Ca2+/calmodulin‐dependent protein kinase II in spinal dorsal horn contributes to the pain hypersensitivity induced by γ‐aminobutyric acid type a receptor inhibition

The fast inhibitory synaptic transmission mediated by the γ‐aminobutyric acid type A receptor (GABA_AR) within spinal dorsal horn exerts a gating control over the synaptic conveyance of nociceptive information from the periphery to higher brain regions. Although a large body of evidence has demonstrated that the impairment of GABAergic inhibition alone is sufficient to elicit pain hypersensitivity in intact animals, the underlying mechanisms remain to be characterized. The present study shows that Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) is an important signaling protein downstream of reduced GABAergic inhibition. We found that pharmacological removal of inhibition by intrathecal application of the GABA_AR antagonist bicuculline significantly enhanced the autophosphorylation of CaMKII at Thr286 in spinal dorsal horn of mice. In addition to increased CaMKII activity, bicuculline also promoted CaMKII interaction with N‐methyl‐D‐aspartate (NMDA)‐subtype glutamate receptors and induced the translocation of CaMKII from cytosolic compartments to the synaptosomal membrane fraction. Immunoblotting analysis revealed that the phosphorylation levels of NMDA receptor NR2B subunit at Ser1303 and of AMPA‐subtype glutamate receptor GluR1 subunit at Ser831, two important CaMKII phosphorylation sites, were substantially enhanced after bicuculline application. Behavioral tests illustrated that intrathecal administration of the CaMKII inhibitor KN‐93, NMDA receptor antagonist D‐APV, or AMPA receptor antagonist GYKI 52466 effectively ameliorated the mechanical allodynia evoked by bicuculline. These data thus indicate that CaMKII signaling is critical for the reduced inhibition to evoke spinal sensitization. © 2013 Wiley Periodicals, Inc.     

Early continuous white noise exposure alters l‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid receptor subunit glutamate receptor 2 and γ‐aminobutyric acid type a receptor subunit β3 protein expression in rat auditory cortex

Auditory experience during the postnatal critical period is essential for the normal maturation of auditory function. Previous studies have shown that rearing infant rat pups under conditions of continuous moderate‐level noise delayed the emergence of adult‐like topographic representational order and the refinement of response selectivity in the primary auditory cortex (A1) beyond normal developmental benchmarks and indefinitely blocked the closure of a brief, critical‐period window. To gain insight into the molecular mechanisms of these physiological changes after noise rearing, we studied expression of the AMPA receptor subunit GluR2 and GABA_A receptor subunit β3 in the auditory cortex after noise rearing. Our results show that continuous moderate‐level noise rearing during the early stages of development decreases the expression levels of GluR2 and GABA_Aβ3. Furthermore, noise rearing also induced a significant decrease in the level of GABA_A receptors relative to AMPA receptors. However, in adult rats, noise rearing did not have significant effects on GluR2 and GABA_Aβ3 expression or the ratio between the two units. These changes could have a role in the cellular mechanisms involved in the delayed maturation of auditory receptive field structure and topographic organization of A1 after noise rearing. © 2009 Wiley‐Liss, Inc.     

Generation of resonance‐dependent oscillation by mGluR‐I activation switches single spiking to bursting in mesencephalic trigeminal sensory neurons

The primary sensory neurons supplying muscle spindles of jaw‐closing muscles are unique in that they have their somata in the mesencephalic trigeminal nucleus (MTN) in the brainstem, thereby receiving various synaptic inputs. MTN neurons display bursting upon activation of glutamatergic synaptic inputs while they faithfully relay respective impulses arising from peripheral sensory organs. The persistent sodium current (I_NaP) is reported to be responsible for both the generation of bursts and the relay of impulses. We addressed how I_NaP is controlled either to trigger bursts or to relay respective impulses as single spikes in MTN neurons. Protein kinase C (PKC) activation enhanced I_NaP only at low voltages. Spike generation was facilitated by PKC activation at membrane potentials more depolarized than the resting potential. By injection of a ramp current pulse, a burst of spikes was triggered from a depolarized membrane potential whereas its instantaneous spike frequency remained almost constant despite the ramp increases in the current intensity beyond the threshold. A puff application of glutamate preceding the ramp pulse lowered the threshold for evoking bursts by ramp pulses while chelerythrine abolished such effects of glutamate. Dihydroxyphenylglycine, an agonist of mGluR1/5, also caused similar effects, and increased both the frequency and impedance of membrane resonance. Immunohistochemistry revealed that glutamatergic synapses are made onto the stem axons, and that mGluR1/5 and Nav1.6 are co‐localized in the stem axon. Taken together, glutamatergic synaptic inputs onto the stem axon may be able to switch the relaying to the bursting mode.     

