Suzanne B. Bausch

Upregulation of L-Type Ca2+ Channels in Reactive Astrocytes after Brain Injury, Hypomyelination, and Ischemia

Anti-peptide antibodies that specifically recognize the α1 subunit of class A–D voltage-gated Ca2+ channels and a monoclonal antibody (MANC-1) to the α2 subunit of L-type Ca2+ channels were used to investigate the distribution of these Ca2+ channel subtypes in neurons and glia in models of brain injury, including kainic acid-induced epilepsy in the hippocampus, mechanical and thermal lesions in the forebrain, hypomyelination in white matter, and ischemia. Immunostaining of the α2 subunit of L-type Ca2+ channels by the MANC-1 antibody was increased in reactive astrocytes in each of these forms of brain injury. The α1C subunits of class C L-type Ca2+ channels were upregulated in reactive astrocytes located in the affected regions in each of these models of brain injury, although staining for the α1 subunits of class D L-type, class A P/Q-type, and class B N-type Ca2+ channels did not change from patterns normally observed in control animals. In all of these models of brain injury, there was no apparent redistribution or upregulation of the voltage-gated Ca2+ channels in neurons. The upregulation of L-type Ca2+ channels in reactive astrocytes may contribute to the maintenance of ionic homeostasis in injured brain regions, enhance the release of neurotrophic agents to promote neuronal survival and differentiation, and/or enhance signaling in astrocytic networks in response to injury.

Seizures, cell death, and mossy fiber sprouting in kainic acid-treated organotypic hippocampal cultures

Sprouting of the mossy fiber axons of the dentate granule cells is a structural neuronal plasticity found in the mature brain of epileptic humans and experimental animals. Mossy fiber sprouting typically arises in experimental animals after repeated seizures and may contribute to the hyperexcitability of the epileptic brain. Investigation of the molecular triggers and spatial cues involved in mossy fiber sprouting has been hampered by the lack of an optimal in vitro model for studying this rearrangement. For an in vitro model to be feasible, the circuitry and receptors involved in convulsant-induced mossy fiber sprouting would have to be localized near the granule cells, rather than being dependent on long-range brain interconnections. However, it is not known whether this is the case. We report here that that application of the convulsant, kainic acid, to organotypic hippocampal explant cultures induces seizures, neuronal cell death, and subsequent dramatic mossy fiber sprouting with a similar laminar preference and time-course to that seen in intact animals. Prolonged (48 h) but not transient (4 h) kainic acid treatment caused regionally selective neuronal cell death. Cultures treated with kainic acid for a prolonged period displayed a time- and dose-dependent increase in supragranular Timm staining reflective of increased mossy fiber innervation to this area. Direct visualization of mossy fiber axons with neurobiotin-labeling revealed that mossy fibers in kainic acid-treated cultures exhibited a dramatic increase in supragranular axonal branch points and synaptic boutons. The cellular and molecular determinants required for kainic acid-induced cell death and subsequent mossy fiber reorganization thus appear to be intrinsic to the hippocampal slice preparation, and are preserved in culture. Given the ease with which functional inhibitors or pharmacological agents may be utilized in this system, slice cultures may provide a powerful model in which to study the molecular components involved in triggering mossy fiber outgrowth and underlying its laminar specificity. Elucidation of these molecular pathways will likely have both specific utility in clarifying the functional consequences of mossy fiber sprouting, as well as general utility in understanding of synaptic reorganization in the mature central nervous system.

The hypothesis that antagonism of fentanyl analgesia by 2-chloroprocaine is mediated by direct action on opioid receptors

