Tae Hee Han

Human umbilical cord blood-derived mesenchymal stem cells protect against neuronal cell death and ameliorate motor deficits in Niemann Pick type C1 mice.

Niemann Pick disease type C1 (NPC) is an autosomal recessive disease characterized by progressive neurological deterioration leading to premature death. In this study, we hypothesized that human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) have the multifunctional abilities to ameliorate NPC symptoms in the brain. To test this hypothesis, hUCB-MSCs were transplanted into the hippocampus of NPC mice in the early asymptomatic stage. This transplantation resulted in the recovery of motor function in the Rota Rod test and impaired cholesterol homeostasis leading to increased levels of cholesterol efflux-related genes such as LXRα, ABCA1, and ABCG5 while decreased levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase were observed in NPC mice. In the cerebrum, hUCB-MSCs enhanced neuronal cell survival and proliferation, where they directly differentiated into electrically active MAP2-positive neurons as demonstrated by whole-cell patch clamping. In addition, we observed that hUCB-MSCs reduced Purkinje neuronal loss by suppression of inflammatory and apoptotic signaling in the cerebellum as shown by immunohistochemistry. We further investigated how hUCB-MSCs enhance cellular survival and inhibit apoptosis in NPC mice. Neuronal cell survival was associated with increased PI3K/AKT and JAK2/STAT3 signaling; moreover, hUCB-MSCs modulated the levels of GABA/glutamate transporters such as GAT1, EAAT2, EAAT3, and GAD6 in NPC mice as assessed by Western blot analysis. Taken together, our findings suggest that hUCB-MSCs might play multifunctional roles in neuronal cell survival and ameliorating motor deficits of NPC mice.

Reduction in synaptic GABA release contributes to target-selective elevation of PVN neuronal activity in rats with myocardial infarction

Neuronal activity in the paraventricular nucleus (PVN) is known to be elevated in rats with heart failure. However, the type of neurons involved and the underlying synaptic mechanisms remain unknown. Here we examined spontaneous firing activity and synaptic currents in presympathetic PVN neurons in rats with myocardial infarction (MI), using slice patch clamp combined with the retrograde labeling technique. In PVN neurons projecting to the rostral ventrolateral medulla (PVN-RVLM), MI induced a significant increase in basal firing rate (1.79 to 3.02 Hz, P < 0.05) and a reduction in the frequency of spontaneous (P < 0.05) and miniature (P < 0.01) inhibitory postsynaptic currents (IPSCs). In addition, MI induced an increase in the paired-pulse ratio of evoked IPSCs (P < 0.05). Bicuculline, a GABA(A) receptor antagonist, increased the firing rate of PVN-RVLM neurons in sham-operated (1.21 to 2.74 Hz, P < 0.05) but not MI (P > 0.05) rats. In contrast, in PVN neurons projecting to the intermediolateral horn of the spinal cord (PVN-IML), MI did not induce any significant changes in the basal firing rate and the properties of spontaneous and miniature IPSCs. The properties of spontaneous excitatory postsynaptic currents (EPSCs) were not altered in either neuron group. In conclusion, our results indicate that MI induces an elevation of firing activity in PVN-RVLM but not in PVN-IML neurons and that the elevated firing rate is largely due to a decrease in GABA release. These results provide evidence for a novel target-selective synaptic plasticity in the PVN that is associated with the sympathetic hyperactivity commonly seen in heart failure.

Serotonin inhibits GABA synaptic transmission in presympathetic paraventricular nucleus neurons.

Activation of serotonin (5-hydroxytryptamine, 5-HT) receptors produces various autonomic and neuroendocrine responses in the hypothalamic paraventricular nucleus (PVN), including increased blood pressure and heart rate. However, the role(s) of 5-HT on the local GABA synaptic circuit have not been well understood in the PVN, where the inhibitory neurotransmitter GABA plays a key role in the modulation of sympathoexcitatory outflow. In the present study, we examined the effects of 5-HT on GABA synaptic transmission in presympathetic PVN neurons projecting to spinal cord using patch-clamp electrophysiology combined with tract-tracing techniques. Bath application of 5-HT (0.01-100 microM) reversibly decreased the frequency of spontaneous GABAergic inhibitory postsynaptic currents (sIPSC) in a concentration dependent manner (IC50, 0.07 microM), with no significant changes in the amplitudes and decay kinetics of sIPSC. The sIPSC inhibition of 5-HT was mimicked by 5-HT1A agonist, 8-OH-DPAT (8-hydroxy-2(di-n-propylamino)tetralin, 10 microM), and blocked by 5-HT1A antagonist WAY-100635 but not by 5-HT1B antagonist SB224289. 5-HT also reduced the frequency of miniature IPSC (mIPSC) (2.59+/-0.51 Hz, control vs. 1.25+/-0.31 Hz, 5-HT, n=16) in similar extent with 5-HT induced reduction of sIPSC frequency (sIPSCs, 55.8+/-6.2%, n=11 vs. mIPSCs, 52.30+/-5.85%, n=16; p>0.5). All together, our results indicate that 5-HT can inhibit presynaptic GABA release via presynaptic 5-HT1A receptors in presympathetic PVN neurons projecting to spinal cord.