Roles for γ‐aminobutyric acid in the development of the paraventricular nucleus of the hypothalamus

The development of the hypothalamic paraventricular nucleus (PVN) involves several factors that work together to establish a cell group that regulates neuroendocrine functions and behaviors. Several molecular markers were noted within the developing PVN, including estrogen receptors (ER), neuronal nitric oxide synthase (nNOS), and brain‐derived neurotrophic factor (BDNF). By contrast, immunoreactive γ‐aminobutyric acid (GABA) was found in cells and fibers surrounding the PVN. Two animal models were used to test the hypothesis that GABA works through GABA_A and GABA_B receptors to influence the development of the PVN. Treatment with bicuculline to decrease GABA_A receptor signaling from embryonic day (E) 10 to E17 resulted in fewer cells containing immunoreactive (ir) ERα in the region of the PVN vs. control. GABA_BR1 receptor subunit knockout mice were used to examine the PVN at P0 without GABA_B signaling. In female but not male GABA_BR1 subunit knockout mice, the positions of cells containing ir ERα shifted from medial to lateral compared with wild‐type controls, whereas the total number of ir ERα‐containing cells was unchanged. In E17 knockout mice, ir nNOS cells and fibers were spread over a greater area. There was also a significant decrease in ir BDNF in the knockout mice in a region‐dependent manner. Changes in cell position and protein expression subsequent to disruption of GABA signaling may be due, in part, to changes in nNOS and BDNF signaling. Based on the current study, the PVN can be added as another site where GABA exerts morphogenetic actions in development. J. Comp. Neurol. 518:2710–2728, 2010. © 2010 Wiley‐Liss, Inc.     

Differential localization of γ‐aminobutyric acid type a and glycine receptor subunits and gephyrin in the human pons,medulla oblongata and uppermost cervical segment of the spinal cord: An immunohistochemical study

Gephyrin is a multifunctional protein responsible for the clustering of glycine receptors (GlyR) and γ‐aminobutyric acid type A receptors (GABA_AR). GlyR and GABA_AR are heteropentameric chloride ion channels that facilitate fast‐response, inhibitory neurotransmission in the mammalian brain and spinal cord. We investigated the immunohistochemical distribution of gephyrin and the major GABA_AR and GlyR subunits in the human light microscopically in the rostral and caudal one‐thirds of the pons, in the middle and caudal one‐thirds of the medulla oblongata, and in the first cervical segment of the spinal cord. The results demonstrate a widespread pattern of immunoreactivity for GlyR and GABA_AR subunits throughout these regions, including the spinal trigeminal nucleus, abducens nucleus, facial nucleus, pontine reticular formation, dorsal motor nucleus of the vagus nerve, hypoglossal nucleus, lateral cuneate nucleus, and nucleus of the solitary tract. The GABA_AR α₁ and GlyR α₁ and β subunits show high levels of immunoreactivity in these nuclei. The GABA_AR subunits α₂, α₃, β_2,3, and γ₂ present weaker levels of immunoreactivity. Exceptions are intense levels of GABA_AR α₂ subunit immunoreactivity in the inferior olivary complex and high levels of GABA_AR α₃ subunit immunoreactivity in the locus coeruleus and raphe nuclei. Gephyrin immunoreactivity is highest in the first segment of the cervical spinal cord and hypoglossal nucleus. Our results suggest that a variety of different inhibitory receptor subtypes is responsible for inhibitory functions in the human brainstem and cervical spinal cord and that gephyrin functions as a clustering molecule for major subtypes of these inhibitory neurotransmitter receptors. J. Comp. Neurol. 518:305–328, 2010. © 2009 Wiley‐Liss, Inc.     

