Expression of Zn transporter subfamily II members Zip-1 and Zip-3 in the hippocampus
containing neurons were visualized in the central nervous system by detecting the expression of an EGFP
reporter engineered into mice doubly homozygous for targeted deletions of these genes (Dufner-Beattie et al, 2006
). These transporters are expressed at low levels throughout the forebrain, brainstem, and cerebellum, however they show a predominant localization within the hippocampal formation. shows the intense EGFP signal found within the hippocampal formation after immunostaining with antiGFP-Alexa 594 (Texas Red) in a coronal section from Zip-1,3
double knockout mouse brain. In comparison, antiGFP-Alexa 594 staining in +/+ brain shows no specific signal such a highlighted staining in the pyramidal cell layers as observed in the −/− mouse brain (data not shown). The fluorescence intensity of EGFP antibody staining in the pyramidal cell layer is about 50% higher than that in the nearby entorhinal cortex after being normalized by the cell density in each region (obtained from densitometry of adjacent Nissl stained sections). demonstrates that the Zip-1
expression in the hippocampal pyramidal cell layer arises from staining of the principal cells. To verify that the strong expression within the cell layer represented intrinsic transporter expression in individual pyramidal cells, a patch pipette was used to load visually identified CA1 pyramidal cells in Zip-1,3
mutant (B) and wild-type (C) mouse brain slices with the fluorescent tracer Alexa 568. In Zip-1,3
null mutants, but not in control wild type mice, significant GFP fluorescence could be detected in pyramidal cells as shown in the corresponding EGFP image in . EGPF staining was also detected in cells at very low intensity similar to neocortex in other regions of brain including, thalamus, pons and cerebellum (data not shown).
Expression of Zip-1,3 Zn transporters in the hippocampus
Ablation of Zip-1 and Zip-3 transporters slows Zn uptake into CA1 pyramidal cells
We used whole cell patch clamp recording combined with fluorescent imaging to examine the potential contribution of Zip-1,3 transporters to Zn uptake into CA1 pyramidal neurons in vitro. We isolated the inward transporter-mediated fraction by adding the NMDA receptor antagonist D-APV and the AMPA receptor antagonist DNQX to the perfusate of hippocampal slices to eliminate possible Zn entry through glutamatergic receptors (GluR). To prevent Zn entry through open voltage-gated Ca2+ channels in the target cell, the intracellular solution contained 5 mM QX-314 to block voltage-gated Na+ channels and the somatic membrane potential was constantly held at -70 mv. To monitor intracellular Zn levels, single CA1 pyramidal neurons were loaded with the membrane impermeable fluorescent tracer Alexa 568 and the Zn sensitive dye FluoZin-3. A pair of Alexa 568 and FluoZin-3 fluorescence images, providing a baseline reference of intracellular Zn activity, was obtained 30 minutes after membrane break-in. We determined that the ratio of F-FluoZin-3/F-Alexa is homogeneous across the cell surface from soma to the proximal one third of the apical dendrite, as demonstrated by a linear relationship between FluoZin-3 fluorescence (F-FluoZin-3) and Alexa fluorescence (F-Alexa) sampled along the line shown in the Alexa image ( left), indicating that cytoplasmic free Zn levels are buffered to a uniform level. To minimize the sampling variance, we measured the ratio of F-FluoZin-3/F-Alexa, instead of F-FluoZin-3 alone, before ( center) and after ( right) exposure of the slice to extracellular Zn2+ in order to calculate Zn uptake. In wild type brain slices, perfusion of 10 μM Zn2+ for 30 minutes increased the ratio of F-FluoZin-3/F-Alexa from 6.7±0.9% to 23.1±8.1% (n=11) ( upper). In (lower), the corresponding result for neurons in Zip-1/3 −/− slices was 4.8±0.9% and 12.1±2.2% (n=9), which represents a significant reduction of approximately 50% (p<0.01) compared to wild-type neurons. This sharp reduction is consistent with the transporter localization obtained with EGFP staining () and supports the notion that Zip-1,3 transporters expressed in hippocampal CA1 pyramidal cells regulate passive Zn homeostasis.
