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, 74 (11), 817-26

Striatal-enriched Protein Tyrosine phosphatase-STEPs Toward Understanding Chronic Stress-Induced Activation of Corticotrophin Releasing Factor Neurons in the Rat Bed Nucleus of the Stria Terminalis

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Striatal-enriched Protein Tyrosine phosphatase-STEPs Toward Understanding Chronic Stress-Induced Activation of Corticotrophin Releasing Factor Neurons in the Rat Bed Nucleus of the Stria Terminalis

Joanna Dabrowska et al. Biol Psychiatry.

Abstract

Background: Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific protein tyrosine phosphatase that opposes the development of synaptic strengthening and the consolidation of fear memories. In contrast, stress facilitates fear memory formation, potentially by activating corticotrophin releasing factor (CRF) neurons in the anterolateral cell group of the bed nucleus of the stria terminalis (BNSTALG).

Methods: Here, using dual-immunofluorescence, single-cell reverse transcriptase polymerase chain reaction, quantitative reverse transcriptase polymerase chain reaction, Western blot, and whole-cell patch-clamp electrophysiology, we examined the expression and role of STEP in regulating synaptic plasticity in rat BNSTALG neurons and its modulation by stress.

Results: Striatal-enriched protein tyrosine phosphatase was selectively expressed in CRF neurons in the oval nucleus of the BNSTALG. Following repeated restraint stress (RRS), animals displayed a significant increase in anxiety-like behavior, which was associated with a downregulation of STEP messenger RNA and protein expression in the BNSTALG, as well as selectively enhancing the magnitude of long-term potentiation (LTP) induced in Type III, putative CRF neurons. To determine if the changes in STEP expression following RRS were mechanistically related to LTP facilitation, we examined the effects of intracellular application of STEP on the induction of LTP. STEP completely blocked the RRS-induced facilitation of LTP in BNSTALG neurons.

Conclusions: Hence, STEP acts to buffer CRF neurons against excessive activation, while downregulation of STEP after chronic stress may result in pathologic activation of CRF neurons in the BNSTALG and contribute to prolonged states of anxiety. Thus, targeted manipulations of STEP activity might represent a novel treatment strategy for stress-induced anxiety disorders.

Keywords: Anxiety; BNST; CRF; STEP; bed nucleus of the stria terminalis; chronic stress; corticotrophin releasing factor; striatal-enriched protein tyrosine phosphatase.

Conflict of interest statement

Conflict of interest statement

All authors report no biomedical financial interests or potential conflicts of interest.

