GABAergic neurons expressing p75 in rat substantia innominata and nucleus basalis.

In vitro findings suggested a role for the p75 neurotrophin receptor in the maturation of GABAergic neurons residing in the basal forebrain (BF), a brain area known to have p75 expression only on cholinergic neurons. We document here the presence of GABAergic neurons which express p75 in the BF in vivo. Colocalization of p75 with the cholinergic marker choline-acetyltransferase (ChAT) and/or the GABAergic marker glutamic acid decarboxylase-67 (GAD67) was investigated in the BF at birth, at two weeks, and in adulthood. A subset of GAD67(+) neurons was p75(+) (p75(+)/GAD67(+)) but ChAT(-) in the substantia innominata and nucleus basalis magnocellularis at birth, whereas all p75(+)/GAD67(+) neurons were also ChAT(+) from two weeks onward. These phenotypic features suggest that a subpopulation of GABAergic neurons could be sensitive to neurotrophins during brain maturation. To unravel this issue, we then pursued a functional analysis by assessing p75 expression profile, and its modulation by nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) in primary BF cell cultures. NGF increased p75 expression exclusively in cholinergic neurons, whereas BDNF induced p75 expression only in a subset of GABAergic neurons (p75(+)/GAD67(+)/ChAT(-)) through a p75- and tyrosine-kinase-dependent mechanism. The latter findings point to a selective role of BDNF in the induction of p75 expression in BF GABAergic neurons. Altogether these results confirm the role of neurotrophins in the developing and mature circuitry of GABAergic neurons in the BF regions.

Evidence for target preferences by cholinergic axons originating from different subdivisions of the basal forebrain.

Possible target preferences of basal forebrain cholinergic neurons were studied in organotypic slice cultures. Cholinergic neurons in slices of medial septum or substantia innominata send axons into both hippocampus and neocortex when co-cultured together. However, septal cholinergic axons course through adjacent slices of neocortex to reach and branch densely in slices of hippocampus, but septal axons seldom grow beyond adjacent hippocampal tissue to reach neocortex. In contrast, cholinergic axons from substantia innominata commonly grow through hippocampus to reach neocortex, and also grow through neocortex to reach hippocampus, with similar branching densities in each target. The greater density of septal axonal branches in hippocampus than in neocortex suggests a preference of septal axons for the hippocampal target.

Maternal glucocorticoid hormone influences nerve growth factor expression in the developing rat brain.

Rat pups nursed from birth by mothers with increased plasma corticosterone show long-lasting biochemical and behavioral modifications. Here we have investigated nerve growth factor (NGF) concentrations in the basal forebrain, prefrontal cortex and hippocampus of both male and female offspring at 11 days of age. Maternal hypercorticosteronemia was achieved by giving corticosterone-enriched water (200 microg/ml) from delivery. There was a significant increase of NGF in the basal forebrain of both sexes and no changes in the prefrontal cortex. In the hippocampus, an increase in NGF was found in males. These results indicate that a moderate increase of corticosterone in the lactating mother modulates NGF in the developing rat. We propose that these effects contribute directly to the long-lasting behavioral and biochemical modifications in pups nursed by hypercorticosteronemic mothers.

Developmental changes of GABA(A) receptor-chloride channels in rat Meynert neurons.

The developmental changes of GABA(A) receptors were investigated in Meynert neurons freshly dissociated from day 0, 2 week-, and 6 month-old rats using both nystatin and gramicidin perforated patch recording modes under voltage-clamp conditions. The age-related changes in the current amplitude and threshold concentration in the concentration-response relationships for GABA indicated the developmental alteration of the GABA(A) receptor subunits and the channel density. The GABA-induced E(Cl-) measured by the gramicidin perforated patch mode shifted to more negative with development. The decay time constant of GABAergic inhibitory postsynaptic spontaneous currents (sIPSCs) in the synaptic active zone accelerated with aging. The GABA-induced currents were potentiated in a concentration dependent manner in the presence of benzodiazepine (BZP) agonists, diazepam (DZP) and zolpidem (ZPM). The potentiation rate of DZP on the GABA(A) response decreased with aging, but not in the case of ZPM, which demonstrated a stronger action in the aging rat neurons. These results suggested that the GABA(A) receptor x Cl- channel complexes may thus change both the assembly and interaction of subunits as well as their functional roles with aging.

