Phosphorescent platinum/palladium coproporphyrins for time-resolved luminescence microscopy

The rostral ventromedial medulla (RVM) is an important mediator of the supraspinal component of opioid antinociception. Previous studies have suggested that activation of the cloned mu- and delta-opioid receptors (MOR1 and DOR1 respectively) in the RVM produces the antinociception mediated by spinally projecting neurons. In the present study, we investigated the expression of mRNA encoding either MOR1 or DOR1 in the RVM of rats. In addition, we examined quantitatively the expression of MOR1 and DOR1 mRNAs in spinally projecting RVM neurons including serotonergic (5HT) cells by using in situ hybridization, immunocytochemistry, retrograde tract-tracing, and the physical disector. Brainstem neurons were labeled in 14 male Sprague-Dawley rats by applying Fluoro-Gold (FG) topically to the dorsal surface of the lumbosacral spinal cord. Five-micrometer-thick cryostat sections were cut and in situ hybridization was performed by using full-length cRNA probes labeled with 35S-UTP. We found that 43% of RVM projection neurons expressed MOR1 mRNA and 83% of RVM projection neurons expressed DOR1 mRNA. Of 192 retrogradely labeled cells in the RVM, 51 cells (27%) were immunoreactive for 5HT. Of this population, half appeared to be labeled for the mRNA encoding MOR1 and over three-fourths appeared to be labeled for the mRNA encoding DOR1. Thus, we conclude that bulbospinal neurons express MOR1 and DOR1; moreover, MOR1 and DOR1 are expressed by significant proportions of 5HT neurons projecting to or through the dorsal spinal cord.     

Origin, course, and laterality of spinocerebellar axons in the North American opossum, Didelphis virginiana

Inhibition of neurons containing gamma-aminobutyric acid (GABA) may underlie some of the excitatory effects of opioids in the central nervous system (CNS). In the present study, we examined the relationship of the cloned mu- and delta-opioid receptors (MOR1 and DOR1, respectively) to GABAergic neurons in brain and spinal cord. This was done by combining immunofluorescent staining for MOR1 or DOR1 with that for GABA or glutamic acid decarboxylase (GAD); fluorescent retrograde tract-tracing was used in some cases to identify neurons with particular projections. In rats, cells double labeled for GABA and MOR1 were observed in layers II-VI of the parietal cortex and in layers II-IV of the piriform cortex. In the hippocampus, double labeling was observed in the dentate gyrus and in regions CA1 and CA3. Double labeling was very prominent in the striatum and in the reticular nucleus of the thalamus; it was also observed in other portions of the diencephalon. However, double labeling for GABA and MOR1 was never observed in the cerebellar cortex. Cells double labeled for GABA and MOR1 were common in the periaqueductal gray (PAG) and the medial rostral ventral medulla (RVM) of both rats and monkeys, suggesting that involvement of GABAergic neurons with supraspinal opioid antinociception may extend to primates. In the RVM of rats, many of those double-labeled neurons were retrogradely labeled from the dorsal spinal cord. In contrast, double-labeled neurons in the PAG were almost never retrogradely labeled from the RVM. No unequivocal examples of double labeling for DOR1 and GAD were found in any region of the CNS that we examined in either rats or monkeys. However, GABAergic neurons were often apposed by DOR1 immunoreactive varicosities. Our findings suggest that activation of mu-opioid receptors directly modulates the activity of GABAergic neurons throughout the CNS, including neurons involved in the supraspinal component of opioid analgesia. In contrast, delta-opioid receptors appear to be positioned to modulate the activity of GABAergic neurons indirectly.     

