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PubMed1999

Expression of the striatal DARPP-32/ARPP-21 phenotype in GABAergic neurons requires neurotrophins in vivo and in vitro.

The medium spiny neuron (MSN) is the major output neuron of the caudate nucleus and uses GABA as its primary neurotransmitter. A majority of MSNs coexpress DARPP-32 and ARPP-21, two dopamine and cyclic AMP-regulated phosphoproteins, and most of the matrix neurons express calbindin. DARPP-32 is the most commonly used MSN marker, but previous attempts to express this gene in vitro have failed. In this study we found that DARPP-32 is expressed in <12% of E13- or E17-derived striatal neurons when they are grown in defined media at high or low density in serum, dopamine, or Neurobasal/N2 (Life Technologies), and ARPP-21 is expressed in <1%. The percentage increases to 25% for DARPP-32 and 10% for ARPP-21 when the same cells are grown in Neurobasal/B27 (Life Technologies) for 7 d. After growth in Neurobasal/B27 plus brain-derived neurotrophic factor (BDNF) for 7 d, E13-derived MSNs are 53.7% DARPP-32-positive and 29. 0% ARPP-21-positive; E17-derived MSNs are 66.8% DARPP-32-positive and 51.5% ARPP-21-positive. The percentage of calbindin-positive neurons also is increased under these conditions. Finally, ARPP-21 expression is reduced in mice with a targeted deletion of the BDNF gene. We conclude that BDNF is required for the maturation of a large subset of patch and matrix MSNs in vivo and in vitro. In addition, we introduce a culture system in which highly differentiated MSNs may be generated, maintained, and studied.

Animals↗
PubMed2001

An activity-dependent neurotrophin-3 autocrine loop regulates the phenotype of developing hippocampal pyramidal neurons before target contact.

Neurotrophin-3 (NT-3), its cognate receptor trkC, and voltage-gated calcium channels are coexpressed by embryonic pyramidal neurons before target contact, but their functions at this stage of development are still unclear. We show here that, in vitro, anti-NT-3 and anti-trkC antibodies blocked the increase, and NT-3 reversed the decrease in the number of calbindin-D(28k)-positive pyramidal neurons induced by, respectively, calcium channel activations and blockades. Similar results were obtained with single-neuron microcultures. In addition, voltage-gated calcium channel inhibition downregulates the extracellular levels of NT-3 in high-density cultures. Moreover, electrophysiological experiments in single-cell cultures reveal a tetrodotoxin-sensitive spontaneous electrical activity allowing voltage-gated calcium channel activation. The mouse NT-3 (-/-) mutation decreases by 40% the number of developing calbindin-D(28k)-positive pyramidal neurons, without affecting neuronal survival, both in vitro and in vivo. Thus, present results strongly support that an activity-dependent autocrine NT-3 loop provides a local, intrinsic mechanism by which, before target contact, hippocampal pyramidal-like neurons may regulate their own differentiation, a role that may be important during early CNS differentiation or after adult target disruption.

Animals↗
PubMed2003

Calcium dynamics, buffering, and buffer saturation in the boutons of dentate granule-cell axons in the hilus.

The axons of dentate gyrus granule cells form synapses in the hilus. Ca(2+) signaling was investigated in the boutons of these axons using confocal fluorescence imaging. Boutons were loaded with various concentrations of the Ca(2+) indicator Oregon Green BAPTA-1 by patch-clamping the cell bodies and allowing the dye to diffuse into the axon. Resting free [Ca(2+)] started at 74 nm, rose to approximately 1 microm immediately after an action potential, and then decayed to rest with a time constant of 43 msec (all extrapolated to a dye concentration of zero). Action potential-induced [Ca(2+)] rises were smaller in larger boutons, consistent with a size-independent Ca(2+) channel density of 45/microm(2). Action potential-induced [Ca(2+)] changes varied with dye concentration in a manner consistent with kappa(E) approximately 20 for the ratio of endogenous buffer-bound Ca(2+) to free Ca(2+). During trains of action potentials, [Ca(2+)] increments summed supralinearly by more than that expected from dye saturation. The amount of endogenous Ca(2+) buffering declined as [Ca(2+)] rose, and this saturation indicated a buffer with a dissociation constant of approximately 500 nm and a concentration of approximately 130 microm. This is similar to the dissociation constant of calbindin-D28K, a Ca(2+)-binding protein that is abundant in dentate granule cells. Thus, calbindin-D28K is a good candidate for the Ca(2+) buffer revealed by these experiments. The saturation of endogenous buffer can generate short-term facilitation by amplifying [Ca(2+)] changes during repetitive activity. Buffer saturation may also be relevant to the presynaptic induction of long-term potentiation at synapses formed by dentate granule cells.

