neuropeptide-y and biocytin

Neurons coexpressing neuropeptide Y, agouti-related peptide, and GABA (NAG) play an important role in ingestive behavior and are located in the arcuate nucleus of the hypothalamus. NAG neurons receive both GABAergic and glutamatergic synaptic inputs, however, the developmental time course of synaptic input organization of NAG neurons in mice is unknown. In this study, we show that these neurons have low numbers of GABAergic synapses and that GABA is inhibitory to NAG neurons during early postnatal period. In contrast, glutamatergic inputs onto NAG neurons are relatively abundant by P13 and are comparatively similar to the levels observed in the adult. As mice reach adulthood (9-10 weeks), GABAergic tone onto NAG neurons increases. At this age, NAG neurons received similar numbers of inhibitory and EPSCs. To further differentiate age-associated changes in synaptic distribution, 17- to 18-week-old lean and diet-induced obesity (DIO) mice were studied. Surprisingly, NAG neurons from lean adult mice exhibit a reduction in the GABAergic synapses compared with younger adults. Conversely, DIO mice display reductions in the number of GABAergic and glutamatergic inputs onto NAG neurons. Based on these experiments, we propose that synaptic distribution in NAG neurons is continuously restructuring throughout development to accommodate the animals' energy requirements.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Age Factors; Animals; Animals, Newborn; Arcuate Nucleus of Hypothalamus; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Female; gamma-Aminobutyric Acid; Inhibitory Postsynaptic Potentials; Lysine; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neurons; Neuropeptide Y; Sodium Channel Blockers; Synapses; Tetrodotoxin; Vesicular Inhibitory Amino Acid Transport Proteins

The shape, structure and connectivity of nerve cells are important aspects of neuronal function. Genetic and epigenetic factors that alter neuronal morphology or synaptic localization of pre- and post-synaptic proteins contribute significantly to neuronal output and may underlie clinical states. To assess the impact of individual genes and disease-causing mutations on neuronal morphology, reliable methods are needed. Unfortunately, manual analysis of immuno-fluorescence images of neurons to quantify neuronal shape and synapse number, size and distribution is labor-intensive, time-consuming and subject to human bias and error. We have developed an automated image analysis routine using steerable filters and deconvolutions to automatically analyze dendrite and synapse characteristics in immuno-fluorescence images. Our approach reports dendrite morphology, synapse size and number but also synaptic vesicle density and synaptic accumulation of proteins as a function of distance from the soma as consistent as expert observers while reducing analysis time considerably. In addition, the routine can be used to detect and quantify a wide range of neuronal organelles and is capable of batch analysis of a large number of images enabling high-throughput analysis.

Topics: Animals; Cells, Cultured; Dendrites; Diagnostic Imaging; Disks Large Homolog 4 Protein; Electronic Data Processing; Guanylate Kinases; Hippocampus; Intracellular Signaling Peptides and Proteins; Lysine; Lysosomal Membrane Proteins; Membrane Proteins; Mice; Mice, Mutant Strains; Microtubule-Associated Proteins; Munc18 Proteins; Neurites; Neurons; Neuropeptide Y; Receptors, Transferrin; Software; Synapses; Synaptic Vesicles; Time Factors; Vesicle-Associated Membrane Protein 2

Depolarization by the neurotransmitter GABA regulates adult neurogenesis. We found interneurons of the neurogliaform cell family to be a primary source of GABA for newborn neurons in mouse dentate gyrus. GABAergic depolarization occurred in concert with reduced synaptic inhibition of mature neurons, suggesting that the local circuitry coordinates the activation of new and pre-existing cells.

