Functional Crosstalk Between Cell-Surface and Intracellular Channels Mediated by Junctophilins Essential for Neuronal Functions

Junctophilins (JPs) contribute to the formation of junctional membrane complexes between the plasma membrane and the endoplasmic/sarcoplasmic reticulum, and provide a structural platform for channel communication during excitation–contraction coupling in muscle cells. In the brain, two neuronal JP subtypes are widely expressed in neurons. Recent studies have defined the essential role of neural JPs in the communication between cell-surface and intracellular channels, which modulates the excitability and synaptic plasticity of neurons in the cerebellum and hippocampus.     

Neurons and glial cells differentially express P2Y receptor mRNAs in the rat dorsal root ganglion and spinal cord

We examined the precise distribution of mRNAs for six cloned rat P2Y receptor subtypes, P2Y1, P2Y2, P2Y4, P2Y6, P2Y12, and P2Y14, in the dorsal root ganglion (DRG) and spinal cord by in situ hybridization histochemistry (ISHH) with 35S-labeled riboprobes. In the DRG, P2Y1 and P2Y2 mRNAs were expressed by 15% and 24% of all neurons, respectively. Although each receptor was evenly distributed between neurofilament-positive and -negative neurons, P2Y2 was rather selectively expressed by TrkA-positive neurons. Schwann cells expressed P2Y2 mRNA, and the nonneuronal cells around the DRG neurons, perhaps the satellite cells, expressed P2Y12 and P2Y14 mRNAs. No ISHH signals for P2Y4 or P2Y6 were seen in any cellular components of the DRG. In the spinal cord, P2Y1 and P2Y4 mRNAs were expressed by some of the dorsal horn neurons, whereas the motor neurons in the ventral horn had P2Y4 and P2Y6 mRNAs. In addition, astrocytes in the gray matter had P2Y1 mRNA, and the microglia throughout the spinal cord expressed P2Y12 mRNA. P2Y14 mRNA was weakly expressed by putative microglia. These findings should provide useful information in interpreting pharmacological and electrophysiological studies in this field given the lack of highly selective antagonists for each P2Y receptor subtype.     

Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs within midbrain dopamine neurons

Many behavioral effects of nicotine result from activation of nigrostriatal and mesolimbic dopaminergic systems. Nicotine regulates dopamine release not only by stimulation of nicotinic acetylcholine receptors (nAChRs) on dopamine cell bodies within the substantia nigra and ventral tegmental area (SN/VTA), but also on presynaptic nAChRs located on striatal terminals. The nAChR subtype(s) present on both cell bodies and terminals is still a matter of controversy. The purpose of this study was to use double-labeling in situ hybridization to identify nAChR subunit mRNAs expressed within dopamine neurons of the SN/VTA, by using a digoxigenin-labeled riboprobe for tyrosine hydroxylase as the dopamine cell marker and (35)S-labeled riboprobes for nAChR subunits. The results reveal a heterogeneous population of nAChR subunit mRNAs within midbrain dopamine neurons. Within the SN, almost all dopamine neurons express alpha2, alpha4, alpha5, alpha6, beta2, and beta3 nAChR mRNAs, with more than half also expressing alpha3 and alpha7 mRNAs. In contrast, less than 10% express beta4 mRNA. Within the VTA, a similar pattern of nAChR subunit mRNA expression is observed except that most subunits are expressed in a slightly lower percentage of dopamine neurons than in the SN. Within the SN, alpha4, beta2, alpha7, and beta4 mRNAs are also expressed in a significant number of nondopaminergic neurons, whereas within the VTA this only occurs for beta4. The heterogeneity in the expression of nAChR subunits within the SN/VTA may indicate the formation of a variety of different nAChR subtypes on cell bodies and terminals of the nigrostriatal and mesolimbic pathways.     

Rat oligodendrocytes and astrocytes preferentially express fibroblast growth factor receptor-2 and -3 mRNAs

Fibroblast growth factors (FGFs) exert various effects on glial cells as well as on neurons in the brain. The mRNAs for four FGF receptors (FGFR-1-FGFR-4) are expressed in the brain. Although FGFR-1 and -4 mRNAs are preferentially expressed in neurons, FGFR-2 and -3 mRNAs are preferentially expressed in glial cells. However, the glial cells that express these receptors remained to be identified. In this study, we found that oligodendrocytes and astrocytes in the brain preferentially expressed FGFR-2 and FGFR-3 mRNAs, respectively. The isoforms of immunoglobulin-like domain III (IIIb and IIIc) of the receptors have crucial roles in ligand binding. We also determined the isoforms of FGFR-2 and FGFR-3 expressed in glial cells to be of type IIIc. The expression of FGFR-2 IIIc and FGFR-3 IIIc with different ligand specificities might play important roles in the various effects of FGFs on oligodendrocytes and astrocytes. © 1996 Wiley-Liss, Inc.     