Background and Objectives. Although 2‐chloroprocaine continues to be a useful drug for epidural anesthesia in obstetrics, it has the anomalous action of decreasing the analgesic effectiveness of subsequently administered epidural fentanyl. Some investigators have suggested that 2‐chloroprocaine may act at an opioid receptor site to antagonize the effects of fentanyl. The purpose of our studies was to investigate this hypothesis. Methods. Radioligiand binding assays using the mu and kappa opioid receptor‐selective radioligands [3H]‐DAMGO and [3H]‐U69,593, respectively, were performed to determine the potencies of lidocaine, 2‐chloroprocaine, and 2‐chloroprocaine metabolites at the mu and kappa opioid receptor sites. Electrophysiologic experiments in in vitro hippocampal slice preparations were then used to examine the effects of 2‐chloroprocaine at these opioid receptor subtypes. Results. Lidocaine caused a partial reduction of [3H]‐DAMGO binding, which was dose‐limited owing to the solubility of lidocaine. 2‐Chloroprocaine caused complete displacement of [3H]‐DAMGO binding, with a median effective concentration of 1.44 ± 0.36 mM. The EC50 values for [3H]‐U69,593 displacement were 177 ± 47 μM for 2‐chloroprocaine and 2.53 ± 0.48 mM for lidocaine. Assuming a competitive interaction between anesthetic and opioid, the Ki value for 2‐chloroprocaine was 435 μM at mu receptors and 49 μM at kappa receptors. In the mu activity bioassay, 2‐chloroprocaine reversed the increased neuronal excitability caused by fentanyl, but this effect was further reduced by naloxone. In addition, 2‐chloroprocaine did not reverse the afterdepolarization caused by fentanyl. In the kappa activity bioassay, 2‐chloroprocaine produced effects similar to the kappa agonist U69,593, but these were not antagonized by naloxone. Conclusions. Although 2‐chloroprocaine has binding affinity at mu and kappa opioid receptor sites, it does not appear to act through an opioid receptor to antagonize the physiologic effects of fentanyl.

A method for triple fluorescence labeling with Vicia villosa agglutinin, an anti-parvalbumin antibody and an anti-G-protein-coupled receptor antibody

The aim of the original study [S.B. Bausch, C. Chavkin, Vicia villosa agglutinin labels a subset of neurons coexpressing both the mu opioid receptor and parvalbumin in the developing rat subiculum, Dev. Brain Res., 97, 1996, 169-177] [3] was to develop a method for identifying a subset of mu opioid receptor-expressing interneurons in the rat subiculum for electrophysiological studies. Previous studies had shown that a subset of parvalbumin-positive neurons in the rat subiculum could be labeled with the lectin, Vicia villosa agglutinin (VVA) [C.T. Drake, K.A. Mulligan, T.L. Wimpey, A. Hendrickson, C. Chavkin, Characterization of Vicia villosa agglutinin-labeled GABAergic neurons in the hippocampal formation and in acutely dissociated hippocampus, Brain Res., 554, 1991, 176-185] [11], and that mu opioid receptor immunoreactivity (-IR) and parvalbumin-IR were colocalized in a subset of neurons in the hippocampus and dentate gyrus [S.B. Bausch, C. Chavkin, Colocalization of mu and delta opioid receptors with GABA, parvalbumin and a G-protein-coupled inwardly rectifying potassium channel in the rodent brain, Analgesia, 1, 1995, 282-285] [2]. We hypothesized that a subset of mu opioid receptor-expressing neurons in the subiculum also would express the calcium binding protein, parvalbumin, and could be labeled with VVA. Labeling of live neurons with VVA [11] then could be used to identify these neurons. This protocol was designed to triple-label neurons expressing the mu opioid receptor, parvalbumin and the carbohydrate group, N-acetylgalactosamine (which binds VVA [S.E. Tollefsen, R. Kornfeld, The B4 lectin from Vicia villosa seeds interacts with N-acetylgalactosamine residues alpha-linked to serine or threonine residues in cell surface glycoproteins, J. Biol. Chem., 258, 1983, 5172-5176][M.P. Woodward, W.W. Young, R.A. Bloodgood, Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation, J. Immunol. Methods, 78, 1985, 143-153] [25, 29]). VVA labeling and immunocytochemistry with an affinity-purified anti-mu opioid receptor antibody [S.B. Bausch, T.A. Patterson, M.U. Ehrengruber, H.A. Lester, N. Davidson, C. Chavkin, Colocalization of mu opioid receptors with GIRK1 potassium channels in rat brain: an immunocytochemical study, Recept. Channels, 3, 1995, 221-241] [4] and an anti-parvalbumin antibody [M.R. Celio, W. Baier, L. Scharer, P.A. de Viragh, C. Gerday, Monoclonal antibodies directed against the calcium binding protein parvalbumin, Cell Calcium, 9, 1988, 81-86] [8] were used to accomplish this goal. Immunofluorescence was used as the detection method; visualization was accomplished with three fluorophores with different excitation/emission spectra and a one laser confocal microscope. This protocol can be modified easily to triple-label neurons for other carbohydrate groups and proteins.