Differential distribution of (GABA)A receptor subunits on bulbospinal serotonergic and nonserotonergic neurons of the ventromedial medulla of the rat

Spinally projecting neurons of the ventromedial medulla (VMM) compose an important efferent pathway for the modulation of nociception. These neurons receive a substantial gamma-aminobutyric acid (GABA)-ergic input, but the GABA receptor that mediates this input is unknown. This study examined the distribution of GABA(A) receptor alpha1 and alpha3 subunits in serotonergic and nonserotonergic neurons of the VMM that project to the dorsal horn in the rat. A pledget of Gelfoam soaked in Fluoro-Gold was placed at the thoracolumbar junction of the spinal cord to label spinally projecting neurons. Alternate sections of the medulla were then incubated with a mixture of antisera to either serotonin and the alpha1 subunit, or to serotonin and the alpha3 subunit of the GABA(A) receptor. Nearly 30% of spinally projecting neurons in the VMM were immunoreactive for the alpha1 subunit. A similar percentage of spinally projecting neurons in the VMM were immunoreactive for the alpha3 subunit, although diffuse cellular labeling combined with intense staining of processes in the neuropil precluded a rigorous semi-quantitative estimation of this population. No alpha1-subunit-immunoreactive neurons colocalized serotonin. In contrast, serotonergic neurons were immunoreactive for the alpha3 subunit. However, these double-labeled neurons were a modest percentage of the serotonergic population. A small percentage of spinally projecting serotonergic neurons was immunoreactive for the alpha3 subunit. These results suggest that significant numbers of spinally projecting serotonergic and nonserotonergic neurons of the VMM possess GABA(A) receptors that differ in their respective subunit compositions and that both classes of neurons may mediate the antinociception produced by the microinjection of GABA(A) receptor antagonists in the VMM.     

Apposition of enkephalin- and neurotensin-immunoreactive neurons by serotonin-immunoreactive varicosities in the rat spinal cord

The descending serotonergic system provides a powerful inhibitory input to the dorsal horn of the spinal cord. Little is known about the chemical identity of the spinal neurons that the serotonergic system innervates, although spinal enkephalinergic neurons are likely candidates. This study investigated the apposition of serotonin-immunoreactive varicosities onto enkephalin- and neurotensin-immunoreactive neurons in the rat lumbosacral spinal cord. Using a double immunofluorescence technique, serotonin-immunoreactive varicosities were observed to abut the soma or proximal dendrites of [Met]enkephalin- and neurotensin-immunoreactive neurons. Nearly 75% of all [Met]enkephalin- and neurotensin-immunoreactive neurons were apposed by serotonin-immunoreactive varicosities in the marginal zone and dorsal gray commissure. In substantia gelatinosa, approximately half of the [Met]enkephalin- and neurotensin-immunoreactive neurons were juxtaposed by serotonin-immunoreactive varicosities. [Met]enkephalin-immunoreactive neurons also were bordered by serotonin-immunoreactive varicosities in the nucleus proprius (65%) and sacral parasympathetic nucleus (75%). The results of this study suggest that the descending serotonergic system mediates nociception via probable contacts with intrinsic enkephalin and neurotensin spinal systems. The mode of action of spinal serotonin on enkephalin and neurotensin neurons may be through "volume" transmission vs synaptic or "wiring" transmission.     

Oxytocinergic and serotonergic innervation of identified lumbosacral nuclei controlling penile erection in the male rat

Penile erection is due to activation of proerectile neurons located in the sacral parasympathetic nucleus of the L6-S1 spinal cord in the rat. Contraction of the ischiocavernosus and bulbospongiosus striated muscles, controlled by motoneurons located in the ventral horn of the L5-L6 spinal cord, reinforces penile erection. Physiological and pharmacological arguments have been provided for a role of oxytocin and serotonin in the spinal regulation of penile erection. Immunohistochemistry of oxytocinergic and serotonergic fibres was performed at the lumbosacral level of the male rat spinal cord, and combined with retrograde tracing from the pelvic nerve or from the ischiocavernosus and bulbospongiosus muscles using wheat germ agglutinin-horseradish peroxidase. Sacral preganglionic neurons retrogradely labelled from the pelvic nerve formed a homogeneous population, predominant at the L6 level. Motoneurons retrogradely labelled from the ischiocavernosus and bulbospongiosus muscles were observed in the medial part of the dorsolateral and in the dorsomedial nuclei. Fibres immunoreactive for oxytocin were mainly distributed in the superficial layers of the dorsal horn, the dorsal gray commissure and the sacral parasympathetic nucleus. Some of these fibres were apposed to retrogradely-labelled sacral preganglionic neurons and at the ultrastructural level, some synapses were evidenced. Fibres immunoreactive for serotonin were largely and densely distributed in the dorsal horn, the dorsal gray commissure, the sacral parasympathetic nucleus and the ventral horn. Some serotonergic fibres occurred in close apposition with retrogradely-labelled sacral preganglionic neurons and motoneurons, and synapses were demonstrated at the ultrastructural level. This study provides morphological support for a role of oxytocin and serotonin on sacral preganglionic neurons innervating pelvic organs and motoneurons innervating the ischiocavernosus and bulbospongiosus muscles.     

