Phosphorylation of neuronal nitric oxide synthase at Ser847 in the nucleus intermediolateralis after spinal cord injury in mice.

We previously demonstrated that Ca2+/calmodulin (CaM)-dependent protein kinase IIalpha (CaM-KIIalpha) can phosphorylate neuronal nitric oxide synthase (nNOS) at Ser847 and attenuate NOS activity in neuronal cells. In the present study we focused on chronological alteration in levels and cellular location of nNOS, phosphorylated (p)-Ser847-nNOS (NP847), CaM-KII and p-Thr286-CaM-KIIalpha following spinal cord injury (SCI) in mice. Western blot analysis showed nNOS to be significantly phosphorylated at Ser847 from 3 h after SCI, peaking at 24 h and gradually decreasing thereafter, and CaM-KII to be colocalized with nNOS after SCI. Immunohistochemical analysis revealed that SCI causes an increase in both NP847 and p-Thr286-CaM-KIIalpha in the nucleus intermediolateralis. These findings suggest that SCI induces p-Thr286-CaM-KIIalpha, which phosphorylates the nNOS at Ser847 in the nucleus intermediolateralis where NO is thought to play a role as a neurotransmitter in autonomic preganglionic neurons. Thus, the NP847 signaling pathway might be involved in the autonomic failure which occurs immediately after SCI.

Protein kinase C gamma-like immunoreactivity in the substantia gelatinosa of the medullary dorsal horn of the rat.

We examined protein kinase C gamma-immunoreactivity (PKCgamma-IR) in the substantia gelatinosa (SG) of the rat medullary dorsal horn (MDH). The density of PKCgamma-IR in the MDH was most intense in the SG. The number of neurons with PKCgamma-IR were also much larger in the SG than in the other layers of the MDH. Double-immunohistochemical studies indicated light and electron microscopically that substance P-containing fibers and I-B4 (isolectin from Bandeiraea simplicifolia)-labeled fibers made synapses on SG neurons with PKCgamma-IR, indicating that SG neurons with PKCgamma might receive nociceptive primary afferent fibers. The results support the notion that PKCgamma in the MDH may contribute to the regulation of the nociception.

Glucocorticoid effects on Fos immunoreactivity and NADPH-diaphorase histochemical staining following spinal cord injury.

Glucocorticoids (GC) provide neuroprotection and early recovery after spinal cord injury (SCI). While several mechanisms were proposed to account for these effects, limited information exists regarding GC actions in sensory areas of the spinal cord. Presently, we studied the time course of Fos expression, and reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemical staining to monitor neuronal responses to SCI with or without GC treatment. Rats with sham-operation or transection at the thoracic level (T7-T8) received vehicle or 5 mg/kg of the GC dexamethasone (DEX) at 5 min post-lesion and were sacrificed 2 or 4 h after surgery. Another group of SCI rats received vehicle or intensive DEX treatment (5 min, 6 h, 18 h and 46 h post-lesion) and were sacrificed 48 h after surgery. The number of NADPH-d positive neurons or Fos immunoreactive nuclei was studied by computer-assisted image analysis in superficial dorsal horn (Laminae I-III) and central canal area (Lamina X) below the lesion. While constitutive Fos immunoreactive nuclei were sparse in controls, SCI increased Fos expression at 2 and 4 h after injury. DEX treatment significantly enhanced the number of Fos positive nuclei in Laminae I-III by 4 h after transection, although the response was not maintained by intensive steroid treatment when tested at 48 h after SCI. NADPH-d positive neurons in Laminae I-III increased at 2 and 4 h after SCI while a delayed increased was found in central canal area (Lamina X). DEX treatment decreased NADPH-d positive neurons to sham-operated levels at all time points examined. Thus, while GC stimulation of Fos suggests activation of neurons involved in sympathetic outflow and/or pain, down-regulation of NADPH-d indicates attenuation of nociceptive outflow, considering the role of enzyme-derived nitric oxide in pain-related mechanisms. Differential hormonal effects on these molecules agree with their localization in different cell populations.

Protein kinase C gamma-like immunoreactivity of trigeminothalamic neurons in the medullary dorsal horn of the rat.

