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Accumulation of cytoplasmic zinc is linked with a cascade of events leading to neuronal death. In many in vivo models of zinc-induced cell death, toxic concentrations of synaptically released zinc enter vulnerable neurons via neurotransmitter- or voltage-gated ion channels. In vitro studies demonstrate, in addition, that zinc can be liberated from intracellular stores following oxidative stress and contribute to cell death processes, including apoptosis. Here we describe accumulation of intracellular zinc in an in vivo model of cell death in the absence of presynaptic zinc release. We focused on the lateral geniculate nucleus (LGN) because LGN neurons undergo apoptosis when separated from their target, the primary visual cortex (V1), and the LGN is mostly devoid of zinc-containing presynaptic terminals. Infant and adult rats and adult mice received unilateral ablation of V1, either by aspiration or kainate injection. One to 14 days later, brain sections were stained with selenium autometallography or fluorescently labeled to localize zinc, or stained immunochemically for activated caspase-3. V1 lesions led to zinc accumulation in LGN neurons in infant and adult subjects. Zinc-containing neurons were evident 1–3 days after aspiration lesions, depending on age, but not until 14 days after kainate injection. Zinc accumulation was followed rapidly by immunostaining for activated caspase-3. Our data indicate that like neurotrauma and excitotoxicity, target deprivation leads to accumulation of zinc in apoptotic neurons. Moreover, zinc accumulation in vivo can occur in the absence of presynaptic zinc release. Together these findings suggest that accumulation of intracellular zinc is a ubiquitous component of the cell death cascade in neurons.
In vivo studies of neuronal injury following global ischemia (Tonder et al., 1990; Koh et al., 1996), trauma (Suh et al., 2000) or seizures (Frederickson et al., 1989; Riba-Bosch & Perez-Clausell, 2004) have implicated cytotoxic elevations of intraneuronal Zn2+ in the patho-physiology of neurodegeneration (Choi & Koh, 1998). In these models, a high concentration of Zn2+ is thought to be released from synaptic vesicles and enter vulnerable postsynaptic neurons via neurotransmitter- and voltage-gated ion channels (Weiss et al., 1993; Koh & Choi, 1994; Sensi et al., 1999). In vitro studies, however, clearly demonstrate that Zn2+ also can be liberated from intracellular stores and contribute to cell death processes, including apoptosis (Aizenman et al., 2000). Thus, oxidative and nitrosative stressors liberate Zn2+ that normally is bound to cysteine residues of intracellular metalloproteins like metallothionein (MT, Pal et al., 2004). Studies with cell-free assays demonstrate that redox agents such as 2,2′-dithiodipyridine (DTDP) can oxidize the cysteine residues of MT to release bound Zn2+ (Maret & Vallee, 1998). In neurons treated with DTDP, liberated Zn2+ can induce neuronal apoptosis via a p38 kinase-dependent process, requiring enhancement of Kv2.1-mediated potassium currents and caspase activation (McLaughlin et al., 2001; Pal et al., 2003). These findings strongly suggest that non-vesicular Zn2+ could have an important role in neurodegeneration, although in vivo conditions wherein Zn2+-associated cell death occurs in the absence of presynaptic Zn2+ release are ill-defined.
It has long been known that destruction of cerebral cortical tissue leads to atrophy of specific thalamic nuclei (von Gudden, 1870). For example, damage to primary visual cortex (V1) results in death of projection neurons in the dorsal lateral geniculate nucleus (LGNd; Lashley, 1941; Matthews, 1973; Agarwala & Kalil, 1998a). It also has been observed that degeneration of thalamic neurons following cortical lesions occurs via apoptosis (Al-Abdulla et al., 1998; Martin et al., 2001; Al-Abdulla & Martin, 2002; Repici et al., 2003), and may result from loss of trophic support after target removal (Eagleson et al., 1990; Agarwala & Kalil, 1998b). Importantly, LGNd neurons, and the corticogeniculate neurons providing reciprocal feedback, do not sequester Zn2+ within their synaptic terminals (Garrett & Slomianka, 1992; Casanovas-Aguilar et al., 1998). Indeed, the LGNd, like most principal thalamic nuclei, is nearly devoid of Zn2+-containing synapses (Mengual et al., 2001). We therefore used LGNd neurons and their trophic dependence on V1 as a model system for investigating the association of non-vesicular Zn2+ with the apoptotic cascade in vivo. Following damage to V1 by aspiration lesion or excitotoxic insult, staining for Zn2+ occurs in the cytoplasm of LGNd neurons, and this is followed rapidly by activation of caspase-3. These findings indicate that target deprivation, like ischemia, trauma and excitotoxicity leads to Zn2+ accumulation in dying neurons.