Differential expression patterns of mRNAs for members of the fibroblast growth factor receptor family,FGFR-1–FGFR-4, in rat brain

We have examined the region-specific expression of mRNAs for four members of rat FGF receptor family, FGFR-1, FGFR-2, FGFR-3, and FGFR-4, in rat brain by in situ hybridization. The FGFR-1, FGFR-2, and FGFR-3 mRNAs were expressed widely but differentially in the brain. However, the FGFR-4 mRNA was not expressed in the brain. The FGFR-1 mRNA was strongly expressed in several regions including the hippocampus, cerebellum, and pedunculoptine tegmental nucleus. The FGFR-2 mRNA expression was high in the choroid plexus, and moderate in the fiber-rich regions (the corpus callosum, external capsule, and internal capsule) and the olfactory bulb. The FGFR-3 mRNA was expressed diffusely in the brain. We have also examined the cellular localization of these mRNAs in the brain. Although the FGFR-1 mRNA was expressed preferentially in neurons, the FGFR-2 and FGFR-3 mRNAs were expressed preferentially in glial cells. The present findings that the FGFR-1, FGFR-2, and FGFR-3 mRNAs were expressed widely but with region-and cell-specificity in the brain indicate that these receptors have different roles in the brain. © 1994 Wiley-Liss, Inc.     

The topographic anatomy of the interpeduncular cistern and endoscopic ventriculocisternostomy in the region of the bottom of the third ventricle

The interpeduncular cistern was microanatomically studied on 14 anatomic specimens of the brain. It was divided into 2 parts: superficial (free) and deep (vascular). The upper interpeduncular cistern wall was divided into hypothalamic and mesencephalic parts. The interpeduncular cistern is connected with the ambient, pretontine, carotid, cerebellopontine, oculomotor, and peduncular cisterns. It is a composite space-occupying, structural formation. Liliequist's membrane is the basic membranous component of a cistern. The proposed division makes it possible to study different parts of the interpeduncular cistern qualitatively and quantitatively and to define clear topographic and anatomic criteria as a guideline in this field.     

Transient expression of GABAA receptor alpha2 and alpha3 subunits in differentiating cerebellar neurons

In the adult mammalian brain, synaptic transmission mediated by gamma-amino butyric acid (GABA) plays a role in inhibition of excitatory synaptic transmission. During brain development, GABA is involved in brain morphogenesis. To clarify how GABA exerts its effect on immature neurons, we examined the expression of the GABAA receptor alpha2 and alpha3 subunits, which are abundantly expressed before alpha1 and alpha6 subunits appear, in the developing mouse cerebellum using in situ hybridization. Proliferating neuronal precursors in the ventricular zone and external granular layer expressed neither alpha2 nor alpha3 subunits. Hybridization signals for the alpha2 and alpha3 subunit mRNAs first appeared in the differentiating zone at embryonic day 13 (E13). The alpha2 subunit was detected in the migrating and differentiating granule cells and cerebellar nucleus neurons until postnatal day 14 (P14). Hybridization signals for the alpha3 subunit mRNA, on the other hand, were localized in the developing Purkinje cells and cerebellar nucleus neurons, and disappeared from Purkinje cells by the end of first postnatal week. Taken together, this indicated that the alpha2 and alpha3 subunits were abundantly expressed in distinct types of cerebellar neurons after completing cell proliferation while forming the neural network. These results suggest that GABA might extrasynaptically activate the GABAA receptors containing alpha2 and/or alpha3 subunits on the differentiating neurons before finishing the formation of synapses and networks, and could be involved in neuronal differentiation and maturation in the cerebellum.     

Distribution and isoform diversity of the organellar Ca2+ pumps in the brain

The gene family of organellar-type Ca²⁺ transport ATPases consists of three members. SERCA1 is expressed exclusively in fast skeletal muscle; SERCA2 is ubiquitously expressed, whereas SERCA3 is considered to be mainly expressed in cells of the hematopoietic lineage and in some epithelial cells. In the brain, the organellar-type Ca²⁺ transport ATPases are almost exclusively transcribed from the SERCA2 gene. Four different SERCA2 mRNAs have been described (classes 1–4). However, unlike in nonneuronal cells, which express the class 1, 2, and 3 splice variants, the main SERCA2 mRNA in the brain is the class 4 messenger. Similar to classes 2 and 3, the class 4 codes for the ubiquitously expressed SERCA2b protein. Recently, we have reported the distribution of the SERCA isoforms in the brain (Baba-Aissa et al., 1996a,b). SERCA2b was present in most neurons of all investigated brain regions. The highest levels were found in the Purkinje neurons of the cerebellum and in the pyramidal cells of the hippocampus. Interestingly, SERCA3 and SERCA2a are coexpressed along with SERCA2b in the Purkinje neurons, but are weakly expressed in the other brain regions if present at all. Since these three protein isoforms have a different affinity for Ca²⁺, their possible roles in relation to Ca²⁺ stores in neurons are discussed.     

