Restoring pars intermedia dopamine concentrations and tyrosine hydroxylase expression levels with pergolide: evidence from horses with pituitary pars intermedia dysfunction.

BACKGROUND: Pituitary pars intermedia dysfunction (PPID) develops slowly in aged horses as degeneration of hypothalamic dopaminergic neurons leads to proliferation of pars intermedia (PI) melanotropes through hyperplasia and adenoma formation. Dopamine (DA) concentrations and tyrosine hydroxylase (TH) immunoreactivity are markedly reduced in PI tissue of PPID-affected equids and treatment with the DA receptor agonist pergolide results in notable clinical improvement. Thus, we hypothesized that pergolide treatment of PPID-affected horses would result in greater DA and TH levels in PI tissue collected from PPID-affected horses versus untreated PPID-affected horses. To test this hypothesis, pituitary glands were removed from 18 horses: four untreated PPID-affected horses, four aged and four young horses without signs of PPID, and six PPID-affected horses that had been treated with pergolide at 2 µg/kg orally once daily for 6 months. DA concentrations and TH expression levels in PI tissues were determined by high performance liquid chromatography with electrochemical detection and Western blot analyses, respectively. RESULTS: DA and TH levels were lowest in PI collected from untreated PPID-affected horses while levels in the pergolide treated horses were similar to those of aged horses without signs of PPID. CONCLUSIONS: These findings provide evidence of restoration of DA and TH levels following treatment with pergolide. Equine PPID is a potential animal model of dopaminergic neurodegeneration, which could provide insight into human neurodegenerative diseases.

Metabolism of Dopamine in Nucleus Accumbens Astrocytes Is Preserved in Aged Mice Exposed to MPTP.

Parkinson disease (PD) is prevalent in elderly individuals and is characterized by selective degeneration of nigrostriatal dopamine (NSDA) neurons. Interestingly, not all dopamine (DA) neurons are affected equally by PD and aging, particularly mesolimbic (ML) DA neurons. Here, effects of aging were examined on presynaptic DA synthesis, reuptake, metabolism and neurotoxicant susceptibility of NSDA and mesolimbic dopamine (MLDA) neurons and astrocyte DA metabolism. There were no differences in phenotypic markers of DA synthesis, reuptake or metabolism in NSDA or MLDA neurons in aged mice, but MLDA neurons displayed lower DA stores. Astrocyte metabolism of DA to 3-methoxytyramine (3-MT) in the striatum was decreased in aged mice, but was maintained in the nucleus accumbens. Despite diminished DA vesicular storage capacity in MLDA neurons, susceptibility to acute neurotoxicant exposure was similar in young and aged mice. These results reveal an age- and neurotoxicant-induced impairment of DA metabolic activity in astrocytes surrounding susceptible NSDA neurons as opposed to maintenance of DA metabolism in astrocytes surrounding resistant MLDA neurons, and suggest a possible therapeutic target for PD.

FosB and ΔFosB expression in brain regions containing differentially susceptible dopamine neurons following acute neurotoxicant exposure.

Parkinson disease (PD) is characterized by progressive neuronal degeneration, in particular nigrostriatal dopamine (NSDA) neurons and consequent deficits in movement. In mice and non-human primates, NSDA neurons preferentially degenerate following exposure to the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Tuberoinfundibular (TI) DA neurons, in contrast, appear to be unaffected in PD and recover following acute MPTP exposure-induced injury (Behrouz et al., 2007; Benskey et al., 2012). The recovery of the TIDA neurons is dependent on de novo protein synthesis and positively correlated with an increase in parkin mRNA and protein expression (Benskey et al., 2012, 2015). Inhibition of parkin upregulation renders TIDA neurons susceptible to degeneration following MPTP exposure. In addition to parkin, other potentially protective proteins are likely to be differentially regulated in TIDA and NSDA neurons following neurotoxicant exposure. The regulation of potential transcription factors for parkin and other neuroprotective pathway genes are of interest since they may provide novel targets for PD disease modifying therapies. As such, we sought to determine if there are time-dependent differences in the expression of AP-1 transcription factors c-Fos, c-Jun, FosB, ΔFosB and JunD in TIDA and NSDA neurons of mice following acute MPTP exposure. We observed that both FosB and ΔFosB expression increase in brain regions containing TIDA, but not NSDA neurons. Furthermore, the nuclear and long-term expression of ΔFosB is consistent with its role as a transcription factor that may influence parkin transcription, which may underlie the unique ability of TIDA neurons to recovery from an injury that leads NSDA neurons to degeneration.