Action Potentials↗
PubMed1991

[Action of 1,25-dihydroxyvitamin D3 in the tooth germ. Modulations of receptor in the development].

The control of tooth development by 1,25-Dihydroxyvitamin D3 is analyzed by light- and electron-microscope immunocytochemistry and Northern-blotting in vitamin D-deficient rats. The receptor for 1,25-Dihydroxyvitamin D3, immunostained at the light microscope in all stem-cells, became immunodetectable only at the ultrastructural level in the ameloblasts which elaborate enamel and odontoblasts which synthetize dentin. Moreover, 1,25-Dihydroxyvitamin D3 induces an up-regulation specifically in these cells. In parallel, the calbindins-D9k, -D28k and osteocalcin, in contrast to the phosphoprotein, appear sensitive to vitamin D-deficiency. A single injection of 1,25-Dihydroxyvitamin D3 led to the increase of steady-state levels of the corresponding calbindin mRNAs. These data show that tooth constitutes a target-organ for 1,25-Dihydroxyvitamin D3, as other components of the phospho-calcic metabolism.

Animals↗
PubMed1991

[Current concepts of odontogenesis].

Tooth growth and development involve complex biochemical and physiological processes. Some vitamins are required as coenzymes, but these molecules are also needed to synthesize specific proteins with structural or transport functions. Collagen synthesis is carried out by ascorbic acid interaction. Vitamin A induces calbindin synthesis. Collagen and calbindins are both involved in the mineralization and development of hard tissues. The formation, eruption and growth of teeth are also mediated by the interaction of several hormones.

Animals↗
PubMed1993

The heterogeneity of the mesostriatal dopaminergic system as revealed in normal and parkinsonian monkeys.

The present immunohistochemical study has shown that the mesostriatal DA system in primates is a highly heterogeneous entity composed of several subsystems displaying variable degrees of vulnerability to MPTP. These subsystems were shown to affect the expression of neuropeptide in the striatofugal neurons in a manner that varies according to the location of the cell bodies in the various striatal territories. Also, a distinct subset of DA neurons in the SN/VTA complex was found to specifically innervate GPi and to be relatively resistant to the neurotoxic effect of MPTP. Furthermore, DA neurons expressing calbindin were found to be much less severely affected than other DA neurons in the SN/VTA complex of PD monkeys. Therefore, in contrast to the situation in other neurodegenerative disorders, such as Alzheimer's and Huntington's diseases, calbindin can be used as a marker for a specific subpopulation of DA neurons that are less prone to degeneration in Parkinson's disease.

Animals↗
PubMed1993

Cultured hippocampal neurons show responses to BDNF, NT-3, and NT-4, but not NGF.

To investigate the possibility of neurotrophins acting directly on hippocampal neurons, we first examined expression of the trk receptors in sections of adult rat brain and in cultures of embryonic rat hippocampus, and then investigated general and specific responses of cultured hippocampal neurons to each of the neurotrophins. In situ hybridization studies indicated high levels of expression of trkB and trkC but not trkA in pyramidal cells, dentate granule neurons, and scattered interneurons. Cultures of embryonic day 18 (E18) hippocampal neurons were also found by Northern analysis to express trkB and trkC but not trkA, indicating potential responsiveness to brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and NT-4, but not NGF. Phosphorylation experiments indeed showed that BDNF, NT-3, and NT-4 produced rapid tyrosine phosphorylation of Trk proteins, as detected by immunoprecipitation using a pan-Trk-specific antibody, whereas NGF produced no detectable tyrosine phosphorylation in hippocampal cultures. Similarly, all of the neurotrophins, except NGF, induced expression of c-fos mRNA and c-fos protein in these cultures. c-Fos protein induction was detectable in approximately 40-50% of the cells. While we observed no major effect of any of the neurotrophins upon the survival of E18 hippocampal neurons, BDNF, NT-3, and NT-4, but not NGF, produced marked increases in the number of neurons expressing detectable levels of either calbindin or AChE. NT-3 produced the greatest increase in the number of calbindin-positive neurons, whereas BDNF and NT-4 produced the greater increase in the number of AChE-positive neurons. Our results suggest that several of the neurotrophins have important effects in the differentiation and maintenance of function of subpopulations of hippocampal neurons.