All procedures were performed in accordance with the National Institutes of Health ‘Guide for the Care and Use of Laboratory Animals’ and were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Long–Evans black-hooded rats (175–200 g), female rats with litters and C57/BL6 mice (17–25 g) were obtained from Harlan Sprague–Dawley (Indianapolis, IN, USA). Adult rats and mice, and postnatal day (P) 10 infant rats were anesthetized with tribromoethanol (232 mg/kg, i.p.) and placed in a stereotaxic device to stabilize the head. Under aseptic conditions, a midline scalp incision was made, and the skin and subcutaneous connective tissue over the right calvarium were reflected. For adult subjects a dental burr was used to make a 4 mm × 4 mm (rat) or 3 mm × 3 mm (mouse) square craniectomy over the right occipital cortex. The medial boundary of the craniectomy was located approximately 0.5 mm lateral to the sagittal suture; the caudal boundary was defined by the lambdoid suture. For P10 rats, a 3 mm × 3 mm craniectomy was made using a #11 scalpel blade to incise the cartilaginous skull. Dura mater was reflected using a 31 gage hypodermic needle and exposed cortical tissue was gently removed by subpial suction with a fire-polished Pasteur pipette (Fig. 1). The lesion cavity was extended ventrally until the alveus surface of the hippocampus was exposed. The location and extent of the lesion was intended to encompass most or all of primary visual cortex (V1; area 17) and adjacent secondary visual areas (Paxinos & Watson, 1986).
Some mice received, in addition, injections of kainic acid (KA) into the left occipital cortex in order to kill cortical neurons (the target of LGNd neurons) without damaging geniculocortical axons (Schwarcz & Coyle, 1977). This allowed us to compare in individual animals the effect of axotomy vs. target-deprivation per se. A 5 mM solution of KA in 0.9% sterile saline was loaded into glass micropipettes (tip diameter ~ 10 μm). A craniectomy similar to that described above was made over the left occipital cortex and the KA-containing micropipette was then inserted through the dura into the cortex to a depth of 0.5–1.0 mm. Approximately 0.2 μL of KA was injected at each site via pressure ejection (Picospritzer, General Valve, Fairfield, NJ, USA), and 20 such injections were made into the exposed cortex in a grid pattern with a center-to-center spacing of 0.5–1.0 mm. A small piece of Gelfoam (Upjohn, Kalamazoo, MI, USA) was placed over the lesion/injection site and the skin was closed with wound clips (adult rats) or with 6.0 sutures. All animals received 3 mg/kg Ketofen (ketoprophin; i.p.; Fort Dodge Animal Health, Fort Dodge, IA, USA), and upon recovering from anesthesia they were returned to their home cages where they remained for 24 h to 14 days.
Autometallographic (AMG) localization of Zn2+ in LGNd neurons was assessed using the selenium method described by Danscher (1982). Multi-element analyses based on particle-induced X-ray emission indicate that selenium precipitates in the mammalian brain stained by this procedure principally contain the metal ion Zn2+ (Danscher et al., 1985). This technique previously has been shown to reveal synaptic Zn2+ as well as intracellular Zn2+ associated with neuronal cell death (Frederickson et al., 1989). Animals received an i.p. injection of the Zn2+ chelator sodium selenite (20 mg/kg) from a freshly prepared stock solution (20 mg/mL in distilled H2O) and were killed by decapitation 1 h later (see Table 1 for distribution of survival times by age and species). The brains were quickly removed, encased in OCT embedding medium (Miles, Elkhart, IN, USA) and rapidly frozen by surrounding them with crushed dry ice. Twenty-micron coronal sections through the brains were cut on a cryostat, collected onto warm (38 °C) microscope slides as a 1: 3 series and stored with a desiccant at −40 °C until they were processed.
