In this study, we have exposed spiral ganglion cultures to neurotrophins, LIF-type cytokines and BMP4 and examined the resulting neuronal survival, morphology and neurite lengths. Under all conditions, monopolar neurons, bipolar neurons and neurons with no neurites comprise over 95% of the neurons in the culture. However, the relative contributions of the different morphologies differs between control, LIF and BMP4 conditions. Specifically, as compared to control, inclusion of LIF type cytokines increases neuronal survival and results in an increase in the number of bipolar neurons; while inclusion of BMP4 increases neuronal survival and results in an increase in the number of monopolar neurons and neurons with no neurites. In addition, bipolar neurons in BMP4 cultures have shorter neurites than those in control or LIF cultures.
We have tested three different cytokines-LIF,CNTF and hOSM – that in other cell types signal through the LIFRβ-gp130 heterodimeric receptor (Turnley and Bartlett, 2000
). LIF and hOSM use the LIFRβ-gp130 receptor directly; CNTF requires the help of an additional binding protein, CNTFRα (Marz et al., 1999
; Turnley and Bartlett, 2000
). All three cytokines showed similar effects on cell survival and morphology, indicating that signaling initiated through the LIFRβ-gp130 receptor increases neuronal survival and exerts a preferential effect on the maintenance of bipolar neurons. That CNTF functions at all in this culture system suggests that the CNTFRα (membrane bound or soluble) is present in the cultures (Marz et al., 1999
). Interestingly, IL6, does not demonstrate similar effects to those of LIF, CNTF or hOSM. This may be due to a lack of the IL6 binding protein in the cultures (Marz et al., 1999
), or a lack of effect of the IL6 type gp130 homodimeric signaling (Turnley and Bartlett, 2000
) on survival and morphology of spiral ganglion neurons.
Inclusion of BMP4 in control cultures not only increases neuronal survival and alters the cellular morphology of the neuronal population, but also results in shorter neurites emanating from bipolar neurons. Even in the presence of LIF, BMP4 effects predominate. Two main possibilities present themselves: either BMP4 and LIF affect different populations of neurons, with BMP4 inhibiting the survival (and hence contribution to the bipolar pool) of LIF responsive neurons; or both support survival of the same population of neurons, with BMP4 acting as an inhibitor of LIF stimulated neurite outgrowth. With the current system, it is not possible to return a growing neuron to the time of plating and look at it a second time under different culture conditions. Thus, there is presently no direct way to determine if LIF and BMP4 exert morphologic effects on the same or different neurons. Whatever the mechanism, the result is that while maintaining survival numbers, the characteristics of the in vitro population shift between predominately bipolar and predominately monopolar and denuded neurons, depending on the factors included in the medium.
Most neurons in adult spiral ganglia in vivo are bipolar. Yet these spiral ganglion cultures return a mix of neuronal morphologies. The aim in cell culture is to induce neurons to regrow their neurites in an observable and quantifiable way. However, this entails the use of a reconstructed environment that by definition has to be different from that from which the neurons were removed, as well as different from that encountered during the initial developmental period. In the broader framework of trying to understand the mechanistic controls on bipolar morphology and neurite growth, the initial mixture of different morphologies we observe in our cultures becomes an advantage to exploit experimentally. For example: Can monopolar neurons be encouraged to regrow the missing axon? Such a mixture may also have some relevance to the in vivo situation in damaged cochleas, when hair cells degenerate and the disconnected peripheral neurites retract toward the cell soma. In particular, if the central axon remains connected to the brain stem, as cochlear implant function would indicate, then during peripheral neurite retraction the neuron can potentially arrive at a state similar to the monopolar neurons in our cultures. The use of bipolar morphology and neurite length as endpoints in our morphologic assay will allow us to examine possible biochemical and other interventions that can shift predominately monopolar populations to bipolar populations of spiral ganglion neurons, a conversion that may have implications in the whole animal.
In other tissues, both LIF and BMP4 exert effects on renewal and differentiation of neural stem/progenitor cells (Bonaguidi et al., 2005
; Bauer and Patterson, 2006
; Chen and Panchision, 2006
). However, preliminary studies in our laboratory failed to observe BrDU labeled neurons under any of our culture conditions (D.S. Whitlon, unpublished), suggesting that the surviving neurons were not derived from cell division. Further, although it can be assumed from prior studies that neurotrophins act directly on the neurons, the present studies do not address the identity of the initial target cells for BMP4 or LIF. This a relevant question given the mixed composition of the dissociated cultures. In other tissues, both LIF type cytokines and BMPs exert effects on multiple cell types (Murphy, 1997
; Dowsing et al., 1999; Turnley and Bartlett, 2000
). Although it is possible that these factors directly bind neurons, it is also conceivable that survival or neurite growth effects of LIF and/or BMP4 are exerted through initial effects on non-neural cell types.
One of the many questions yet to be answered about the regrowth of spiral ganglion neurites is whether the two bipolar neurites are equivalent or different in vitro. In vivo
, both processes of spiral ganglion cells are morphologically axons, and in fact there is very little to differentiate them in situ
other than their position and a small narrowing at the initiation of the peripheral neurite (Brown et al., 1988
; Goycoolea et al., 1990
). Interestingly, we find that bipolar neurons in culture are not, in general, symmetrical with respect to neurite size. One neurite is usually longer than the other. Under control conditions, 50% of the longer neurites of bipolar neurons are longer than 150 μm, while only 15% of the shorter neurites of bipolar neurons are longer than 150 μm. It might seem reasonable that this difference in length represents differences in neurite identity and that the neurite emanating from the monopolar neurons, since similar in size, are similar in identity to the longer neurites of bipolar neurons. While this may ultimately be proven true, there is evidence in other neuronal cell types that the microtubules defining neurites are nucleated at the centrosome (Ahmad and Baas, 1995
), which in cerebellar granule cells alternates between different sides of the cell soma to extend growth of different axons (Zmuda and Rivas, 1998
). Therefore, without specific markers for peripheral or central fibers, it cannot yet be known whether the sizes of the neurites are statistical results of the position of the centrosome or are actually manifestations of differential neurite identities.
In the present work, we use LIF-type cytokines and BMP4 as triggers to our final endpoints –changes in survival, morphology or neurite length. The purpose is to initiate cellular mechanisms that promote or fail to promote neurite growth and to make them visible enough to measure and analyze. Although this work does not address any issues surrounding the possible use of LIF-type cytokines or BMP4 as therapeutic agents in vivo, these factors are known to be endogenous to the cochlea during development or after tissue damage (Oh et al., 1996
; Takemura et al., 1996
; Malgrange et al., 1998
; Morsli et al., 1998
; Cole et al., 2000
; Cho et al., 2004
; Li et al., 2005
; Pujades et al., 2006
). Whether there are times in development or damage that BMP4 or LIF-like mechanisms can come into play in regulating neuronal morphology, neuronal development or neurite growth are questions for further study.