Dennis J. Goebel

NMDA receptor subunit gene expression in the rat brain: a quantitative analysis of endogenous mRNA levels of NR1Com, NR2A, NR2B, NR2C, NR2D and NR3A

Electrophysiological recordings have shown NMDA receptors to be heterogenous structures capable of responding to selected antagonists and agonists in multiple ways. This diversity in functional response has led investigators to conclude that these channels are comprised of unique combinations of receptor subunits which determine a cells functional NMDA-signature [H. Meguro, H. Mori, K. Araki, E. Kushiya, T. Kutsuwada, M. Yamazaki, T. Kumanishi, M. Arakawa, K. Sakimura, M. Mishina, Functional characterization of a heteromeric NMDA receptor channel expressed from cloned cDNAs, Nature (London) 357 (1992) 70-74; T. Ishii, K. Moriyoshi, H. Sugihara, K. Sakurada, H. Kadotani, M. Yokoi, C. Akazawa, R. Shigemoto, N. Mizuno, S. Nakanishi, Molecular characterization of the family of the N-methyl-d-aspartate receptor subunits, J. Biol. Chem. 268 (1993) 2836-2843; K.A. Wafford, C.J. Bain, B. Le Bourdelles, P.J. Whiting, J.A. Kemp, Preferential co-assembly of recombinant NMDA receptors composed of three different subunits, NeuroReport 4 (1993) 1347-1349; T. Priestley, P. Laughton, J. Myers, B. Le Bourdelles, J. Kerby, P.J. Whiting, Pharmacological properties of recombinant human N-methyl-d-aspartate receptors comprising NR1a/NR2A and NR1a/NR2B subunit assemblies expressed in permanently transfected mouse fiberblast cells, Mol. Pharmacol. 48 (1995) 841-848; P.H. Seeburg, N. Burnashev, G. Kohr, T. Kuner, R. Sprengel, H. Monyer, The NMDA receptor channel: molecular design of a coincidence detector, Recent Prog. Horm. Res. 50 (1995) 19-34; A.L. Buller, D.T. Monagahan, Pharmacological heterogeneity of NMDA receptors: characterization of NR1a/NR2D heteromers expressed in Xenopus oocytes, Eur. J. Pharmacol. 320 (1997) 87-94]. In situ hybridization and immunocytochemical studies have shown that there is a spatio-temporal level of expression throughout the brain for each of the receptor subunits with some regions showing a strong preference for a particular subunit. Although these studies collectively show that there are regional differences with respect to NMDA receptor subunit expression in the brain, it has not been determined at what level(s) these genes are expressed or whether each region displays a unique NMDA-subunit signature. The present study was undertaken to examine the level of gene expression for the NR1, NR2A, NR2B, NR2C, NR2D and NR3A receptor subunits in isolated regions of rat brain using the nuclease protection assay. Results show that each of the brain regions examined expresses all six NMDA receptor subunits. The level of message expression for NR1 greatly exceeded that of the other subunits combined, with values ranging from 67-88% of the total subunit gene expression. The relative proportions of the other subunits (NR2A-D and NR3A) varied widely, suggesting that NMDA receptor composition is unique to each region of the brain.

Impairment of TrkB-PSD-95 signaling in Angelman syndrome.

Brain-derived neurotrophic factor signaling is defective in Angelman syndrome and can be rescued by disruption of Arc/PSD95 binding.

GABA-immunoreactivity in ganglion cells of the rat retina☆

Ganglion cells in the rat retina were labeled with the fluorescent dye, Diamidino-yellow, by retrograde transport from the superior colliculus and subsequently reacted for GABA-like immunoreactivity with a rhodamine-conjugated antiserum. Examination of sectioned retinas by fluorescence microscopy showed double labeling in approximately 6% of the ganglion cells. The presence of GABA in these neurons suggests that they may be involved in providing direct inhibitory input to the rat tectum.

Immunocytochemical evidence for the involvement of glycine in sensory centers of the rat brain.

Glycine-like immunoreactivity was localized to a number of sites in the rat brain which are involved in processing sensory information. In the auditory and vestibular systems, glycine immunoreactivity was seen in dorsal and ventral cochlear nuclei, superior olive, trapezoid body, medial and lateral vestibular nuclei, and inferior colliculus. Staining in the visual system was seen in retina, dorsal lateral geniculate nucleus, and superior colliculus. The olfactory system exhibited staining in the olfactory bulb and accessory olfactory formation. Somatosensory centers with glycine immunoreactivity included the dorsal column nuclei, spinal trigeminal nucleus, principal sensory nucleus of V, reticular formation, and periaqueductal gray. Glycine-immunoreactive neurons were also seen in cerebellar cortex, deep cerebellar nuclei, hippocampus, cerebral cortex, and striatum. The distribution of staining indicates that glycine plays a major role in sensory centers with actions at both strychnine-sensitive and strychnine-insensitive receptors.

Immunocytochemical demonstration of glycine in retina

An antiserum against glycine (Gly) conjugated to bovine serum albumin was raised and used to localize Gly-containing neurons in the retina. Reactive cells were also seen in the brainstem. This provides the first direct confirmation of the Gly content of these cells.

