Tomio Ono

Activity-dependent Expression of Parathyroid Hormone-related Protein (PTHrP) in Rat Cerebellar Granule Neurons REQUIREMENT OF PTHrP FOR THE ACTIVITY-DEPENDENT SURVIVAL OF GRANULE NEURONS

To identify genes whose expression is neuronal activity-dependent, we used an mRNA differential display technique and discovered that parathyroid hormone-related protein (PTHrP) is expressed in an activity-dependent manner in primary cultures of rat cerebellar granule neurons. PTHrP mRNA was expressed as early as 1 h by the addition of KCl to a final concentration of 25 mm to the culture medium. This expression was induced by Ca2+ influx through voltage-dependent L-type Ca2+ channels and regulated at the transcriptional step. PTHrP mRNA was persistently expressed before and after the time of commitment of granule neurons to apoptosis when they are cultured in the presence of 25 mmKCl or both 150 μm N-methyl-d-aspartic acid and 15 mmKCl, both of which promote the survival of these neurons. PTHrP was rapidly secreted into the culture medium in a depolarization-dependent manner. Parathyroid hormone/PTHrP receptor mRNA was also expressed in the primary cultures, and its expression was up-regulated by KCl and/orN-methyl-d-aspartic acid. The addition of anti-PTHrP antiserum to the culture medium resulted in a reduction of the activity-dependent survival of the granule neurons. These results suggest that PTHrP is involved in an autocrine loop and required for the survival of granule neurons.

Hippocalcin protects hippocampal neurons against excitotoxin damage by enhancing calcium extrusion.

Hippocalcin, which is a member of the neuronal calcium-sensor protein family, is highly expressed in hippocampal pyramidal cells. Recently, it was demonstrated that hippocalcin deficit caused an increase in neuronal cell death in the field CA3 of Ammons horn (CA3) region of the hippocampus following the systemic injection of kainic acid. Treatment with kainic acid results in seizure-induced cell death in CA3. In the present study, we injected quinolinic acid, which is an N-methyl-d-aspartate receptor agonist, into the hippocampal field CA1 of Ammons horn (CA1) region in hippocalcin-knockout (-/-) mice, a procedure which mimics transient ischemia. Although significant pyknotic changes were observed at the injected site in wild-type (+/+) mice 24 h after injection, the area of pyknotic cells extended throughout the hippocampus in -/- mice. The quantification of cell numbers in Nissl-stained sections indicated that the cell damage in -/- mice was more severe than that in +/+ mice. The density of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling-positive cells roughly paralleled that of Nissl-stained pyknotic cells. Primary cultures of hippocampal neurons showed that the number of surviving neurons from -/- mice after 7 days in culture was smaller than the number from +/+ mice. The measurement of intracellular calcium concentrations in single cells revealed that the calcium extrusion from -/- neurons was slower than that from +/+ neurons. The involvement of hippocalcin in the upkeep of calcium extrusion was confirmed using hippocalcin-expressing COS7 cells. These results suggest that hippocalcin plays an important role in calcium extrusion from neurons and, in turn, helps to protect them against calcium-dependent excitotoxin damage in the hippocampus.

The Calpain Proteolytic System in Neonatal Hypoxic‐Ischemiaa

Neonatal rats were subjected to transient cerebral hypoxic-ischemia (HI, unilateral occlusion of the common carotid artery +7.70% O2 for 100 min) and allowed to recover for up to 14 days. Calpain caseinolytic activity was found to increase in both hemispheres for at least 20 hr. Hypoxic exposure per se increased the activity of calpains, more pronounced in a membrane-associated fraction, probably through interaction with cellular components, whereas HI introduced a loss of activity, most likely through consumption and loss of proteases. Consecutive tissue sections were stained with antibodies against calpastatin, alpha-fodrin, the 150-kDa breakdown product of alpha-fodrin (FBDP, marker of calpain proteolysis) or microtubule-associated protein 2 (MAP-2, marker of dendrosomatic neuronal injury). Areas with brain injury displayed a distinct loss of MAP-2, which clearly delineated the infarct. FBDP accumulated in injured and borderline regions ipsilaterally, and a less conspicuous, transient increase in FBDP also occurred in the contralateral hemisphere, especially in the white matter. The cytosolic fraction (CF) and the membrane and microsomal fraction (MMF) of cortical tissue were subjected to Western blotting and stained with antibodies against calpain, calpastatin and the 150-kDa breakdown product of alpha-fodrin (FBDP). Calpain immunoreactivity decreased bilaterally in the CF during the insult (62-68% of controls) and remained significantly lower during early recovery, whereas the MMF showed no significant changes. This translocation of calpains coincided with the appearance of FBDP in the ipsilateral, HI hemisphere, displaying a significantly higher level of FBDP from immediately after the insult until at least 1 day of recovery (204-292% of controls). No significant changes in FBDP were found in the contralateral, undamaged hemisphere, despite translocation of calpains in both hemispheres, a prerequisite for calpain activation. This discrepancy may be related to changes in the endogenous inhibitor, calpastatin. Calpastatin protein was found to decrease during and shortly after HI in the ipsilateral, but not the contralateral, hemisphere. The inhibitory activity of calpastatin also tended to decrease after HI, indicating that a reduction of calpastatin may be necessary for extensive calpain activation to occur. The mRNA of m-calpain increased in the HI hemisphere 48 hr after the insult (167%, p < 0.001), a time point when the protein was also increased. In summary, our findings indicate that calpains are activated during HI and in the early phase of reperfusion after HI, preceding neuronal death.