Noradrenergic input to nociceptive modulatory neurons in the rat rostral ventromedial medulla

Within the rostral ventromedial medulla (RVM), there are two classes of putative pain modulation neurons: ON cells and OFF cells, which respectively burst or pause prior to withdrawal reflexes elicited by noxious stimulation. Alpha-adrenergic agonists injected into the RVM produce changes in the latency of spinal nocifensive reflexes and, when iontophoretically applied, alter the firing of RVM ON but not OFF cells. To provide further information about the contribution of norepinephrine to RVM neuron function, we analyzed the distribution of tyrosine hydroxylase immunoreactive (TH-ir) appositions upon RVM ON and OFF cells. In the lightly anesthetized rat, seven ON and five OFF cells were identified by changes in their discharge rate in relation to nociceptive withdrawal reflexes and were labeled by intracellular injection of neurobiotin. Sections containing labeled cells were visualized by using avidin conjugated to a Texas Red fluorophore. Tissue with labeled cells was subsequently processed for TH-ir by using a Bodipy fluorophore conjugated secondary antibody. The distribution of the Bodipy-labeled fibers and terminals upon the Texas Red-labeled neurons was mapped using a confocal laser-scanning microscope. All the labeled neurons exhibited close TH-ir appositions. Appositions were of two types: swellings and fibers. Although the numbers and density of appositions varied among the cells, there were no consistent differences that correlated with physiological properties. Thus the overall density of appositions for ON cells (29.0 +/- 22.2 x 10(4) microns2) did not differ significantly from that for OFF cells (25.4 +/- 22.2 x 10(4) microns2). Tyrosine hydroxylase immunoreactive (TH-ir) appositions upon ON and OFF cells varied with their location along the dorso-ventral axis with more ventral neurons having a greater density of TH-ir swelling-type appositions. In a separate study, TH-ir and dopamine-beta-hydroxylase-like immunoreactivity (DBH-ir) were mapped in the same sections by using confocal microscopy. Nearly 97% of the TH-ir profiles co-localized with DBH-ir. These observations provide evidence that both ON and OFF cells in the RVM are targeted by noradrenergic inputs.     

A procedure for in situ hybridization combined with retrograde labeling of neurons: application to the study of cell adhesion molecule expression in Dil-labeled rat pyramidal neurons

We have devised a simple method that combines retrograde labeling of projecting neurons and in situ hybridization histochemistry to examine mRNA expression in the retrogradely labeled neurons. First, projecting neurons were retrogradely labeled in vivo by injection of the lipophilic neuronal tracer Dil. The fluorescence of the labeled neurons in the brain slices was photoconverted into stable DAB precipitate by green light illumination. The slices were cut into thinner sections and processed for detection of specific mRNA by in situ hybridization. Using this highly sensitive method, we demonstrate here that the corticospinal tract neurons in newborn rats express mRNA for the cell adhesion molecule L1. TAG-1 mRNA was not detected in these neurons. Therefore, the present method provides an important tool to study the molecular expression of projection neurons during the development of neuronal circuitry.     

