![]() The fetuses were decapitated and the whole brain was transferred into fresh cold Hibernate E/ B27/ Glutamax ™/ Antibiotic-Antimycotic. After C-sectioning, the fetuses were collected in ice cold Hibernate E/ B27/ Glutamax ™/ Antibiotic-Antimycotic. ![]() The timed pregnant rats (fetal day 18) were euthanized using carbon dioxide. However, they lose this ability to synthesize multiple neurotransmitters as they mature.ġ8 day-old fetuses were obtained from timed pregnant rats. The neurons, at this early stage of development, then follow a complex combinatorial strategy of electrochemical coding in order to determine their final fate. Based on these findings, we are proposing that at a very early developmental stage, neurons have the ability to synthesize multiple neurotransmitters. In cultured neonatal and adult hippocampal neurons, co-localization of VGLUT1 and ChAT proteins was not observed. On the other hand, fetal hippocampal neurons, which expressed GABA, did not express ChAT. Our studies have shown that in the fetal rat, hippocampal neuron culture most of the glutamatergic neurons, which expressed glutamate vesicular transporter (VGLUT1), also co-expressed choline acetyltransferase (ChAT) proteins. A serum-free culture medium and patternable, synthetic silane substrate was utilized to grow the cells and perform the immunocytochemical studies. In this study, the aim was to identify and quantify the origin of this rare population of ChAT-immunoreactive cells in the cultured hippocampal neurons that were obtained from fetal, neonatal and adult rats. However, the origin of these endogenous ChAT-immunoreactive cells in the hippocampus is not clear. Furthermore, it has been observed that these small populations of cholinergic neurons in the rat hippocampus do not compensate for the loss of septo-hippocampal cholinergic fibers ( Frotscher, 1988). Interestingly, these endogenous cholinergic neurons, present in the hippocampus, have a functional role in the induction of long-term potentiation (LTP) at mossy fiber-CA3 synapses ( Maeda et al., 1994). This small number of ChAT-immunoreactive cells in the hippocampus are the intrinsic source of hippocampal cholinergic innervation, in addition to the well-established septo-hippocampal cholinergic projection ( Baisden et al., 1984 Colom et al., 2005 De Lacalle et al., 1994 Ferencz et al., 2001 Frazier et al., 1996 Frotscher and Leranth, 1985 Frotscher et al., 1986 Gahwiler et al., 1987 Gilad et al., 1987 Ishimaru et al., 1995 Linke et al., 1994 Nilsson et al., 1992 Ransmayr et al., 1989 Ransmayr et al., 1992 Schwegler et al., 1996 Sotty et al., 2003 Sugaya et al., 1997 Vincent and McGeer, 1981 Williams et al., 1989). In the adult mammalian hippocampus, Choline acetyltransferase (ChAT) immunoreactive cells are rare, but are observed in all layers of the hippocampus ( Frotscher et al., 1986). ![]() One possible explanation for this observation is that the neurons have the ability to synthesize multiple neurotransmitters at a very early stage of development and then with time follows a complex, combinatorial strategy of electrochemical coding to determine their final fate. Colocalization of VGLUT1 and ChAT in these relatively more mature neurons was not observed. Neonatal and adult rat hippocampal neurons were also cultured to verify whether these more mature neurons also co-express VGLUT1 and ChAT proteins in culture. On the contrary, when the cultures were double stained with GABA and ChAT, colocalization was not observed. Interestingly, most of the glutamatergic neurons which expressed glutamate vesicular transporter (VGLUT1) also co-expressed choline acetyltransferase (ChAT) proteins. Using this method a large proportion of glutamatergic (glutamate vesicular transporter, VGLUT1-immunoreactive) neurons, a small fraction of GABAergic (GABA-immunoreactive) neurons and a large proportion of cholinergic (ChAT-immunoreactive) neurons were observed in culture. This study aimed at quantifying and identifying the origin of this small population of ChAT-immunoreactive cells in the hippocampus at early developmental stages, by culturing the fetal hippocampal neurons in serum-free culture and on a patternable, synthetic silane substrate N-1 diethylenetriamine (DETA). This is the intrinsic source of the hippocampal cholinergic innervation, in addition to the well-established septo-hippocampal cholinergic projection. A very small population of Choline acetyltransferase (ChAT) immunoreactive cells is observed in all layers of the adult hippocampus.
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