Evidence of functional duplicity of Nestin expression in the adult mouse midbrain.

Whether or not neurogenesis occurs in the adult substantia nigra pars compacta (SNc) is an important question relevant for developing better treatments for the motor symptoms of Parkinson's disease (PD). Although controversial, it is generally believed that dividing cells here remain undifferentiated or differentiate into glia, not neurons. However, there is a suggestion that Nestin-expressing neural precursor cells (NPCs) in the adult SNc have a propensity to differentiate into neurons, which we sought to confirm in the present study. Adult (>8-weeks old) transgenic NesCreERT2/GtROSA or NesCreERT2/R26eYFP mice were used to permanently label Nestin-expressing cells and their progeny with ß-galactosidase (ß-gal) or enhanced yellow fluorescent protein (eYFP), respectively. Most ß-gal+ or eYFP+ cells were found in the ependymal lining of the midbrain aqueduct (Aq) and in the midline ventral to Aq. Smaller but significant numbers were in the periaqueductal gray (PAG), the ventral tegmental area (VTA), and in SNc. Low-level basal proliferation was evidenced by a modest increase in number of ß-gal+ or eYFP+ cells over time, fewer ß-gal+ or eYFP+ cells when mice were administered the anti-mitotic agent Cytarabine, and incorporation of the proliferation marker bromodeoxyuridine (BrdU) in a very small number of ß-gal+ cells. No evidence of migration was found, including no immunoreactivity against the migration markers doublecortin (DCX) or polysialic acid neural cell adhesion molecule (PSA-NCAM), and no dispersal of ß-gal+ or eYFP+ cells through the midbrain parenchyma over time. However, ß-gal+ or eYFP+ cells did increase in size and express higher levels of mature neuronal genes over time, indicating growth and neuronal differentiation. In mice whose SNc dopamine neurons had been depleted with 6-hydroxy-dopamine, a model of PD, there were ~2-fold more ß-gal+ cells in SNc specifically, although the proportion that were also NeuN+ was not affected. Remarkably, as early as 4days following putative Nestin-expression, many ß-gal+ or eYFP+ cells had mature neuronal morphology and were NeuN+. Furthermore, mature neuronal ß-gal+ cells were immunoreactive against the self-renewal or pluripotency marker sex determining region Y-box 2 (Sox2). Overall, our data support the notion that some Nestin-expressing, presumably NPCs, have a limited capacity for proliferation, no capacity for migration, and a propensity to generate new neurons within the microenvironment of the adult midbrain. However, our data also suggest that significant numbers of extant midbrain neurons express Nestin and other classical neurogenesis markers in contexts that are presumably not neurogenic. These findings foreshadow duplicitous roles for Nestin and other molecules that are traditionally associated with neurogenesis in the adult midbrain, which should be considered in future PD research.

Tamsulosin modulates, but does not abolish the spontaneous activity in the guinea pig prostate gland.

AIMS: To examine the effects of the α1A -adrenoceptor antagonist, tamsulosin, on spontaneous contractile and electrical activity in the guinea-pig prostate gland. METHODS: The effects of tamsulosin (0.1 and 0.3 nM) were investigated in adult and ageing male guinea pig prostate glands using conventional tension recording and electrophysiological intracellular microelectrode recording techniques. RESULTS: Tamsulosin reduced spontaneous activity, and had different age-dependent effects on adult and ageing guinea pigs at different concentrations. 0.1 nM tamsulosin caused a significantly greater reduction of spontaneous contractile and electrical activity in ageing guinea pigs in comparison to adult guinea pigs. In contrast, 0.3 nM tamsulosin had a significantly greater reduction of spontaneous contractile and electrical activity in adult guinea pigs in comparison to ageing guinea pigs. CONCLUSIONS: This study demonstrates that tamsulosin can modulate spontaneous myogenic stromal contractility and the underlying spontaneous electrical activity; tamsulosin does not block spontaneous activity. This reduction in spontaneous activity suggests that downstream cellular mechanisms underlying smooth muscle tone are being targeted, and these may represent novel therapeutic targets to better treat benign prostatic hyperplasia.

Effects of imatinib mesylate on the spontaneous activity generated by the guinea-pig prostate.

