Transient postnatal NMDA receptor blockade by phencyclidine (PCP), ketamine, or MK-801 induces developmental neuroapoptosis and adult behavioral deficits, which resemble abnormal human behaviors typically present in schizophrenia. This study tested the hypothesis that PCP-induced developmental apoptosis causes a specific deficit of GABAergic interneurons containing parvalbumin (PV), calretinin (CR), or calbindin (CB). Young adult (PND56) rats that were given a single dose of PCP (10 mg/kg) on PND7 exhibited no densitometric change of either CR or CB neurons in any brain region studied, but demonstrated a selective deficit of PV-containing neurons in the superficial layers (II-IV) of the primary somatosensory (S1), motor (M), and retrosplenial cortices, but not in the striatum (CPu) or hippocampus. Further, CR and CB neurons, which were expressed at the time of PCP administration, showed no colocalization with cellular markers of apoptosis (terminal dUTP nick-end labeling (TUNEL) of broken DNA or cleaved caspase-3), indicating that CR- and CB-containing neurons were protected from the toxic effect of PCP and survived into adulthood. This suggests that the deletion of PV neurons occurred during development, but cleaved caspase-3 showed no colocalization with BrdU, a specific marker of S-phase proliferation. These data suggest that the loss of PV-containing neurons was not due to an effect of PCP on proliferating neurons, but rather an effect on post-mitotic neurons. The developmental dependence and neuronal specificity of this effect of PCP provides further evidence that this model may be valuable in exploring the pathophysiology of schizophrenia.

This study determined the role of caspase-3 in phencyclidine (PCP)-induced neurodegeneration in postnatal rats. PCP administration to postnatal day 7 rats induced a dose-dependent increase in caspase-3 enzymatic activity in frontal cortex, striatum, and hippocampus. Enzymatic activation was present at 4 h, peaked between 6 and 12 h, and disappeared by 24 h. Further, cleaved caspase-3-immunoreactive neurons were detected as early as 2 h in the cortex, and were found throughout the brain, including, in addition, the thalamus and striatum. Within the cingulate, frontal, parietal, and retrosplenial cortices, immunoreactivity was specific for layers II-IV (especially layer II). Neurons positive for both silver staining and terminal deoxynucleotidyl transferase biotin-d-UTP nick-end labeling (TUNEL) were found in the same brain regions and subregions. Double labeling experiments confirmed that cleaved caspase-3 and TUNEL were coexpressed in many neurons in all brain regions and subregions studied. Temporal studies revealed that procaspase-3 cleavage preceded TUNEL staining by about 3 h, with many neurons being positive for both caspase-3 and TUNEL 9 h after PCP treatment. In organotypic corticostriatal slices, PCP caused a concentration-and time-dependent cleavage of procaspase-3 that was also colocalized with TUNEL staining in layers II-IV of the parietal cortex. Caspase-3 activation again preceded PCP-induced DNA damage assessed by TUNEL. PCP-induced neuronal death in vitro as measured by TUNEL staining was blocked 85% by Ac-AAVALLPAVLLALLAPDEVD-CHO, a cell-permeable selective caspase-3 inhibitor. These data demonstrate that caspase-3 activation plays a necessary role in the regionally selective neuronal death induced by PCP in the developing rat brain.

Oxidative stress may be involved in the cellular damage and tissue destruction as burn wounds continues to progress after abatement of the initial insult. Since iron and calcium ions play key roles in oxidative stress, this study tested whether topical application of Livionex formulation (LF) lotion, that contains disodium EDTA as a metal chelator and methyl sulfonyl methane (MSM) as a permeability enhancer, would prevent or reduce burn injury. Methods We used an established brass comb burn model with some modifications. Topical application of LF lotion was started 5 minutes post-burn, and repeated every 8 hours for 3 consecutive days. Rats were euthanized and skin harvested for histochemistry and immunohistochemistry. Formation of protein adducts of 4-hydroxynonenal (HNE), malonadialdehyde (MDA) and acrolein (ACR) and expression of aldehyde dehydrogenase (ALDH) isozymes, ALDH1 and ALDH2 were assessed. Results LF lotion-treated burn sites and interspaces showed mild morphological improvement compared to untreated burn sites. Furthermore, the lotion significantly decreased the immunostaining of lipid aldehyde-protein adducts including protein -HNE, -MDA and -ACR adducts, and restored the expression of aldehyde dehydrogenase isozymes in the unburned interspaces. Conclusion This data, for the first time, demonstrates that a topically applied EDTA-containing lotion protects burn injury progression with a concomitant decrease in the accumulation of reactive lipid aldehydes and protection of aldehyde dehydrogenase isozymes. Present studies are suggestive of therapeutic intervention of burn injury by this novel lotion.

