The present study asks two fundamental questions. First, does the Ca2+
-dependent exocytosis of IGF-1 we recently described in olfactory bulb neurons (Cao et al., 2011
) represent a standard pathway of neuropeptide secretion similar to the well-characterized pathway of LDCV exocytosis (Winkler and Fischer-Colbrie, 1998
), or does it constitute a novel distinct secretory pathway? Second, is the triggering of IGF-1 secretion mediated by Ca2+
-binding to Syt10 (but not to the synaptotagmins normally implicated in neuropeptide secretion) dependent on complexins similar to neurotransmitter secretion and chromaffin granule exocytosis induced by Ca2+
-binding to Syt1, Syt2, Syt7, or Syt9?
Our data show that the vesicles mediating IGF-1 secretion are distinct in location and size from those mediating neuropeptide secretion ( and ), but that the exocytosis of these IGF-1-containing vesicles is nevertheless dependent on complexins (-). Moreover, our data demonstrate that complexin functions in IGF-1 secretion as an activator that enables rapid Ca2+-triggered exocytosis of IGF-1 containing vesicles. Thus, olfactory bulb neurons (most likely mitral neurons) generate two classes of peptidergic secretory vesicles whose Ca2+-triggered exocytosis depends on different isoforms of synaptotagmins but nevertheless uniformly requires complexins.
The evidence for our conclusions is as follows:
- Immunocytochemically, Syt10 and IGF-1 are localized to the same secretory vesicles in neurons, whereas Syt1 and the LDCV markers ANF, and phogrin are localized to a different class of secretory vesicles ( and )
- Morphologically, vesicles containing Syt10 and IGF-1 are significantly larger (cumulative distribution analysis, Kolmogorov–Smirnov test, P<0.01) than vesicles containing Syt1, ANF, and phogrin ()
- In both olfactory bulb and cortical neurons, KD of complexin increased spontaneous mini-release but decreased evoked neurotransmitter release, whereas only in olfactory bulb neurons KD of complexin also changed the capacitance and input resistance consistent with a change in IGF-1 secretion ( and ).
- Direct analysis of IGF-1 secretion revealed that the complexin KD severely impaired Ca2+-triggered IGF-1 exocytosis ()
- Comparison of the Ca2+-dependence of neurotransmitter release and IGF-1 secretion revealed a similar Ca2+-dependence of exocytosis ()
- Optical analyses of the effect of the complexin KD on the kinetics of IGF-1 exocytosis monitored via pHluorin-tagged Syt10 demonstrated that the complexin KD broadly suppressed exocytosis ( and )
- Structure-function studies of complexin revealed that the activating role of complexin is essential for both neurotransmitter release and IGF-1 exocytosis ( and )
Based on these data, we conclude that olfactory bulb neurons contain at least two independent and distinct pathways of Ca2+
-regulated polypeptide secretion – a previously characterized LDCV pathway for neuropeptide secretion utilizing Syt1 as a Ca2+
-sensor (Walch-Solimena et al., 1993
; Zhu et al., 2007
), and a novel pathway for IGF-1 secretion using Syt10 as a Ca2+
-sensor (Cao et al., 2011
). Both pathways are dependent on complexins, despite the fact that Syt1 and Syt10 are functionally distinct and non-redundant (Cao et al., 2011
). Thus, complexins may generally cooperate with synaptotagmins in exocytosis, and not only function as co-factors for Syt1-dependent exocytosis in synaptic and chromaffin exocytosis (Reim et al., 2001
; Cai et al., 2008
; Xue et al., 2007
; Maximov et al., 2009
), suggesting a much broader role in exocytosis than previously envisioned. Thus, the present data indicate that the phenotype of genetic complexin manipulations in mice, flies, and worms may be due to a general if partial impairment of multiple types of exocytosis. The general role of complexins in exocytosis is likely to be primarily as a positive activator of SNARE complexes prior to fusion-pore opening since this function appears to be more essential than the clamping function of complexin in synaptic vesicle exocytosis (Yang et al., 2010
) and IGF-1 exocytosis ( and ). It should be noted, however, that complexin – different from synaptotagmin – does not seem to be absolutely required for neurotransmitter release or IGF-1 exocytosis, even when analyzed in double complexin-1/2 KO neurons (Reim et al., 2000). Thus, complexin acts more as a co-factor for synaptotagmins that improves the secretory reaction than as a building block of the secretory machinery.
Interestingly, the clamping activity of complexin seems to have no significant role in IGF-1 secretion even though it appears to exert a significant control of neurotransmitter release at synapses. It is possible that the clamping function of complexin does not play a major role in controlling IGF-1 exocytosis, or that the rate of spontaneous IGF-1 exocytosis is too small to allow detection of spontaneous IGF-1 release events even after unclamping. The sensitivity of IGF-1 ELISA assays is much lower than that of mEPSC measurements which allow detection of single vesicle exocytosis events, strongly supporting the second hypothesis.
Our results complement recent findings that complexin is required for postsynaptic AMPA-receptor exocytosis triggered by LTP, and suggest that postsynaptic regulated exocytosis may also be dependent on a synaptotagmin (Ahmad et al., 2012
). Furthermore, given the fact that complexin and at least Syt14, Syt15, and Syt16 appear to be universally expressed in all cells at low levels (McMahon et al., 1995
; Fukuda, 2003a
; Herrero-Turrion et al., 2006
), our results suggest that a complexin/synaptotagmin-dependent pathway may operate in all cells to mediate an as yet unidentified type of exocytosis that could be regulated by a signal different from Ca2+
. The universal role for complexin in exocytosis thus emerging is consistent with the evolutionary conservation of complexins and synaptotagmins in all animals including sponges, and suggests that manipulating complexin function may be a general approach to influence regulated exocytosis in cells.
Why do olfactory bulb neurons develop a specific, activity-dependent pathway of IGF-1 exocytosis? A first clue to this question came from studies demonstrating that IGF-1 performs an essential role in the continuous activity-dependent assembly of the neural circuitry of the olfactory bulb, which needs to be re-wired throughout life because of adult neurogenesis of granule cells (Scolnick et al., 2008
; Hurtado-Chong et al., 2009
; Vicario-Abejon et al., 2003
; Pixley et al., 1998
). Moreover, a recent study revealed that neuronal diversity in the olfactory bulb may in part result from a neuron's activity-dependent adaptation to the local neural circuits (Angelo et al., 2012
). However, the underlying molecular mechanisms are unclear. Our results may explain how activity adjusts the properties of the neurons in the circuitry of the olfactory bulb. In the olfactory bulb, IGF-1 secreted from a mitral cell may, in a paracrine manner, only activate adjacent mitral cells sharing an identical glomerulus while leaving other, more distant, mitral cells unaffected. Since IGF-1 has been widely implicated in a variety of neuronal and circuit functions (Blair and Marshall 1997
; Ramsey et al., 2005
; Xing et al., 2007
; Man et al., 2000
; Wang et al., 2000; Kim et al., 2007
; Tropea et al., 2006
), our data suggest that the Ca2+
-triggered IGF-1 exocytosis mediated by complexins and synaptotagmin may be involved the regulation of mitral cell diversity in olfactory bulb. Moreover, similar mechanisms could potentially operate in other brain areas, linking activity-dependent exocytosis to the development of specific neuronal properties and neural circuits.