Synaptic plasticity in the hippocampus shows resistance to acute ethanol exposure in transgenic mice with astrocyte-targeted enhanced CCL2 expression

Abstract

It has been shown that ethanol exposure can activate astrocytes and microglia resulting in the production of neuroimmune factors, including the chemokine CCL2. The role of these neuroimmune factors in the effects of ethanol on the central nervous system has yet to be elucidated. To address this question, we investigated the effects of ethanol on synaptic transmission and plasticity in the hippocampus from mice that express elevated levels of CCL2 in the brain and their non-transgenic littermate controls. The brains of the transgenic mice simulate one aspect of the alcoholic brain, chronically increased levels of CCL2. We used extracellular field potential recordings in acutely isolated hippocampal slices to identify neuroadaptive changes produced by elevated levels of CCL2 and how these neuroadaptive changes affect the actions of acute ethanol. Results showed that synaptic transmission and the effects of ethanol on synaptic transmission were similar in the CCL2-transgenic and non-transgenic hippocampus. However, long-term potentiation (LTP), a cellular mechanism thought to underlie learning and memory, in the CCL2-transgenic hippocampus was resistant to the ethanol-induced depression of LTP observed in the non-transgenic hippocampus. Consistent with these results, ethanol pretreatment significantly impaired cued and contextual fear conditioning in non-transgenic mice, but had no effect in CCL2-transgenic mice. These data show that chronically elevated levels of CCL2 in the hippocampus produce neuroadaptive changes that block the depressing effects of ethanol on hippocampal synaptic plasticity and support the hypothesis that CCL2 may provide a neuroprotective effect against the devastating actions of ethanol on hippocampal function.

Fig. 1

Protein expression in CCL2-tg and non-tg hippocampus. (A-B) Levels of cellular (A) and synaptic (B) proteins determined by Western blot analysis in CCL2-tg and non-tg hippocampus. Graphs show the mean normalized values. The number of animals studied for each protein is marked in the corresponding bar. Inserts above the graphs show representative Western blots. * Indicates a significant difference from non-tg hippocampus (unpaired t-test).

Fig. 2

Input-output (I/O) curves measured in the CA1 region of hippocampal slices from CCL2-tg and non-tg mice. (A) Representative recordings of data for I/O relationship. (B-D) I/O curves constructed from the mean values of the presynaptic volley (PSV) amplitude (B), fEPSP slope (C), and population spike (PS) amplitude (D). No differences were observed in the amplitude of the PSV or the slope of the fEPSP for CCL2-tg hippocampal slices compared with non-tg hippocampal slices. The PS amplitude was significantly larger in CCL2-tg hippocampal slices compared to non-tg hippocampal slices (p=0.024, repeated measures ANOVA). Data are derived primarily from ACSF only exposed slices shown in Figure 3.

Fig. 3

Affect of acute ethanol on input-output (I/O) curves measured in the CA1 region of hippocampal slices from CCL2-tg and non-tg mice. (A-B) I/O curves constructed from the mean values of the fEPSP slope in the presence of 20 mM (open circles) and 60 mM ethanol (closed triangles) in non-tg (A) and CCL2-tg (B) hippocampal slices. 20 mM acute ethanol had no effect on the I/O relationship for the fEPSP slope in either the non-tg (A) or CCL2-tg (B) hippocampal slices. 60 mM acute ethanol significantly decreased the I/O relationship for the fEPSP slope in both non-tg (p=0.033) and CCL2-tg hippocampal slices (p=0.048). (C-D) I/O curves constructed from the mean values of the population spike (PS) amplitude in the presence of 20 mM (open circles) and 60 mM ethanol (closed triangles) in non-tg hippocampal slices. 20 mM acute ethanol had no effect on the I/O relationship for the PS in either the non-tg (C) or CCL2-tg (D) hippocampal slices. 60 mM acute ethanol significantly decreased the I/O relationship for the PS in both non-tg (p=0.003) and CCL2-tg (p=0.001) hippocampal slices. Representative recordings of data for the I/O relationship are shown above each graph. Statistical analysis was determined using repeated measures ANOVA. * Indicates a significant difference between ethanol and ACSF treated hippocampal slices.