Acetylcholinesterase↗
PubMed1993

Vitamin D receptor zinc finger region binds to a direct repeat as a dimer and discriminates the spacing number between each half-site.

1 alpha,25-Dihydroxyvitamin D3, the most active metabolite of vitamin D3, is a multifunctional agent. The actions of 1 alpha,25-dihydroxyvitamin D3 are mediated through its receptor that activates the specific genes in a ligand-dependent manner. In order to investigate the details of DNA binding properties of vitamin D receptor, we have developed the overexpression and purification system of vitamin D receptor DNA binding domain. The purified peptide could specifically bind to the osteopontin-derived vitamin D responsible element (VDRE) but not to the osteocalcin and the calbindin D-9k-derived VDREs, as determined by bandshift analysis. The osteopontin VDRE contains a direct repeat of GGTTCA motif separated by 3 nucleotides, whereas the osteocalcin and calbindin D-9k VDREs have inadequate direct repeat. Further analyses using synthetic oligonucleotides revealed that vitamin D receptor DNA binding domain could discriminate the spacing number between the consensus steroid-responsible element motif and had different affinities to direct repeats that consisted of various related sequences. These studies give insight into ways in which vitamin D receptor mediates the signal of 1 alpha,25-dihydroxyvitamin D3.

Animals↗
PubMed1996

Are the interlaminar zones of the ferret dorsal lateral geniculate nucleus actually part of the perigeniculate nucleus?

The ferret dorsal lateral geniculate nucleus (LGNd) contains interneurons within the interlaminar zones situated between the laminae corresponding to the ipsi- and contralateral eyes. We found that a subset of these neurons exhibits electrophysiological properties similar to those previously reported for perigeniculate (PGN) neurons, including the generation of rhythmic sequences of rebound low-threshold Ca2+ spikes at a frequency of 1-4 Hz after the intracellular injection of a hyperpolarizing current pulse. These "PGN-like" interlaminar interneurons innervated restricted regions of the A-laminae, inhibited thalamocortical cells through GABAA, and perhaps GABAB, receptors, and were excited by axon collaterals from thalamocortical cells. This reciprocal relationship is identical to that formed by PGN cells and allowed the PGN-like interlaminar neurons to participate in the generation of spindle waves and other network oscillations. Pharmacologically, PGN-like interlaminar interneurons were also similar to PGN neurons: both generated a prolonged depolarization in response to the local application of serotonin, 1S,3R-ACPD, and CCK8S, and a rapid depolarization followed by a more prolonged hyperpolarization in response to acetylcholine. Examination of parvalbumin and calbindin staining in the ferret LGNd revealed that both PGN and a subset of interlaminar neurons were parvalbumin-positive. In contrast, calbindin-positive cells were relatively absent in the PGN and sparsely present in the interlaminar zones, but were numerous in the A and C laminae. Our results indicate that the interlaminar zone in between laminae A and A1 and A1 and C in the ferret LGNd possesses a cell type that is electrophysiologically, pharmacologically, anatomically, immunocytochemically, and functionally similar to neurons in the PGN.

Animals↗
PubMed1998

Neurite outgrowth from bipolar and horizontal cells after experimental retinal detachment.

PURPOSE: To study the responses of horizontal cells and rod bipolar cells, the second-order neurons in the retina, to the degeneration induced by experimental retinal detachment. METHODS: Retinas from the eyes of domestic cats were examined 1, 3, 7, and 28 days after detachment using immunocytochemical and electron microscopic analyses. Retinal sections were labeled with antibodies to synaptophysin, calbindin D, and protein kinase C (PKC), proteins that serve as markers for synaptic terminals, horizontal cells, and rod bipolar cells, respectively. RESULTS: Beginning 1 day after detachment, the outer plexiform layer becomes disorganized and synaptophysin-labeled photoreceptor terminals are detected among the cell bodies of photoreceptors in the outer nuclear layer (ONL). At the same time, horizontal and rod bipolar cell processes grow into the ONL. In some cases, these processes contact photoreceptor terminals that have withdrawn deep into the ONL. Double-labeling experiments with antibodies to glial fibrillary acidic protein (Müller cell labeling) and phosphodiesterase gamma (cone labeling) demonstrate that the calbindin D- and PKC-positive neurite outgrowths are not derived from either Müller cells or cone photoreceptors. CONCLUSIONS: Horizontal and rod bipolar cell processes lengthen after retinal detachment, perhaps in response to a withdrawal of their presynaptic targets, the photoreceptor synaptic terminals. This apparent attempt to maintain synaptic contact after injury demonstrates a plasticity in the adult retina that may be of importance for the recovery of vision in human patients.

