Coexistence of NADPH diaphorase with GABA,glycine, and acetylcholine in rat spinal cord

The enzyme NADPH diaphorase is present in many spinal neurons, and is thought to correspond to nitric oxide synthase. In order to determine which types of neuron in the spinal cord contain this enzyme, we have carried out a combined enzyme histochemical and immunocytochemical study with antibodies to GABA, glycine, and choline acetyltransferase. Two hundred and twenty-four NADPH diaphorase-positive neurons in midlumbar spinal cord from four rats were tested for GABA- and glycine-like immunoreactivity. The majority of these neurons (207/224) were GABA-immunoreactive and 139 were also glycine-immunoreactive. NADPH diaphorase-positive neurons in laminae I and II generally showed both types of immunoreactivity, while those in deeper laminae of the dorsal horn and around the central canal either showed both types or else were only GABA-immunoreactive. Since GABA and acetylcholine are thought to coexist in spinal neurons, NADPH diaphorase staining was combined with immunostaining for choline acetyltransferase. Immunoreactive neurons in laminae III and IV were all NADPH diaphorase-positive, while only some of those around the central canal and in the deeper laminae of the dorsal horn were positive. Choline acetyltransferase-immunoreactive neurons in the intermediolateral cell column (presumed sympathetic preganglionic neurons) were often NADPH diaphorase-positive, whereas those in the ventral horn (presumed motorneurons) were not. NADPH diaphorase-positive cells in the intermediolateral cell column were not immunoreactive with GABA or glycine antibodies. These results suggest that NADPH diaphorase is largely restricted to GABAergic neurons in the lumbar spinal cord, and that it is mainly present in those neurons in which GABA coexists with glycine or acetylcholine. Since nitric oxide has been implicated in pain processing and hyperalgesia, while GABA, glycine, and acetylcholine are thought to be involved in analgesia and prevention of hyperalgesia, it is likely that nitric oxide synthase-containing GABAergic neurons in dorsal horn have dual actions in transmission of nociceptive information. © 1993 Wiley-Liss, Inc.     

Changes of NADPH-diaphorase reactivity in lumbar spinal cord of short-term streptozotocin induced diabetic rats

Diabetes is an endocrine and metabolic disorder often associated with erectile dysfunction and peripheral neuropathy. Among other factors, penile erection is induced by activation of nitric oxide synthase (NOS). Hypothalamic paraventricular nuclei neurons produce NO and project to spinal cord areas implicated in penile reflexes. These nuclei have shown an increase of NOS in streptozotocin-induced diabetic rats. NOS-containing neurons are identical to the populations of neurons selectively stained for NADPH-diaphorase activity. Using this technique, we have evaluated changes of NOS in the lumbar spinal cord of diabetic rats with or without insulin treatment. Positive staining was found in motoneurons, dorsal horn neurons (layer II), neurons surrounding the ependimus (layer X) and neurons at the intermediolateral cell column (ILCC). Diabetic animals showed significant decrease in reactive area and increase of the histochemical reaction in motoneurons from the sexual dimorphic nuclei and in neurons of the ILCC. A marked decrease of the number of reactive neurons was also observed in layer II. Morphologic alterations were observed in neurons of layer X as an increase in the percentage of multipolar neurons and a decrease in the number and length of secondary processes. The alterations observed in these animals were absent in the insulin treated diabetic animals. These results show the plasticity of lumbar spinal cord neurons, suggesting a direct participation of NO synthesis in the physiopathology of the erection dysfunction in diabetes.     

