Localization of M(2) muscarinic acetylcholine receptor protein in cholinergic and non-cholinergic terminals in rat hippocampus.

The muscarinic receptor family (M(1)-M(4)) mediates cholinergic modulation of hippocampal transmission. Pharmacological and physiological studies have indicated that a presynaptic receptor on cholinergic terminals plays a key role in regulating ACh release, although the molecular identity of this subtype is uncertain. In this study, the localization of the M(2) receptor is described in detail for the pyramidal cell layer in the CAl region of the hippocampus. Electron microscopic analysis of M(2) immunoreactivity in this area revealed mainly presynaptic expression of this subtype. Double-labeling experiments using antibodies to M(2) and to the vesicular acetylcholine transporter, a novel, specific marker of cholinergic terminals, were used to investigate the nature of these presynaptic receptors. These studies have revealed that M(2) is located in cholinergic and non-cholinergic terminals. This is the first direct anatomical evidence that suggests that M(2) may indeed function as a cholinergic autoreceptor in the hippocampus. The distribution of the M(2) receptor in non-cholinergic terminals also suggests functional roles for M(2) as a presynaptic heteroreceptor.

Expression of m1-m4 muscarinic acetylcholine receptor proteins in rat hippocampus and regulation by cholinergic innervation.

A family of muscarinic ACh receptor genes are expressed in hippocampus, but little is known about the localization of the encoded proteins and their regulation by cholinergic innervation. Subtype-specific antibodies were used to localize m1-m4 proteins in the hippocampal formation by immunocytochemistry and to determine the alterations in the subtypes following deafferentation. Each of the receptors is differentially localized in Ammon's horn and dentate gyrus, with highly complementary distributions. m1 is widely expressed in somata and dendrites of pyramidal neurons and granule cells in dentate gyrus. m2 immunoreactivity is expressed mostly in nonpyramidal neurons, and in several discrete bands of fibers and puncta surrounding pyramidal neurons and other layers. m3 is enriched in pyramidal neurons, the neuropil in stratum lacunosum-moleculare and the outer third of the molecular layer of dentate gyrus. m4 is enriched in nonpyramidal neurons, in fiber pathways (alveus, fimbria, and hippocampal commissure), and in the inner third of the molecular layer. Fimbria-fornix lesions decreased ipsilateral m2- and m4-immunoreactive axons in the fimbria, with no apparent changes in the distribution of any of the receptors in hippocampus. 192-IgG immunotoxin lesions of the cholinergic septohippocampal projections, which spare noncholinergic projections, produced a small decrease in m2-immunoreactive fibers in the fimbria with no other major changes in the distribution of subtypes. Immunoprecipitation studies at 3-28 d following fimbria-fornix lesions revealed a 25% loss of m2 at 3 d in hippocampus, and upregulation of both m1 (20-29% at 7-14 d) and m4 (44% at 28 d). Thus, the vast majority of muscarinic receptor subtypes are intrinsic to the hippocampal formation and/or nonseptal hippocampal afferents. A subset of m2 and m4 are presynaptically localized, with m2 in cholinergic axons and m2 and m4 possibly in noncholinergic axons that comprise the septohippocampal pathway. The unique laminar and regional distributions of m1-m4 in the hippocampus reflect differential cellular and subcellular distributions of the subtypes and/or selective association of receptor subtypes with certain afferent and intrinsic connections. These results indicate that each subtype likely has a different role in cholinergic modulation of excitatory and inhibitory hippocampal circuits.

Light and electron microscopic study of m2 muscarinic acetylcholine receptor in the basal forebrain of the rat.

The m2 muscarinic acetylcholine receptor gene is expressed at high levels in basal forebrain, but the paucity of information about localization of the encoded receptor protein has limited the understanding of cellular and subcellular mechanisms involved in cholinergic actions in this region. The present study sought to determine the cellular localization of m2 protein, its relationship to cholinergic neurons, and its pre- and postsynaptic distribution in the rat medial septum-diagonal band complex using immunocytochemistry with polyclonal rabbit antibodies and a newly developed rat monoclonal antibody specific to the m2 receptor. Light microscopic colocalization studies demonstrated that m2 was present in a subset of choline acetyltransferase immunoreactive neurons, in choline acetyltransferase-negative neurons, and in more neuropil elements than was choline acetyltransferase. Intraventricular injections of 192 IgG-saporin, an immunotoxin directed to the low-affinity nerve growth factor receptor, resulted in depletion of choline acetyltransferase-immunoreactive neurons in the medial septum-diagonal band complex, whereas m2 immunoreactivity in neurons and in the neuropil was unchanged. By electron microscopy, m2 receptor in medial septum-diagonal band complex was localized to the plasmalemma of a small population of small to medium-sized neurons, and it was also found in dendrites, axons, and axon terminals in the neuropil. Neurons expressing m2 immunoreactivity received synaptic contacts from unlabelled axon terminals. A small distinct subpopulation of large neurons, unlabelled by m2 immunoreactivity, received synaptic contacts from m2-immunoreactive terminals. Thus, m2 receptor is situated to mediate the local effects of acetylcholine on basal forebrain cholinergic and noncholinergic neurons and, also, at both pre- and postsynaptic sites.

