Use of Confocal Microscope for the Cellular Analysis of the Glycine Synaptic Receptor

Abstract

The presence of glycine receptors was examined with a monoclonal antibody and indirect immunofluorescence on reticular neurons of the goldfish (Carassius auratus) brainstem. Images of thin (0.6μm) optical sections were recorded from 80μm thick specimen with a confocal microscope thus obviating the need for mechanical slicing. Due to the reduced out-of-focus noise, high resolution was obtained. Lookthrough projections were computer generated. Compared with classical methods involving serial sectioning, this approach allowed the analysis of the subcellular distribution of this receptor with a considerable gain of time and increased resolution. On the Mauthner cell, an identified reticulo-spinal neuron, we found, that the size of glycine receptor microdomains varies depending on the cellular localization, i.e. somatic or dendritic. Furthermore the intensity of fluorescence was uneven within individual clusters, probably reflecting differences in receptor concentration. These heterogeneities may influence the variance of synaptic inhibitory noise in different regions of the Mauthner cell.     

Identification and localization of ryanodine binding proteins in the avian central nervous system

Ryanodine binding proteins of the CNS have been identified using monoclonal antibodies against avian skeletal muscle ryanodine binding proteins. These proteins were localized to intracellular membranes of the dendrites, perikarya, and axons of cerebellar Purkinje neurons using laser confocal microscopy and immunoelectron microscopy. Ryanodine binding proteins were not found in dendritic spines. Immunoprecipitation and [3H]epiryanodine binding experiments revealed that the cerebellar ryanodine binding proteins have a native molecular weight of approximately 2000 kd and are composed of two high molecular weight (approximately 500 kd) polypeptide subunits. A comparable protein having a single high molecular weight polypeptide subunit was observed in the remainder of the brain. If the ryanodine binding proteins in muscle and nerve are similar in function, then the neuronal proteins may participate in the release of calcium from intracellular stores that are mechanistically and spatially distinct from those gated by inositol trisphosphate receptors.     

Prostaglandin E₂ regulates AMPA receptor phosphorylation and promotes membrane insertion in preoptic area neurons and glia during sexual differentiation

Sexual differentiation of the rodent brain is dependent upon the organizing actions of the steroid hormone, estradiol. In the preoptic area, a brain region critical for the expression of adult reproductive behavior, there are twice as many dendritic spine synapses per unit length on newborn male neurons compared to female neurons and this sex difference correlates with the expression of adult male copulatory behavior. The sex difference in the POA is achieved via estradiol''s upregulation of the membrane-derived lipid signaling molecule prostaglandin E₂ (PGE₂); PGE₂ is necessary and sufficient to masculinize both dendritic spine density and adult sexual behavior in rats. We have previously shown that PGE₂ activates EP₂ and EP₄ receptors which increases protein kinase A (PKA) activity and that masculinized dendritic spine density and sex behavior are both dependent upon PKA as well as activation of AMPA type glutamate receptors. In the current experiments, we build upon this signaling cascade by determining that PGE₂ induces phosphorylation of the AMPA receptor subunit, GluR1, which leads to increased AMPA receptor insertion at the membrane. Treating female pups on the day of birth with PGE₂ induced the phosphorylation of GluR1 at the PKA-sensitive site within 2 hours of treatment, an effect that was blocked by co-administration of the PKA/AKAP inhibitor, HT31 with PGE₂. Brief treatment of mixed neuronal/glial POA cultures with PGE₂ or the cAMP/PKA stimulator, forskolin, increased membrane associated GluR1 in both neurons and glia. We speculate that PGE₂ induced increases in AMPA receptor associated with the membrane underlies our previously observed increase in dendritic spine density and is a critical component in the masculinization of rodent sex behavior.     

