Brain Development Laboratory, The Howard Florey Institute, University of Melbourne, Parkville, 3010, Victoria, Australia
Address correspondence to Seong-Seng Tan, Brain Development Laboratory, The Howard Florey Institute, The University of Melbourne, Parkville, Victoria 3010, Australia. Email: stan{at}hfi.unimelb.edu.au.
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Abstract |
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Introduction |
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GABAergic interneurons are distributed in all layers of the gray matter where they form intricate networks of synaptic contacts with both pyramidal and nonpyramidal neurons (Somogyi et al., 1983, 1998
; Meskenaite, 1997
). These local neuronal circuits are important for the proper function of mature cortical neurons. For instance, GABAergic inhibition of pyramidal neurons serves to prevent excessive firing in response to peripheral stimuli (Benardo and Wong, 1995
). However, nonpyramidal innervation of other nonpyramidal neurons plays an important role to inhibit inhibitory neurons (Tamas et al., 1998
) and the effect of this disinhibition is thought to synchronize pyramidal cell activity (Jefferys et al., 1996
). Indeed, interconnected GABAergic neurons have been shown to exert synchronous output activity in modulating the operation of pyramidal neuron networks (Traub et al., 1996
; Galarreta and Hestrin, 2001
). The generation of synchronicity requires the formation of GABAergic networks between developing interneurons and the types of GABAergic interneurons involved have been defined in vivo and in vitro (Tamas et al., 1998
; Beierlein et al., 2000
; Voigt et al., 2001
; Amitai et al., 2002
). However, little is known concerning the generic and specific mechanisms that promote arborization of immature interneurons after their migration into the neocortex. For example, at what stages in development are GABAergic interneurons most actively involved in neurite outgrowth and branching? What is the influence of interneuron cell to cell contact on these processes? What chemotrophic factors may influence these activities?
The purpose of the current study is to follow the early events of interneuron neurite outgrowth and branching in a three-dimensional collagen gel environment. The collagen gel system provides neurite outgrowth conditions that mostly close mimics conditions in vivo (Harris et al., 1985). Previous in vitro studies have established that the differentiation of GABAergic neurons closely resembles the development of their in vivo counterparts, suggesting that tangential migration per se is not absolutely necessary and the in vitro system provides an accessible model for studying interneuron arborization (de Lima and Voight, 1997
). By combining a low cell density culture environment with the collagen gel matrix, it is possible to directly visualize individual neurites (axons and dendrites) from the cell body to the growth cone, permitting individual branches to be correctly assigned to the appropriate cell body (Wang et al., 1999
). The current study covers the entire neurogenetic period when immature GABAergic interneurons are known to be entering the cerebral wall. Using the three-dimensional gel system, we report the development of branch points and neurite outgrowth between interneurons of various developmental stages. In addition, we compare the effects of interneuron cellcell contact with single interneurons that did not make contact. Finally, we report on the effects of Slit protein and brain-derived neurotrophic factor (BDNF), two known chemobranching molecules, on these interneurons.
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Materials and Methods |
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In experiments to test the effects of Slit protein, growth medium was used to culture human embryonic kidney (HEK) 293 cells that had been stably transfected with Xenopus Slit (xSlit) carrying the six-myc epitope tag or with control vector plasmid (Li et al., 1999). Conditioned medium was collected after 72 h from cells expressing Slit or control plasmid, and used to reconstitute the collagen gel cultures. In testing for the effects of BDNF, a recombinant protein (gift from Dr T. Hughes, University of Melbourne) was added to growth medium at a final concentration of 5 ng/ml.
Cultures were incubated at 37°C in a CO2 atmosphere for 24 h before fixation and processing for GABA immunocytochemistry. Primary antibody against GABA was raised in rabbit (Sigma, Australia) and used at a 1:200 dilution. GABA immunoreactivity was revealed by an Alexa-conjugated donkey anti-rabbit IgG (1:200, Molecular Probes, Eugene, OR). To reveal cell nuclei, cells were stained with bisbenzamide.
Fluorescent images of individual neurons were captured (x40 objective) with a Spot Digital Camera (Diagnostic Instrument Inc., Sterling Heights, MI) and the magnified images used for analysis of neurite length and branching. Only neurons with unambiguous extension of neurites from the cell soma were included in the analysis (Image-Pro-Plus software; Media Cybernetics, Silver Spring, MD). The length of the longest neurite, presumed to be the axon, was calculated from the captured image. Neurites projecting from the soma were individually counted as branches, as were neurites found extending from other shafts. In each category, 120 GABAergic neurons were randomly selected for analysis until this number was reached. For studying the effects of interneuron cell to cell contact, 60 pairs of GABA+ cells were randomly chosen and the same measurements applied. Statistical comparisons between different age groups were performed using the one-way ANOVA test. To compare neurite length and branching between contacting and non-contacting GABAergic interneurons, the Students t-test was used.