UNLABELLED: What's known on the subject? and what does the study add?: Several studies have examined the functional role of tyrosine kinase receptors in the generation of spontaneous activity in various segments of the gastrointestinal and urogenital tracts through the application of its inhibitor, imatinib mesylate (Glivec®), but results are fairly inconsistent. This is the first study detailing the effects of imatinib mesylate on the spontaneous activity in the young and ageing prostate gland. As spontaneous electrical activity underlies the spontaneous rhythmic prostatic contractions that occur at rest, elucidating the mechanisms involved in the regulation of the spontaneous electrical activity and the resultant phasic contractions could conceivably lead to the identification of better targets and the development of more specific therapeutic agents to treat prostate conditions. OBJECTIVE: To investigate the effect of imatinib mesylate, a tyrosine kinase receptor inhibitor, in the generation of spontaneous electrical and contractile activity in the young and ageing guinea-pig prostate. MATERIALS AND METHODS: Standard tension and intracellular recording were used to measure spontaneous contractions and slow waves, respectively from the guinea-pig prostate at varying concentrations of imatinib mesylate (1-50 µm). RESULTS: Imatinib mesylate (1-10 µm), did not significantly affect slow waves recorded in the prostate of both age groups but at 50 µm, the amplitude of slow waves from the ageing guinea-pig prostate was significantly reduced (P < 0.05, n = 5). In contrast, the amplitude of contractions across all concentrations in the young guinea-pig prostate was reduced to between 35% and 41% of control, while the frequency was reduced to 15.7% at 1 µm (n = 7), 49.8% at 5 µm (n = 10), 46.2% at 10 µm (n = 7) and 53.1% at 50 µm (n = 5). Similarly, imatinib mesylate attenuated the amplitude and slowed the frequency of contractions in ageing guinea-pigs to 5.15% and 3.3% at 1 µm (n = 6); 21.1% and 20.8% at 5 µm (n = 8); 58.4% and 8.8% at 10 µm (n = 11); 72.7% and 60% at 50 µm (n = 5). CONCLUSIONS: A significant reduction in contractions but persistence of slow waves suggests imatinib mesylate may affect the smooth muscle contractile mechanism. Imatinib mesylate also significantly reduced contractions in the prostates of younger guinea pigs more than older ones, which is consistent with the notion that the younger guinea-pig prostate is more reliant on the tyrosine-dependent pacemaker ability of interstitial cells of Cajal-like prostatic interstitial cells.

Nitric oxide signaling pathways involved in the inhibition of spontaneous activity in the guinea pig prostate.

PURPOSE: We investigated nitric oxide mediated inhibition of spontaneous activity recorded in young and aging guinea pig prostates. MATERIALS AND METHODS: Conventional intracellular microelectrode and tension recording techniques were used. RESULTS: The nitric oxide donor sodium nitroprusside (10 µM) abolished spontaneous contractions and slow wave activity in 5 young and 5 aging prostates. Upon adding the nitric oxide synthase inhibitor L-NAME (10 µM) the frequency of spontaneous contractile and electrical activity was significantly increased in each age group. This increase was significantly larger in 4 to 8 preparations of younger vs aging prostates (about 40% to 50% vs about 10% to 20%, 2-way ANOVA p<0.01). Other measured parameters, including the duration, amplitude and membrane potential of spontaneous electrical and contractile activity, were not altered from control values. The guanylate cyclase inhibitor ODQ (10 µM) significantly increased the frequency of spontaneous activity by 10% to 30% in 6 young guinea pig prostates (Student paired t test p<0.05). However, it had no effect on aging prostates. The cGMP analogue 8-Br-GMP (1 µM) and the PDE5 inhibitor dipyridamole (1 µM) significantly decreased the frequency of contractile activity by about 70% in 4 to 9 young and older prostates (Student paired t test p<0.05). CONCLUSIONS: The decrease in the response to L-NAME in spontaneous contractile and slow wave activity in aging prostate tissue compared to that in young prostates suggests that with age there is a decrease in nitric oxide production. This may further explain the increase in prostatic smooth muscle tone observed in age related prostate specific conditions, such as benign prostatic hyperplasia.

Contractility and pacemaker cells in the prostate gland.

PURPOSE: We focused on the current opinion on mechanisms generating stromal tone in the prostate gland. MATERIALS AND METHODS: We selected the guinea pig as the main species for investigation since its prostate has a high proportion of smooth muscle that undergoes age related changes similar in many respects to that in humans. The main techniques that we used were tension recording and electrophysiology. RESULTS: We previously reported distinct electrical activity and cell types in the prostate, and speculated on their functional roles. We believe that a specialized group of c-kit immunoreactive prostatic interstitial cells that lie between glandular epithelium and smooth muscle stroma have a role similar to that of gastrointestinal interstitial cells of Cajal, generating the pacemaker signal that manifests as slow wave activity and triggers contraction in smooth muscle cells in guinea pig prostates. CONCLUSIONS: Since changes in muscle tone are involved in the etiology of age dependent prostate specific conditions such as benign prostatic hyperplasia, knowledge of the electrical properties of the various prostatic cell types and their interactions with each other, with nerves and with the hormonal environment, and how these factors change with age is of considerable medical importance.