Distribution of vesicular glutamate transporters 1 and 2 in the rat spinal cord, with a note on the spinocervical tract

To evaluate whether the organization of glutamatergic fibers systems in the lumbar cord is also evident at other spinal levels, we examined the immunocytochemical distribution of vesicle glutamate transporters 1 and 2 (VGLUT1, VGLUT2) at several different levels of the rat spinal cord. We also examined the expression of VGLUTs in an ascending sensory pathway, the spinocervical tract, and colocalization of VGLUT1 and VGLUT2. Mainly small VGLUT2-immunoreactive varicosities occurred at relatively high densities in most areas, with the highest density in laminae I-II. VGLUT1 immunolabeling, including small and medium-sized to large varicosities, was more differentiated, with the highest density in the deep dorsal horn and in certain nuclei such as the internal basilar nucleus, the central cervical nucleus, and the column of Clarke. Lamina I and IIo displayed a moderate density of small VGLUT1 varicosities at all spinal levels, although in the spinal enlargements a uniform density of such varicosities was evident throughout laminae I-II in the medial half of the dorsal horn. Corticospinal tract axons displayed VGLUT1, indicating that the corticospinal tract is an important source of small VGLUT1 varicosities. VGLUT1 and VGLUT2 were cocontained in small numbers of varicosities in laminae III-IV and IX. Anterogradely labeled spinocervical tract terminals in the lateral cervical nucleus were VGLUT2 immunoreactive. In conclusion, the principal distribution patterns of VGLUT1 and VGLUT2 are essentially similar throughout the rostrocaudal extension of the spinal cord. The mediolateral differences in VGLUT1 distribution in laminae I-II suggest dual origins of VGLUT1-immunoreactive varicosities in this region.     

Localization of NADPH diaphorase in the lumbosacral spinal cord and dorsal root ganglia of the cat

The distribution of NADPH-d activity in the spinal cord and dorsal root ganglia of the cat was studied to evaluate the role of nitric oxide in lumbosacral afferent and spinal autonomic pathways. At all levels of the spinal cord NADPH-d staining was present in neurons and fibers in the superficial dorsal horn and in neurons around the central canal and in the dorsal commissure. In addition, the sympathetic autonomic nucleus in the rostral lumbar segments exhibited prominent NADPH-d cellular staining whereas the parasympathetic nucleus in the sacral segments was not well stained. The most prominent NADPH-d activity in the sacral segments occurred in fibers extending from Lissauer's tract through laminae I along the lateral edge of the dorsal horn to lamina V and the region of the sacral parasympathetic nucleus. These fibers were very similar to VIP-containing and pelvic nerve afferent projections in the same region. They were prominent in the S₁–S₃ segments but not in adjacent segments (L₆–L₇ and Cx₁) or in thoracolumbar and cervical segments. NADPH-d activity and VIP immunoreactivity in Lissauer's tract and the lateral dorsal horn were eliminated or greatly reduced after dorsal-ventral rhizotomy (S₁–S₃), indicating the fibers represent primary afferent projections. A population of small diameter afferent neurons in the L₇–S₂ dorsal root ganglia were intensely stained for NADPH-d. The functional significance of the NADPH-d histochemical stain remains to be determined; however, if NADPH-d is nitric oxide synthase then this would suggest that nitric oxide may function as a transmitter in thoracolumbar sympathetic preganglionic efferent pathways and in sacral parasympathetic afferent pathways in the cat. © 1994 Wiley-Liss, Inc.     

Light and electron microscopic immunocytochemical localization of AMPA-selective glutamate receptors in the rat spinal cord

