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Anomalous mole-fraction effects (AMFE) were studied, using the inside-out configuration of the patchclamp technique, in both recombinant wild-type alpha-homomeric rat olfactory adenosine 3',5'-cyclic monophosphate (cAMP)-gated channels (rOCNC1) expressed in human embryonic kidney cells (HEK 293) and native cyclic nucleotide-gated (CNG) channels in acutely isolated rat olfactory receptor neurons. Single-channel and macroscopic currents were activated by 200 microM and 500 microM cAMP, respectively. Macroscopic currents, measured with mixtures of Na(+)-NH(4)(+) or Cs(+)-Li(+) in the cytoplasmic bathing solution, displayed AMFE in the rOCNC1 channels at both positive and negative membrane potentials. The rOCNC1 single-channel conductance showed a distinct minimum (or maximum) in an 80% Na(+)-20% NH(4)(+) mixture (or a 60% Cs(+)-40% Li(+) mixture), but only at positive membrane potentials. Macroscopic measurements in native olfactory CNG channels with mixtures of Na(+)-NH(4)(+) indicated similar AMFE. These results suggest that both native CNG channels and recombinant alpha-homomeric channels allow several ions to be present simultaneously within the channel pore. They also further validate the dominant role of the alpha-subunit in permeation through these channels, provide the first evidence to suggest that rOCNC1 channels have multi-ion properties and further justify the use of the rOCNC1 channel as an effective model for structure-function studies of ion permeation and selectivity in olfactory CNG channels.

Calbindin-D28k (calbindin) is a calcium-binding protein that is distributed widely in the rat brain. The localisation of calbindin immunoreactivity in the medulla oblongata and its colocalisation with adrenaline-synthesising neurons [phenylethanolamine-N-methyltransferase-immunoreactive (PNMT-IR)] was examined (Granata and Chang [1994] Brain Res. 645:265-277). However, detailed information about the distribution of calbindin-IR neurons in the reticular formation of the medulla oblongata in particular is lacking. In this report, the authors address this issue with an emphasis on the quantitation of calbindin-IR neurons, catecholamine neurons [tyrosine hydroxylase (TH)-IR, or PNMT-IR], and spinally projecting neurons in the ventral brainstem. Rats received injections of the retrograde tracing agent cholera toxin B (CTB) into the thoracic spinal cord or into the superior cervical ganglion. Immunocytochemistry was used to reveal calbindin, TH, PNMT, and CTB immunoreactivity. Ten calbindin-IR cell groups were identified within the pontomedullary reticular formation. Seven previously undescribed but distinct clusters of calbindin-IR neurons were found. Within the ventral pons, a population of calbindin-IR neurons occurred dorsal but adjacent to the A5 cell group. These calbindin-IR neurons did not contain either TH or PNMT immunoreactivity, and few if any of these neurons projected to the spinal cord. A distinct group of calbindin-IR neurons was present in the ventral medulla. Seventy-five percent of these calbindin-IR neurons contained TH immunoreactivity, 45% contained PNMT immunoreactivity, and 21% were spinally projecting neurons. Spinally projecting, calbindin-IR neurons were a subpopulation of PNMT-IR cells. In the caudal ventral medulla, no TH-IR or PNMT-IR cells were calbindin-IR. In the intermediolateral cell column, close appositions of calbindin-IR terminals on identified sympathetic preganglionic neurons as well as calbindin-IR synapses indicated that these neurons may affect directly the sympathetic outflow. The results demonstrate for the first time the existence of a new subpopulation of spinally projecting, PNMT-IR neurons in the rostral ventrolateral medulla., (Copyright 2000 Wiley-Liss, Inc.)

Our laboratory is interested in characterizing the neurotransmitter and hormonal phenotype of neurons in the rat hypothalamus expressing novel neuropeptide receptors of the neuropeptide Y and galanin families. In this review, we describe a technique combining nonradioactive in situ hybridization to detect mRNA and fluorescence immunohistochemistry to detect protein antigens. We examined paraffin sections of rat hypothalamus using confocal microscopy to determine whether mRNA for the galanin receptor, GALR2, was colocalized at the cellular level of resolution with somatostatin or tyrosine hydroxylase immunoreactivity. We found that many neurons in the hypothalamus expressed both GALR2 mRNA and either somatostatin or tyrosine hydroxylase immunoreactivity. The simultaneous detection of mRNA and protein immunoreactivity in individual neurons using the confocal microscope for visualization is an excellent tool for the analysis of newly characterized genes in the central nervous system., (Copyright 1999 Academic Press.)

One of the structural posttranslational modifications contributing to the formation of insoluble, and protease-resistant protein deposits in Alzheimer's disease (AD), such as neurofibrillary tangles (NFT) and beta-amyloid plaques are 'advanced glycation endproducts' (AGE). Using a polyclonal antibody against AGE in frozen sections of fixed brain tissue from Alzheimer's disease patients, AGE were identified in a further characteristic protein deposit in AD, namely in Hirano bodies. AGE are localized to ovoid, spherical, and rod-like Hirano bodies in the hippocampus, particularly numerous in the stratum lacunosum-moleculare of CA1. Since Hirano bodies are known to contain mainly cytoskeletal and cytoplasmic components and are localized within the soma of neurons our study suggests that AGE formation and intracellular protein crosslinking represent early stages during neuronal degeneration.

Both the cyclic adenosine 3',5'-monophosphate and the phosphoinositide second messenger systems are involved in olfactory signal transduction. The inositol 1,4,5-trisphosphate receptor is one of the principal intracellular calcium channels responsible for mobilizing stored calcium. The precise location of the 1,4,5-trisphosphate receptor (endoplasmic reticulum vs surface) and its role in the events of olfactory signal transduction need to be defined. By light microscopic and confocal immunohistochemistry we show expression of the olfactory-enriched G-protein, G(olf), associated with cyclic AMP responses, and of the inositol 1,4,5-trisphosphate receptor in the dendritic projections and cilia of essentially all olfactory receptor neurons, indicating that individual neurons express components of both second messenger systems. By immunoelectron microscopy, we demonstrate that the inositol 1,4,5-trisphosphate receptor is localized to the surface membrane of rat olfactory cilia. This is the first morphological demonstration of the surface membrane localization of inositol 1,4,5-trisphosphate receptor in olfactory cilia. Our findings, taken in conjunction with electrophysiological data from other workers, are supportive of the inositol 1,4,5-trisphosphate receptor playing a novel role in regulating calcium flux at the ciliary surface membrane.