One series of sections through each brain was processed with AMG according to a modification of the Danscher method (Dyck et al., 1993; Land & Akhtar, 1999). Briefly, slides were thawed, allowed to dry at room temperature, and fixed and hydrated in a descending series of ethanol (i.e. 95%, 15 min; 70%, 50%, 2 min each), after which they were rinsed in distilled H2O (3 × 2 min). Slides were then dipped in 0.5% gelatin, dried and immersed in a physical developer prepared from 50% Acacia Gum (120 mL), 2.0 M sodium citrate buffer (20 mL), 0.5 M hydroquinone (30 mL) and 37 mM silver lactate (30 mL). The latter two reagents were added to the incubation medium in the dark. The slides were incubated in the dark for 90–180 min with agitation. After development, the slides were washed for 30 min in warm (40 °C) running tap water in order to remove gelatin and any adhering precipitates, then rinsed in distilled H2O (3 × 2 min). The slides were fixed in 5% sodium thiosulphate for 12 min, rinsed in distilled H2O (4 × 2 min) and postfixed in 70% alcohol for 30 min. Some section series were further dehydrated in ascending alcohols and coverslipped with Permount (Histologic Mounting Media, Fisher Scientific, FairLawn, NJ, USA). Other series were counterstained with 0.1% thionin or 1% acid fuchsin (Lees, 1989) after the AMG reaction to reveal degeneration-related changes in LGNd neurons, as well as to determine the cellular localization of silver staining.
In some brains we visualized free Zn2+ using a fluorescent Zn2+ indicator, Zinpyr-1 (NeuroBioTex, Galveston, TX, USA), that became available during the course of this study. Zinpyr compounds were specifically developed to facilitate the visualization of intracellular Zn2+ in biological samples, and they exhibit strong fluorescence in response to nanomolar concentrations of Zn2+ in the presence of millimolar concentrations of Ca2+ and Mg2+ (Burdette et al., 2003; Frederickson et al., 2004a). Indeed, other common metal ions like Ca2+, Mg2+, K+ and Na+ elicit no appreciable change in the emission spectra of Zinpyr reagents (Frederickson et al., 2004a). We did not repeat all experiments using Zinpyr, but the data confirm and extend our findings on cytoplasmic Zn2+ accumulation revealed by the AMG method (see Results). Cryostat sections were prepared as above. Sections were immersed for 1–5 min in a 10 μM solution of Zinpyr-1 prepared in 0.9% NaCl. Without rinsing, sections were coverslipped with glycerin and photographed under epifluorescence illumination. Coverglasses were removed, and the sections were washed in phosphate buffer containing 0.9% sodium chloride (phosphate-buffered saline, PBS; pH 7.3; 3 × 10 min) and fixed for 2 min in 4% paraformaldehyde. After rinsing in PBS, sections were exposed to 4′,6-diamidino-2-phenylindole (DAPI; 1: 1000; Sigma) for 1 min, rinsed in 0.1 M phosphate buffer and coverslipped again with glycerin.
Metal response element (MRE)-binding transcription factor-1 (MTF-1)-deficient dko7 cells were a gift from W. Schaffner (ESBATech, Zurich, Switzerland). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 24 U/mL penicillin, 24 U/mL streptomycin and 2 mM L-glutamine, and transfected with the MTF-1 expression vector (pCMV-MTF-1-FLAG; a gift from G. Andrews, University of Kansas Medical Center, Kansas City, KS, USA) or a blank vector, as previously described (Hara & Aizenman, 2004). Briefly, cells were seeded onto 11-mm glass coverslips at a cell density of 3.5 × 104 cells/well in a 24-well plate. The next day dko7 cells were transfected with 1.5 μg DNA using LipofectAMINE (Gibco/BRL). Twenty-four hours later cells were exposed to 100 μM Zn2+ for 10 min in MEM (plus 0.01% bovine serum albumin and 25 mM HEPES). After exposure cells were incubated with MEM for 2.5 h, and subsequently with 6 μM Zynpyr-1 in MEM for 5 min, fixed with 10% formaldehyde and mounted on microscope slides for visualization.
