γ-Aminobutyric acidA receptors on a bistratified amacrine cell type in the rabbit retina

γ-Aminobutyric acid (GABA) is considered to be a major inhibitory neurotransmitter in the inner plexiform layer of the retinas of all vertebrate species. It is contained in and released from nearly 40% of the amacrine cells and is known to play a major role in many aspects of visual processing. By using well-characterized antibodies to several subunits of the GABA_A receptor, we have analyzed their localization on the cell bodies and dendritic trees of two amacrine cell populations in the rabbit retina, which have been either filled intracellularly with Lucifer yellow or stained immunohistochemically. Both populations are selectively stained by intravitreal injection of the fluorescent nuclear dye 4′,6-diaminidin-2-phenylindoldihydrochloride (DAPI). We have found that the most significant concentration of the α₁ and β_2/3 GABA_A receptor subunits is localized to the DAPI-3 type amacrine cell. The perikarya of the DAPI-3 cells are found in the proximal inner nuclear layer and send their processes into two sublayers in sublaminae a and b of the inner plexiform layer. These processes abut but do not directly overlap those of the two mirror-symmetric populations of starburst amacrine cells. Because the cell bodies of the DAPI-3 cells are the only ones in the inner nuclear layer that stain strongly for either the α₁ or β_2/3 subunits, such staining is a diagnostic feature of these cells. Their processes also constitute the most strongly staining ones found within the inner plexiform layer. The dendritic trees of DAPI-3 cells, which range from about 150 μm up to about 300 μm, exhibit recurvate looping processes reminiscent of those described for directionally selective ganglion cells. In contrast to the DAPI-3 cell, we have also shown that the starburst amacrine cells exhibit no immunoreactivity for the α₁ GABA_A receptor subunit and very little for the β_2/3 subunit. Thus, we have shown that the DAPI-3 cells contain the highest concentrations of the α₁ and β_2/3 GABA_A receptor subunits in the rabbit retina. These cells, which costratify near the processes of both the starburst amacrine cells and the ON-OFF directionally selective ganglion cells, thus, are situated both anatomically and by virtue of their receptor content to potentially interact. J. Comp. Neurol. 393:309–319, 1998. © 1998 Wiley-Liss, Inc.     

Methamphetamine‐induced up‐regulation of α2/δ subunit of voltage‐gated calcium channels is regulated by DA receptors

This study was carried out to determine the roles of dopamine D1 and D2 receptors on the up‐regulation of α₂/δ subunit of voltage‐gated Ca²⁺ channels (VGCCs) induced by methamphetamine (METH). In the conditioned place preference paradigm, METH‐induced place preference suppressed with gabapentin, an antagonist for α₂/δ subunit. Under these conditions, the increase in α₂/δ subunit expression was found in the frontal cortex and limbic forebrain. In addition, the METH‐induced place preference was significantly attenuated by dopamine D1 and D2 receptor antagonists, SCH23390 and sulpiride, respectively. The expression of α₂/δ subunit protein and its mRNA was significantly enhanced in the METH‐treated cortical neurons. These increases in protein and mRNA of α₂/δ subunit were completely abolished by SCH23390 and sulpiride with simultaneous exposure to METH. These findings indicate that up‐regulation of α₂/δ subunit is regulated through the activation of dopamine D1 and D2 receptors during METH treatment. Synapse 64:822–828, 2010. © 2010 Wiley‐Liss, Inc.     

GABAA Receptor subunits have differential distributions in the rat retinae: In situ hybridization and immunohistochemistry

The distributions of nine different subunits of the gamma-aminobutyric acid_A (GABA_A) receptor (α₁, α₂, α₃, ₅; β₁, β₂, β₃; γ₂; δ) were investigated in the rat retina using immunocytochemistry and in situ hybridization. With the exception of the α₅ subunit, all subunits could be localized. Each subunit was expressed in characteristic strata within the inner plexiform layer (IPL). Some subunits (e.g., γ₂) showed a ubiquitous distribution, while others (e.g., δ) were restricted to narrow sublayers. Double labeling experiments using different combinations of the subunit-specific antibodies revealed colocalizations of subunits within individual neurons. Additionally, GABA_A receptor subunits were mapped to distinct populations of retinal neurons by coapplication of defined immunocytochemical markers and subunit-specific antibodies. Cholinergic amacrine cells were found to express the α₂, β1, β_2/3 and δ subunits, while dopaminergic amacrine cells express the α₂, α₃ and γ₂ subunits. Dissociated rod bipolar cells express the γ₂ subunits. In summary, this study provides evidence for the existence of multiple GABA_A receptor subtypes in the retina. The distinct stratification pattern of the subunits in the IPL suggests that different functional circuits involve specific subtypes of GABAA receptors. © 1995 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.     