Increased Density of Dystrophin Protein in the Lateral Versus the Vermal Mouse Cerebellum

Dystrophin, present in muscle, also resides in the brain, including cerebellar Purkinje neurons. The cerebellum, although historically associated with motor abilities, is also implicated in cognition. An absence of brain dystrophin in Duchenne muscular dystrophy (DMD) and in the mdx mouse model results in cognitive impairments. Localization studies of cerebellar dystrophin, however, have focused on the vermal cerebellum, associated with motor function, and have not investigated dystrophin distribution in the lateral cerebellum, considered to mediate cognitive function. The present study examined dystrophin localization in vermal and lateral cerebellar regions and across subcellular areas of Purkinje neurons in the mouse using immunohistochemistry. In both vermal and lateral cerebellum, dystrophin was restricted to puncta on somatic and dendritic membranes of Purkinje neurons. The density of dystrophin puncta was greater in the lateral than the vermal region. Neither the size of puncta nor the area of Purkinje neuron somata differed between regions. Results support the view that cognitive deficits in the DMD and the mdx model may be mediated by the loss of dystrophin, particularly in the lateral cerebellum. Findings have important implications for future studies examining the neurophysiological sequelae of neuronal dystrophin deficiency and the role of the lateral cerebellum in cognition.     

Antibodies Specific for α-Subunit Subtypes of GABAA Receptors Reveal Brain Regional Heterogeneity

Abstract: Antisera were produced in rabbits against synthetic peptides based on subtype-specific regions of the cDNA sequences of the α1, α2, α3, and α4 (also termed α5) subunits of mammalian GABA_A receptors. The antigen peptides were chosen from the putative cytoplasmic loop between the proposed third and fourth membrane spanning helices; they were not only subtype-specific sequences, but also their hydrophilicity and predicted secondary structures suggested high potential antigenicity. In all cases, antipeptide antisera recognized on western blots the corresponding α-subunit polypeptide of the GABA_A receptors purified from bovine brain by benzodiazepine-affinity chromatography, and were able to immunoprecipitate binding activity from detergent-solubilized purified receptors. The four antisera each recognized a unique polypeptide, and only one, in the purified receptor, with α1, α2, α3, and α4 identified at 51, 52, 56, and 57 kDa, respectively. This represents the first identification of the α4 gene product on a gel. Both the relative amount of staining in immunoblots and the fraction of receptor binding that could be immunoprecipitated by saturating concentrations of each of the four subtypespecific antibodies varied in a consistent manner between receptors purified from different brain regions. Thus, cerebral cortex receptor contained all four α polypeptides on western blots, and significant activity could be precipitated by all four. Hippocampal receptor lacked α3 immunoreactivity on blotting and by immunoprecipitation; α1 was less, whereas both α2 and α4 were more abundant in hippocampus than in cortex by both techniques. Cerebellum receptor contained only α1 of the four α subunits tested, and the anti-α1 antibodies immunoprecipitated >90% of the binding activity. The variable amounts of staining and immunoprecipitation from the three brain areas by the four antisera demonstrate the presence of heterooligomeric receptor complexes with different α-subunit constituents in cortex, hippocampus, and cerebellum. The sum of cortical receptor activity precipitated individually by the four anti-α antisera was > 150%, indicating that some heterooligomers are likely to contain more than one class of α subtype, although most receptor complexes probably contain only one α subtype. These α-subunit subtype-specific antibodies should be useful in analyzing structure, function, and localization of GABA_A/benzodiazepine receptors in mammalian brain.     

Contribution of extrasynaptic N-methyl-d-aspartate and adenosine A1 receptors in the generation of dendritic glutamate-mediated plateau potentials

Thin basal dendrites can strongly influence neuronal output via generation of dendritic spikes. It was recently postulated that glial processes actively support dendritic spikes by either ceasing glutamate uptake or by actively releasing glutamate and adenosine triphosphate (ATP). We used calcium imaging to study the role of NR2C/D-containing N-methyl-d-aspartate (NMDA) receptors and adenosine A1 receptors in the generation of dendritic NMDA spikes and plateau potentials in basal dendrites of layer 5 pyramidal neurons in the mouse prefrontal cortex. We found that NR2C/D glutamate receptor subunits contribute to the amplitude of synaptically evoked NMDA spikes. Dendritic calcium signals associated with glutamate-evoked dendritic plateau potentials were significantly shortened upon application of the NR2C/D receptor antagonist PPDA, suggesting that NR2C/D receptors prolong the duration of calcium influx during dendritic spiking. In contrast to NR2C/D receptors, adenosine A1 receptors act to abbreviate dendritic and somatic signals via the activation of dendritic K⁺ current. This current is characterized as a slow-activating outward-rectifying voltage- and adenosine-gated current, insensitive to 4-aminopyridine but sensitive to TEA. Our data support the hypothesis that the release of glutamate and ATP from neurons or glia contribute to initiation, maintenance and termination of local dendritic glutamate-mediated regenerative potentials.     

