The influence of salt loading on vasopressin gene expression in magno- and parvocellular hypothalamic neurons: an immunocytochemical and in situ hybridization analysis

Arginine vasopressin peptide and messenger RNA expression were examined at the cellular level in the magnocellular and parvocellular neurons in the rat paraventricular nucleus after dehydration and rehydration, employing immunocytochemistry and in situ hybridization histochemistry on the same tissue sections. Most magnocellular vasopressinergic neurons of control animals expressed both vasopressin-like immunoreactivity and messenger RNA. However, neurons negative for vasopressin-like immunoreactivity but expressing messenger RNA were also detected, and their number increased during dehydration. In contrast, almost all of the parvocellular vasopressinergic neurons of dehydrated animals expressed vasopressin messenger RNA alone, with continued increase in their number after rehydration, despite return of the number of magnocellular vasopressinergic neurons to the control level. Vasopressin messenger RNA and corticotropin releasing factor-like immunoreactivity were co-localized in the same parvocellular neurons, and vasopressin-immunoreactive nerve terminals were detected in the external zone of the median eminence. These findings suggest that magno- and parvocellular vasopressinergic neurons are differentially activated during dehydration/rehydration. Osmotic stimuli activate all magnocellular vasopressinergic neurons, but the effect is not simultaneous in all of these neurons. Parvocellular vasopressinergic neurons are also activated by the stress of dehydration which effect appears to last longer than in the magnocellular system.     

Estrogen receptor α is involved in the estrogenic regulation of arginine vasopressin immunoreactivity in the supraoptic and paraventricular nuclei of ovariectomized rats

The ovarian hormone estradiol regulates the expression of arginine vasopressin gene and the release of arginine vasopressin by magnocellular hypothalamic neurons. Magnocellular neurons express estrogen receptor β and are contacted by afferent neurons that express estrogen receptor α. In this study we have assessed the effect of selective ligands for estrogen receptors to determine the subtype of estrogen receptor involved in the regulation of arginine vasopressin immunoreactivity in the supraoptic and paraventricular nuclei of ovariectomized rats. The volume fraction occupied by arginine vasopressin immunoreactive material was significantly increased in both nuclei in the animals treated with estradiol compared to the animals injected with vehicle. A similar result was obtained with an estrogen receptor α selective agonist. In contrast, the administration of an estrogen receptor β selective agonist did not significantly affect arginine vasopressin immunoreactivity. This finding suggests that estradiol may regulate arginine vasopressin levels on the supraoptic and paraventricular nuclei by acting on afferent neurons expressing estrogen receptor α.     

Calcitonin gene-related peptide in primary afferent neurons of rat: co-existence with fluoride-resistant acid phosphatase and depletion by neonatal capsaicin

Immunohistochemical and histochemical techniques were used to re-examine the extent to which neonatal capsaicin treatment depletes calcitonin gene-related peptide in the dorsal horn of the spinal cord, to determine the localization of calcitonin gene-related peptide in relation to that of fluoride-resistant acid phosphatase in lumbar dorsal root ganglia, and to compare the distribution of these primary afferent markers in the dorsal horn. A substantial depletion of calcitonin gene-related peptide was observed in the dorsal horn of adult rats treated neonatally with capsaicin suggesting that a large proportion of this peptide in the dorsal horn is contained within capsaicin-sensitive primary afferent fibers. In dorsal root ganglia 30% of all or 44% of small- and medium-sized calcitonin gene-related peptide-immunoreactive cells were positive for fluoride-resistant acid phosphatase. Conversely, 50% of cells positive for the phosphatase enzyme also displayed immunoreactivity for the peptide. In lamina II of the dorsal horn calcitonin gene-related peptide and fluoride-resistant acid phosphatase were found to have an overlapping distribution. The presence of fluoride-resistant acid phosphatase in a substantial proportion of neuropeptide-containing primary sensory neurons suggests a lack of segregation of sensory neuronal populations into peptide- and non-peptide-containing subgroups at least on the basis of non-peptide neurons defined as those containing fluoride-resistant acid phosphatase.     

