The distribution of vasopressin-, oxytocin-, and neurophysin-producing neurons in the guinea pig brain

Summary The location, cytology and projections of vasopressin-, oxytocin-, and neurophysin-producing neurons in the guinea pig were investigated using specific antisera against vasopressin, oxytocin or neurophysin in the unlabeled antibody enzyme immunoperoxidase method. Light microscopic examination of the neurons of the supraoptic and paraventricular nuclei shows that hormone is transported not only in axons, but also in processes having the characteristics of dendrites. Neurons were found to contain only vasopressin or oxytocin; all neurons containing neurophysin appear to contain either vasopressin or oxytocin. In the neural lobe, vasopressin and oxytocin terminals are intermingled. In the median eminence, vasopressin and oxytocin fibers are intermingled in the internal zone. In a caudal portion of the median eminence, a number of vasopressin and neurophysin (but few oxytocin) axons enter the external zone from the internal zone, and surround portal capillaries. In the supraoptic nucleus, vasopressin neurons outnumber oxytocin neurons with a ratio of at least 5:1. The paraventricular nucleus is separated into two distinct groups of neurons, a lateral group consisting of only vasopressin neurons, and a medial group consisting of only oxytocin neurons. In addition to axons passing to the neurohypophysis, a number of axons appear to interconnect the supraoptic and paraventricular nuclei.Supported by the Deutsche Forschungsgemeinschaft (SFB 51, C/21 and C/27), (We 608/3)Acknowledgements. The authors are greatly indebted to Mmes. R. Köpp-Eckmann, B. Reijerman, A. Scheiber, I. Wild and Mr. U. Schrell for technical assistance, to Mmes. P. Campbell and U. Wolf for editorial assistance, and to Dr. R.R. Dries and Ferring Pharmaceuticals, Kiel, for the generous provision of high quality peptides     

Simultaneous immunogold labeling of GABAergic terminals and vasopressin-containing neurons in the rat paraventricular nucleus

Summary The GABAergic innervation of vasopressin-containing cells in the magnocellular part of the paraventricular nucleus was studied at the electron-microscope level using antibodies against GABA and vasopressin. The detection of both GABA and vasopressin on the same ultrathin section, performed with a double-labeling immunogold method, revealed GABAergic terminals in symmetrical synaptic contact with vasopressin-containing neurons. These GABAergic terminals displayed mitochondria, clear synaptic vesicles and varying numbers of electron-dense vesicles. Vasopressin-immunoreactivity was associated with neurosecretory granules, whereas GABA-immunoreactivity was found above mitochondria, clear synaptic vesicles and some electron-dense vesicles. This study, demonstrating the extensive participation of GABA in the innervation of magnocellular vasopressin-secreting neurons, suggests that this inhibitory neurotransmitter regulates vasopressin secretion at the level of the paraventricular nucleus.     

Simultaneous monoamine histofluorescence and neuropeptide immunocytochemistry: III. Ontogeny of catecholamine varicosities and neurophysin neurons in the rat supraoptic and paraventricular nuclei

The ontogeny of the rat supraoptic (SON) and paraventricular (PVN) nuclei was studied using a combined fluorescence-immunocytochemical technique for the simultaneous localization of catecholamines (CA) and neurophysin (NP). NP neurons and CA varicosities were first detected in the SON and PVN at 17 days postcoitus. The development of NP neurons which included increases in immunoreactivity in both nuclei proceeded through fetal and neonatal stages, approaching maturity by 21–28 days postnatal; the maturation of the PVN lagged behind that of the SON. CA varicosities appeared to make contact with NP neurons beginning at 21–22 days postcoitus. An apparent increase in varicosity-perikaryal contacts with age was observed in both nuclei; by 14–21 days postnatal adult-like patterns were established. The prenatal dominance of NP stain relative to CA fluorescence may suggest a possible neurotrophic role for magnocellular neurons and/or their products upon ingrowing noradrenergic axons.     