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors are probably the most widespread excitatory neurotransmitter receptors of the central nervous system, and they play a role in most normal and pathological neural activities. However, previous detailed studies of AMPA subunit distribution have been limited mainly to the brain. Thus, a comprehensive study of AMPA receptor subunit distribution was carried out on sections of rat spinal cord and dorsal root ganglia, which were immunolabeled with antibodies made against peptides corresponding to C-terminal portions of the AMPA receptor subunits: GluR1, GluR2/3, and GluR4. In the spinal cord. Labeling was most prominent in the superficial dorsal horn, motoneurons, and nuclei containing preganglionic autonomic neurons. Immunostaining also was observed in neurons in other regions including those known to contain Renshaw cells and Ia Inhibitory cells. Although overall immunostaining was lighter with antibody to GluR1 than with GluR2/3 and 4, there were neurons were neurons that preferentially stained with antibody to GluR1. These “GluR1 intense” neurons were usually fusiform and most concentrated in lamina X. In dorsal root ganglia, immunostaining of ganglion cell bodies was moderate to dense with antibody to GluR2/3 and light to moderate with antibody to GluR4. Possible neuroglia in the spinal cord (mainly GluR2/3 and 4) and satellite cells in dorsal root ganglia (GliR4) were immunostained. Electron microscopic studies of the the superficial dorsal horn and lateral motor column showed staining that was restricted mainly to postsynaptic densities and associated dendritic and cell body cytoplasm. In dorsal horn, colocalization of dense-cored vesicles with clear, round synaptic vesicles was observed in unstained presynaptic terminals apposed to stained postsynaptic densities. Subsynaptic dense bodies (Taxi-bodies) were associated with some stained postsynaptic densities in the superficial dorsal horn and lateral motor column. Based on several morphological features including vesicle structure and presence of Taxi-bodies, it is likely that at least some of the postsynaptic staining seen in this study is apposed to glutamatergic input from primary sensory afferent terminals. © 1994 Wiley-Liss, Inc.     

A comparison of the ultrastructure of substance P and enkephalin-immunoreactive elements in the nucleus of the dorsal lateral funiculus and laminae I and II of the rat spinal cord

The ultrastructure of substance P (SP)- and enkephalin (ENK)-immunoreactive elements in the nucleus of the dorsal lateral funiculus (NDLF) and in laminae I and II of the spinal cord was examined in the rat using the peroxidate-antiperoxidase (PAP) technique. Electron-microscopic observations were made of a large number of immunolabelled terminals (n = 428; many followed in serial sections), axons, and immunoreactive cell bodies and dendrites which were occasionally encountered. Morphometric analysis was used to describe and compare the fine structural features of immunolabelled elements. Both SP- and ENK-immunoreactive terminals contained clear synaptic vesicles and dense-cored vesicles of similar size but the ENK-immunoreactive profiles contained significantly more dense-cored vesicles than SP-immunolabelled profiles. Both SP- and ENK-immunoreactive profiles in the dorsal laminae of the dorsal horn contacted mainly smaller dendritic elements. Only rarely were axo-axonic interactions noted. The NDLF contains widely scattered cell bodies dispersed within a neuropil which is rich in synaptic complexes and is interdigitated between fascicles of myelinated and unmyelinated axons. Numerous SP- and ENK-immunoreactive profiles were observed in the NDLF, many of which made asymmetric synaptic contacts with NDLF neurons. Although both the dorsal gray and NDLF contain large numbers of SP- and ENK-immunoreactive elements which are similar in morphology in both regions, the NDLF can be distinguished from laminae I and II by a number of criteria, including the nature of the neuropil, principle sources of SP innervation, and the termination patterns of ascending projections.     

Estrogen receptor-immunoreactive neurons are present in the female rat lumbosacral spinal cord

Presence of an estrogen receptor is crucial for cells to respond to estrogen; thus, estrogen-responsive neurons should be identifiable by immunohistochemically staining for the estrogen receptor (ER). Even though spinal neurons are involved in sexual behaviors and innervation of genital organs, little information is available about ER-containing neurons in the spinal cord. Consequently, we have undertaken a study of ER-containing neurons in the female rat lumbosacral cord, an area involved in reproductive functions and predicted to contain estrogen-responsive neurons. In addition, since parasympathetic preganglionic neurons in the lumbosacral cord produce nitric oxide (NO), we also sought to determine if ER-immunoreactive (-IR) neurons contain the enzymes for NO production. Finally, we compared the distribution of ER-IR neurons to the presence of uterine cervix-related neurons. Uterine cervix-related neurons were identified by expression of FOS-immunoreactivity after vaginocervical mechanostimulation (VCS). The lumbosacral spinal cords were removed from intact, ovariectomized, and VCS-treated rats and sections stained by immunohistochemistry. ER-IR was present in the nuclei of neurons located predominately in the dorsal one-half of the spinal cord. Specific sites include the dorsal horn, lamina V, the sacral parasympathetic nucleus (SPN) (which contains preganglionic parasympathetic neurons) and extending into the lateral funiculus, and lamina X. Some ER-IR neurons were NADPH-d-positive and were localized in laminae V and VII. FOS-IR neurons had a distribution pattern similar to the distribution of neurons containing ER. The presence of ER neurons in these regions suggest that they are responsive to circulating estrogen. © 1996 Wiley-Liss, Inc.     