We examined protein kinase C gamma-like immunoreactivity (PKCgamma-LI) of trigeminothalamic neurons in the rat medullary dorsal horn (MDH) after injecting a retrograde tracer, Fluoro-Gold (FG), into the thalamus. Over 90% of FG-labeled neurons in the marginal layer (lamina I) and a few FG-labeled neurons in the superficial part of the magnocellular layer (lamina III) showed PKCgamma-LI. No PKCgamma-neurons in the substantia gelatinosa (lamina II) were labeled with FG. PKCgamma-mediated regulation of trigeminothalamic neurons may contribute to the changes in MDH activity during persistent pain.

FRAP-positive and capsaicin-sensitive terminals in the substantia gelatinosa of the mouse spinal trigeminal nucleus caudalis.

FRAP (fluoride-resistant acid phosphatase)-reactivity in the substantia gelatinosa of the mouse spinal trigeminal nucleus caudalis (STNC) was examined by light and electron microscopy. Degenerated figures of terminals caused by capsaicin were compared with the FRAP-positive terminals. Scalloped (fan-like) or indented, sinuous, slender, and cap-like figures with closely packed agranular synaptic vesicles of various sizes were common to both FRAP-positive and capsaicin-sensitive terminals. These terminals had glomerular or nonglomerular endings. Sometimes FRAP-positive and capsaicin-sensitive glomerular terminals made presynapses with surrounding dendrites. Frequently, both nonglomerular terminals were in direct contact with the neuronal soma. The terminal features of FRAP-positive and capsaicin-sensitive ones in the mouse STNC are the same as those seen in the superficial dorsal horn of the spinal cord. These findings suggest that some of the FRAP-positive terminals are capsaicin-sensitive, thereby indicating their nociceptive primary afferent.

Low-frequency stimulation of afferent Adelta-fibers induces long-term depression at primary afferent synapses with substantia gelatinosa neurons in the rat.

Impulses in primary afferent nerve fibers may produce short- or long-lasting modifications in spinal nociception. Here we have identified a robust long-term depression (LTD) of synaptic transmission in substantia gelatinosa neurons that can be induced by low-frequency stimulation of primary afferent Adelta-fibers. Synaptic transmission between dorsal root afferents and neurons in the substantia gelatinosa of the spinal cord dorsal horn was examined by intracellular recording in a transverse slice dorsal root preparation of rat spinal cord. Conditioning stimulation of dorsal roots with 900 pulses given at 1 Hz (10 V, 0.1 msec) produced LTD of EPSP amplitudes in substantia gelatinosa neurons to 41 +/- 10% of control that lasted for at least 2 hr. When A- and C-fibers were recruited, conditioning stimulation was as effective as A-fiber stimulation alone. After LTD, synaptic strength could be increased to its original level by applying a second, high-frequency tetanic stimulus to the dorsal root, indicating that LTD is reversible and not attributable to damage of individual synapses. Bath application of the GABAA receptor antagonist bicuculline and glycine receptor antagonist strychnine did not affect LTD. When NMDA receptors were blocked by bath application of D-2-amino-5-phosphonovaleric acid, LTD was abolished or strongly reduced. Loading substantia gelatinosa neurons with Ca2+ chelator BAPTA also blocked or reduced LTD. After incubation of slices with calyculin A, a selective and membrane permeable inhibitor of protein phosphatases 1 and 2A, LTD was not attenuated. We propose that this form of LTD may be relevant for long-lasting segmental antinociception after afferent stimulation.

Fluoride-resistant acid phosphatase (FRAP)-positive afferent terminals make synaptic contact with interneuronal soma in the substantia gelatinosa of the mouse spinal dorsal horn.

Fluoride-resistant acid phosphatase (FRAP)-reactive terminals making contact with interneuronal soma are found in the substantia gelatinosa of the mouse spinal dorsal horn. About one half of the interneuronal somata receive FRAP-positive boutons. By electron microscopy, these FRAP-positive terminals appear small, dark, slender, roundish, cap-like, ellipsoid or sinuous and electron-dense, scalloped (fan-like) contours with clear spherical synaptic vesicles of variable size, some large dense-core vesicles and mitochondria. All these features are very similar to those of capsaicin-sensitive terminals. Thus they are considered to be nociceptive primary afferent endings. Therefore, some of the FRAP-positive terminals are suggested to have a modulatory role in the nociceptive circuit in the substantia gelatinosa.

