Sang-Soep Nahm

Circadian Regulation and Function of Voltage-Dependent Calcium Channels in the Suprachiasmatic Nucleus

Individual neurons within the suprachiasmatic nuclei (SCNs) are capable of functioning as autonomous clocks and generating circadian rhythms in the expression of genes that form the molecular clockworks. Limited information is available on how these molecular oscillations in individual clock cells are coordinated to provide for the ensemble rhythmicity that is normally observed from the entire SCN. Because calcium influx via voltage-dependent calcium channels (VDCCs) has been implicated in the regulation of gene expression and synchronization of rhythmicity across the population of SCN clock cells, we first examined the rat SCN and an immortalized line of SCN cells (SCN2.2) for expression and circadian regulation of different VDCC α1 subunits. The rat SCN and SCN2.2 cells exhibited mRNA expression for all major types of VDCC α1 subunits. Relative levels of VDCC expression in the rat SCN and SCN2.2 cells were greatest for L-type channels, moderate for P/Q- and T-type channels, and minimal for R- and N-type channels. Interestingly, both rat SCN and SCN2.2 cells showed rhythmic expression of P/Q- and T-type channels. VDCC involvement in the regulation of molecular rhythmicity in SCN2.2 cells was then examined using the nonselective antagonist, cadmium. The oscillatory patterns of rPer2 and rBmal1 expression were abolished in cadmium-treated SCN2.2 cells without affecting cellular morphology and viability. These findings raise the possibility that the circadian regulation of VDCC activity may play an important role in maintaining rhythmic clock gene expression across an ensemble of SCN oscillators.

Effects of early postnatal ethanol intubation on GABAergic synaptic proteins

Fetal alcohol syndrome includes brain damage from aberrant synaptogenesis, altered cell-cell signaling and blunted plasticity in surviving neurons. Distortion of neurotrophic GABA signals by ethanol-mediated allosteric modulation of GABA(A) receptor (GABA(A)R) activity during brain maturation may play a role. In this regard, early postnatal binge-like ethanol treatment on postnatal days (PDs) 4-9 acutely inhibits whole cell GABA(A)R Cl(-) current and subsequently blunts GABA(A)R function in medial septum/diagonal band (MS/DB) neurons and cerebellar Purkinje cells [Dev. Brain Res. 130 (2001) 25-40; Brain Res. 810 (1998) 100-113; Brain Res. 832 (1999) 124-135]. In light of these functional changes, we hypothesized that ethanol treatment also would decrease levels of proteins important for assembly of GABAergic synapses in maturing brain. To test this relationship, binge-like ethanol intubation was administered to rat pups on PDs 4-9 producing peak blood ethanol concentrations in the range of 302.5+/-6.3 mg/dl. GABAergic synaptic proteins were measured in brain tissue on PDs 13-14 when GABA(A)R currents in individual MS/DB neurons are reduced, but those of cerebellar Purkinje neurons are not yet altered [Dev. Brain Res. 130 (2001) 25-40; Brain Res. 810 (1998) 100-113; Brain Res. 832 (1999) 124-135]. Surprisingly, ethanol did not decrease protein levels of GABA(A)R alpha1/beta2 subunits, GAD(67) or gephyrin in MS/DB at this time when whole cell recordings indicate GABA(A)R function is impaired in acutely dissociated individual neurons. However, in cerebellum where ethanol treated Purkinje cell GABA(A)R function remains normal on PDs 13-14 [Brain Res. 832 (1999) 124-135], reduced levels of several GABAergic synaptic proteins including: GAD(67), GABA(A)R alpha1 subunit, ClC-2 a voltage-gated Cl(-) channel, synaptotagmin a synaptic vesicle protein, and N-cadherin, a synapse associated cell adhesion molecule, were found. These results indicate that binge-like ethanol exposure differentially decreases GABAergic synaptic proteins in some brain areas in a pattern that does not parallel reductions in GABA(A)R function of individual neurons that survive this ethanol insult.

Single-cell RT-PCR detects shifts in mRNA expression profiles of basal forebrain neurons during aging

