Cholinergic innervation of cerebral cortex in organotypic slice cultures: Sustained basal forebrain and transient striatal cholinergic projections (original) (raw)
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Neuroscience, 2002
AbstractöDistributions of somata and neurites of cholinergic neurons were studied after seeding dissociated cells onto organotypic slice cultures. Slice cultures were made from hippocampal formation and adjacent cortical regions from rats or mice. Dissociated cell suspensions of basal forebrain tissue from rat or mouse fetuses were seeded onto the slice cultures. Combined cultures were maintained for 1^21 days in vitro. Cultures processed for acetylcholinesterase (AChE) histochemistry demonstrated non-random patterns of cholinergic cells and their neurites. Labeled cells appeared most frequently in the molecular layer of the dentate gyrus, and in the deeper layers of cortical regions adjacent to the hippocampus. Neurites extending from these labeled cells appeared to target the dentate molecular layer and the cortical subplate layer. By 4 days in vitro, AChE-positive basal forebrain cells display several short and thick neurites that appear to be dendrites, and one long process that appears to be an axon. By 5 days in vitro, dendrites are well developed; by 7 days the presumed axon has extended widely over the cortical target zone. These neurites are maintained through 3 weeks in culture. Distributions of cells varied with the age of the slice. AChE-labeled cells were not seen overlying hippocampal tissue when dissociated cells were seeded on slice cultures made from day 0 rats, but a few labeled cells were seen when seeded on slices from day 2 rats. Clear non-random patterns of labeled cells and neurite outgrowth were seen on slice cultures from day 5 or older pups. The non-random distribution seen with AChE-positive neurons was not seen using other techniques that labeled all cells (non-selective £uorescent labels) or all neurons; these techniques resulted in labeled cells scattered apparently homogenously across the slice culture.
Evidence for a cholinergic projection to neocortex from neurons in basal forebrain
Proceedings of the …, 1979
Unilateral stereotaxic injection of 3.5 nmol of kainic acid into the ventral globus pallidus of rats reduced biochemical cholinergic neuronal markers by 45-50% and virtually eliminated histochemical staining for acetylcholinesterase in neocortex ipsilateral to the lesion. At the lesion site, the large, multipolar neurons that stain densely for acetylcholinesterase were absent when compared with the uninjected side. Kainate was as effective as electrocoagulation for reducing cholinergic markers although it did not affect aminergic projections ascending through the lesioned area. The conclusion that the cholinergic projection originated in neuronal perikarya at the lesion site was supported by the failure of kainate or electrolytic lesions in contiguous regions to produce similar effects. These studies provide strong evidence for a cholinergic projection to neocortex from neurons in the forebrain in the nucleus basalis.
Cholinergic innervation of mouse forebrain structures
The Journal of Comparative Neurology, 1994
Using choline acetyltransferase (C U T) immunocytochemistry and acetylcholinesterase (AChE) histochemistry, we investigated regional and laminar differences in cholinergic innervation in the cerebral cortex, hippocampus, amygdala, and thalamus of mice. In mice, unlike rats, the patterns of CUT-immunostained and AChE-positive fibers are virtually identical in the cortex and are organized in a trilaminar pattern with cholinergic processes prominent in layers I and IV and within the lower portion of layer V and upper segment of layer VI. CUT-immunoreactive cells were not seen in cortex. In the amygdala, the basolateral nucleus showed the highest density of cholinergic processes. In the hippocampus, a thin, dense band of CUT-labeled processes was present in the inner segment of the molecular layer of the dentate gyrus and within the stratum oriens of CA1-3, adjacent to the basal aspect of pyramidal cells. Within the thalamus, anteroventral, mediodorsal (lateral portion), intralaminar, and reticular nuclei showed high densities of cholinergic processes. The results of this study provide the basis for examining the effects of transgenes and age on forebrain cholinergic systems.