Activation of vasopressinergic and oxytocinergic neurons during aging in the Wistar rat

The activity of the hypothalamo-neurohypophyseal system (HNS) was determined in male Wistar rats from 3 to 32 months of age. Plasma levels of vasopressin (AVP) and oxytocin (OXT) were measured by means of a radioimmunoassay. In addition, the distribution of the Golgi apparatus marker enzyme thiamine-pyrophosphatase (TPP-ase) was measured as a parameter for neurosecretory activity in the hypothalamic supraoptic and paraventricular nuclei (SON and PVN). Plasma levels of radioimmunoassayable AVP were increased in the 32-month-old animals. Plasma levels of radioimmunoassayable OXT in 32-month-old animals did not differe from the levels found in the youngest group, but were higher than in 11-month-old animals. Neurosecretory activity in the SON was similar in 3- and 32-month-old animals, whereas in the PVN neurosecretory activity was increased in the 32-month-old animals. Urine excretion decreased between 6 and 11 months of age and remained on the same level until 32 months of age. In other words, instead of a loss of HNS function as has been suggested in the literature, an increased neurosecretory activity was observed in aged rats.     

P2X receptors are differentially expressed on vasopressin- and oxytocin-containing neurons in the supraoptic and paraventricular nuclei of rat hypothalamus

In the present study, the distribution of P2X receptor protein and colocalization of P2X receptors with vasopressin and oxytocin in the supraoptic and paraventricular nuclei of rat hypothalamus was studied using double-labeling fluorescence immunohistochemistry. The results showed that vasopressin-containing neurons expressed P2X₂, P2X₄, P2X₅ and P2X₆ receptor and oxytocin-containing neurons expressed P2X₂, P2X₄ and P2X₅ receptors in the supraoptic nucleus. In the paraventricular nucleus, vasopressin-containing neurons expressed P2X₄, P2X₅ and P2X₆ receptors, while oxytocin-containing neurons expressed P2X₄ receptors. This study provides the first evidence that P2X receptor subunits are differentially expressed on vasopressin- and oxytocin-containing neurons in the supraoptic and paraventricular nuclei, and hence, provides a substantial neuroanatomical basis for possible functional interactions between the purinergic and vasopressinergic systems, and the purinergic and oxytocinergic systems in the rat hypothalamus. Wei Guo and Jihu Sun contributed equally to this work.     

Effects of Morris water maze testing on the neuroendocrine stress response and intrahypothalamic release of vasopressin and oxytocin in the rat

Adult male Wistar rats were trained in the Morris water maze (MWM) on 3 consecutive days to find a visible platform. Concomitantly, microdialysis samples from the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei were collected in order to monitor local release of the neuropeptides vasopressin (AVP) and oxytocin (OXT), respectively, during controllable swim stress. Additionally, a separate set of animals was equipped with chronic jugular venous catheters to collect blood samples for analyzing plasma concentrations of corticotropin (ACTH) and corticosterone during training in the MWM. As measured by microdialysis, swimming in the MWM caused a significantly increased release of AVP within the PVN and of OXT within the SON on each of the 3 test sessions. In contrast to OXT in the SON, basal AVP concentrations in the PVN tended to rise from day to day. Plasma ACTH and corticosterone were found to be similarly elevated in response to MWM exposure on each of the test sessions. Taken together, these data demonstrate that testing in the MWM is not only associated with a significant activation of the hypothalamo-pituitary-adrenal axis but also with an intrahypothalamic release of AVP and OXT. If compared with findings using repeated forced swimming as an uncontrollable stressor (Wotjak, C.T., Ganster, J., Kohl, G., Holsboer, F., Landgraf, R., Engelmann, M., 1998. Dissociated central and peripheral release of vasopressin, but not oxytocin, in response to repeated swim stress: new insights into the secretory capacities of peptidergic neurons. Neuroscience 85, 1209-1222), the present results suggest that (1) similarities in the release profiles of AVP in the PVN and plasma hormone levels are fairly independent from the controllability of the stressor and seem, thus, to primarily relate to the physical demands of the task, whereas (2) the different intra-SON OXT release profiles might be linked to the controllability of the stressor.     

