The natural axis of transmitter receptor distribution in the human cerebral cortex.

Transmitter receptors constitute a key component of the molecular machinery for intercellular communication in the brain. Recent efforts have mapped the density of diverse transmitter receptors across the human cerebral cortex with an unprecedented level of detail. Here, we distill these observations into key organizational principles. We demonstrate that receptor densities form a natural axis in the human cerebral cortex, reflecting decreases in differentiation at the level of laminar organization and a sensory-to-association axis at the functional level. Along this natural axis, key organizational principles are discerned: progressive molecular diversity (increase of the diversity of receptor density); excitation/inhibition (increase of the ratio of excitatory-to-inhibitory receptor density); and mirrored, orderly changes of the density of ionotropic and metabotropic receptors. The uncovered natural axis formed by the distribution of receptors aligns with the axis that is formed by other dimensions of cortical organization, such as the myelo- and cytoarchitectonic levels. Therefore, the uncovered natural axis constitutes a unifying organizational feature linking multiple dimensions of the cerebral cortex, thus bringing order to the heterogeneity of cortical organization.

Mouse Retinal Cell Atlas: Molecular Identification of over Sixty Amacrine Cell Types.

Amacrine cells (ACs) are a diverse class of interneurons that modulate input from photoreceptors to retinal ganglion cells (RGCs), rendering each RGC type selectively sensitive to particular visual features, which are then relayed to the brain. While many AC types have been identified morphologically and physiologically, they have not been comprehensively classified or molecularly characterized. We used high-throughput single-cell RNA sequencing to profile >32,000 ACs from mice of both sexes and applied computational methods to identify 63 AC types. We identified molecular markers for each type and used them to characterize the morphology of multiple types. We show that they include nearly all previously known AC types as well as many that had not been described. Consistent with previous studies, most of the AC types expressed markers for the canonical inhibitory neurotransmitters GABA or glycine, but several expressed neither or both. In addition, many expressed one or more neuropeptides, and two expressed glutamatergic markers. We also explored transcriptomic relationships among AC types and identified transcription factors expressed by individual or multiple closely related types. Noteworthy among these were Meis2 and Tcf4, expressed by most GABAergic and most glycinergic types, respectively. Together, these results provide a foundation for developmental and functional studies of ACs, as well as means for genetically accessing them. Along with previous molecular, physiological, and morphologic analyses, they establish the existence of at least 130 neuronal types and nearly 140 cell types in the mouse retina.SIGNIFICANCE STATEMENT The mouse retina is a leading model for analyzing the development, structure, function, and pathology of neural circuits. A complete molecular atlas of retinal cell types provides an important foundation for these studies. We used high-throughput single-cell RNA sequencing to characterize the most heterogeneous class of retinal interneurons, amacrine cells, identifying 63 distinct types. The atlas includes types identified previously as well as many novel types. We provide evidence for the use of multiple neurotransmitters and neuropeptides, and identify transcription factors expressed by groups of closely related types. Combining these results with those obtained previously, we proposed that the mouse retina contains ∼130 neuronal types and is therefore comparable in complexity to other regions of the brain.

Classifying neuronal subclasses of the cerebellum through constellation pharmacology.

A pressing need in neurobiology is the comprehensive identification and characterization of neuronal subclasses within the mammalian nervous system. To this end, we used constellation pharmacology as a method to interrogate the neuronal and glial subclasses of the mouse cerebellum individually and simultaneously. We then evaluated the data obtained from constellation-pharmacology experiments by cluster analysis to classify cells into neuronal and glial subclasses, based on their functional expression of glutamate, acetylcholine, and GABA receptors, among other ion channels. Conantokin peptides were used to identify N-methyl-d-aspartate (NMDA) receptor subtypes, which revealed that neurons of the young mouse cerebellum expressed NR2A and NR2B NMDA receptor subunits. Additional pharmacological tools disclosed differential expression of α-amino-3-hydroxy-5-methyl-4-isoxazloepropionic, nicotinic acetylcholine, and muscarinic acetylcholine receptors in different neuronal and glial subclasses. Certain cell subclasses correlated with known attributes of granule cells, and we combined constellation pharmacology with genetically labeled neurons to identify and characterize Purkinje cells. This study illustrates the utility of applying constellation pharmacology to classify neuronal and glial subclasses in specific anatomical regions of the brain.