Electrophysiological and Morphological Properties of Neurons Acutely Isolated from the Rostral Gustatory Zone of the Rat Nucleus of the Solitary Tract

The biophysical and morphological characteristics of acutelyisolated neurons from the rostral nucleus of the solitary tract(rNST) were investigated under current clamp conditions andcompared with the results obtained from neurons recorded inbrain slices. The passive membrane properties of the isolatedneurons were similar to rNST neurons in brain slices and theneurons maintained their morphological characteristics althoughtheir dendritic tree was truncated. The isolated neurons alsoretained their characteristic repetitive firing properties.In addition we also noted developmental changes in the intrinsicmembrane properties of the isolated neurons, such as a shorteningin action potential duration, decrease in membrane time constantand input resistance, that occurred when these parameters werecompared in neurons isolated from young (5–10 days) andolder animals. These enzymatically dispersed neurons thereforeretained both the membrane properties and morphology observedin the intact brainstem and in vitro brain slice preparation.The use of this isolated neuron preparation provides a basisfor further study of rNST neurobiology. Chem. Senses 21: 729–737,1996     

Masking of epitopes in tissue sections

Summary Antisera to chicken brain antigen (CBA) isolated by hydroxyapatite chromatography from 8 M urea extracts following repeated extractions with phosphate buffer selectively decorate neurofilaments (NF) in neuronal perikarya, dendrites and axons. The antisera also reacted with GFA protein, the astrocyte-specific intermediate filament protein, as indicated by the adsorption of NF immunoreactivity following passage of the antisera through columns prepared with purified GFA protein. Moreover, the antisera stained the polypeptides of the NF triplet (70 kd, 150 kd, 200 kd) and GFA protein by the immunoblotting procedure. Monoclonal antibodies selectively decorating NF in tissue sections were isolated from a fusion of mouse myeloma cells with spleen cells of mice immunized with CBA. By the immunoblotting procedure the antibodies decorated the 150 kd NF polypeptide and GFA protein. No staining of glial filaments or any other structure on tissue sections was also observed with antibodies derived from another fusion strongly reacting with GFA protein on immunoblots. All antibodies (monoclonal and polyclonal) appeared to react with the same region of the GFA polypeptide as indicated by immunoblots of cleavage products.     

Effects of hCG on reduced numbers of hCG receptors in the prefrontal cortex and cerebellum of rat models of Alzheimer’s disease

ABSTRACT

Age-associated changes in the levels of luteinizing hormone and human chorionic gonadotropin (hCG) are potential risk factors for Alzheimer’s disease (AD); hCG concentration is related to the incidence of AD. The highest density of hCG receptors is in zones of the brain that are vulnerable to AD and streptozotocin (STZ) can decrease the density of this receptor. We investigated the effects of different doses of hCG on hCG receptor density in the prefrontal cortex and cerebellum in a rat model of STZ-induced AD. AD was induced by intracerebroventricular injection of 3 mg/kg STZ. The resulting AD rats were treated for 3 days with 50, 100 or 200 IU/200 μl hCG, or with saline as a control. Sections of prefrontal cortex and cerebellum were stained immunohistochemically and hCG receptor-immunoreactive (ir) neurons were counted. STZ injected into the lateral ventricles of rat brains reduced the density of hCG receptor-ir neurons in the prefrontal cortex and cerebellum. hCG administration resulted in a significant dose-dependent increase in the number of hCG receptor-ir neurons in the prefrontal cortex and cerebellum. The maximum increase in the number of receptors occurred following the 200 IU dose of hCG. Administration of hCG ameliorated the lowered density of hCG receptor-ir neurons in the cerebellum and prefrontal cortex in STZ-induced AD rats.     