Neurons and plaques of Alzheimer's disease patients highly express the neuronal membrane docking protein p42IP4/centaurin alpha

The protein p42(IP4) (also called centaurin alpha), identified as a brain-specific InsP4/PtdInsP3 (PIP3)-binding protein, has been shown to be localized in human brain, specifically expressed in neurons. Several casein kinases have been found to be involved in Alzheimer's disease (AD) pathology. Since casein kinase I was reported to possess a binding domain for p42(IP4), we here investigated the expression and localization of p42(IP4) in AD brains. In cortical neurons of AD brains intracellular immunostaining for p42(IP4) exceeded the level seen in these neurons of normal brain. Statistically, significantly more p42(IP4)-immunoreactive neurons were found in temporal and angular cortex of AD patients as compared to control brain. Mostly impressively, neuritic plaques displayed a very prominent signal. Thus, we suggest that the up-regulated p42(IP4) in AD neurons may serve as a docking protein to recruit signaling molecules such as different subtypes of casein kinase I to the plasma membrane. This is the first indication for a functional interaction of these protein in possible neuronal damage. Therefore proteins such as p42(IP4), central players in signaling, may be appropriate targets for preventing neurodegenerative processes.     

Molecular cloning of rat cDNAs for β and γ subtypes of 14-3-3 protein and developmental changes in expression of their mRNAs in the nervous system

We isolated cDNAs to β and γ subtypes of 14-3-3 protein, a putative regulatory protein for protein kinase C, from the brain and clarified a high homology in sequences of nucleotides and deduced amino acids between the two rat subtypes and the bovine counterparts and even reciprocally between the two rat subtypes. In Northern blot analysis, the gene expression of the two subtypes was detected weakly at E13, increased progressively after birth and reached a maximum at P7–P14. Thereafter it decreased slightly. In situ hybridization analysis allowed detection of the β but not the γ subtype in the matrix cells of the ventricular germinal zone of the neural wall. In post-mitotic neurons in the mantle zone and maturing brain loci, genes of the two subtypes were expressed in patterns similar to each other, and three neuron types were identified: type I neurons with high levels of expression throughout development; type II neurons showing high expression during the early developmental stages with a subsequent decrease in the expression at maturing and adult stages; and type III neurons showing consistently low levels of expression throughout development. The wider and more highly-patterned expression of the 14-3-3 protein family than expected suggests that this protein may be involved in the elaborate regulation of some fundamental cellular activities and differentiation of neurons.     

Scorpion alpha and alpha-like toxins differentially interact with sodium channels in mammalian CNS and periphery

Scorpion alpha-toxins from Leiurus quinquestriatus hebraeus, LqhII and LqhIII, are similarly toxic to mice when administered by a subcutaneous route, but in mouse brain LqhII is 25-fold more toxic. Examination of the two toxins effects in central nervous system (CNS), peripheral preparations and expressed sodium channels revealed the basis for their differential toxicity. In rat brain synaptosomes, LqhII binds with high affinity, whereas LqhIII competes only at high concentration for LqhII-binding sites in a voltage-dependent manner. LqhII strongly inhibits sodium current inactivation of brain rBII subtype expressed in HEK293 cells, whereas LqhIII is weakly active at 2 microM, suggesting that LqhIII affects sodium channel subtypes other than rBII in the brain. In the periphery, both toxins inhibit tetrodotoxin-sensitive sodium current inactivation in dorsal root ganglion neurons, and are strongly active directly on the muscle and on expressed muI channels. Only LqhII, however, induced repetitive end-plate potentials in mouse phrenic nerve-hemidiaphragm muscle preparation by direct effect on the motor nerve. Thus, rBII and sodium channel subtypes expressed in peripheral nervous system (PNS) serve as the main targets for LqhII but are mostly not sensitive to LqhIII. Toxicity of both toxins in periphery may be attributed to the direct effect on muscle. Our data elucidate, for the first time, how different toxins affect mammalian central and peripheral excitable cells, and reveal unexpected subtype specificity of toxins that interact with receptor site 3.     