Development of the time coding pathways in the auditory brainstem of the barn owl.

The barn owl's head grows after hatching, causing interaural distances to more than double in the first 3 weeks posthatch. These changes expose the bird to a constantly increasing range of interaural time cues. We have used Golgi and ultrastructural techniques to analyze the development of the connections and cell types of the nucleus magnocellularis (NM) and the nucleus laminaris (NL) with reference to the growth of the head. The time coding circuit is formed but immature at the time of hatching. In the month posthatch, the auditory nerve projection to the NM matures, and appears adult-like by posthatch day (P)21. NM neurons show a late growth of permanent dendrites starting at P6. Over the first month, these dendrites change in length and number, depending upon rostrocaudal position, to establish the adult pattern in which high best frequency neurons have few or no dendrites. These changes are not complete by P21, when NM neurons still have more dendrites than in the adult owl. The neurons of NL have many short dendrites before hatching. Their number is greatly reduced by P6, and then does not change during later development. Like NM neurons, NL neurons and dendrites grow in the first month posthatch, and at P21, NL dendrites are longer than those in the adult owl. Thus, the auditory brainstem circuits grow in the first month after hatching, but are not yet mature at the time the head reaches its adult size.

An indirect cholinesterase inhibitor, metrifonate, increases neocortical EEG arousal in rats.

We investigated the ability of a cholinesterase inhibitor, metrifonate, to desynchronize cortical EEG activity. Metrifonate suppressed immobility-related high voltage spindling activity in young and aged rats at doses of 30 and 60 mg kg-1, p.o., and 10, 30 and 60 mg kg-1, p.o., respectively. The increase in EEG 1-20 Hz amplitude induced by scopolamine (0.2 mg kg-1, i.p.) was fully alleviated by metrifonate (30 and 100 mg kg-1, p.o.) and partially alleviated by a reference cholinesterase inhibitor, THA (3 and 6 mg kg-1, i.p.). Nucleus basalis (NB) lesions induced by quisqualic acid decreased frontal cortical choline acetyltransferase activity by 80% and increased cortical EEG slow waves. Metrifonate and THA did not reverse NB lesion-induced EEG abnormality. We conclude that metrifonate enhances cholinergic desynchronization of cortical EEG waves and that a severe defect of presynaptic NB cholinergic fibres limits the therapeutic effects of metrifonate.

Neuroprotection against NMDA induced cell death in rat nucleus basalis by Ca2+ antagonist nimodipine, influence of aging and developmental drug treatment.

In the current study the neuroprotective effect of the L-type calcium channel antagonist nimodipine in rat brain was investigated in N-methyl-D-aspartate-induced neuronal degeneration in vivo. In the present model NMDA was unilaterally injected in the magnocellular nucleus basalis and the neurotoxic impact assessed by measuring cortical cholinergic fibre loss as a percentage of fibre density of the intact control hemisphere. This procedure proved to be a reproducible model in which the degree of damage was almost linearly proportional to the NMDA dose. Neuroprotection by nimodipine was determined in a number of conditions. First, the effect of nimodipine treatment in adult animals starting two weeks prior to neurotoxic injury was compared with neuroprotection provided by perinatal treatment of the mother animals with the calcium antagonist. Surprisingly, the degree of protection was in both cases similar, yielding almost 30% reduction of fibre loss. The neuroprotective effect in adulthood of perinatal nimodipine treatment may be explained by developmentally enhanced calcium binding proteins or persistent developmental changes in calcium channel characteristics. Protection by nimodipine was also investigated in aged, 26 month old rats. Compared to young adult cases, aged animals proved to be less vulnerable to NMDA exposure, while nimodipine application was more potent, thus yielding a reduction of nearly 50% in nerve fibre damage induced by NMDA infusions. Possible mechanisms of differential calcium influx in the various experimental conditions will be discussed.

Dysfunction of the brain cholinergic system during aging and after lesions of the nucleus basalis of Meynert.

In order to establish how closely the models mimic aging and Alzheimer's disease, a comparison was made, using the extensive literature available, between brain cholinergic dysfunction in aging animals and man, and between that in animals with lesions of the nucleus basalis of Meynert and in subjects affected by Alzheimer's disease. It is concluded that cholinergic dysfunction in the aging rat closely resembles that in aging man. A similarity can also be found between the cortical dysfunction induced by lesions of the nucleus basalis and that occurring in patients with Alzheimer's disease. However, cholinergic dysfunction only represents a limited aspect of the neorotransmitter deficits and neuropathological alterations of the disease.

