Neurons In Organotypic Slice Cultures Research Articles

The influence of target and non-target brain regions on the development of mid-brain dopaminergic neurons in organotypic slice culture

The development and regeneration of rat dopaminergic neurons of the ventral mesencephalon was studied in organotypic slice cultures. Single ventral mesencephalon cultures and co-cultures of ventral mesencephalon with striatum (a target region) or cerebellum (a non-target region) were prepared from postnatal day 1 Wistar rats. Cultures were processed for tyrosine hydroxylase and glial fibrillary acidic protein immunoreactivity, at two day intervals, for an overall incubation period of 20 days. Analysis of these cultures revealed that the striatal target tissue, exerted neither a trophic nor a tropic influence on the tyrosine hydroxylase immunoreactive neurons. In both single and co-cultures, tyrosine hydroxylase immunoreactive neurites projected radially from the ventral mesencephalon slice. However, in striatal co-cultures, tyrosine hydroxylase immunoreactive neurites were seen penetrating the striatal slice, whereas in cerebellar co-cultures no tyrosine hydroxylase immunoreactive neurites entered the cerebellar tissue. Glial fibrillary acidic protein positive cells actively migrated from the tissue sections, however tyrosine hydroxylase immunoreactive neurite outgrowth was not guided by these glial cells. Tyrosine hydroxylase immunoreactive neurites terminated once they had penetrated the striatal slice. This retardation of neurite growth by a target region could be important in establishing and reinforcing synaptic connections in the developing nigro-striatal pathway.

The effect of acetylcholinesterase on outgrowth of dopaminergic neurons in organotypic slice culture of rat mid-brain

This study has investigated the possibility that acetylcholinesterase could play a non-classical role as an adhesion factor or growth factor in the development of dopaminergic neurons in organotypic slice culture of postnatal day 1 rats. When the culture medium was supplemented with acetylcholinesterase (3 U/ml), outgrowth of tyrosine hydroxylase-immunoreactive neurites was significantly enhanced. Addition of a specific inhibitor of acetylcholinesterase, BW284c51, caused a decrease in the number of tyrosine hydroxylase neurons and a reduction in the cell body size and extent of neurite outgrowth of remaining neurons. However, echothiophate which also inhibits AChE activity, did not produce these effects. Therefore acetylcholinesterase could act as a growth enhancing factor for dopaminergic neurons, and disruption of an as yet unidentified site on the acetylcholinesterase molecule by BW284c51 could decrease the survival and outgrowth of these neurons.

The effect of acetylcholinesterase on outgrowth of dopaminergic neurons in organotypic slice culture of rat mid-brain

The effect of acetylcholinesterase on outgrowth of dopaminergic neurons in organotypic slice culture of rat mid-brain

Activity-dependent regulation of dendritic spine density on cortical pyramidal neurons in organotypic slice cultures.

In order to examine the effects of activity on spine production and/or maintenance in the cerebral cortex, we have compared the number of dendritic spines on pyramidal neurons in slices of P0 mouse somatosensory cortex maintained in organotypic slice cultures under conditions that altered basal levels of spontaneous electrical activity. Cultures chronically exposed to 100 microM picrotoxin (PTX) for 14 days exhibited significantly elevated levels of electrical activity when compared to neurons in control cultures. Pyramidal neurons raised in the presence of PTX showed significantly higher densities of dendritic spines on primary apical, secondary apical, and secondary basal dendrites when compared to control cultures. The PTX-induced increase in spine density was dose dependent and appeared to saturate at 100 microM. Cultures exhibiting little or no spontaneous activity, as a result of growth in a combination of PTX and tetrodotoxin (TTX), showed significantly fewer dendritic spines compared to cultures maintained in PTX alone. These results demonstrate that the density of spines on layers V and VI pyramidal neurons can be modulated by growth conditions that alter the levels of spontaneous electrical activity.

Neurotrophin-4 selectively promotes survival of striatal neurons in organotypic slice culture

The neurotrophins (NGF, BDNF, NT-3, and NT-4) provide trophic support to subpopulations of neurons in the central and peripheral nervous systems. We examined organotypic slices of neonatal mouse striatum maintained in medium supplemented with neurotrophins or with CNTF to determine which of these factors influence the survivability of striatal neurons. Neuron counts at the end of the culture period revealed that NT-4 was the only factor that had a significant effect on neuronal survival, suggesting that NT-4 is a trophic factor for striatal neurons in organotypic slices.