Heterotrimeric guanosine triphosphate‐binding protein‐coupled modulatory actions of motilin on K+ channels and postsynaptic γ‐aminobutyric acid receptors in mouse medial vestibular nuclear neurons

Some central nervous system neurons express receptors of gastrointestinal hormones, but their pharmacological actions are not well known. Previous anatomical and unit recording studies suggest that a group of cerebellar Purkinje cells express motilin receptors, and motilin depresses the spike discharges of vestibular nuclear neurons that receive direct cerebellar inhibition in rats or rabbits. Here, by the slice‐patch recording method, we examined the pharmacological actions of motilin on the mouse medial vestibular nuclear neurons (MVNs), which play an important role in the control of ocular reflexes. A small number of MVNs, as well as cerebellar floccular Purkinje cells, were labeled with an anti‐motilin receptor antibody. Bath application of motilin (0.1 μm ) decreased the discharge frequency of spontaneous action potentials in a group of MVNs in a dose‐dependent manner (K_d, 0.03 μm ). The motilin action on spontaneous action potentials was blocked by apamin (100 nm ), a blocker of small‐conductance Ca²⁺‐activated K⁺ channels. Furthermore, motilin enhanced the amplitudes of inhibitory postsynaptic currents (IPSCs) and miniature IPSCs, but did not affect the frequencies of miniature IPSCs. Intracellular application of pertussis toxin (PTx) (0.5 μg/μL) or guanosine triphosphate‐γ‐S (1 mm ) depressed the motilin actions on both action potentials and IPSCs. Only 30% of MVNs examined on slices obtained from wild‐type mice, but none of the GABAergic MVNs that were studied on slices obtained from vesicular γ‐aminobutyric acid transporter‐Venus transgenic mice, showed such a motilin response on action potentials and IPSCs. These findings suggest that motilin could modulate small‐conductance Ca²⁺‐activated K⁺ channels and postsynaptic γ‐aminobutyric acid receptors through heterotrimeric guanosine triphosphate‐binding protein‐coupled receptor in a group of glutamatergic MVNs.     

Vesicular glutamate transporter‐3 in the rodent brain: Vesicular colocalization with vesicular γ‐aminobutyric acid transporter

Vesicular glutamate transporters (VGLUT1–3) carry glutamate into synaptic vesicles. VGLUT3 has been reported to be localized in nonglutamatergic neuronal populations in the brain. However, detailed subcellular localization of VGLUT3 has not been shown. In particular, the identity of synaptic vesicles expressing VGLUT3 remains to be revealed. Here we present novel electron microscopic postembedding immunogold data from mouse and rat brains showing that small, clear, and round synaptic vesicles in γ‐aminobutyric acid (GABA)‐ergic nerve terminals contain labeling for both VGLUT3 and the vesicular GABA transporter (VGAT). Immunoisolation of synaptic vesicles confirmed the immunogold data and showed vesicular colocalization of VGLUT3 and VGAT. Moreover, we show that gold particles signaling VGLUT3 are present in synaptic vesicles in acetylcholinergic nerve terminals in the striatum. Quantitative immunogold analyses reveal that the density of VGLUT3 gold particles is similar in GABAergic terminals in the hippocampus and the neocortex to that in cholinergic terminals in the striatum. In contrast to in the hippocampus and the neocortex, VGLUT3 was absent from VGAT‐positive terminals in the striatum. The labeling pattern produced by the VGLUT3 antibodies was found to be specific; there was no labeling in VGLUT3 knockout tissue, and the observed labeling throughout the rat brain corresponds to the known light‐microscopic distribution of VGLUT3. From the present results, we infer that glutamate is released with GABA from inhibitory terminals and acetylcholine from cholinergic terminals. J. Comp. Neurol. 521: 3042–3056, 2013. © 2013 Wiley Periodicals, Inc.     

Immunocytochemical localization of the α6 subunit of the γ-aminobutyric acidA receptor in the rat nervous system

The localization in the rat central nervous system and retina of the α₆ subunit peptide of the γ-aminobutyric acid (GABA_A) receptor has been studied by light microscopy immunocytochemistry with a specific anti-α₆ antibody. The α₆ subunit was present in the granule cells of the cerebellum, the granule cells of the dorsal cochlear nucleus, axons of the olfactory nerve including the glomerular endings, layer II of the dorsal horn of the spinal cord, and in the retinal synaptic layers, particularly the inner plexiform layer. Thus, contrary to the general belief, the α₆ subunit is not exclusively localized in the granule cells of the cerebellum. It is also expressed in some sensory neurons and other neurons involved in the initial processing of sensory information. © 1996 Wiley-Liss, Inc.     

