A substrate trapping mutant form of striatal-enriched protein tyrosine phosphatase prevents amphetamine-induced stereotypies and long-term potentiation in the striatum

Abstract

Background: Chronic, intermittent exposure to psychostimulant drugs results in striatal neuroadaptations leading to an increase in an array of behavioral responses on subsequent challenge days. A brain-specific striatal-enriched tyrosine phosphatase (STEP) regulates synaptic strengthening by dephosphorylating and inactivating several key synaptic proteins. This study tests the hypothesis that a substrate-trapping form of STEP will prevent the development of amphetamine-induced stereotypies.

Methods: A substrate-trapping STEP protein, TAT-STEP (C-S), was infused into the ventrolateral striatum on each of 5 consecutive exposure days and 1 hour before amphetamine injection. Animals were challenged to see whether sensitization to the stereotypy-producing effects of amphetamine developed. The same TAT-STEP (C-S) protein was used on acute striatal slices to determine the impact on long-term potentiation and depression.

Results: Infusion of TAT-STEP (C-S) blocks the increase of amphetamine-induced stereotypies when given during the 5-day period of sensitization. The TAT-STEP (C-S) has no effect if only infused on the challenge day. Treatment of acute striatal slices with TAT-STEP (C-S) blocks the induction of long-term potentiation and potentates long-term depression.

Conclusions: A substrate trapping form of STEP blocks the induction of amphetamine-induced neuroplasticity within the ventrolateral striatum and supports the hypothesis that STEP functions as a tonic break on synaptic strengthening.

Figure 1. Schematic design of experiments

(A) Six days after bilateral cannula implantation into ventrolateral striatum (VLS) or cortex, rats were infused with TAT-STEP (C-S) or TAT-myc one hour before an injection of amphetamine (5 mg/kg), and this was repeated for 5 consecutive days. The first drug challenge took place after 6 days of no injections and consisted of a single injection of amphetamine (2 mg/kg) (Day 12). In the second part of this experiment, rats were again injected with d-amphetamine (5 mg/kg) for 5 days, but without any infusion of TAT-STEP (C-S) or TAT-myc. Six days later, animals received a challenge dose of amphetamine (2 mg/kg). (B) Six days after bilateral cannula implantation, animals were injected with amphetamine (5 mg/kg) for 5 consecutive days. On day 12, rats were injected with amphetamine (2 mg/kg) 1 hour after being infused with TAT-STEP (C-S) or TAT-myc.

Figure 2. TAT-STEP (C-S) infusions into VLS

(A) Schematic diagram of TAT-STEP (C-S) indicating the positions of the TAT-peptide, myc and six histidine (His) tags, the KIM domain, and the C-S mutation site within the phosphatase domain. (B) Immunocytochemical staining was performed with anti-myc antibody at different time points (30 min, 60 min and 6 hours).

Figure 3. TAT-STEP (C-S) infusion into VLS blocks the development of amphetamine-induced stereotypies

(A) Challenge day 1 (behavioral score): rats were infused with TAT-STEP (C-S) (n=6) or TAT-myc (control; n=6) in VLS 1 hr before intraperitoneal injection of d-amphetamine (5 mg/kg) for 5 days. After 6 days of no injections, rats received a challenge dose of d-amphetamine (2 mg/kg). Rats infused with TAT-STEP (C-S) showed significant differences in the behavioral score. Rats infused into the cortex showed no significant differences (B) Up and down head movements were significantly different as well. Crossovers and rearings were not different. (A) and (B) Mean ± S.E. ^***p<0.0001, ^**p<0.005 (C) Histological verification of cannula tip placements in rats infused with either TAT-STEP (C-S) (triangles) or TAT-myc into VLS (circles). (D) Cannula tip placement analyses in rats infused with either TAT-STEP (C-S) (triangles) or TAT-myc (circles) into cortex.

Figure 4. TAT-STEP infusion did not cause permanent damage in VLS

(A) Rats that had had a previous infusion with the indicated compound were injected with amphetamine (5 mg/kg) for 5 days, but without infusion of TAT-STEP (C-S) or TAT-myc. There were now no significant differences between the two groups with the challenge dose of amphetamine (2 mg/kg). (B) There were no significant differences in the other behavioral measures.

Figure 5. TAT-STEP infusion only on the challenge day does not block amphetamine-induced stereotypies

(A) Animals received 5 days of d-amphetamine injections (5 mg/kg). Infusions occurred only on the challenge day; TAT-STEP (C-S) (n=6) or TAT-myc (control; n=6). No significant differences were found in behavioral scores between groups. (B) No significant differences were found for up and down head movements, crossover, and rearing scores. (C) Histological verification of cannula placements in rats infused with either TAT-STEP (C-S) (triangles) or TAT-myc (circles).

Figure 6. Effects of TAT-STEP on corticostriatal plasticity

(A) Preincubation of striatal slices with TAT-STEP (C-S) (300 nM) blocked HFS-induced LTP in striatal spiny neurons. LTP induction was unaffected by TAT-myc (300 nM). Traces on the right are examples of EPSPs recorded before (upper) and 10 minutes after HFS (lower) from neurons treated with TAT-myc and TAT-STEP (C-S). (B) LFS protocol fails to induce long-lasting changes of corticostriatal transmission in TAT-myc-bathed slices, while it causes LTD in slices preincubated with TAT-STEP (C-S). Traces on the right are examples of EPSPs recorded before and 10 minutes after the end of LFS protocol.