To establish the temporal relationship between occurrence of intracellular Zn2+ and neuronal apoptosis following cortical ablation we prepared a parallel series of animals wherein we immunohisto-chemically localized activated caspase-3. Twenty-four hours to 14 days post-lesion (see Table 1), rats and mice were anesthetized with isoflurane (IsoFlo, Abbott Laboratories, North Chicago, IL, USA) and perfused transcardially with 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.3). Brains were removed, postfixed overnight then transferred to 30% sucrose in phosphate buffer for cryoprotection. Frozen sections were cut in the coronal plane at 30 μm on a sledge microtome and collected in phosphate buffer. A 1: 3 series of sections was washed in PBS and preincubated for 1 h at room temperature in blocking serum (Blotto) consisting of PBS, 4% W/V non-fat dry milk and 0.4% Triton X-100. Sections were then incubated for 18 h at 4 °C in primary antisera from one of several sources directed against activated caspase-3, diluted in Blotto (BioVision, Mountain View, CA, USA, 1: 100 dilution; Cell Signalling Technology, Beverly, MA, USA, 1: 250 dilution; Merck-Frosst, Montreal, Quebec, Canada, 1: 16 000 dilution). Blocking peptide (e.g. the large fragment of activated caspase-3 resulting from cleavage) was used to test antibody specificity. Sections subsequently were washed 3 × 10 min in Blotto and transferred to Blotto containing biotinylated secondary antisera (1: 200; Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. Sections then were washed in 0.1 M phosphate buffer and incubated in the Avidin/Biotin peroxidase complex (Vector) according to the manufacturer’s instructions. After rinsing 3 × 10 min in phosphate buffer, sections were incubated in 0.5 mg/mL 3,3′-diaminobenzidine tetrahydrochloride (DAB) with 0.01% H2O2. In some cases (see below), nickel sulphate (2.5% W/V) was included in the reaction mixture to produce a black reaction product. DAB-stained sections were washed in phosphate buffer, mounted onto slides, dehydrated and coverslipped. Some series also were counterstained with 0.1% thionin.
During the course of this study we noted a relatively sparse ‘background’ immunostaining of cell nuclei throughout the brains of control and lesioned rats with all of the antibodies against activated caspase-3 (see also below). The comparatively small and consistent size of most stained nuclei suggested that they were non-neuronal. To test this we stained brain sections with anti-caspase-3 antibodies as described above, using nickel sulphate in the DAB step. After extensive washing in buffer (3 × 30 min) we then double-stained the sections with antibodies to GFAP, a marker for astroglial cells. Primary antisera (Polysciences, Warrington, PA, USA) were used at a dilution of 1: 8000. Remaining steps were as described above except that nickel sulphate was not included in the DAB reaction.
To characterize the time-course of Zn2+ accumulation in the LGNd we determined the number of Zn2+-containing cells per unit area at different post-lesion survival times. We selected from each Zn2+-stained brain a section at the rostrocaudal midpoint of the LGNd ipsilateral to cortical lesion and used Neurolucida (Microbrightfield, Williston, Vermont, USA) to measure the cross-sectional area of the nucleus. We then used the Meander Scan function to plot the location of Zn2+-containing cells and calculate their number per mm2. In all cases there were no Zn2+-containing cells in the LGN contralateral to cortical lesions (Figs 1 and and22).
We observed a low level of activated caspase-3 immunostaining in LGNd of rats ipsilateral and contralateral to cortical lesion at all survival times (e.g. Fig. 4). Such ‘background’ staining indeed was observed throughout each brain, was limited to the nuclear region of a small proportion of cells, and was completely blocked by preincubation of primary antiserum with immunizing peptide. As we show below, many of these nuclei appear to be associated with astroglial cells. Following cortical ablation there was a visible increase in the number of caspase-immunoreactive elements. In particular, this included the occurrence of cytoplasmic immunostaining in LGNd neurons following cortical lesions (see Results). However, in the absence of double-immunostaining, there was some ambiguity whether an individual stained element could be attributed to ‘background’ staining or to lesion-induced caspase activation. Thus, to characterize the time-course of activated caspase-3 expression, and to account for the potential presence of background immunostaining, we used Neurolucida as above to determine the number of immunoreactive elements per unit area in matched section through the LGNd ipsilateral and contralateral to cortical lesion. The data then are expressed as a ratio of IPSI/CONTRA labeling.