The α1, α2, α3, and γ2 subunits of GABAA receptors show characteristic spatial and temporal expression patterns in rhombencephalic structures during normal human brain development

γ‐Aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in adult mammalian brain, mediating its actions chiefly via a pentameric chloride ion channel, the GABA_A receptor. Nineteen different subunits (α1‐6, β1‐3, γ1‐3, δ, ε, π, θ, ρ1‐3) can give rise to multiple receptor subtypes that are the site of action of many clinically important drugs. In the developing brain, however, GABA_A receptors mediate excitatory actions due to an increased chloride concentration within neurons and seem to control cell proliferation, migration, differentiation, synapse maturation, and cell death. Little is known about the distribution of single subunits in the human brain. Here we describe developmental changes in the immunohistochemical distribution of four subunits (α1, α2, α3, and γ2) in the human rhombencephalon. The γ2 was the most abundant subunit in all rhombencephalic structures during development and in adults, whereas α subunits showed a structure‐ and age‐characteristic distribution. The α1 was expressed prenatally in the molecular and Purkinje cell layer, but only postnatally in the granule cell layer and the dentate nucleus. Expression was completely absent in the inferior olivary nucleus. The α2 gradually increased during development, showing some layer specificity in the cerebellar cortex. The α3‐immunoreactivity in the cerebellar cortex was relatively weak, but it was abundantly observed in different cell populations in the subcortical cerebellar structures. Structure‐ and age‐characteristic colocalization between subunits during development suggests differences in GABA_A receptor composition. Interestingly, subunit expression in several instances differed between human and rodent brain, underlining the importance of immunohistochemical studies in humans. J. Comp. Neurol. 524:1805–1824, 2016. © 2015 Wiley Periodicals, Inc.     

Differential localization of Ca2+ channel α1 subunits in the enteric nervous system: Presence of α1B channel-like immunoreactivity in intrinsic primary afferent neurons

Immunocytochemistry was employed to locate calcium (Ca²⁺) channel proteins in the enteric nervous system (ENS) of the rat and guinea pig. Anti-peptide antibodies that specifically recognize the α₁ subunits of class A (P/Q-type), B (N-type), C and D (L-type) Ca²⁺ channels were utilized. α_1B channel-like immunoreactivity was abundant in both enteric plexuses, the mucosa, and circular and longitudinal muscle layers. Immunoreactivity was predominantly found in cholinergic varicosities, supporting a role for Ca²⁺ channels, which contain the α_1B subunit, in acetylcholine release. Immunoreactivity was also associated with the cell soma of calbindin-immunoreactive submucosal and myenteric neurons, cells that have been proposed to be intrinsic primary afferent neurons. α_1C channel-like immunoreactivity was distributed diffusely in the cell membrane of a large subset of neuronal cell bodies and processes, whereas α_1D was found mainly in the cell soma and proximal dendrites of vasoactive intestinal polypeptide-immunoreactive neurons in the guinea pig gut. α_1A channel-like immunoreactivity was found in a small subset of cell bodies and processes in the rat ENS. The differential localization of the α₁ subunits of Ca²⁺ channels in the ENS implies that they serve distinct roles in neuronal excitation and signaling within the bowel. The presence of α_1B channel-like immunoreactivity in putative intrinsic primary afferent neurons suggested that class B Ca²⁺ channels play a role in enteric sensory neurotransmission; therefore, we determined the effects of the N-type Ca²⁺ channel blocker, ω-conotoxin GVIA (ω-CTx GVIA), on the reflex-evoked activity of enteric neurons. Demonstrating the phosphorylation of cyclic AMP (cAMP)-responsive element-binding protein (pCREB) identified neurons that became active in response to distension. Distension elicited hexamethonium-resistant pCREB immunoreactivity in calbindin-immunoreactive neurons in each plexus; however, in preparations stimulated in the presence of ω-CTx GVIA, pCREB immunoreactivity was found only in calbindin-immunoreactive neurons in the submucosal plexus and not in myenteric ganglia. These data confirm that intrinsic primary afferent neurons are located in the submucosal plexus and that N-type Ca²⁺ channels play a role in sensory neurotransmission. J. Comp. Neurol. 409:85–104, 1999. © 1999 Wiley-Liss, 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.     