Neurokinin B receptor (NK3)-containing neurons in the paraventricular and supraoptic nuclei of the rat hypothalamus synthesize vasopressin and express Fos following intravenous injection of hypertonic saline.

Subnuclear localization of neurokinin B receptor (NK3) in the paraventricular and supraoptic nuclei of the hypothalamus was immunohistochemically investigated in the rat. In the paraventricular hypothalamic nucleus, intense neurokinin B receptor-like immunoreactivity was found in the posterior magnocellular part, moderate to weak neurokinin B receptor-like immunoreactivity was seen in the other parts. In the supraoptic nucleus, intense neurokinin B receptor-like immunoreactivity was distributed in its principal part, and a few neurons with neurokinin B receptor-like immunoreactivity were found in the retrochiasmatic part. Co-localization of neurokinin B receptor-like immunoreactivity with vasopressin-like immunoreactivity was examined through serial adjacent sections. Neurons with both neurokinin B receptor-like immunoreactivity and vasopressin-like immunoreactivity were primarily found in the supraoptic nucleus and posterior magnocellular part of the pavaventricular nucleus. A small number of neurons with neurokinin B receptor-like immunoreactivity and vasopressin-like immunoreactivity were also seen in the circular nucleus and the region surrounding blood vessel in the anterior hypothalamus. Many neurokinin B receptor-containing neurons in the paraventricular and supraoptic nuclei, as well as in circular nucleus and the region surrounding the blood vessel, expressed Fos-like immunoreactivity after intravenous injection of hypertonic saline. The present study demonstrated that a large proportion of neurokinin B receptor-like immunoreactive neurons in the paraventricular hypothalamic and supraoptic nuclei contained vasopressin-like immunoreactivity, and expressed Fos-like immunoreactivity after intravenous administration of hypertonic saline. The results suggest that neurokinin B receptor in the two nuclei may be involved in modulation of the release of vasopressin when the internal environment is disturbed.     

Depletion of substance P from rat primary sensory neurons by ATP, an implication of P2X receptor-mediated release of substance P.

Effects of ATP on substance P immunoreactivity were examined in cultured dorsal root ganglion neurons. We found that treatment of dorsal root ganglion neurons with ATP significantly depleted substance P immunoreactivity on the neurites and somata of the neurons. The effects of ATP were significantly inhibited by the purinergic P2 receptor antagonists suramin (30 microM) and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (10 microM). We also showed that ATP-induced depletion of substance P immunoreactivity from dorsal root ganglion neurons depended on the entry of Ca(2+). In a spinal cord slice preparation, we also found the internalization of neurokinin-1/substance P receptors in many dorsal horn neurons following the application of ATP or alpha,beta-methylene-ATP.Together these results indicate that activation of P2X receptors may result in release of substance P from primary afferent neurons.     

Immunohistochemical studies of substance P, cholecystokinin-octapeptide and somatostatin in dorsal root ganglia of the rat

Immunofluorescence histochemistry was used to determine the distribution of substance P, somatostatin and cholecystokinin-octapeptide-immunoreactive perikarya in C6, T6, T10, L2 and S1 dorsal root ganglia of rat. Five different categories of immunoreactive primary afferent neurons were distinguished on the basis of cell size, cytology and peptide immunoreactivities. The population of small cells (diameter less than 20 microns) included three groups which were identified as containing somatostatin, substance P, or substance P + cholecystokinin-octapeptide. Two groups of cells were identified in an intermediate size range (diameter 21-43 microns) as containing cholecystokinin-octapeptide or cholecystokinin-octapeptide + substance P. These categories may reflect four distinct populations of primary afferent neurons. The relative abundance of dorsal root ganglion cells containing substance P, cholecystokinin-octapeptide or somatostatin immunoreactivities was significantly different within segmental levels. More neurons were immunoreactive for cholecystokinin-octapeptide than substance P in ganglia C6, T6 and T10. Somatostatin-containing cells were fewest in number regardless of level. The number of immunoreactive cells also varied among spinal ganglia. L2 contained the greatest number of immunoreactive cells; S1 contained the fewest. These studies are relevant to our understanding of dorsal root ganglia in two ways. Firstly, the data document significant variation in the distribution of peptide-containing neurons among spinal ganglia associated with various cord levels. The variation in peptide-containing cell populations among spinal ganglia may reflect differences in populations of modality-specific primary afferent fibers as well as in populations of somatic and visceral primary afferent fibers at each level. Furthermore, the data indicate that the relative abundance of a population of peptide-containing primary afferent neurons cannot be extrapolated from the examination of spinal ganglia from a single level. Secondly, substance P and cholecystokinin-octapeptide did not co-exist in all spinal ganglion cells as previously reported. In conjunction with immunostaining characteristics and cell size, the differential distribution of the two peptides defined four cell types, raising the possibility that each cell type may mediate a different modality.     

