The potential effect of altered dendritic morphology on basic physiological properties in VEGFD deficient CA1 pyramidal neurons was investigated (Figure 3-3). To this end, single cells were recorded in whole cell configuration. The capacitance of VEGFD downregulated cells was not altered compared to control cells confirming the morphological findings. Although basal and apical dendrites showed abnormal changes in length and complexity, the overall dendritic size did not change and this is reflected in the membrane capacitance. This supports the hypothesis of the involvement of homeostatic mechanisms. Further, the result is in line with the current literature. Hemstedt et al. (2017) reported also no difference in membrane capacitance values in VEGFD downregulated cells. However, the values measured by Hemstedt and colleagues are smaller compared to our results (shSCR median: 144.3 pF; shVEGFD cells median: 139.15 pF, Hemstedt et al.: shSCR mean: 84 pF; shVEGFD cells mean: 80 pF). We determined the membrane capacitance in current clamp (section 2.8.1). It was shown, that this method yields higher capacitance values than measurements in voltage clamp (Golowasch et al. 2009), which was used by Hemstedt et al. . This explains the different values obtained in the two studies. Besides, both methods were developed for equipotential model systems, which is not the case for biological neurons and can lead to incorrect measurements of the capacitance particularly in dendritic
compartments that are further away from the soma. Mauceri et al. (2011) showed a decreased membrane capacitance in cultured VEGFD downregulated neurons, which is in line with the lower dendritic surface area observed in vitro. However, in the experiments carried out in hippocampal slices presented here, it was not possible to confirm this observation. One possible reason might be that cultured neurons do not have a polarity (basal/apical) and any laminated structure and therefore the homeostatic effect from external factors do not apply.
This study observed a significant increase of input resistance after VEGFD downregulated neurons from acute hippocampal slices. The input resistance is inversely proportional to the membrane surface area. Since a balance between basal and apical dendritic length was observed and membrane capacitance was therefore not altered, changes in input resistance were not expected. However, it is possible that basal dendrites have a higher impact on the input resistance since they are directly connected to the soma. Because basal dendrites are shorter in VEGFD downregulated cells this can cause a higher input resistance. The experiments from Mauceri et al. (2011) in cultured VEGFD knockdown cells revealed a slight increase of the input resistance, confirming the shortening of all dendrites.
Through shorter basal dendrites and a higher input resistance caused by VEGFD downregulation, it is possible that synaptic currents at the basal dendrites could be translated more efficiently into postsynaptic potentials and somatic depolarization. In turn, this could lead to increased action potential output after basal dendritic input (Johnston et al. 1996). To test this, the action potential firing threshold was determined for VEGFD downregulated cells. The firing threshold was not different, which indicates no higher excitability of shVEGFD cells. However, further parameters need to be investigated to gain a better understanding of the excitability of these cells, for example firing frequency or firing mode and more. The action potential firing threshold and excitability of cells determine strongly the integration of a neuron into neuronal networks. Since this is crucial for the single cell as well as neuronal networks, strong homeostatic mechanism have been proposed to control the action-potential threshold and excitability (Beck and Yaari 2008). Buzsaki, for example, reported homeostatic mechanisms regulating the neuronal excitability by bursting discharge in order to maintain synaptic strength and subsequent firing rate (Buzsaki 2002). A higher excitability of the cells due to changed dendritic morphology could lead to abnormal circuit synchronization and thus cognitive dysfunction, as shown for e.g. Alzheimer’s disease (Palop et al. 2007).
Dendritic ion channels regulate excitability and information integration. They can influence the degree of the attenuation of EPSPs propagating along dendrites. In this study, possible changes of the h-current (Ih) in VEGFD downregulated cells was
hypothesized. The Ih current is mediated through HCN channels 1 and 2, which are
et al. 2002). Ih in dendrites causes an attenuation of EPSP summation and contributes
to the separation of dendritic compartments (Magee 1998). It has been shown, that a downregulation of Ih in CA1 pyramidal cells causes a hyperpolarizing resting membrane
potential, a higher input resistance and an increase in EPSP summation (Shah et al. 2004; Jung et al. 2007). Further, it has been shown that HCN-1 null mutant mice have motor learning and memory deficits (Nolan et al. 2003). An increase in Ih was
hypothesized since VEGFD downregulated cells exhibit longer and more complex apical dendrites and thus potentially more HCN 1 and 2 channels. The sag amplitude was measured as a parameter for Ih current. However, the sag amplitude did not change
after VEGFD downregulation. This indicates that less HCN 1 and 2 channels are expressed due to intrinsic plasticity processes, in order to keep the Ih constant. Several
mechanisms could underlie this process. Different rates of expression and insertion influences the density of channels and changes in mRNA and protein levels have been shown for HCN channels (Shah et al. 2004). Additionally, the channels could be modulated by tyrosine-kinase mediated phosphorylation, which can be trigger by changed stimulation patterns (Hoffman and Johnston 1998). The mechanism underlying intrinsic plasticity are not well understood and need further investigation. The fact that VEGFD downregulated cells do not show a difference in the sag amplitude, could also be due to technical issues. The cells were recorded at the soma in whole-cell configuration, while Ih current is mediated through HCN channels expressed away from
the soma. Due to space clamp issues, the recording of the sag amplitude might be inaccurate.