**4. Discussion**

The experiments presented in this work, performed at the hippocampal mossy fiber synapses from CA3 area, allow the comparison of autofluorescence signals, recorded from non-incubated slices, with fluorescence zinc changes obtained from NG-loaded slices, after subtraction of the autofluorescence component. At these synapses, it

### **Figure 6.**

*Schematic representation of the major cellular mechanisms and ionic fluxes involved in KCl depolarization (20 mM) at the hippocampal mossy fibers-CA3 pyramidal cells synapses.*

### *FAD-Linked Autofluorescence and Chemically-Evoked Zinc Changes at Hippocampal Mossy… DOI: http://dx.doi.org/10.5772/intechopen.100898*

was observed that chemically induced depolarization by KCl (20 mM), evoked clear autofluorescence changes that recovered partially during the 30 min period following washout. These changes have a lower amplitude and a slower time course than those of the similarly evoked fluorescence signals observed in slices incubated with the zinc indicator Newport Green. The latter signals, corresponding to the total fluorescence changes in this work, have previously been described by Bastos et al. [22]. The application of 20 mM KCl promotes the depolarization of the presynaptic membrane, mediated by voltage dependent potassium channels (VDKCs), to a resting value of about −54 mV [13]. This increase in the membrane potential activates presynaptic VDCCs, triggering glutamate and zinc co-release, as illustrated in **Figure 6**.

### **Figure 7.**

*Diagram of the principal mechanisms and ionic movements involved in TEA depolarization at the hippocampal mossy fiber-CA3 synaptic system.*

Subsequently, after diffusion in the cleft and binding to specific pre- and postsynaptic sites, zinc flows into the postsynaptic region through several channels and receptors, including NMDA and calcium permeable AMPA/KA receptors and L- and T-type VDCCs [3–5, 8, 34]. There is also experimental evidence that zinc can be released from intracellular sources following blockade of ERs [35, 36]. Consequently, at the postsynaptic region, calcium and zinc entry through both glutamate receptor channels and VDCCs leads to cytosolic calcium and zinc accumulation that may cause the flow of both ions to mitochondria, through the activation of the mitochondrial Ca-uniporter [30, 37–43].

TEA also causes membrane depolarization that may trigger various cellular processes, as described in **Figure 7**.

In this work, the autofluorescence changes evoked by a single or by consecutive identical TEA applications were all similar. Interestingly, the time course of these changes is similar to that of the corresponding fluorescence signals recorded from Newport Green loaded slices. The TEA triggered fluorescence changes are depressed during the perfusion of TEA and recover to or above the baseline level upon washout, as previously reported [24]. In the present study, after subtracting the autofluorescence component, the real zinc signals evoked by TEA have about half the amplitude of that of the total fluorescence traces.

The changes of both autofluorescence and total fluorescence TEA induced signals are the opposite of those evoked by KCl. As previously mentioned, the membrane depolarization is higher in the presence of KCl than in TEA. The blockade of presynaptic VDKCs by TEA evokes a weak depolarization [23], followed by glutamate and zinc release and the activation of KATP channels by this ion. This leads to membrane hyperpolarization with a lower amplitude than that of the KCl evoked depolarization. Consequently, the hyperpolarizing effect of the zinc induced activation of presynaptic KATP channels can be occulted by the large increase in the resting potential, due to the strong KCl evoked depolarization, mediated by VDKCs [13].

### **5. Conclusions**

The amount of calcium entry is related to the intensity of autofluorescence because increased intracellular calcium and zinc can trigger an increase in FAD (flavoprotein) and NAD, as well as in the oxidation of FADH2 and NADH [28, 44]. In the present experimental conditions (excitation wavelength of 480 nm, emission light collected above 500 nm) and taking into account the spectral properties of FAD, the autofluorescence detected is considered to have FAD origin. As previously mentioned, KCl depolarization causes the entry of both calcium and zinc ions to the postsynaptic region [10, 22, 24, 45, 46]. When increases in cytosolic zinc concentration are high, zinc ions enter the mitochondria, and if in excess may have neurotoxic effects [4, 47, 48]. Thus, the origin of the KCl evoked autofluorescence signals is, under our experimental conditions and based on previous studies, the flavoproteins. For example, Pancani [49], have found, using the complex I inhibitor rotenone a KCl induced NADH fluorescence decrease of mitochondrial origin. This is in agreement with the observed FAD enhancement since the NADH and FAD fluorescence changes are opposite [29, 44, 50].

### **Acknowledgements**

We thank CNC - Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal, for providing the rat brains. Work funded by strategic *FAD-Linked Autofluorescence and Chemically-Evoked Zinc Changes at Hippocampal Mossy… DOI: http://dx.doi.org/10.5772/intechopen.100898*

project UID/NEU/04539/2013. All authors are aware of and have approved the manuscript as submitted. All authors are aware they must each submit a completed License to Publish form before the manuscript can be published. (Each author will receive an email with instructions).