Origin of quantal size variation and high-frequency miniature postsynaptic currents at the Caenorhabditis elegans neuromuscular junction.

The neuromuscular junction (NMJ) of Caenorhabditis elegans has proved to be a very useful model synapse for investigating molecular mechanisms of synaptic transmission. Intriguingly, miniature postsynaptic currents (minis) at this synapse occur at an unusually high frequency (50-90 Hz in wild-type worms) and show large variation in quantal size (from <10 pA to >200 pA). It is important to understand the cellular and molecular bases for these properties of minis in order to interpret electrophysiological data from this synapse properly. Existing data suggest that several factors may contribute to the high frequency and quantal size variation, including 1) the establishment of multiple NMJs with each body-wall muscle cell, 2) diversity of postsynaptic receptors (two acetylcholine receptors and one GABA receptor), 3) association of one presynaptic site with several body-wall muscle cells, 4) effects of Ca(2+) at the presynaptic site, and 5) a possibly elevated (less negative) resting membrane potential in motoneurons. Neither the frequency nor the quantal size of minis is affected by electrical coupling of body-wall muscle cells. Furthermore, quantal size variation is not due to synchronized multivesicular release. Analyses of the C. elegans NMJ may lead to a better understanding of the mechanisms controlling the frequency and quantal size of minis of other synapses as well.

Receptor classes and the transmitter-gated ion channels.

Transmitter-gated channels, which can be selective for cations or for anions, form an important class among the membrane receptors responsible for signal transduction. Thirteen principal types of these channels can now be recognized and most of these are available for analysis in recombinant form. It is instructive to contrast their characteristic structural features with those of the two other primary classes of the signal-transducing receptors of membranes.

Glutamate receptor subtypes may be classified into two major categories: a study on Xenopus oocytes injected with rat brain mRNA.

Three major subtypes of glutamate receptors that are coupled to cation channels are known. Recently an additional subtype that is coupled to G proteins and stimulates inositol phospholipid metabolism (the metabotropic glutamate receptor) has been proposed. The pharmacological characteristics of this receptor have now been examined. Although it shares some agonists with N-methyl-D-aspartate- and quisqualate-subtype receptors, it shares virtually no antagonists with any of the three cation channel-coupled receptor subtypes. Thus the metabotropic glutamate receptor belongs to a receptor category that is completely different from that of the other three receptor subtypes, not only functionally, but also pharmacologically.

Presynaptic lonotropic receptors.

The release of transmitters through vesicle exocytosis from nerve terminals is not constant but is subject to modulation by various mechanisms, including prior activity at the synapse and the presence of neurotransmitters or neuromodulators in the synapse. Instantaneous responses of postsynaptic cells to released transmitters are mediated by ionotropic receptors. In contrast to metabotropic receptors, ionotropic receptors mediate the actions of agonists in a transient manner within milliseconds to seconds. Nevertheless, transmitters can control vesicle exocytosis not only via slowly acting metabotropic, but also via fast acting ionotropic receptors located at the presynaptic nerve terminals. In fact, members of the following subfamilies of ionotropic receptors have been found to control transmitter release: ATP P2X, nicotinic acetylcholine, GABA(A), ionotropic glutamate, glycine, 5-HT(3), andvanilloid receptors. As these receptors display greatly diverging structural and functional features, a variety of different mechanisms are involved in the regulation of transmitter release via presynaptic ionotropic receptors. This text gives an overview of presynaptic ionotropic receptors and briefly summarizes the events involved in transmitter release to finally delineate the most important signaling mechanisms that mediate the effects of presynaptic ionotropic receptor activation. Finally, a few examples are presented to exemplify the physiological and pharmacological relevance of presynaptic ionotropic receptors.

Neurotransmitter receptor heteromers and their integrative role in 'local modules': the striatal spine module.