Enhanced cell proliferation and neuroblast differentiation in the rat hippocampal dentate gyrus following myocardial infarction.

Basic and clinical studies have revealed that depression is frequently observed following myocardial infarction (MI). We observed changes in neurons in the subgranular zone of the hippocampal dentate gyrus (DG) 14 days after chronic cardiac ischemia. Cresyl violet staining was conducted to examine neurodegeneration. Cresyl violet-positive neurons in the hippocampus in the MI-operated group were similar to those in the sham-operated group, and Fluoro-Jade B-positive cells were not observed in either group. Next, we observed changes in cell proliferation using Ki67 and in the differentiation of neuroblasts using doublecortin (DCX) in the DG. The number of Ki67- and DCX-positive cells in the subgranular zone of the DG in the MI-operated group was significantly increased compared to that in the sham-operated group. In addition, DCX-positive processes were prominent in the MI group. These results suggest that MI may influence cell proliferation and affect neuroblast differentiation in the subgranular zone of the DG.

Adenosine reduces GABAergic IPSC frequency via presynaptic A1 receptors in hypothalamic paraventricular neurons projecting to rostral ventrolateral medulla

Adenosine is an inhibitory modulator of neuronal transmission, including GABAergic transmission in the hypothalamus. It is known that the local GABAergic inputs tonically inhibit the hypothalamic paraventricular neurons projecting to the rostral ventrolateral medulla (RVLM; PVN-RVLM neurons) which regulate sympathetic outflow. In this study, we examined the effects of adenosine on GABAergic synaptic transmission in the PVN-RVLM neurons using whole cell patch-clamp combined with the retrograde labeling technique. Adenosine (100 μM) reversibly decreased the frequency of miniature IPSCs (from 3.41 ± 0.75 to 2.19 ± 0.49 Hz) in a concentration-dependent manner (IC₅₀ = 1.0 μM) without affecting the amplitude and the decay time constant of miniature IPSCs. Adenosine increased the paired-pulse ratio of evoked IPSCs from 1.19 ± 0.05 to 2.28 ± 0.09 (P<0.001). The effects of adenosine was mimicked by a selective A₁ receptor agonist (CHA, 10 μM), and blocked by a selective A₁ receptor antagonist (DPCPX, 2 μM), but not by a selective A₂ receptor antagonist (DMPX, 10 μM). In conclusion, the results showed that adenosine inhibits synaptic GABA release via presynaptic A₁ receptors in the PVN-RVLM neurons, indicating a potential of adenosine A₁ receptors in regulating sympathetic tone in normal and disease states.

Direct Corticosteroid Modulation of GABAergic Neurons in the Anterior Hypothalamic Area of GAD65-eGFP Mice

Corticosterone is known to modulate GABAergic synaptic transmission in the hypothalamic paraventricular nucleus. However, the underlying receptor mechanisms are largely unknown. In the anterior hypothalamic area (AHA), the sympathoinhibitory center that project GABAergic neurons onto the PVN, we examined the expression of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) of GABAergic neurons using intact GAD65-eGFP transgenic mice, and the effects of corticosterone on the burst firing using adrenalectomized transgenic mice. GR or MR immunoreactivity was detected from the subpopulations of GABAergic neurons in the AHA. The AHA GABAergic neurons expressed mRNA of GR (42%), MR (38%) or both (8%). In addition, in brain slices incubated with corticosterone together with RU486 (MR-dominant group), the proportion of neurons showing a burst firing pattern was significantly higher than those in the slices incubated with vehicle, corticosterone, or corticosterone with spironolactone (GR-dominant group; 64 vs. 11~14%, p< 0.01 by χ(2)-test). Taken together, the results show that the corticosteroid receptors are expressed on the GABAergic neurons in the AHA, and can mediate the corticosteroid-induced plasticity in the firing pattern of these neurons. This study newly provides the experimental evidence for the direct glucocorticoid modulation of GABAergic neurons in the AHA in the vicinity of the PVN.