Morphology and somatotopic organization of the central terminals of hindlimb hair follicle afferents in the rat lumbar spinal cord

The morphology of the central collateral arborizations of 24 A-beta hair follicle afferents (HFAs) innervating different regions of the skin of the hindlimb were studied by the intra-axonal injection of horseradish peroxidase (HRP) in adult rats. A total of 236 collaterals were recovered. These fell into three classes--complex, simple, and blind-ending--based on numbers of boutons and terminal branch patterns. The morphology of the HFA central arbors innervating the lateral and medial leg and dorsum of the foot was flame-shaped. Afferents with receptive fields on the glabrous-hairy skin border consistently had extra terminal branches running ventromedially into laminae IV/V. Differences in the width of terminal arbors were found. HFA terminals innervating the lateral leg formed narrower sheets than those innervating the dorsum of the foot and toes. The somatotopic organization of the collaterals and terminal arborizations of individual afferents were analyzed both by considering all the collaterals along an axon's rostrocaudal extent and by only examining arbors with boutons (the complex and simple arbors). Thirty-seven percent of blind-ending and 18% of simple collaterals were found to overlap in the rostrocaudal direction with the complex arborizations of afferents whose receptive fields were in a different cutaneous nerve territory. There was no overlap between complex arborizations of afferents from different nerve territories. However, the complex arbors of afferents with receptive fields within a particular nerve territory showed considerable terminal overlap even if they had nonadjacent peripheral receptive fields. The topographical organization of the central terminals of HFAs, forms a coarse somatotopic map of overlapping terminals whereby a particular region of dorsal horn has a maximal, but not exclusive, input from a particular area of skin.     

p75NTR expression in rat urinary bladder sensory neurons and spinal cord with cyclophosphamide-induced cystitis

A role for nerve growth factor (NGF) in contributing to increased voiding frequency and altered sensation from the urinary bladder has been suggested. Previous studies have examined the expression and regulation of tyrosine kinase receptors (Trks) in micturition reflexes with urinary bladder inflammation. The present studies examine the expression and regulation of another receptor known to bind NGF, p75(NTR), after various durations of bladder inflammation induced by cyclophosphamide (CYP). CYP-induced cystitis increased (P < or = 0.001) p75(NTR) expression in the superficial lateral and medial dorsal horn in L1-L2 and L6-S1 spinal segments. The number of p75(NTR)-immunoreactive (-IR) cells in the lumbosacral dorsal root ganglia (DRG) also increased (P < or = 0.05) with CYP-induced cystitis (acute, intermediate, and chronic). Quantitative, real-time polymerase chain reaction also demonstrated significant increases (P < or = 0.01) in p75(NTR) mRNA in DRG with intermediate and chronic CYP-induced cystitis. Retrograde dye-tracing techniques with Fastblue were used to identify presumptive bladder afferent cells in the lumbosacral DRG. In bladder afferent cells in DRG, p75(NTR)-IR was also increased (P < or = 0.01) with cystitis. In addition to increases in p75(NTR)-IR in DRG cell bodies, increases (P < or = 0.001) in pericellular (encircling DRG cells) p75(NTR)-IR in DRG also increased. Confocal analyses demonstrated that pericellular p75(NTR)-IR was not colocalized with the glial marker, glial fibrillary acidic protein (GFAP). These studies demonstrate that p75(NTR) expression in micturition reflexes is present constitutively and modified by bladder inflammation. The functional significance of p75(NTR) expression in micturition reflexes remains to be determined.     

