Distribution of serotonin 2A, 2C and 3 receptor mRNA in spinal cord and medulla oblongata.

It is known that 5-HT receptors have significant roles in nociceptive and motor functions. We have compared the cellular localization of the mRNAs encoding serotonin 5-HT(2A,) 5-HT(2C,) 5-HT(3) receptor subtypes within different levels of the rat spinal cord and medulla. In the spinal cord, 5-HT(2C) receptor mRNA is expressed at high levels in most of the gray matter, except for lamina II. In contrast, 5-HT(2A) receptor mRNA is expressed exclusively in lamina IX. 5-HT(3) receptor mRNA has a low level and diffuse pattern of expression increasing towards the ventral horn. In both gray and white matter, there is a characteristic presence of a few highly stained cells. For each subtype, the expression pattern is similar in all four levels of the spinal cord. In the medulla, 5-HT(2C) receptor mRNA is at high levels in many nuclei including the hypoglossal nucleus, the gigantocellular reticular nucleus alpha and the parvocellular reticular nucleus alpha, the spinal nucleus of the trigeminal tract, the facial, and the dorsal medullary reticular field. Moderate to low levels of expression are seen in the spinal vestibular nucleus, the vagus, the solitary nuclei and the raphe. 5-HT(2A) receptor is expressed at high levels in some nuclei such as the hypoglossal nucleus, the intercalate nucleus, the inferior olive and the lateral reticular nucleus. Moderate to low levels of expression are seen in the facial, the medial vestibular nuclei, the nucleus ambiguous, the vagus, and the gigantocellular reticular nucleus. 5-HT(3) receptor mRNA is present at low levels in most of the nuclei examined, with a few scattered strongly labeled cells. The results show a distinct distribution of the three subtypes of receptors supporting their physiological roles and will help to understand the mechanisms of nociception and motor function.

GABA(A) receptor-mediated miniature postsynaptic currents and alpha-subunit expression in developing cortical neurons.

Previous studies have described maturational changes in GABAergic inhibitory synaptic transmission in the rodent somatosensory cortex during the early postnatal period. To determine whether alterations in the functional properties of synaptically localized GABA(A) receptors (GABA(A)Rs) contribute to development of inhibitory transmission, we used the whole cell recording technique to examine GABAergic miniature postsynaptic currents (mPSCs) in developing cortical neurons. Neurons harvested from somatosensory cortices of newborn mice showed a progressive, eightfold increase in GABAergic mPSC frequency during the first 4 wk of development in dissociated cell culture. A twofold decrease in the decay time of the GABAergic mPSCs, between 1 and 4 wk, demonstrates a functional change in the properties of GABA(A)Rs mediating synaptic transmission in cortical neurons during development in culture. A similar maturational profile observed in GABAergic mPSC frequency and decay time in cortical neurons developing in vivo (assessed in slices), suggests that these changes in synaptically localized GABA(A)Rs contribute to development of inhibition in the rodent neocortex. Pharmacological and reverse transcription-polymerase chain reaction (RT-PCR) studies were conducted to determine whether changes in subunit expression might contribute to the observed developmental alterations in synaptic GABA(A)Rs. Zolpidem (300 nM), a subunit-selective benzodiazepine agonist with high affinity for alpha1-subunits, caused a reversible slowing of the mPSC decay kinetics in cultured cortical neurons. Development was characterized by an increase in the potency of zolpidem in modulating the mPSC decay, suggesting a maturational increase in percentage of functionally active GABA(A)Rs containing alpha1 subunits. The relative expression of alpha1 versus alpha5 GABA(A)R subunit mRNA in cortical tissue, both in vivo and in vitro, also increased during this same period. Furthermore, single-cell RT-multiplex PCR analysis revealed more rapidly decaying mPSCs in individual neurons in which alpha1 versus alpha5 mRNA was amplified. Together these data suggest that changes in alpha-subunit composition of GABA(A)Rs contribute to the maturation of GABAergic mPSCs mediating inhibition in developing cortical neurons.

A simple enzyme histochemical method for the simultaneous demonstration of acetylcholinesterase and monoamine oxidase in fixed-frozen sections.

We describe an enzyme histochemical technique for the simultaneous demonstration of acetylcholinesterase (AChE) and monoamine oxidase (MAO) (Types A, B, or A+B) in fixed-frozen sections. Several regions in the mesencephalon and brainstem were examined for both somatic and neuropil labeling. The results obtained are equivalent or superior to those obtained using previous methods for the individual localization of these enzymes. The simultaneous visualization of AChE and MAO in the same section allows the relationship of the two enzymes to be easily assessed with brightfield microscopy.

Human intestinal folate transport: cloning, expression, and distribution of complementary RNA.

BACKGROUND & AIMS: Despite intensive investigations, very little is known about the molecular identity(ies) of the intestinal folate transport system(s), especially in humans. The aim of this study was to isolate a functional human intestinal folate carrier complementary DNA (cDNA) clone and determine the distribution of complementary RNA at the tissue and cellular levels. METHODS: Hybridization screening, modified Marathon cDNA amplification, expression in Xenopus oocytes, Northern analysis, and in situ hybridization were used. RESULTS: The hIFC-1 cDNA contains an open reading frame for 591 amino acids (relative molecular mass = 64,826, pI = 9.4, 12 transmembrane domains, three protein kinase C phosphorylation sites, and one N-glycosylation site) with 74% DNA and 66% amino acid sequence homologies with the mouse cDNA counterpart. Xenopus oocytes injected with hIFC-1 cRNA show induced folate uptake that was (1) saturable with substrate concentration (apparent Michaelis constant = 0.71 +/- 0.06 micromol/L; maximum velocity = 128 +/- 3 fmol x h(-1) x oocyte(-1)), (2) inhibited by methotrexate, folinic acid, and folic acid (Ki = 0.84 micromol/L, 0.71 micromol/L, and 10 micromol/L, respectively), and (3) sensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (Ki = 0.29 mmol/L). Northern analysis showed wide distribution of hIFC1-complementary messenger RNA species in various human tissues. In situ hybridization on sections of human jejunum showed preferential hIFC-1 expression in epithelial cells, especially in the upper half of the villi. CONCLUSIONS: These results represent the first molecular characterization of a human small intestinal folate carrier.

Laminar distributions of choline acetyltransferase and acetylcholinesterase activities in the inner plexiform layer of rat retina.

Choline acetyltransferase and acetylcholinesterase activities were measured in samples taken at 7-micron increments through the inner plexiform layer of rat retina. These enzyme activities were not uniformly distributed through the depth of the inner plexiform layer. Peaks of choline acetyltransferase activity occurred at about one-third and peaks of acetylcholinesterase activity at about one-fifth of the depth into the inner plexiform layer from either side. The positions of the two peaks of choline acetyltransferase activity most likely correspond to the locations of processes from cholinergic amacrine somata in the inner nuclear layer, which spread in sublamina a, and processes from cholinergic amacrine somata "displaced" in the ganglion cell layer which spread in sublamina b of the inner plexiform layer. The peaks of acetylcholinesterase activity may in addition correspond to the processes of cholinoceptive amacrine and ganglion cells. The magnitudes of choline acetyltransferase and acetylcholinesterase activities are as high as found anywhere in rat brain, emphasizing the important role of cholinergic mechanisms in visual processing through the rat inner plexiform layer.