Sevoflurane neurotoxicity in neonatal rats is related to an increase in the GABAAR α1/GABAAR α2 ratio

Exposure of neonatal rat to sevoflurane leads to neurodegeneration and deficits of spatial learning and memory in adulthood. However, the underlying mechanisms remain unclear. The type A γ‐aminobutyric acid receptor (GABA_AR) is a target receptor for sevoflurane. The present study intends to investigate the changes in GABA_AR α1/α2 expression and its relationship with the neurotoxicity effect due to sevoflurane in neonatal rats. After a dose–response curve was constructed to determine minimum alveolar concentration (MAC) and safety was guaranteed in our 7‐day‐old neonatal rat pup mode, we conducted two studies among the following groups: (A) the control group; (B) the sham anesthesia group; and (C) the sevoflurane anesthesia group and all three groups were treated in the same way as the model. First, poly(ADP‐ribose) polymerase‐1 protein (PARP‐1) expression was determined in the different brain areas at 6 hr after anesthesia. Second, the expression of PARP‐1 and GABA_AR α1/GABA_AR α2 in the hippocampus area was tested by Western blotting at 6 hr, 24 hr, and 72 hr after anesthesia in all three groups. After 4 hr, with 0.8 MAC (2.1%) sevoflurane anesthesia, the PARP‐1 expression was significantly higher in the hippocampus than the other brain areas (p < .05). Compared with Groups A and B, the expression of PARP‐1 in the hippocampus of Group C significantly increased at 6 hr after sevoflurane exposure (216% ± 15%, p < .05), and the ratio of the α1/α2 subunit of GABA_AR surged at 6 hr (126% ± 6%), 24 hr (127% ± 8%), and 72 hr (183% ± 22%) after sevoflurane exposure in the hippocampus (p < .05). Our study showed that sevoflurane exposure of 0.8 MAC (2.1%)/4 hr was a suitable model for 7‐day‐old rats. And the exposure to sevoflurane could induce the apoptosis of neurons in the early stage, which may be related to the transmission from GABA_AR α2 to GABA_AR α1.     

Differential distributions of the NMDA receptor channel subunit mRNAs in the mouse retina

In the retina, the ε2 and ζ1 subunit mRNAs of the NMDA receptor channel were expressed from embryonic stages and found in ganglion cell layer and whole layer of inner nuclear layer at postnatal day 21 (P21). The ε1 subunit mRNA appeared postnatally and was distributed in ganglion cell layer and an inner third of inner nuclear layer at P21. These findings suggest that molecular organization of the NMDA receptor channel may alter during the retinal development.     

Metformin increases peroxisome proliferator–activated receptor γ Co‐activator‐1α and utrophin a expression in dystrophic skeletal muscle

Introduction: Metformin (MET) stimulates skeletal muscle AMP‐activated protein kinase (AMPK), a key phenotype remodeling protein with emerging therapeutic relevance for Duchenne muscular dystrophy (DMD). Our aim was to identify the mechanism of impact of MET on dystrophic muscle. Methods: We investigated the effects of MET in cultured C₂C₁₂ muscle cells and mdx mouse skeletal muscle. Expression of potent phenotypic modifiers was assessed, including peroxisome proliferator–activated receptor (PPAR)γ coactivator‐1α (PGC‐1α), PPARδ, and receptor‐interacting protein 140 (RIP140), as well as that of the dystrophin‐homolog, utrophin A. Results: In C₂C₁₂ cells, MET augmented expression of PGC‐1α, PPARδ, and utrophin A, whereas RIP140 content was reciprocally downregulated. MET treatment of mdx mice increased PGC‐1α and utrophin A and normalized RIP140 levels. Conclusions: In this study we identify the impact of MET on skeletal muscle and underscore the timeliness and importance of investigating MET and other AMPK activators as relevant therapeutics for DMD. Muscle Nerve 52 : 139–142, 2015     

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.     