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Results |
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Interneuron Branching and Neurite Outgrowth Is Promoted by CellCell Contact
GABA immunoreactivity was detected in immature neurons from as early as E13.5 onwards following 24 h culture (Fig. 1A). By using a low to medium plating density, the morphology of individual GABA immunoreactive neurons could be easily observed. Interneurons were frequently present as single cells but in many cases, pairs of contacting GABAergic neurons were also seen (Fig. 1AD
). Their cell bodies were invariably ovoid-shaped or fusiform, and the overall morphology suggests a migrating phenotype with dissimilar neurites resembling leading and trailing processes (Fig. 1AD
). Overall, there was no overt differences in the appearance of contacting (Fig. 1E
, white cell bodies), compared to non-contacting neurons (Fig. 1E
, dark cell bodies). For non-contacting GABA neurons, the number of branch points per neuron did not change appreciably between the various time-points examined, varying between two and three branch points per neuron (Fig. 1F
, bottom curve). Similarly, non-contacting neurons showed an average longest neurite lengths of
30 µm although for the age group P1.5 this had increased to
40 µm (Fig. 1G
, bottom curve).
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The Effects of Exogenous Slit Protein on Neurite Branching and Outgrowth
In a previous study, we demonstrated that old but not young interneurons in vitro respond to Slit by increased branching and neurite lengths (Sang et al., 2002). Young interneurons, isolated from E13.5 embryos displayed suppressed neurite lengths and branching in respond to Slit protein present in conditioned media. In contrast, old interneurons (in vitro equivalent of E18.5 cells) showed increased neurite outgrowth and branching following exposure to Slit protein for 24 h. This Slit-mediated activity was attenuated in the presence of excess quantity of the Slit-receptor, RoboN. We concluded that Slit protein is an important accessory for interneuron arborization among mature, but not juvenile, GABAergic neurons.
In the present work, we extended our study by examining cultured interneurons taken from four different stages, and also compared the effects of Slit protein on contacting versus non-contacting interneurons. As before, contacting interneurons increased their number of branch points and have greater neurite lengths (Figs 2A,D,E and 3A,D,E), but these effects in the E13.5, E15.5 and P1.5 cohorts were counteracted in the presence of Slit protein (Figs 2B,D,E
and 3B,D,E). In contrast, the E17.5 cohort showed a totally different picture in the presence of Slit (Fig. 3B,D
). In this age group, Slit protein promoted neurite branching and neurite outgrowth in both contacting and non-contacting interneurons (Figs 3D
and 4A,B). Thus it would appear that a positive response to Slit protein was only found within interneurons isolated from E17.5 embryos, not younger or older stages. In the cortical plate, Slit1 is expressed from E15.5 onwards and is present until birth (Whitford et al., 2002
).
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BDNF has been shown in other studies to regulate GABAergic interneuron development, including the promotion of neurite outgrowth and their maturation, stimulation of GABA expression and other calcium-binding proteins (Huang et al., 1999; Mizuno et al., 1994
; Widmer and Hefti, 1994
). In the presence of BDNF, branching activity among E13.5 and E15.5 interneurons was suppressed. This was observed with both contacting and non-contacting interneurons in these two age groups (Figs 2C,D,E
and 4A). Neurite length was slightly increased, but not significantly (Fig. 4B
). In this respect, BDNF differed from Slit in promoting neurite extension despite inhibiting neurite branching (Fig. 4A,B
). In contrast, the E17.5 cohort of interneurons showed a different response to BDNF. In the E17.5 group, branching activity in response to BDNF was significantly increased, exceeding the effects seen with Slit (Fig. 4A
). After birth, BDNF did not elicit a similar branching activity from P1.5 interneurons (Fig. 4A
). Paradoxically, BDNF had an opposite effect on neurite outgrowth of E17.5 neurons (Fig. 3D
). So whilst E17.5 neurons displayed maximal branching in the presence of BDNF (Fig. 3C,D
), they showed less neurite outgrowth compared with neurons cultured in the presence of Slit (Fig. 4A,B
). A similar suppression of neurite extension was also seen with P1.5 cells (Fig. 4B
). Thus, immature interneurons responded differently to BDNF depending upon their age of culture. Younger interneurons responded to BDNF by suppression of neurite branching whilst E17.5 interneurons responded by increasing branching activity but decreasing neurite outgrowth.