Compartmentation of NADPH-diaphorase activity in the mouse cerebellar cortex

The mammalian cerebellum is built around an array of parasagittal bands of Purkinje cells that can be demonstrated by immunocytochemical staining for the differentiation antigen zebrin II. Climbing and Mossy fiber afferents also terminate in bands, and the afferent terminal fields and the Purkinje cell bands are aligned. The convergence of mossy and climbing fiber pathways onto the Purkinje cells, which are the sole output of the cerebellar cortex, is a characteristic feature of cerebellar circuitry. Previous studies showed that when both afferent pathways are activated synchronously there develops a long-term depression of synaptic efficacy at the parallel fiber-Purkinje cell synapse. Two second messenger pathways mediate long-term depression: one involves diacylglyceroland protein kinase C, and the other involves nitric oxide that is generated by a nitric oxide synthase. We have studied the distribution of nitric oxide synthase in the adult Mouse cerebellum by using nicatinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry. NADPH-diaphorase activity is found mainly in the granule and basket cells. Within the granular layer NADPH-diaphorase activity is expressed nonuniformly patches of granule cells and synaptic glomeruli. The patches are yseen in all lobules, are reproducible from individual to individual, and are topographically ordered with respect to the Purkinje cell compartments as revealed by using anti-zebrin II immunocytochemistry. These data imply that nitric oxide-dependent, long-term depression may only involve a subset of mossy fiber/granule cell projections, and that one role for nitric oxide may be to refine cerebellar receptive fields. © 1994 Wiley-Liss, Inc.     

A quantitative study of spinothalamic neurons in laminae I, III, and IV in lumbar and cervical segments of the rat spinal cord

The major ascending outputs from superficial spinal dorsal horn consist of projection neurons in lamina I, together with neurons in laminae III-IV that express the neurokinin 1 receptor (NK1r) and have dendrites that enter the superficial laminae. Some neurons in each of these populations belong to the spinothalamic tract, which conveys nociceptive information via the thalamus to cortical areas involved in pain. A projection from the cervical superficial dorsal horn to the posterior triangular nucleus (PoT) has recently been identified. PoT is at the caudal end of the thalamus and was not included in injection sites in many previous retrograde tracing studies. We have injected various tracers (cholera toxin B subunit, Fluoro-Gold, and fluorescent latex microspheres) into the thalamus to estimate the number of spinothalamic neurons in each of these two populations, and to investigate their projection targets. Most lamina I and lamina III/IV NK1r-immunoreactive spinothalamic neurons in cervical and lumbar segments could be labeled from injections centered on PoT. Our results suggest that there are 90 lamina I spinothalamic neurons per side in C7 and 15 in L4 and that some of those in C7 only project to PoT. We found that 85% of the lamina III/IV NK1r-immunoreactive neurons in C6 and 17% of those in L5 belong to the spinothalamic tract, and these apparently project exclusively to the caudal thalamus, including PoT. Because PoT projects to second somatosensory and insular cortices, our results suggest that these are major targets for information conveyed by both these populations of spinothalamic neurons.     

NADPH-diaphorase localization in the CNS and peripheral tissues of the predatory sea-slug Pleurobranchaea californica