Optimized alcoholytic deacetylation of N-acetyl-blocked polypeptides for subsequent Edman degradation

Myenteric neurons projecting to the mucosa of the guinea pig proximal colon were identified using the combination of a neuronal tracing method and immunohistochemical techniques. The tracer DiI (1, 1'didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) was applied onto the mucosa of a specimen of proximal colon which was then placed in organotypic culture to allow retrograde transport of the dye. After culture, the myenteric plexus was stained with antisera raised against choline acetyltransferase (ChAT) and calbindin (Calb). Of the myenteric neurons labeled with DiI, 99% had smooth cell bodies with Dogiel Type II morphology. Of these neurons, 70% projected in the longitudinal direction and the majority of them (65%) were located anally from the DiI application site, i.e., had ascending projections. Ascending neurons projected over significantly shorter distances than descending ones (3.1+/-0.5 mm vs. 4.6+/-1.2 mm, respectively; P<0.01). Of the labeled myenteric neurons, 98% were ChAT immunoreactive. Of these neurons, 78% were also immunoreactive for Calb and were preferentially ascending neurons. ChAT-immunoreactive but Calb-negative neurons did not have preferential projection. This study revealed the presence of two populations of myenteric neurons projecting to the mucosa of the guinea pig proximal colon. Morphological characteristics and neurochemical coding were suggestive for a putative sensory function for these neurons.     

Projections from the lateral mammillary nucleus to the anterodorsal thalamic nucleus in the rat

There has been an abundance of research on the connections of the mammillary bodies but the projections from the lateral mammillary nucleus to the anterodorsal thalamic nucleus has remained a gray area due to a dearth of material which directly addresses the details of this pathway. This study seeks to further define the nature of this particular nerve connection within the mammillothalmic tract. The technique employed is fluorescent nerve tract tracing using two fluorescent tracers implanted separately into each anterodorsal thalamic nucleus then followed retrogradely to the soma of the neurons in the lateral mammillary nucleus. Fluorescent photomicrography allowed us to document the single and double labeled cells of the lateral mammillary nucleus. The single labeled cells can be categorized into ipsilaterally projecting neurons and contralaterally projecting neurons. About half of all labeled cells were bilaterally projecting double-labeled, a third was ipsilaterally projecting single-labeled and the remainder were contralaterally projecting single labeled-cells. There were no labeled cells traced to the medial mamillary nucleus. The mammillary bodies play an important role in the limbic circuitry and a part of the so-called "Papez Circuit". The pathway by which the mammillary body projects to the other structures of the limbic system and the way it connects the limbic system to other parts of the brain like the tegmentum is not fully understood. This clarification of the connection between the lateral mammilary nucleus and the anterodorsal thalamic nucleus is but one of the contemplated pathways.     

GABA- and glutamate-immunoreactive synapses on sympathetic preganglionic neurons projecting to the superior cervical ganglion

