Methamphetamine-induced cell death: selective vulnerability in neuronal subpopulations of the striatum in mice

Abstract

Methamphetamine (METH) is an illicit and potent psychostimulant, which acts as an indirect dopamine agonist. In the striatum, METH has been shown to cause long lasting neurotoxic damage to dopaminergic nerve terminals and recently, the degeneration and death of striatal cells. The present study was undertaken to identify the type of striatal neurons that undergo apoptosis after METH. Male mice received a single high dose of METH (30 mg/kg, i.p.) and were killed 24 h later. To demonstrate that METH induces apoptosis in neurons, we combined terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining with immunohistofluorescence for the neuronal marker neuron-specific nuclear protein (NeuN). Staining for TUNEL and NeuN was colocalized throughout the striatum. METH induces apoptosis in approximately 25% of striatal neurons. Cell counts of TUNEL-positive neurons in the dorsomedial, ventromedial, dorsolateral and ventrolateral quadrants of the striatum did not reveal anatomical preference. The type of striatal neuron undergoing cell death was determined by combining TUNEL with immunohistofluorescence for selective markers of striatal neurons: dopamine- and cAMP-regulated phosphoprotein, of apparent Mr 32,000, parvalbumin, choline acetyltransferase and somatostatin (SST). METH induces apoptosis in approximately 21% of dopamine- and cAMP-regulated phosphoprotein, of apparent Mr 32,000-positive neurons (projection neurons), 45% of GABA-parvalbumin-positive neurons in the dorsal striatum, and 29% of cholinergic neurons in the dorsal-medial striatum. In contrast, the SST-positive interneurons were refractory to METH-induced apoptosis. Finally, the amount of cell loss determined with Nissl staining correlated with the amount of TUNEL staining in the striatum of METH-treated animals. In conclusion, some of the striatal projection neurons and the GABA-parvalbumin and cholinergic interneurons were removed by apoptosis in the aftermath of METH. This imbalance in the populations of striatal neurons may lead to functional abnormalities in the output and processing of neural information in this part of the brain.

Fig. 1

Schematic of the striatum indicating the four regions selected for cell counts. DM, VM, DL, and VL. Black boxes indicate the 0.26 mm² area where cells were manually counted after immunostaining. Reproduced from Hof et al. (2000).

Fig. 2

Estimation of the number of NeuN-positive neurons in the four quadrants of the striatum. Coronal sections through the striatum (bregma 0.38±0.1 mm) were processed for immunohistofluorescence with Cy3-labeled antibodies against the NeuN. NeuN-positive neurons were counted from 20 μm thick coronal sections in an area of 0.26 mm² for each of the four quadrants of the caudate-putamen (CPu, DM, DL, VM, and VL). The mean number of NeuN-positive neurons was taken from an average of 30 sections from six control animals (five serials sections from each animal). No significant deviation in total cell counts was observed between animals or between regions of the striatum.

Fig. 3

Induction of cell death by METH as a function of dose. Mice were injected once with METH (i.p.) at the doses indicated. The mice were killed 24 h after METH and sections of striatal tissue were processed for TUNEL immunohistofluorescence. (A) Scanning confocal micrographs of TUNEL staining in the striatum of mice treated with increasing doses of METH (a–e). Scale bar=50 μm. (B) Percentage of striatal neurons displaying TUNEL staining for DM, VM, DL, and VL regions of the striatum (mean S.E.M.). * P<0.005, ** P<0.01, *** P<0.05 compared with saline control. (C) Scatter graphs indicate percentage of TUNEL-positive neurons for each animal within each treatment group. Each dot represents one animal. Blue=saline, red=METH 10 mg/kg, green=METH 20 mg/kg, purple=METH 30 mg/kg, yellow=METH 40 mg/kg; n=10–11 per experimental group.

Fig. 3

Induction of cell death by METH as a function of dose. Mice were injected once with METH (i.p.) at the doses indicated. The mice were killed 24 h after METH and sections of striatal tissue were processed for TUNEL immunohistofluorescence. (A) Scanning confocal micrographs of TUNEL staining in the striatum of mice treated with increasing doses of METH (a–e). Scale bar=50 μm. (B) Percentage of striatal neurons displaying TUNEL staining for DM, VM, DL, and VL regions of the striatum (mean S.E.M.). * P<0.005, ** P<0.01, *** P<0.05 compared with saline control. (C) Scatter graphs indicate percentage of TUNEL-positive neurons for each animal within each treatment group. Each dot represents one animal. Blue=saline, red=METH 10 mg/kg, green=METH 20 mg/kg, purple=METH 30 mg/kg, yellow=METH 40 mg/kg; n=10–11 per experimental group.

Fig. 4

METH induces apoptosis in some striatal neurons of the striatum. Double-labeled epifluorescent micrographs of striatal tissue stained with Cy3-labeled antibodies against NeuN and TUNEL with FITC-conjugated dUTPs in control (a–c) and METH-treated (30 mg/kg, i.p., killed 24 h post-treatment) animals (d–i). Bottom panels (c, f, i) are overlays of both TUNEL and NeuN staining. Higher magnification of METH-treated animals indicates that NeuN-positive neurons overlap with TUNEL-positive cells (g–i). White arrows point to overlapping TUNEL and NeuN positive cells. Scale bar=100 μm (a–f), 20 μm (g–i).