Localization of metabotropic glutamate receptors 2/3 on primary afferent axons in the rat

The goal of the present study is to determine the relationship of metabotropic glutamate receptors 2/3 (mGluR2/3) to dorsal root ganglion cells, peripheral primary afferent fibers in digital nerves and central primary afferent fibers in the spinal cord. We demonstrate that approximately 40% of L4 and L5 dorsal root ganglion cells contain mGluR2/3-like immunoreactivity. These mGluR2/3-positive cells are small in diameter (23 microm) and 76% stain for the isolectin Griffonia simplicifolia (I-B4), while 67% of I-B4 cells have mGluR2/3-like immunoreactivity. Electron microscopic analyses of mGluR2/3-like immunoreactivity in axons in digital nerves indicate that 32% of unmyelinated and 28% of myelinated axons are labeled. In the lumbar dorsal horn, mGluR2/3-like immunoreactivity is localized preferentially in lamina IIi with lighter staining in laminae III and IV. The dense mGluR2/3-like immunoreactivity in lamina IIi is consistent with the localization of these receptors in I-B4-labeled dorsal root ganglion cells. Elimination of primary afferent input following unilateral dorsal rhizotomies significantly decreases the mGluR2/3-like immunoreactivity density in the dorsal horn although some residual staining does remain, suggesting that many but not all of these receptors are located on primary afferent processes.The finding that mGluR2/3s are located on peripheral sensory axons suggests that they are involved in peripheral sensory transduction and can modulate transmission of sensory input before it reaches the spinal cord. This offers the possibility of altering sensory input, particularly noxious input, at a site that would avoid CNS side effects. Since many but not all of these receptors are located on primary afferent terminals, these receptors may also influence primary afferent transmission in the dorsal horn through presynaptic mechanisms and glutamatergic transmission in general through both presynaptic and postsynaptic mechanisms. Since these receptors are concentrated in lamina IIi and also largely co-localized with I-B4, they may have considerable influence on nociceptive processing by what are considered to be non-peptidergic primary afferent neurons.     

Analysis of parvalbumin and calbindin D28k-immunoreactive neurons in dorsal root ganglia of rat in relation to their cytochrome oxidase and carbonic anhydrase content

Histochemical and immunohistochemical techniques were used to determine relationships between the parvalbumin or calbindin D28k content and the cytochrome oxidase or carbonic anhydrase activity of neurons in lumbar dorsal root ganglia in rat. Subpopulations of dorsal root ganglion neurons that displayed parvalbumin- or calbindin D28k-immunoreactivity were classified as containing either light, moderate or dense histochemical reaction product for cytochrome oxidase and either a positive or negative reaction for carbonic anhydrase. It was found that approximately 90% of all parvalbumin and calbindin D28k-immunoreactive cells exhibited dense staining for cytochrome oxidase and that 87% of parvalbumin- and 76% of calbindin D28k-immunoreactive cells were positive for carbonic anhydrase. Conversely, 85% of all cells with a dense cytochrome oxidase reaction contained parvalbumin and calbindin D28k. Although not quantified, it appeared that many, but not all, carbonic anhydrase-positive cells contained parvalbumin or calbindin D28k. These results indicate the existence of a subpopulation of primary sensory neurons that contains parvalbumin and calbindin D28k and that expresses high levels of cytochrome oxidase and carbonic anhydrase activity. It is suggested that primary afferent neurons with this cytochemical profile transmit a sensory modality that requires them to discharge rapidly and/or frequently. The existence of a subpopulation of carbonic anhydrase-positive cells that lack immunoreactivity for parvalbumin or calbindin D28k suggests that the role of carbonic anhydrase in some sensory neurons is unrelated to functions requiring these calcium binding proteins.     