Reduced Noradrenergic Signaling in the Spleen Capsule in the Absence of CB1 and CB2 Cannabinoid Receptors.

The spleen is a visceral organ that contracts during hypoxia to expel erythrocytes and immune cells into the circulation. Spleen contraction is under the control of noradrenergic sympathetic innervation. The activity of noradrenergic neurons terminating in the spleen capsule is regulated by α2-adrenergic receptors (AR). Interactions between endogenous cannabinoid signaling and noradrenergic signaling in other organ systems suggest endocannabinoids might also regulate spleen contraction. Spleens from mice congenitally lacking both CB1 and CB2 cannabinoid receptors (Cnr1 -/- /Cnr2 -/- mice) were used to explore the role of endocannabinoids in spleen contraction. Spleen contraction in response to exogenous norepinephrine (NE) was found to be significantly lower in Cnr1 -/- /Cnr2 -/- mouse spleens, likely due to decreased expression of capsular α1AR. The majority of splenic Cnr1 mRNA expression is by cells of the spleen capsule, suggestive of post-synaptic CB1 receptor signaling. Thus, these studies demonstrate a role for CB1 and/or CB2 in noradrenergic splenic contraction.

The effects of gestational and chronic atrazine exposure on motor behaviors and striatal dopamine in male Sprague-Dawley rats.

This study sought to investigate the effects of environmentally relevant gestational followed by continued chronic exposure to the herbicide, atrazine, on motor function, cognition, and neurochemical indices of nigrostriatal dopamine (DA) activity in male rats. Dams were treated with 100 µg/kg atrazine, 10mg/kg atrazine, or vehicle on gestational day 1 through postnatal day 21. Upon weaning, male offspring continued daily vehicle or atrazine gavage treatments for an additional six months. Subjects were tested in a series of behavioral assays, and 24h after the last treatment, tissue samples from the striatum were analyzed for DA and 3,4-dihydroxyphenylacetic acid (DOPAC). At 10mg/kg, this herbicide was found to produce modest disruptions in motor functioning, and at both dose levels it significantly lowered striatal DA and DOPAC concentrations. These results suggest that exposures to atrazine have the potential to disrupt nigrostriatal DA neurons and behaviors associated with motor functioning.

Comparison of the structure, function and autophagic maintenance of mitochondria in nigrostriatal and tuberoinfundibular dopamine neurons.

A pathological hallmark of Parkinson׳s disease (PD) is progressive degeneration of nigrostriatal dopamine (NSDA) neurons, which underlies the motor symptoms of PD. While there is severe loss of midbrain NSDA neurons, tuberoinfundibular (TI) DA neurons in the mediobasal hypothalamus (MBH) remain intact. In the present study, confocal microscopic analysis revealed that mitochondrial content and numbers of mitophagosomes were lower in NSDA neuronal cell bodies in the substantia nigra pars compacta (SNpc) compared to TIDA neuronal cell bodies in the arcuate nucleus (ARC) of C57BL/6J male mice. Mitochondrial respiration, mass, membrane potential and morphology were determined using bioenergetic, flow cytometric and transmission electron microscopic analyses of synaptosomes isolated from discrete brain regions containing axon terminals of NSDA and TIDA neurons. Maximum and spare respiratory capacities, and mitochondrial mass were lower in synaptosomal mitochondria derived from the striatum (ST) as compared with the MBH, which correlated with lower numbers of mitochondria per synaptosome in these brain regions. In contrast, there was no regional difference in mitochondrial basal, maximum or spare respirations following inhibition of Complex I activity with rotenone. These results reveal that higher numbers of viable mitochondria are correlated with more extensive autophagic mitochondrial quality maintenance in TIDA neurons as compared with NSDA neurons.

Methylmercury impairs canonical dopamine metabolism in rat undifferentiated pheochromocytoma (PC12) cells by indirect inhibition of aldehyde dehydrogenase.