Fig. 5

The number of DARPP-32-containing projection neurons is decreased by METH. Mice received one injection of METH (i.p.) at 30 mg/kg of body weight and were killed 24 h later. Sections of striatal tissue were processed for immunohistofluorescence. (A) Double-labeled epifluorescent micrographs of striatal tissue stained with Cy3-labeled antibodies against DARPP-32 and TUNEL with FITC-conjugated dUTPs in control (a–c) and METH-treated animals (d–i). Bottom panels (c, f, I, l) are overlays of both TUNEL and DARPP-32 staining. Higher magnification of METH-treated animals indicates that the two chromophores do not overlap (g–i). Scale bar=100 μm (a–f), 20 μm (g–i). (B) Counts of DARPP-32-positive neurons (mean±S.E.M.) demonstrate a significant decrease in all four quadrants of the striatum after exposure to METH. * P<0.05 compared with corresponding regions of saline control. CPu, caudate-putamen.

Fig. 6

Immunohistofluorescence of parvalbumin interneurons is diminished by METH. Mice received a single injection of METH (i.p.) at a dose of 30 mg/kg and were killed 24 h after the treatment. Sections of striatal tissue were processed for immunohistofluorescence. (A) Double-labeled epifluorescent micrographs of striatal tissue stained with Cy3-labeled antibodies against parvalbumin and TUNEL with FITC-conjugated dUTPs in control (a–c) and METH-treated animals (d–f). Bottom panels (c, f, i, l) are overlays of both TUNEL and parvalbumin staining. Higher magnification of tissue from METH-treated animals demonstrated that the two chromophores do not overlap (g–i), although cell bodies showed significant loss of dendritic arborizations (j–l). Scale bar=100 μm (a–f), 20 μm (g–l). (B) Counts of parvalbumin-positive neurons (mean±S.E.M.) demonstrate a significant decrease of immunohistofluorescence in the dorsal regions of the striatum. * P<0.0001, ** P<0.005, *** P<0.05 compared with corresponding regions of saline control. ^! P<0.0001, ^!! P<0.001 compared with the DM region of saline. ^† P<0.005 compared with the DL region of saline. CPu, caudate-putamen.

Fig. 7

Some cholinergic interneurons are vulnerable to METH. Animals were injected once with METH (i.p., 30 mg/kg) and killed 24 h after METH. Coronal sections through the striatum were processed for immunohistofluorescence. (A) Double-labeled epifluorescent micrographs of striatal tissue stained with Cy3-labeled antibodies against ChAT and TUNEL with FITC-conjugated dUTPs in control (a–c) and METH-treated mice (d–i). Bottom panels (c, f, i) are overlays of both TUNEL and ChAT staining. Higher magnification of METH-treated group demonstrates the lack of colocalization between the two chromophores (g–i). Scale bar=100 μm (a–f), 20 μm (g–i). (B) Counts of ChAT-positive neurons (mean±S.E.M.) demonstrate a significant decrease in the DM region of the striatum. * P<0.005 compared with the corresponding regions of saline control. ^! P<0.0001, ^† P<0.005 compared with the DL region of saline. ^!! P<0.0005 compared with DM region of saline. ^# P<0.05 compared with the DM of METH. ^% P<0.05 compared with the DL region of METH. CPu, caudate-putamen.

Fig. 8

SST interneurons are refractory to METH-induced apoptosis. (A) Double-labeled epifluorescent micrographs of striatal tissue stained with antibody against SST (Cy3) and TUNEL with FITC-conjugated dUTPs in control (a–c) and METH-treated (30 mg/kg, i.p., killed 24 h post-treatment) mice (d–i). Bottom panels (c, f, i) are overlays of both TUNEL and SST staining. Higher magnification of METH-treated mice shows absence of overlap between the two chromophores (g–i). Scale bar=100 μm (a–f), 20 μm (g–i). (B) Counts of SST-positive neurons (mean±S.E.M.) demonstrate no statistical significance between the two treatment groups or between the regions within the treatment groups. CPu, caudate putamen.

Fig. 9

Effect of METH treatment on the cell density of the striatum. Mice were treated with a single METH 30 mg/kg (i.p.) injection and killed 24 h later. Coronal sections of the striatum were stained with Cresyl Violet. Light micrographs of Nissl staining in saline (A) and METH 30 mg/kg (B) treated animals. (C) Nissl-stained cells were counted in 20 μm thick coronal sections of the striatum in an area of 0.26 mm² for each of the four quadrants of the caudate-putamen (CPu, DM, DL, VM, and VL). Results represent the mean±S.E.M. number of cells counted in 10 animals per experimental group. * P<0.05 compared with controls of each corresponding striatal region. No significance was found between regions.