Characterization of the peptidergic afferent innervation of the stomach in the rat, mouse and guinea-pig

Retrograde tracing of the fluorescent marker, True Blue, has been used together with immunohistochemistry employing antibodies to substance P, calcitonin gene-related peptide, somatostatin, vasoactive intestinal polypeptide and morphine-modulating peptide to study the afferent innervation of the stomach in rat, mouse and guinea-pig. Up to 85% of spinal afferents to the stomach in all three species contained immunoreactive calcitonin gene-related peptide, and up to 50% contained substance P. In all three species less than 10% of vagal afferents to the stomach reacted with antibodies to calcitonin gene-related peptide, or substance P. Cacitonin gene-related peptide-immunoreactive fibres were found in the myenteric plexus, circular muscle and around submucosal blood vessels in the stomach. In the rat, removal of the coeliac ganglion, splanchnic nerve section, or capsaicin treatment virtually abolished calcitonin gene-related peptide immunoreactivity in the stomach. Capsaicin and splanchnic section also abolished the staining of immunoreactive calcitonin gene-related peptide fibres in the coeliac ganglion. The same treatments abolished substance P staining of fibres around submucosal blood vessels, but in the myenteric plexus and circular smooth muscle there were still abundant immunoreactive fibres, presumably arising from intrinsic cell bodies. No somatostatin-containing visceral afferents could be found, although somatostatin was localized to cell bodies in rat dorsal root ganglia. Immunoreactive vasoactive intestinal polypeptide-containing dorsal root ganglia neurons were not found; although antibodies to morphine-modulatory peptide revealed immunoreactive nerve cell bodies, we were unable to exclude the possibility that this result is attributable to cross reactivity with calcitonin gene-related peptide. These results provide direct evidence that calcitonin gene-related peptide is a marker for a major subset of visceral primary afferent neurons and suggest that this population of spinal afferents makes a major contribution to the total gastric content of calcitonin gene-related peptide.     

Co-localization of calcitonin gene-related peptide-like immunoreactivity with substance P in cutaneous, vascular and visceral sensory neurons of guinea pigs

Calcitonin gene-related peptide (CGRP) has been immunohistochemically co-localized with substance P (SP) in capsaicin-sensitive, varicose axons supplying the skin, viscera and cardiovascular system of the guinea pig. After treatment with colchicine in vitro, 82% of SP neurons in the dorsal root ganglia contained CGRP-like immunoreactivity while 96% of CGRP neurons were immunoreactive for SP. Both CGRP- and SP-like immunoreactive material are transported peripherally and centrally from dorsal root ganglia. Thus, in tissues such as the gut where there are intrinsic nerves containing SP but lacking CGRP, CGRP-like immunoreactivity is a useful means of specifically labelling axons of most sensory neurons containing SP.     

Differences in the chemical expression of rat primary afferent neurons which innervate skin, muscle or joint

The fluorescent dye Fast Blue was injected in anaesthetized rats into either skin, muscle or knee joint of the hindlimb. Following retrograde transport of the dye to lumbar dorsal root ganglia, the cell bodies of primary afferent neurons innervating these different target tissues were identified in ganglion sections by fluorescence microscopy. The sections were processed to demonstrate activity of the enzyme thiamine monophosphatase, or immunoreactivity to calcitonin gene-related peptide, substance P, or somatostatin, in Fast Blue labelled neurons. In all cases immunoreactivity to the antineurofilament antibody RT97 was used to classify dorsal root ganglion cells as being either small dark (RT97 negative, unmyelinated axons) or large light (RT97 positive, myelinated axons). The proportion of small dark cells labelled from each target decreased in the order: skin, muscle, joint. Thiamine monophosphatase and somatostatin were present only in small dark cells, while calcitonin gene-related peptide and substance P were found in both small dark and large light cells. In large light cells of all three targets, more contained calcitonin gene-related peptide than substance P. Among small dark cells, thiamine monophosphatase and somatostatin were found predominantly in skin afferents, while calcitonin gene-related peptide and substance P were more common in muscle and joint afferents. The chemical expression of primary afferents is therefore characteristic of the peripheral target they innervate. This could reflect either a maintained influence of the target on the afferents, or the factors which operate only during development.     