The environmental neurotoxicant methylmercury (MeHg) disrupts dopamine (DA) neurochemical homeostasis by stimulating DA synthesis and release. Evidence also suggests that DA metabolism is independently impaired. The present investigation was designed to characterize the DA metabolomic profile induced by MeHg, and examine potential mechanisms by which MeHg inhibits the DA metabolic enzyme aldehyde dehydrogenase (ALDH) in rat undifferentiated PC12 cells. MeHg decreases the intracellular concentration of 3,4-dihydroxyphenylacetic acid (DOPAC). This is associated with a concomitant increase in intracellular concentrations of the intermediate metabolite 3,4-dihydroxyphenylaldehyde (DOPAL) and the reduced metabolic product 3,4-dihydroxyethanol. This metabolomic profile is consistent with inhibition of ALDH, which catalyzes oxidation of DOPAL to DOPAC. MeHg does not directly impair ALDH enzymatic activity, however MeHg depletes cytosolic levels of the ALDH cofactor NAD(+), which could contribute to impaired ALDH activity following exposure to MeHg. The observation that MeHg shunts DA metabolism along an alternative metabolic pathway and leads to the accumulation of DOPAL, a reactive species associated with protein and DNA damage, as well as cell death, is of significant consequence. As a specific metabolite of DA, the observed accumulation of DOPAL provides evidence for a specific mechanism by which DA neurons may be selectively vulnerable to MeHg.

The role of parkin in the differential susceptibility of tuberoinfundibular and nigrostriatal dopamine neurons to acute toxicant exposure.

Parkinson disease causes degeneration of nigrostriatal dopamine (DA) neurons, while tuberoinfundibular DA neurons remain unaffected. A similar pattern is observed following exposure to 1-methy-4-phenyl-1,2,3,6-tetrahydropyradine (MPTP). The mechanism of tuberoinfundibular neuronal recovery from MPTP is associated with up-regulation of parkin protein. Here we tested if parkin mediates tuberoinfundibular neuronal recovery from MPTP by knocking-down parkin in tuberoinfundibular neurons using recombinant adeno-associated virus (rAAV), expressing a short hairpin RNA (shRNA) directed toward parkin. Following knockdown, axon terminal DA and tyrosine hydroxylase (TH) concentrations were analyzed 24h post-MPTP administration. rAAV-shRNA-mediated knockdown of endogenous parkin rendered tuberoinfundibular neurons susceptible to MPTP induced terminal DA loss, but not TH loss, within 24h post-MPTP. To determine if the neuroprotective benefits of parkin up-regulation could be translated to nigrostriatal neurons, rAAV expressing human parkin was injected into the substantia nigra of mice and axon terminal DA and TH concentrations were analyzed 24h post-MPTP. Nigral parkin over-expression prevented loss of TH in the axon terminals and soma of nigrostriatal neurons, but had no effect on terminal DA loss within 24h post-MPTP. These data show that parkin is necessary for the recovery of terminal DA concentrations within tuberoinfundibular neurons following acute MPTP administration, and parkin can rescue MPTP-induced decreases in TH within nigrostriatal neurons.

Enhanced humoral immunity in mice lacking CB1 and CB2 receptors (Cnr1-/-/Cnr2-/- mice) is not due to increased splenic noradrenergic neuronal activity.

Peripheral sympathetic noradrenergic neurons originating in the celiac mesenteric plexus have axons that terminate in close proximity to antibody-producing B cells in the spleen. Norepinephrine (NE) released from these neurons is reported to augment antibody production in response to an immune challenge via an action at the ß2-adrenergic receptor (ß2AR). Cannabinoids are immunosuppressive, and mice lacking CB1 and CB2 receptors (Cnr1(-/-)/Cnr2(-/-) mice) have augmented cell-mediated immune responses. The purpose of this study was to determine if Cnr1(-/-)/Cnr2(-/-) mice also exhibit enhanced humoral immunity and if that is associated with corresponding changes in noradrenergic neurons terminating in the spleen. The results reveal that IgM and IgG are enhanced in Cnr1(-/-)/Cnr2(-/-) mice as compared to WT both in immunologically naïve and lipopolysaccharide (LPS)-treated mice. While the elevated antibody production was correlated with increased expression of ß2AR on splenic B cells and increased splenic capsule NE concentrations, the activity of noradrenergic neurons was suppressed in spleens from Cnr1(-/-)/Cnr2(-/-) mice as compared with WT controls. Together, these results suggest that Cnr1(-/-)/Cnr2(-/-) mice exhibit enhanced NE vesicular storage in axon terminals in these neurons, which might limit the NE available to bind ß2AR on target cells, such as B cells. The results also demonstrate that enhanced antibody responses in the absence of CB1 and CB2 receptors are not due to increased sympathetic noradrenergic neuronal activity in the spleen.