Catecholamine distribution and relationship to magnocellular neurons in the paraventricular nucleus of the rat

Summary The distribution of catecholamine synthesizing enzymes within the paraventricular nucleus of the rat hypothalamus is elucidated immunocytochemically by use of antibodies to tyrosine hydroxylase, dopamine -hydroxylase, and phenylethanolamine-N-methyltransferase. Tyrosine hydroxylase-immunostained cell bodies are localized in the periventricular stratum and adjacent parvocellular regions, but rarely in magnocellular subnuclei of the paraventricular nucleus. Tyrosine hydroxylase-immunostained fibers are present in greatest density in the periventricular zone, and moderate density in the parvocellular and magnocellular subnuclei. Dopamine -hydroxylase-immunostained fibers are remarkably dense in the posterior magnocellular division of the paraventricular nucleus, especially in the dorso-lateral portion where vasopressin-containing cells predominate. Noradrenergic fiber input to these magnocellular neurons is likely since phenylethanolamine-N-methyltransferase-immunostained fibers are sparse in magnocellular subnuclei of the paraventricular nucleus. Dual immunocytochemical staining of thick and thin tissue sections demonstrates with clarity an anatomical association of dopamine -hydroxylase-immunostained fibers and magnocellular neurons. Dopamine -hydroxylase-immunostained and phenylethanolamine-N-methyltransferase-immunostained fibers are dense in the medial parvocellular component of the paraventricular nucleus; distinct features of both antisera are presented.     

Two populations of somatostatin-immunoreactive neurons in the guinea pig striatum

Summary Two populations of neurons displaying somatostatin-like immunoreactivity were detected immunohistochemically in the guinea pig striatum using a monoclonal antibody. Sparse, well-stained neurons similar to those described in other species were observed throughout the guinea pig caudate-putamen. These neurons contained both neuropeptide Y and NADPH-diaphorase in addition to somatostatin. A second large population of somatostatin immunoreactive neurons in which these other substances did not coexist was found within the putamen.     

Immunocytochemical evidence for the presence of a mutant vasopressin precursor in the supraoptic nucleus of the homozygous Brattleboro rat

Summary CP-14, a tetradecapeptide from the predicted mutant vasopressin precursor in the homozygous Brattleboro rat was detected immunocytochemically in the supraoptic nucleus of homozygous Brattleboro but not normal rats. The staining was localized to the periphery of the perikarya. CP-14 immunoreactivity was not found in the neural lobes, paraventricular nuclei, accessory nuclei or suprachiasmatic nuclei of either homozygous Brattleboro or normal rats. Vasopressin immunoreactivity was found in the neural lobe and in the perinuclear region of neurons of the supraoptic, paraventricular, suprachiasmatic and accessory nuclei of normal rats. Vasopressin immunoreactivity was also found in homozygous Brattleboro rats, mainly in the ventral part of the supraoptic nucleus: densely stained solitary cells were found amongst other faintly stained perikarya. In both cell-types the staining was mainly in the periphery of the perikarya. No vasopressin immunoreactivity was detected in the paraventricular nuclei, suprachiasmatic nuclei, accessory nuclei or neural lobe of homozygous Brattleboro rats.CP-14 and vasopressin immunoreactivities were found to be co-localized; both were present in the periphery of the same perikarya of the supraoptic nuclei of homozygous Brattleboro rats. Differential staining was found with antioxytocin serum in both normal rats and homozygous Brattleboro rats: separate neurons were stained for either oxytocin or vasopressin and CP-14. Immunoreactive oxytocin was found mainly in the perinuclear region of the neurons from the supraoptic, paraventricular and accessory nuclei.     

Glucagon-like immunoreactivity in hypothalamic neurons of the rat

Summary Antisera specific for three different regions of pancreatic proglucagon were used to examine the distribution of such immunoreactivity in rat hypothalamus. Neurons in the supraoptic and paraventricular nuclei were immunoreactive with an antiserum against glucagon, but not with antisera directed towards the aminoterminal region of proglucagon (glicentin) or the glucagon-like peptide I sequence in the carboxyl-terminal region of proglucagon. These findings confirm a previous report of glucagon-like immunoreactivity in the supraoptic and paraventricular nuclei, but indicate that, while this material is immunochemically related to glucagon, it is not derived from a proglucagon-like precursor.     