'Local module' is a fundamental functional unit of the central nervous system that can be defined as the minimal portion of one or more neurons and/or one or more glial cells that operates as an independent integrative unit. This review focuses on the importance of neurotransmitter receptor heteromers for the operation of local modules. To illustrate this, we use the striatal spine module (SSM), comprised of the dendritic spine of the medium spiny neuron (MSN), its glutamatergic and dopaminergic terminals and astroglial processes. The SSM is found in the striatum, and although aspects such as neurotransmitters and receptors will be specific to the SSM, some general principles should apply to any local module in the brain. The analysis of some of the receptor heteromers in the SSM shows that receptor heteromerization is associated with particular elaborated functions in this local module. Adenosine A(2A) receptor-dopamine D(2) receptor-glutamate metabotropic mGlu(5) receptor heteromers are located adjacent to the glutamatergic synapse of the dendritic spine of the enkephalin MSN, and their cross-talk within the receptor heteromers helps to modulate postsynaptic plastic changes at the glutamatergic synapse. A(1) receptor-A(2A) receptor heteromers are found in the glutamatergic terminals and the molecular cross-talk between the two receptors in the heteromer helps to modulate glutamate release. Finally, dopamine D(2) receptor-non-alpha(7) nicotinic acetylcholine receptor heteromers, which are located in dopaminergic terminals, introduce the new concept of autoreceptor heteromer.

Neurotransmitter receptor genotypes associated with mental and behavioral disorders.

AIM: Investigation of association studies within the field of mental and behavioral disorders is of value given their complex molecular etiology including epistatic interactions of multiple genes with small effects. MATERIALS & METHODS: Utilizing biomedical text mining, associations are uncovered for all mental and behavioral conditions listed in Diagnostic and Statistical Manual of Mental Disorders Text Revision. Specifically, a computational pipeline is designed to retrieve neurotransmitter receptor variations from biomedical literature with a text mining approach, where unique polymorphisms are also mined. RESULTS: Analyses of 1337 unique neurotransmitter receptors and 465 distinct conditions yield 1568 unique gene-disease associations. CONCLUSION: This study takes an unconventional approach to association studies and generates a novel dataset of associations for disorders such as major depression and schizophrenia, which provides a global perspective for their genetic etiology.

Two distinct receptor subtypes for mammalian bombesin-like peptides.

The mammalian bombesin-like peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB), are structurally related neuropeptides that elicit a wide spectrum of biological activities including regulation of smooth muscle contraction, stimulation of secretion, modulation of neural activity, and growth regulation. Earlier studies have shown that GRP and NMB are expressed in different regions of both the CNS and peripheral organs. Recent ligand-binding and molecular-cloning studies have revealed two pharmacologically distinct G-protein-coupled receptor subtypes for mammalian bombesin-like peptides that have different relative affinities for GRP, NMB and bombesin receptor antagonists. Similar to the peptide ligands, the two receptor subtypes are expressed in a distinct but overlapping set of CNS regions, some of which have been identified in functional studies as sites where bombesin peptides elicit defined biological responses. Delineation of these peptide ligands and receptor subtypes will be important in future studies that explore the molecular basis for the heterogeneous nature of the responses to bombesin observed in mammalian systems.

G-protein-independent signaling by G-protein-coupled receptors.

Two classes of receptors transduce neurotransmitter signals: ionotropic receptors and heptahelical metabotropic receptors. Whereas the ionotropic receptors are structurally associated with a membrane channel, a mediating mechanism is necessary to functionally link metabotropic receptors with their respective effectors. According to the accepted paradigm, the first step in the metabotropic transduction process requires the activation of heterotrimeric G-proteins. An increasing number of observations, however, point to a novel mechanism through which neurotransmitters can initiate biochemical signals and modulate neuronal excitability. According to this mechanism metabotropic receptors induce responses by activating transduction systems that do not involve G-proteins.

Different receptors mediate the electrophysiological and growth cone responses of an identified neuron to applied dopamine.

Neurotransmitters are among the many cues that may guide developing axons toward appropriate targets in the developing nervous system. We have previously shown in the mollusk Lymnaea stagnalis that dopamine, released from an identified pre-synaptic cell, differentially affects growth cone behavior of its target and non-target cells in vitro. Here, we describe a group of non-target cells that also produce an inhibitory electrophysiological response to applied dopamine. We first determined, using pharmacological blockers, which receptors mediate this physiological response. We demonstrated that the dopaminergic electrophysiological responses of non-target cells were sensitive to a D2 receptor antagonist, as are known target cell responses. However, the non-target cell receptors were linked to different G-proteins and intracellular signaling pathways than the target cell receptors. Despite the presence of a D2-like receptor at the soma, the growth cone collapse of these non-target cells was mediated by D1-like receptors. This study shows that different dopamine receptor sub-types mediated the inhibitory physiological and growth cone responses of an identified cell type. We therefore not only provide further evidence that D2- and D1-like receptors can be present on the same neuron in invertebrates, but also show that these receptors are likely involved in very different cellular functions.