Sustained resistance to acute MPTP toxicity by hypothalamic dopamine neurons following chronic neurotoxicant exposure is associated with sustained up-regulation of parkin protein.

Hypothalamic tuberoinfundibular dopamine (TIDA) neurons remain unaffected in Parkinson disease (PD) while there is significant degeneration of midbrain nigrostriatal dopamine (NSDA) neurons. A similar pattern of susceptibility is observed following acute exposure to the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and the resistance of TIDA neurons to MPTP is associated with increased expression of parkin and ubiquitin carboxy-terminal hydrolase L-1 (UCHL-1). In the present study, the response of TIDA and NSDA neurons to acute MPTP administration following chronic MPTP exposure was examined. Mice were treated with ten injections of either MPTP (20mg/kg; s.c.; every 3.5 days) or saline vehicle (10 ml/kg; s.c.; every 3.5 days). Following a 21 day recovery period, chronic saline- and MPTP-treated mice received an additional injection of either saline (10 ml/kg; s.c.) or MPTP (20mg/kg; s.c.) and were sacrificed 24h later. NSDA neurons displayed significant axon terminal degeneration (as reflected by decreases in DA, tyrosine hydroxylase (TH) and DA transporter concentrations in the striatum) as well as loss of TH-immunoreactive (IR) neurons in the substantia nigra (SN) following MPTP, whereas TIDA neurons revealed no overt axon terminal pathology or loss of TH-IR cell bodies. NSDA neuronal pathology was associated with transient decreases in concentrations of parkin and UCHL-1 protein in the SN, which returned to normal levels by 21 days following cessation of chronic neurotoxicant exposure. Resistance of TIDA neurons to MPTP toxicity was correlated with a transient increase in UCHL-1 and a sustained elevation in parkin in the arcuate nucleus. TIDA neurons represent a DA neuron population with a unique and inherent ability to adapt to acute and chronic toxicant administration with a sustained elevation of the neuroprotective protein parkin. The correlation between the ability to increase parkin and UCHL-1 expression and the resistance of DA neurons to neurotoxicant exposure is consistent with a functional link between these features and an underlying differential susceptibility to toxicant-associated neurodegeneration.

The role of de novo catecholamine synthesis in mediating methylmercury-induced vesicular dopamine release from rat pheochromocytoma (PC12) cells.

The purpose of this study was to characterize methylmercury (MeHg)-induced dopamine (DA) release from undifferentiated pheochromocytoma (PC12) cells and to examine the potential role for DA synthesis in this process. MeHg caused a significant increase in DA release that was both concentration- and time-dependent. DA release was significantly increased by 2µM MeHg at 60min and by 5µM MeHg at 30min; 1µM MeHg was without effect. Because DA release induced by 5µM MeHg was associated with a significant percentage of cell death at 60 and 120min, 2µM MeHg was chosen for further characterization of release mechanisms. MeHg-induced DA release was attenuated but not abolished in the absence of extracellular calcium, whereas the vesicular content depleting drug reserpine (50nM) abolished release. Thus, MeHg-induced DA release requires vesicular exocytosis but not extracellular calcium. MeHg also increased intracellular DA and the rate of DA storage utilization, suggesting a role for DA synthesis in MeHg-induced DA release. The tyrosine hydroxylase inhibitor α-methyltyrosine (300µM, 24h) completely abolished MeHg-induced DA release. MeHg significantly increased DA precursor accumulation in cells treated with 3-hydroxybenzylhydrazine (10µM), revealing that MeHg increases tyrosine hydroxylase activity. Overall, these data demonstrate that MeHg facilitates DA synthesis, increases intracellular DA, and augments vesicular exocytosis.

Comparison of the D2 receptor regulation and neurotoxicant susceptibility of nigrostriatal dopamine neurons in wild-type and CB1/CB2 receptor knockout mice.

