Gamma- and UV-induced DNA synthesis in neurons of the rat neocortex

Nuclear DNA synthesis in neocortex neurons of neonatal 14- and 60-day rats after in vitro irradiation of isolated sections was estimated by the incorporation of a labeled precursor into DNA. gamma- and UV-radiation increased the rate of DNA synthesis in the cells of animals of all studied age groups. However, the level of the UV-induced synthesis sharply dropped during the postnatal ontogenesis while gamma-radiation-induced synthesis decreased slightly. The peculiarities revealed in the repair DNA synthesis seem to be influenced by the process of postnatal differentiation of a neuron accompanied by the nucleosome length shortening and the decrease in the DNA-polymerase alpha content.     

Low level incorporation of tritiated thymidine into the nuclear DNA of Purkinje neurons of adult mice.

Adult mice were pulse labeled with tritiated thymidine [3H]TdR and killed 9 hr later. A low level incorporation of [3H]TdT into the nuclear DNA of Purkinje neurons was found in autoradiographs. Enzymatic digestions with DNase and with RNase in combination with autoradiographic grain counts indicate that a portion of nuclear DNA is not stable in the Purkinje nucleus. These results are discussed in light of reports of the stable nature of DNA in Purkinje neurons of adult mice.     

Purkinje cell maturation in the light of RNA synthesis

Wistar rats 1- to 90-day-old received an injection of 3H-uridine and were killed 20 min to 44 h later. Autoradiographic examination revealed the highest grain count densities in Purkinje cell nuclei around postnatal day (PD) 6 while the incidence of labelled nuclei stayed at the peak values till PD 15. Silver staining of Purkinje cell nuclei showed that the expression of nucleolar r-RNA coding genes is maximal at PD 15; in some cells it even slightly exceeds adult values. After PD 15, the percentage of labelled Purkinje cell nuclei declined; this was more pronounced in the nucleolar region than outside the nucleolus. The percentage of cells with cytoplasmic labelling culminated on PD 15. The highest grain counts were found in Purkinje cell cytoplasm on PD 6 at 44 h p.i. interval. Reversal in nuclear grain counts at 2 and 6 h p.i. intervals observed between PD 15 and PD 25 suggests faster degradation, or processing and export, of a newly synthesized nuclear RNA in these age groups. Frequency distribution analysis of grain count densities revealed a small group of Purkinje cells with higher incorporation of 3H-uridine both in the nucleolar region and the whole nucleus at PD 15. In situ hybridization of 3H-r-RNA revealed a slight binding excess to DNA of some Purkinje cell nuclei but not in granule cells of 1-month-old rats. These data, together with those published recently by Brodsky et al. (1985), indicate an uneven structural organization and partial overexpression of the genom coding r-RNA synthesis in the population of Purkinje cells.     

Impaired calcium release in cerebellar Purkinje neurons maintained in culture

Cerebellar Purkinje neurons demonstrate a form of synaptic plasticity that, in acutely prepared brain slices, has been shown to require calcium release from the intracellular calcium stores through inositol trisphosphate (InsP(3)) receptors. Similar studies performed in cultured Purkinje cells, however, find little evidence for the involvement of InsP(3) receptors. To address this discrepancy, the properties of InsP(3)- and caffeine-evoked calcium release in cultured Purkinje cells were directly examined. Photorelease of InsP(3) (up to 100 microM) from its photolabile caged analogue produced no change in calcium levels in 70% of cultured Purkinje cells. In the few cells where a calcium increase was detected, the response was very small and slow to peak. In contrast, the same concentration of InsP(3) resulted in large and rapidly rising calcium responses in all acutely dissociated Purkinje cells tested. Similar to InsP(3), caffeine also had little effect on calcium levels in cultured Purkinje cells, yet evoked large calcium transients in all acutely dissociated Purkinje cells tested. The results demonstrate that calcium release from intracellular calcium stores is severely impaired in Purkinje cells when they are maintained in culture. Our findings suggest that cultured Purkinje cells are an unfaithful experimental model for the study of the role of calcium release in the induction of cerebellar long term depression.     