Animals↗
PubMed1998

Organizing principles of cortical integration in the rat neostriatum: corticostriate map of the body surface is an ordered lattice of curved laminae and radial points.

The neuroanatomic organizing principles underlying integrative functions in the striatum are only partially understood. Within the three major subdivisions of the striatum-sensorimotor, associative, and limbic--longitudinal zones of axonal plexuses from the cerebral cortex end in bands and clusters that innervate cell groups. To identify organizing principles of the corticostriate bands and clusters, we localized somatosensory cortical cells receptive to light touch on the hindlimb, forelimb, or vibrissae by extracellular recording, and we labeled their projections by iontophoretic application of dextran anterograde tracers. The results show that cortical cells in columnar groups project to the striatum in the form of successive strips, or laminae, that parallel the curve of the external capsule. The vibrissae somatosensory cortex projects to the most lateral lamina. Just medial to the vibrissae projection, the major axonal arborizations arising from hindlimb and forelimb somatosensory cortex are organized within a common lamina, where they interdigitate and overlap as well as remain separate. In addition, the hindlimb and forelimb cortex send small projections to the vibrissae lamina, and vice versa, forming broken, radially oriented lines of points across the laminar strips. The major somatosensory projections are in the dorsolateral, calbindin-poor sensorimotor striatum, whereas the radially oriented projection points extend into the medial, calbindin-rich associative striatum. Extending previous studies of corticostriate projections, this report shows a grid translation of columnar somatosensory cortical inputs into striatum and a detailed map for the rat sensorimotor zone. The lattice-like grid is a novel functional/neuroanatomic organization that is ideal for distributing, combining, and integrating information for sensorimotor and cognitive processing.

Animals↗
PubMed1999

Graft-host connections in long-term full-thickness embryonic rabbit retinal transplants.

PURPOSE: To establish neuronal connections in the rod and cone pathway between laminated rabbit retinal transplants and the host retina. METHODS: Fourteen adult rabbits received a complete full-thickness embryonic transplant. After survival times of 3 to 10 months, the retinas were studied under light microscope and with immunohistochemistry. Antibodies against protein kinase C (PKC), parvalbumin, and calbindin were used to label rod bipolar cells, AII amacrine cells, and cone bipolar cells, respectively. The AB5 antibody was used to label ganglion cells. RESULTS: The transplants displayed laminated morphology with layers parallel to the host retinal pigment epithelium. In the oldest specimens (10 months after surgery), laminated layers of graft and host approached each other and almost reconstructed the normal retinal appearance. The ganglion and cone bipolar cells of the host survived well, as was seen with AB5 and calbindin double-labeling. Connections between cone bipolar cells in the graft and ganglion cells in the host were not common. PKC-labeled rod bipolar cells and parvalbumin-labeled AII amacrine cells of host and graft showed sprouting activity directed toward an intermediate plexiform layer located between the graft and host. In specimens double-labeled with PKC and parvalbumin, this intermediate plexiform layer was seen to contain numerous PKC- and parvalbumin-labeled processes. Direct connections between rod bipolar and AII amacrine cells in host and graft were seen in the 10-month specimens. CONCLUSIONS: Full-thickness embryonic transplants survive for at least 10 months, and normal laminated morphology develops. Host and graft fuse and together contribute nerve cell processes to an intermediate plexiform layer. Direct graft-host contacts are also present between neuronal types that in the normal retina participate in the rod pathway.

Animals↗
PubMed1999

Immunocytochemical localization of cannabinoid CB1 receptor and fatty acid amide hydrolase in rat retina.