Motor dysfunctions of Parkinson Disease (PD) are due to the progressive loss of midbrain nigrostriatal dopamine (NSDA) neurons. Evidence suggests a role for cannabinoid receptors in the neurodegeneration of these neurons following neurotoxicant-induced injury. This work evaluates NSDA neurons in CB1/CB2 knockout (KO) mice and tests the hypothesis that CB1/CB2 KO mice are more susceptible to neurotoxicant exposure. NSDA neuronal indices were assessed using unbiased stereological cell counting, high pressure liquid chromatography coupled with electrochemical detection or mass spectrometry, and Western blot. Results reveal that CB1 and CB2 cannabinoid receptor signaling is not necessary for the maintenance of a normally functioning NSDA neuronal system. Mice lacking CB1 and CB2 receptors were found to be equally susceptible to the neurotoxicant 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP). These studies support the use of CB1/CB2 KO mice for investigating the cannabinoid receptor-mediated regulation of the NSDA neuronal system in models of PD.

Recovery of hypothalamic tuberoinfundibular dopamine neurons from acute toxicant exposure is dependent upon protein synthesis and associated with an increase in parkin and ubiquitin carboxy-terminal hydrolase-L1 expression.

Hypothalamic tuberoinfundibular dopamine (TIDA) neurons remain unaffected in Parkinson disease (PD) while there is significant degeneration of midbrain nigrostriatal dopamine (NSDA) neurons. A similar pattern of susceptibility is observed in acute and chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse and rotenone rat models of degeneration. It is not known if the resistance of TIDA neurons is a constitutive or induced cell-autonomous phenotype for this unique subset of DA neurons. In the present study, treatment with a single injection of MPTP (20 mg/kg; s.c.) was employed to examine the response of TIDA versus NSDA neurons to acute injury. An acute single dose of MPTP caused an initial loss of DA from axon terminals of both TIDA and NSDA neurons, with recovery occurring solely in TIDA neurons by 16 h post-treatment. Initial loss of DA from axon terminals was dependent on a functional dopamine transporter (DAT) in NSDA neurons but DAT-independent in TIDA neurons. The active metabolite of MPTP, 1-methyl, 4-phenylpyradinium (MPP+), reached higher concentration and was eliminated slower in TIDA compared to NSDA neurons, which indicates that impaired toxicant bioactivation or distribution is an unlikely explanation for the observed resistance of TIDA neurons to MPTP exposure. Inhibition of protein synthesis prevented TIDA neuron recovery, suggesting that the ability to recover from injury was dependent on an induced, rather than a constitutive cellular mechanism. Further, there were no changes in total tyrosine hydroxylase (TH) expression following MPTP, indicating that up-regulation of the rate-limiting enzyme in DA synthesis does not account for TIDA neuronal recovery. Differential candidate gene expression analysis revealed a time-dependent increase in parkin and ubiquitin carboxyl-terminal hydrolase-L1 (UCH-L1) expression (mRNA and protein) in TIDA neurons during recovery from injury. Parkin expression was also found to increase with incremental doses of MPTP. The increase in parkin expression occurred specifically within TIDA neurons, suggesting that these neurons have an intrinsic ability to up-regulate parkin in response to MPTP-induced injury. These data suggest that TIDA neurons have a compensatory mechanism to deal with toxicant exposure and increased oxidative stress, and this unique TIDA neuron phenotype provides a platform for dissecting the mechanisms involved in the natural resistance of central DA neurons following toxic insult.

Opioid-mediated regulation of A11 diencephalospinal dopamine neurons: pharmacological evidence of activation by morphine.

Dopamine (DA) neurons of the A11 diencephalospinal system represent the sole source of DA innervation to the spinal cord in mice, serving neuromodulatory roles in the processing of nociceptive input and movement. These neurons originate in the dorso-caudal diencephalon and project axons unilaterally throughout the rostrocaudal extent of the spinal cord, terminating predominantly in the dorsal horn. The density of A11 DA axon terminals in the lumbar region is greater in males compared to females, while in both sexes the activity of neurons terminating in the thoracic spinal cord is greater than those terminating in the lumbar region. The present study was designed to test the hypothesis that A11 DA neurons are activated by opioids. To test this hypothesis, male and female mice were systemically treated with agonists or antagonists acting at the µ-opioid receptor, and spinal cord concentrations of DA and its metabolite DOPAC were determined in the thoracic and lumbar spinal cord using high performance liquid chromatography coupled with electrochemical detection. Systemic administration of the µ-opioid agonist morphine led to a dose- and time-dependent increase in spinal cord DOPAC/DA ratio (an estimate of DA neuronal activity) in both male and female mice, with greater changes occurring in the lumbar segment. Blockade of opioid receptors with the opioid antagonist naloxone reversed the stimulatory effects of morphine on A11 DA neurons in both male and female mice, but had little to no effect on the activity of these neurons when administered alone. Present findings are consistent with the conclusion that spinal cord-projecting axon terminals of A11 DA neurons are activated by opioids in both male and female mice, most likely through a dis-inhibitory mechanism.