Slowed Zn uptake in mice lacking Zip-1,3 transporters
Protection from seizure-induced cell injury in the CA1 area of Zip-1,3 −/− mutants
To determine whether this two fold reduction in the contribution of transporter-mediated Zn entry during intense synaptic activation could influence cell survival following injury in vivo, we employed a widely used hippocampal convulsant that generates extensive excitotoxic pyramidal cell damage while allowing survival of the animal. Zip-1,3 mutants and wild-type littermates were injected intraperitoneally with kainic acid KA (15mg/kg), monitored by videoEEG to quantify seizure activity, and early hippocampal cell injury was evaluated 24 hours later with Fluoro-Jade (FJ) staining. shows representative images of FJ staining in the hippocampus of Zip-1/3 −/− and +/+ brains analyzed one day after KA injection. The Fluoro-Jade staining is largely confined to the pyramidal cell layers except for the CA2 area. The dentate granule layer was not stained at all. This pattern of Fluoro-Jade staining is consistent with the cell death pattern in the hippocampus following excitotoxic insults.
Attenuated neurodegeneration in the hippocampal CA1 area in Zip-1,3 −/− mutants after KA-induced seizures
In a large sample of mice from both genotypes, disruption of Zip-1,3 transporters produced clear protection in the hippocampal CA1 region. We found positive cellular FJ staining in 21/41 injected +/+ mice in the CA1 area; in contrast, 11/43 injected −/− mutants showed positive FJ staining in the same area. We calculated a Fluoro Jade index (FJI) for hippocampal pyramidal subregions in each section to quantify cell injury in the mutant and littermate control genotypes. We found evidence that the two hippocampal subregions exhibit different vulnerability profiles to kainate-induced injury. The average FJI in CA1 for the −/− group was 9±19% (n=43) of its maximal value, which is significantly smaller than the value for the +/+ group (23±32%, n=41, p=0.01). Only 5/43 Zip-1,3 −/− mice fell in the upper 60 percentile of FJIs compared to 13/41 from the +/+ group. These data demonstrate a striking reduction in early CA1 injury in the −/− group after KA injection compared with wild-type mice. The pattern of selective protection of CA1 neurons in −/− mice was even more evident when cell damage from both sub-regions is directly compared, as shown in . In wild type mice, a group of mice (8/41) exhibits a characteristic pattern of vulnerability exclusively confined to the CA1 pyramidal cell region ( left panel). These mice display extensive CA1 FJ staining (>40% FJImax) while the CA3 region is mostly spared (<10% FJImax). In the −/− group ( right panel), the number of mice with exclusive CA1 cell damage was greatly diminished. In contrast, due to significant neuroprotection in the CA1 region, Zip-1,3 null mutants show damage largly confined to the CA3 region, with minor amounts of CA1 injury. Only 2/43 mutants showed evidence of isolated CA1 damage. The FJI for the CA3 area was positive in a similar proportion of Zip-1,3 null mice (26/43) compared to wild type mice (26/41), however the average FJI for CA3 in Zip 1,3 null mice was 29±27% (n=43) of its maximal value, which is significantly larger than the average value from the +/+ group (19±23%, n=41, p=0.04), indicating more CA3 cell damage occurs in the −/− group. summarizes cell injury in each sub-region of hippocampus.
