Here we examined the role of TrkB in regulating the synaptic structures of hippocampal dentate granule cells. Both post-synaptic and presynaptic structures were assessed to gain insight into whether TrkB modifies the morphological substrates of granule cell connectivity. Both were altered. Postsynaptically, granule cells exhibited fewer primary dendrites whereas dendritic spines on these dendrites were larger in area in TrkB−/− mice relative to wildtype littermates. Presynaptically, conditional deletion of TrkB increased the number of filopodia per giant mossy fiber bouton. In contrast, the size and density of giant mossy fiber boutons was unchanged. Together, these findings suggest altered cortical and associational/commissural input to dentate granule cells and enhanced feedforward inhibition of CA3 pyramidal cells.
The present study relies on anatomical methods to assess presynaptic and postsynaptic terminals in TrkB−/− mice and littermate controls. Changes in the number and/or structure of spines and boutons guide predictions as to the functional consequences of TrkB elimination. Physiological studies, however, are ultimately required to test these predictions. Importantly, it is unclear whether the changes identified in the TrkB−/− mice are triggered by the absence of TrkB within granule cells themselves or from afferents or targets of the granule cells or from synaptically remote locales. Likewise, it is unclear whether the changes found are an immediate consequence of the absence of TrkB or due to some homeostatic response to the absence of TrkB. With these caveats in mind, the present results do provide clear insights into the impact of TrkB on granule cell connectivity.
TrkB does not appear to be required for granule cell survival or gross morphological stability. Degeneration of cortical neurons has been reported following conditional deletion of TrkB (Xu et al., 2000
). Granule cells, on the other hand, appeared surprising normal after the elimination of TrkB. Previous analyses of Nissl-stained sections likewise revealed no overt abnormalities or obvious loss of granule cells (He et al., 2004
). Similarly, in the present study degenerating GFP-labeled granule cells were not observed, and no increase in Fluoro-Jade B labeling of dying granule cells was apparent in two-month-old animals. Importantly, in the Thy-GFP expressing mouse line studied here, dentate granule cells do not begin to express GFP until they are about four-weeks-old (Walter et al., 2007
), the obvious implication being that the cells examined in the present study survived for at least this long. We did not conduct stereological cell counts, so we cannot exclude the possibility of more subtle differences in cell numbers (Lähteinen et al., 2003
), nor can we rule out the possibility that death increases in older animals (von Bohlen und Halbach et al., 2003
; Baquet et al., 2004
). The majority of granule cells, however, appear to survive into adulthood. Similarly, granule cell polarity was not disrupted in these animals. Dendrites were found to project from the top of the soma into the molecular layer, and granule cell mossy fiber axons projected into the hilus. The area, density, and spatial distribution of giant mossy fiber boutons were also not altered in TrkB−/−
mice. The present results contrast with prior analyses of CNS histology and mossy fiber terminals in conventional TrkB knockout mice. With conventional knockouts, TrkB is eliminated from every cell in the animal for the entire lifespan. In contrast, the conditional strategy used here temporally and spatially restricts the elimination of TrkB to CNS neurons following expression of synapsin I-driven Cre recombinase. Conventional elimination of TrkB results in numerous nervous system lesions and neonatal death within two to three weeks (Alcántara et al., 1997
). Early death is likely a consequence of myocardial pathology in these animals (Donovan et al., 2000
). At a subcellular level, 11 to 12-day-old conventional TrkB knockout mice exhibit reduced numbers of mossy fiber boutons, with remaining boutons being smaller in size and possessing fewer synaptic terminals (Otal et al., 2005
). Whether the earlier elimination, broader tissue distribution, broader cellular distribution, and/or the poor health of conventional TrkB knockouts account for differences between the current and prior studies is not clear. That said, our findings suggest that granule cell survival and the persistence of gross structural characteristics does not require TrkB.