Topics: Action Potentials; Animals; Animals, Newborn; Bacterial Proteins; Biophysical Phenomena; Cell Adhesion Molecules, Neuronal; Electric Stimulation; Excitatory Amino Acid Antagonists; Extracellular Matrix Proteins; GABA Antagonists; Glutamic Acid; Green Fluorescent Proteins; Interneurons; Iontophoresis; Luminescent Proteins; Lysine; Mice; Mice, Inbred C57BL; Mice, Transgenic; Models, Neurological; Nerve Net; Nerve Tissue Proteins; Neural Inhibition; Neurogenesis; Neuropeptide Y; Nitric Oxide Synthase Type I; Patch-Clamp Techniques; Picrotoxin; Pro-Opiomelanocortin; Quinoxalines; Reelin Protein; Serine Endopeptidases; Stem Cell Niche; Synaptic Potentials; Time Factors; Valine

We investigated the properties of neostriatal neuropeptide Y (NPY)-expressing interneurons in transgenic GFP (green fluorescent protein)-NPY reporter mice. In vitro whole-cell recordings and biocytin staining demonstrated the existence of a novel class of neostriatal NPY-expressing GABAergic interneurons that exhibit electrophysiological, neurochemical, and morphological properties strikingly different from those of previously described NPY-containing, plateau-depolarization low-threshold spike (NPY-PLTS) interneurons. The novel NPY interneuron type (NPY-neurogliaform) differed from previously described NPY-PLTS interneurons by exhibiting a significantly lower input resistance and hyperpolarized membrane potential, regular, nonaccommodating spiking in response to depolarizing current injections, and an absence of plateau depolarizations or low-threshold spikes. NPY-neurogliaform interneurons were also easily distinguished morphologically by their dense, compact, and highly branched dendritic and local axonal arborizations that contrasted sharply with the sparse and extended axonal and dendritic arborizations of NPY-PLTS interneurons. Furthermore, NPY-neurogliaform interneurons did not express immunofluorescence for somatostatin or nitric oxide synthase that was ubiquitous in NPY-PLTS interneurons. IPSP/Cs could only rarely be elicited in spiny projection neurons (SPNs) in paired recordings with NPY-PLTS interneurons. In contrast, the probability of SPN innervation by NPY-neurogliaform interneurons was extremely high, the synapse very reliable (no failures were observed), and the resulting postsynaptic response was a slow, GABA(A) receptor-mediated IPSC that has not been previously described in striatum but that has been elicited from NPY-GABAergic neurogliaform interneurons in cortex and hippocampus. These properties suggest unique and distinctive roles for NPY-PLTS and NPY-neurogliaform interneurons in the integrative properties of the neostriatum.

Topics: Animals; Bicuculline; Cell Count; Cerebral Cortex; Corpus Striatum; Electric Stimulation; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; Green Fluorescent Proteins; In Vitro Techniques; Inhibitory Postsynaptic Potentials; Interneurons; Lysine; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nerve Growth Factor; Neural Pathways; Neuropeptide Y; Nitric Oxide Synthase; Patch-Clamp Techniques; Quinoxalines; Somatostatin

The introduction of a reporter gene into bacterial artificial chromosome (BAC) constructs allows a rapid identification of the cell type expressing the gene of interest. Here we used BAC transgenic mice expressing a tau-sapphire green fluorescent protein (GFP) under the transcriptional control of the neuropeptide Y (NPY) genomic sequence to characterize morphological and electrophysiological properties of NPY-GFP interneurons of the mouse juvenile primary somatosensory cortex. Electrophysiological whole-cell recordings and biocytin injections were performed to allow the morphological reconstruction of the recorded neurons in three dimensions. Ninety-six recorded NPY-GFP interneurons were compared with 39 wild-type (WT) NPY interneurons, from which 23 and 19 were reconstructed, respectively. We observed that 91% of the reconstructed NPY-GFP interneurons had developed an atypical axonal swelling from which emerge numerous ramifications. These abnormalities were very heterogeneous in shape and size. They were immunoreactive for the microtubule-associated protein tau and the lysosomal-associated membrane protein 1 (LAMP1). Moreover, an electron microscopic analysis revealed the accumulation of numerous autophagic and lysosomal vacuoles in swollen axons. Morphological analyses of NPY-GFP interneurons also indicated that their somata were smaller, their entire dendritic tree was thickened and presented a restricted spatial distribution in comparison with WT NPY interneurons. Finally, the morphological defects observed in NPY-GFP interneurons appeared to be associated with alterations of their electrophysiological intrinsic properties. Altogether, these results demonstrate that NPY-GFP interneurons developed dystrophic axonal swellings and severe morphological and electrophysiological defects that could be due to the overexpression of tau-coupled reporter constructs.