Fig. 4

Short-term plasticity in CCL2-tg and non-tg hippocampal slices in the absence and presence of acute ethanol. (A-B) Representative traces illustrating paired-pulse facilitation (PPF) of the fEPSP (A) and the PP ratio for the PS (B). Traces R1 and R2 are superimposed responses to paired stimuli separated by 40 ms for the fEPSP and 10 ms for the PS. (C) Summarized results for PPF at 40, 100, and 200 ms paired-pulse intervals. No differences in PPF of the fEPSP slopes were observed for CCL2-tg hippocampal slices compared to non-tg hippocampal slices. (D) Summarized results for the PP ratio at 10 and 20 ms paired-pulse intervals. No differences in the PP ratio for the PS were observed in the CCL2-tg hippocampal slices compared to non-tg hippocampal slices. (E-F) Summarized results for 10 ms (E) and 20 ms (F) paired-pulse intervals for the PS in the presence of 20 mM and 60 mM acute ethanol. Acute ethanol application did not affect the PP ratio of the PS in either CCL2-tg hippocampal slices or non-tg hippocampal slices at the 10 ms interval (E). 20 mM acute ethanol application produced a significant increase in the paired-pulse ratio of the PS in the CCL2-tg hippocampal slices at the 20 ms interval compared with ACSF control CCL2-tg hippocampal slices that were not exposed to ethanol (^@, p=0.017). 60 mM acute ethanol application produced a significant increase in the paired-pulse ratio in non-tg hippocampal slices compared with ACSF control non-tg hippocampal slices (*, p=0.004) an effect that was not observed in CCL2-tg hippocampal slices. Thus, there was a significant difference between genotypes in the presence of 60 mM acute ethanol (#, p=0.048). Statistical analysis was determined using the unpaired t-test. The number of slices studied is marked in the corresponding bar.

Fig. 5

Synaptic plasticity measurements following TBS in hippocampal slices from CCL2-tg and non-tg mice in the absence and presence of acute ethanol. (A) Representative dendritic fEPSP traces illustrating post-tetanic potentiation (PTP) 1–3 minutes following TBS and longterm potentiation (LTP) 50–60 minutes following TBS compared to baseline traces recorded prior to TBS. (B) Synaptic plasticity measurements in hippocampal slices from CCL2-tg and non-tg mice expressed as percent of baseline fEPSP slope. High-frequency stimulation (TBS) was used to elicit synaptic plasticity and occurs at time zero. No differences in synaptic plasticity were observed between CCL2-tg and non-tg hippocampal slices (p=0.968). (C-F) Synaptic plasticity measurements in the presence and absence of acute 20 mM and 60 mM ethanol in non-tg (C and D) and CCL2-tg (E and F) hippocampal slices. A decrease in synaptic plasticity was observed in the presence of 20 mM (C, p < 0.0001) and 60 mM (D, p < 0.0001) ethanol in non-tg hippocampal slices when compared to the ACSF treated non-tg hippocampal slices. An enhancement in synaptic plasticity was observed in the presence of 20 mM ethanol (E) in CCL2-tg hippocampal slices when compared to the ACSF treated CCL2-tg hippocampal slices (p < 0.0001). There was no effect of acute 60 mM ethanol on synaptic plasticity in the CCL2-tg hippocampal slices (F) when compared to the ACSF treated CCL2-tg hippocampal slices (p=0.292). Statistical analysis was determined using repeated measures ANOVA. * Indicates a significant difference between ethanol and ACSF treated hippocampal slices.

Fig. 6

Effect of acute ethanol on cued and contextual fear conditioning. For fear conditioning, mice were treated with ethanol (1 g/kg) or saline prior to the conditioning trial. (A) Freezing responses during habituation and during exposure to contextual cues. (B) Freezing responses before the cue was activated (pre-cue) and during cue exposure in the conditioned stimulus (CS+ test). Data are expressed as mean ± SEM of the time (s) spent freezing. Non-tg mice treated with ethanol showed significantly less freezing behavior during both the context test (p=0.007) and CS+ test (p=0.037) compared to control non-tg mice treated with saline. The number of animals studied for each test is marked in the corresponding bar. * Indicates a significant difference between ethanol and saline treated mice of the same genotype.

Fig. 7

Ethanol recovery assessed with rotarod testing. Rotarod test recovery times following 2 g/kg ethanol. There was no significant difference between non-tg and CCL2-tg mice in neither this behavioral effect of ethanol, nor the blood ethanol levels determined at the time of recovery.