Activity-dependent survival of rat cerebellar granule neurons is not associated with sustained elevation of intracellular Ca2+

Ca2+ plays a pivotal role for the activity-dependent survival of neurons. In primary culture of cerebellar granule neurons, we found that there is no significant difference in intracellular Ca2+ level in the survival-promoting condition (cultures in the presence of 25 mM KCl) and that in the apoptosis-inducing condition (cultures in the presence of 5 mM KCl). This was not due to the inactivation of voltage-dependent L-type Ca2+ channels in the survival-promoting condition, but due to the enhanced rate of the influx and the efflux of Ca2+ in the survival-promoting condition compared to that in the apoptosis-inducing condition. These results suggest that the activity-dependent survival of the granule neurons is not associated with sustained rise of intracellular Ca2+ but associated with the enhanced turnover rate of Ca2+.

Activity-dependent survival and enhanced turnover of calcium in cultured rat cerebellar granule neurons

Neurons survive when their activity is maintained. An influential hypothesis on the cellular mechanism underlying this phenomenon is that there is an appropriate range of intracellular Ca2+ concentration ([Ca2+]i) for survival. The rat cerebellar granule neuron in culture serves as the most often used model system for the analysis of activity-dependent survival, since it does not survive unless an excitant (KCl or glutamate) is added to the culture medium. Against the above-mentioned hypothesis, we found in our previous examination no difference between steady-state [Ca2+]i in granule neurons cultured under high KCl (i.e., survival) and low KCl (i.e., death) conditions. In this report, we present the quantitative background of unchanged [Ca2+]i between the two culture conditions. Influx of Ca2+ due predominantly to L-type voltage-dependent calcium channels was higher in high KCl cultures than in low KCl cultures. At the same time, efflux of Ca2+ due to the activity of Ca2+/Na+ antiport was also higher in high KCl cultures. Additionally, we found that the endocytotic activity was greater in high KCl cultures than in low KCl cultures, as monitored by the rate of uptake of horseradish peroxidase added to medium. Since the uptake was blocked by an internal Ca2+ chelator, the increased endocytotic activity in high KCl cultures might be a consequence of the enhanced Ca2+ turnover.

Mice deficient in alivin1/amigo2 show enhanced locomotor activity

The amygdala and serotonergic innervation thereonto are considered to cooperatively regulate emotional states and behaviors. In the present experiments, we investigated how the serotonergic input modulates the excitability of lateral amygdala (LA) neurons by whole cell recordings and voltage-sensitive dye imaging in rat brain slices. Bath-application of serotonin (5-HT) induced a slow afterdepolarization (sADP) in LA neurons. This sADP lasted for more than 5 s. This sADP was also induced by synaptic stimulations. These results suggest that 5-HT enhances the excitability of amygdala neurons by inducing sADP. On the other hand, by using voltage imaging, we observed that bath-application of 5-HT suppressed the excitatory synaptic propagation from LA to endopiriform nucleus, perirhinal cortex, piriform cortex and basolateral amygdala. The suppression by 5-HT was also observed in patch clamp recording from the neurons in endopiriform nucleus and basolateral amygdala. Taken together, it is suggested that 5-HT has two opposite effects, enhancing the excitability of LA and suppressing the synaptic propagation from LA.