Met-enkephalin is preferentially transported into the peripheral processes of primary afferent fibres in both control and HSV1-driven proenkephalin A overexpressing rats

The demonstration of preproenkephalin A gene expression in rat dorsal root ganglia has raised the question of the physiological role of met-enkephalin-containing primary afferent fibres. Recently, we showed that systemic infection with a recombinant Herpes simplex virus encoding preproenkephalin A (HSVLatEnk1) yielded a marked increase in the density of met-enkephalin-like material synthesising neurons in rat dorsal root ganglia. This study further investigated the synthesis, transport and release of met-enkephalin-like material in the central and/or peripheral processes of primary afferent fibres in HSVLatEnk1-infected and control rats. In controls, dorsal root ganglia neurons containing met-enkephalin-like material were scarce and only a few positively labelled processes were seen at the peripheral output of the dorsal root ganglia. Met-enkephalin-like material accumulated at the proximal side of ligatured sciatic nerve, but not in ligatured L4-L5 dorsal roots. In HSVLatEnk1-infected rats with numerous somas and fibres stained for met-enkephalin-like material in dorsal root ganglia, met-enkephalin immunoreactive material largely accumulated at the proximal side of the ligatured sciatic nerve and few positively stained fibres were also observed in ligatured dorsal roots. Electrical stimulation of L4-L5 dorsal roots attached to a dorsal slice of the lumbar enlargement produced an overflow of met-enkephalin-like material which was approximately 70% higher in HSVLatEnk1-infected rats compared to controls. At the periphery, subcutaneous microdialysis showed higher basal levels of met-enkephalin-like material in the interstitial fluid of hindpaw plantar area in HSVLatEnk1-infected rats, and electrical stimulation of the ipsilateral sciatic nerve resulted in an approximately three-fold-higher overflow of this material than in control rats.These data demonstrated that met-enkephalin synthesised in dorsal root ganglion of both control and preproenkephalin A overexpressing rats is preferentially transported into the peripheral processes of primary afferent fibres where the peptide reaches a releasable compartment, thus providing a neuronal source of peripheral met-enkephalin.     

Stretch receptor-associated expression of alpha 3 isoform of the Na+, K+-ATPase in rat peripheral nervous system

Expression of the neuronal alpha(3) isoform of the Na(+),K(+)-ATPase (alpha(3) Na(+),K(+)-ATPase) was studied in the rat peripheral nervous system using histological and immunohistochemical techniques. Non-uniform expression of the alpha(3) Na(+),K(+)-ATPase was observed in L5 ventral and dorsal roots, dorsal root ganglion, sciatic nerve and its branches into skeletal muscle. The alpha(3) Na(+),K(+)-ATPase was not detected in nerve fibers in skin, saphenous and sural nerves. In dorsal root ganglion 12+/-2% of neurons were immunopositive for alpha(3) Na(+),K(+)-ATPase and all these neurons were large primary afferents that were not labeled by Griffonia simplicifolia isolectin B4 (marker of small primary sensory neurons). In dorsal and ventral roots 27+/-3% and 40+/-3%, respectively, of myelinated axons displayed immunoreactivity for alpha(3) Na(+),K(+)-ATPase. In contrast to the dorsal roots, strong immunoreactivity in ventral roots was observed only in myelinated axons of small caliber, presumably gamma-efferents. In the mixed sciatic nerve alpha(3) Na(+),K(+)-ATPase was detected in 26+/-5% of myelinated axons (both small and large caliber). In extensor hallicus proprius and lumbricales hind limb muscles alpha(3) Na(+),K(+)-ATPase was detected in some intramuscular axons and axonal terminals on intrafusal muscle fibers in the spindle equatorial and polar regions (regions of afferent and efferent innervation of the muscle stretch receptor, respectively). No alpha(3) Na(+),K(+)-ATPase was found in association with innervation of extrafusal muscle fibers or in tendon-muscle fusion regions. These data demonstrate non-uniform expression of the alpha(3) isoform of the Na(+),K(+)-ATPase in rat peripheral nervous system and suggest that alpha(3) Na(+),K(+)-ATPase is specifically expressed in afferent and efferent axons innervating skeletal muscle stretch receptors.     