[3H]phorbol 12,13-dibutyrate/PKC binding in thoracic spinal cord: no change in amyotrophic lateral sclerosis.

[3H]phorbol 12,13-dibutyrate ([3H]PDBu), a selective ligand for various protein kinase C isozymes (PKC), was used to investigate the distribution of [3H]PDBu/PKC binding sites in thoracic spinal cords of patients who died with amyotrophic lateral sclerosis (ALS) and subjects free of neurological disease. In controls, binding of [3H]PDBu was mostly concentrated in the substantia gelatinosa and to a lesser extent in other dorsal horn regions (laminae III and IV). [3H]PDBu binding sites were also present in the ventral horn and laminae X but in somewhat lower quantities. The distribution of [3H]PDBu binding in thoracic spinal cords of patients who died with ALS was unchanged compared to controls and no significant differences were observed in the amount of specific binding in ALS. The present results on the distribution of [3H]PDBu binding sites in human spinal cord are consistent with previous studies of [3H]PDBu binding in rodents indicating that high levels of PKC are present in the dorsal horn. The results also suggest that levels of PKC isozymes are not altered in ALS spinal cord.

NADPH diaphorase in the spinal cord of rats.

To identify spinal neurons that may synthesize nitric oxide, cells and fibers histochemically stained for NADPH diaphorase (a nitric oxide synthase) were studied in the spinal cord of rats. The histochemical reaction gave an image similar to the best Golgi impregnations, staining cells down to their finest processes. Transverse, horizontal, and parasagittal 50 and 100 microns sections were used to follow dendritic and axonal arborizations of stained neurons. Major cell groups were identified in the superficial dorsal horn and around the central canal (at all spinal levels), and in the intermediolateral cell column (at thoracic and sacral levels). Scattered positive cells were also found in deeper dorsal horn, ventral horn, and white matter. In some cases, axons of cells in the dorsal horn could be traced into the white matter; many of these cells resembled neurons projecting to various supraspinal targets. Stained cells in the intermediolateral column, which sent their axons into the ventral root, were presumed to be preganglionic autonomic neurons. Dense plexes of fibers were stained in laminae I and II and in the intermediolateral column. A large number of NADPH diaphorase-positive neurons in the spinal cord appear to be involved in visceral regulation. Fibers of the intermediolateral system had a special relationship with vasculature, suggesting that nitric oxide may help to couple neural activity with regional blood flow in the spinal cord. The abundance of NADPH diaphorase-positive neurons and fibers in the superficial dorsal horn suggests that nitric oxide may also be involved in spinal sensory processing.

Distribution of glomeruli with fluoride-resistant acid phosphatase (FRAP)-containing terminals in the substantia gelatinosa of the rat.

In the cervical enlargement of the rat spinal cord, fluoride-resistant acid phosphatase (FRAP) occurs in most of the small dark sinuous primary afferent central terminals (CI-terminals) of type I-synaptic glomeruli of lamina II and is lacking in the large light roundish primary afferent CII-terminals of type II-glomeruli. Reactive CI-terminals are heterogeneously distributed across the dorsoventral thickness of lamina II, with maximal frequency in the zone heavily stained for FRAP in light micrographs, which roughly corresponds to the dorsalmost portion of lamina IIi. In the rest of lamina II many CI-terminals do not contain FRAP. Since all CI-terminals appear to originate from unmyelinated, presumably nociceptive, primary afferents, it is proposed that the FRAP-reactive ones arise from the FRAP-containing subpopulation of DRG small cells, while those lacking FRAP, which have a distinct area of termination in the dorsal horn, belong to the peptide-containing subpopulation.

Morphine blocks the increase in acid phosphatase in the substantia gelatinosa during pain.