Effect of penicillin on an excitatory synapse

When penicillin, an epileptogenic agent, was applied to the neuromuscular junctions of the superficial flexor muscles of crayfish, the excitatory junctional potential (EJP) amplitudes were increased by 50–200%. This effect of the drug was not due to changes in the passive electrical properties of the muscle cell membrane or to an increase in its chemical sensitivity to acetylcholine (ACh), the presumed transmitter at the junction studied. Inactivating the penicillin with the enzyme penicillinase, or substituting acetate for penicillin in the test solutions eliminated the effect on EJPs, showing that the penicillin ion was the active agent. Penicillin ions did decrease the frequency of spontaneous miniature EJPs and increase the amplitude or presynaptic spikes recorded extracellularly, suggesting that augmentation of EJPs may have been due to alterations at the presynaptic nerve terminals.     

Regulation of RGS proteins by chronic morphine in rat locus coeruleus

The present study explored a possible role for RGS (regulators of G protein signalling) proteins in the long term actions of morphine in the locus coeruleus (LC), a brainstem region implicated in opiate physical dependence and withdrawal. Morphine influences LC neurons through activation of micro -opioid receptors, which, being Gi/o-linked, would be expected to be modulated by RGS proteins. We focused on several RGS subtypes that are known to be expressed in this brain region. Levels of mRNAs encoding RGS2, -3, -4, -5, -7, -8 and -11 are unchanged following chronic morphine, but RGS2 and -4 mRNA levels are increased 2-3-fold 6 h following precipitation of opiate withdrawal. The increases in RGS2 and -4 mRNA peak after 6 h of withdrawal and return to control levels by 24 h. Immunoblot analysis of RGS4 revealed a striking divergence between mRNA and protein responses in LC: protein levels are elevated twofold following chronic morphine and decrease to control values by 6 h of withdrawal. In contrast, levels of RGS7 and -11 proteins, the only other subtypes for which antibodies are available, were not altered by these treatments. Intracellular application of wild-type RGS4, but not a GTPase accelerating-deficient mutant of RGS4, into LC neurons diminished electrophysiological responses to morphine. The observed subtype- and time-specific regulation of RGS4 protein and mRNA, and the diminished morphine-induced currents in the presence of elevated RGS4 protein levels, indicate that morphine induction of RGS4 could contribute to aspects of opiate tolerance and dependence displayed by LC neurons.     

Axotomy increases plasma membrane Ca2+ pump isoform4 in primary afferent neurons

The expression of plasma membrane Ca(2+)-ATPase, a calcium pump located in cell membrane regulating intracellular Ca(2+) levels by Ca(2+) extrusion from cells, was examined in dorsal root ganglion neurons in naive rats and after spinal nerve ligation. The mRNAs and proteins in plasma membrane Ca(2+)-ATPase 1-3 were expressed in all size neurons with intense labeling in medium to large neurons. After spinal nerve ligation, these three isoforms showed downregulation of their expression. In contrast, plasma membrane Ca(2+)-ATPase 4 was expressed mainly in small neurons, and the number and signal intensity were significantly increased after spinal nerve ligation. These data suggest that plasma membrane Ca(2+)-ATPase isoforms have a distinct pattern of expression and regulation by axotomy in dorsal root ganglion neurons in normal and pathological conditions.     

Expression of m1-m4 muscarinic acetylcholine receptor immunoreactivity in septohippocampal neurons and other identified hippocampal afferents

Muscarinic cholinergic transmission plays an important role in modulating hippocampal activity and many higher brain functions. Many of the modulatory effects of acetylcholine on hippocampal function result from direct effects in the hippocampus or from actions on the hippocampal afferent neurons. At each site, the differential expression of a family of five distinct but related receptor substypes governs the nature of the response. The aim of the present study was to identify the subtypes expressed in the hippocampal afferent neurons by combining retrograde tracing with immunocytochemistry. The retrograde tracer, wheat germ agglutinin conjugated to horseradish peroxidase, was injected into the hippocampus unilaterally to label afferent neurons, and was combined with muscarinic (m) acetylcholine (ACh) receptors (mAChRs) with immunocytochemistry to identify the m1-m4 subtypes expressed. The retrogradely labeled cells in the basal forebrain that contribute to the septohippocampal pathway were found to express m2, m3, and, to a lesser extent, m1. Commissural/associational pathway neurons, which were identified by retrogradely labeled cells in the ipsi- and contralateral dentate gyrus, expressed m1, m3, and m4. The retrogradely labeled cells in the entorhinal cortex of the perforant pathway expressed predominantly m1 and m3, with fewer neurons expressing m2 and m4. Raphe-hippocampal cells were found to express m1. Thus, this study provides evidence for the diversity of mAChR subtypes expressed in neurons that project to the hippocampus. The complex modulation by acetylcholine of hippocampal function, therefore, is governed not only by the variety of mAChRs expressed in the hippocampus but also by their differential expression in extrinsic hippocampal afferents. © 1996 Wiley-Liss, Inc.