Characterisation of axon terminals in the rat dorsal horn that are immunoreactive for serotonin 5-HT3A receptor subunits

Serotonin 5-HT₃ receptors are abundant in the superficial dorsal horn and are likely to have an involvement in processing of nociceptive information. It has been shown previously that 5-HT₃ receptors are present on primary afferent terminals and some dorsal horn cells. The primary aim of the present study was to determine what classes of primary afferent possess 5-HT₃A receptor subunits. We performed a series of double- and triple-labelling immunofluorescence experiments. Subunits were labelled with an anti-peptide antibody and primary afferent axons were identified by the presence of calcitonin gene-related peptide (CGRP) and binding of the lectin IB4. Quantitative confocal microscopic analysis revealed that approximately 10% of axons displaying 5-HT₃A immunoreactivity were also labelled for CGRP but that only 3% of these fibres bind IB4. We also investigated the relationship between immunoreactivity for the subunit and descending serotoninergic systems, axons originating from inhibitory neurons that contain glutamic acid decarboxylase, and axons of a subpopulation of excitatory neurons that contain neurotensin. None of these types of axon was associated with immunoreactivity for receptor subunits. Ultrastructural studies confirmed that punctate immunoreactive structures observed with the light microscope were axon terminals. These terminals invariably formed asymmetric synaptic junctions with dendritic profiles and often contained a mixture of granular and agranular vesicles. Some terminals formed glomerular-like arrangements. Immunoreactive cells were also examined and were found to contain intense patches of reaction product within the cytoplasm. We conclude that the majority (about 87%) of dorsal horn axons that are immunoreactive for 5-HT₃A receptor subunits do not originate from the subtypes of primary afferent fibres that bind IB4 or contain CGRP. It is likely that most of these axons have an excitatory action and they may originate from dorsal horn interneurons and/or fine myelinated primary afferent fibres. Electronic Publication     

The expression and localization of somatostatin receptors in dorsal root ganglion neurons of normal and monoarthritic rats

Somatostatin has antinociceptive effects by acting on somatostatin (sst) receptors in primary afferent neurons. Five sst receptor subtypes (sst(1-5)) have been identified. In the present study we assessed the expression and localization of the sst receptor subtypes in lumbar dorsal root ganglia of normal rats and of rats with unilateral antigen-induced arthritis (AIA) in the knee joint. We used polymerase chain reaction (PCR) of material from dorsal root ganglia and immunohistochemistry in dorsal root ganglion paraffin sections. PCR data show that sst(1), sst(2(a)), sst(2(b)), sst(3), and sst(4) receptors are expressed in lumbar dorsal root ganglia of the rat. The sst(5) receptor was expressed in a few samples. Available antibodies revealed sst(2(a)) and sst(2(b)) receptor-like immunoreactivity in the vast majority of neurons, and sst(4) receptor-like immunoreactivity in about 40% of the dorsal root ganglion neurons and in some satellite cells. Real time PCR at 3, 10 and 21 days after induction of AIA did not reveal changes in receptor expression. Immunohistochemistry showed that a similar high proportion of neuronal profiles expressed sst(2(b)) receptor-like IR in control and AIA rats, but the proportion of neuronal profiles with sst(2(a)) receptor-like IR was significantly lower in acute and chronic AIA rats than in control rats. Although the proportion of neuronal profiles with sst(4) receptor-like IR was significantly higher at 21 days than at 3 days values at 3 or at 21 days were not significantly different from control. These data show that the majority of dorsal root ganglion neurons exhibit somatostatin receptor-like IR thus suggesting a high potential for inhibition by somatostatin. The reduction in the proportion of neuronal profiles with sst(2(a)) immunoreactivity suggests that inhibition of neuronal activity by somatostatin is reduced during painful arthritis.     