The distribution of putative nitric oxide synthase (NOS)-containing cells in the opisthobranch mollusc Pleurobranchaea californica was studied histochemically via NADPH-diaphorase (NADPH-d) reduction of Nitro Blue Tetrazolium (NTB). Whole mounts and cryostat sections were prepared from the central nervous system and peripheral organs, including the buccal muscles, esophagus, salivary glands, foot, mantle, and gills. NADPH-d-positive neurons were localized predominantly to the buccal and pedal ganglia as well as to distinct areas of the cerebropleural and visceral ganglia. A variety of identified neurons were positive for NADPH-diaphorase in various central ganglia, including the metacerebral cells of the cerebropleural ganglion, putative locomotor neurons of the pedal ganglia, and buccal motoneurons. Specific staining was observed only in somata of central neurons, whereas neuropil areas remained unstained. However, NADPH-d-reactive axons were dense in buccal ganglion nerves, whereas peripheral nerves and connectives of other ganglia had few or no NADPH-d positive terminals. In the periphery, NADPH-d activity was detected only in a few neurons of the rhinophore and tentacle ganglia. NADPH-d staining was marked in the salivary glands and gills, but there was no or very little staining in the esophagus, buccal mass, and foot. Histochemical stain production required the presence of both β-NADPH and NBT; α-NADPH could not substitute for β-NADPH. The inhibitor of NOS, 2,6-dichlorophenol-indophenol, at 10⁻³ M, totally abolished NADPH-d-positive staining. The apparent high activity of central NADPH-d contrasts with much lower activity in the ganglia of the related gastropod Tritonia. These data suggest a role for nitric oxide as a signal molecule in the central nervous system of Pleurobranchaea. © 1996 Wiley-Liss, Inc.     

Distribution of dopamine immunoreactivity in the rat,cat, and monkey spinal cord

In the present study, the distribution of dopamine (DA) was identified light microscopically in all segments of the rat, cat, and monkey spinal cord by using immunocytochemistry with antibodies directed against dopamine. Only fibers and (presumed) terminals were found to be immunoreactive for DA. Strongest DA labeling was present in the sympathetic intermediolateral cell column (IML). Strong DA labeling, consisting of many varicose fibers, was found in all laminae of the dorsal horn, including the central canal area (region X), but with the exception of the substantia gelatinosa, which was only sparsely labeled, especially in rat and monkey. In the motoneuronal cell groups DA labeling was also strong and showed a fine granular appearance. The sexually dimorphic cremaster nucleus and Onuf's nucleus (or its homologue) showed a much stronger labeling than the surrounding somatic motoneurons. In the parasympathetic area at sacral levels, labeling was moderate. The remaining areas, like the intermediate zone (laminae VI-VIII), were only sparsely innervated. The dorsal nucleus (column of Clarke) showed the fewest DA fibers, as did the central cervical nucleus, suggesting that cerebellar projecting cells were avoided by the DA projection. In all species, the descending fibers were located mostly in the dorsolateral funiculus, but laminae I and III also contained many rostrocaudally oriented fibers. It is concluded that DA is widely distributed within the spinal cord, with few differences between species, emphasizing that DA plays an important role as one of the monoamines that influences sensory input as well as autonomic and motor output at the spinal level. © 1996 Wiley-Liss, Inc.     

Light and electron microscope study of GABA-immunoreactive neurones in lamina III of rat spinal cord.

In order to determine whether different morphological types of neurone in lamina III of rat spinal dorsal horn contain different neurotransmitters, a combined Golgi and immunocytochemical study was performed. Semithin sections through the cell bodies of 52 Golgi-impregnated neurones in this lamina were tested with antisera to GABA and glycine. Thirty of these cells were immunoreactive with anti-GABA antiserum and 25 of these also showed glycine-like immunoreactivity. These cells had dendrites which were oriented along the rostrocaudal axis and occupied lamina III, with some extension into lamina IV and the ventral half of lamina II. Although some of the nonimmunoreactive cells had similar morphology, many of them had dendrites which passed in a dorsal and/or ventral direction and crossed laminar boundaries. Three of the neurones which were immunoreactive with both antisera were examined with the electron microscope. These cells received a variety of synapses including some from axons which resembled low threshold myelinated mechanoreceptive primary afferents. These results indicate that there is a relationship between morphology and function for neurones in lamina III. It is suggested that the inhibitory neurones which contain both GABA and glycine selectively regulate the transmission of information from low threshold mechanoreceptive primary afferents to other dorsal horn neurones.     