Neonatal androgen-dependent sex differences in lumbar spinal cord dopamine concentrations and the number of A11 diencephalospinal dopamine neurons.

A(11) diencephalospinal dopamine (DA) neurons provide the major source of DA innervation to the spinal cord. DA in the dorsal and ventral horns modulates sensory, motor, nociceptive, and sexual functions. Previous studies from our laboratory revealed a sex difference in the density of DA innervation in the lumbar spinal cord. The purpose of this study was to determine whether sex differences in spinal cord DA are androgen dependent, influenced by adult or perinatal androgens, and whether a sex difference in the number of lumbar-projecting A(11) neurons exists. Adult male mice have significantly higher DA concentrations in the lumbar spinal cord than either females or males carrying the testicular feminization mutation (tfm) in the androgen receptor (AR) gene, suggesting an AR-dependent origin. Spinal cord DA concentrations are not changed following orchidectomy in adult male mice or testosterone administration to ovariectomized adult female mice. Administration of exogenous testosterone to postnatal day 2 female mice results in DA concentrations in the adult lumbar spinal cord comparable to those of males. Male mice display significantly more lumbar-projecting A(11) DA neurons than females, particularly in the caudal portion of the A(11) cell body region, as determined by retrograde tract tracing and immunohistochemistry directed toward tyrosine hydroxylase. These results reveal an AR-dependent sex difference in both the number of lumbar-projecting A(11) DA neurons and the lumbar spinal cord DA concentrations, organized by the presence of androgens early in life. The AR-dependent sex difference suggests that this system serves a sexually dimorphic function in the lumbar spinal cord.

Increased cellular turnover in response to fluoxetine in neuronal precursors derived from human embryonic stem cells.

Previous reports have shown that antidepressants increase neuronal cell proliferation and enhance neuroplasticity both in vivo and in vitro. This study investigated the direct effects of one such antidepressant, fluoxetine , on cell proliferation and on the production of neurotrophic factors in neuronal precursors derived from human embryonic stem cells (hESCs; H9). Fluoxetine induced the differentiation of neuronal precursors, strongly enhancing neuronal characteristics. The rate of proliferation was higher in fluoxetine -treated cells than in control cells, as determined by MTT [3(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide] assay. The CPDL (cumulative population doubling level) of the fluoxetine-treated cells was significantly increased in comparison to that of control cells (p<.001). Bromodeoxyuridine incorporation and staurosporine-induced apoptosis assays were elevated in fluoxetine-treated cells. Quantitative RT-PCR analysis revealed no significant differences in the expression of neurotrophic factors, brain-derived neurotrophic factor (BDNF);glial-derived neurotrophic factor (GDNF) and cAMP-responsive element-binding protein (CREB) between cells treated with fluoxetine for two weeks and their untreated counterparts. These results may help elucidate the mechanism of action of fluoxetine as a therapeutic drug for the treatment of depression. Data presented herein provide more evidence that, in addition to having a direct chemical effect on serotonin levels, fluoxetine can influence hESC-derived neuronal cells by increasing cell proliferation, while allowing them to maintain their neuronal characteristics.

Estradiol and song affect female zebra finch behavior independent of dopamine in the striatum.

Female songbirds display preferences for certain song characteristics, but the neural and hormonal mechanisms mediating these preferences are not fully clear. The present study sought to further explore the role of estradiol, as well as assess potential roles of dopaminergic systems, on behavioral responses to song. Adult female zebra finches were treated with estradiol and exposed to tutored or untutored song or silence. Behavior was quantified and neurochemistry of the nucleus accumbens and striatum was examined with high performance liquid chromatography. As a control, the responses of these two systems to treatment with raclopride, a specific D2 receptor antagonist, were also evaluated. This manipulation did not affect dopamine (DA), but did increase DOPAC and the DOPAC/DA ratio. Estradiol reduced the display of two behaviors, distance calls and visual scanning, but had no effect on dopaminergic responses. Auditory stimulus exposure affected other vocalizations, but song presentation did not modulate the levels of DA or its metabolite, DOPAC in the nucleus accumbens or striatum. Collectively, the results suggest that both estradiol and auditory stimuli can modify the behavioral responses of adult zebra finches, but they may not change DA concentration or turnover in striatal dopamine neurons.