Light- and electron-microscopic study of substance P-immunoreactive neurons in the guinea pig retina

Substance P (SP) immunoreactivity in the guinea pig retina was studied by light and electron microscopy. The morphology and distribution of SP-immunoreactive neurons was defined by light microscopy. The SP-immunoreactive neurons formed one population of amacrine cells whose cell bodies were located in the proximal row of the inner nuclear layer. A single dendrite emerged from each soma and descended through the inner plexiform layer toward the ganglion cell layer. SP-immunoreactive processes ramified mainly in strata 4 and 5 of the inner plexiform layer. SP-immunoreactive amacrine cells were present at a higher density in the central region around the optic nerve head and at a lower density in the peripheral region of the retina. The synaptic connectivity of SP-immunoreactive amacrine cells was identified by electron microscopy. SP-labeled amacrine cell processes received synaptic inputs from other amacrine cell processes in all strata of the inner plexiform layer and from bipolar cell axon terminals in sublamina b of the same layer. The most frequent postsynaptic targets of SP-immunoreactive amacrine cells were the somata of ganglion cells and their dendrites in sublamina b of the inner plexiform layer. Amacrine cell processes were also postsynaptic to SP-immunoreactive neurons in this sublamina. No synaptic outputs onto the bipolar cells were observed.     

Morphological analysis of the neurons in the area of the hypothalamic magnocellular dorsal nucleus of the guinea pig

Summary In the guinea-pig hypothalamus, a group of enkephalinergic cells forms a well-circumscribed nuclear area called the magnocellular dorsal nucleus (MDN). This nucleus gives rise to a prominent projection to the lateral spetum: the hypothalamo-septal enkephalinergic pathway. In the present study, MDN neurons visualized by Golgi impregnation were subjected to morphological analysis in order to define the potential segregation of cellular types within the MDN. This study was complemented by additional observations of MDN neurons intracellularly injected by Lucifer yellow (LY) or horseradish peroxidase (HRP) during the in vitro incubation of hypothalamic slices. The following results were obtained from the analysis of 200 neurons: 163 Golgi-impregnated cells plus 37 injected cells (LY=14; HRP=23). Thirteen HRP-injected cells were precisely located in the MDN and 10 were located in the perifornical area surrounding the MDN. Four different cellular types were identified. Type-I neurons (41%) displayed a globular perikaryon, a variable number of primary dendrites that were poorly ramified, no preferential orientation, and an axon emerging from the perikaryon. Type-II neurons (30.5%) had a triangular perikaryon, three well-ramified primary dendrites, an orientation perpendicular to the third ventricle, and an axon emerging from the perikaryon. Type-III neurons (22%) exhibited a spindle-shaped perikaryon, two opposed well-ramified primary dendrites, an orientation perpendicular to the third ventricle, and an axon emerging from a primary dendrite. Type-IV neurons (6.5%), showed a globular perikaryon, a variable number of primary dendrites, poorly ramified dendrites, an orientation parallel to the third ventricle, and an axon whose orientation could not be identified. Neurons labeled after intracellular injection belonged to the first three cellular types.     

Ontogeny of vasopressin and oxytocin in the fetal rat: Early vasopressinergic innervation of the fetal brain

The content and distribution of vasopressin and oxytocin were determined during fetal development in the rat brain and pituitary by means of radioimmunoassay and immunocytochemistry. The vasopressin content in the fetal brain showed a gradual rise from day 16 of pregnancy onwards, while pituitary vasopressin rapidly increased from fetal day 19 until birth. The oxytocin content in the fetal brain was considerably lower than the vasopressin content. A decrease in oxytocin content was seen between day 16 and day 18 while from day 18 of pregnancy onwards a slight increase was found. The pituitary oxytocin content starts to rise between day 17 and 18 of pregnancy, but at term the pituitary oxytocin content was only 1/20 of the vasopressin value. Immunocytochemistry revealed that vasopressin levels in the fetal rat brain were not only due to the presence of the classical hypothalamoneurohypophyseal system, but also to the early development of exohypothalamic fibers. Vasopressin containing cells were seen from fetal day 16 in the supraoptic nucleus, and from fetal day 18 in the paraventricular nucleus. The fiber outgrowth of these cells towards the pituitary and extrahypothalamic brain sites seems to be well synchronized, as on day 17 vasopressin containing fibers could be demonstrated in the olfactory bulb as well as in the median eminence. No positive staining for oxytocin could be obtained in the fetal rat, while during the entire fetal period no positive staining was found in cell bodies in the region of the suprachiasmatic nucleus. The early peptidergic innervation of the brain, which enabled the tracing of the source of some exohypothalamic fibers, might be related to several central processes among which brain development itself is included.     