International Journal of Developmental Neuroscience - INT J DEV NEUROSCI, 2001
The distribution of acetylcholinesterase histochemistry and choline-O-acetyltransferase immunohistochemistry in the basal forebrain was studied in newborn mice (P0) and until 60 days of postnatal life (P60). A weak acetylcholinesterase activity was found at P0 and P2 in the anterior and intermediate parts of the basal forebrain, and higher in the posterior region. The intensity of labeling, neuronal size and dendritic growth seems to increase progressively in all regions of basal forebrain from P4 to P10. The AChE+ cell count shows that in the anterior portion of the magnocellular basal nucleus the number of cells does not vary significantly from birth to the second month of postnatal life. However, in the intermediate and posterior portions of the nucleus the mean number of labeled cells increases significantly from birth to the end of the second week of postnatal life (P13). The choline-acetyltransferase immunoreactivity appears only detectable at the end of the first week (P6) as...
Developmental Brain Research, 1985
Measurements of choline acetyltransferase (CHAT) activity were made during the development of the neocortical cholinergic innervation, and correlated with the development of the acetylcholinesterase (ACHE) staining pattern in mouse cerebral cortex and several other areas of the forebrain between the time of initial onset and maturity. ChAT activity can first be measured on postnatal day 6 (P6). The enzyme reaches 40% of adult activity by P18 and adult values by 7 weeks postnatal. The onset of AChE staining varies for different regions of the forebrain and for various areas within the cerebral cortex. The earliest appearance of AChE is seen in several basal forebrain nuclei including the striatum, the ventromedial region of the globus pallidus and the hypothalamus on embryonic day 18 (El8). In neocortex and olfactory cortex, AChE-stained axons are seen in the white matter before birth, but do not enter cingulate cortex and hippocampus until P2. By P2. almost all areas of the basal forebrain and diencephalon have acquired some AChE staining pattern. The adult distribution of AChE staining is reached by 3 weeks postnatal in all areas of the forebrain. Adult cerebral cortex shows a characteristic pattern of alternating AChE dense and AChE sparse bands which vary in depth depending on the cortical area. The cortical banding pattern develops in an 'inside-out' fashion, starting in layer VI and gradually entering more superficial layers. In parallel with the AChE pattern of development in cortex, transient AChE staining can be observed in some thalamic nuclei and in some forebrain fiber systems. In the neostriatum patches of intense AChE staining first develop along the ventrolateral border, then spread throughout the whole nucleus and finally coalesce to a uniform high density over the entire neostriatum. We discuss the close spatial and temporal correspondence between AChE pattern development and reported data on synapse formation, and speculate on the role of the cortical cholinergic system in development.
Brain Research Bulletin, 1997
Unilateral AMPA lesions of the nucleus basalis magnocellularis (nbm) produced a nearly complete loss of cholinergic markers in the ipsilaterel frontal and parietal cortices with no recovery at 6 months. The loss was associated with compensatory increases in AChE-posi'dve fibre density in the contraleteral cortex, in ipsilateral cortical regions not receiving their cholinergic innervation from the nbm and in the size of cholinergic magnocellular neurones in the contralateral nbm. The hypertrophy and increase in AChE-positive fibre density were apparent at 4-6 weeks after lesion and increased with time. Cholinergic transplants to cholinergically deafferented cortex prevented development of the compensatory increases in AChE-positive fibre density and restored AChE-positive fibre density and ChAT activity to control levels in ipsilateral cholinergically deafferented regions, partially after 6-8 weeks and completely after 6 months. In contrast, when cholinergic grafts were placed into unlesioned cortex, axonal outgrowth was localized to the vicinity of the transplant and did not develop with time. These results support the concept that vacant synapses promote and direct axonal outgrowth from transplanted neurones and that grafted cholinergic neurones integrate into the lesioned forebrain choiinergic projections system and prevent the lesion-induced changes in AChE-positive fibre density and ChAT activity.