Determinants of postsynaptic Ca2+ signaling in Purkinje neurons

Neuronal integration in Purkinje neurons involves many forms of Ca2+ signaling. Two afferent synaptic inputs, the parallel and the climbing fibers, provide a major drive for these signals. These two excitatory synaptic inputs are both glutamatergic. Postsynaptically they activate alpha-amino-3-hydroxy-5-methyl-4-propionic acid (AMPA) receptors (AMPARs) and metabotropic glutamate receptors (mGluRs). Unlike most other types of central neurons, Purkinje neurons do not express NMDA (N-methyl-D-aspartate) receptors (NMDARs). AMPARs in Purkinje neurons are characterized by a low permeability for Ca2+ ions. AMPAR-mediated synaptic depolarization may activate voltage-gated Ca2+ channels, mostly of the P/Q-type. The resulting intracellular Ca2+ signals are shaped by the Ca2+ buffers calbindin and parvalbumin. Ca2+ clearance from the cytosol is brought about by Ca2+-ATPases in the plasma membrane and the endoplasmic reticulum, as well as the Na+-Ca2+-exchanger. Binding of glutamate to mGluRs induces postsynaptic Ca2+-transients through two G protein-dependent pathways: involving (1) the release of Ca2+ ions from intracellular Ca2+ stores and (2) the opening of the cation channel TRPC1. Homer proteins appear to play an important role in postsynaptic Ca2+ signaling by providing a direct link between the plasma membrane-resident elements (mGluRs and TRPC1) and their intracellular partners, including the IP3Rs.     

Phenothiazine antagonism of the noradrenergic inhibition of cerebellar Purkinje neurons.

Phenothiazine derivatives were examined as potential antagonists of the inhibitory noradrenergic synapses from the nucleus locus coeruleus to rat cerebellar Purkinje cells. Fluphenazine, and its thioxanthine analogue, flupenthixol, antagonized the inhibitory action of norepinephrine, when iontrophoretically applied to single cells. Alpha-flupenthixol was generally more active than the beta isomer. Fluphenazine had no appreciable effect on inhibitions induced by iontophoresis of GABA or cyclic AMP. Parenteral fluphenazine also blocked the inhibition of Purkinje cells produced by the stimulation of the noradrenergic pathway from locus coeruleus, but basket and stellate cell inhibitory inputs to Purkinje cells were unaffected. These data suggest that fluphenazine can specifically block a known central adrenergic inhibitory pathway.     

Myosin-Va transports the endoplasmic reticulum into the dendritic spines of Purkinje neurons

Extension of the endoplasmic reticulum (ER) into dendritic spines of Purkinje neurons is required for cerebellar synaptic plasticity and is disrupted in animals with null mutations in Myo5a, the gene encoding myosin-Va. We show here that myosin-Va acts as a point-to-point organelle transporter to pull ER as cargo into Purkinje neuron spines. Specifically, myosin-Va accumulates at the ER tip as the organelle moves into spines, and hydrolysis of ATP by myosin-Va is required for spine ER targeting. Moreover, myosin-Va is responsible for almost all of the spine ER insertion events. Finally, attenuation of the ability of myosin-Va to move along actin filaments reduces the maximum velocity of ER movement into spines, providing direct evidence that myosin-Va drives ER motility. Thus, we have established that an actin-based motor moves ER within animal cells, and have uncovered the mechanism for ER localization to Purkinje neuron spines, a prerequisite for synaptic plasticity.     

Physiological role of dendrotoxin-sensitive K+ channels in the rat cerebellar Purkinje neurons

To understand the contribution of potassium (K+) channels, particularly alpha-dendrotoxin (D-type)-sensitive K+ channels (Kv.1, Kv1.2 or Kv1.6 subunits), to the generation of neuronal spike output we must have detailed information of the functional role of these channels in the neuronal membrane. Conventional intracellular recording methods in current clamp mode were used to identify the role of alpha-dendrotoxin (alpha-DTX)-sensitive K+ channel currents in shaping the spike output and modulation of neuronal properties of cerebellar Purkinje neurons (PCs) in slices. Addition of alpha-DTX revealed that D-type K+ channels play an important role in the shaping of Purkinje neuronal firing behavior. Repetitive firing capability of PCs was increased following exposure to artificial cerebrospinal fluid (aCSF) containing alpha-DTX, so that in response to the injection of 0.6 nA depolarizing current pulse of 600 ms, the number of action potentials insignificantly increased from 15 in the presence of 4-AP to 29 action potentials per second after application of DTX following pretreatment with 4-AP. These results indicate that D-type K+ channels (Kv.1, Kv1.2 or Kv1.6 subunits) may contribute to the spike frequency adaptation in PCs. Our findings suggest that the activation of voltage-dependent K+ channels (D and A types) markedly affect the firing pattern of PCs.     

Large-conductance cationic channels in the nuclear envelope of Purkinje neurons from the rat cerebellum

Using a patch-clamp technique, we studied the biophysical properties of large-conductance channels in the nuclear envelope of rat cerebellar Purkinje neurons. Our experiments showed that channels with identical conductance, selectivity, and kinetics are expressed in the external and internal nuclear membranes of these cells. These channels connect the perinuclear space with the cyto-and nucleoplasm; they are not channels of the complex of the nuclear pores for passive diffusion of ions and small molecules, as was believed earlier [17]. We hypothesize that large-conductance cationic channels in the membranes of the nuclear envelope are identical to ion channels of the endoplasmic reticulum and are necessary for functioning of the intermembrane space of the envelope as a calcium store. Neirofiziologiya/Neurophysiology, Vol. 39, No. 2, pp. 113–118, March–April, 2007.     