Progress in the development of potent bombesin receptor antagonists.

Bombesin and the mammalian-related peptides gastrin-releasing peptide (GRP), GRP and neuromedin B have been shown to have numerous actions in the CNS, gastrointestinal tract and on growth. However, the role of the peptides in various physiological processes has remained unclear because of the lack of potent antagonists. Recent in vitro studies have described four different classes of bombesin receptor antagonist, some of which are active in the nanomolar range and in vivo. Robert Jensen and David Coy describe recent insights into peptide structural determinants of biological activity. Evidence from structure-function studies have resulted in identification of some analogues that function as potent antagonists in all systems examined. Furthermore, various subtypes of bombesin receptors can now be differentiated by these various classes of antagonist.

Profiling neurotransmitter receptor expression in mouse gonadotropin-releasing hormone neurons using green fluorescent protein-promoter transgenics and microarrays.

The definition of neurotransmitter receptors expressed by individual neuronal phenotypes is essential for our understanding of integrated neural regulation. We report here a single-neuron strategy using green fluorescent protein (GFP)-promoter transgenic mice and oligonucleotide microarrays that has enabled us to provide a qualitative profile of the neurotransmitter receptors expressed by the gonadotropin- releasing hormone (GnRH) neurons, critical for the neural regulation of fertility. Acute brain slices were prepared from adult female GnRH-GFP transgenic mice and single GnRH neurons identified and patched. The contents of GnRH neurons underwent reverse transcription and cDNA amplification using the switch mechanism at the 5' end of RNA templates system, and hybridization to mouse gene oligonucleotide arrays. Fifty different neurotransmitter receptor subunit mRNAs were detected in GnRH neurons. Many of the classical amino acid and aminergic receptors were present in addition to 14 distinct, and in most cases novel, neuropeptidergic receptor signaling families. Four of the latter were selected for functional validation with gramicidin-perforated patch-clamp electrophysiology. Galanin, GnRH and neuromedin B were all found to exert direct depolarizing actions upon GnRH neurons whereas somatostatin induced a potent hyperpolarizing response. These studies demonstrate a relatively straightforward approach for transcriptome profiling of specific neuronal phenotypes. The stimulatory actions of GnRH and galanin upon GnRH neurons found here indicate that positive ultrashort feedback loops exist among the GnRH neuronal population.

Binding of a iodinated substance P analog to a NK-1 receptor on isolated cell membranes from rat anterior pituitary.

The characteristics of binding sites for substance-P (SP) on rat anterior pituitary membranes were studied, using 125I-labeled Bolton-Hunter SP as a ligand. The binding of [125I]Bolton-Hunter SP was saturable and reversible, and reached a plateau of maximal binding after approximately 12 min of incubation. Scatchard and Hill analyses of specific binding revealed a single class of noninteracting binding sites with a high affinity (Kd = 0.72 nM) and a moderate density (binding capacity = 32.16 fmol/mg protein). The biologically active tachykinins, which, in addition to SP, consist of physalaemin, eledoisin, kassinin, neurokinin-A, and neurokinin-B, could inhibit the binding in a concentration-dependent manner. Based on titration curves, the IC50 values of the tachykinins were measured, and the receptor was classified as a NK-1 receptor. These results demonstrate that a population of specific SP-binding sites is present in rat anterior pituitary and further support evidence that this peptide has an influence on pituitary function.

Noradrenergic receptor function and aging: beyond the binding.

Quantitative studies of noradrenergic binding sites with aging probably underestimate the functional alterations in this, and other, central synaptic systems. Transmitters like norepinephrine, whose actions are based on amplification and interaction of interneuronal signalling capacity, rely on interactions among receptor subtypes for their complete effect. Thus, slight losses of one or both receptor subtypes with normal aging may have far more devastating effects.

Glutamate and GABA receptors in vertebrate glial cells.

Glial cells of the central nervous system express receptors for the main inhibitory and excitatory neurotransmitters, GABA and glutamate. The glial GABA and glutamate receptors share many properties with the neuronal GABAA and kainate/quisqualate receptors, but are molecularly and, in some aspects, pharmacologically distinct from their neuronal counterparts. The functional role of these receptors is as yet speculative: They have been proposed to control proliferation of astrocytes, serve to balance ion changes at GABAergic synapses, or they could enable the glial cell to detect neuronal synaptic activity.