Chemical modulation of the monoamine neurotransmitter systems involving dopamine (DA), serotonin (5-HT), and norepinephrine (NE) provides an important means to control certain neurological disorders such as depression, 1 anxiety, 2 alcoholism, 3 chronic pain, 4 eating disorders, 5 and obsessive compulsive disorders. 6 For example, a major pharmaceutical approach to the treatment of depression has come about through the development of agents that interfere with the primary mechanism of removal of 5-HT or NE from the synapse. 7 As the result, the selective 5-HT reuptake inhibitors (SSRIs) such as fluoxetine (Prozac) 8 and paroxetine (Paxil), 9 among others, have been developed for the treatment of depression and related psychological disorders. Despite these recent clinical developments, a detailed understanding of the structural factors that govern the potency and selectivity of ligands at the specific monoamine transporters is still evolving. During our efforts to discover ligands of possible use as medications, 10 we discovered a rather interesting aspect of the 5-HT transporter (SERT) structure activity relationships (SAR), namely that significant selectivity and potency can be achieved through the use of a bivalent ligand approach (Figure 1).The rationale for employing the bivalent ligand approach stems from the possibility that dimeric structures may be capable of bridging independent recognition sites on the transporters resulting in a thermodynamically more favorable binding interaction than the monovalent binding of two molecules. 11 As such proximal binding sites are likely to differ in their location for the three monoaminergic transporters the length of the linker connecting the two binding moieties could thus provide a means to finetune transporter selectivity profiles. Empirical support for such a possibility stems from the enhanced potency and selectivity reported for both bivalent narcotic antagonists containing the naltrexamine pharmacophore 12 and for serotonin-based bivalent 5-HT 1B/1D agonists. 13 We have recently reported on the chemistry and pharmacology of some 3,4-disubstituted piperidine-based ligands that show reasonable potency at the dopamine transporter (DAT). 14 On the basis of these and other studies 15 it had become apparent to us that 3,4-disubstituted piperidines are structurally related to drugs such as femoxetine and paroxetine that exhibit high potency and selectivity for the SERT. 16 One of these piperidines was therefore chosen as the starting monomer for the assembly of bivalent ligands that were postulated to exhibit potent and selective SERT activity.Briefly, piperidine-based ligands 4-16 were prepared in optically pure form from arecoline (1) 14 (Scheme 1) and were evaluated for their ability to inhibit high affinity uptake of DA, 5-HT, and NE using rat synaptosomal nerve endings. 17 The uptake data expressed as K i values and the selectivity profile (ratio of K i values) for these compounds are provided in Table 1. In general, all dimers (7-16) exhibit substantially ...

SummaryPhencyclidine is an N-methyl D-aspartate receptor (NMDAR) blocker that has been reported to induce neuronal apoptosis during development and schizophrenia-like behaviors in rats later in life. Brain derived neurotrophic factor (BDNF) has been shown to prevent neuronal death caused by NMDAR blockade, but the precise mechanism is unknown. This study examined the role of the phosphatidylinositol-3 kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) pathways in BDNF protection of PCP-induced apoptosis in corticostriatal organotypic cultures. It was observed that BDNF inhibited PCP-induced apoptosis in a concentration dependent fashion. BDNF effectively prevented PCP-induced inhibition of the ERK and PI-3K/Akt pathways and suppressed GSK-3β activation. Blockade of either PI-3K/Akt or ERK activation abolished BDNF protection. Western blot analysis revealed that the PI-3K inhibitor LY294002 prevented the stimulating effect of BDNF on the PI-3K/Akt pathway, but had no effect on the ERK pathway. Similarly, the ERK inhibitor PD98059 prevented the stimulating effect of BDNF on the ERK pathway, but not the PI-3K/Akt pathway. Co-application of LY294002 and PD98059 had no additional effect on BDNF-evoked activation of Akt or ERK. However, concurrent exposure to PD98059 and LY294002 caused much greater inhibition of BDNF-evoked phosphorylation of GSK-3β at serine 9 than did LY294002 alone. Finally, either BDNF or GSK-3β inhibition prevented PCP-induced suppression of cyclic-AMP response element binding protein (CREB) phosphorylation. These data demonstrate that the protective effect of BDNF against PCP-induced apoptosis is mediated by parallel activation of the PI-3K/Akt and ERK pathways, most likely involves inhibition of GSK-3β and activation of CREB. KeywordsPCP (phencyclidine); NMDAR (N-methyl-D-aspartate receptor); BDNF (brain-derived neurotrophic factor); PI-3K (phosphoinositide-3 kinase); ERK (extracellular signal-regulated kinase); GSK-3β (Glycogen Synthase Kinase 3 β);