Optical Imaging of Cytosolic Calcium, Electrophysiology, and Ultrastructure in Pyramidal Neurons of Organotypic Slice Cultures from Rat Hippocampus

Organotypic slice cultures from rat hippocampal cortex grown in an interface between culture medium and a CO2-enriched atmosphere maintained much of the morphological connectivity characteristic of the hippocampus in situ and thinned out considerably, facilitating optical measurements of fluorescent dyes sensitive to Ca2+ in individual neurons. Pyramidal neurons of the CA3 region presented morphological features of differentiated cells, including complex dendritic arborization and large numbers of dendritic spines. The fine cytoskeletal substructure at the postsynaptic density, below the plasma membrane, and within the core of the head and neck of dendritic spines in rapidly frozen slice cultures presents the characteristic morphology previously described for Purkinje cell dendritic spines in acutely dissected cerebellar cortex slices after rapid freezing. CA3 neurons responded to intracellular current injection with a train of action potentials, spike frequency adaptation, and a slow afterhyperpolarization. These spike trains caused rapid increases in dendritic [Ca2+]i that decayed to resting levels after termination of the current pulse. Dendritic spines were clearly observed in proximal dendrites of CA3 neurons in live preparations. [Ca2+]i transients in these dendritic spines closely followed the changes observed in the main dendritic shaft. Orthodromic synaptic stimulation from the dentate hilus generated long-lasting synaptic potentials accompanied by large [Ca2+]i transient in CA3 pyramidal neurons. The [Ca2+]i response was first observed in the proximal dendrites, after which the soma exhibited a [Ca2+]i increase, returning to resting levels within 10 s after the synaptic stimulus. Slice cultures thus provide a favorable opportunity to investigate [Ca2+]i responses in individual neurons maintained in an organotypic synaptic environment, taking advantage of high-resolution optical techniques.

Tyrosine hydroxylase immunoreactive neurons in organotypic slice cultures of the rat striatum and neocortex

Organotypic slice cultures of striatum and neocortex were prepared from newborn to seven day old rats and cultured for three to 60 days. When processed for tyrosine hydroxylase (TH) immunocytochemistry medium-sized, aspiny TH immunoreactive (TH-i) neurons with a similar morphology were revealed in the striatum and the neocortex. The neurons had a very similar morphology in both tissues and were present both when the two tissues were grown separately as single cultures and when grown together either en bloc as part of the same tissue slices or as co-cultures. In order to examine whether innervation by dopaminergic fibers would affect the expression of TH-i neurons in the striatal slice cultures, co-cultures of ventral mesencephalon (VM) and striatum were prepared, but the ingrowth of TH-i fibers from the VM did not alter the expression of TH immunoreactivity by a subpopulation of striatal neurons.

Continuous presence of nerve growth factor is required for maintenance of cholinergic septal neurons in organotypic slice cultures

In co-cultures of rat septum and hippocampus, cholinergic neurons, identified by immunocyto-chemical techniques using antibodies against choline acetyltransferase, were found to be exclusively located in septal tissue. The presence of nerve growth factor during the entire growth period of four weeks increased the activities of acetylcholinesterase and choline acetyltransferase about 10-fold and strongly increased the number of acetylcholinesterase-positive neurons. Application of nerve growth factor yielded different effects depending on the age of the cultures. During the first two weeks in vitro, nerve growth factor enhanced the number of acetylcholinesterase-positive neurons, an effect which was no longer observed following later applications of nerve growth factor. Nerve growth factor increased the activities of cholinergic enzymes during all phases of in vitro development, but the effects of one-week applications were always considerably smaller than those observed following continuous application of nerve growth factor. The results of different application schedules suggest that the continuous presence of nerve growth factor is needed for maximal increases in cholinergic enzyme activities and maintenance of cholinergic neurons in septohippocampal co-cultures.

Development of vasoactive intestinal polypeptide (VIP)-containing neurons in organotypic slice cultures from rat visual cortex

Using immunohistochemistry we have been studying the postnatal maturation of vasoactive intestinal polypeptide (VIP)-positive neurons in organotypic slice cultures from rat visual cortex. The development in vitro is compared with the occurrence of VIP-containing cells in vivo, where they are first observed around postnatal day 5. A further increase in number and morphological maturation occurs within the following 3 weeks. In cultures prepared from 1- or 2-day-old rats, i.e. before VIP is expressed in vivo. VIP-containing neurons appear after about 5 days and gradually increase in number over the next 2 weeks. Thus the time course of postnatal expression of VIP in vitro and the morphology of VIP-immunoreactive neurons in culture closely matches the situation in vivo. These observations suggest that the maturation of VIP-containing neurons occurs independently of cortical afferents and that the intrinsic connectivity and activity is sufficient for their postnatal maturation. Therefore organotypic slice cultures should be a suitable system to study mechanisms of neurochemical maturation in the cortex.