Some nerve endings in the rat pelvic paracervical autonomic ganglia and varicosities in the uterus contain calcitonin gene-related peptide and originate from dorsal root ganglia

The pelvic paracervical autonomic ganglia of female rats were studied for a subpopulation of nerve endings that could be derived from sensory nerve fibers. Immunohistochemical staining using an antiserum against the synaptic-terminal protein synapsin I was used to identify terminal boutons, while an antiserum against the neuropeptide calcitonin gene-related peptide was used to reveal a subpopulation of sensory nerve fibers. The uterine cervix was also examined for the existence of calcitonin gene-related peptide and synapsin I immunoreactivity in nerve fiber varicosities. In addition, the location of nerve endings in the paracervical ganglion was compared to that in the superior cervical ganglion. Synapsin I immunoreactivity was present in the paracervical ganglion in abundant boutons around neuron somata and in the cervix in varicose nerve fibers of the myometrium, vasculature and epithelium. Double labeling immunocytochemistry revealed calcitonin gene-related peptide-like immunoreactivity in subpopulations of synapsin I-immunoreactive endings in ganglia and nerve varicosities in the cervix. Injection of a retrograde axonal tracer, fluorogold, into the paracervical ganglion produced labeled neurons in dorsal root ganglia and spinal cord; however, fluorogold-labeled neurons containing calcitonin gene-related peptide immunoreactivity were visualized only in dorsal root ganglia. Injections of fluorogold into the uterine cervix produced labeled neurons in the paracervical ganglion and dorsal root ganglia; however, only those in dorsal root ganglia contained immunoreactivity for calcitonin gene-related peptide. These results suggest that immunoreactivity for calcitonin gene-related peptide is present in a subpopulation of nerve endings in the paracervical ganglion and not merely in fibers of passage. The nerve endings in the ganglion and varicosities in the uterine cervix originate from sensory neurons in dorsal root ganglia. The arrangement of endings in the ganglia could play a role in sensory/autonomic interactions for modulation of visceral activity.     

Calcium-binding proteins calbindin and parvalbumin in the superficial dorsal horn of the rat spinal cord

Neurons containing the calcium-binding proteins, calbindin or parvalbumin, were studied by immunohistochemistry in the superficial dorsal horn of the rat spinal cord. Calbindin-containing cells were found in laminae I, II and III, being more abundant in laminae I and II. Some of the neurons in lamina I containing calbindin projected to the supraspinal area. Parvalbumin-containing neurons were mainly distributed in laminae IIi and III. Calbindin and parvalbumin were not detected in the same cells. Some 75% of the neurotensin-like immunoreactive neurons contained calbindin, which corresponded to 13% of the calbindin-containing neurons. Calbindin was sometimes found in the same cells with substance P, enkephalin or somatostatin but less frequently (44-46% of the peptide-containing neurons). Parvalbumin was not found together with these peptides. Electron microscopy showed that the immunoreactive products of calbindin or parvalbumin were mostly in the dendrites or cell bodies. Immunoreactive axon terminals were relatively few. In rhizotomized animals, neurons containing one of these proteins in laminae II and III were found to receive direct inputs of primary afferent fibers. These findings indicate that neurons containing these two proteins belong to different subpopulations of dorsal horn neurons. They may be important in primary afferent processing.     

Aspartate-like immunoreactivity in primary afferent neurons

There is now good evidence that amino acids act as neurotransmitters in primary afferent neurons of dorsal root ganglia. Glutamate is the primary candidate for such a role, and there are reasons to believe that release of glutamate may be accompanied by the release of other neuroactive substances. Using immunocytochemical techniques, we have tested the hypothesis that some dorsal root ganglion neurons contain elevated levels of aspartate as well as glutamate. Antisera raised against conjugates of aspartate or glutamate were used for this purpose. Blocking experiments confirmed that these antibodies were specific to their antigens in cryostat sections of dorsal root ganglia.