Ryanodine and inositol trisphosphate receptors coexist in avian cerebellar Purkinje neurons

Two intracellular calcium-release channel proteins, the inositol trisphosphate (InsP3), and ryanodine receptors, have been identified in mammalian and avian cerebellar Purkinje neurons. In the present study, biochemical and immunological techniques were used to demonstrate that these proteins coexist in the same avian Purkinje neurons, where they have different intracellular distributions. Western analyses demonstrate that antibodies produced against the InsP3 and the ryanodine receptors do not cross-react. Based on their relative rates of sedimentation in continuous sucrose gradients and SDS-PAGE, the avian cerebellar InsP3 receptor has apparent native and subunit molecular weights of approximately 1,000 and 260 kD, while those of the ryanodine receptors are approximately 2,000 and 500 kD. Specific [3H]InsP3- and [3H]ryanodine-binding activities were localized in the sucrose gradient fractions enriched in the 260-kD and the approximately 500-kD polypeptides, respectively. Under equilibrium conditions, cerebellar microsomes bound [3H]InsP3 with a Kd of 16.8 nM and Bmax of 3.8 pmol/mg protein; whereas, [3H]ryanodine was bound with a Kd of 1.5 nM and a capacity of 0.08 pmol/mg protein. Immunolocalization techniques, applied at both the light and electron microscopic levels, revealed that the InsP3 and ryanodine receptors have overlapping, yet distinctive intracellular distributions in avian Purkinje neurons. Most notably the InsP3 receptor is localized in endomembranes of the dendritic tree, in both the shafts and spines. In contrast, the ryanodine receptor is observed in dendritic shafts, but not in the spines. Both receptors appear to be more abundant at main branch points of the dendritic arbor. In Purkinje neuron cell bodies, both the InsP3 and ryanodine receptors are present in smooth and rough ER, subsurface membrane cisternae and to a lesser extent in the nuclear envelope. In some cases the receptors coexist in the same membranes. Neither protein is observed at the plasma membrane, Golgi complex or mitochondrial membranes. Both the InsP3 and ryanodine receptors are associated with intracellular membrane systems in axonal processes, although they are less abundant there than in dendrites. These data demonstrate that InsP3 and ryanodine receptors exist as unique proteins in the same Purkinje neuron. These calcium-release channels appear to coexist in ER membranes in most regions of the Purkinje neurons, but importantly they are differentially distributed in dendritic processes, with the dendritic spines containing only InsP3 receptors.     

Interspike background activity in extracelullarly recorded Purkinje neurons: spectral analysis

The aim of this study was to investigate the spectral characteristics of Purkinje cell interspike background activity caused by the occurrence of particular action potentials or by electrically induced enhancement of cerebellar inhibitory and excitatory input drive. Spontaneously active Purkinje neurons were extracellularly recorded in anesthetized rats before and after cessation of stimulation from the inferior olive (10) or locus coeruleus (LC). After A/D conversion (30 kHz), direct spectral analysis of extracted interspike background activity was done. Our results have shown that, in contrast to simple spikes, the occurrence of complex spikes induces changes in the spectra of interspike background activity. The different spectral changes of interspike background activity induced by LC and 10 stimulation also indicated the importance of this extracellularly recorded phenomenon.     

Specific localized expression of cGMP PDEs in Purkinje neurons and macrophages

As cGMP hydrolyzing cyclic nucleotide phosphodiesterases (PDEs) have diverse regulatory and catalytic properties, the specific cGMP PDEs a cell expresses will determine the duration and intensity of a cGMP signal. This, in turn, results in different cellular responses between cell types and tissues. Therefore, identifying which cGMP PDEs are expressed in different tissues and cell types could increase our understanding of physiological and pathological processes. The brain is one area where large numbers of diverse cGMP PDEs are expressed in specific regions and cell types. A case in point is differential expression of cGMP PDEs in neuronal cells. For example, we have recently found that PDE5 is expressed in all Purkinje neurons while PDE1B is expressed in only a subset of these neurons. The expression of PDE2 has also been found to be selective for discrete populations of neurons. Another example of selective cGMP PDE expression is seen with cytokine-induced differentiation of monocytes to macrophages. We have recently discovered that monocyte differentiation with the cytokine macrophage colony-stimulating factor (M-CSF) causes an upregulation of PDE2 and a small increase in PDE1B while granulocyte-macrophage colony-stimulating factor (GM-CSF) causes a large increase in PDE1B but a decrease in PDE2. These same cytokines can influence the phenotype of microglial cells and are likely to affect their expression of cGMP PDEs. In this report, we present recent results from our laboratory and review earlier findings illustrating the concept of highly specific expression of cGMP PDEs and discuss how this may be important for understanding brain function and dysfunction.     

