Prenatal cocaine exposure reduced NMDA/Glycine and K⁺-depolarization induced BDNF and proBDNF release in hippocampi (a) and PFCX (b).

<p>Hippocampal and PFCX slices prepared from P21 prenatal cocaine- and saline-treated rats were used to determine spontaneous BDNF/proBDNF efflux as well as BDNF and proBDNF release induced by 10-min 10 μM NMDA/1 μM glycine in LMKR or 1-min 65 mM K⁺-depolarization in a superfusion system. BDNF and proBDNF in the perfusate were then immunoprecipitated with immobilized anti- BDNF and determined by Western blotting with anti-BDNF. The brain slices were collected, homogenized and solubilized and the level of β-actin in the brain slices was determined by Western blotting to illustrate equal quantities of tissues. The blots were quantified by densitometric scanning. Data are expressed as means ± s.e.m. of the ratios of BDNF or proBDNF optical intensity to the optical intensity of β-actin. n = 6. **p < 0.05, *p < 0.01 compared to LMKR-treated in the same group. ##p < 0.05, #p < 0.01 compared to respective protein in the saline-treated group.</p

Prenatal cocaine exposure did not alter the expression levels of full-length (145-KDa) and truncated (95-KDa) TrkB, p75^NTR, pro-BDNF and BDNF, Akt1 and ERK2, N-Shc and Shc, as well as NR1 and PLC-γ1 in both hippocampus and prefrontal cortex.

<p>The expression levels of 145- and 95-KDa TrkB (a). P75NTR (b), pro-BDNF and BDNF (c) as well as Akt1 and ERK2(d), Shc and N-Shc (e), NR1 and PLC-γ1(f), in 50 μg post-mitochondrial synaptosome-enriched fractions prepared from hippocampi and PFCX of P21 rats exposed to saline or cocaine <i>in utero</i> were compared by Western blotting. The blots were stripped and re-probed with anti-β-actin to validate equal loading. Densitometric quantification of blots revealed no discernible differences in 145- and 95-KDa TrkB, P75NTR, proBDNF and BDNF, Akt1, ERK2, Shc and N-Shc, NR1 and PLC-γ1 expression levels. n = 4. Data are mean ± s.e.m. of the ratio of 145-, 95-KDa TrkB, p75^NTR, proBDNF, BDNF, Akt1, ERK2, N-Shc, Shc, NR1 or PLC-γ1to β-actins optical intensities.</p

Prenatal cocaine exposure reduced proBDNF release in hippocampi (top) and PFCX (bottom).

<p>Brain slices treated with 10 μM of MMP-9 inhibitor I and tPA inhibitor were used to assess proBDNF released spontaneously and induced by 10-min 10 μM NMDA/1 μM glycine or 1-min 65 mM K⁺-depolarization. ProBDNF in the perfusate were then immunoprecipitated with immobilized anti-BDNF and the level of proBDNF was determined by Western blotting with specific anti-proBDNF. The brain slices were collected, homogenized, and solubilized and the level of β-actin in the brain slices was determined by Western blotting. The blots were quantified by densitometric scanning. Data are means ± s.e.m. of the ratios of proBDNF optical intensity to the optical intensity of β-actin that serves to verify equal amounts of tissues. n = 5 (3 males and 2 females). **p < 0.05, *p < 0.01 compared to LMKR-treated in the same group. ##p < 0.05, #p < 0.01 compared to respective protein in the saline-treated group.</p

Prenatal cocaine exposure did not alter the expression level of tPA in both hippocampi (a) and PFCX (b).

<p>The expression level of tPA in 50 μg post-mitochondrial (P2) fractions prepared from 10 μM NMDA/1 μM glycine or 65 mM K⁺ superfused hippocampal and PFCX slices of P21 prenatal cocaine- and saline-exposed rats were compared by Western blotting. Brain slices (100 μm x 100 μm x 3 mm) were superfused with 0.2 ml/min LMKR for 30 min or with 10 μM NMDA/1 μM glycine in LMKR for 10 min or 65 mM K+-depolarization for 1 min followed by 9 min with LMKR. Slices were collected, homogenized and centrifuged to obtain post-mitochondrial fractions. The blots were stripped and re-probed with anti-β-actin to validate equal loading. Densitometric quantification of blots revealed no discernible differences in tPA expression level. n = 4 (2 males and 2 females). Data are mean ± s.e.m. of the ratio of tPA to β-actin optical intensities.</p

Prenatal cocaine exposure increased BDNF binding affinity for TrkB.

