MDMA decreases glutamic acid decarboxylase (GAD) 67-immunoreactive neurons in the hippocampus and increases seizure susceptibility: Role for glutamate

Abstract

3,4-Methylenedioxy-methamphetamine (MDMA) is a unique psychostimulant that continues to be a popular drug of abuse. It has been well documented that MDMA reduces markers of 5-HT axon terminals in rodents, as well as humans. A loss of parvalbumin-immunoreactive (IR) interneurons in the hippocampus following MDMA treatment has only been documented recently. In the present study, we tested the hypothesis that MDMA reduces glutamic acid decarboxylase (GAD) 67-IR, another biochemical marker of GABA neurons, in the hippocampus and that this reduction in GAD67-IR neurons and an accompanying increase in seizure susceptibility involve glutamate receptor activation. Repeated exposure to MDMA (3×10mg/kg, ip) resulted in a reduction of 37-58% of GAD67-IR cells in the dentate gyrus (DG), CA1, and CA3 regions, as well as an increased susceptibility to kainic acid-induced seizures, both of which persisted for at least 30days following MDMA treatment. Administration of the NMDA antagonist MK-801 or the glutamate transporter type 1 (GLT-1) inducer ceftriaxone prevented both the MDMA-induced loss of GAD67-IR neurons and the increased vulnerability to kainic acid-induced seizures. The MDMA-induced increase in the extracellular concentration of glutamate in the hippocampus was significantly diminished in rats treated with ceftriaxone, thereby implicating a glutamatergic mechanism in the neuroprotective effects of ceftriaxone. In summary, the present findings support a role for increased extracellular glutamate and NMDA receptor activation in the MDMA-induced loss of hippocampal GAD67-IR neurons and the subsequent increased susceptibility to evoked seizures.

Keywords: Excitotoxicity; GABA; Glutamate; MDMA.

Figure 1

MDMA selectively decreases DAB-labeled GAD67-IR in the rat hippocampus. A) Rats were treated with MDMA (1x or 3×10 mg/kg, ip) or vehicle 7 or 30 days prior to sacrifice. GAD67-IR neurons were counted in 6 right and left images of each brain region from 6 rats/treatment group. *Indicates p<0.05 compared to vehicle. B) Representative images of DAB-labeled GAD67 cells in the CA1, CA3, and DG in rats treated with vehicle or MDMA (3×10 mg/kg, ip).

Figure 2

Glutamate mediates MDMA-induced reductions in GAD67-IR. Animals received ceftriaxone (CEF) (200 mg/kg, ip, daily) for 7 days prior to MDMA, MK-801 (0.3mg/kg, sc, 30 min prior to each injection of MDMA) or vehicle prior to either MDMA (3 × 10 mg/kg, ip) or vehicle for a total of six treatment groups. Animals were sacrificed 7 days after treatment with MDMA, and GAD67-IR was assessed using a stereological technique in the CA1 (A), CA3 (B), and DG (C). GAD67-IR neurons were counted in 2 sections in each brain region from 6–8 rats/treatment group. *Indicates p<0.05 compared to the values for vehicle treated rats.

Figure 3

MDMA increases susceptibility to kainic acid-induced seizures. Rats were given kainic acid (8mg/kg, sc) 7 or 30 days following the administration of either vehicle or MDMA (3×10mg/kg, ip). A) The percentage of rats exhibiting stage 3–5 seizures is depicted. N=8–19 rats/group. B) Of the animals that seized in panel A (n=5–17 rats/group), latency to seizure was recorded as the time (min) at which the animal first exhibited a stage 3–5 behavior. *Indicates p<0.05 compared to the values for vehicle treated rats.

Figure 4

MK-801 and ceftriaxone prevent the MDMA-induced increase in seizure susceptibility. Rats were treated with A) MK-801 (0.3mg/kg, sc) or vehicle 30 min prior to each injection of either MDMA (3×10 mg/kg, ip) or vehicle. B) Ceftriaxone (CEF) (200mg/kg, i.p.) or vehicle was given daily for 7 days prior to either MDMA (3 × 10 mg/kg, ip) or vehicle. Kainic acid (8mg/kg, sc) was administered 7 days after MDMA treatment. The percentage of rats exhibiting stage 3–5 seizures is depicted. N=5–10 rats/group. *Indicates (p<0.05) compared to VEH/VEH. #Indicates (p<0.05) compared to VEH/MDMA.

Figure 5

MDMA-induced hyperthermia was maintained in MK-801- and Ceftriaxone- treated animals. Core body temperatures were recorded every 30 min beginning 1 hr prior and ending 7 hr after the first injection of MDMA. A) Rats were treated with either a single daily injection of ceftriaxone (CEF) (200mg/kg, i.p.) or vehicle for 7 days prior to either MDMA (3 × 10 mg/kg, ip) or vehicle. B) Rats were treated with either MK-801 (0.3mg/kg, sc) or vehicle 30 min prior to each injection of either MDMA (3×10 mg/kg, ip) or vehicle. The values for animals that received MDMA in addition to either CEF or MK-801 were not significantly different from the values for MDMA-treated rats (p>0.05). N=6–8 rats/group.

Figure 6

Ceftriaxone prevents the MDMA-induced increase in extracellular glutamate. Extracellular glutamate was determined in the hippocampus in rats treated daily with ceftriaxone (CEF), (200 mg/kg, ip,) or vehicle for 7 days prior to either MDMA (3 × 10 mg/kg, ip, as indicated by the arrows) or vehicle. N=7–11 rats/group. *Indicates values that differ significantly (p<0.05) from the corresponding average baseline values for vehicle treated animals.