Our previous work suggests that virtually all of the synapses on sympathetic preganglionic neurons projecting to the rat adrenal medulla are immunoreactive for either the inhibitory amino acid, gamma-aminobutyric acid (GABA) or the excitatory amino acid, L-glutamate. To investigate whether or not this is true for other groups of sympathetic preganglionic neurons, and to determine whether or not the proportion of inputs containing each type of amino acid neurotransmitter is the same for different groups of sympathetic preganglionic neurons, we retrogradely labelled rat and rabbit sympathetic preganglionic neurons projecting to the superior cervical ganglion and used post-embedding immunogold on ultrathin sections to localise GABA- and glutamate-immunoreactivity. The cell bodies and dendrites of both rat and rabbit sympathetic preganglionic neurons projecting to the superior cervical ganglion received synapses and direct contacts from nerve fibres immunoreactive for GABA and from nerve fibres immunoreactive for glutamate. In the rat, GABA was present in 48.9% of the inputs to sympathetic preganglionic neurons projecting to the superior cervical ganglion, and glutamate was present in 51.7% of inputs. Double immunogold labelling for glutamate and GABA on the same section, as well as labelling of consecutive serial sections for the two antigens, indicated that GABA and glutamate occur in separate populations of nerve fibres that provide input to rat sympathetic preganglionic neurons projecting to the superior cervical ganglion. We now have shown that GABA or glutamate is present in virtually all of the inputs to sympathetic preganglionic neurons projecting to the superior cervical ganglion and in essentially all of the inputs to sympathetic preganglionic neurons supplying the adrenal medulla. These findings are consistent with the hypothesis that all fast synaptic transmission in central autonomic pathways may be mediated by either excitatory or inhibitory amino acids. Furthermore, we showed a statistically significant difference in the proportion of glutamate-immunoreactive inputs between sympathetic preganglionic neurons projecting to the superior cervical ganglion and sympathoadrenal neurons (data from Llewellyn-Smith et al. [Llewellyn-Smith, I.J., Phend, K.D., Minson, J.B., Pilowsky, P.M., Chalmers, J.P., 1992. Glutamate immunoreactive synapses on retrogradely labelled sympathetic neurons in rat thoracic spinal cord. Brain Res. 581, 67-80]), with preganglionics supplying the adrenal medulla receiving more excitatory inputs than those supplying the superior cervical ganglion. This increased excitatory input to sympathoadrenal neurons may explain the predominant activation of these neurons following baroreceptor unloading.     

Projections of GABAergic and cholinergic basal forebrain and GABAergic preoptic-anterior hypothalamic neurons to the posterior lateral hypothalamus of the rat

Within the basal forebrain, gamma-aminobutyric acid (GABA)-synthesizing neurons are codistributed with acetylcholine-synthesizing neurons (Gritti et al. [1993] J. Comp. Neurol. 329:438-457), which constitute one of the major forebrain sources of subcortical afferents to the cerebral cortex. In the present study, descending projections of the GABAergic and cholinergic neurons were investigated to the lateral posterior hypothalamus (LHp) through which the medial forebrain bundle passes and where another major forebrain source of subcortical afferents is situated. Retrograde transport of cholera toxin b subunit (CT) from the LHp was combined with immunohistochemical staining for glutamic acid decarboxylase (GAD) and choline acetyl transferase (ChAT) using a sequential peroxidase-antiperoxidase (PAP) technique. A relatively large number of GAD+ neurons (estimated at approximately 6,200), which represented > 15% of the total population of GAD+ cells in the basal forebrain (estimated at approximately 39,000), were retrogradely labeled from the LHp. These cells were distributed through the basal forebrain cell groups, where ChAT+ cells are also located, including the medial septum and diagonal band nuclei, the magnocellular preoptic nucleus, and the substantia innominata, with few cells in the globus pallidus. In these same nuclei, a small number of ChAT+ cells were retrogradely labeled (estimated at approximately 800), which represented only a small percentage (< 5%) of the ChAT+ cell population in the basal forebrain (estimated at approximately 18,000). Both the GAD+ and ChAT+ LHp-projecting neurons represented a small subset of their respective populations in the basal forebrain, distinct from the magnocellular, presumed cortically projecting, basal neurons. In addition to the GAD+ cells in the basal forebrain, GAD+ cells in the adjacent preoptic and anterior hypothalamic regions were also retrogradely labeled in significant numbers (estimated at approximately 5,500) and proportion (> 20%) of the total population (estimated at approximately 30,000) from the LHp. The retrogradely labeled GAD+ neurons were distributed in continuity with those in the basal forebrain through the lateral preoptic area, medial preoptic area, bed nucleus of the stria terminals, and anterior and dorsal hypothalamic areas. Of the large number of cells that project to the LHp in the basal forebrain and preoptic-anterior hypothalamic regions (estimated at approximately 66,000), the GAD+ neurons represented a significant proportion (> 15%) and the ChAT+ neurons a very small proportion (approximately 2%). The relative magnitude of the GABAergic projection suggests that it may represent an important inhibitory influence of the descending efferent output from the basal forebrain and preoptic-anterior hypothalamic regions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dopamine neurons make glutamatergic synapses in vitro