Cannabinoids have major effects on central nervous system function. Recent studies indicate that cannabinoid effects on the visual system have a retinal component. Immunocytochemical methods were used to localize cannabinoid CB1 receptor immunoreactivity (CB1R-IR) and an endocannabinoid (anandamide and 2-arachidonylglycerol) degradative enzyme, fatty acid amide hydrolase (FAAH)-IR, in the rat retina. Double labeling with neuron-specific markers permitted identification of cells that were labeled with CB1R-IR and FAAH-IR. CB1R-IR was observed in all cells that were protein kinase C-immunoreactive (rod bipolar cells and a subtype of GABA-amacrine cell) as well as horizontal cells (identified by calbindin-IR). There was also punctate CB1R-IR in the distal one-third of the inner plexiform layer (IPL) that could not be assigned to a cell type. FAAH-IR was most prominent in large ganglion cells, whose dendrites projected to a narrow band in the proximal IPL. Weaker FAAH-IR was observed in the soma of horizontal cells (identified by calbindin-IR); the soma of large, but not small, dopamine amacrine cells (identified by tyrosine hydroxylase-IR); and dendrites of orthotopic- and displaced-starburst amacrine cells (identified by choline acetyltransferase-IR) but in less than 50% of the starburst amacrine cell somata. The extensive distribution of CB1R-IR on horizontal cells and rod bipolar cells indicates a role of endocannabinoids in scotopic vision, whereas the more widespread distribution of FAAH-IR indicates a complex control of endocannabinoid release and degradation in the retina.

Amidohydrolases↗
PubMed2000

Thapsigargin induces a calmodulin/calcineurin-dependent apoptotic cascade responsible for the death of prostatic cancer cells.

BACKGROUND: New agents are required for the treatment of androgen-independent prostate cancer. Due to the low rate of proliferation of these malignant cells, agents which can activate the apoptotic death of these cells without requiring the cells being in the proliferative cell cycle are critically required. Thapsigargin (TG), via its ability to perturb intracellular free calcium [Ca(2+)](i), is such a cell proliferation-independent cytotoxic agent. The present study focuses on more completely describing the biochemical cascade during the apoptotic death of androgen-independent prostate cancer cells induced by TG and on the mechanistic requirements for this death. METHODS: A variety of cell and molecular biology techniques (e.g., time-lapse video, fluorescence image analysis, Northern and Western blotting) were used to examine the temporal relationship between changes in [Ca(2+)](i), GADD 153 transcription, translocation of the NFATc transcription factor to the nucleus, translocation of BAD from the cytosol to the mitochondria, caspase 9 activation, DNA fragmentation, and the loss of clonogenic survival induced by TG treatment of both human TSU-prl and rat AT3.1 prostate cancer cells in vitro. Additional studies using both microinjection of inhibitors of calmodulin and DNA transfections to induce expression of Ca(2+) binding proteins, e.g., calbindin, were performed to evaluate the causal relationship between [Ca(2+)](i) elevation, calmodulin/calcineurin activation, and apoptosis of prostate cancer cells. RESULTS: Using simultaneous fluorescence ratiometric and phase contrast image analysis in individual cells followed longitudinally for several days, it was documented that TG induced early (1-12 hr) moderate (i.e., <500 nM) elevation in [Ca(2+)](i). During this early rise in [Ca(2+)](i), genes like GADD 153 are induced at the transcriptional level. This early rise is followed by a return of [Ca(2+)](i) to baseline (i.e., approximately 50 nM) before the induction of a delayed (i.e., >12 hr) secondary rise ( approximately 10 microM) in [Ca(2+)](i). During the secondary rise in [Ca(2+)](i), Ca(2+) binds to calcineurin and calmodulin, allowing these proteins to form a complex which activates calcineurin's latent phosphatase activity. Once activated, calcineurin dephosphorylates NFATc and BAD, allowing translocation of these proteins to the nucleus and mitochondria, respectively. BAD translocation induces the release of cytochrome C from the mitochondria into the cytoplasm, which results in activation of caspase 9 and DNA fragmentation. If the TG-induced rise in [Ca(2+)](i) is blocked by overexpressing calbindin, or if calmodulin function is inhibited, these apoptotic events are prevented. CONCLUSIONS: TG induces the apoptotic death of prostate cancer cells via the activation of a reversible signaling phase induced by a transient nanomolar rise in [Ca(2+)](i), which involves new gene transcription and translation. This reversible signaling phase is followed by an irreversible commitment to undergo the execution phase which is induced by a secondary micromolar rise in [Ca(2+)](i). This secondary [Ca(2+)](i) rise irreversibly commits the cell to a calmodulin/calcineurin-dependent cascade, which results in DNA and cellular fragmentation into apoptotic bodies.