Localisation of Formyl-Peptide Receptor 2 in the Rat Central Nervous System and Its Role in Axonal and Dendritic Outgrowth

Arachidonic acid and docosahexaenoic acid (DHA) released by the action of phospholipases A₂ (PLA₂) on membrane phospholipids may be metabolized by lipoxygenases to the anti-inflammatory mediators lipoxin A4 (LXA4) and resolvin D1 (RvD1), and these can bind to a common receptor, formyl-peptide receptor 2 (FPR2). The contribution of this receptor to axonal or dendritic outgrowth is unknown. The present study was carried out to elucidate the distribution of FPR2 in the rat CNS and its role in outgrowth of neuronal processes. FPR2 mRNA expression was greatest in the brainstem, followed by the spinal cord, thalamus/hypothalamus, cerebral neocortex, hippocampus, cerebellum and striatum. The brainstem and spinal cord also contained high levels of FPR2 protein. The cerebral neocortex was moderately immunolabelled for FPR2, with staining mostly present as puncta in the neuropil. Dentate granule neurons and their axons (mossy fibres) in the hippocampus were very densely labelled. The cerebellar cortex was lightly stained, but the deep cerebellar nuclei, inferior olivary nucleus, vestibular nuclei, spinal trigeminal nucleus and dorsal horn of the spinal cord were densely labelled. Electron microscopy of the prefrontal cortex showed FPR2 immunolabel mostly in immature axon terminals or ‘pre-terminals’, that did not form synapses with dendrites. Treatment of primary hippocampal neurons with the FPR2 inhibitors, PBP10 or WRW4, resulted in reduced lengths of axons and dendrites. The CNS distribution of FPR2 suggests important functions in learning and memory, balance and nociception. This might be due to an effect of FPR2 in mediating arachidonic acid/LXA4 or DHA/RvD1-induced axonal or dendritic outgrowth.     

The Visualization of Neuronal Benzodiazepine Receptors in the Brain by Autoradiography and Immunohistochemistry

Abstract

Recent methodological improvements in receptor autoradiography have enabled the in vitro and in vivo binding of the benzodiazepines in the brain to be visualized and pharmacologically characterized with an anatomical resolution unattainable by biochemical radioligand binding assays. This approach, combined with computerized microdensitometry, can be used not only to map the distribution of benzodiazepine receptors in the brain but also to quantify their regional densities. Furthermore, immunohistochemical studies, using monoclonal antibodies directed against the solubilized and purified GABA/benzodiazepine receptor-ionophore complex, have revealed the distribution of antigenic sites on brain neurons and their processes. The brain regions of intense immunoreactivity are known to contain a high density of GABA-ergic efferents and neuronal-type benzodiazepine receptors. Current trends and prospects in this area of receptor research are briefly reviewed.     

Brainstem Neurons Survive the Identical Ischemic Stress That Kills Higher Neurons: Insight to the Persistent Vegetative State

Global ischemia caused by heart attack, pulmonary failure, near-drowning or traumatic brain injury often damages the higher brain but not the brainstem, leading to a ‘persistent vegetative state’ where the patient is awake but not aware. Approximately 30,000 U.S. patients are held captive in this condition but not a single research study has addressed how the lower brain is preferentially protected in these people. In the higher brain, ischemia elicits a profound anoxic depolarization (AD) causing neuronal dysfunction and vasoconstriction within minutes. Might brainstem nuclei generate less damaging AD and so be more resilient? Here we compared resistance to acute injury induced from simulated ischemia by ‘higher’ hippocampal and striatal neurons versus brainstem neurons in live slices from rat and mouse. Light transmittance (LT) imaging in response to 10 minutes of oxygen/glucose deprivation (OGD) revealed immediate and acutely damaging AD propagating through gray matter of neocortex, hippocampus, striatum, thalamus and cerebellar cortex. In adjacent brainstem nuclei, OGD-evoked AD caused little tissue injury. Whole-cell patch recordings from hippocampal and striatal neurons under OGD revealed sudden membrane potential loss that did not recover. In contrast brainstem neurons from locus ceruleus and mesencephalic nucleus as well as from sensory and motor nuclei only slowly depolarized and then repolarized post-OGD. Two-photon microscopy confirmed non-recoverable swelling and dendritic beading of hippocampal neurons during OGD, while mesencephalic neurons in midbrain appeared uninjured. All of the above responses were mimicked by bath exposure to 100 µM ouabain which inhibits the Na⁺/K⁺ pump or to 1–10 nM palytoxin which converts the pump into an open cationic channel.Therefore during ischemia the Na⁺/K⁺ pump of higher neurons fails quickly and extensively compared to naturally resilient hypothalamic and brainstem neurons. The selective survival of lower brain regions that maintain vital functions will support the persistent vegetative state.     