GABAA and GABAC receptors on mammalian rod bipolar cells

Rod bipolar (RB) cells of mammalian retinae receive synapses from different γ-aminobutyric acid (GABAergic) amacrine cells in the inner plexiform layer (IPL). We addressed the question whether RB cells of the rabbit and of the rat retina express different types of GABA receptors at these synapses. RB cells were immunolabeled in vertical sections of rat retinae with an antibody against protein kinase C (PKC). The sections were double-labled for the α1, α2, α3, or γ2 subunits of the GABA_A receptor. Punctate immunofluorescence, which represents synaptic localization, was found for all four subunits. Many of the α1-, α3-, or γ2-immunoreactive puncta coincided with the axon terminals of the PKC-immunolabeled RB cells. Sections and wholemounts of rabbit retinae were also double labeled for PKC and the ρ subunits of the GABA_C receptor. Rabbit RB cells were decorated by many ρ-immunoreactive puncta, which were shown by electron microscopy to represent synaptic localization. Previous work from our laboratory has shown that the α1, α2, α3, and ρ subunits are not found within the same synapse but are expressed at different synaptic sites. Taken together, these results suggest that RB cells of mammalian retinae express at least three different types of GABA receptors at synaptic sites in the IPL: GABA_C receptors, GABA_Areceptors containing the α1 subunit, and GABA_A receptors containing the α3 subunit. J. Comp. Neurol. 396:351–365, 1998. © 1998 Wiley-Liss, Inc.     

Neurons of the chick brain and retina expressing both α-bungarotoxin-sensitive and α-bungarotoxin-insensitive nicotinic acetylcholine receptors: an immunohistochemical analysis

Immunohistochemical methods were used to study the possible co-localization of two α-bungarotoxin-sensitive (α7 and α8) and two α-bungarotoxin-insensitive (β2 and α3) subunits of the nicotinic acetylcholine receptors in neurons of the chick brain and retina. Several structures contained neurons that were doubly-labeled with antibodies against the α7 subunit and the β2 subunit. These structures included, for example, the interpeduncular nucleus, nucleus spiriformis lateralis, optic tectum, pretectal visual nuclei, and the lateral hypothalamus. Double-labeling with antibodies against the α7 and α8 subunits was also seen in several regions, which included the interpeduncular nucleus, visual pretectum, lateral hypothalamus, dorsal thalamus, and the habenular complex. In the retina, many cells in the inner nuclear layer were observed to contain α8 and α3 subunits, whereas neurons in the ganglion cell layer were seen to contain α7 and α8 or, less frequently, α7 and α3 subunits. These results indicate that α-bungarotoxin-sensitive and α-bungarotoxin-insensitive subunits of the nicotinic receptors are co-expressed by neurons of the chick brain and retina.     

Signaling mechanism underlying α2A‐adrenergic suppression of excitatory synaptic transmission in the medial prefrontal cortex of rats

Stimulation of α_2A‐adrenoceptors (ARs) in the prefrontal cortex (PFC) produces a beneficial effect on cognitive functions such as working memory. A previous study in our laboratory showed that α_2A‐AR stimulation suppresses excitatory synaptic transmission in layer V‐VI pyramidal cells of the rat medial PFC (mPFC). However, the intracellular mechanism underlying the α_2A‐AR suppression remains unclear. In the present study, we recorded evoked excitatory postsynaptic current (eEPSC) in layer V‐VI pyramidal cells of the mPFC, using whole‐cell patch‐clamp recording. We found that the α_2A‐AR agonist guanfacine significantly suppresses eEPSC in mPFC pyramidal cells. The α_2A‐AR inhibition is mediated by the Gi‐cAMP‐PKA‐PP1‐CaMKII‐AMPAR signaling pathway, as such inhibition no longer exists when each step of this pathway is blocked with NF023, Rp‐cAMP, PKI_5–24 or H89, tautomycin, and KN‐62 or KN‐93, respectively.     