We used subpial aspiration of occipital cortex, including V1 and surrounding cortical areas, in P10 rats to induce degeneration of neurons in the ipsilateral LGNd and to investigate the association of cytoplasmic Zn2+ with the cell death process (Fig. 1A). This procedure previously was shown to result in rapid apoptotic loss of LGNd neurons (Cunningham et al., 1979; Repici et al., 2003). The LGNd, like most thalamic nuclei, has very low levels of chelatable Zn2+, and somata of LGNd neurons normally do not contain free Zn2+ that can be revealed by AMG or other methods (Fig. 1B). By contrast, ablation of occipital cortex quickly leads to accumulation of chelatable Zn2+ within the cytoplasm of LGNd neurons whose cortical target was removed (Fig. 1C). Punctate staining within LGNd neurons can be detected by 24 h after cortical damage at P10, and is persistently observed in the LGNd for up to 5 days post-lesion, by which time a majority of neurons have degenerated (see also Cunningham et al., 1979).
Chelatable Zn2+ in the LGNd ipsilateral to the ablated cortex is closely associated with other hallmarks of neuronal damage. For example, acid fuchsin counterstaining of sections processed with AMG reveals numerous acidophilic cells in the affected LGNd, most of which also contain chelatable Zn2+ (Fig. 2A and B). Silver deposits are largely coextensive with the cytoplasmic acidophilia of the axotomized neurons (i.e. Fig. 2B). Intracellular Zn2+ staining similarly is observed within cells exhibiting nuclear condensation, or pycnosis (Fig. 2C and D). In Nissl-stained sections, a cloud of silver-stained puncta typically surrounds the small, darkly stained nucleus obscuring other cellular detail (Fig. 2D). Chelatable Zn2+ thus accumulates in the cytoplasm of LGNd cells that are dead or are dying as a result of neonatal cortical lesion.
Recent studies indicate that visual cortex ablation in adult rats and mice also results in apoptosis of LGNd neurons (Al-Abdulla et al., 1998; Martin et al., 2001; Al-Abdulla & Martin, 2002), although over a longer time-course than in neonatal animals (Repici et al., 2003). To evaluate the possible association of intracellular Zn2+ accumulation and cell death in adult LGNd, and to establish the temporal relationship between accumulation of cytoplasmic Zn2+ and the apoptotic response of mature neurons to loss of their cortical target, we performed occipital cortical ablations in adult rats and mice (see also below). We then stained sections through the LGNd to visualize cellular Zn2+, as above, or with immunohistochemistry for activated caspase-3, the main executioner protease in the apoptotic pathway in mammalian neurons (Kuida et al., 1996).
Ablation of occipital cortex leads to accumulation of chelatable Zn2+ in the cytoplasm of mature LGNd neurons, as it does in neonatal neurons. In contrast with observations in infant rats, we first detected intracellular Zn2+ in mature neurons at 5 days post-lesion using autometallography. AMG staining can be observed in the affected LGNd for up to 14 days, the latest time point that we investigated (Fig. 3).
The apparently delayed onset of Zn2+ accumulation following adult vs. neonatal cortical lesions could reflect a reduced sensitivity of the AMG method in tissue from older animals. Alternatively, the time-course of Zn2+ accumulation may be modified as neurons mature. To address this in more detail we prepared several adult rats with cortical lesions, and stained sections through the LGNd with a recently developed high-affinity fluorescent Zn2+ indicator, Zinpyr-1 (Burdette et al., 2003). Zinpyr-1 reveals a diffuse cohort of Zn2+-containing cells in the LGNd and surrounding structures as early as 3 days post-lesion, but not at 1 or 2 days as we saw in infant rats (Fig. 4A and B). In confirmation of our AMG data, numerous labeled cells are seen at 5 days (Fig. 4C) and thereafter. Thus, in terms of Zn2+ accumulation, the cellular response to target loss is delayed by several days in adult compared with neonatal neurons, but nevertheless appears to begin somewhat earlier than what the AMG data reveal.
Notably, at 3 days post-lesion Zinpyr-1 staining is associated with cellular nuclei, a location that never is stained by the AMG method (Fig. 4D–F). At later survival times Zinpyr-1 also labels cytoplasmic Zn2+, as does autometallography (Fig. 4G–I). This suggests an early response of LGNd cells to cortical damage that selectively targets Zn2+ to the cell nucleus, and a later response wherein Zn2+ accumulates in the cytoplasm. The early response could reflect Zn2+ binding by MTF-1, which is a selective intracellular Zn2+ sensor that rapidly translocates to the nucleus upon binding free cytoplasmic Zn2+ (Westin & Schaffner, 1988; Palmiter, 1994). To test whether Zinpyr-1 can reveal the presence of MTF-associated Zn2+ we transfected dko7 cells, derived from mouse embryos having homozygous knockout of the MTF-1 gene (Heuchel et al., 1994), with either empty vector or with MTF-1 expression plasmid, and then challenged the cells with 100 μM Zn2+ for 10 min. Whereas the nuclei of dko7 cells transfected with empty vector are relatively unstained 2.5 h following the Zn2+ exposure (Fig. 4J), nuclei of cells transfected with MTF-1 become intensely stained with Zinpyr-1 following similar exposure conditions (Fig. 4K). This indicates that Zinpyr-1 can reveal Zn2+ that has been delivered to the nucleus as a result of MTF-1 translocation to that organelle.