Altered pattern of vasopressin distribution in the hypothalamus of rats subjected to immobilization stress

Summary Generally, the CRF-like activity of vasopressin is studied in experiments involving adrenalectomy and corticosteroid replacement. In order to avoid this complex type of stress, male and female (diestrus, estrus) rats were exposed for 5min to immobilization stress and sacrificed 5, 15, and 30 min thereafter. After a survival period of 5 min the vasopressin-synthesizing part of the paraventricular nucleus exhibited an increased activity. Vasopressin-reactive axons in the pericapillary layer of the median eminence and among the solid cell clusters of the pars tuberalis became more conspicuous and increased in number. In this group of experimentally treated animals the prechiasmatic division of the supraoptic nucleus did not show any changes in immunoreactivity. The same holds true for the neurohypophyses in all experimental groups. In animals with increased survival times the supraoptic nucleus exhibited a slightly increased activity, whereas the staining intensity of the paraventricular nucleus decreases gradually. From these results it can be concluded that the paraventricular nucleus is involved in the first phase of the stress response. The problem of vasopressin or a very similar peptide synthesized in this nucleus and exerting a CRF-releasing function is discussed.The author wishes to express her sincere gratitude to Dr. A. Weindl, Munich, for supplying the antivasopressin and to Dr. L.A. Sternberger, Edgewood Arsenal for generously supplying the PAP-complex. The skillful technical assistance of Mrs. Helga Prien is thankfully acknowledgedDedicated to Prof. Dr. Helmut Leonhardt on the occasion of his 60th birthdaySupported by the Deutsche Forschungsgemeinschaft (Grant Nr. Kr569/1) and Stiftung Volkswagen-werk     

Changes in oxytocin content in the magnocellular neurons of the rat hypothalamus following water deprivation or estrogen treatment

Summary The effect of water deprivation or estrogen treatment on the oxytocin content of rat hypothalamic cells was examined using a quantitative immunohistological technique. Oxytocin-containing cells were visualized using the immunoperoxidase technique of Sternberger and a primary antiserum directed against oxytocin. The optical density of the darkest 3.2 m diameter spot in the cytoplasm of a cell was used as a measure of the oxytocin content of that cell. Water deprivation produced a significant decrease in anti-oxytocin staining in the anterior commissural nucleus of males and females. There was a similar decrease in the paraventricular nucleus of males, but not in the paraventricular nucleus of females or the supraoptic nucleus of either males or females. Estrogen treatment of ovariectomized female rats produced a fall in anti-oxytocin staining in the anterior commissural, but not paraventricular or supraoptic nuclei.     

Ontogeny of vasopressin and oxytocin in the fetal rat: Early vasopressinergic innervation of the fetal brain

The content and distribution of vasopressin and oxytocin were determined during fetal development in the rat brain and pituitary by means of radioimmunoassay and immunocytochemistry. The vasopressin content in the fetal brain showed a gradual rise from day 16 of pregnancy onwards, while pituitary vasopressin rapidly increased from fetal day 19 until birth. The oxytocin content in the fetal brain was considerably lower than the vasopressin content. A decrease in oxytocin content was seen between day 16 and day 18 while from day 18 of pregnancy onwards a slight increase was found. The pituitary oxytocin content starts to rise between day 17 and 18 of pregnancy, but at term the pituitary oxytocin content was only of the vasopressin value. Immunocytochemistry revealed that vasopressin levels in the fetal rat brain were not only due to the presence of the classical hypothalamoneurohypophyseal system, but also to the early development of exohypothalamic fibers. Vasopressin containing cells were seen from fetal day 16 in the supraoptic nucleus, and from fetal day 18 in the paraventricular nucleus. The fiber outgrowth of these cells towards the pituitary and extrahypothalamic brain sites seems to be well synchronized, as on day 17 vasopressin containing fibers could be demonstrated in the olfactory bulb as well as in the median eminence. No positive staining for oxytocin could be obtained in the fetal rat, while during the entire fetal period no positive staining was found in cell bodies in the region of the suprachiasmatic nucleus. The early peptidergic innervation of the brain, which enabled the tracing of the source of some exohypothalamic fibers, might be related to several central processes among which brain development itself is included.     