Immunocytochemical localization of the β2 subunit of the gamma-aminobutyric acidA receptor in the rat brain

An antiserum to the β₂ subunit of the rat gamma-aminobutyric acid (GABA_A) receptor was prepared by immunizing a rabbit with a fusion protein expressed in bacteria. The fusion protein had the large, intracellular loop expanding between the putative M3 and M4 transmembrane domains of the β₂ subunit fused to staphylococcal protein A (SPA). The antiserum immunoprecipitated both the solubilized and the affinity-purified GABA_A receptors. The anti-β₂ antibodies were affinity purified on immobilized β₂ intracellular loop peptide. The antibodies recognized a 55–57 kDa peptide in immunoblots of either crude membranes from rat cerebral cortex or affinity-purified GABA_A receptors from bovine cerebral cortex. Immunocytochemistry with the affinity-purified antibody has revealed for the first time the localization of the β₂ subunit in the rat brain. A comparative study of the regional and cellular immunoreactivities of the affinity-purified anti-β₂ antibody and the monoclonal antibody 62-3G1 (which recognizes both β₂ and β₃ subunits) is presented. The procedure described for generating and preparing specific anti-β₂ subunit antibodies that are valuable for immunocytochemistry could be extended to other GABA_A receptor subunits. © 1994 Wiley-Liss, Inc.     

Distinct properties of murine α5 γ‐aminobutyric acid type a receptors revealed by biochemical fractionation and mass spectroscopy

γ‐Aminobutyric acid type A receptors (GABA_ARs) that contain the α5 subunit are expressed predominantly in the hippocampus, where they regulate learning and memory processes. Unlike conventional postsynaptic receptors, GABA_ARs containing the α5 subunit (α5 GABA_ARs) are localized primarily to extrasynaptic regions of neurons, where they generate a tonic inhibitory conductance. The unique characteristics of α5 GABA_ARs have been examined with pharmacological, immunostaining, and electrophysiological techniques; however, little is known about their biochemical properties. The aim of this study was to modify existing purification and enrichment techniques to isolate α5 GABA_ARs preferentially from the mouse hippocampus and to identify the α5 subunit by using tandem mass spectroscopy (MS/MS). The results showed that the detergent solubility of the α5 subunits was distinct from that of α1 and α2 subunits, and the relative distribution of the α5 subunits in Triton X‐100‐soluble fractions was correlated with that of the extracellular protein radixin but not with that of the postsynaptic protein gephyrin. Mass spectrometry identified the α5 subunit and showed that this subunit associates with multiple α, β, and γ subunits, but most frequently the β3 subunit. Thus, the α5 subunits coassemble with similar subunits as their synaptic counterparts yet have a distinct detergent solubility profile. Mass spectroscopy now offers a method for detecting and characterizing factors that confer the unique detergent solubility and possibly cellular location of α5 GABA_ARs in hippocampal neurons. © 2009 Wiley‐Liss, Inc.     

Developmental regulation of connexins 26, 32, 36, and 43 in trigeminal neurons

The transition from sucking to chewing during postnatal development is accompanied by changes in masticatory muscle activity patterns. We previously demonstrated that changes in numerous parameters of chemical synapses among neurons, and intrinsic membrane properties of neurons, comprising brainstem oral-motor circuits are coincident with changes in masticatory muscle activity patterns. Considering recent findings that implicate a role for gap junctions in early locomotor and respiratory behaviors, our present study focuses on the developmental regulation of connexin proteins in trigeminal neurons as a first step in understanding a role for gap junctions in developing oral-motor circuits used for ingestive behaviors. We conducted immunohistochemistry studies to examine connexin (Cx) 26, 32, 36, and 43 expression in trigeminal motor and mesencephalic trigeminal nuclei during postnatal development at the light and electron microscopic levels. Postnatal days (P) 1, 6, 14, 21, and adult mice were used. Cx32, 36, and 43 expression was developmentally regulated in the trigeminal motor nucleus, while Cx26 expression remained high throughout postnatal development. In the mesencephalic trigeminal nucleus, Cx26, 32, and 43 expression was intense throughout development, with only Cx36 showing a developmental regulation. Ultrastructural examination of neonatal trigeminal motoneurons and mesencephalic trigeminal neurons revealed connexin expression in cell membranes, cytoplasm, and cell nuclei (Cx43, Cx32). Our results show that connexin proteins are differentially regulated between trigeminal motoneurons and mesencephalic trigeminal neurons during development, and suggest a possible role for gap junctions in the development of trigeminal neurons and the function and maturation of oral-motor circuits.     