Role of connexin 43 in the maintenance of spontaneous activity in the guinea pig prostate gland.

BACKGROUND AND PURPOSE: To investigate the role of connexin 43 in the maintenance of spontaneous activity in prostate tissue from young and old guinea pigs. EXPERIMENTAL APPROACH: Conventional intracellular microelectrode and tension recording techniques, coupled with Western blot analysis and immunohistochemistry for connexin 43 (CX43) were used. The effects of three gap junction uncouplers, 18ß glycyrrhetinic acid (10 µM, 40 µM), carbenoxolone (10 µM, 50 µM) and octanol (0.5 mM, 1 mM), were studied in cells displaying slow wave activity and on spontaneously contracting tissue from prostate glands of young (2-5 months) and old (9-16 months) guinea pigs. KEY RESULTS: 18ß Glycyrrhetinic acid (40 µM), carbenoxolone (50 µM) or octanol (0.5 mM) abolished slow wave activity in prostate tissue from young and old guinea pigs and depolarized membrane potential by approximately 5 mV. These treatments also abolished all contractions in both sets of prostate tissue. These effects were reversed upon washout. Western blot analysis and CX43 immunohistochemistry showed that there was no age-related difference in the expression and distribution of CX43 in prostate tissues. CONCLUSION AND IMPLICATIONS: When gap junctional communication via CX43 was disrupted, spontaneous activity was abolished at a cellular and whole tissue level; CX43 is therefore essential for the maintenance of spontaneous slow wave activity and subsequent contractile activity in the guinea pig prostate gland.

Spontaneous electrical waveforms in aging guinea pig prostates.

PURPOSE: We characterized spontaneous electrical activity in the aging guinea pig prostate. MATERIALS AND METHODS: Membrane potential recordings were made using conventional single microelectrode recording techniques. RESULTS: Three types of spontaneous waveforms were recorded, including spikes, slow waves and spontaneous transient depolarizations. Spikes were classified as hyperactive or active. Active cells showed a mean +/- SEM frequency of 5.06 +/- 0.63 minutes(-1), significantly different from that in hyperactive cells (362.05 +/- 151.82 minutes(-1), p <0.05). After hyperpolarization amplitude was also significantly different in the active and hyperactive groups (17.80 +/- 1.98 vs 9.96 +/- 1.05 mV). Spike activity was abolished by 1 microM nifedipine in 7 preparations (p <0.05). Slow wave activity occurred at a frequency of 5.2 +/- 0.5 minutes(-1). The spike component of slow wave activity was abolished by 1 microM nifedipine, although the depolarizing transient remained unaltered from control values (8.1 +/- 3.1 mV, paired Student's t test p >0.05). Spontaneous transient depolarizations were recorded in the presence of slow waves in 10 preparations and of spikes in 13, and in quiescent cells in 9. Spontaneous transient depolarization frequency was highest in otherwise quiescent cells (24.55 +/- 6.48 minutes(-1)) compared to that in the presence of slow waves or spikes. Adding 1 microM nifedipine in 5 preparations did not significantly affect any measured parameters (p >0.05). Pacemaker potentials were not recorded in the aging prostate. CONCLUSIONS: With increased age there is an increase in spike activity, which could conceivably explain the increased prostatic tone that accompanies aging. Spike activity and the spike component of the slow wave were abolished by nifedipine, suggesting a role for L-type channels. Finally, spontaneous transient depolarizations were unaffected by nifedipine, suggesting that mechanisms other than Ca(2+) entry via L-type channels are responsible for their generation and maintenance.

Spontaneous electrical activity in the prostate gland.

The cellular mechanisms that underlie the initiation, maintenance and propagation of electrical activity in the prostate gland remain little understood. Intracellular microelectrode recordings have identified at least two distinct electrical waveforms: pacemaker potentials and slow wave activity. By analogy with the intestine, we have proposed that pacemaker activity arises from a morphologically distinct group of c-Kit positive interstitial cells that lie mainly between the glandular epithelium and smooth muscle layers. We speculate that pacemaker activity arising from the prostatic interstitial cells (PICs) is likely to propagate and initiate slow wave activity in the smooth muscle cells resulting in contraction of the stromal smooth muscle wall. While spontaneous electrical activity in the prostate gland is myogenic in origin, it is clear that nerve-mediated agents are able to modulate this activity. Excitatory agents such as histamine, phenylephrine and a raised potassium saline all increase slow wave discharge. In contrast, nitric oxide donors reduce or abolish the spontaneous electrical events. However, the cellular mechanisms underlying the action of various endogenously released agents remain to be elucidated.