Viability Assessment of Primo-node Slices From Organ Surface Primo-vascular Tissues in Rats

The primo-vascular system is a novel thread-like structure which is recently rediscovered, but its cellular properties are largely unknown. In this study, a slice preparation for primo-nodes was developed to facilitate study of the cellular properties of primo-node cells in vitro. Slices (4-8 slices; 200 μm thick) were sectioned from single primo-nodes collected from the abdominal organ surface of rats and incubated in oxygenated Krebs solution at 25°C or 31°C for up to 7 hours. Trypan blue staining and whole-cell patch-clamp recordings were performed to estimate the viability of cells in the slices. Viability was largely maintained during the first 3 hours, but subsequently decreased (from 80% to 21%, p < 0.001). In addition, the viability of slices incubated at 31°C was higher than those incubated at 25°C (80%vs. 47%, p < 0.001). In whole-cell patch-clamp experiments, high resistance seals readily formed and primo-node cells showed a mean resting membrane potential (-38 mV) comparable to that recorded with sharp electrodes and outwardly-rectifying current-voltage relationships. The results show that the primo-node slices developed in this study maintained viability for up to 4 hours in vitro.

Time course of diurnal rhythm disturbances in autonomic function of rats with myocardial infarction

The diurnal rhythm of the autonomic function is known to be blunted in heart failure, but the timing of this blunting is not well understood. We examined the time course of the alterations in autonomic function in rats with myocardial infarction (MI) by analyzing heart rate variability (HRV). MI was induced by coronary artery ligation, and HRV was analyzed at 2, 4, 6, and 8 weeks post-MI. Diurnal rhythm in heart rate (HR) was maintained over the study period. However, diurnal rhythm in the standard deviation of averages of normal R-R intervals (SDANN) and the ratio between low and high frequency band powers (LF/HF ratio) were disrupted in MI rats at 2 weeks, which persisted up to 8 weeks, with the exception of 4 weeks. The dark-light differences in the LF/HF ratio changed from negative to positive values between weeks 2 and 4 in the MI rats. We also found decreases in HR, SDANN, and the LF/HF ratio in the dark phase at weeks 6 and 8 and an increase in plasma norepinephrine (NE) level at week 8. Collectively, the results indicate that the timing of the disturbance of diurnal rhythm in SDANN and the LF/HF ratio is different from those in HR and in plasma NE level, suggesting that the mechanisms underlying these changes are different. In addition, there is a transition from the compensatory to the decompensatory phase between 4 and 6 weeks post-MI. These findings may help to understand the progression and pathophysiology of heart failure.

Basic Electrophysiological Properties of Cells in the Organ Surface Primo Vascular Tissues of Rats

The primo vascular system (PVS) is a novel circulatory system composed of primo vessels and primo nodes, and its morphological and functional properties are largely unknown. In this study, we characterized basic electrophysiological properties of the cells in the primo vessels and primo nodes on the surface of abdominal organs. The electrophysiological activities of the cells in the primo vessels and nodes were studied by intracellular recording technique with sharp electrodes and by whole-cell slice patch recording techniques, respectively. The cells of the primo vessels and nodes did not exhibit spontaneous activities or action potentials. The resting membrane potentials of the primo vessel and node cells were −21.14 ± 2.24 mV (n = 35) and −36.69 ± 1.38 mV, respectively (n = 67). The current-voltage relations of the primo vessel cells were linear, but those of the primo node cells were outwardly rectifying. Morphologically, most primo vessel cells showed a round shape, but a small portion of cells showed a longitudinal one. In contrast, most cells in the primo node slices were round-shaped, and the longitudinal cells were not observed. Taken together, the results indicate that the cells in the PVS tissues are electrophysiologically and morphologically heterogenous, and that the small round cells that are most abundant in the primo vessels and nodes are nonexcitable.

Increased αB-crystallin in hypothalamic paraventricular nucleus of rats with myocardial infarction

The hypothalamus plays an important role in maintaining a homeostasis of the body against stress response. In particular, the paraventricular nucleus of the hypothalamus is a critical region for disorders related to the autonomic nervous system, such as congestive heart failure and hypertension. αB-crystallin is a family of heat shock proteins that are widely expressed in the brain, including in glial cells, astrocytes, oligodendrocytes, and neurons. Many studies have demonstrated that expression level of αB-crystallin is up-regulated and involved in protecting cells from pathological conditions. In the present study, we examined the expression and potential role of αB-crystallin in the paraventricular nucleus (PVN) regions of rats with myocardial infarction (MI). Our results demonstrate that mRNA encoding αB-crystallin and protein for both native and phosphorylate forms (Ser-59) of αB-crystallin was significantly increased in the PVN during MI.