Quantitation, cellular localization and regulation of neurokinin receptor gene expression within the rat substantia nigra

The diverse biological effects of substance P and related peptides are mediated by multiple neurokinin receptors. The CNS sites of neurokinin receptor biosynthesis have not been fully elucidated and little is known about the regulation of neurokinin receptor gene expression. In the present study, the abundance of neurokinin-1, neurokinin-2 and neurokinin-3 receptor messenger RNAs in various rat brain regions was quantitated using a sensitive solution hybridization assay. Midbrain neurokinin receptor gene expression was then examined in detail. In situ hybridization experiments localized high levels of neurokinin-3 receptor messenger RNA to presumptive dopamine neurons, as evidenced by sensitivity to 6-hydroxydopamine lesions and the presence of tyrosine hydroxylase messenger RNA in serial sections. Lesions of nigral afferent (including substance P-containing) pathways from the caudate-putamen increased both nigral neurokinin-3 and neurokinin-1 receptor messenger RNA levels two- to three-fold. These data provide the anatomical substrate for physiological data suggesting that substance P (released from striatonigral neurons) may act on nigral cells through neurokinin-1 receptors, while the substance P co-transmitter neurokinin A may act preferentially on dopamine neurons through neurokinin-3 receptors. The magnitude of denervation-induced changes in neurokinin receptor messenger RNAs suggests significant plasticity of neurokinin receptor gene expression.

Quantitation of rat dopamine transporter mrna effects of cocaine treatment and withdrawal

Abstract: Dopamine transporter mRNA levels in the rat substantia nigra were quantified using a sensitive nuclease protection assay with a highly homologous human dopamine transporter cDNA clone. The same probe was also used to visualize dopamine transporter mRNA in the substantia nigra by in situ hybridization. Repeated cocaine administration (15 mg/kg, twice a day for 6.5 days) resulted in a >40% decrease in nigral dopamine transporter mRNA levels. In contrast, dopamine transporter mRNA levels were unchanged after either acute treatment (4 h before death) or repeated cocaine treatment followed by a 72‐h withdrawal period. Thus, blockade of the dopamine transporter by repeated cocaine administration may result in the down‐regulation of dopamine transporter gene expression in dopamine neurons.

Colocalization of substance P and γ-aminobutyric acid in amacrine cells of the cat retina

Abstract Substance P and γ-aminobutyric acid (GABA) were colocalized by immunocytochemistry in two subpopulations of amacrine cells in the cat retina. All of the cells which stained for substance P also showed GABA reactivity. However, there were many GABA-immunoreactive cells which did not stain for substance P. The presence of neuropeptides provides a basis for additional neurochemical characterization of the multiple populations of GABA immunoreactive cells.

Calretinin in the cat retina: colocalizations with other calcium-binding proteins, GABA and glycine.

Immunocytochemical techniques were used to determine the distribution of the calcium-binding protein calretinin in the cat retina. Comparisons were made with parvalbumin and calbindin as well as with the inhibitory neurotransmitters GABA and glycine. Calretinin immunoreactivity was seen in horizontal cells and multiple subpopulations of amacrine and ganglion cells. Cone outer segments were also stained. Calbindin immunoreactivity was present in cone photoreceptors, horizontal cells, at least two subtypes of cone bipolar cell, numerous amacrine cells, and cells residing in the ganglion cell layer. Heavy staining for parvalbumin was found in both A- and B-type horizontal cells, distinct subpopulations of amacrine and ganglion cells, and a small population of cone photoreceptor cells. To confirm the identity of cone photoreceptors, comparisons were made with retinas stained for opsins specific for red/green or blue cones (Szél et al., 1986). The localization of parvalbumin corresponded with that of blue-type cones only whereas calretinin and calbindin staining showed the same distribution as both red/green and blue cones. Double-label immunofluorescence studies revealed colocalization of all three of the calcium-binding proteins in a number of neurons including horizontal cells and AII amacrine cells. To assess a possible transmitter-specific relationship for calretinin, double-label studies were carried out with GABA and glycine. However, the staining patterns for each of these inhibitory amino acids differed substantially from that of calretinin. The possibility remains that calretinin and other calcium-binding proteins may play a role in neurotransmission through interactions with receptors or second-messenger agents.

Autoradiographic studies of [3H]-glycine, [3H]-GABA, and [3H]-muscimol uptake in the mudpuppy retina

Autoradiographic studies showed selective accumulation of [3H]-glycine, [3H]-GABA, and the GABA agonist [3H]-muscimol by neurons of the mudpuppy retina. [3H]-Glycine was taken up by bipolar cells, amacrine cells, and displaced amacrine or ganglion cells. Both [3H]-GABA and [3H]-muscimol were also accumulated by bipolar cells, amacrine cells and ganglion layer cells. However, the [3H]-GABA uptake pattern differed from that of [3H]-muscimol in showing labeling of horizontal cells, an increased percentage of cells in the ganglion cell layer, and a band in the most proximal portion of the inner plexiform layer. Variations in grain density suggested the presence of multiple subpopulations of [3H]-glycine- and [3H]-GABA-labeled amacrine cells. The labeled cells may play a role in inhibitory pathways in the inner retina.