Toxic gases and fumes have been shown to be injurious to the upper airways. Repair of this injury involves proliferation and migration of surviving nonciliated cells, followed by differentiation to a normal phenotype. Because recent results suggested that growth factors could improve the outcome of an airway injury, we undertook this study to determine the efficacy of these materials as an initial treatment to accelerate the healing process. In 24 anesthetized sheep, a portion of the trachea was exposed to smoke from smouldering cotton cooled to 37 degrees C. Twelve received aerosolized epidermal growth factor plus platelet derived growth factor, while twelve received placebo. At 10 days after injury, nonciliated and ciliated cells were totally absent in the injured trachea receiving the placebo. In animals receiving growth factors, nonciliated and ciliated cells, however, were present (56% and 31% of uninjured trachea, respectively). At 13 days after injury, nonciliated and ciliated cell counts in those receiving placebo were 67% and 33% of uninjured, respectively. In sheep receiving growth factors, tracheal nonciliated and ciliated cell counts had increased to 105% and 64% of uninjured trachea, respectively. We conclude that growth factors therapy after airway injury stimulates cell proliferation and differentiation, and this therapeutic intervention to accelerate the repair process in acute airway injury is an approach applicable to humans.

Acute and subchronic administration of N-methyl-D-aspartate antagonists to rats in the early postnatal period has been reported to produce widespread and selectively cortical neurotoxicity, respectively. To resolve this apparent discrepancy, we sought to clarify these data by determining the dose and temporal and regional characteristics of acute and subchronic phencyclidine (PCP)-induced neurotoxicity. Measurement of degenerating neurons with the cupric silver technique following a single dose of PCP on postnatal day (PN) 7 revealed that neurodegeneration increased in all areas measured (frontal, parietal and cingulate cortices, striatum, hippocampus, subiculum, and thalamus) within 9 hr. Silver staining peaked at 9-16 hr and was then not detectable or was greatly reduced after 24 hr depending on the specific region. Dose-response analysis at 9 hr showed that the lowest effective dose was 1, 3, and 10 mg/kg for the frontal cortex, hippocampus, and striatum, respectively. However, repeated PCP administration (10 mg/kg) on PN 7, 9, and 11 elicited an increase in silver staining only in the frontal cortex. To determine whether the loss of effect in the striatum and hippocampus was due to a "tolerance" mechanism or to a developmental phenomenon, we compared the effects of PCP given on PN 7, 9, or 11 with those of two doses given on PN 7 and 9 or three doses administered on PN 7, 9, and 11. Analysis of these experiments shows that both developmental factors and unknown mechanisms of tolerance underlie the apparent selective cortical neurotoxicity observed following subchronic PCP administration in perinatal rat pups.

A series of novel conformationally constrained tricyclic tropane derivatives containing a biaryl moiety, (Z)-9-(biarylylmethylene)-7-azatricyclo[4.3.1.0(3,7)]decanes, were synthesized and evaluated for their ability to inhibit reuptake of dopamine (DA), serotonin (5-HT), and norepinephrine (NE) by the DA, 5-HT, and NE transporters. Most of the compounds containing a methoxycarbonyl substituent at C-10 exhibit moderate to high inhibitory activity at the NET but lower activity at the DAT and SERT. Among these new compounds, some potent, NET-selective ligands were identified. The p-methoxy derivative 11a has a K(i) value of 39 nM for uptake inhibition at the NET and moderate to high selectivity over the SERT (100-fold) and the DAT (20-fold). Compound 11f exhibits a remarkable potency (K(i) = 9.7 nM) at the NET and a 25-fold selectivity over both the SERT and the DAT. Analogue 23 containing a thiophene ring as a bioisosteric replacement of the phenyl ring Ar(1) displays a high activity (K(i) = 10.3 nM) for the NET and similar selectivity over the SERT (50-fold) and the DAT (37-fold). The selectivity profile of biaryl analogues differs from that of the monoaryl series, as most members of that series display excellent potency at and selectivity for the SERT (J. Med. Chem. 2002, 45, 1930). This finding suggests that the different shape and size of the lipophilic recognition pocket that encompasses the aryl ring(s) of these tropanes are major determinants of a ligand's transporter activity at either the NET or the SERT. Some of the compounds in this series may also be valuable in sorting out the contribution of the individual transporters to cocaine's reinforcing properties.