Nitric oxide synthase and NADPH-diaphorase after acute hypobaric hypoxia in the rat caudate putamen

Changes in the production system of nitric oxide (NO), a multifunctional biological messenger known to participate in blood-flow regulation, neuromodulation, and neuroprotection or neurotoxicity, were investigated in the caudate putamen of adult rats submitted to hypobaric hypoxia. Employing immunohistochemistry, Western blotting, enzymatic assay, and NADPH-diaphorase staining, we demonstrate that neuronal nitric oxide synthase (nNOS) expression and constitutive nitric oxide synthase (cNOS) activity were transiently activated by 7 h of exposure to a simulated altitude of 8325 m (27,000 ft). In addition, endothelial nitric oxide synthase (eNOS) immunoreactivity and blood vessel NADPH-diaphorase staining peaked immediately after the hypoxic stimulus, whereas inducible nitric oxide synthase (iNOS) expression and activity remained unaltered. Nitrotyrosine formation, a marker of protein nitration, was evaluated by immunohistochemistry and Western blotting, and was found to increase parallel to nitric oxide synthesis. We conclude that the nitric oxide system undergoes significant transient alterations in the caudate putamen of adult rats submitted to acute hypobaric hypoxia.     

Synaptic relationships between hair follicle afferents and neurones expressing GABA and glycine-like immunoreactivity in the spinal cord of the rat

gamma-Aminobutyric acid (GABA) and glycine have been implicated in the inhibition of sensory pathways in the dorsal horn of the spinal cord. The object of this study is to investigate the interactions between neurones immunoreactive for GABA and/or glycine and hair follicle afferent terminals labelled by intracellular injection with neurobiotin. GABA and glycine-like immunoreactivity in axons and dendrites in synaptic contact with the afferent terminals was demonstrated by using a postembedding immunogold method, and serial section reconstruction was used to show the distribution and nature of these interactions in lamina III of the dorsal horn. Most afferent boutons (94%) were postsynaptic at axo-axonic synapses: 67% of presynaptic boutons presynaptic to the afferent terminals were immunoreactive for GABA and glycine, 24% for GABA alone, and 7% for glycine alone. Only a small percentage of dendrites postsynaptic to afferent boutons appeared to belong to inhibitory interneurones: 3% were immunoreactive for GABA and glycine, 10% for glycine alone, but 87% were immunoreactive for neither antibody. Many afferent boutons were the central terminals of what appeared to be type IIb glomeruli and were involved triadic synaptic arrangements at which boutons presynaptic to an afferent terminal also made axodendritic contacts with dendrites postsynaptic to the afferent. Many of the presynaptic boutons involved in the triads were immunoreactive for GABA and glycine. Because afferent terminals do not themselves express glycine receptors (Mitchell et al. [1993] J. Neurosci. 13:2371-2381), glycine may therefore act on dendrites postsynaptic to hair follicle afferent terminals at these triads.     

Effect of suckling on NADPH-diaphorase (Nitric oxide synthase, NOS) reactivity and NOS gene expression in the paraventricular and supraoptic nuclei of lactating rats

This study examined the effect of suckling on nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d, a histochemical marker for nitric oxide synthase, NOS) reactivity and neuronal NOS mRNA expression in the paraventricular (PVN) and supraoptic (SON) nuclei of lactating rats. Freely nursing (non-separated) dams and those separated from pups for 12 h and then reunited for 0, 15, 30, 60, 90, 120 and 180 min were used for the study. Dams separated from pups and sacrificed at time zero (without reunion) showed a significant decrease in NADPH-d staining and NADPH-d positive cells as well as in the NOS mRNA expression in the PVN and SON compared to that observed in non-separated dams. Reunion with pups and restoration of suckling significantly increased NADPH-d reactivity after 15, 30, 60 min, but not after 90, 120 and 180 min compared to non-reunited pups-deprived dams. A pattern of NADPH-d reactivity and neuronal NOS mRNA expression indistinguishable from that observed during free lactation was reinstated shortly (15 min) after the restoration of suckling stimulus, suggesting that the NADPH-d reactivity in lactation depends on the presence of the suckling stimulus. These results show that suckling stimulus may play a modulatory role in the regulation of NOS reactivity in the magnocellular neurones of the hypothalamic PVN and SON during lactation.     