Brain Research Bulletin, 1989
Magnocellular regions of the basal forebrain contain cholinergic neurons that project to the cerebral cortex. Neurons in the same basal forebrain regions innervate the brainstem. The present study investigated whether these brainstem projecting neurons are cholinergic, project also to the cortex, and share similar physiological properties as cortically projecting neurons. Data with retrograde tracing from various regions of the pons, medulla, and cortex combined with choline acetyhransferase immunofluorescence indicated that: 1) brainstem projecting neurons are usually segregated from cortically projecting and/or cholinergic neurons in the basaf forebrain, 2) virtuaily no brainstem projecting neurons in the basal forebrain are cholinergic, and 3) only rarely do basal forebrain neurons have axon collaterals that project to both cortex and brainstem. Extracellutar recordings from basal forebrain neurons confirmed the paucity of axonal collateralization and the topographic segregation between cortically and brainstem projecting basal forebrain neurons, and, in addition, showed that brainstem projecting neurons have a slower mean conduction velocity than cortically projecting neurons. These observations suggest that basal forebrain neurons projecting to the brainstem (pans, medulla) and the cortex represent separate cell populations in terms of projections, neurotransmitter content, distribution, and physiological properties. Basal forebrain Brainstem Choiinergic Retrograde tracing Immuno~uo~~ence Extracellular recordings
Neuroscience, 1993
Slices of striatal tissue from newborn to eight-day-old rats were cultured for six to 47 days. Cholinergic neurons and fibres were then visualized by histochemical staining for acetylcholinesterase or immunoeytochemical staining for choline acetyltransferase. GABA-containing neurons and fibres were visualized by immunocytochemical staining for glutamate decarboxylase or GABA. Corresponding to the normal postnatal development in vivo, acetylcholinesterase staining of the striatal tissue progressed from a "patchy" distribution in the six to 14 days old cultures to an almost even distribution of high acetylcholinesterase activity after 18-27 days. Extrinsic afferents were accordingly not necessary for the maintenance of a patch-matrix-like, acetylcholinesterase distribution during the first one to two weeks in culture, just as a subsequent, normal developmental change of the acetylcholinesterase staining pattern into a more homogeneous distribution also occurred without such afferents. Cholinerglc, choline acetyltransferase-immunoreactive neurons were evenly distributed within the cultured striatal tissue, like in vivo, but the density of the neurons appeared to be higher in the cultures. The neurons had a morphology corresponding to the "classical", large-sized, aspiny, cholinergic interneurons in the adult rat striatum. Glutamate decarboxylase-immunoreactive and GABA-immunoreactive neurons were either lightly or darkly stained and of medium size, but some large, lightly stained glutamate decarboxylase-immunoreactive and GABA-immunoreactive neurons were also found. The difference in staining density among the medium-sized cells was observed with both antisera and hence provide evidence for the existence of two populations of medium-sized GABAergic neurons, which in vivo are intensely stained internenrons and more weakly stained, spiny projection neurons. Fibres stained better for glutamate decarboxylase than for GABA and outgrowth of glutamate decarboxylase-immunoreactive nerve fibres from the striatal slice cultures onto the coverslip was often observed. The presence at all culture periods of "protospines" on cell bodies and proximal dendrites of some glutamate deearboxylase-immunoreactive, and in particular some GABA-immunoreactive neurons, suggested that at least some developmental characteristics might be maintained for extended periods in culture. In several cultures, groups of small GABA-immunoreactive cells were observed. Similar groups were also found by staining for glutamate decarboxylase, but a smaller proportion of the cells were then positively stained. In view of their immature appearance with few or no processes, the known presence of GABA in neuroblast-like cells, and the recent demonstration of neuronal and glial progenitor cells in the adult mouse striatum, the small cells might belong to a population of undifferentiated cells surviving in the slice cultures. In conclusion, the study demonstrates that striatal cholinergic interneurons and GABA-containing interneurons and projection neurons can survive in slice cultures for at least seven weeks without the reinstated or continous presence of normal extrinsic afferents. During this period the neurons attain a basically normal morphological and neurochemieal differentation. Based on the results we will proceed to use slice cultures for experimental neurotoxic and neurotrophic studies, related to striatal neurodegenerative diseases, like Huntington's chorea, Parkinson's disease and cerebral ischemia. Tissue slices from various brain regions, including neocortex, hippocampus, striatum, cerebellum, ven