In order to compare the time-course of zinc accumulation following cortex ablation with other components of the cell death cascade in this paradigm, we used immunohistochemistry for the cleaved, active form of caspase-3 to look for caspase activation in LGNd neurons. Activation of caspase-3, like accumulation of cytoplasmic Zn2+, requires several days to become apparent in adult LGNd neurons ipsilateral to cortical damage. Antisera from several sources (see Materials and methods) produced sparse staining of cellular nuclei throughout the brains of operated and control rats (Fig. 5A and C). This ‘background’ nuclear staining, like the cytoplasmic staining described below, is completely blocked by preincubation of primary antisera with immunizing peptide (data not shown). Double-immunostaining with antisera against GFAP revealed that most stained nuclei are associated with astroglial processes (Fig. 5D). Caspase-3 expression within nuclei of astrocytes is not accompanied by chelatable zinc, as both the AMG and Zinpyr methods produce labeling only within the affected LGNd of operated subjects.
In addition to nuclear staining within astroglia, we could detect robust cytoplasmic labeling for cleaved caspase-3 in the LGNd ipsilateral to cortical damage beginning at 5 days after cortex ablation (Fig. 5B). Immunoreactivity for cleaved caspase-3 is observed within somata and neurites of what appear to be LGNd projection neurons (Fig. 5E). The number of caspase-3-immunoreactive cells increases for several days, with the highest numbers reached at about 7 days (Fig. 6). The number of caspase-3-immunoreactive cells declines somewhat thereafter, although stained elements still can be observed after 14 days survival, the latest time point we investigated.
To address whether intracellular Zn2+ accumulation and apoptotic cell death of LGNd neurons results from loss of cortically derived neurotrophic factors or other molecules essential for neuronal survival, or from direct mechanical injury of LGNd neurons we prepared a series of adult mice that received unilateral cortical ablation as above. Upon completion of the cortical lesion, the mice then received injections of KA into the opposite hemisphere to produce excitotoxic death of occipital cortical neurons without damaging LGNd axons (see Materials and methods; Schwarcz & Coyle, 1977).
Chelatable Zn2+ first can be detected in adult mouse LGNd cells ipsilateral to subpial cortex aspiration about 5 days after surgery, as described above for rats, and Zn2+-containing cells can be detected there for at least 14 days (Fig. 7C, F and I). In the contralateral hemisphere that received cortical kainate injection the LGNd remains essentially free of AMG reaction product for at least 11 days (Fig. 7B and E). There also is little evidence of cortical degeneration around the injection sites until that time (compare Fig. 7A with D). However, 11–14 days after kainate injection occipital cortical tissue shows histological changes that include thinning of the cortical mantle and loss of Zn2+ staining associated with cortical intrinsic circuits in the area of injection (Fig. 7D and G; Garrett & Slomianka, 1992). Simultaneously, LGNd neurons in the injected hemisphere begin to contain chelatable Zn2+ (Fig. 7H). It seems likely therefore that expression of cytoplasmic Zn2+ is a prominent feature of a neuron’s response to target deprivation per se.
Neurons in the mammalian LGNd atrophy and die if they are disconnected from their target, V1. Here we show that an early and consistent feature of neuronal degeneration in the developing and mature LGNd as a result of V1 ablation is accumulation of intracellular Zn2+ in affected neurons. The presence of Zn2+ in dying LGN cells was corroborated by two independent methods, each of which has high selectivity for the metal ion (Danscher et al., 1985; Frederickson et al., 2004a). Our results demonstrate that Zn2+ accumulates in LGNd neurons despite a paucity of Zn2+-containing presynaptic terminals in that nucleus, and is followed rapidly by activation of caspase-3. We also provide evidence that this Zn2+-associated neurodegeneration results from target deprivation, per se, and does not require direct damage to LGNd cells. Thus, accumulation of intracellular Zn2+ appears to be a ubiquitous and naturally occurring component of the cell death process in neurons (Pal et al., 2004). Indeed, the elevation in the concentration of cytoplasmic Zn2+, in and of itself, may be an important trigger of the neurodegenerative process, as a large amount of evidence now supports the role of this metal in initiating a variety of cell death-inducing cascades in neurons (Koh, 2001; see also Frederickson et al., 2004b).