Morphology and somatotopy of the central arborizations of rapidly adapting glabrous skin afferents in the rat lumbar spinal cord

The central arborizations in the dorsal horn of the spinal cord of 23 rapidly adapting (RA) A-beta primary afferent neurons innervating different regions of the glabrous skin of the hindpaw were studied by the intra-axonal injection of horseradish peroxidase in adult rats. A total of 284 arbors of the complex, simple, and blind-ending variety were recovered. The arbors of RA afferents innervating the toes, paw pads, and non-pad hindpaw differed from each other in branch pattern and dimensions. The simple and complex arbors, which are both bouton-containing, were distributed mainly in laminae III–V, although some complex arbors projected dorsally into lamina IIi. The hindpaw glabrous skin afferent terminals were located in the lumbar enlargement from caudal L3 to rostral L6. A crude somatotopic organization was observed such that toes 1–5 were represented successively in more caudal positions from mid-L4 to caudal L5. The paw pads were organized in a rostrocaudal sequence moving from the paw pads proximal to toe 1 across the foot to the paw pads proximal to toe 5, from caudal L3 to mid-L5. Non-pad hindpaw afferents were located in caudal L5. Overlap between toe, paw pad and non-pad afferent central fields was present, however, and the central terminals of afferents with non-adjacent peripheral receptive fields were shown to occupy the same region of the dorsal horn. © 1993 Wiley-Liss, Inc.     

The sprouting of saphenous nerve terminals in the spinal cord following early postnatal sciatic nerve section in the rat

Transganglionic labelling of the saphenous nerve in rats with WGA-HRP revealed the central distribution of its terminals in the lumbar dorsal horn. The terminal field was clearly defined and consistent in rats aged between day 6 and day 90. If, however, the sciatic nerve was sectioned on day 1 of postnatal life, the saphenous terminal field expanded to occupy twice the normal area (measured between the L2 and L4 boundaries). The spread was caudal, medial, and lateral into areas normally occupied by sciatic nerve terminals. This sprouting was very weak if the sciatic nerve was sectioned later in postnatal life, on day 5, and nonexistent if sectioning took place on day 10. Crushing the sciatic nerve on day 1 also triggered the effect but the spread of the terminal field was less than that produced by section of the sciatic nerve. There was no evidence of sprouting from the contralateral intact sciatic nerve. The results demonstrate the necessity of intact afferent input during a critical period of neonatal life in order to maintain the precise somatotopic termination pattern of dorsal root afferents.     

The contralateral input to the dorsal horn of the spinal cord in the decerebrate spinal rat

Input from the contralateral limb and tail was examined in the lumbar dorsal horn of decerebrate spinal rats. Fifty-three cells were recorded from laminae 4, 5 and 6 and classified according to their ipsilateral response to natural and electrical stimulation. Twenty-nine (54%) of these cells were found to have inhibitory contralateral fields. This inhibition was evoked by noxious pinching or heating of the skin. In most cases the inhibitory field was a mirror image of the excitatory ipsilateral field although it also often included the tail. Activity evoked by natural and electrical stimulation as well as spontaneous activity was inhibited by contralateral skin stimulation. Noxious specific and wide dynamic range cells displayed these fields but low threshold mechanoreceptive cells did not. Twenty-six cells (49%) received direct short-latency excitatory input from the contralateral sciatic nerve; this correlated well with the presence of contralateral fields. Trains of stimuli applied to the contralateral sciatic nerve at Aδ- and C-fibre strength resulted in inhibition of the cell whereas trains of Aβ strength had no effect. The results demonstrate the existence of segmental contralateral control over dorsal horn cell activity, not involving supraspinal pathways.