Pattern of afferents to the lateral septum in the guinea pig

Summary The septal region represents an important telencephalic center integrating neuronal activity of cortical areas with autonomous processes. To support the functional analysis of this brain area in the guinea pig, the afferent connections to the lateral septal nucleus were investigated by the use of iontophoretically applied horseradish peroxidase (HRP). Retrogradely labeled perikarya were located in telencephalic, diencephalic, mesencephalic and metencephalic sites. The subnuclei of the lateral septum (pars dorsalis, intermedia, ventralis, posterior) receive afferents from the (i) medial septal nucleus, diagonal band of Broca (pars horizontalis and pars ventralis), and the principal nucleus of the stria terminalis, the hippocampus, and amygdala (nucleus medialis); (ii) the medial habenular nucleus, and the para- (peri-) ventricular, parataenial and reuniens nuclei of the thalamus; the anterior, lateral and posterior hypothalamic areas in particular, the medial and lateral preoptic, suprachiasmatic, periventricular, paraventricular, arcuate, premammillary, and supramammillary nuclei; (iii) the periaquaeductal grey, ventral tegmental area, nucleus interfascicularis, nucleus reticularis linearis, central linear nucleus, interpeduncular nucleus; (iv) dorsal and medial raphe complex, and locus coeruleus. Each subnucleus of the lateral septum displays an individual, differing pattern of afferents from the above-described regions. Based on a double-labeling method, the vasopressinergic and serotonergic afferents to the lateral septum were found to originate in the nucleus paraventricularis hypothalami and the raphe nuclei, respectively.Abbreviations ARC arcuate nucleus - BNST bed nucleus of the stria terminalis - CL central linear nucleus - DBBh diagonal band of Broca (pars horizontalis) - DBBv diagonal band of Broca (pars ventralis) - DR dorsal raphe nucleus - HC hippocampus - IF interfascicular nucleus - IP interpeduncular nucleus - LC locus coeruleus - LDT laterodorsal tegmental nucleus - LHA lateral hypothalamic area - LPO lateral preoptic area - LSN lateral septal nucleus - MA medial amygdaloid nucleus - MH medial habenular nucleus - MPO medial preoptic region - MR medial raphe nucleus - MSN medial septal nucleus - PAG periaquaeductal grey - PEN periventricular nucleus - PHA posterior hypothalamic area - PMd premammillary region (pars dorsalis) - PMv premammillary region (pars ventralis) - PT parataenial nucleus - PVN paraventricular hypothalamic nucleus - PVT paraventricular thalamic nucleus - RE nucl. reuniens - RL nucl. reticularis linearis - SCN suprachiasmatic nucleus - SMl supramammillary region (pars lateralis) - SMm supramammillary region (pars medialis) - SUB subiculum - TS triangular septal nucleus - VTA ventral tegmental area - ac anterior commissure - bc brachium conjunctivum - bp brachium pontis - cc corpus callosum - fr fasciculus retroflexus - fx fornix - ml medial lemniscus - mlf fasciculus longitudinalis medialis - mp mammillary peduncle - mt mammillary tract - oc optic chiasm - on optic nerve - pc posterior commissure - pt pyramidal tract - sm stria medullaris - st stria terminalis - vhc ventral hippocampal commissure Supported by the Deutsche Forschungsgemeinschaft (Nu 36/2-1)     

Co-existence and origin of peptidergic and adrenergic nerves in the guinea pig uterus