Accumulation of intracellular Zn2+ and activation of caspase-3 in LGNd neurons observed in the present study parallel, and to some extent presage, other morphological and molecular sequelae that have been described in the LGNd following cortical damage. Based upon examination of Nissl-stained material, neurons in adult rat LGNd ipsilateral to cortex ablation appear unremarkable and indistinguishable from normal for the first 3 days after surgery (Agarwala & Kalil, 1998a; Al-Abdulla et al., 1998). However, our findings using the fluorescent Zn2+ indicator Zinpyr-1 indicate that Zn2+ already is beginning to accumulate within neuronal nuclei at this survival time, in advance of any gross cytological changes. One possibility is that at about 3 days after injury Zn2+ is transported to the nucleus as a result of binding to the transcription factor MTF-1, from which it can be displaced by Zinpyr-1 but not by autometallography. Two days later, on the fifth post-lesion day, we observed both Zinpyr-1 stained and histochemically reactive Zn2+ throughout the cytoplasm of dying cells; Zn2+-containing cells were present until the end of the second week. This coincides with a period during which there is profound atrophy and loss of LGNd projection neurons following cortical ablation (Agarwala & Kalil, 1998a; Al-Abdulla et al., 1998).
Increased immunostaining for the active (cleaved) form of caspase-3 in the ipsilateral vs. contralateral LGNd followed a similar, though temporally shifted, time-course compared with intracellular Zn2+ accumulation. Thus, the number of caspase-3-immunoreactive elements in the ipsilateral LGNd rose markedly above background on the fifth post-lesion day and continued to increase until 1 week after the lesion. The number of caspase-3-immunoreactive elements declined during the second post-lesion week, presumably due to death and removal of most LGNd projection neurons (Lashley, 1941; Matthews, 1973; Agarwala & Kalil, 1998a). These data are consistent with the findings of Martin et al. (2001) who showed that protein levels of pro-caspase-3 (i.e. the inactive pro-enzyme) are significantly elevated in soluble fractions of LGNd homogenates by 4 days after cortex ablation, which was followed by a peak in protein levels for the activated form of caspase-3 1 day later, at 5 days post-lesion. Moreover, levels of p53, a transcription factor that regulates expression of pro-apoptotic genes (Levine, 1997), and Bax, a pro-apoptotic regulatory protein (Deckwerth et al., 1996), showed similar prominent increases between the fourth and fifth days following surgery (Martin et al., 2001). Together these findings point to an earlier, upstream event, perhaps associated with Zn2+ accumulation, which leads to neurodegeneration in the LGNd after cortex ablation. Specifically, using Zinpyr-1 we were able to detect intracellular Zn2+ at 3 days post-surgery. This is 2 days prior to the detection of the active form of caspase-3 at 5 days. This sequence parallels previous in vitro findings showing that oxidant-induced release of the metal from intracellular binding stores not only precedes but is required for caspase activation (McLaughlin et al., 2002).
We also observed accumulation of cytoplasmic zinc in LGNd neurons of infant rats (i.e. P10–P14) following ablation of V1. In contrast with findings in adult rats, where we first detected intracellular Zn2+ after 3 days, numerous Zn2+-containing cells already were evident in the LGNd of infant rats by 24 h post-lesion, the earliest time point examined with the histochemical technique. Interestingly, Repici et al. (2003) recently showed that caspase-3 activation in LGNd neurons of perinatal rats first can be detected 24 h after cortex lesion (though not earlier), with peak activation occurring at 2 days post-lesion, a finding we have confirmed in preliminary studies (Land & Aizenman, unpublished). There is therefore a close temporal relationship between accumulation of cytoplasmic Zn2+ and caspase activation in infant rats as there is in adults. The more rapid onset of these events in young animals may reflect a greater dependence of immature neurons on their synaptic targets (Jones & Lavell, 1986; see also Martin et al., 1998).
