Table 2.

Acetylcholine-dependent Ca²⁺ activity in hypothalamic cultures raised under various cell culture conditions^2-a

Culture condition	Total number of neurons responding to bicuculline	Number of neurons with the cholinergic response (%)^2-b	Ca²⁺ response to bicuculline is reduced by AChR antagonists, nmCa²⁺
Culture condition	Total number of neurons responding to bicuculline	Number of neurons with the cholinergic response (%)^2-b	Bicuculline	Bicuculline + AChR antagonists
20 μm AP5 + 2 μmCNQX	n = 30 of 80	n = 27 of 80 (34%)	166.9 ± 9.1	13.3 ± 2.1***^2-c
100 μmAP5	n = 69 of 90	n = 66 of 90 (73%)	221.2 ± 13.6	22.4 ± 2.6***
5 μmCPP	n = 79 of 106	n = 73 of 106 (69%)	173.3 ± 9.5	19.8 ± 2.3***
10 μmCNQX	n = 2 of 233	n = 2 of 233 (1%)	156.5 ± 6.5	14.0 ± 4.0*
100 μmAP5 + 10 μm CNQX + 20 mm KCL	n= 0 of 341	n = 0 of 341 (0%)	0	0
1 μm TTX	n = 0 of 109	n = 0 of 109 (0%)	0	0
CONTROL^2-d	n = 0 of 174	n = 0 of 174 (0%)	0	0
BLOCK^2-e (18 DIV/14 DIB)	n = 150 of 169	n = 143 of 169 (85%)	170.7 ± 8.1	21.6 ± 1.6***
10 d after BLOCKADE removal	n = 0 of 119	n = 0 of 119 (0 %)	0	0
10 d after BLOCKADE reintroduction	n = 67 of 105	n = 57 of 105 (54%)	112.2 ± 5.2	19.7 ± 2.8***
BLOCK (10 DIV/6 DIB)	n = 9 of 59	n = 7 of 59 (12%)	135.4 ± 18.2	12.8 ± 5.0**
BLOCK (74 DIV/70 DIB)	n= 157 of 167	n = 149 of 167 (89%)	142.3 ± 6.2	25.8 ± 1.9***
BLOCK (74 DIV/14 DIB)	n= 120 of 210	n = 107 of 210 (51%)	150.1 ± 5.8	11.9 ± 1.0***

↵F2-a All cells were tested with bicuculline (50 μm) in the presence of 100 μm AP5 and 10 μm CNQX in the medium.
↵F2-b This column presents the number of neurons that reduced (by ≥50%) bicuculline-mediated Ca²⁺response during the application of AChR antagonists, atropine and mecamylamine (100 μm each). The average amplitude of the response measured in these neurons before and during administration of AChR antagonists is shown in the next column.
↵F2-c Significance of differences (Student'st test) was calculated relative to the response mediated by bicuculline alone: ***p < 0.0001, **p< 0.001, *p < 0.05. The measurements of Ca²⁺ response were done relative to the initial background Ca²⁺ level.
↵F2-d CONTROL, Cells from control cultures (33 DIV). The experiment is illustrated in Figure 8g.
↵F2-e BLOCK, Cells from cultures chronically incubated in the presence of 100 μm AP5 and 10 μm CNQX. The time of incubation is shown in parentheses.