A novel function for the ER retention signals in the C-terminus of kainate receptor subunit,GluK5

Classically, endoplasmic reticulum (ER) retention signals in secreted integral membrane proteins impose the requirement to assemble with other cognate subunits to form functional assemblies before they can exit the ER. We report that GluK5 has two ER retention signals in its cytoplasmic C-terminus: an arginine-based signal and a di-leucine motif previously thought to be an endocytic motif. GluK5 assembles with GluK2, but surprisingly GluK2 association does little to block the ER retention signals. We find instead that the ER retention signals are blocked by two proteins involved in intracellular trafficking, SAP97 and CASK. We show that SAP97, in the presence of CASK and the receptor complex, assumes an extended conformation. In the extended conformation, SAP97 makes its SH3 and GuK domains available to bind and sterically mask the ER retention signals in the GluK5 C-terminus. SAP97 and CASK are also necessary for sorting receptor cargoes into the local dendritic secretory pathway in neurons. We show that the ER retention signals of GluK5 play a vital role in sorting the receptor complex in the local dendritic secretory pathway in neurons. These data suggest a new role for ER retention signals in trafficking integral membrane proteins in neurons.SignificanceWe present evidence that the ER retention signals in the kainate receptors containing GluK5 impose a requirement for sorting into local dendritic secretory pathways in neurons, as opposed to traversing the somatic Golgi apparatus. There are two ER retention signals in the C-terminus of GluK5. We show that both are blocked by physical association with SAP97 and CASK. The SH3 and GuK domains of SAP97, in the presence of CASK, bind directly to each ER retention signal and form a complex. These results support an entirely new function for ER retention signals in the C-termini of neuronal receptors, such as NMDA and kainate receptors, and define a mechanism for selective entry of receptors into local secretory pathways.     

Regulation of Glial Na+/K+-ATPase by Serotonin: Identification of Participating Receptors

The purpose of the present study was the characterization of the receptors participating in the regulatory mechanism of glial Na⁺/K⁺-ATPase by serotonin (5-HT) in rat brain. The activity of the Na⁺ pump was measured in four brain regions after incubation with various concentrations of serotoninergic agonists or antagonists. A concentration-dependent increase in enzyme activity was observed with the 5-HT_1A agonist R (+)-2-dipropylamino-8-hydroxy-1,2,3, 4-tetrahydronaphthalene hydrobromide (8-OH-DPAT) in homogenates or in glial membrane enriched fractions from cerebral cortex and in hippocampus. Spiperone, a 5-HT_1A antagonist, completely inhibited the response to 8-OH-DPAT but had no effect on Na⁺/K⁺-ATPase activity in cerebellum where LSD, a 5-HT₆ agonist, elicited a dose-dependent response similar to that of 5-HT. In brainstem, a lack of reponse to 5-HT and other agonists was confirmed. Altogether, these results show that serotonin modulates glial Na⁺/K⁺-ATPase activity in the brain, apparently not through only one type of 5-HT receptor. It seems that the receptor system involved is different according to the brain region. In cerebral cortex, the response seems to be mediated by 5-HT_1A as well as in hippocampus but not in cerebellum where 5-HT₆ appears as the receptor system involved.     

Decrease in occurrence of fast startle responses after selective Mauthner cell ablation in goldfish (Carassius auratus )

A single action potential in one of a pair of reticulospinal neurons, the Mauthner cells, precedes a short-latency electromyographic response of the trunk and tail musculature on the opposite side of the body and a fast startle response in goldfish. It has been postulated that not only the Mauthner cell, but also an array of neurons can trigger or participate in fast startle responses (Eaton et al. 1991). We have selectively ablated the Mauthner cells in goldfish to study how neurons of the brainstem fast startle response network interact. The probability of eliciting a fast startle response was significantly less in fish with double Mauthner cell ablations, as compared to the responsiveness of control fish. The finding that there is a significant decrease in the occurrence of fast startle responses in animals with no Mauthner cells, implies that the Mauthner cell may play a role in triggering the involvement of the other network elements in fast startle responses. We hypothesize that Mauthner cell activation may be important in bringing those reticulospinal neurons that are “primed” by the behavioral context to threshold and provides the basis for studies focused on the interactive nature of the brainstem startle response network. Accepted: 4 November 1998     