Figures

Figure 1
Figure 1. STEP and CRF immunoreactivity co-localize in neurons of the CeAl, BNSTALG, but not PVN
A-A″ CRF (A, red, closed arrow) and STEP (A′, green, open arrow) show high somatodendritic immunoreactivity in the CeAl. Dual-label immunofluorescence revealed almost complete co-localization of STEP with CRF in neurons of the CeAl (A″, double arrow). B-B″ A similar pattern of co-localization was observed in neurons of the BNSTALG subdivision BNSTov, but not BNSTjxt. C-C″ STEP did not co-localize with CRF in neurons of the PVN. (S-Striatum, LV-Lateral ventricle, IC-Internal capsule, AC-Anterior commissure, BNSTALG-Anterolateral cell group of the BNST, BNSTov-Oval nucleus of the BNST, BNSTjxt-Juxtacapsular nucleus of the BNST, BNSTal-Anterolateral nucleus of the BNST, CeAl-Lateral division of the central nucleus of the amygdala, BLA-Basolateral nucleus of the amygdala, 3V-3rd ventricle, 10x, scale bar 100 μm).
Figure 2
Figure 2. In the BNSTov STEP immunoreactivity co-localizes with CRF but not NPY or ENK
A-A″ At higher magnification CRF-positive cells (A, closed arrows) and STEP-positive (A′, open arrows) showed almost complete co-localization in the BNSTov (A″, double arrows). However, there are occasional STEP-immunoreactive neurons which do not appear to co-localize CRF (A″, open arrows). B-B″ STEP-positive neurons (B′, green, open arrows) do not co-localize with NPY (B, red, closed arrows), or ENK (C-C″). ENK-positive processes are observed to make perisomatic contacts with STEP-expressing neurons (C″, double arrow). (NPY-Neuropeptide Y, ENK-Enkephalin, 63x, scale bar 10 μm).
Figure 3
Figure 3. STEP-positive neurons in the BNSTov co-localize several known STEP substrates
A-A″ All STEP-positive neurons (A, green, open arrows) co-localize GluN1 (A′, red, closed arrows) in the BNSTALG (A″, merged, double arrows) but the majority of GluN1-immunoreactive neurons do not co-localize STEP (A″, merged, closed arrows). B-B″ Virtually all STEP-positive neurons in the BNSTALG (B, open arrows) co-localize p38 (B′, red, closed arrows). However, numerous p38-positive neurons do not co-localize STEP (B″, closed arrows). C-C″ Nearly all STEP-positive neurons of the BNSTALG (green, open arrows, C) co-express ERK1/2 (C′, closed arrows). However, the highest ERK1/2 immunoreactivity was observed in BNST neurons that do not express STEP (C″, closed arrows). In addition, some STEP-positive cells do not express ERK 1/2 (C″, open arrows).
Figure 4
Figure 4. Repeat restraint stress (RRS) causes an increase in anxiety-like behavior and a concomitant decrease in STEP mRNA and protein expression in the BNSTALG
A: A plot of the acoustic startle response (ASR) before (pretest, day 0) and 10 days after RRS onset (posttest). On day 10 (six days after the termination of RRS), stressed rats exhibited a significant increase in ASR as compared to NS rats (F(59, 180)=1.94, p=0.0005, n=24, two-way ANOVA). There was also a significant effect of time on ASR (F(1, 180)=14.70, p=0.0002, n=24, two-way ANOVA), but there was no significant interaction between time and treatment (F(29, 180)=0.64, p=0.9748, two-way ANOVA). B: A histogram showing the effects of RRS on STEP mRNA expression levels in the BNSTALG. STEP mRNA was measured by real-time RT-PCR normalized to 18S RNA expression, thus we have analyzed mean fold change expressed as ΔΔCts between NS and RRS groups. Quantitative RT-PCR revealed that RRS caused a significant (6-fold) decrease in STEP mRNA levels compared to non-stressed rats (p<0.05, n=4). C: A histogram showing the effect of RRS of STEP protein levels in the BNSTALG. Western blot analysis showed that RRS significantly reduced expression of the cytosolic 46 kDa STEP isoform, but not the 61 kDa membrane-bound STEP isoform (p<0.005, n=4).
Figure 5
Figure 5. A . RRS significantly down-regulates STEP protein content in the BNSTALG. B–C. RRS up-regulates protein content of the NMDAR subunits in the synaptic membranes of the BNSTALG neurons
Representative Western blot showing that RRS significantly reduced expression of the cytosolic STEP46 isoform (p<0.005, n=4, A). RRS caused a significant up-regulation in protein expression of the GluN1 subunit of the NMDAR in synaptic membranes fraction of the BNSTALG neurons on day 10 (n=8, p<0.05, B), and non-significant increase in GluN2B subunit of the NMDAR (n=8, p=0.08, C).
Figure 6
Figure 6. RRS significantly facilitates long-term potentiation (LTP) in Type III but not Type I or II neurons, and this effect could be blocked by exogenous STEP application
A: Effect of high frequency stimulation of the stria terminalis (HFS, five 100 Hz, 1s trains, interval 20s) on EPSC amplitude in Type I BNSTALG neurons. EPSC amplitude was normalized to the mean amplitude of the 15 min baseline eEPSC and expressed as the percentage of baseline. In Type I neurons RRS had no effect on the magnitude of LTP level at any time point (F (39, 520) = 0.19, p=1.00, Two-way ANOVA). B: RRS caused no enduring change in LTP magnitude at any time point in Type II neurons (F (39, 520) = 0.31, p=1.00, Two-way ANOVA) in comparison to NS group. C: In Type III neurons, RRS significantly increased the magnitude of LTP (F (1, 440) = 93.88, p<0.001, Two-way ANOVA). D: Administration of exogenous wild-type STEP protein (300 nM) in the patch-recording pipette completely blocked the RRS-induced facilitation of LTP (p<0.05). Application of the wild-type STEP protein had no effect on the magnitude of LTP induced in non-stressed rats.
Figure 7
Figure 7
A schematic showing the proposed signaling cascade by which activation of STEP could regulate the development of synaptic plasticity in CRF neurons of the BNSTALG. Following NMDA receptor-dependent activation, STEP dephosphorylates tyrosine residues in the activation domains of 1) NMDA receptors, 2) AMPA receptors, 3) p-38, 4) ERK1/2, and 5) Fyn kinase. Dephosphorylation of these key STEP substrates would put a significant temporal constraint on the induction of synaptic plasticity in these neurons. Green arrows = activation. Red lines = inhibition.

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