Interactions between dopamine and glutamate play prominent roles in memory, addiction, and schizophrenia. Several lines of evidence have suggested that the ventral midbrain dopamine neurons that give rise to the major CNS dopaminergic projections may also be glutamatergic. To examine this possibility, we double immunostained ventral midbrain sections from rat and monkey for the dopamine-synthetic enzyme tyrosine hydroxylase and for glutamate; we found that most dopamine neurons immunostained for glutamate, both in rat and monkey. We then used postnatal cell culture to examine individual dopamine neurons. Again, most dopamine neurons immunostained for glutamate; they were also immunoreactive for phosphate-activated glutaminase, the major source of neurotransmitter glutamate. Inhibition of glutaminase reduced glutamate staining. In single-cell microculture, dopamine neurons gave rise to varicosities immunoreactive for both tyrosine hydroxylase and glutamate and others immunoreactive mainly for glutamate, which were found near the cell body. At the ultrastructural level, dopamine neurons formed occasional dopaminergic varicosities with symmetric synaptic specializations, but they more commonly formed nondopaminergic varicosities with asymmetric synaptic specializations. Stimulation of individual dopamine neurons evoked a fast glutamatergic autaptic EPSC that showed presynaptic inhibition caused by concomitant dopamine release. Thus, dopamine neurons may exert rapid synaptic actions via their glutamatergic synapses and slower modulatory actions via their dopaminergic synapses. Together with evidence for glutamate cotransmission in serotonergic raphe neurons and noradrenergic locus coeruleus neurons, the present results suggest that glutamatergic cotransmission may be the rule for central monoaminergic neurons.     

An immunocytochemical investigation of the relationship between substance P and the neurokinin-1 receptor in the lateral horn of the rat thoracic spinal cord

The relationship between substance P-containing axons and sympathetic preganglionic neurons possessing the neurokinin-1 receptor was investigated in the lateral horn of the rat thoracic spinal cord. Sympathetic preganglionic neurons were labelled retrogradely with Fluorogold. Sections containing labelled cells were reacted with antibodies against choline acetyltransferase, substance P and the neurokinin-1 receptor and examined with three-colour confocal laser scanning microscopy. In all, 95 sympathetic preganglionic neurons were examined and 79% of these were immunoreactive for the neurokinin-1 receptor. Substance P-immunoreactive axons not only made contacts with preganglionic neurons which were immunoreactive for the receptor but also made contacts with cells which did not express the receptor. Dendrites, labelled with immunoreactivity for choline actyltransferase, also received contacts from substance P-immunoreactive varicosities but this was not related to the presence or the absence of receptor. An electron microscopic analysis was performed to investigate the relationship between substance P-containing boutons and dendrites possessing the neurokinin-1 receptor. Immunoreactivity for substance P was detected with peroxidase immunocytochemistry and immunoreactivity for the receptor was detected with the silver-intensified gold method. Substance P-containing boutons made synapses with dendrites which were positively and negatively labelled for the receptor. Receptor immunoreactivity was not usually present at synapses formed by substance P boutons with neurokinin-1-immunoreactive dendrites. It is concluded that substance P may modulate much of the activity of sympathetic preganglionic neurons through an indirect non-synaptic mechanism.     

Does licensing of drugs in industrialized countries guarantee drug quality and safety for Third World countries? The case of norplant licensing in Finland