Topics: Animals; Axons; Dendrites; Fluorescent Antibody Technique; In Vitro Techniques; Interneurons; Luminescent Proteins; Lysine; Lysosomal Membrane Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Immunoelectron; Neurodegenerative Diseases; Neuropeptide Y; Patch-Clamp Techniques; Reverse Transcriptase Polymerase Chain Reaction; Somatosensory Cortex; tau Proteins

A fundamental property of neuronal networks in Ammon's horn is that each area comprises a single glutamatergic cell population and various types of GABAergic neurons. Here we describe an exception to this rule, in the form of granule cells that reside within the CA3 area and function as glutamatergic nonprincipal cells with distinct properties. CA3 granule cells in normal, healthy rats, similarly to dentate gyrus granule cells, coexpressed calbindin and the homeobox protein Prox1. However, CA3 granule cells were located outside of the dentate gyrus, often hundreds of micrometers from the hilar border, in the lucidum and radiatum layers. CA3 granule cells were present in numbers that were comparable to the rarer GABAergic neuronal subtypes, and their somato-dendritic morphology, intrinsic properties, and perforant path inputs were similar to those of dentate gyrus granule cells. CA3 granule cell axons displayed giant mossy fiber terminals with filopodial extensions, demonstrating that not all mossy fibers originate from the dentate gyrus. Somatic paired recordings revealed that CA3 granule cells innervated CA3 pyramidal and GABAergic cells similarly to conventional mossy fiber synapses. However, CA3 granule cells were distinct in the specific organization of their GABAergic inputs. They received GABAergic synapses from cholecystokinin-expressing mossy fiber-associated cells that did not innervate the dentate granule cell layer, and these synapses demonstrated unusually strong activity-dependent endocannabinoid-mediated inhibition of GABA release. These results indicate that granule cells in the CA3 constitute a glutamatergic, nonprincipal neuronal subtype that is integrated into the CA3 synaptic network.

Topics: Animals; Animals, Newborn; CA3 Region, Hippocampal; Calbindins; Cannabinoid Receptor Modulators; Cholecystokinin; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; gamma-Aminobutyric Acid; Homeodomain Proteins; In Vitro Techniques; Lysine; Membrane Potentials; Microscopy, Electron, Transmission; Nerve Net; Neurons; Neuropeptide Y; Patch-Clamp Techniques; Quinoxalines; Rats; Rats, Wistar; S100 Calcium Binding Protein G; Synapses; Tumor Suppressor Proteins

The basal forebrain (BF) contains cholinergic as well as different types of non-cholinergic corticopetal neurons and interneurons, including neuropeptide Y (NPY) containing cells. BF corticopetal neurons constitute an extrathalamic route to the cortex and their activity is associated with an increase in cortical release of the neurotransmitter acetylcholine, concomitant with low voltage fast cortical EEG activity. It has been shown in previous studies (Duque et al. in J Neurophysiol 84:1627-1635, 2000) that in anesthetized rats BF cholinergic neurons fire mostly during low voltage fast cortical EEG epochs, while increased NPY neuronal firing is accompanied by cortical slow waves. In this paper, electrophysiologically and neurochemically characterized cholinergic and NPY-containing neurons were 3D reconstructed from serial sections and morphometrically analyzed. Cholinergic and NPY-containing neurons, although having roughly the same dendritic surface areas and lengths, were found to differ in dendritic thickness and branching structure. They also have distinct patterns of dendritic endings. The subtle differences in dendritic arborization pattern may have an impact on how synaptic integration takes place in these functionally distinct neuronal populations. Cholinergic neurons exhibited cortically projecting axons and extensive local axon collaterals. Elaborate local axonal arbors confined to the BF also originated from NPY-containing neurons. The presence of local axon collaterals in both cholinergic and NPY neurons indicates that the BF is not a mere conduit for various brainstem inputs to the cortex, but a site where substantial local processing must take place.