Zip-1,3 null mutants exhibit a lower seizure threshold to kainate
The extent of hippocampal cell injury in experimental seizure models is strongly dependent on the severity of seizure activity. It is therefore possible that if developmental absence of Zip-1,3 transporters lowered the underlying network excitability, weaker seizures, rather than reduced vulnerability to Zn entry, might explain the differences in cellular damage between the mutant and wild type brain. To compare seizure severity between the genotypes, both behavioral and EEG seizure monitoring was continuously performed in mice from both groups during the period of KA exposure and accurately quantified by frequency analysis of the digitized EEG trace. Zip-1,3 null mutants display normal patterns of EEG activity during routine monitoring, but reveal a latent hyperexcitability upon administration of the glutamate agonist. shows EEG activity recorded from mice (−/− n=8; +/+ n=6) before, and during the first 4 hours after KA injection. Zip-1,3 mutants exhibited more synchronized high frequency spike bursting (marked in red in ) than wild-type mice, and 3/8 mutant mice showed essentially constant EEG seizure activity, lasting even beyond the 4 hour experimental monitoring window. Among the 6 +/+ mice recorded for EEG activity, none displayed seizures beyond 4 hours, and the EEG seizure discharges were discontinuous. The average latency for the initial onset of high frequency EEG spiking was 11±9 minutes (n=8) in the −/− group, which is less than one half that in the +/+ group (26±17 minutes, n=6, p=0.05). Seizure-induced lethality also differed between genotypes; 17% of the tested mutants died during a prolonged seizure, compared to only 4% in the +/+ group after injection of 15 mg/kg KA. At a higher dose (30 mg/kg), the difference in mortality was even greater (mortality rate was 66% and 22% respectively for −/− and +/+ mice). These results demonstrate that less seizure-induced neurodegeneration occurs in the CA1 area of Zip-1,3 −/− mice even though they experience far more severe seizure activity after KA injection than the +/+ group.
During seizures, glutamate excitotoxicity, particularly mediated by NMDA receptors, also contributes to hippocampal pyramidal cell injury. If Zip −/− mice express a lower NMDA receptor density, then less neuronal cell damage in the −/− group could be explained by a reduction in glutamate excitotoxicity. To evaluate this possibility, we compared the expression levels of NMDA receptors by western blot between +/+ and −/− mice. shows NMDA receptor expression in the cortex and hippocampus of +/+ and Zip-1,3 −/− mouse brain. Comparable immunoreactive bands corresponding to the 120 kDa NMDAR1 subunit were detected in cortex and hippocampus, indicating unaltered global NMDA receptor expression in Zip-1,3 −/− mouse. This result also suggests that the increased seizure severity in Zip-1,3 −/− brain is unlikely to be explained by secondary changes in NMDA receptor density.
Unaltered NMDA receptor expression in Zip-1,3 −/− mouse brain
Less neuronal cell damage in Znt-3 −/− mouse with KA injection
If postsynaptic Zn uptake contributes to pyramidal cell vulnerability, then reduction of extracellular Zn content during seizure activity might resemble the effect of Zip-1,3 transporter deletion. We examined early neurodegeneration in mice lacking synaptic Zn by injecting Znt-3 −/− mice with KA (15 mg/kg) in parallel studies. The same dose of KA, which usually evokes a full body convulsion in either Zip-1,3 −/−or +/+ group within the first 30 minutes, only caused forelimb tremors in Znt-3 −/− mice within the first hour of the injection. After a second dose of 15 mk/Kg KA injection one hour later, Znt-3 −/− mice started to develop full body convulsions and were sacrificed for Fluoro Jade staining 24 hours later. summarizes the result for the Znt-3 −/− group. There is much less cell damage in both the CA3 and CA1 areas after two injections of 15 mg/kg KA in comparison to the +/+ group with one injection of 15 mg/kg KA. The overwhelming majority of mice did not show any cell damage in the CA1 area. Only 3/34 Znt-3 −/− mice fell in the upper 60 percentile of FJIs. The mean FJI in the CA1 area was 8±17% (n=34) which is significantly less than that in the +/+ group (p=0.01). In the CA3 area, the average FJI was 9±12% (n=34) which is also significantly smaller than the average value from the +/+ group (p=0.03). As shown in , cell injury for both CA1 and CA3 remained in a smaller range compared to the +/+ group. Interestingly, EEG monitoring of Znt-3 −/− mice after only one injection of KA at 15 mg/kg showed more seizure activity than the +/+ group even though their behavioral seizure activity scores were low. Although their average latency for the initial onset of high frequency EEG spiking was not significantly different from mice in the +/+ group, Znt-3 −/− mice displayed longer, on average, seizure episodes of high frequency EEG spiking. Our results indicate that reduction of Zn uptake, either by knock-out of Zip-1,3 transporters or abolishment of synaptic Zn, can protect neurons from neurodegeneration following prolonged seizures..
Less hippocampal cell damage in Znt3 −/− mouse brain following KA injection