Acid phosphatase activity in the rat substantia gelatinosa has been shown to increase in response to a formalin-induced painful stimulus. In the rat the substantia gelatinosa is the location of the first synapse in the pain pathway. One site of morphine's analgesic effect is at this first synapse. The present study shows that morphine blocks the previously observed increase in acid phosphatase activity during a painful stimulus. Naloxone antagonizes the morphine effect. These results point to a possible functional role of acid phosphatase in the afferent transmission of pain signals.

The confronting cisternae in neurons in the substantia gelatinosa of the spinal cord of rats and Japanese monkeys.

The neurons in the substantia gelatinosa of the spinal cord of the rat and Japanese monkey were investigated electron microscopically and ultracytochemically. The confronting cisternae observed in the cytoplasm of the majority of gelatinosal neurons in both species were composed of closely apposed parallel cisternae with electron-dense flocculent material, and a continuity with the cisternae of the rough endoplasmic reticulum was often observed. Ultracytochemically, the p-nitrophenylphosphatase (p-NPPase) activity was present in the confronting cisternae, but thiamine monophosphatase (TMPase) and thiamine pyrophosphatase (TPPase) activities were absent. These results indicate that the confronting cisternae are a variant of the rough endoplasmic reticulum.

Histochemical localization of acetylcholinesterase in the glycogen body (sinus rhomboidalis) of common brown dove, Streptopelia senegalensis and house sparrow, Passer domesticus.

Acetylcholinesterase (AChE) activity was studied in the glycogen bodies of the spinal cords of 2 birds namely Streptopelia senegalensis and Passer domesticus. A possible functional significance of AChE in the light of relative enzymatic localization especially in Hoffmann-Kolliker nuclei (motor cell groups), substantia gelatinosa and other regions of gray matter of 2 avian glycogen bodies has been discussed.

Changes in acid phosphatase activity in the substantia gelatinosa in response to pain.

Two-day and 15-day-old offspring of capsaicin-pretreated Wistar rats were given subcutaneous injections of a 5% formalin solution to the dorsal aspect of the right forepaw. One hour after injection, acid phosphatase activity of the substantia gelatinosa of formalin/capsaicin cervical sections was significantly greater bilaterally than in saline/capsaicin 15-day-old animals. In 2-day-old animals the formalin/control right cervical sections had significantly higher acid phosphatase activity than saline/control. These results are further support for a direct functional relation between activity in the substantia gelatinosa and chemogenic pain stimulus.

[Oblongata in the spinal trigeminal nucleus of the adult rat (author's transl)].

The substantia gelationsa has been noted as a region of central pain control, In 1979, we reported that an intense thiamine monophosphatase (TMPase) activity was specifically localized on the plasma membrane of the synaptic glomeruli, especially dense sinusoid axon terminals (DSA). In the present investigation, the ultracytochemical localization of TMPase activity was studied in the spinal trigeminal nucleus of the adult rat. Vibratome sections of aldehyde-fixed specimens were incubated in the incubation medium (veronal-acetate buffer, pH 5. 8, 27 mM, thiamine monophosphoric acid chloride 2mM, lead nitrate 2.7 mM, sucrose 146 mM, final pH 5.4) for 30 min at 37 degrees C, and processed for light as well as electron microscopy. At the light microscopic level, the reaction products, lead sulfide, were observed to be localized in the substantia gelatinosa of the caudal medulla oblongata in the spinal trigeminal nucleus. At the electron microscopic level, the TMPase activity was positive on the plasma membrane of DSA in the region where is particularly concerned with the relay of thermal and noxious stimuli. It has been suggested by us previously that there may be some relationship between the TMPase activity and pain, mechanism in the spinal cord, and the results that the situation seems to be hold true in the spinal trigeminal nucleus, also.

Appearance of acid phosphatase in neonatal rat substantia gelatinosa.

The onset of acid phosphatase activity was observed in neonatal rat substantia gelatinosa using the Gomori method. Although acid phosphatase activity was not present at birth it appeared during the first day postnatally. By six to ten days postnatally enzyme activity appeared to reach its adult level. The activity was quantified using atomic absorption spectrophotometry which showed that acid phosphatase activity reached its adult level by 6 days postnatally. Acid phosphatase in the substantia gelatinosa is fluoride resistant from its first appearance.