Quantitation and three-dimensional reconstruction of Ch4 nucleus in the human basal forebrain.

The basal nucleus of Meynert, incorporating the Ch4 group of cholinergic neurons, was examined in six patients with no signs of neurological abnormalities. The ages of the patients ranged from 20 to 80 years. Despite a number of descriptions of these neurons, few age-related studies have been dedicated to the analysis of the entire anteroposterior extent of the nucleus. Staining with cresyl violet and acetylcholinesterase histochemistry, alone or in combination, was used to identify the cytoarchitectural organization of the Ch4. Computer-assisted morphometry was used for three-dimensional visualization and quantitation. The three-dimensional computer reconstructions revealed a continuous ribbon of neurons with a highly variable density. Four distinct subregions could be clearly identified in all cases by their cytoarchitecture and cellular morphology, although these subgroups were different to those previously described. There were no quantitative differences between the hemispheres in volume, density or cell number of the Ch4, although equivalent levels varied in area and density. The measures were similar in all cases with the exception of the case aged 80 years old. The data demonstrate individual variability in three dimensions and confirm previous studies that found only a mild decline of the Ch4 in old age.

Modulatory action of cholinergic drugs on N-methyl-D-aspartate response in dissociated rat nucleus basalis of Meynert neurons.

The effect of cholinergic drugs on N-methyl-D-aspartate (NMDA)-activated responses in neurons dissociated freshly from the nucleus basalis of Meynert (nBM) of immature and mature rats were investigated with the whole-cell patch-clamp technique. The NMDA (10(-4) M)-evoked inward current (INMDA) consisted of transient peak and successive steady-state components. In mature neurons, the peak component was suppressed by pretreatment with acetylcholine (ACh, 10(-12)-10(-5) M) but facilitated by higher concentrations (greater than 10(-5) M). Regardless of the concentration, ACh had no effect on INMDA of immature neurons. The results suggest that ACh behaves as either inhibitory or excitatory modulator of glutaminergic transmission in nBM neurons of adult mammals depending on ACh concentration.

Early development of the nucleus basalis-cortical projection but late expression of its cholinergic function.

The aim of this study was to examine the development of the basalocortical pathway by using choline acetyltransferase and nerve growth factor receptor immunocytochemistry, acetylcholinesterase histochemistry and retrograde axonal transport. The observations were made in the ferret because in this species brain development occurs over a much more protracted period than in the rat. Staining for choline acetyltransferase immunoreactivity in the brain was minimal before birth. Adult levels of staining for the enzyme were not seen in cell bodies until three weeks after birth and in axons up to six weeks after birth. This, however, did not mean that presumptive cholinergic pathways are absent early in development. There was strong staining for nerve growth factor receptor in basal forebrain neurons from at least two weeks before birth. Positive staining for acetylcholinesterase was found in axons that begin to invade the cerebral cortex a week before birth. The retrograde axonal transport technique showed that the basalocortical pathway has a normal organization in the neonate. The conclusion is that cholinergic pathways form early in the prenatal period in the ferret but express their transmitter function late in postnatal development.

NGF receptor immunoreactivity in aged rat brain.

The cellular distribution of nerve growth factor (NGF) receptor (NGFR) immunoreactivity in 3 cholinergic nuclei (medial septal nucleus, nucleus of the diagonal band and nucleus basalis magnocellularis) of the aged rat brain was compared to that of young-adult animals. In young-adult rats, NGFR immunoreactivity was strong in the neuronal body and in the whole dendritic tree. In aged animals, NGFR immunoreactivity was weak in both cell body and dendrites and was practically absent in the dendrite's distal portion. The loss of dendritic NGFR may play a critical role in the decline of neuronal function in the aging brain.

[Postnatal ontogenesis of cholinergic fibers in the rat cerebral cortex]. / Ontogenetische Untersuchungen der cholinergen Innervation der Grosshirnrinde bei Ratten.