Insulin withdrawal-induced cell death in adult hippocampal neural stem cells as a model of autophagic cell death.

The term "autophagic cell death" was coined to describe a form of cell death associated with the massive formation of autophagic vacuoles without signs of apoptosis. However, questions about the actual role of autophagy and its molecular basis in cell death remain to be elucidated. We recently reported that adult hippocampal neural stem (HCN) cells undergo autophagic cell death following insulin withdrawal. Insulin-deprived HCN cells exhibit morphological and biochemical markers of autophagy, including accumulation of Beclin 1 and the type II form of microtubule-associated protein 1 light chain 3 (LC3) without evidence of apoptosis. Suppression of autophagy by knockdown of Atg7 reduces cell death, whereas promotion of autophagy with rapamycin augments cell death in insulin-deficient HCN cells. These data reveal a causative role of autophagy in insulin withdrawal-induced HCN cell death. HCN cells have intact apoptotic capability despite the lack of apoptosis following insulin withdrawal. Our study demonstrates that autophagy is the default cell death mechanism in insulin-deficient HCN cells, and provides a genuine model of autophagic cell death in apoptosis-intact cells. Novel insight into molecular mechanisms of this underappreciated form of programmed cell death should facilitate the development of therapeutic methods to cope with human diseases caused by dysregulated cell death.

Effects of sildenafil on nigrostriatal dopamine neurons in a murine model of Parkinson's disease.

The objective of this study was to determine if the phosphodiesterase 5 (PDE-5) inhibitor, sildenafil, could be used as a neuroprotective agent in a chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) murine model of Parkinson's disease. The underlying hypothesis of these studies is that blockade of PDE-5 catabolism of cGMP will attenuate the loss of nigrostriatal dopamine (NSDA) neurons following chronic neurotoxin exposure. Chronic MPTP-treated mice were administered sildenafil using three different regimens. Animals were: 1) treated with sildenafil and then exposed to chronic MPTP; 2) treated concurrently with sildenafil and MPTP; and 3) first exposed to MPTP and subsequently treated with sildenafil. End points of neurotoxicity included dopamine (DA) and tyrosine hydroxylase (TH) concentrations in NSDA axon terminals in the striatum, and stereological cell counts of TH immunoreactive neurons in the substantia nigra. Results reveal that sildenafil did not prevent neurotoxicity produced by chronic MPTP exposure regardless of the treatment paradigms employed. On the other hand, sildenafil did not produce any deleterious effect on NSDA neuron function nor did it potentiate the neurotoxic effects of MPTP. These results suggest that sildenafil would not accelerate DA cell loss when used as a treatment for erectile dysfunction in men diagnosed with Parkinson's disease.

Autophagic death of adult hippocampal neural stem cells following insulin withdrawal.

Novel therapeutic approaches using stem cell transplantation to treat neurodegenerative diseases have yielded promising results. However, survival of stem cells after transplantation has been very poor in animal models, and considerable efforts have been directed at increasing the viability of engrafted stem cells. Therefore, understanding the mechanisms that regulate survival and death of neural stem cells is critical to the development of stem cell-based therapies. Hippocampal neural (HCN) stem cells derived from the adult rat brain undergo cell death following insulin withdrawal, which is associated with downregulation of antiapoptotic Bcl-2 family members. To understand the type of cell death in HCN cells following insulin withdrawal, apoptosis markers were assessed. Of note, DNA fragmentation or caspase-3 activation was not observed, but rather dying cells displayed features of autophagy, including increased expression of Beclin 1 and the type II form of light chain 3. Electron micrographs showed the dramatically increased formation of autophagic vacuoles with cytoplasmic contents. Staurosporine induced robust activation of caspase-3 and nucleosomal DNA fragmentation, suggesting that the machinery of apoptosis is intact in HCN cells despite the apparent absence of apoptosis following insulin withdrawal. Autophagic cell death was suppressed by knockdown of autophagy-related gene 7, whereas promotion of autophagy by rapamycin increased cell death. Taken together, these data demonstrate that HCN cells undergo a caspase-independent, autophagic cell death following insulin withdrawal. Understanding the mechanisms governing autophagy of adult neural stem cells may provide novel strategies to improve the survival rate of transplanted stem cells for treatment of neurodegenerative diseases. Disclosure of potential conflicts of interest is found at the end of this article.