A knowledge of the anatomy of medullary serotonergic cells is critical to understanding local and brainstem circuits in which these cells participate. Serotonergic neurons (n = 16) were identified, as previously described (Mason [1997] J. Neurophysiol. 77:1087-1098) by their slow and steady background discharge in halothane anesthetized rats. Neurons were then intracellularly labeled with Neurobiotin and visualized with 3,3'diaminobenzidine. The validity of the physiological identification of serotonergic cells was confirmed by processing two neurons that were physiologically characterized as serotonergic for serotonin immunoreactivity; both tested cells contained immunoreactive serotonin. The dendrites and axon of each labeled cell were reconstructed by using a three-dimensional computerized system. Somata were small or medium in size and had fusiform, triangular, or multipolar shapes. The dendritic arbor was constricted with most dendrites extending for less than 500 microm from the soma. All labeled axons projected caudally and travelled in the ventrolateral medulla, either dorsal or ventral to the lateral reticular nucleus. Most cells had collaterals and/or dense axonal swellings in the nucleus reticularis gigantocellularis, nucleus reticularis magnocellularis, raphe magnus, and the ventrolateral medulla. Non-local collaterals and swellings were also observed in the nucleus reticularis gigantocellularis and in the ventrolateral medulla at all medullary levels. The results demonstrate that 1) the dendrites of serotonergic cells are restricted to raphe magnus and the ventral part of nucleus reticularis magnocellularis; and 2) serotonergic cells project to medullary nuclei that contain bulbospinal cells which project to dorsal, intermediate, and ventral horns. Serotonergic cell projections to brainstem sites may mediate the integration of sensory, autonomic, and motor modulation at the brainstem level.     

Antinociception mediated by the periaqueductal gray is attenuated by orphanin FQ

Orphanin FQ or nociceptin (OFQ/N(1-17)) is a recently discovered peptide which, upon intracerebroventricular administration, reverses opioid-mediated analgesias. OFQ/N(1-17) terminals are located in the periaqueductal gray (PAG), a structure known to be involved in pain modulation, suggesting that the functional anti-opioid effects of OFQ/N(1-17) are mediated by PAG neurons. To test this, subsequent microinjections of morphine or kainic acid and OFQ/N(1-17) were made into the PAG of awake rats. Administration of OFQ/N(1-17) attenuated the tail flick inhibition produced by both morphine and kainic acid microinjection. OFQ/N(1-17) attenuation of antinociception produced by a neuroexcitant indicates that OFQ/N(1-17) reverses opioid antinociception by inhibiting PAG output neurons.     

Dual ultrastructural immunocytochemical labeling of mu and delta opioid receptors in the superficial layers of the rat cervical spinal cord

The delta opioid receptor (DOR) and mu opioid receptor (MOR) are abundantly distributed in the dorsal horn of the spinal cord. Simultaneous activation of each receptor by selective opiate agonists has been shown to result in synergistic analgesic effects. To determine the cellular basis for these functional associations, we examined the electron microscopic immunocytochemical localization of DOR and MOR in single sections through the superficial layers of the dorsal horn in the adult rat spinal cord (C2-C4). From a total of 270 DOR-labeled profiles, 49% were soma and dendrites, 46% were axon terminals and small unmyelinated axons, and 5% were glial processes. 6% of the DOR-labeled soma and dendrites, and < 1% of the glial processes also showed MOR-like immunoreactivity (MOR-LI). Of 339 MOR-labeled profiles, 87% were axon terminals and small unmyelinated axons, 12% were soma and dendrites, and 2% were glial processes. 21% of the MOR-labeled soma and dendrites, but none of the axon terminals also contain DOR-LI. The subcellular distributions of MOR and DOR were distinct in axon terminals. In axon terminals, both DOR-LI and MOR-LI were detected along the plasmalemma, but only DOR-LI was associated with large dense core vesicles. DOR-labeled terminals formed synapses with dendrites containing MOR and conversely, MOR-labeled terminals formed synapses with DOR-labeled dendrites. These results suggest that the synergistic actions of selective MOR- and DOR-agonists may be attributed to dual modulation of the same or synaptically linked neurons in the superficial layers of the spinal cord.     