γδ T cells: The overlooked T‐cell subset in demyelinating disease

γδ T cells represent a small subpopulation of T cells expressing a restricted repertoire of T‐cell receptors and, unlike αβ T cells, function more as cells of the innate immune system. These cells are found in skin and mucosal sites as well as secondary lymphoid tissues and frequently act as first line of defense sentinels. γδ T cells have been implicated in the pathogenesis of demyelinating disease, although little was known regarding their trafficking and effector functions. In this Mini‐Review, we highlight recent studies demonstrating that γδ T cells migrate rapidly to the CNS during experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis. γδ T‐cell trafficking to the CNS is independent of β₂‐integrins and occurs well before onset of clinical signs of disease, peaking early during the acute phase of disease. γδ T‐cell‐mediated production of inflammatory cytokines, including interferon‐γ and tumor necrosis factor‐α, appears critical for EAE development, suggesting that these cells may set the stage for activation of other subsets of infiltrating effector cells. These data suggest that γδ T cells or subsets of γδ T cells may represent a new therapeutic target in demeylinating disease. © 2009 Wiley‐Liss, Inc.     

α-tocopherol protects PC12 cells From hyperoxia-induced apoptosis

A rat clonal pheochromocytoma cell line (PC12) was cultured under normoxic (21% O₂) and hyperoxic (50% O₂) conditions. PC12 cells underwent apoptotic cell death when they were cultured in charcoal-stripped medium in a high-oxygen atmosphere. Vitamin E homologs, α-tocopherol (αT), β-tocopherol (βT), γ-tocopherol (γT), and δ-tocopherol (δT), were added to the culture medium to study their biological activities. αT was more effective than γT and δT in preventing hyperoxia-induced cell death. Addition of exogenous αT to charcoal-treated medium prevented lactate dehydrogenase (LDH) leakage from PC12 cells and also inhibited the apoptosis, which was accompanied by DNA fragmentation. Additional αT was rapidly concentrated in PC12 cells, suggesting that it exerts antioxidant effects. Our data show that PC12 cell death under high-oxygen conditions is due to apoptosis and that, among the vitamin E homologs, αT most effectively prevents hyperoxic apoptosis. J. Neurosci. Res. 52:184–191, 1998. © 1998 Wiley-Liss, Inc.     

A critical role of TRPM2 channel in Aβ42‐induced microglial activation and generation of tumor necrosis factor‐α

Amyloid β (Aβ)‐induced neuroinflammation plays an important part in Alzheimer's disease (AD). Emerging evidence supports a role for the transient receptor potential melastatin‐related 2 (TRPM2) channel in Aβ‐induced neuroinflammation, but how Aβ induces TRPM2 channel activation and this relates to neuroinflammation remained poorly understood. We investigated the mechanisms by which Aβ₄₂ activates the TRPM2 channel in microglial cells and the relationships to microglial activation and generation of tumor necrosis factor‐α (TNF‐α), a key cytokine implicated in AD. Exposure to 10–300 nM Aβ₄₂ induced concentration‐dependent microglial activation and generation of TNF‐α that were ablated by genetically deleting (TRPM2 knockout ;TRPM2‐KO) or pharmacologically inhibiting the TRPM2 channel, revealing a critical role of this channel in Aβ₄₂‐induced microglial activation and generation of TNF‐α. Mechanistically, Aβ₄₂ activated the TRPM2 channel via stimulating generation of reactive oxygen species (ROS) and activation of poly(ADPR) polymerase‐1 (PARP‐1). Aβ₄₂‐induced generation of ROS and activation of PARP‐1 and TRPM2 channel were suppressed by inhibiting protein kinase C (PKC) and NADPH oxidases (NOX). Aβ₄₂‐induced activation of PARP‐1 and TRPM2 channel was also reduced by inhibiting PYK2 and MEK/ERK. Aβ₄₂‐induced activation of PARP‐1 was attenuated by TRPM2‐KO and moreover, the remaining PARP‐1 activity was eliminated by inhibiting PKC and NOX, but not PYK2 and MEK/ERK. Collectively, our results suggest that PKC/NOX‐mediated generation of ROS and subsequent activation of PARP‐1 play a role in Aβ₄₂‐induced TRPM2 channel activation and TRPM2‐dependent activation of the PYK2/MEK/ERK signalling pathway acts as a positive feedback to further facilitate activation of PARP‐1 and TRPM2 channel. These findings provide novel insights into the mechanisms underlying Aβ‐induced AD‐related neuroinflammation.     