Aspartate immunoreactivity was found in approximately 30% of neurons in cervical dorsal root ganglia. The relation between cell size and staining intensity for aspartate was examined using quantitative video microscopy: the great majority of cells immunopositive for aspartate were small (15–30 μm in diameter); about 85% of these cells stained for aspartate, although staining intensities varied over a wide range. By reacting consecutive sections with anti-aspartate and anti-glutamate it was shown that elevated levels of aspartate were found in the same cells which contained elevated levels of glutamate. By measuring the staining intensity of individual cells for both aspartate and glutamate, it was also shown that there was a positive correlation between staining intensity and, presumably, concentration of the two amino acids. The presence of high levels of aspartate in terminals located in the superficial laminae of the dorsal horn was verified by pre- and post-embedding immunocytochemistry with the electron microscope. Aspartate was demonstrated in scalloped terminals, including dark scalloped terminals believed to be associated with unmyelinated fibers of nociceptors.

This evidence supports the hypothesis that aspartate as well as glutamate is present in the cell bodies and terminals of nociceptive primary afferents, and may be released by the terminals of these afferents to activate neurons in the superficial laminae of the dorsal horn.     

Properties of glutaminase of crayfish CNS: implications for axon-glia signaling

Previous studies have shown that arginine vasopressin is an important neuropeptide that can modulate the reflex control of blood pressure and heart rate. The nucleus ambiguus, where cardiac parasympathetic neurons are located, receives dense arginine vasopressin projections. However the mechanisms by which arginine vasopressin alters cardiac parasympathetic activity are unknown. We tested the hypothesis that arginine vasopressin can alter the activity of cardiac parasympathetic neurons by altering the spontaneous GABAergic input to these neurons. Experiments were conducted using whole cell patch clamp recordings of cardiac parasympathetic neurons in an in vitro slice preparation in rats. The results of this study demonstrate that arginine vasopressin increases the frequency and amplitude of GABAergic inhibitory post-synaptic currents in cardiac parasympathetic neurons. Arginine vasopressin did not alter the GABAergic currents evoked by exogenous application of GABA. Similarly, in the presence of tetrodotoxin, arginine vasopressin did not alter the frequency, amplitude or decay time of GABAergic miniature synaptic events evoked by high osmolarity. These results indicate that arginine vasopressin likely acts on neurons precedent to cardiac parasympathetic neurons and that arginine vasopressin likely acts not at the synaptic terminal but at the soma or dendrites of the precedent neuron. Oxytocin and agonists for the V(2)-arginine vasopressin and V(1b)-arginine vasopressin receptors had no effect. By contrast, the arginine vasopressin-evoked responses were completely abolished by a selective V(1a)-arginine vasopressin receptor antagonist indicating arginine vasopressin responses are mediated by V(1a)-arginine vasopressin receptors.We conclude that the V(1a)-arginine vasopressin receptor-mediated increase in frequency and amplitude of inhibitory GABAergic activity to cardiac parasympathetic neurons may be at least one mechanism by which central arginine vasopressin may increase heart rate and inhibit reflex bradycardia.     

Differential expression of synaptoporin and synaptophysin in primary sensory neurons and up-regulation of synaptoporin after peripheral nerve injury

Synaptoporin and synaptophysin are integral membrane components of synaptic vesicles. The distribution of synaptoporin and its relationship with synaptophysin in sensory afferent fibers remain unclear. In the present study, we showed that in the rat dorsal root ganglia synaptoporin was expressed in subsets of small neurons that contain either calcitonin gene-related peptide or isolectin B4, and was distributed in their afferent terminals in laminae I-II of the spinal cord. Synaptophysin was expressed in 57% of synaptoporin-containing small dorsal root ganglion neurons and in large dorsal root ganglion neurons. In the spinal dorsal horn, synaptophysin-immunolabeling was weak in the afferent fibers in lamina I, outer lamina II and the dorsal part of inner lamina II, but strong in the afferent fibers in laminae III-IV. However, a subpopulation of isolectin B4-positive small dorsal root ganglion neurons expressed both synaptoporin and synaptophysin, and their afferent fibers were mainly distributed in the ventral part of inner lamina II. After peripheral nerve injury, synaptoporin expression was up-regulated in small dorsal root ganglion neurons, and synaptoporin level was increased in their afferent terminals. Thus, synaptoporin and synaptophysin have topographically distinct distributions in afferent fibers. Synaptoporin is a major synaptic vesicle protein in Adelta- and C-fibers in both physiological and neuropathic pain states.