Localization of muscarinic m3 receptor protein and M3 receptor binding in rat brain.

A family of receptor subtypes, defined either by molecular (m1-m5) or pharmacological (M1-M4) analysis, mediates muscarinic cholinergic neurotransmission in brain. The distribution and functions of the m3 receptor protein in brain and its relation to M3 ligand binding sites are poorly understood. To better characterize the native brain receptors, subtype-specific antibodies reactive with the putative third inner loops were used: (i) to measure the abundance of m3 protein and its regional distribution in rat brain by immunoprecipitation; (ii) to determine the cellular and subcellular distribution of m3 protein by light microscopic immunocytochemistry; and (iii) to compare the distribution of m3 immunoreactivity with the autoradiographic distribution of M3 binding sites labeled by [3H]4-diphenylacetoxy-N-methyl piperidine methioxide in the presence of antagonists selective for the other receptor binding sites. The m3 protein, measured by immunoprecipitation, accounted for 5-10% of total solubilized receptors in all brain regions studied. Immunocytochemistry also revealed a widespread distribution of m3-like immunoreactivity, and localized the subtype to discrete neuronal populations and distinct subcellular compartments. The distribution of m3 protein was consistent with the messenger RNA expression, and like M3 binding sites, the protein was enriched in limbic cortical regions, striatum, hippocampus, anterior thalamic nuclei, superior colliculus and pontine nuclei. However, m3 immunoreactivity and M3 binding were differentially localized in regions and lamina of cortex and hippocampus. The results confirm the presence of m3 protein in brain, its low abundance compared to other muscarinic receptor subtypes, and provide the first immunocytochemical map of its precise localization. The distribution of m3 suggests that it mediates a wide variety of cholinergic processes in brain, including possible roles in learning and memory, motor function and behavioral state control. However, since the distribution of the molecularly-defined receptor protein is distinct from the pharmacologically-defined M3 binding site, investigations of the functions of m3 in brain must await development of more selective ligands or use of non-pharmacological approaches.

Further evidence that Retzius-Cajal cells transform to nonpyramidal neurons in the developing rat visual cortex.

Electron microscopy and tritiated thymidine autoradiographic techniques were used to study the life history of Retzius-Cajal cells in the developing visual cortex of the rat, a subject which has long been debated by investigators. The findings show unequivocally that at least some of these characteristic cells of the immature animals remain in the adult cortex in the form of typical nonpyramidal neurons.

Retzius-Cajal cells: an ultrastructural study in the developing visual cortex of the rat.

The ontogenesis of Retzius-Cajal cells, a unique feature of developing cortical layer I in a variety of mammalian species, was examined with the electron microscope in coronal or tangential sections of the visual cortex of rats whose ages were closely spaced in time between day 17 of gestation and adulthood. At 17 days of gestation, Retzius-Cajal cells already display a characteristic appearance and some of the cytoplasmic organelles by which they are identified in the perinatal period. At birth they are recognized by their large size, horizontally oriented long processes, dark cytoplasmic ground substance and abundance of tightly packed organelles. One feature which is most typical of these cells at this, and later stages of development, is the presence in the cytoplasm of numerous wide cisterns of granular endoplasmic reticulum filled with electron-opaque material. Synapses are rarely seen on the perikarya and processes during the first week of postnatal life but become more frequent later in development. A pattern of modifications becomes noticeable in the morphology of these cells during the first postnatal week with the appearance of growth cone-like differentiations and new processes of varying sizes. Furthermore, their cytoplasm slowly acquires a lighter appearance, and the thickness of the characteristically long processes diminishes. The frequency of Retzius-Cajal cells decreases with age and at the end of the third postnatal week only very few can be recognized with certainty. Careful examination of a large series of sections during subsequent days revealed that the morphological characteristics of Retzius-Cajal cells continue to change until these cells can on longer be distinguished from classical layer I nonpyramidal neurons.