Animals↗
PubMed1994

Multiple neuropeptide immunoreactivities in a renin-producing human paraganglioma.

BACKGROUND: A case of a renin-producing paraganglioma of adrenal origin with metastases to the retroperitoneal area, paravaginal area, and the ovary is reported with immunohistochemical findings indicating expression of multiple neuropeptide immunoreactivities. The patient was 23 years old at the time of diagnosis, and died from metastatic spread of the tumor 7 years later. METHODS: Tumor tissue was examined by light microscopy, indirect immunohistochemistry, and electron microscopy. RESULTS: The tumor tissue investigated contained several cells exhibiting opioid peptide-like immunoreactivities (i.e., enkephalin and dynorphin-like immunoreactivity [LI]). A lower number of cells displayed neuropeptide Y-, galanin-, somatostatin-, neurotensin-, substance P-, peptide histidine-isoleucine-, cholecystokinin-, renin-, and calbindin-LI. CONCLUSION: To the authors' knowledge, galanin, dynorphin, peptide histidine-isoleucine, cholecystokinin, and calbindin have not been reported previously to occur in paraganglioma, and renin has been reported to occur very rarely. A review of recent literature suggest that enkephalin-, neuropeptide Y-, and somatostatin-like immunoreactivities may be useful as diagnostic markers for paragangliomas.

Adrenal Gland Neoplasms↗
PubMed2002

Localisation of cannabinoid CB(1) receptor immunoreactivity in the guinea pig and rat myenteric plexus.

Activation of cannabinoid CB(1) receptors inhibits gastrointestinal motility, propulsion, and transit, whereas selective antagonism of these receptors has the opposite effects, suggesting the presence of endocannabinoid tone. Supporting evidence for presynaptic CB(1) receptors on myenteric neurons has been found in vitro. In this study, selective CB(1) receptor antibodies and neuronal markers were used to identify and characterise myenteric neurons expressing cannabinoid receptors. Whole mounts of rat and guinea pig myenteric preparations were dually labelled with antibodies against the CB(1) receptor and choline acetyltransferase, neurofilament proteins, calbindin, calretinin, synapsin I, microtubule-associated protein-2, calcitonin gene-related peptide, or substance P. The pattern of CB(1) receptor labelling and the neurochemical classification of CB(1) receptor-positive cells were markedly influenced by the species and fixation procedure. Virtually all choline acetyltransferase-immunoreactive myenteric neurons expressed CB(1) receptors in ganglia from both species. Subpopulations of neurons identified with calbindin, calretinin, and microtubule-associated protein-2 did not express CB(1) receptors. A few calcitonin gene-related peptide- and substance P-positive somata coexpressed CB(1) receptor immunoreactivity but showed little colocalisation on individual fibres. There was a close association between CB(1) receptor immunoreactivity and fibres labelled for synaptic protein, suggesting a role in the modulation of transmitter release. Functional responses to cannabinoids in the presence of hexamethonium suggest further that CB(1) receptors occur on excitatory motoneurons. In conclusion, CB(1) receptors are expressed on a variety of cholinergic sensory, interneuronal, and motor neurons in myenteric ganglia.

Animals↗
PubMed2003

Parvocellular subparafascicular thalamic nucleus in the rat: anatomical and functional compartmentalization.