Degeneration of LGNd neurons following cortex ablation can be ameliorated by application of basic fibroblast growth factor, ciliary neurotrophic factor or conditioned medium from co-cultures of thalamic and cortical neurons to the region of the lesion cavity (Eagleson et al., 1990, 1992; Agarwala & Kalil, 1998b). The neuroprotective effect of such agents suggests that LGNd cell death results from loss of specific trophic factors synthesized by cortical target neurons. Our results comparing effects of cortex ablation vs. cortical kainate injections, which kills cortical neurons without damaging axons of LGNd cells, support this idea. Both procedures led to Zn2+ accumulation in LGNd neurons, but in kainate-injected hemispheres the occurrence of cytoplasmic Zn2+ was delayed an additional week, coinciding with the time when cortical target neurons had degenerated. These findings further indicate that, like ischemia (Tonder et al., 1990; Koh et al., 1996), neurotrauma (Suh et al., 2000) and excitotoxicity (Frederickson et al., 1989; Choi & Koh, 1998; Riba-Bosch & Perez-Clausell, 2004), target deprivation leads to accumulation of Zn2+ in apoptotic neurons. Our findings suggest, however, that this phenomenon occurs even in the absence of a presynaptic source of vesicular zinc.
The fact that Zn2+ accumulates in dying LGNd neurons in the apparent absence of vesicular Zn2+ release adds to a growing body of evidence that Zn2+ from non-synaptic sources is associated with neuronal cell death (Aizenman et al., 2000). For example, infusion of compounds that generate nitric oxide (NO) into cerebellar cortex, portions of which like the LGNd have very low levels of chelatable zinc, leads to intense Zn2+ staining in somata of Purkinje neurons (Frederickson et al., 2002). NO, and NO-derived species, are potent liberators of Zn2+ from intracellular stores like MT and other Zn2+-containing proteins (Aravindakumar et al., 1999; Pearce et al., 2000; Pal et al., 2004), and one possibility is that NO leads to increased cytoplasmic staining for Zn2+ by displacing it from protein binding sites. That non-vesicular Zn2+ is associated with neuronal cell death also is evident from studies of seizure-induced neuronal damage in mice with targeted disruption of the genes encoding the vesicular zinc transporter, ZnT-3, or MT. Thus, kainate-induced seizures in ZnT-3 null mice that lack synaptic vesicle zinc result in Zn2+ accumulation in degenerating hippocampal neurons, as it does in wild-type mice (Lee et al., 2000). In this case cytoplasmic Zn2+ presumably is derived from non-synaptic sources. Kainate treatment also leads to Zn2+-mediated neurodegeneration in thalamic nuclei that, as noted above, contain little or no synaptic zinc. Importantly, Zn2+-associated thalamic cell death is nearly abolished in mice lacking the MT gene, strongly suggesting that Zn2+ released from intracellular stores may be a critical contributor to the cell death process (Lee et al., 2003). It remains to be determined, however, whether the cytoplasmic Zn2+ observed in all pathophysiological, slowly developing conditions like target deprivation is derived from MT itself, or other sources.
Our results suggest that accumulation of intracellular Zn2+ in thalamic neurons deprived of their cortical targets may be an early and critical sign of the impending demise of these cells. Based on earlier work, the accumulation of Zn2+ in neurons destined to die may be a reflection of the liberation of this metal from intracellular binding proteins following an oxidative or nitrosative signal (Aizenman et al., 2000; Sensi et al., 2003; Bossy-Wetzel et al., 2004; Pal et al., 2004). As target and growth factor deprivation-induced apoptosis models have implicated oxidative stress as an important component of the cell death cascade (Estevez et al., 1998; Martin et al., 2003; Pandey et al., 2003), it is quite conceivable that a similar event has taken place in our LGNd model. What the sources of this oxidative signal are, or the mechanisms by which these signals are triggered in afflicted neurons are unclear at this time. Nonetheless, the newfound importance of non-synaptic Zn2+ sources as a trigger of neuronal cell death provides several novel targets in the ongoing, strategic battle against neurodegenerative processes in the brain (see also Frederickson et al., 2004b).
We thank Lorraine Shamalla-Hannah and Karen Hartnett for most excellent technical assistance. The contribution of Giovanni Di Pino was not insignificant. This work was supported by NIH grant NS43277.