Dysfunction of amyloid precursor protein signaling in neurons leads to DNA synthesis and apoptosis

The classic neuropathological diagnostic markers for AD are amyloid plaques and neurofibrillary tangles, but their role in the etiology and progression of the disease remains incompletely defined. Research over the last decade has revealed that cell cycle abnormalities also represent a major neuropathological feature of AD. These abnormalities appear very early in the disease process, prior to the appearance of plaques and tangles; and it has been suggested that neuronal cell cycle regulatory failure may be a significant component of the pathogenesis of AD. The amyloid precursor protein (APP) is most commonly known as the source of the beta-amyloid (Abeta) peptides that accumulate in the brains of patients with AD. However, a large body of work supports the idea that APP is also a signaling receptor. Most recently, it has been shown that familial AD (FAD) mutations in APP or simple overexpression of wild type APP cause dysfunction of APP signaling, resulting in initiation of DNA synthesis in neurons and consequent apoptosis. In this article, we review the evidence that APP has the potential to activate aberrant neuronal cell cycle re-entry in AD, and we describe a signal transduction pathway that may mediate this abnormal activation of the cell cycle.     

Stable reprogrammed heterokaryons form spontaneously in Purkinje neurons after bone marrow transplant

Heterokaryons are the product of cell fusion without subsequent nuclear or chromosome loss. Decades of research using Sendai-virus or polyethylene glycol (PEG)-mediated fusion in tissue culture showed that the terminally differentiated state of a cell could be altered. But whether stable non-dividing heterokaryons could occur in animals has remained unclear. Here, we show that green fluorescent protein (GFP)-positive bone-marrow-derived cells (BMDCs) contribute to adult mouse Purkinje neurons through cell fusion. The formation of heterokaryons increases in a linear manner over 1.5 years and seems to be stable. The dominant Purkinje neurons caused the BMDC nuclei within the resulting heterokaryons to enlarge, exhibit dispersed chromatin and activate a Purkinje neuron-specific transgene, L7-GFP. The observed reprogrammed heterokaryons that form in brain may provide insights into gene regulation associated with cell-fate plasticity.     

A long-term depression of AMPA currents in cultured cerebellar Purkinje neurons.

Cerebellar long-term depression (LTD) is a model of synaptic plasticity in which conjunctive stimulation of parallel fiber and climbing fiber inputs to a Purkinje neuron induces a persistent depression of the parallel fiber-Purkinje neuron synapse. We report that an analogous phenomenon may be elicited in the cultured mouse Purkinje neuron when iontophoretic glutamate application and depolarization of the Purkinje neurons are substituted for parallel fiber and climbing fiber stimulation, respectively. The induction of LTD in these cerebellar cultures requires activation of both ionotropic (AMPA) and metabotropic quisqualate receptors, together with depolarization in the presence of external Ca2+. This postsynaptic alteration is manifest as a depression of glutamate or AMPA currents, but not aspartate or NMDA currents. These results strengthen the contention that the expression of cerebellar LTD is at least in part postsynaptic and provide evidence that activation of both ionotropic and metabotropic quisqualate receptors are necessary for LTD induction.     

Calcium requirement of long-term depression and rebound potentiation in cerebellar Purkinje neurons

Cerebellar Purkinje neurons (PNs) receive two main excitatory inputs, from climbing fibers and parallel fibers, and inhibitory inputs, from GABAergic interneurons. The synapses formed by parallel fibers and by inhibitory interneurons on PNs are able to undergo long-lasting in efficacy. Thus, the excitatory parallel fiber-PN synapse undergoes long-term fibers. Synaptic inhibition can be potentiated by climbing fiber activity by a mechanism named rebound potentiation, resulting in a more powerful inhibitory effect of GABAergic interneurons. The induction of both long-term depression and rebound potentiation requires a transient elevation of the cytoplasmic calcium concentration ([Ca²⁺]_i). The [Ca²⁺]_i-transient is caused by Ca²⁺ entry through voltage-gated Ca²⁺ channels and, possibly, by release of Ca²⁺ from IP_3- and ryanodine-sensitive stores. Direct Ca²⁺ entry through synaptic AMPA receptor channels seems not to contribute significantly to the Ca²⁺ signal mediating the induction of both long-term depression and rebound potentiation.