The parvocellular subparafascicular thalamic nucleus (SPFp) is located in the posterior thalamus, consists of horizontally oriented cells, and extends from rostromedial to caudolateral, fusing with the posterior intralaminar nucleus and the peripeduncular nucleus. The present study demonstrates a chemoarchitechtonic and functional parcellation of the rat SPFp. Analysis of the distributions of the neuropeptides galanin, calcitonin gene related peptide (CGRP), substance P, and calbindin revealed the existence of a medial and lateral subdivision within SPFp, and a possible intermediate subdivision. The medial subdivision contains a dense population of galanin-immunoreactive fibers, originating from galanin neurons in the lumbosacral spinal cord. In contrast, the lateral subdivision contains CGRP-positive fibers and neurons. The presence of substance P and calbindin immunoreactivity throughout the entire nucleus suggests that these are separate subdivisions of SPFp, rather than different subnuclei. The present study also investigated the functional association of the separate subdivisions of SPFp for male and female rat sexual behavior. In the medial subdivision, Fos-positive neurons were activated in males by display of ejaculation and in females by vaginocervical stimulation. Thus, Fos induction in medial SPFp appears to reflect processing of inputs related to those events. In contrast, sexual behavior did not induce Fos in the lateral SPFp. Taken together, the present results indicate the existence of separate subdivisions in SPFp that are involved in different behavioral functions. The medial SPFp may process inputs important for sexual behavior, whereas the lateral SPFp may be involved in convergence of auditory and nociceptive inputs important for conditioned fear responses.

Animals↗
PubMed2004

Mechanically evoked reflex electrogenic chloride secretion in rat distal colon is triggered by endogenous nucleotides acting at P2Y1, P2Y2, and P2Y4 receptors.

Mechanical activation of the mucosal lining of the colon by brush stroking elicits an intestinal neural reflex and an increase in short circuit current (Isc) indicative of electrogenic chloride ion transport. We tested whether endogenous nucleotides are physiologic regulators of mucosal reflexes that control ion transport. The brush stroking-evoked Isc response in mucosa and submucosa preparations (M-SMP) of rat colon was reduced by the P2Y1 receptor (R) antagonist 2'deoxy-N6-methyl adenosine 3',5'-diphosphate diammonium salt (MRS 2179) and further blocked by tetrodotoxin (TTX). M-SMP Isc responses to serosal application of the P2Y1 R agonist 2-methylthioadenosine-diphosphate (2MeSADP) or the P2Y2/P2Y4 R agonist 5'uridine-triphosphate (UTP) were reduced but not abolished by TTX. The potency profile of nucleotides for increasing Isc was 5'adenosine-triphosphate (ATP; effective concentration at half maximal response [EC50] 0.65 x 10(4) M) congruent with UTP (EC50 1.0 x 10(-4) M) congruent with 2MeSADP (EC50 = 1.60 x 10(-4) M). Mucosal touch and distention-induced Ca2+ transients in submucous neurons were reduced by apyrase and prevented by blocking the P2Y1 R with MRS 2179 and TTX; denervation of the mucosa. It did not occur by touching a ganglion directly. 2MeSADP Ca2+ responses occurred in subsets of neurons with or without substance P (SP) responses. The potency profile of nucleotides on the neural Ca2+ response was 2MeSADP (5 x 10(-7) M) > UTP (6 x 10(-6) M) > ATP (9 x 10(-5) M). The expression of P2Y R immunoreactivity (ir) in nerve cell bodies was in the order of P2Y1 R > P2Y4 R >> P2Y2 R. P2Y1R ir occurred in the cell somas of more than 90% of neuronal nitric oxide synthase, vasoactive intestinal peptide (VIP), calretinin, or neuropeptide Y (NPY)-ir neurons, 78% of somatostatin neurons, but not in calbindin or SP neurons. P2Y2 R ir was expressed in a minority of SP, VIP, NPY, vesicular acetylcholine transporter, and calcitonin gene-related peptide-ir varicose fibers (5-20%) and those surrounding calbindin (5-20%) neurons. P2Y4 ir occurred mainly in the cell somas of 93% of NPY neurons. Reverse transcriptase polymerase chain reaction of the submucosa demonstrated mRNA for P2Y1R, P2Y2, P2Y4, P2Y6, and P2Y12 Rs. Expression of P2Y1, P2Y2, and P2Y4 protein was confirmed by western blots. In conclusion, endogenous nucleotides acting at P2YRs transduce mechanically evoked reflex chloride ion transport in rat distal colon. Nucleotides evoke reflexes by acting primarily at postsynaptic P2Y1 Rs and P2Y4 R on VIP+/NPY+ secretomotor neurons, at P2Y2 Rs on no more than 2% of VIP+ secretomotor neurons, and 2Y2 Rs mainly of extrinsic varicose fibers surrounding putative intrinsic primary afferent and secretomotor neurons. During mucosal mechanical reflexes, it is postulated that P2Y1 R, P2Y2 R, and P2Y4 R are activated by endogenous ATP, UTP, and 5'uridine-diphosphate.

Animals↗