Copper-uptake is critical for the down regulation of synapsin and dynamin induced by neocuproine: modulation of synaptic activity in hippocampal neurons

Abstract

Extracellular and intracellular copper and zinc regulate synaptic activity and plasticity, which may impact brain functionality and human behavior. We have found that a metal coordinating molecule, Neocuproine, transiently increases free intracellular copper and zinc levels (i.e., min) in hippocampal neurons as monitored by Phen Green and FluoZin-3 fluorescence, respectively. The changes in free intracellular zinc induced by Neocuproine were abolished by the presence of a non-permeant copper chelator, Bathocuproine (BC), indicating that copper influx is needed for the action of Neocuproine on intracellular Zn levels. Moreover, Neocuproine decreased the mRNA levels of Synapsin and Dynamin, and did not affect the expression of Bassoon, tubulin or superoxide dismutase (SOD). Western blot analysis showed that protein levels of synapsin and dynamin were also down regulated in the presence of Neocuproine and that these changes were accompanied by a decrease in calcium transients and neuronal activity. Furthermore, Neocuproine decreased the number of active neurons, effect that was blocked by the presence of BC, indicating that copper influx is needed for the action of Neocuproine. We finally show that Neocuproine blocks the epileptiform-like activity induced by bicuculline in hippocampal neurons. Collectively, our data indicates that presynaptic protein configuration and function of primary hippocampal neurons is sensitive to transient changes in transition metal homeostasis. Therefore, small molecules able to coordinate transition metals and penetrate the blood-brain barrier might modify neurotransmission at the Central Nervous System (CNS). This might be useful to establish therapeutic approaches to control the neuronal hyperexcitabiltity observed in brain conditions that are associated to copper dyshomeotasis such as Alzheimer's and Menkes diseases. Our work also opens a new avenue to find novel and effective antiepilepsy drugs based in metal coordinating molecules.

Keywords: copper; dynamin; epileptiform-like activity; hyperexcitability; neocuproine; synapsin; synaptic activity; zinc.

Figure 1

Neocuproine increases intracellular Zn²⁺ [Zn²⁺]_i by promoting copper influx in hippocampal neurons. (A) The traces show the intracellular changes of Phen Green fluorescence after extracellular acute application of CuCl₂ (3 μM) (quenching), EDTA (10 μM) (recovery) or Neocuproine (Neo, 10 μM) (quenching). Traces in color correspond to the signal recorded in a single cell. The black trace corresponds to the typical background signal recorded outside of cells. (B) Confocal microphotographs of hippocampal cultures loaded with Phen Green, untreated (a and c) or treated with Neo (10 μM) for 10 min (b and d). Pseudocolor images shown in “c” and “d”, correspond to fluorescence photographs shown in “a” and “b”, respectively. Bar represents 100 μm. (C) The traces show the intracellular changes of FluoZin-3 fluorescence under control conditions or after the acute application of the agents indicated in the figure (Pyr/Zn = 30/10 μM; TPEN = 10 μM). Each trace corresponds to the signal recorded in a single cell. (D) Confocal microphotographs of hippocampal cultures loaded with FluoZin-3, before (time = 0) and after of a short application (up to 8 min) of Neo (10 μM). Scale bar, 250 μm. At the right of this panel is shown a confocal zoom of two neurons from the experiment described in “(D)”. The graph summarizes the intracellular Zn²⁺ changes recorded in the soma of single neurons treated as shown in “(D)”. Values are mean ± SEM (N = 3). (E) The traces show the rapid intracellular changes of FluoZin-3 fluorescence after the acute application of Neocuproine (Neo, 10 μM). (F–H) Each colored trace corresponds to the FluoZin-3 fluorescent signal recorded on the soma of single hippocampal neuron under the treatments indicated in the figures (Neocuproine, Neo = 10 μM; Bathocuproine, BC = 10 μM; TPEN = 10 μM). The black traces correspond to the typical background signal recorded outside of cells.

Figure 2

Acute application of Neocuproine does not alter synaptic transmission. (A) Calcium transients of hippocampal neurons in the absence or presence of Neocuproine (Neo, 10 μM). (B) Typical miniature synaptic currents obtained in the absence or presence of Neocuproine (Neo, 10 μM). The graphs summarize the frequency (C) and the amplitude (D) values of miniature synaptic currents recorded in three independent experiments performed under the conditions described in “(B)”. In both cases values are mean ± SEM (N = 3).

Figure 3

Chronic application of Neocuproine decreases the number of active neurons. (A) The top traces show typical calcium transients observed in active hippocampal neurons in culture. The bottom traces show the lack of calcium transients in inactive hippocampal neurons in culture. Each trace corresponds to the signal recorded in a single cell. (B) The graph summarizes the percentage of active hippocampal neurons under different treatments: untreated (control) or treated with Neocuproine (Neo, 10 μM) in the absence or presence of Bathocuproine (BC, 10 μM) for 12 h. Values are mean ± SEM (N = 3). **, p < 0.01.

Figure 4

Chronic application of Neocuproine down-regulates synapsin and dynamin. (A) mRNA levels of dynamin I (dyI), dynamin II (dyII), synapsin, Bassoon (Bas), SNAP29, Superoxide Dismutase (SOD) and Tubulin (Tub) expressed by rat hippocampal cultures (11 DIV) treated in the absence or presence of Neocuproine (Neo, 10 μM) for 12 h. Values are mean ± SEM (N = 3). *, p < 0.05. (B) Protein levels of synapsin and dynamin I expressed by rat hippocampal cultures (11 DIV) treated in the absence or presence of Neo (up to 30 μM) for 12 h. Whole brain homogenate (H) of 15 days post-natal rat was used as positive control. (C,D) The graphs summarize the data obtained in experiments described in “(B)”. Values are mean ± SEM (N = 3). *, p < 0.05; **, p < 0.01.

Figure 5

Bathocuproine blocks the decrease of synapsin induced by Neocuproine treatments. (A) Microphotographs of rat hippocampal neurons treated with Neocuproine (Neo, 10 μM and 30 μM) for 12 h. Synapsin immunoreactivity is shown in green, and MAP2 in red. Scale bar, 25 μm. (B) Protein levels of synapsin expressed by rat hippocampal cultures (11 DIV) treated in the absence or presence of Neo (up to 30 μM), and Neo (10 μM) + Bathocuproine (BC, 10 μM) for 12 h. (C) The graph summarize the data obtained in the experiment described in “(B)”. Values are mean ± SEM (N = 3). * p < 0.05.

Figure 6

Neocuproine blocks the epileptiform-like activity induced by bicuculline in hippocampal neurons. (A) Representative traces of miniature postsynaptic currents illustrate the effect of control and chronic (24 h) bicuculline (5 μM) treatment in the absence or presence of Neocuproine (Neo, 10 μM) in hippocampal neurons. (B,C) The graphs summarize the effect of the different treatments on the frequency (Hz) and amplitude (pA) of the records obtained in three independent experiments performed under the conditions described in “(A)”. Values are mean ± SEM obtained from the indicated number of neurons. ***, p < 0.001.