Topics: Acetylcholine; Animals; Axons; Basal Nucleus of Meynert; Brain Mapping; Cell Polarity; Cell Shape; Choline O-Acetyltransferase; Cholinergic Fibers; Dendrites; Electrophysiology; Image Cytometry; Immunohistochemistry; Lysine; Male; Neural Pathways; Neurons; Neuropeptide Y; Presynaptic Terminals; Rats; Rats, Sprague-Dawley; Software; Staining and Labeling

Reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, electrophysiological recording, and intraneuronal injection of the neuronal tracer biocytin were integrated in a study of the functional expression of corticotropin-releasing factor (CRF) receptors in the guinea pig enteric nervous system. RT-PCR revealed expression of CRF1 receptor mRNA, but not CRF2, in both myenteric and submucosal plexuses. Immunoreactivity for the CRF1 receptor was distributed widely in the myenteric plexus of the stomach and small and large intestine and in the submucosal plexus of the small and large intestine. CRF1 receptor immunoreactivity was coexpressed with calbindin, choline acetyltransferase, and substance P in the myenteric plexus. In the submucosal plexus, CRF1 receptor immunoreactivity was found in neurons that expressed calbindin, substance P, choline acetyltransferase, or neuropeptide Y. Application of CRF evoked slowly activating depolarizing responses associated with elevated excitability in both myenteric and submucosal neurons. Histological analysis of biocytin-filled neurons revealed that both uniaxonal neurons with S-type electrophysiological behavior and neurons with AH-type electrophysiological behavior and Dogiel II morphology responded to CRF. The CRF-evoked depolarizing responses were suppressed by the CRF1/CRF2 receptor antagonist astressin and the selective CRF1 receptor antagonist NBI27914 and were unaffected by the selective CRF2 receptor antagonist antisauvagine-30. The findings support the hypothesis that the CRF1 receptor mediates the excitatory actions of CRF on neurons in the enteric nervous system. Actions on enteric neurons might underlie the neural mechanisms by which stress-related release of CRF in the periphery alters intestinal propulsive motor function, mucosal secretion, and barrier functions.

Topics: Action Potentials; Aniline Compounds; Animals; Calbindins; Choline O-Acetyltransferase; Corticotropin-Releasing Hormone; Enteric Nervous System; Gastrointestinal Tract; Guinea Pigs; Lysine; Male; Myenteric Plexus; Neurons; Neuropeptide Y; Peptide Fragments; Pyrimidines; Receptors, Corticotropin-Releasing Hormone; RNA, Messenger; S100 Calcium Binding Protein G; Stress, Physiological; Submucous Plexus; Substance P

The role of interneurons in neurovascular coupling was investigated by patch-clamp recordings in acute rat cortical slices, followed by single-cell reverse transcriptase-multiplex PCR (RT-mPCR) and confocal observation of biocytin-filled neurons, laminin-stained microvessels, and immunodetection of their afferents by vasoactive subcortical cholinergic (ACh) and serotonergic (5-HT) pathways. The evoked firing of single interneurons in whole-cell recordings was sufficient to either dilate or constrict neighboring microvessels. Identification of vasomotor interneurons by single-cell RT-mPCR revealed expression of vasoactive intestinal peptide (VIP) or nitric oxide synthase (NOS) in interneurons inducing dilatation and somatostatin (SOM) in those eliciting contraction. Constrictions appeared spatially restricted, maximal at the level of neurite apposition, and were associated with contraction of surrounding smooth muscle cells, providing the first evidence for neural regulation of vascular sphincters. Direct perfusion of VIP and NO donor onto the slices dilated microvessels, whereas neuropeptide Y (NPY) and SOM induced vasoconstriction. RT-PCR analyses revealed expression of specific subtypes of neuropeptide receptors in smooth muscle cells from intracortical microvessels, compatible with the vasomotor responses they elicited. By triple and quadruple immunofluorescence, the identified vasomotor interneurons established contacts with local microvessels and received, albeit to a different extent depending on interneuron subtypes, somatic and dendritic afferents from ACh and 5-HT pathways. Our results demonstrate the ability of specific subsets of cortical GABA interneurons to transmute neuronal signals into vascular responses and further suggest that they could act as local integrators of neurovascular coupling for subcortical vasoactive pathways.