<p>Prenatal cocaine exposure increased BDNF binding affinity for TrkB by 18- and 15- fold (dotted lines) in biotinylated hippocampal and PFCX synaptic membranes, respectively from P21 prenatal cocaine- and saline-treated rats. In the control membranes, there were two BDNF binding sites, a high-affinity site (KD1) and a low-affinity site (KD2) (solid lines). Prenatal cocaine exposure also increased the low-affinity binding of BDNF by 82- and 12-fold. Nonlinear regression data curve fit was performed using Prism. Data points are the means and vertical bars are the s.e.m. derived from 6 independent rats (3 males and 3 females) in each treatment group.</p

Prenatal cocaine exposure altered 145-KDa TrkB conformation in hippocampi (top) and PFCX (bottom).

<p>The conformational states of the immunopurified 145-KDa TrkB was analyzed by separating on pH3-10 isoelectric focusing gels and then Western blotted with anti-TrkB. Blots were quantified by densitometric scanning. Data are means ± s.e.m. of the pI 6.1 and pI 6.9. n = 5 (3 males and 2 females). p < 0.01 compared to respective protein in the saline-treated group.</p

The concentration-response relationships of BDNF-induced TrkB and p75^NTR activation.

<p>Concentration-response relationships of BDNF-induced TrkB and p75^NTR activation indicate that p75^NTR is far less sensitive to BDNF than TrkB. (a) The magnitudes of TrkB and p75NTR activation induced by a 30-min incubation with 50–200 ng/ml BDNF were assessed in hippocampal and PFCX slices prepared from naïve P21 rats using pY TrkB and TRAF2/6 recruitment to p75^NTR, respectively, as the guides. (b) Densitometric quantification of pY 145- and 95-KDa TrkB and TRAF2/6 blots revealed that while TrkB was activated by 50–200 ng/ml of BDNF in a dose-dependent manner, p75NTR is activated only by 200 ng/ml of BDNF in both hippocampus and PFCX. n = 4. Data are mean ± s.e.m. of the ratios of pY to total 145-, 95-KDa TrkB or TRAF2 and TRAF6 to p75^NTR optical intensities. *p < 0.01 compared to the basal level of respective protein.</p

Prenatal cocaine exposure heightened TrkB-NMDAR interaction in hippocampi and PFCX.

<p>Prenatal cocaine exposure heightened TrkB-NMDAR interaction in hippocampi and PFCX as indicated by the Western blot detection of increased level of obligatory subunit of the NMDARs, NR1 in the anti-TrkB immunoprecipitates (top). Western blots were analyzed by densitometric quantification. The data are expressed as the ratios of NR1 optical intensity normalized by the optical intensity of total 145-KDa TrkB. n = 6 (3 males and 3 females). Data are means ± s.e.m. of the ratios of NR1 to 145-KDa TrkB optical intensities. *p < 0.01, compared to respective protein in the saline-treated group.</p

<i>Ex vivo</i> exposure to 50–200 ng/ml BDNF did not alter the expression of full-length (145-KDa) and truncated (95-KDa) TrkB and p75NTR in BDNF-incubated hippocampal and prefrontal cortical slices.

<p>The abundance of 145- and 95-KDa TrkB (a, b) as well as p75NTR (c, d) in 50 μg post-mitochondrial synaptosome-enriched fractions prepared from hippocampal and PFCX slices of P21 prenatal cocaine- and saline-exposed rats following 30-min incubation with 50–200 ng/ml BDNF were compared by Western blotting. The blots were stripped and re-probed with anti-β-actin to validate equal loading. Densitometric quantification of blots revealed no discernible differences in 145- and 95-KDa TrkB as well as p75NTR expression levels as a result of incubation with BDNF. n = 4. Data are mean ± s.e.m. of the ratio of 145-, 95-KDa TrkB, p75NTR to β-actin optical intensities.</p

Prenatal cocaine exposure increased 50 ng/ml BDNF-induced TrkB activation in hippocampi and PFCX.

<p>(a) Representative blots showing prenatal cocaine exposure increased 50 ng/ml BDNF-induced TrkB activation in hippocampi and PFCX evidenced by higher activated (tyrosine-phosphorylated [pY]) TrkB. (b) Summary of the densitometric quantification of the pY and total 145- and 95-KDa TrkB. The data are expressed as the ratios of pY- full-length (145-KDa) and truncated TrkB (95-KDa) optical intensity normalized by the optical intensity of total 145- and 95-KDaTrkB, respectively. n = 6 (3 females and 3 males). Data are reported as means ± s.e.m. of the ratio of pY-TrkB to TrkB optical intensities. *p < 0.01, **p < 0.05 compared to respective protein in the saline-treated group.</p