In vitro and in vivo physiology of low nanomolar concentrations of Zn2+ in artificial cerebrospinal fluid

Abstract

Artificial cerebrospinal fluid (ACSF), i.e., brain extracellular medium, which includes Ca²⁺ and Mg²⁺, but not other divalent cations such as Zn²⁺, has been used for in vitro and in vivo experiments. The present study deals with the physiological significance of extracellular Zn²⁺ in ACSF. Spontaneous presynaptic activity is suppressed in the stratum lucidum of brain slices from young rats bathed in ACSF containing 10 nM ZnCl₂, indicating that extracellular Zn²⁺ modifies hippocampal presynaptic activity. To examine the in vivo action of 10 nM ZnCl₂ on long-term potentiation (LTP), the recording region was perfused using a recording electrode attached to a microdialysis probe. The magnitude of LTP was not modified in young rats by perfusion with ACSF containing 10 nM ZnCl₂, compared to perfusion with ACSF without Zn²⁺, but attenuated by perfusion with ACSF containing 100 nM ZnCl₂. Interestingly, the magnitude of LTP was not modified in aged rats even by perfusion with ACSF containing 100 nM ZnCl₂, but enhanced by perfusion with ACSF containing 10 mM CaEDTA, an extracellular Zn²⁺ chelator. The present study indicates that the basal levels of extracellular Zn²⁺, which are in the range of low nanomolar concentrations, are critical for synaptic activity and perhaps increased age-dependently.

Figure 1. Suppression of spontaneous mossy fiber exocytosis of young rats in the presence of 10 nM ZnCl₂.

(A) Schematic illustration of mossy fiber exocytosis assessed by attenuation of FM4-64 fluorescence. (B) FM4-64 fluorescence images in the CA3 after loading FM4-64 into slices bathed in ACSF, ACSF containing 1 nM Zn²⁺, and ACSF containing 10 nM Zn²⁺, and FM4-64 fluorescence image in the CA3 after loading of FM4-64 into slices bathed in ACSF and transferring the slices to ACSF containing 1 μM TTX (time 0 sec) (upper). Each point and line (mean ± SEM) represents the changes in FM4-64 fluorescence in the stratum lucidum (SL) of brain slices bathed in ACSF (n = 17), 1 nM ZnCl₂ (n = 5), 10 nM ZnCl₂ (n = 8) and 1 μM TTX (n = 6) after loading FM4-64 (time 0), which is expressed as 100%. (n = 6) (middle). Each bar and line (the mean ± SEM) represents the rate of the decreased FM4-64 fluorescence at time 300 sec (lower). *p < 0.05; **p < 0.01, vs. ACSF (Tukey’s test).

Figure 2. No effect of 10 nM CuCl₂ and 10 nM FeCl3 on spontaneous mossy fiber exocytosis of young rats.

FM4-64 fluorescence images in the CA3 after loading FM4-64 into slices bathed in ACSF and ACSF containing 10 nM Cu²⁺ or ACSF containing 10 nM Fe³⁺ (time 0 sec) (upper). Each point and line (mean ± SEM) represents the changes in FM4-64 fluorescence in the stratum lucidum (SL) of brain slices bathed in ACSF (n = 10), 10 nM CuCl₂ (n = 6), and 10 nM FeCl₃ (n = 6) after loading FM4-64 (time 0), which is expressed as 100%. (n = 6) (lower).

Figure 3. Intracellular Ca²⁺ imaging in the hippocampus of young rats with calcium orange AM.

Intracellular calcium orange fluorescence was imaged to estimate the basal levels of cytosolic Ca²⁺ in the hippocampus of brain slices bathed in ACSF (n = 7) and 10 nM ZnCl₂ in ACSF (n = 7). SR, stratum radiatum; SL, stratum lucidum; DML. dentate molecular layer (left). Each bar and line (the mean ± SEM) represents fluorescent intensity in the SR, SL, and DML.

Figure 4. Action of extracellular Zn²⁺ and extracellular Zn2⁺ chelator in dentate gyrus LTP of young and aged rats.

(A) Recording region was perfused with ACSF in young rats and then 10 nM (n = 5) and 100 nM (n = 4) ZnCl₂ in ACSF as shown by the shaded bar and HFS (10 trains of 20 pulses at 200 Hz separated by 1 s) was delivered at time 0 min (left). Each bar and line (mean ± SEM) represents the averaged PS amplitude of the last 10 min (time 50–60 min) (middle). ***p < 0.001, vs. control (ACSF) (n = 10) (Tukey’s test). Representative fEPSP recordings at time −70 (black line), −30 (grey line) and 50–60 min (red line) are shown (right). (B) Recording region was perfused with ACSF in aged rats and then 100 nM (n = 18) and 10 mM CaEDTA (n = 7) ZnCl₂ in ACSF as shown by the shaded bar and HFS (10 trains of 20 pulses at 200 Hz separated by 1 s) was delivered at time 0 min (left). Each bar and line (mean ± SEM) represents the averaged PS amplitude of the last 10 min (time 50–60 min) (middle). *p < 0.05, vs. control (ACSF) (n = 26) (Tukey’s test). Representative fEPSP recordings at time −70 (black line), −30 (grey line) and 50–60 min (red line) are shown (right).