Olivocochlear innervation of inner and outer hair cells during postnatal maturation: evidence for a waiting period

Reconstructions of the efferent innervation of the hamster (Mesocricetus auratus) cochlea were done during postnatal development. Efferent neurons were labeled via injections of biocytin and horseradish peroxidase into the crossed olivocochlear (OC) bundles using an in vitro brainstem technique. Such injections retrogradely labeled cell bodies in ventral periolivary regions of the superior olive consistent with their being medial OC neurons. Anterogradely labeled axons were traced to the cochlea, where they terminated on or below inner hair cells (IHCs) prior to postnatal day 5 (P5). After P5, labeled axons terminated on IHCs and outer hair cells (OHCs) and after P10, the majority of labeled axons terminated on the OHCs. In the electron microscope, small labeled terminals containing densely packed synaptic vesicles were found both adjacent to IHCs (axosomatic) as well as apposed to afferent and efferent fibers below IHCs prior to P5. By P10, large labeled terminals were axosomatic to OHCs and no longer found on IHCs. Consistent with previous reports, these data suggest that medial OC axons form part of an early primary innervation on and below IHCs before terminating on OHCs. This raises the possibility that OC neurons demonstrate a period of waiting below an intermediate target similar to that described in the development of thalamocortical projections.     

Dose-dependent pain-facilitatory and -inhibitory actions of neurotensin are revealed by SR 48692, a nonpeptide neurotensin antagonist: influence on the antinociceptive effect of morphine

Neurotensin has bipolar (facilitatory and inhibitory) effects on pain modulation that may physiologically exist in homeostasis. Facilitation predominates at low (picomolar) doses of neurotensin injected into the rostroventral medial medulla (RVM), whereas higher doses (nanomolar) produce antinociception. SR 48692, a neurotensin receptor antagonist, discriminates between receptors mediating these responses. Consistent with its promotion of pain facilitation, the minimal antinociceptive responses to a 30-pmol dose of neurotensin microinjected into the RVM were markedly enhanced by prior injection of SR 48692 into the site (detected using the tail-flick test in awake rats). SR 48692 had a triphasic effect on the antinociception from a 10-nmol dose of neurotensin. Antinociception was attenuated by femtomolar doses, attenuation was reversed by low picomolar doses (corresponded to those blocking the pain-facilitatory effect of neurotensin) and the response was again blocked, but incompletely, by higher doses. The existence of multiple neurotensin receptor subtypes may explain these data. Physiologically, pain facilitation appears to be a prominent role for neurotensin because the microinjection of SR 48692 alone causes some antinociception. Furthermore, pain-facilitatory (i.e., antianalgesic) neurotensin mechanisms dominate in the pharmacology of opioids; the response to morphine administered either into the PAG or systemically was potentiated only by the RVM or systemic injection of SR 48692. On the other hand, reversal of the enhancement of antinociception occurred under certain circumstances with SR 48692, particularly after its systemic administration.     

Development of serotoninergic chick retinal neurons

Numerous neurotransmitters have been studied in detail in the developing retina. Almost all known neurotransmitters and neuromodulators were demonstrated in vertebrate retinas using formaldehyde-induced fluorescence, uptake autoradiography or immunohistochemistry procedures. Serotoninergic (5HT) amacrine neurons were described in the inner nuclear layer (INL) of the retina with their dendrites spreading within the inner plexiform layer (IPL). The present work describes the morphological pattern of development of serotoninergic amacrine neurons with a stratified dendritic branching pattern in the chick retina from embryonic day 12 to postnatal day 7. Serotoninergic-bipolar neurons are also described. SHT-amacrine neurons have round or pear-shaped somata and primary dendritic trees oriented toward the IPL that runs through the INL, showing several varicosities. Secondary dendrites then go through the INL, without any collateral branch. At the outer and inner margin of the IPL the primary and secondary dendrites originate an outer and an inner serotoninergic network, respectively. When the primary dendritic tree reaches the IPL it deflects laterally in sublayer 1-the outer serotoninergic network. Tertiary branches then arise from the secondary dendrite and deflect in the innermost sublayer of the IPL-the inner serotoninergic network. The final pattern of branching of 5HT amacrine cells was present at embryonic day 14 and was completely developed at hatching. Serotoninergic (5HT) bipolar neurons were also present in the INL at hatching. They are weakly immunoreactive and are probably a subset of bipolar cells that accumulate serotonin from the intersynaptic cleft and are not "true" 5HT neurons.