Topics: Animals; Cerebral Cortex; Cerebrovascular Circulation; Cytoplasm; gamma-Aminobutyric Acid; In Vitro Techniques; Interneurons; Lysine; Microcirculation; Muscle, Smooth, Vascular; Neuropeptide Y; Nitric Oxide Donors; Nitric Oxide Synthase; Patch-Clamp Techniques; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptors, Neuropeptide; Signal Transduction; Somatostatin; Vasoactive Intestinal Peptide; Vasoconstriction; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

Four types of neurons have previously been identified by neurochemical markers in the submucosal ganglia of the guinea-pig small intestine, and functional roles have been ascribed to each type. However, morphological differences among the classes have not been determined, and there is only partial information about their projections within the submucosa. In the present work, we used intracellular microelectrodes to fill neurons of each type with biocytin, which was then converted to a permanent dye, so that the shapes of the neurons could be determined and their projections within the submucosa could be followed. Cell bodies of noncholinergic secretomotor/ vasodilator neurons had Dogiel type I morphology. These neurons, which are vasoactive intestinal peptide immunoreactive, had single axons that ran through many ganglia without providing terminals around other neurons. Cholinergic secretomotor neurons with neuropeptide Y immunoreactivity had Stach type IV morphology, and cholinergic secretomotor/vasodilator neurons had stellate cell bodies. The axons of these two types ran short distances in the plexus and did not innervate other submucosal neurons. Neurons of the fourth type, intrinsic primary afferent neurons, had cell bodies with Dogiel type II morphology and their processes supplied networks of varicose processes around other nerve cells. It is concluded that each functionally defined type of submucosal neuron has a characteristic morphology and that intrinsic primary afferent neurons synapse with secretomotor neurons to form monosynaptic secretomotor reflex circuits.

Topics: Acetylcholine; Animals; Axons; Bodily Secretions; Cell Size; Dendrites; Female; Guinea Pigs; Immunohistochemistry; Intestine, Small; Lysine; Male; Neural Pathways; Neurons; Neuropeptide Y; Submucous Plexus; Vasoactive Intestinal Peptide; Vasodilation; Vasomotor System

Biocytin, recently introduced in neuroanatomical studies, was used as a retrograde tract tracer in combination with immunofluorescence in order to analyse the neurochemical characters of some central neuronal projections to the pars intermedia in two amphibian species, the anuran Rana esculenta and the urodele Triturus carnifex. After biocytin insertions in the pars intermedia, neurons became retrogradely labelled in the suprachiasmatic hypothalamus and the locus coeruleus of the brainstem in both species. Some scattered biocytin-labelled neurons were observed in the preoptic area. Moreover, working on the same sections, immunofluorescence revealed a number of codistributions and, in some cases, colocalization in the same neurons of biocytin labellings and immunopositivity for (1) tyrosine hydroxylase in the suprachiasmatic hypothalamus and the locus coeruleus of Rana and Triturus, (2) gamma-aminobutyric acid in the suprachiasmatic hypothalamus of Rana and Triturus and (3) neuropeptide Y in the suprachiasmatic hypothalamus of Rana. The specificity of such colocalizations was fully confirmed using dual-channel confocal laser scanning microscopy analysis.