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Discussion |
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The question then follows regarding the nature of the stimulus and how this may lead to the back propagation of trophic signals to the cell machinery. One possibility is for interneuronal cell contact to establish gap junction coupling that may facilitate the passage of micromolecular signals between cells (Connors et al., 1983). These signals may initiate cellular changes leading to increased neuronal arborization. These may also represent the initial steps of establishing interneuronal circuits between GABAergic neurons (Tamas et al., 1998
; Voigt et al., 2001
). Another possibility is for interneuronal contact to trigger receptorligand complexes which in turn may lead to transcriptional changes favoring neurite outgrowth. A classic example of this is found in the neocortical pyramidal neurons grown at high density. Following neurite to neurite contact, there is activation of the Notch receptorligand complex leading to the generation of signals to inhibit further neurite outgrowth (Sestan et al., 1999
).
A third possibility concerns the GABA neurotransmitter. There is evidence that GABA plays a trophic role in the immature brain (Lauder et al., 1986), including stimulation of cell migration (Behar et al., 1998
), and regulation of its dendritic morphology via GABA transmission and release (Matsutani and Yamamoto, 1998
). Activation of GABA receptors in migrating interneurons causes a rise in intracellular calcium (Soria and Valdeolmillos, 2002
). The precise mechanisms are unclear but it has been suggested that GABAergic transmission causes BDNF release from target cells which have paracrine effects on interneuron soma size and dendritic arborization (Marty et al., 1996
). Both BDNF and its receptor TrkB are expressed in the rodent cortical plate from the earliest phases of cortical neurogenesis (Fukumitsu et al., 1998
). BDNF has been found to promote dendritic elongation of interneurons (Matsutani and Yamamoto, 1998
), but the responses are not uniform, with increases in dendritic arbors among 1 week old interneurons but quite the reverse in older interneurons (Marty et al., 1996
). In the current study, BDNF has the general property of increasing neurite length among interneurons at embryonic but not at postnatal stages. However, the standout effect of BDNF is found among E17.5 interneurons where the branching effect is maximal, exceeding the effect of Slit. By P1.5, the branch-promoting effects of BDNF was no longer present. The precise reason for the stage-specific effect of BDNF at E17.5 is unclear, but this stage is coincident with the invasion of thalamic axons (that lie waiting in the underlying subplate) into the cortical plate (Molnar and Blakemore, 1995
). This leads us to propose the hypothesis that thalamic innervation of maturing pyramidal neurons in the cortical plate may create an environment whereby interneurons are stimulated to extend their arbors. Following contact with target pyramidal neurons, transient GABA transmission from interneurons may then elicit BDNF release from the target cells, providing further trophic stimuli for local circuit arborization.
Slit belongs to a family of secreted proteins with chemorepellant activity and is known to be expressed in the striatal cortical regions through which interneurons migrate (Nguyen Ba-Charvet et al., 1999; Yuan et al., 1999
; Whitford et al., 2002
). In addition to a chemorepulsive role for interneuron migration (Zhu et al., 1999
), Slit has been shown to repel extending GABAergic neurites (Sang et al., 2002
). In addition, we and others have shown that Slit has a chemobranching effect on cortical interneurons (Sang et al., 2002
; Whitford et al., 2002
). In agreement with our previous study, the current results emphasize that not all interneuron cohorts respond in a similar fashion to Slit. Whereas interneuron cultures established from early stages (E13.5 and 15.5) showed suppressed neurite outgrowth, interneurons taken from E17.5 brains responded positively by increasing arborization. The effect was not seen at postnatal P1.5.
Taken together, the above results indicate increased neurite growth and arborization following contact between neighboring cells in culture. Interneurons from different developmental stages respond differently to BDNF and Slit, with repression of branching at earlier time-points but accelerated branching and neurite extension at E17.5. Translating these findings to the in vivo cortical environment would suggest a critical period during late corticogenesis when interneurons are most responsive to chemotrophic stimuli. This period corresponds with the near-completion of cortical layering and arrival of most interneurons following long distance tangential migration (Anderson et al., 2001). One may therefore hypothesize that the capacity to respond to local chemotrophic signals is delayed until E17.5 when the full complement of interneurons have filled the neocortical space and nearest-neighbor distances between interneurons are undergoing adjustment. Therefore inappropriate arborization before E17.5 would exclude late-arriving interneurons from tiling the cortical landscape. This developmental mechanism has been shown to occur for amacrine cells, another interneuron population in the retina known to respond to short-range interactions during the creation of neuronal mosaics (Galli-Resta et al., 1997
). While there is still no evidence that cortical interneurons obey similar rules for mosaic arrangement, they are known regularly dispersed and the spatial rules for electrical coupling between interneurons are beginning to emerge (Amitai et al., 2002
). In this context, neurite extension and branching from the immature interneuron may be mechanisms for positional adjustment and sampling of neuronal space.
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Acknowledgments |
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