Although gross disruption of granule cell structure was not observed, granule cells from TrkB−/−
mice did exhibit fewer primary dendrites relative to cells from control mice. This finding is consistent with previous studies in which BDNF overexpression, both in vivo (Tolwani et al., 2002
) and in vitro (Danzer et al., 2002
), led to an increase in primary dendrite number among granule cells. Together, these studies support the idea that TrkB activation promotes primary dendrite formation of the granule cells. Interestingly, only granule cells located along the molecular layer border exhibited decreases. Primary dendrite number for granule cells located along the hilar border was equivalent between genotypes. Granule cells located on the molecular border are in general older than cells on the hilar border (Altman and Das, 1965
; Altman and Bayer, 1990
), and typically possess more primary dendrites than their younger neighbors (which usually have only a single primary dendrite). Together this suggests that activation of TrkB during the maturation of granule cells contributes to formation of its dendritic arbor. Finally, the functional consequences of increased or decreased numbers of primary dendrites are not clear. Changes in primary dendrite number could reflect changes in total dendritic length or a redistribution of dendritic structures. Either scenario might result in dramatic changes in the number and/or type of synaptic inputs received by a cell. Similarly, changes in dendritic structure would also alter passive electrotonic properties. Changes in EPSP summation or transmission to the axon hillock would be likely consequences.
Further evidence for a role of TrkB in regulating granule cell synaptic inputs comes from analysis of dendritic spines. Although spine density was unchanged in TrkB−/−
mice, spine area was significantly increased along dendritic segments in the inner and outer molecular layers. These findings are consistent with recent studies examining TrkBflox/flox
CaMKII-CRE mice, in which granule cell spine density was not altered, but mean spine length was significantly increased (von Bohlen und Halbach et al., 2006
). Interestingly, changes in granule cell spine structure may result from alterations in entorhinal and associational/commissural inputs (Frotscher et al., 2000
). These inputs exhibited reduced numbers of collaterals and varicosities in conventional TrkB−/−
mice (Martinez et al., 1998
). Alternatively, the absence of TrkB within the spine itself may result in reduced spine area, perhaps via reduced calcium entry through the transient receptor potential channel (TRPC) family of ion channels (Tyler and Pozzo-Miller, 2003
; Pozzo-Miller, 2006
; Amaral and Pozzo-Miller, 2007
). Finally, whether increased spine area reflects reduced synaptic function, (Gonzalez et al., 1999
; Kovalchuk et al., 2002
; Elmariah et al., 2004
) remains to be determined.
The present data suggest that elimination of TrkB altered the number of granule cell-inhibitory interneuron synapses but not granule cell-CA3 pyramidal cell synapses. In TrkB−/−
mice, the number of filopodial extensions per mossy fiber bouton was significantly increased. These filopodia make synaptic contact with inhibitory interneurons, which in turn provide feedforward inhibition to CA3 pyramidal cells. Increased numbers of filopodia suggest a strengthening of this inhibitory pathway. In contrast, neither the size nor density of granule cell-CA3 pyramidal cell synapses were altered in TrkB−/−
mice. Whether other parameters of this synapse are modulated by TrkB deletion is not known. Our findings are consistent; however, with recent physiological studies indicating normal basal synaptic activation of CA3 pyramidal cells by mossy fibers in slices from TrkB−/−
mice (Huang et al., in press
). These results are also consistent with work by De Paola et al. (2003)
, in which they demonstrated in vitro that BDNF treatment led to rapid changes in filopodia dynamics, while giant boutons were comparatively stable. Interestingly, a number of studies now demonstrate a role for TrkB in regulating GABAergic synaptic transmission (Bolton et al., 2000
; Carmona et al., 2003
; Carmona et al., 2006
; Singh et al., 2006
; Swanwick et al., 2006
; Kohara et al., 2007
). The present study extends these findings by demonstrating that TrkB can also act upstream of the GABAergic neuron, regulating excitatory input to these cells. Conditional deletion of the TrkB gene leads to a striking impairment of epileptogenesis in the kindling model of epilepsy (He at al., 2004
). A disproportionate increase in the strength of feedforward inhibition compared to excitation of CA3 pyramidal cells by mossy fiber axons of TrkB−/−
mice may contribute to this impairment.