synaptic junctions the interior presynaptic layer is not divided into dense projections. 4. The intermembranous material consists of 2 median situated intracleft lines, which are more or less fused with each other. 5. The staining intensity of the paramembranous material of the symmetric junctions stained with E-PTA corresponds to the intensity of the material of the asymmetric synaptic junctions.

Histochemical detection of anionic components in the cephalopod brain.

Brains obtained from three species of mediterranean cephalopods (Loligo, Sepia, Octopus) were fixed in Bouin's solution. Paraffin sections were cut sequentially at the frontal plane and used for Alcian blue staining (critical electrolyte concentration method), colloidal iron hydroxide staining procedure, the periodic acid Schiff's reagent method, and the lead tetra-acetate-Schiff method. The stained sections were evaluated at 2 regions of different histological composition: the palliovisceral ganglion and the optic lobe. A high concentration of anionic components was found in synaptic regions of the neuropil whereas neuronal cell bodies showed a relatively weak staining of these constituents. There was a significant reaction of the perineuronal glia nets in the cellular rind of the palliovisceral ganglion. From the comparison of staining patterns obtained with the 4 methods in this study and literature data it can be concluded that the detected anionic sites are mainly carboxyl groups of acidic proteins and/or glycoproteins. Sulphate groups may be present in lower concentrations. Their distribution reveals that the role of anionic components other than sialic acids in the invertebrate brain might be discussed in the context of synaptic transmission similar to that in vertebrates. The possible involvement of the glia cell population has to be taken into consideration.

The synthesis and transport of fucosylated glycans in the immature mouse cerebellum. An autoradiographic and microchemical study of differentiating cell and tissue compartments.

A study of 3H-fucose incorporation in the immature mouse parenchyma revealed that the rate of synthesis of fucosylated glycans in the neuropil of more differentiated internal granular layer, as well as in the PURKINJE cell bodies is higher than in the less mature external granular layer. The 3H-fucose incorporation into the choroid plexus, meninges, and blood vessel walls exceeds highly incorporation into the cerebellar parenchyma. A follow-up autoradiographic study revealed remarkable differences in the radioactivity of incorporated 3H-fucose in the "cell-body rich" and "cell-fibre rich" layers indicating transport of newly synthesized glycans from the perikarya of differentiating cells into their processes extending mainly into the molecular layer and the cerebellar medulla. It is assumed that the increased rate of 3H-fucose incorporation reflect 1. increasing complexity of the fucosylated glycans of differentiating cells 2. increase in the turnover of cell membrane components, 3. accelerated export of glycosylated compounds into the outgrowing fibres of differentiating cerebellar cells.

Hypoxia and hypercapnia increase the sympathoadrenal medullary functions in anesthetized, artificially ventilated rats.

Graded hypoxia (FETO2 14-6%) and hypercapnia (FETCO2 6-10%), which were applied for 45s and 2 min, respectively, to urethane anesthetized and artificially ventilated rats produced an increase in adrenal sympathetic efferent nerve activity in parallel with increases in adrenaline and noradrenaline secretion measured in the adrenal venous effluent. Percentage increases in adrenaline and noradrenaline were almost equal. In rats whose carotid sinus nerves (CSN) were bilaterally cut, hypoxia did not produce any effect on adrenal sympathetic nerve activity or catecholamine secretion. In contrast, excitatory adrenal nerve and catecholamine secretory responses to hypercapnia remained unchanged in CSN denervated rats. After severing a splanchnic nerve whose branches innervated the adrenal gland, while maintaining the resting level of catecholamine secretion by low-frequency stimulation of the peripheral end of the splanchnic nerve, hypoxia did not produce any increase in catecholamine secretion. Hypercapnia (FETCO2 8 and 10%), however, induced catecholamine secretion from denervated adrenal medulla, although the magnitude of the response was significantly lower than that in animals with adrenal nerve intact. It is concluded that hypoxia stimulates the adrenal medulla via the carotid chemoreceptor reflex whereas hypercapnia acts mainly via mechanisms besides carotid chemoreceptors such as central chemoreceptors with some direct stimulatory effect on the adrenal medulla. The functional significance of these dual mechanisms of sympathoadrenal excitation during hypoxia and hypercapnia is discussed.