Topics: Afferent Pathways; Animals; Axonal Transport; Coloring Agents; Fluorescent Antibody Technique, Indirect; gamma-Aminobutyric Acid; Locus Coeruleus; Lysine; Microscopy, Confocal; Microscopy, Fluorescence; Nerve Tissue Proteins; Neuropeptide Y; Pituitary Gland, Posterior; Preoptic Area; Rana esculenta; Species Specificity; Suprachiasmatic Nucleus; Triturus; Tyrosine 3-Monooxygenase

Hilar mossy cells of the mouse were shown recently to display calretinin immunoreactivity (Liu et al. [1996] Exp Brain Res 108:389-403). The morphological and connectional characteristics of these cells are poorly understood. In the present study, we used immunohistochemical, electron microscopic, and neuronal tracing techniques to describe their distribution, morphology, and connectivity. The distribution of calretinin-immunoreactive mossy cells varied significantly along the dorsoventral axis of the hilus. At dorsal levels, calretinin immunoreactivity was limited largely to a subpopulation of interneurons. At mid-dorsoventral and ventral levels, however, most if not all mossy cells displayed calretinin immunoreactivity. We found that most hilar mossy cells are calretinin immunoreactive but lack gamma-aminobutyric acid, as demonstrated by postembedding immunostaining of alternate semithin sections. Calretinin-immunoreactive mossy cells typically had two to three thick dendrites covered with complex spines (thorny excrescences). Electron microscopy revealed that these spines received multiple asymmetric contacts from mossy fibres. Axons arising from these cells formed a strong belt of calretinin immunoreactivity restricted to the inner third of the dentate molecular layer. This immunoreactivity was equally dense throughout the dorsoventral length of the dentate gyrus, suggesting that axons of calretinin-immunoreactive mossy cells located in the ventral levels diverge greatly and are capable of innervating distant regions of the dentate gyrus. Ultrastructural examination showed that calretinin-immunoreactive boutons made asymmetric synaptic contacts primarily on spines and, occasionally, on dendritic shafts of granule cells and accounted for the majority of asymmetrical synapses in the inner molecular layer. Injections of the retrograde tracer wheatgerm agglutinin-gold into the dentate gyrus demonstrated that calretinin-immunoreactive mossy cells concentrated in the ventral hilus project massively to both the dorsal and ventral aspect of the contralateral dentate gyrus. A small proportion of retrogradely labelled cells showed immunoreactivity for neuropeptide Y or somatostatin. If mossy cells of the ventral hilus receive the majority of their input from ventral granule cells, one may expect ventral granule cells to be more efficient in recruiting large numbers of granule cells during synchronous activity patterns than dorsal granule cells. Spontaneous acti

Topics: Animals; Calbindin 2; Dendrites; Dentate Gyrus; gamma-Aminobutyric Acid; Immunohistochemistry; Lysine; Male; Mice; Microscopy, Electron; Nerve Tissue Proteins; Neural Pathways; Neurons; Neuropeptide Y; S100 Calcium Binding Protein G; Silver Staining; Somatostatin

Analogs of Neuropeptide Y (NPY) were synthesized with conventional Boc/benzyl protective group strategy. Instead of Asn7 in the native sequence, Boc-Lys(Alloc)-OH was incorporated. At the end of the synthesis the Alloc group was selectively removed by palladium-catalyzed hydrostannolysis and biotin coupled to the epsilon-amino group of Lys7. After cleavage and characterization with plasma desorption mass spectrometry the N epsilon,7-biotinyl-[Lys7]-NPY and the nonbiotinylated analog [Lys7]-NPY were investigated as ligands to the NPY receptor from rat cerebral cortex. Both analogs were found to be high affinity ligands to the NPY receptor and bound with essentially the same affinity as unmodified NPY.

Topics: Animals; Biotin; Cerebral Cortex; Lysine; Male; Mass Spectrometry; Neuropeptide Y; Rats; Rats, Sprague-Dawley; Receptors, Neuropeptide Y