Fig. 3.

Endogenous neuromodulators localize to presynaptic sites in hippocampal neurons. Dual-color image showing that puncta stained by an antibody against endogenous BDNF (red) (A) are present in presynaptic clusters (green) along axons of fixed 7-DIV hippocampal neurons expressing synaptophysin-EGFP. Analogous image of endogenous tPA (red) (B) in fixed 10-DIV hippocampal neurons expressing synaptophysin-EGFP (green). Quantification of data obtained from these types of images revealed that the probability that a bouton houses a DCG containing endogenous BDNF is 0.41 ± 0.04 (n = 171 SV clusters, 65 DCGs). The analogous result for endogenous tPA is 0.39 ± 0.07 (n = 382 SV clusters, 171 DCGs). Each of these endogenous localization probabilities is statistically indistinguishable from its analogous exogenous localization probability. Specifically, results from the Student's t-test yielded large p values for both BDNF (p = 0.40 > 0.05) and tPA (p = 0.49 > 0.05). Scale Bar = 2 μm for A and B.

In past work, several groups (including ours) also have used immunostaining to compare levels of expression of endogenous and exogenous neuromodulators. These analyses reveal that, on average, exogenous neuromodulators are expressed at levels that are ~2–5× endogenous levels (Kolarow et al., 2007, Lochner et al., 2008, Matsuda et al., 2009) and that the density of DCGs containing exogenous neuromodulators is less than twice that of DCGs containing endogenous neuromodulators. We tend to find exogenous protein chimeras expressed at the lower end of this range, perhaps because we image neurons quickly after viral delivery of DNAs encoding the chimeras. The important conclusion from these studies is that overexpression of chimeras is modest and thus is unlikely to introduce major artifacts into our results, such as extensive labeling of DCGs that do not contain endogenous neuromodulators.