Somatic and neuritic spines on tyrosine hydroxylase–immunopositive cells of rat retina

Dopamine‐ and tyrosine hydroxylase–immunopositive cells (TH cells) modulate visually driven signals as they flow through retinal photoreceptor, bipolar, and ganglion cells. Previous studies suggested that TH cells release dopamine from varicose axons arborizing in the inner and outer plexiform layers after glutamatergic synapses depolarize TH cell dendrites in the inner plexiform layer and these depolarizations propagate to the varicosities. Although it has been proposed that these excitatory synapses are formed onto appendages resembling dendritic spines, spines have not been found on TH cells of most species examined to date or on TH cell somata that release dopamine when exposed to glutamate receptor agonists. By use of protocols that preserve proximal retinal neuron morphology, we have examined the shape, distribution, and synapse‐related immunoreactivity of adult rat TH cells. We report here that TH cell somata, tapering and varicose inner plexiform layer neurites, and varicose outer plexiform layer neurites all bear spines, that some of these spines are immunopositive for glutamate receptor and postsynaptic density proteins (viz., GluR1, GluR4, NR1, PSD‐95, and PSD‐93), that TH cell somata and tapering neurites are also immunopositive for a γ‐aminobutyric acid (GABA) receptor subunit (GABA_AR_α1), and that a synaptic ribbon‐specific protein (RIBEYE) is found adjacent to some colocalizations of GluR1 and TH in the inner plexiform layer. These results identify previously undescribed sites at which glutamatergic and GABAergic inputs may stimulate and inhibit dopamine release, especially at somata and along varicose neurites that emerge from these somata and arborize in various levels of the retina. J. Comp. Neurol. 525:1707–1730, 2017. © 2016 Wiley Periodicals, Inc.     

17β‐estradiol regulation of the mRNA expression of t‐type calcium channel subunits: Role of estrogen receptor α and estrogen receptor β

Low‐voltage‐activated (T‐type) calcium channels are responsible for burst firing and transmitter release in neurons and are important for exocytosis and hormone secretion in pituitary cells. T‐type channels contain an α1 subunit, of which there are three subtypes, Cav3.1, ‐3.2, and ‐3.3, and each subtype has distinct kinetic characteristics. Although 17β‐estradiol (E2) modulates T‐type calcium channel expression and function, little is known about the molecular mechanisms involved. We used real‐time PCR quantification of RNA extracted from hypothalamic nuclei and pituitary in vehicle and E2‐treated C57BL/6 mice to elucidate E2‐mediated regulation of Cav3.1, ‐3.2, and ‐3.3 subunits. The three subunits were expressed in both the hypothalamus and the pituitary. E2 treatment increased the mRNA expression of Cav3.1 and ‐3.2, but not Cav3.3, in the medial preoptic area and the arcuate nucleus. In the pituitary, Cav3.1 was increased with E2 treatment, and Cav3.2 and ‐3.3 were decreased. To examine whether the classical estrogen receptors (ERs) were involved in the regulation, we used ERα‐ and ERβ‐deficient C57BL/6 mice and explored the effects of E2 on T‐type channel subtypes. Indeed, we found that the E2‐induced increase in Cav3.1 in the hypothalamus was dependent on ERα, whereas the E2 effect on Cav3.2 was dependent on both ERα and ERβ. However, the E2‐induced effects in the pituitary were dependent on only the expression of ERα. The robust E2 regulation of T‐type calcium channels could be an important mechanism by which E2 increases the excitability of hypothalamic neurons and modulates pituitary secretion. J. Comp. Neurol. 512:347–358, 2009. © 2008 Wiley‐Liss, Inc.