Endosomal location of dopamine receptors in neuronal cell cytoplasm

Five subtypes of dopamine receptor exist in two subfamilies: two D₁-like (D₁ and D₅) and three D₂-like (D₂, D₃ and D₄). We produced novel monoclonal antibodies against all three D₂-like receptors and used them to localize receptors in Ntera-2 (NT-2) cells, the human neuronal precursor cell line. Most of the immunostaining for all three receptors colocalized with mannose-6-phosphate receptor, a marker for late endosomes formed by internalization of the plasma membrane. This result was obtained with antibodies against three different epitopes on the D₃ receptor, to rule out the possibility of cross-reaction with another protein, and controls without primary antibody or in the presence of competitor antigen were completely negative. In rat cerebral cortex and hippocampus, some of the dopamine receptor staining was found in similar structures in neuronal cell cytoplasm. Only some of the neurons were positive for dopamine receptors and the pattern was consistent with previously-reported patterns of innervation by dopamine-producing neurons. Endosomal dopamine receptors may provide a useful method for identifying cell bodies of dopamine-responsive neurons to complement methods that detect only active receptors in the neuronal cell membrane.     

A Gaba/Benzodiazepine Receptor Complex from Bovine Brain: Purification,Reconstitution and Immunological Characterization

Abstract

A GABA/benzodiazepine receptor complex was purified from bovine cerebral cortex. The receptor fraction displayed binding sites for benzodiazepines as well as high and low affinity binding sites for GABA which are characteristics of the membrane-bound receptor. Two monoclonal antibodies of which one was directed against the 50 kd and the other against the 55 kd subunit were used for immunoprecipitation studies. Both of them were shown to quantitatively precipitate the entire receptor population. These results indicate that the binding sites for benzodiazepines and GABA (high and low affinity sites) reside on the same receptor complex containing a mixture of 50 kd and 55 kd subunits. Reconstitution of the receptor in phospholipid vesicles was achieved.     

GABAA receptor subunit polypeptides increase in parallel but exhibit distinct distributions in the developing rat cerebellum

The GABA_A receptor, a multisubunit ligand-gated ion channel, plays a central role in cell–cell communication in the developing and adult nervous system. Although the developmental expression of mRNAs encoding many subunit isoforms has been extensively characterized throughout the central nervous system, little is known concerning the relationship between subunit mRNA and polypeptide expression. To address this issue, we examined the developmental expression of the α1, β2/3, and γ2 subunit polypeptides, subunits that are thought to coassemble in many brain regions. Western blot analysis using subunit-specific antibodies revealed that the levels of these polypeptides in both the cerebral cortex and cerebellum increased severalfold during the second postnatal week. Whereas polypeptide expression in the cerebellum paralleled that of the corresponding subunit mRNAs, increase in β2/3 and γ2 polypeptide expression in the cerebral cortex occurred in the absence of detectable changes in the mRNA levels. To determine whether the increases in subunit polypeptide expression in the cerebellum were accompanied by changes in distribution, immunohistochemistry was performed. These studies demonstrated that the subunits exhibited different but partially overlapping distributions that remained constant throughout postnatal development. Our findings suggest that although GABA_A receptor subunit polypeptide expression may be regulated primarily at the level of the mRNA, additional regulatory mechanisms may play role. Furthermore, the observation that subunit distribution remains constant in the cell bodies of cerebellar Purkinje neurons, which express the α1, β2, β3, and γ2 subunit mRNAs exclusively, suggests that GABA_A receptor subunit composition in this cell population does not change during postnatal maturation. 1994 John Wiley & Sons, Inc.     

Autoradiographic Localization and Characterization of Adenosine Receptor Subtypes in Mammalian Brain

Abstract

Quantitative receptor autoradiography studies have shown that adenosine A₁ receptors are heterogeneously distributed in the rat brain with high concentrations found in the forebrain and cerebellum. In contrast, high affinity A₂ receptors appear to be exclusively localized in the striatum. These observations are discussed in relation to the putative neuromodulatory role of the purine in central neurotransmission.