γ‐Aminobutyric acid type a receptor inhibition triggers a nicotinic neuroprotective mechanism

Nicotinic acetylcholine receptor (nAChR)‐mediated neuroprotection has been implicated in the treatment of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases and hypoxic ischemic events as well as other diseases hallmarked by excitotoxic and apoptotic neuronal death. Several modalities of nicotinic neuroprotection have been reported. However, although this process generally involves α4β2 and α7 subtypes, the underlying mechanisms are largely unknown. Interestingly, both activation and inhibition of α7 nAChRs have been reported to be neuroprotective. We have shown that inhibition of α7 nAChRs protects the function of acute hippocampal slices against excitotoxicity in an α4β2‐dependent manner. Neuroprotection was assessed as the prevention of the N‐methyl‐D‐aspartate‐dependent loss of the area of population spikes (PSs) in the CA1 area of acute hippocampal slices. Our results support a model in which α7 AChRs control the release of γ‐aminobutyric acid (GABA). Blocking either α7 or GABA_A receptors reduces the inhibitory tone on cholinergic terminals, thereby promoting α4β2 activation, which in turn mediates neuroprotection. These results shed light on how α7 nAChR inhibition can be neuroprotective through a mechanism mediated by activation of α4β2 nAChRs. © 2012 Wiley Periodicals, Inc.     

Transganglionic transport of wheat germ agglutinin-HRP and choleragenoid-HRP in rat trigeminal primary sensory neurons

Horseradish peroxidase conjugates of either the lectin wheat germ agglutinin (WGA-HRP) or choleragenoid (B-HRP) have been shown to be sensitive neuroanatomical tracers. In the present study a comparison was made between these two conjugates as transganglionic tracers in trigeminal primary sensory neurons following injection into the rat mystacial vibrissae skin. Differences between the two tracers were observed in the labeling of cell bodies in the trigeminal ganglion. Injection of WGA-HRP resulted in labeling of predominantly small cell bodies, whereas B-HRP gave rise to labeling of somewhat larger cell bodies. By increasing the concentration of the injected WGA-HRP solution the number of labeled cells increased substantially, while a corresponding increase in the concentration of B-HRP resulted in a relatively small increase in the number of labeled cells. WGA-HRP injection resulted in labeling of primary afferents mainly in the substantia gelatinosa of the trigeminal subnucleus caudalis. When the concentration of the injected WGA-HRP solution was increased, labeling was also observed in the marginal and magnocellular zones. Following B-HRP injection, labeling was only observed in the magnocellular zone and innermost part of the substantia gelatinosa. This general pattern of labeling was the same when the concentration of the B-HRP solution was increased.     

Differential localization and expression of α and β isoenzymes of protein kinase c in the rat retina

Expression and cellular localization of three isoenzymes of Ca²⁺-dependent protein kinase C (PKCα, PKCβ, and PKCγ) in the adult rat retina were revealed by immunohistochemistry and in situ hybridization histochemistry with isoenzyme-specific antibodies and cRNA probes. Immunoreactivities and mRNA signals for PKCα were conspicuous in rod bipolar cells. A subgroup of amacrine cells expressed PKCα. The cells in the ganglion cell layer also displayed PKCα gene products. Positive immunoreactivities for PKCβ were localized as stripe patterns in the inner plexiform layer, corresponding to the stratification levels of axon terminals of cone bipolar cells. The somata of cone bipolar cells expressed PKCβ. Amacrine cells and retinal ganglion cells also displayed PKCβ gene products. The results obtained by immunohistochemistry were confirmed with colocalization of mRNA signals for PKCα and PKCβ on the somata. The cell membranes showed stronger immunoreactivities than did the cytoplasms for both PKCα and PKCβ. Neither immunoreactivities nor mRNA signals for PKCγ were detected in all retinal regions. The differential roles of Ca²⁺-dependent PKC isoenzymes could be revealed in physiological defined retinal neurons. J. Neurosci. Res. 54:655–663, 1998. © 1998 Wiley-Liss, Inc.