The medial septum and nucleus of the diagonal band (MS/nDB) contain cholinergic and GABAergic neuronal populations that have been identified based on immunohistochemical staining and/or electrophysiological properties. We explored the molecular diversity of MS/nDB neurons using single-cell reverse transcription-polymerase chain reaction (scRT-PCR) to assess gene expression profiles during aging in individual neurons acutely isolated from young (2-4 months) and aged (26-27 months) F344 rats. Neuronal gene expression profiles were characterized by detection of mRNAs for choline acetyltransferase (ChAT, cholinergic) and glutamate decarboxylase (GAD67, GABAergic), as well as mRNAs for calcium binding proteins (CaBPs) calbindin-D28k, calretinin and parvalbumin. Four major neuronal populations were identified: ChAT-positive (ChAT+) cells, GAD-positive (GAD+) cells, ChAT+/GAD+ cells and ChAT negative/GAD negative (ChAT-/GAD-) cells. With age, the percentage of cells expressing ChAT mRNA decreased from 53% in young to 40%, and the expression of GAD67 mRNA was reduced from 56 to 35% of the cells tested. The percentage of cells with detectable levels of both ChAT and GAD67 mRNA was reduced from 24% in young to 9% in aged. Concomitantly, the percentage of ChAT-/GAD- cells increased from 15 to 34% with age. Of the CaBPs, calretinin expression was observed most frequently in this study, and its detection decreased from 33 to 22% of the cells with age. Observations concerning the CaBPs were confirmed using in situ hybridization. These results suggest a shift in the mRNA expression profiles of MS/nDB neuronal populations during aging and exemplify the molecular diversity of cholinergic and GABAergic cells.

Postnatal apoptosis in cerebellar granule cells of homozygous leaner (tgla/tgla) mice

Leaner mice carry a homozygous, autosomal recessive mutation in the mouse CACNA1A gene encoding the α1A subunit of P/Q-type calcium channels, which results in an out-of-frame splicing event in the carboxy terminus of the α1A protein. Leaner mice exhibit severe ataxia, paroxysmal dyskinesia and absence seizures. Functional studies have revealed a marked decrease in calcium currents through leaner P/Q-type channels and altered neuronal calcium ion homeostasis in cerebellar Purkinje cells. Histopathological studies of leaner mice have revealed extensive postnatal cerebellar Purkinje and granule cell loss. We examined the temporospatial pattern of cerebellar granule cell death in the leaner mouse between postnatal days (P) 10 and 40. Our observations clearly indicate that leaner cerebellar granule cells die via an apoptotic process and that the peak time of neuronal death is P20. We did not observe a significant increase in microglial and astrocytic responses at P20, suggesting that glial responses are not a cause of neuronal cell death. We propose that the leaner cerebellar granule cell represents anin vivo animal model for low intracellular [Ca2+]-induced apoptosis. Since intracellular [Ca2+] is critical in the control of gene expression, it is quite likely that reduced intracellular [Ca2+] could activate a lethal cascade of altered gene expression leading to the apoptotic granule cell death in the leaner cerebellum.

Neuronal nitric oxide synthase expression in cerebellar mutant mice.

Nitric oxide (NO) is a diffusible, multifunctional signaling molecule found in many areas of the brain. NO signaling is involved in a wide array of neurophysiological functions including synaptogenesis, modulation of neurotransmitter release, synaptic plasticity, central nervous system blood flow and cell death. NO synthase (NOS) activity regulates the production of NO and the cerebellum expresses high levels of nitric oxide synthase (NOS) in granule, stellate and basket cells. Cerebellar mutant mice provide excellent opportunities to study changes of NO/NOS concentrations and activities to gain a greater understanding of the roles of NO and NOS in cerebellar function. Here, we have reviewed the current understanding of the functional roles of NO and NOS in the cerebellum and present NO/NOS activities that have been described in various cerebellar mutant mice and NOS knockout mice. NO appears to exert neuroprotective effects at low to moderate concentrations, whereas NO becomes neurotoxic as the concentration increases. Excessive NO production can cause oxidative stress to neurons, ultimately impairing neuronal function and result in neuronal cell death. Based on their genetics and cerebellar histopathology, some of cerebellar mutant mice display similarities with human neurological conditions and may prove to be valuable models to study several human neurological disorders, such as autism and schizophrenia.

Insulin-like growth factor-I improves cerebellar dysfunction but does not prevent cerebellar neurodegeneration in the calcium channel mutant mouse, leaner

The effects of insulin-like growth factor-I (IGF-I) on cerebellar dysfunction and neurodegeneration were investigated in leaner mice, which exhibit cerebellar ataxia and neurodegeneration related to P/Q-type calcium channel mutations. Leaner mice showed significantly reduced serum and cerebellar IGF-I concentrations compared to wild-type mice at postnatal day 30. Behavioral assessment of leaner mice injected with IGF-I subcutaneously for 4 weeks showed partially improved cerebellar function. Histological analysis of IGF-I treated leaner cerebella showed no difference in the number of dying Purkinje cells compared to control leaner cerebella. These results further support potential use of IGF-I as a therapeutic aid for cerebellar ataxia related to calcium channel mutations. Nonetheless, IGF-I administration does not rescue dying cerebellar neurons, which suggests that the beneficial effects of IGF-I may have been achieved through surviving cerebellar neurons.