Summary The occurrence and distribution of peptidergic nerves in the guinea pig uterus was studied by means of immunocytochemistry using numerous neuropeptide antisera. Neuropeptide Y (NPY)-immunoreactive (IR) nerves were the most abundant, whereas substance P (SP)-, calcitonine gene-related peptide (CGRP)-, and neurokinin A (NKA)-IR nerves were less frequent, and peptide histidine isoleucine (PHI)-IR nerves were the most sparse. Chemical sympathectomy by means of 6-hydroxydopamine, and capsaicin treatment revealed the division of the peptidergic nerves into three separate populations: (1) NPY-IR nerves, which co-existed with adrenergic nerves, (2) SP-, CGRP-and NKA-IR nerves, which mutually co-existed, and (3) PHI-IR nerves. Parallel-running adrenergic/NPY-IR and SP-IR nerves could be found with very similar although not completely identical morphological appearance. Paracervical ganglia contained neurotensin-and dynorphin A-IR cell bodies in addition to cell bodies with immunoreactivities similar to those in prevertebral ganglia. Combined retrograde tracing with True blue and immunocytochemistry showed that the adrenergic and NPY-IR uterine nerves originate in paracervical and prevertebral ganglia. In the prevertebral ganglia the cellular origin was the same for adrenergic and NPY-IR nerves. In contrast, SP-, CGRP-,and NKA-IR nerves originated in dorsal root ganglia. At full-term pregnancy all the neuropeptide immunoreactivities had vanished, probably reflecting a fetus-induced general nerve degeneration.     

Expression of sodium channel Nav1.6 in cholinergic myenteric neurons of guinea pig proximal colon

We wished to establish the functional identity of Na_v1.6-expressing myenteric neurons of the guinea pig proximal colon by determining the extent of colocalization of Na_v1.6 and selected neurochemical markers. Na_v1.6-like immunoreactivity (-li) was primarily localized to the hillock and initial segments of myenteric neurons located near junctions with internodal fiber tracts. Immunoreactivity for Na_v1.6 was co-localized with choline-acetyltransferase-li, representing 96% of Na_v1.6-immunoreactive neurons; about 5% of these neurons showed co-localization with calretinin-li, but none with substance-P-li. Cholinergic neurons expressing Na_v1.6 were amongst the smallest (somal area <300 μm²) of all cholinergic myenteric neurons observed. Only three of 234 Na_v1.6-immunoreactive neurons exhibited nNOS-li, and none co-localized with calbindin-li. These data suggest that Na_v1.6 is expressed in a small uniform population of cholinergic myenteric neurons that lie within the guinea pig proximal colon and that are likely to function as excitatory motor neurons.This work was supported in part by grants from the Autzen Endowment and Cadeau Foundation. A.C. Bartoo was supported by a grant from the Poncin Foundation.     

Expression of the sodium channel Nav1.2 in chemically identified myenteric neurons in the guinea pig

Our purpose was to identify Na_v1.2-expressing myenteric neurons of the small and large intestine of the guinea pig by using antibodies directed against Na_v1.2 and selected neurochemical markers. Na_v1.2-like immunoreactivity (-li) co-localized with immunoreactivity for choline acetyltransferase in all regions, representing 45%–67% of Na_v1.2-positive neurons. Na_v1.2-li co-localized with immunoreactivity for the neural form of nitric oxide synthase more frequently in the colon (20% of neurons exhibiting Na_v1.2-li) than in the ileum (8%). Co-localization of Na_v1.2-li with immunoreactivity for a form of neurofilament (NF145) was infrequently observed in the ileum and colon. Enkephalin-immunoreactive cell bodies co-localized with Na_v1.2-li in all regions. Few myenteric cell bodies immunoreactive for neuropeptide Y were observed in the ileum, but all co-localized with Na_v1.2-li. This and our previous data suggest that Na_v1.2 is widely expressed within the guinea pig enteric nervous system, including the three main classes of myenteric neurons (sensory, motor, and interneurons), and is involved in both excitatory and inhibitory pathways. Notable exceptions include the excitatory motor neurons to the longitudinal smooth muscle, the ascending interneurons of the ileum, and the myenteric neurons immunoreactive for NF145, few of which are immunoreactive for Na_v1.2. This work was supported in part by grants from the Autzen Endowment and Cadeau Foundation. A.C. Bartoo was supported by a grant from the Poncin Foundation.