Approximately 80% of the activities of cortical cholinergic marker enzymes are restricted to afferent fibres and terminals of the magnocellular basal nucleus of Meynert (NbmM) (Wenk et al. 1980). In the evolution this nucleus strongly follows a phylogenetic gradation (Brockhaus 1942) which is positively correlated to the cerebralization, i.e. the neocortical development which an animal species has reached (Gorry 1963). Moreover, in contrast to the brain stem, in vertebrate brains of different phylogenetic levels only the telencephalon showed a clear progressive representation of cholinergic markers (Wächtler 1980, 1981). However, the increased cholinergic activity of the telencephalon in higher forms can not be ascribed to a special hemispheric component (Wächtler 1982). Using the quantitative-histochemical evaluation of the cholinergic marker enzyme acetylcholinesterase (AChE) in the epithalamus (nucleus medialis habenulae) and in different cortical areas of rats during postnatal ontogenesis it is shown that (i) the cholinergic differentation in the thalamic structure seems to be already finished immediately after birth and (ii) the cholinergic synaptogenesis in the cerebral cortex does not start until birth, but takes place postnatally in phylogenetic graduated cortices in temporal echeloned steps (paleo- greater than archi- greater than neocortex). These ontegenetic results correspond to the phylogenetic trends and reflect - in accordance with the biogenetic basic rule - the phylogenetic gradation in the development of the cholinergic basal nucleus of Meynert and its cortical projections shortened in ontogenesis.

Histogenesis of the striopallidal system in the rat. Neurogenesis of its neurons.

This study discusses the histogenesis of the structures of the extrapyramidal system. The first part based on [3H]thymidine autoradiography provides data on the time of origin of the various neuronal populations that characterize different structures of the extrapyramidal system. Such data are essential to any further study dealing with the localization of the different sites of origin of the neurons along the ependymal matrix and with their migration during the histogenetic sequences leading to their definitive pattern of adult distribution. The neurons of globus pallidus and of the entopeduncular nucleus are generated on days 12-15 and days 11-14, respectively. A peak of neurogenesis occurs on day 12 for the entopeduncular nucleus and on day 14 for the globus pallidus. In the pallidum, the first neurons to form on day 12 settle caudally, while neurons generated on day 15 settle in the rostral extremity. The genesis of the medium-sized neurons of the neostriatum extends from day 12 to at least postnatal day 2. A peak is obvious on day 15. Although the neurogenesis of these neurons follows a mild caudorostral gradient, a more careful examination reveals four different patterns of settlement according to the area involved and the period of gestation. At the level of the caudal neostriatum, the neurons display a clear mediolateral spatiotemporal gradient. More rostrally, the neurons generated on days 13, 14 and 15 show two patterns of settlement. On the one hand, many neurons settle rather densely along the external capsule on day 13, occupying more rostral levels on days 14 and 15. On the other hand, in the body of the neostriatum, clusters of isochronically generated neurons are obvious. Later, newly generated neurons display a rather homogeneous distribution in the structure. A parallel is drawn between these patterns of development and the patterns of distribution of afferent terminals or neurotransmitters. The large chromophilic neurons of the neostriatum appear exclusively during the early period. Two peaks of neurogenesis are apparent. The one on day 13 comprises neurons that settle caudally. It is contemporaneous to the neurogenesis of the adjacent basal nucleus. The second peak occurs on day 15 and corresponds to that of the medium-sized neurons.

Guidance of acetylcholinesterase-containing fibres by target tissue in co-cultured brain slices.

Slices of various brain regions were prepared from newborn and from 7-day old rats and co-cultured in different combinations. In the majority of co-cultures of septal and hippocampal slices, acetylcholinesterase-positive fibres originating in the septal nuclei invaded the adjacent hippocampal slice. A similar pattern of hippocampal ingrowth by acetylcholinesterase-positive fibres occurred with slices prepared from the nucleus basalis of Meynert and from spinal cord. Septal neurones also projected to cortical slices, an effect which even occurred in the presence of their natural target tissue. In contrast to these massive projections to brain areas which in situ receive cholinergic inputs, no significant acetylcholinesterase-positive fibre ingrowth was observed in tissues which lack major cholinergic afferents in situ (hypothalamus, substantia nigra and cerebellum). These results indicate that under our culture conditions, acetylcholinesterase-positive fibres selectively invade cholinergic target areas. This effect is independent of the brain area from which the cholinergic neurones were derived.