Central G-Protein Coupled Receptors (GPCR)s as molecular targets for the treatment of obesity: assets, liabilities and development status.

In the last decade, the G-Protein-Coupled Receptor (GPCR) superfamily has emerged as a very promising and enriched source of therapeutic targets for the treatment of obesity. GPCRs represent the largest family of mammalian proteins, with approximately 1000 members. It is estimated that the GPCR family may comprise greater than 1% of the human genome and is the molecular target for approximately 30% of currently marketed drugs. Human GPCRs are modulated by a large variety of ligands, including peptides, lipids, neurotransmitters, nucleotides, ions and external sensory signals such as pheromones, tastes or odors. Many of the above ligands have been implicated in the physiological control of energy balance. This article will examine the biological rationale, assets, identified liabilities and current drug development status of these receptors as anti-obesity drug targets.

Antinociceptive activity of the bradykinin B1 and B2 receptor antagonists, des-Arg9, [Leu8]-BK and HOE 140, in two models of persistent hyperalgesia in the rat.

There has been recent evidence linking bradykinin (BK) receptors with inflammation. This study has investigated the involvement of BK receptors in two models of persistent inflammatory hyperalgesia in rats. In a Freund's adjuvant-induced hyperalgesia model and an ultraviolet (UV)-induced hyperalgesia model in rats the specific B2 antagonist, D-Arg[Hyp3, Thi5, D-Tic7, Oic8]-BK (HOE 140), was either ineffective or weakly active in reversing hyperalgesia. The specific B1 antagonist, des-Arg9, [Leu8]-BK, was effective in reversing or preventing the development of hyperalgesia in both Freund's adjuvant-induced hyperalgesia and UV-induced hyperalgesia. The B1 agonist, des-Arg9-BK, produced a small exacerbation of hyperalgesia in both models. Data suggest that in persistent inflammatory conditions in the rat bradykinin B1 receptors are involved in the accompanying hyperalgesia.

Cerebellar excitatory amino acid binding sites in normal, granuloprival, and Purkinje cell-deficient mice.

Using quantitative autoradiography, the cellular localization and characterization of cerebellar excitatory amino acid binding sites in normal, Purkinje cell-deficient and granuloprival (granule cell-deficient) mouse cerebella were investigated. In the molecular layer of normal mouse cerebellum, the quisqualate subtype of excitatory amino acid receptor (assayed by [3H](RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate, quisqualate-sensitive L-[3H]glutamate, and [3H]6-cyano-7-nitroquinoxaline-2,3-dione binding) predominated. In the granule cell layer of the cerebellum, N-methyl-D-aspartate-sensitive L-[3H]glutamate and [3H]glycine binding sites were predominant. In the molecular layer of Purkinje cell-deficient mutant mice, [3H](RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate binding sites and [3H]6-cyano-7-nitro-quinoxaline-2,3-dione binding were reduced to 24% (P less than 0.01) and 36% (P less than 0.001) of control, respectively, while quisqualate-sensitive [3H]glutamate binding sites were reduced to 54% of control (P less than 0.01). N-Methyl-D-aspartate-sensitive [3H]glutamate and [3H]glycine binding were unchanged. In the granule cell layer of these mouse cerebella, there was no change in excitatory amino acid receptor binding. In the molecular layer of granuloprival mouse cerebella, [3H](RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate binding was increased to 205% of control (P less than 0.01), [3H]6-cyano-7-nitro-quinoxaline-2,3-dione binding was increased to 136% of control (P less than 0.02), and quisqualate-sensitive [3H]glutamate binding was increased to 152% of control (P less than 0.01). N-Methyl-D-aspartate-sensitive [3H]glutamate and [3H]glycine binding were unchanged. In areas of granule cell depletion N-methyl-D-aspartate-sensitive [3H]glutamate and [3H]glycine binding were reduced to 68% (P less than 0.01) and 59% (P less than 0.01) of control, respectively. In the granule cell layer, binding to quisqualate receptors was not significantly different from binding in controls with any of the ligands tested. These results suggest that three different receptor assays: [3H](RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate, quisqualate-sensitive L-[3H]glutamate, and [3H]6-cyano-7-nitro-quinoxaline-2,3-dione binding can be used to demonstrate that quisqualate receptor specific binding sites are located on Purkinje cell dendrites in the molecular layer of cerebellum, and that these binding sites apparently up-regulate in response to granule cell ablation and Purkinje cell deafferentation.